Derivatives of peptides, the method of production thereof, farmcampsite and method of inhibiting thrombin in a mammal

 

(57) Abstract:

The peptide derivative of General formula I

X - Y - NH - (CH2)r- G

or its pharmaceutically acceptable salt, or MES, where the values of X, Y, r, G, specified in paragraph 1 of the claims, which may find use in inhibiting thrombin in a mammal. Describes the method of obtaining them, farmcampsite and method of inhibiting thrombin in a mammal. 4 S. and 2 C.p. f-crystals, 2 tab.

The present invention relates to inhibitors of thrombin, which are used as anticoagulants in mammals. In particular, it relates to a peptide derivative having high antithrombotic activity, anticoagulant activity and oral bioavailability.

The process of blood coagulation, thrombosis, is called a complex proteolytic cascade leading to the formation of thrombin. Thrombin proteoliticeski destroys the activation peptides of A-chains and B-chains of fibrinogen, which is soluble in blood plasma, initiating the formation of insoluble fibrin.

Anticoagulation usually reach by introducing heparins and coumarins.

Parenteral pharmacological control megastat on thrombin by accelerating inhibitory effects of endogenous anti-thrombin-III (main physiological inhibitor of thrombin). Because the levels of anti-thrombin-III in plasma changes and due to the fact that the boundary surface of thrombin, apparently, taken in relation to this indirect mechanism, heparins can be inefficient. Since the tests for coagulation is considered an effective and safe levels of heparin should be monitored through analysis of coagulation (especially the determination of activated partial thromboplastin time (APTT/). Coumarins inhibit the generation of thrombin by blocking post-translational gamma-carboxylation in the synthesis of prothrombin and other proteins of this type. Due to their mechanism, the effects of coumarins may occur only slowly, after 6-24 h after injection. Moreover, they are non-selective anticoagulants. Therefore, coumarins require monitoring through analysis of coagulation (especially the analysis of the prothrombin time /PT/).

Currently increased interest in small synthetic peptides that are recognized by proteolytic enzymes in the same way as natural substrates. Tripeptide-aldehydes such as D-Phe-Pro-Arg-H, Boc-D-Phe-Pro-Arg-H, D-MePhe-Pro-Arg-H, Bajuszetal. , J. Med.Chem., 33, 1729-1735 (1990), show a strong direct ing what coagulants people described Simoons and others, Circulation, 90, 1-231, Abstr. 1241 (1994). Numerous researchers have synthesized analogues in an attempt to obtain pharmaceutical agents, as, for example, Shuman and others, J. Med. Chem., 36, 314-319 (1993). In U.S. patent N 4346078 provides a number of anticoagulant peptides containing agmatinase (1-amino-4-guanidinate) group. Agmatine derivatives and related compounds described in PCT application with international publication number WIPO 93/11152, and in European patent application, publication N 601459 of 15 June 1994, These compounds differ from the previous series, in which agmatinase compounds do not contain carbonyl groups present in these compounds with Arg-group.

Although the heparins and coumarins are effective anticoagulants, still not appeared medicinal product on the basis of known Tripeptide-aldehyde, despite the fact that the term for this class of compounds, there is therefore a need for anticoagulants, which selectively affect thrombin and, regardless of anti-thrombin-III, have an inhibitory effect soon after administration, preferably oral route, and not interfere with the lysis of blood clots, necessary to maintain hemostasis, as indicated below, are strong inhibitors of thrombin, can have high bioavailability when administered orally. In addition, some compounds of the present invention can also cause inhibition of factor Xa, which is involved in coagulants cascade.

Therefore, the first object of the present invention are new peptide derivatives, which are strong inhibitors of thrombin, suitable as anticoagulants.

Other objects, features and advantages will be clear to experts from the following descriptions and claims.

The present invention relates to thrombin inhibitory compound having the formula I,

X-Y-NH-(CH2)r-G (I)

where X denotes Proline; homopolymer; Rm-(CH2)g-NH-CH2-C(O)-,

< / BR>
< / BR>
where Rd- carboxy or methylsulphonyl;

Re- OTHERc, NHCORcor NHCOORcwhere Rc- C1-C10alkyl, C3-C8cycloalkyl or (C3-C8)cycloalkyl(C1-C6)alkyl with 4 to 10 carbon atoms;

T - C3-C8cycloalkyl, C1-C8alkyl,

< / BR>
a = 0, 1 or 2 and

Q denotes-OH, C1-C4and the B>-Rm;

g = 1, 2, or 3;

B is hydrogen or (C1-C4)-alkyl;

R' is hydrogen or (C1-C4)-alkyl;

R" - C1-C4alkyl, C1-C4perfluoroalkyl, -(CH2)d-Rmor unsubstituted or substituted aryl, where aryl refers to phenyl, naphthyl, 5 - or 6-membered unsubstituted or substituted aromatic heterocyclic ring containing one or two heteroatoms that are the same or different and which are selected from sulfur, oxygen and nitrogen, or a 9 - or 10-membered, unsubstituted or substituted condensed-bicyclic aromatic heterocyclic group with one or two heteroatoms that are the same or different and which are selected from sulfur, oxygen or nitrogen;

Rm- A-COORb, SO2(C1-C4)-alkyl, -SO3H, -P(O)(ORb)2or tetrazol-5-yl;

Rn- A-COORbor tetrazol-5-yl;

each Rbindependently from each other hydrogen or C1-C4alkyl;

d = 1, 2, or 3;

m = 0, 1, or 2;

n = 1 or 2; and

Z is hydrogen, C1-C4alkyl, C1-C4alkoxy, hydroxy, halogen, or RaSO2NH, where Ra- C1-C4alkyl;

Y means:

< / BR>
where BR>Rpis hydrogen, C1-C6alkyl, C3-C8cycloalkyl or -(CH2)p-L-(CH2)q-T';

where p = 0, 1, 2, 3 or 4;

L is a bond, -O-, -S - or-NH-;

q = 0, 1, 2 or 3;

T' is hydrogen, C1-C4alkyl, C3-C8cycloalkyl, -COOH, -CONH2or Ar, where Ar denotes unsubstituted or substituted aryl, where aryl is phenyl, naphthyl; a 5 - or 6-membered unsubstituted or substituted aromatic heterocyclic ring with one or two heteroatoms that are the same or different and selected from sulfur, oxygen and nitrogen; or a 9 - or 10-membered unsubstituted or substituted condensed-bicyclic aromatic heterocyclic group with one or two heteroatoms that are the same or different and selected from sulfur, oxygen and nitrogen;

RY- -CH2-, -O-, -S - or-NH-;

RZlink, or together with RYand the three neighboring carbon atoms form a saturated carbocyclic ring of 5-8 atoms, one atom can be O-, -S - or-NH-;

r = 1, 2, or 3 and

G IS -(CH2)s-R, where s = 0-5; -CH=CH-(CH2)t-R, where t = 0-3; or G represents

< / BR>
where D and E are each independently of one another, N or CH;

k = 0 or 1;

b = 0 or 1;
G denotes

< / BR>
and where from one to all of unsubstituted carbon atoms of the aromatic or heterocyclic rings:

< / BR>
may contain fluorine as a substituent;

or its pharmaceutically acceptable salt; or to pharmaceutically acceptable MES of the compounds or its salts;

provided that A does not denote a hydrogen or tert-butyloxycarbonyl, when G represents -(CH2)s-NH-C(NH)NH2Y denotes an unsubstituted Proline (Rpis hydrogen) and T stands for:

< / BR>
and provided further, that R is amino or guanidino,

when r = 1 and s = 0;

and provided, further, that A does not denote hydrogen, C1-C4alkyl, methylsulphonyl or -(CH2)g-Rmand when G represents (CH2)s-R, where R denotes

< / BR>
Y denotes

< / BR>
the unsubstituted Proline (Rp= hydrogen) or 4-hydroxyproline (Rp=OH), R' denotes hydrogen;

T denotes cyclohexyl and Q denotes - NH-A;

and provided further, that R"SO2doesn't mean arylsulfonyl, when G represents -(CH2)s-R, where R denotes

< / BR>
Y denotes an unsubstituted Proline (Rpis hydrogen) or 4-methylthiophenyl (Rp
< / BR>
T stands for (C1-C8)-alkyl,

< / BR>
and Q denotes - NH-A.

A specific group of the above compounds of formula I include compounds of formula I, where

X denotes Proline, homopolymer;

< / BR>
< / BR>
T represents C3-C8cycloalkyl, C1-C8alkyl,

< / BR>
a is 0 or 1;

Q IS-OH, C1-C4alkoxy, or - NH-A;

A is hydrogen, C1-C4alkyl, R"SO2-, R"OC(O)-, R"C(O)- or -(CH2)g-COOH;

g = 1, 2, or 3;

B is hydrogen or C1-C4alkyl;

R' is hydrogen or C1-C4alkyl;

R" - C1-C4alkyl, C1-C4perfluoroalkyl, -(CH2)d-COOH or unsubstituted or substituted aryl, where aryl refers to phenyl, naphthyl; a 5 - or 6-membered unsubstituted or substituted aromatic heterocyclic ring with one or two heteroatoms that are the same or different and selected from sulfur, oxygen and nitrogen; or a 9 - or 10-membered, unsubstituted or substituted condensed-bicyclic aromatic heterocyclic group with one or two heteroatoms, which are identical or different and are chosen from among sulfur, oxygen and nitrogen;

d = 1, 2, or 3;

m = 0, 1, or 2;

O2NH, where Ra- C1-C4alkyl; Y represents:

< / BR>
where Rg- C1-C6alkyl, C3-C8cycloalkyl or - (CH2)p-L-(CH2)q-T';

Rpis hydrogen, C1-C6alkyl, C3-C8cycloalkyl or -(CH2)p-L-(CH2)q-T';

where p = 0, 1, 2, 3 or 4;

L is a bond, -O-, -S - or-NH-;

q = 0, 1, 2 or 3; and T' is hydrogen, C1-C4alkyl, C3-C8cycloalkyl, -COOH, -CONH2or Ar, where Ar is unsubstituted or substituted aryl, where aryl refers to phenyl, naphthyl; a 5 - or 6-membered unsubstituted or substituted aromatic heterocyclic ring with one or two heteroatoms that are the same or different and which are selected from sulfur, oxygen and nitrogen; or a 9 - or 10-membered unsubstituted or substituted condensed-bicyclic aromatic heterocyclic group with one or two heteroatoms that are the same or different and selected from sulfur, oxygen and nitrogen;

RY- -CH2-, -O-, -S - or-NH-; and

RZlink, or together with RYand the three neighboring carbon atoms form a saturated carbocyclic ring with 5 to 8 atoms, one atom of which may represent-O-, -S - or-NH-;and each E is independently - N or CH;

k = 0 or 1;

b = 0 or 1;

M - S, O or NH;

each W is independently N or CH; and

R denotes-NH2< / BR>
< / BR>
or their pharmaceutically acceptable salts, or pharmaceutically acceptable solvate of the above compounds or their salts;

provided that A does not denote a hydrogen or tert - butyloxycarbonyl, when G represents -(CH2)s-NH-C(NH)NH2Y is an unsubstituted Proline (Rp- hydrogen) and T stands for

< / BR>
and provided further, that R does not denote amino or guanidino, when r = 1 and s = 0;

and provided further, that A does not denote hydrogen, C1-C4-alkyl, methylsulphonyl or -(CH2)g-COOH, when G represents -(CH2)s-R, where R denotes

< / BR>
Y denotes

< / BR>
the unsubstituted Proline (Rp- hydrogen) or 4-hydroxyproline (Rp- OH);

R' is hydrogen;

T - cyclohexyl and Q is-NH-A;

and provided further, that R"SO2doesn't mean arylsulfonyl, when G represents -(CH2)s-R R -

< / BR>
Y is an unsubstituted Proline (Rp- hydrogen) or 4-methyl-thioproline (Rp- -SCH3), and Q = -NH-A;

and provided further, that not A denotes R"SO2- when G denotes

< / BR>
T - C1-C8alkyl,

< / BR>
and Q = notitiam, containing the compound of formula (I) in combination with a pharmaceutically acceptable carrier, diluent or excipient.

The present invention also relates to a method for inhibiting thrombosis in a mammal which comprises the administration to a mammal in need of treatment, antithrombotic doses of the compounds of formula I.

The present invention further relates to a method of inhibiting thrombin, which includes an introduction to a mammal in need of treatment, inhibiting thrombin dose of the compounds of formula I.

The invention relates to new inhibitors of thrombin, pharmaceutical compositions containing the compounds as active ingredients, and to the use of the compounds as anticoagulants for prophylaxis and treatment of thromboembolic disorders such as venous thrombosis, pulmonary embolism, arterial thrombosis, in particular myocardial ischemia, myocardial infarction and cerebral thrombosis; state General and local hypercoagulable such, which come as a result of angioplasty and coronary bypass surgery; and to generalize tissue damage, as it relates to the inflammatory process.

Ter is osvetleni chain containing a specified number of carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl and sec-butyl. The term "perfluoroalkyl" by itself or as part of another substituent refers to an alkyl radical with a linear or branched chain containing the specified number of carbon atoms, where each atom of hydrogen replaced by fluorine atom, such as trifluoromethyl, perforated, per-fluoro-n-propyl, perforator, PERFLUORO-n-butyl, PERFLUORO-tert-butyl, perforator and PERFLUORO-sec-butyl.

The term "C3-C8cycloalkyl" refers to saturated alicyclic rings with 3-8 carbon atoms, such as cyclopropyl, methylcyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 4-methylcyclohexyl, cyclooctyl etc.

The term "alkoxy" denotes an alkyl radical with a linear or branched chain having the specified number of carbon atoms associated with another part of the molecule by an oxygen atom. The term "halogen" denotes chlorine, fluorine, bromine or iodine. The term "acetyl" refers CH3-C(O). The term "tert-butyloxycarbonyl" means (CH3)3C-O-C(O)- and usually abbreviated as "Boc". The term "benzyloxycarbonyl" represents C6H5CH2-O-C(O the em any 5 - or 6-membered ring, which has a stable structure and contains one or two nitrogen atom, one sulfur atom, one oxygen atom, one nitrogen atom and one sulfur atom; or one nitrogen atom and one oxygen atom, 5-membered ring contains one or two double bonds and the 6-membered ring containing two or three double bonds. Such heterocyclic systems include furyl, thienyl, pyrrolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolin, pyranyl, pyridinyl, pyrimidinyl, pyrazinyl, oxazinyl and triazinyl.

The term "9 - or 10-membered heterocyclic ring" refers to any bicyclic group in which any of the above 5 - or 6-membered rings condensed with a benzene ring or another 6-membered heterocycle, as defined above, having a stable structure. These heterocyclic systems include indolyl, benzothiazyl, benzofuran, benzoxazole, benzisoxazole, benzimidazolyl, chinoline, ethenolysis, benzimidazolyl and benzothiazolyl.

You should take into account that many of the above compounds may exist in tautomeric form. All such forms are within the scope of the present invention.

All listed to determine Ar or R" is an aromatic or generalcontractor independently selected from halogen, hydroxyl, C1-C4of alkyl, C1-C4alkoxyl, amino (-NH2), mono-(C1-C4alkyl)amino, -(CH2)j-COOH, mercapto, -S(O)h(C1-C4-alkyl), -NHS(O)h(C1-C4the alkyl), -NHC(O) (C1-C4the alkyl), -S(O)hNH2, -S(O)hNH(C1-C4the alkyl) or-S(O)hN(C1-C4alkyl)2where

h = 0, 1 or 2 and

j = 0, 1, 2, 3, or 4.

Especially preferred for the substituent R"(C)is 1-methylindol-2 oil.

In the formula I of the carbonyl radical X is associated with the amine radical Y. Carbonyl radical Y is then attached to the amino group shown in the formula I.

Group

< / BR>
where Z and A are both hydrogen, refers here to phenylglycine and is denoted by Phg. Connection, where A, for example, methyl, belong to N- methylvinylpyridine group and denoted MePhg. Substituted compounds where Z is other than hydrogen, are the type and position of the substituent for the substituted group, for example, 3'-chlorophenylglycine or Phg (3-Cl).

Group

< / BR>
where Z and A are both hydrogen, refers to, i.e. phenylalanyl and is denoted by Phe. Connection, where A, for example, methyl, belong to N- metilfenidato Deputy, to the substituted group, for example, 3'-chlorophenylalanine or Phe(3-Cl).

Group

< / BR>
where R' is hydrogen, refers to the 1 - and 3-tetrahydro-isoquinoline-carbonyl, respectively, which are respectively abbreviations 1-Tiq and 3-Tiq.

Group

< / BR>
where R' is hydrogen, refer to the 1 - and 3-perhydroanthracene, respectively, and are, respectively, the abbreviations 1-Piq and 3-Piq. As indicated by wavy lines, there are various forming by condensation cycles, isomers of these deputies; the present invention includes each individual isomer, and combinations thereof.

Group

< / BR>
such as Proline, azetidine-2-carbonyl, respectively, and are, respectively, the abbreviation Pro and Azt.

Group

< / BR>
denotes a saturated bicyclic system 4,5; 5,5; 6,5; a 7.5 or 8.5 - type. The stereochemistry at position 3a is a CIS - relative to the carbonyl; another prednostima connection can be either CIS - or TRANS-communication, except for the 4.5 - and 5.5-systems, which may be CIS-pragmatically communication. Define RYand RZprovide that changed the ring, which includes the aforementioned three carbon atoms is a saturated carbocyclic to be heteroatom, selected from-O-, -S - and-NH-. This definition includes the preferred component originating from octahedron-2-carboxylic acid, the abbreviation "Ohi", as represented by formula

< / BR>
Different CIS - and TRANS-forms of this structural units included in the scope of the present invention.

A sign in the form of stars in the radical Y denotes a chiral center, which is (L). The asterisk in the radical X represents a chiral center, which is a (D) or (DL); # sign in the radical X represents a chiral center, which is a (L).

In addition, depending on the branching of the alkyl substituents may exist as diastereomers. Compounds of the present invention include a mixture of two or more diastereomers, and each individual isomer.

Preferred compounds of the present invention include compounds of formula I, where

< / BR>
homopolymer, 1 - or 3-Tig, or 1-or 3-Pig, and

Y - prolinol; and their pharmaceutically acceptable salt and solvate. In particular, preferred all connections, where Q is NHA and A is hydrogen or a sulfonamide (e.g., A= R"SO2-), R' is hydrogen, Z is hydrogen and B is hydrogen. Also preferred are those compounds where R is guanidines such where G is R-substituted phenyl (i.e., D=E=CH, k = 0); in particular, preferred compounds where G is a 4 - amidinophenoxy group.

A preferred group of such compounds in which from one to all different unsubstituted carbon atoms of the aromatic or heteroaromatic rings:

< / BR>
contain as a substituent fluorine, is a group in which a fluorine-Deputy no in - or-position to D or E, when D or E is nitrogen.

Another group of preferred compounds of the present invention include compounds of formula I, as defined above, where X denotes

< / BR>
where T is cyclohexyl, a = 1, R' is hydrogen and Q is-NH-A. One particular subgroup is one in which A stands for hydrogen. The second special subgroup is one in which A - R"SO2- in particular, when R" is ethyl. The third special subgroup is one in which A is -(CH2)g-COOH; preferably g = 1.

Special values Y for the compounds of formula I, in which X, r and G have the abovementioned meanings, are (L)-Proline (Pro), (S)-CIS-octahydro-1H-indole-2 - carbonyl (Ohi) and N-(2-phenylethyl)glycyl [NPhCH2CH2Gly].

For the compounds of formula I, in which R denotes-NH2, prefer G -

< / BR>
where aniline ring may contain one or two fluorine atom as substituents;

b) r = 1 and G denotes

< / BR>
C) r = 1 or 2 and G denotes

< / BR>
One preferred group of compounds of formula I are those in which Y denotes (L)-prolinol, r = 1 and G denotes

< / BR>
where each of D and E represents CH, k = 0 and R - amidino, and which may be represented by formula (Ia):

< / BR>
where benzamidine ring is unsubstituted or may contain one or two fluorine atom as substituents, preferably in meta-position to medinova the radical, and X is any of the above values.

The most preferred value for the compounds of formula Ia is where benzamidine ring is unsubstituted.

Another especially preferred group of compounds of formula (I) are those in which Y - (L)-prolinol, r = 1 and G denotes

< / BR>
where M is sulfur, each W is CH and R - amidino, and which may be represented by formula (Ib)

< / BR>
where X is any of the above values.

An additional preferred group of compounds of formula I are those in which Y - (L)-prolinol, r = 1 and G denotes

< / BR>
where D is nitrogen or CH, k is acini and D - nitrogen or CH.

The preferred value for X in the compound of formula Ia, Ib or Ic is

< / BR>
where R' is hydrogen, a = 1, T is cyclohexyl or phenyl, and Q denotes-NH-A. More preferably, A is hydrogen, ethylsulfonyl or carboxymethyl. One especially preferred value for X is N-carboxymethyl-D-cyclohexylethyl. Another preferred value for X is N-carboxymethyl-D-i.e. phenylalanyl.

Specific compounds of formula I of the invention described in the examples. The preferred connection type, which can be used as a pharmaceutically acceptable salt or MES, may be selected from the compounds obtained in examples 15, 18, 23, 44, 45, 46, 48, 49, 51, 52, 56, 65, 66, 68-72, 80, 86, 87, 88 and 92. More preferred compounds may be selected among the compounds described in the examples 45, 46, 48, 51, 65, 70-72. One of the most preferred types, based on their unexpectedly superior properties shown in example 48. Another very preferred type of connection is the type in example 65.

As indicated above, the invention includes pharmaceutically acceptable salts of the compounds delivered above formula I. Specific compound of the present invention can containing the number of inorganic and organic acids with the formation of pharmaceutically acceptable salts. Commonly used acids to obtain salts of accession are inorganic acids such as hydrochloric acid, Hydrobromic acid, itestosterone acid, sulfuric acid, phosphoric acid, etc. and organic acids such as p-toluensulfonate, methanesulfonate, oxalic acid, p-bromophenylacetate, carboxylic acid, succinic acid, citric acid, benzoic acid, acetic acid, etc. are Examples of such pharmaceutically acceptable salts are salts, such as sulfate, persulfate, bisulfate, sulfite, bisulfite, phosphate, monohydratefast, dihydrophosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, kaprilat, acrylate, methoxybenzoate, formate, isobutyrate, caproate, heptanoate, propionate, oxalate, malonate, succinate, suberate, sebacina, fumarate, maleate, Butin-1,4 - diet, hexyne-1,6-diet, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, phthalate, sulfonate, xylene-sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, etc. are Preferred such as hydrochloric acid, Hydrobromic acid and sulfuric acid.

Compounds of the present invention can form a hydrate and a solvate with appropriate solvents. Preferred solvents for obtaining solvate forms are water, alcohols, tetrahydrofuran, DMF and DMSO. The preferred alcohols are methanol and ethanol. Depending on the size of the solvent molecules can be selected other appropriate solvents. Small molecules of the solvent is preferred to facilitate appropriate education of MES. MES or hydrate usually formed during recrystallization or in the process of education of salt. A suitable reference in relation to the solvate is Sykes, Peter, A Guidebook to mechanism in Organic Chemistry, 6th ed., (1986, John Villey and Sons, new York). As used in the present invention, the term "MES" includes hydrated forms, such as monohydrate and dihydrate.

The compounds of formula I get the known methods of binding peptides. According to one such method, an acid of the formula P-X'-COOH, where X'is-C(O)- indicates-X-having specified for formula I, value, and P denotes aminosidine group, if necessary, is introduced into the reaction accession with carboxyls is given in (unprotect or deesterification) and svobodnaia the form of the dipeptide (b) associated with protecting reagent (d). The above reaction sequence is illustrated by the following scheme 1:

< / BR>
where G' has the same meaning as G, except R represents-CN, - NHP

< / BR>
each P represents aminosidine group, if necessary, P' - H or P; alk is a lower alkyl or a similar protective carboxylate group; and Y' has the same meaning as Y, is represented with amino - and carboxyl functional groups, i.e. Y has the same meaning as - N-Y'-C(O)-.

If it has, cyano-group in G' is transferred to the value of R; and the protective group in (c) then remove known in the art methods, such as hydrogenation in the presence of a metal catalyst to obtain compounds of formula I.

Binding of P-X'-COOH derived from HN-Y'-COO-alk conduct, first protecting the amino group of the amino acids, if it exists. Use regular aminosidine group commonly used for temporary protection or blocking the amino group.

Aminosidine group refers to substituents of the amino group commonly used to block or protect the amino group in the process of interaction with other functional groups of the compounds. Examples of such aminosidine groups are formyl gropina group; blocking groups, urethane type, such as benzyloxycarbonyl, tert - butoxycarbonyl, 4-phenylbenzoxazole, 2-methylbenzyloxycarbonyl, 4-methoxybenzenesulfonyl, 4-forantimicrobial, 4-chlorobenzenesulfonyl, 3-chlorobenzylidene, 2-chlorobenzenesulfonyl, 2,4-dichlorobenzenesulfonyl, 4-bromobenzyloxycarbonyl, 3-bromobenzyloxycarbonyl, 4-nitrobenzenesulfonyl, 4-cyanobenzeneboronic, 2-(4-xenil)isopropoxycarbonyl, 1,1-diffenret-1-jocstarbunny, 1,1-diphenylprop-1-jocstarbunny, 2-phenylprop-2 - jocstarbunny, 2-(p-toluyl)-prop-2-jocstarbunny, cyclopentanecarbonyl, 1-methylcyclopentadienyl, cyclohexyloxycarbonyl, 1-methylcyclohexanecarboxylic, 2-methylcyclohexanecarboxylic, 2-(4-toluensulfonyl)etoxycarbonyl, 2-(methylsulphonyl)etoxycarbonyl, 2-(triphenylphosphino)etoxycarbonyl, 9-oftenlooklikethis ("FMOC"), 2-(trimethylsilyl)etoxycarbonyl, allyloxycarbonyl, 1-(trimethylsilylmethyl)-prop-1-relaxerror, 5-benzisoxazoles, 4-acetoxybenzoic, 2,2,2-trichlorocyanuric, 2-ethinyl-2-propoxycarbonyl, cyclopropanecarbonyl, 4-(decyloxy)benzyloxycarbonyl, isobornylacitate, 1 piperidinylcarbonyl and the like aminosidine group. The type aminosidine group is not limiting, only the substituted amino group must be stable under the conditions subsequent (s) interaction (s) on other positions of the molecule and can be removed at the appropriate time without affecting the rest of the molecule. Preferred aminosidine groups are benzyloxycarbonyl, allyloxycarbonyl, tert-butoxycarbonyl, trityl. Such aminosidine groups used in the cephalosporin, penicillin and peptide, therefore, covered by the above terms. Further examples of groups belonging to the above terms are described by J. W. Barton. "Protective Groups in Organic Chemistry", J. G. WcOmie, Ed., Plenum Press, New York, N. Y., 1973, Chapter 2, and T. W. Greene, "Protective Groups in Organic Synthesis", John Wiley and Sons, New York, N. Y., 1981, Chapter 7. The related term "protected amino group" defines an amino group, a substituted protective for the amine function group discussed above.

When performing binding assays, for HN-Y'-COOH use ester protective group which is removed under conditions which aminosidine group remains unaffected. Aminosidine group alleluya acid P-X'-COOH is thus saved for the protection of amino groups within Pospolitaya in the description, refers to one of the ester derivatives of carboxylic acid, typically used for binding or protection of the carboxyl group of reactions on other functional groups of the compounds. Examples of such protective carboxylic acid groups include C1-C4alkyl, benzyl, 4-nitrobenzyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl, 2,4-dimethoxybenzyl, 2,4,6-trimethoxybenzyl, 2,4,6-trimethylbenzyl, pentamethylbenzyl, 3,4-methylenedioxybenzyl, benzhydryl, 4,4'-dimethoxybenzil, 2,2', 4,4'-tetramethoxybenzene, tert-butyl, tert-amyl, trityl, 4-methoxytrityl, 4,4'-dimethoxytrityl, 4,4', 4"-trimethoxytrityl, 2-phenylprop-2-yl, trimethylsilyl, tert-butyl dimethylsilane, phenacyl, 2,2,2-trichlorethyl, - (trimethylsilyl)ethyl, - (di(n-butyl)methylsilyl)ethyl, p-toluensulfonate, 4-nitrobenzenesulfonate, allyl, cinnamyl, 1-(trimethylsilylmethyl)-prop-1-EN-3-yl, etc. groups. The type used for carboxyterminal group is not determinative, because the converted carboxylic acid is stable to the conditions of the serial (s) reactions (reactions) on other positions of the molecule, and the protective group can be removed at the appropriate time without affecting the rest of the molecule. In particular,

important reduction conditions using catalysts based on vysokokachestyovennogo metal, such as Raney Nickel. (Such harsh conditions for removal of the protective group should be avoided when removing aminosidine groups, as discussed below). In addition, examples of these groups are y E. Haslam, "Protecrive Groups in Organic Chemistry", J. G. W. McOmie, Ed., Plenum Press, New York, N. Y., 1973. Chapter 5, and T. W. Greene, "Protective Groups in Organic Synthesis", John Wiley and Sons, New York, N. Y., 1981, Chapter 5.

The compounds of formula I can also be obtained by synthesis of the first amide predecessor HN-Y'-CONH(CH2)r-G' and then reaction with the protected X-group. According to one such method,

(d) receive and connect with PN-Y'-COOH (g), as shown below, to obtain the amide (h)

< / BR>
where P denotes aminosidine group, such as benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), p-toluensulfonyl, etc. are Preferably used aminosidine group is removed by hydrogenation or treatment with a weak acid (for example, triperoxonane acid) or a strong acid (such as HCl). Examples of other suitable aminosidine groups are given in "Protective Groups in Organic Synthesis", second edition, T. W. Greene and Peter G. M. Wuts, Chapter 7, pages 309-405 (1991), John Wiley and Sons, Inc. Group Vos or other suitable protective group is removed from the amino nitrogen of the Y residue, which is then acelerou with p the group if it's in G', transform and protective groups in (c) is removed as described above.

The binding of compounds P-X'-COOH is carried out by first protecting the amino group of the amino acids, if it exists. Apply standard aminosidine group, usually used for temporary protection or blocking of amino groups. Examples of such protective groups described above.

The above binding assays carried out under cooling, preferably at temperatures between about -20oC and 15oC. binding assays carried out in an inert organic solvent, such as dimethylformamide, dimethylacetamide, tetrahydrofuran, methylene chloride, chloroform, etc., and as in the usual solvents, and mixtures of such solvents. Usually, when the coupling reaction using active ester alleluya acid, apply anhydrous conditions.

Intermediate products (d) and (g) are obtained by standard methods of organic chemistry, as shown in the following diagrams:

< / BR>
where-K-R is -(CH2)r-G.

According to the above sequences, protected guanidine can be obtained by double protection S-methylisothiazoline. Prefer istia S-methylisothiazoline in the presence of di-tert - BUTYLCARBAMATE. Often use the resulting salt of the acid with S-Methylisothiazolinone, which can be converted into the free base in situ by dissolving the salt in water and processing water base. Di-tert-BUTYLCARBAMATE then injected into the reaction in mixing with the water solvent, such as tert-butanol, to obtain the double-protected S-Methylisothiazolinone. The desired double-protected guanidine then treated with the appropriate diamine H2N-K-NH2in an inert solvent or combination of solvents. Usually effectively use miscible with water solvents, such as dimethylformamide or water, or mixtures thereof. This reaction usually ends completely after 3-72 hours the resulting protected guanidine can then be connected, as described above, to obtain the protected intermediate products of compounds of the formula I, where

< / BR>
For compounds of formula I, where R=-NH2intermediate compound is odnozameshchenny diamine. In most cases, this intermediate product can be obtained simply by the interaction of unprotected diamine with one mole equivalent of protective reagent. Other methods of obtaining the target amine (R= -NH2 is clonally group, for example, nitro - or cyano group. In the case of the nitro-group transformation of the amino group is usually carried out on such substances, where the nitro group directly attached to an aromatic ring, particularly phenyl group. In such cases nitroaniline group reduced to the corresponding aniline using any of many well-known specialist methods. One particularly effective method is the handling of nitrocompounds by hydrosulfite sodium directionspanel a solvent such as ethanol, water, or mixtures thereof. When nitrosoaniline refluxed in a mixture of water with ethanol in the presence of hydrosulfite sodium, the restoration was completed in a few hours. Cyano group can be recovered, if desired, in the presence of a reducing agent, such as sociallyengaged, borane in a solvent such as tetrahydrofuran, or by reduction with sodium borohydride, promoted by metal.

Amidine according to the present invention

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can also be obtained from the nitrile precursor. The specialist there are many ways to implement this transformation. In particular, the use of hydrogen sulfide the Oia in methanol is a preferred and effective way of performing this conversion. Alternatively, to implement this transformation is also possible to use heating nitrile hydrochloride a hydroxyl base, such as N, N-diisopropylethylamine, containing a hydroxyl group in a solvent such as ethanol, with subsequent catalytic hydrogenation (for example, hydrogenation on palladium-on-coal). This way we obtain hydroxyamide

< / BR>
in the form of an intermediate product, which can be selected, if desired.

Other compounds used as starting materials in the synthesis of compounds of the present invention, are well known and, in the absence of their sales, easily synthesized by conventional methods commonly used for this purpose by the specialist.

4-substituted Proline (Rp= C1-C6alkyl, C3-C8cycloalkyl or -(CH2)p-L-(CH2)q-T'), which is used to produce compounds of the present invention, all have the CIS-configuration of the substituent in position 4 relative to a carbonyl group. Intermediate compounds for the introduction of this functional group in the compounds of formula I get the usual ways.

For example, 4-substituted prolinnova derivatives, Gee, as follows:

< / BR>
where R4= R3CH2= Rp-group containing methylene group in place of joining prolinnova ring.

4-Hydroxyproline (as CIS-and TRANS-forms are commercially available) first protect aminosidine group, and in this case especially suitable Cbz-group. The resulting intermediate compound is then atrificial to complex ether (particularly suitable methyl and especially ethyl esters) and then oxidized to obtain the corresponding ketone. This oxidation is carried out in any of the many ways oxidation, such as oxidation by Jones or using pyridinesulfonamide, especially suitable for this transformation is the use of pyridinesulfonamide dry directionspanel a solvent such as dichloromethane. When the reaction is carried out within 8-16 hours, then at room temperature interaction is usually completely ends. This multifunctional ketone intermediate compound is then injected into interaction with an appropriate Wittig reagent to obtain the desired olefin. Usually appropriate, Rp-substituted triphenylethylene add to dry IRD potassium). Introducing ketone after about three hours at room temperature, can be selected desired intermediate olefin. With the aim of obtaining good yields of olefin, preferably of 0.4-0.6-molar excess of Wittig reagent relative to the ketone. The olefin is then reduced to the desired Rp-substituted Proline using standard recovery techniques. Catalytic hydrogenation is the easiest way to implement this transformation in the laboratory. Hydrogenation of the olefin in the presence of a catalyst (for example, 5% palladium-on-coal) in an inert solvent, such as ethanol, effectively at atmospheric pressure. In the case of such intermediate products, which aminosidine group is Cbz, the hydrogenation removes the protective group, which leads to a connection that can be used to bind to P-X'-COOH. How to account for the specialist, this method may not be effective for producing compounds where Rpgroup attached to prolinnova ring through a heteroatom or are aromatic ring. Thus, in the diagram above, R3must mean alkyl, aralkyl (e.g., benzyl), (cycloalkyl)alkyl, etc.

According to the presented reaction scheme alternative to the above reaction the Wittig and suitable for producing compounds which cannot be obtained by Wittig reagents. Thus, to obtain intermediate products, where Ra denotes an alkyl, phenyl, etc., intermediate pyrrolidine enter into interaction with the corresponding Grignard reagent. Usually use a slight molar excess of Grignard reagent, usually working at a low temperature (for example, from -80 to -60oC) antifreeze inert solvent such as tetrahydrofuran. After addition of the reagents, the reaction mixture can be heated to room temperature, after which the reaction is usually complete within a few hours. The obtained intermediate compound dehydration, for example, processing triperoxonane acid. Intermediate 3,4-dihydrocodeinone then reduced to the desired intermediate CIS-compounds under the same conditions recovery that described above for recovery of olefinic intermediate product.

Intermediate compounds, where hetero-L-group is oxygen and it is directly attached to prolinnova ring (i.e. p=0), can be obtained by Mitsunobu reaction (Mitsunobu, Synthesis, 1 (1981)):

< / BR>
In this reaction, the ester of TRANS-hydroxypyrrolidine acid to approximately 0oC and add diethylazodicarboxylate. After heating to room temperature the reaction mixture was treated to give the desired intermediate CIS-product. While the above diagram represents the reaction for compounds where L = -O-, p = q = 0 and T=Ar, this reaction sequence suitable for the production of other compounds, where p=0 and L= -O-.

Intermediate compounds, where L denotes a sulfur attached directly to the ring, can be obtained by first converting the hydroxy group in tosylate or other similar removable group and the subsequent replacement of tiolet-anion (see , for example, Kropcho etc., J. Med. Chem., 31, 1148-1160 (1988); Smith and others, J. Med.Chem., 31 875-855 (1988)).

Intermediate products, where L denotes the nitrogen attached directly to the ring, can be obtained by first converting the hydroxy group in tosylate or other similar removable group and subsequent substitution by azide. Azide you can recover the known methods and then alkilirovanii with obtaining the desired functional group (see, for example, Smith and others, J. Med.Chem., 31, 875-855 (1988)).

Compounds of the present invention, containing CIS-Ohi-functional group is obtained by synthesis of ethyl is 2)) and restore this intermediate product by hydrogenation over 5% Pd/C in ethanol to obtain a complex ether octahedron-2-carboxylic acid, usually called Ohi-ether, as presented below:

< / BR>
Compounds of the present invention, containing TRANS-Ohi-functional group, receive according to the method of Vincent and others, Drug Desugn and Discovery, so 9, S. 11-28 (1992)). This synthesis is presented in the diagram below:

< / BR>
The compound of the present invention, containing a bicyclic system with a heteroatom or without it, can be obtained according to the method Teetz and others, Tetrahedron Letters, 25, 4479 (1984). The usual scheme for the following:

< / BR>
where P denotes a protective group, and Rx- alkyl.

Intermediate compounds for the introduction of N-substituted glycine functional group (Y) that is used to obtain the compounds of the present invention, receive conventional methods.

For example, a complex halogenoacetyl ester such as tert-butylbromide can be converted into the desired substituted compound by treatment with the appropriate primary amine:

BrCH2COO-tert-butyl + RgNH2---> HNRgCH2COO-tert-butyl.

Tert-Butyl-bromoacetate subjected to interaction with the corresponding amine by themselves or, preferably, in directionspanel a solvent such as alcohol. Prefer is about, the reaction mixture also contains directionspanel acid acceptor, such as at least a molar equivalent of triethylamine. While the reagents usually combine cooling (for example, 0oC), the reaction is usually carried out by heating to room temperature, after which the reaction is finished completely for 24 hours. Although bromoacetate is preferred for this transformation, you can use other halogenated, such as iodoacetate and chloracetate. Can be similarly used other ester groups. Tert-Butyl ester is preferred because it can later be easily removed by subsequent treatment with anisole and triperoxonane acid.

The second way of obtaining these intermediates represented by the following reaction scheme:

R0-CHO+H2NCH2COO-ethyl ---> R0-CH=NCH2COO ethyl ---> R0-CH2- NHCH2COO-ethyl,

where R0-CH2means Rggroup containing unsubstituted methylene group adjacent to the place of connection to the glycine group.

In the above reaction scheme, the corresponding aldehyde is mixed with glycine complex air mportant glycine ester, it may be added molar equivalent of a base, such as potassium hydroxide, to obtain the free base complex aminoether. The interaction of the aldehyde with complex glycine ether leads to the formation of the intermediate Schiff's base, which you can then restore in situ by treatment with a reducing agent, such as cyanoborohydride sodium. The formation of Schiff bases is usually within less than one hour; recovery usually ends after 10-15 hours a Complicated methyl or ethyl esters, are suitable as these groups can be removed (unprotect) by treatment with lithium hydroxide in aqueous dioxane. The use of the corresponding ketone rather than an aldehyde R0-CHO leads to the production of intermediate products, where a methylene group that is associated with glycinamido substituted.

Alternatively, and especially for those compounds where Rg- Ar (i.e., without the introduction of alkyl groups), preferably to obtain an intermediate compound P-X'-CONHAr conventional methods (for example, by reacting an activated form P-X'-COOH with ArNH2and then the interaction of this intermediate compound with alkylhalogenide, as described above, to obtain the P-X'-CONHAr-CH2Anastasia invention or intermediate products for their production can be vzaimoprevrascheny standard methods. For example, aryl compounds, which are substituted by a nitro-group, can be recovered (for example, in the presence of hydrosulfite sodium directionspanel a solvent such as ethanol, water or a mixture). When nitrosoaniline refluxed in a mixture of water/ethanol in the presence of hydrosulfite sodium, recovery is usually completed within a few hours. The resulting amine may be present in the target product; if the amine present in the intermediate product, it may be desirable education in its target a desired shape (for example, acylation with getting acylated amine or protection to avoid adverse reactions in the implementation sequence of chemical reactions. If the desired connection is free amine, in this case particularly suitable is the Cbz protective group. Other transformations and interconversion of this type are known to chemists organikum.

As it is clear to experts, the above transformation can be done using the original substances listed above, or in most cases, it is also possible to obtain intermediate di - or tripeptides containing the same line is cnyh groups; therefore, the order and type of chemical reactions is dictated by the need and type protective groups, as well as the consistency of the responses. Also expert it is clear that it is possible to choose other protective groups in order to serve the purpose of protecting the functional groups in the process sequence of chemical reactions, but can also be removed under appropriate conditions and in the appropriate order according to the sequence of transformations. For example, in the diagram above, 1 G' includes substituents, where R is-CN; this nitrile group can be converted to amidin or restored to an amine, which may be optionally further converted into guanidine of the present invention.

Compounds of the present invention is best to highlight in the form of salts accession acids. Salts of compounds of formula I, formed from acids such as those mentioned above, are suitable as pharmaceutically acceptable salts for introduction as antithrombotic agents for the preparation of formulations of these agents. Other salts accession acids can be obtained and used for isolation and purification of peptides. For example, can also be used soliloquist.

The compound of the formula I get:

a) by simultaneous or sequential removal of the protective group P from the corresponding compounds of formula II:

(P)X-Y-NH-(CH2)r-G(P), (II)

where (P)X is the radical X, which may contain one or more protective groups P, independently selected from aminosidine group P for the compounds of formula I, where X includes the main NH radical, and carboxyterminal group P for the compounds of formula I, where X includes carboxylates and G(P) denotes the radical of G, which may contain one or more independently selected aminosidine groups P; or

b) for compounds of formula I, where R stands for

< / BR>
by hydrogenation of the corresponding compounds of formula I, where

R stands for

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and then, when the desired salt of the compounds of formula I, a salt with a pharmaceutically acceptable acid.

It may be preferable to holding method b) simultaneously with method a). For the compounds of formula I, in which kislotnosti group is tert-butyl ester and/or aminosidine groups are tert-butyloxycarbonyl, protective (s) group (s) can be removed by treatment with strong acid, such as triperoxonane the following anisole. For the compounds of formula I, in which kislotnosti group is a complex benzyl ether and/or aminosidine group is benzyloxycarbonyl, protective (s) group (s) can be removed by hydrogenation is usually carried out in ethanolic hydrogen chloride in the presence of palladium-on-coal as a catalyst.

The preferred method of purification of compounds of formula I, while obtaining a desired stable salt form, a method described in U.S. patent 5 250 660. According to this method, a stable sulfates or hydrochloride obtained by purification by preparative chromatography with C18inverted phase in which the aqueous component contains sulfuric acid or hydrochloric acid at pH 2.5 and the organic component is acetonitrile. the pH of the acidic mobile phase was adjusted to a value of from about pH 4 to about 6 using anion-exchange resin in the hydroxyl form, such as Bio-Rad AG 1X8. After establishing the pH of the salt solution of sulphate of Tripeptide or hydrochloride Tripeptide lyophilized obtaining pure salt in dry powder form. For example, according to the method, the raw D-Phe-Pro-p-NHCH2C6H4C(NH)NH2the sulfate can be dissolved in water and/SUB>SO4; B = acetonitrile) during the 10 o'clock Multiple fractions are collected and those containing the product, as determined using analytical R P HPLC, pooled. the pH of the combined fractions was adjusted to 4.0 to 4.5 using resin AG-1X8 in hydroxide form (Bio-Rad, 3300 Ragatto Blvd. , Richmond, 94804). The solution is filtered and the filtrate lyophilized obtaining pure D-, L-diamide in the form of a sulfate salt.

Optically active isomers diastereomers by radical X are also part of the present invention. Such optical active isomers can be obtained from the appropriate optically active precursors by the above methods, or by separation of racemic mixtures. This separation can be accomplished by obtaining derivatives with chiral reagent and subsequent chromatographytandem or by re-crystallization. The removal of the chiral auxiliary compound by conventional methods leads essentially to the optically pure isomers of the compounds of the present invention or their precursors. Details concerning divisions can be set from Jacques and others, Enantiomers, Racemares, and Resolutions, John Wiley and Sons, 1981.

The compounds used as starting materials in the synthesis somepossible, usually used for this purpose specialists.

The following examples further describe the invention and to the corresponding comparative examples, but they do not limit the scope of protection of the invention.

Used in the present description, the abbreviations have the following meanings:

Amino acid residues: Arg = arginyl; Glu = glutamyl; Cly = glycyl; Pro = shed; hPro = gamopolis; Azt = azetidin-2-carbonyl; Phg = phenylglycyl; Phe = i.e. phenylalanyl; hPhe = homophenylalanine; 1-Tiq = 1,2,3,4-tetrahydroisoquinoline-1-carbonyl; 3-Tiq = 1,2,3,4-tetrahydroisoquinoline-3-carbonyl; Cha = - cyclohexylethyl; hCha = - amino -- cyclohexylmethyl; NM1 = N-methylindol-2 oil; Ohi = CIS-octahedron-2 oil; 1-Piq = perhydro-isoquinoline-1-carbonyl; 3-Piq = perhydrosqualene-3-carbonyl; Met = national; Met (O2) = S,S-cioccolatini. Agm - agmatine; Boc is tert-butyloxycarbonyl; Bn is benzyl; Cbz - benzyloxycarbonyl; DC - dicyclohexylcarbodiimide; DMF is dimethylformamide; Et is ethyl; DMSO is dimethylsulfoxide; EtOAc is ethyl acetate; Et2O - diethyl ether; EtOH is ethanol; Fmoc - 9-fluorenylmethoxycarbonyl; FAB-MS mass spectrum obtained by fast atom bombardment; FD-MS mass spectrum with field desorption; 1S-MS mass spectrum, obtained by applying an electron beam; effektivnaya liquid chromatography with reversed phase; Ph = phenyl; TFA = TPA = triperoxonane acid; THF is tetrahydrofuran; TLC = thin-layer chromatography.

Use the following parameters for high-performance liquid chromatography with reversed phases:

solvent A: 0.05% of an aqueous solution of hydrochloric acid (1.5 ml of concentrated hydrochloric acid in 3 l of water);

solvent B: acetonitrile;

gradient: as specified in each example;

Method 1: column: 2.5 x 25 cm, phase Vydac C18; volumetric flow rate: 5 ml/min;

Method 2: column: 5 cm x 25 cm; phase: Vydac C18; volumetric flow rate: 10 ml/min;

Method 3: column: 2.5 cm x 50 cm; phase Vydac C18; volumetric flow rate: 10 ml/min

Unless nothing else, the establishment and achievement of the pH is carried out using aqueous solutions of acids or bases.

In the examples, which specifies1H-NMR, the product obtained in the reaction described data proton magnetic resonance confirming that you have received the specified connection; IR without providing data indicates that you have received a satisfactory infrared spectrum. HRMS is used to confirm the exact weight compounds, for which no satisfactory elemental analysis is="ptx2">

Example 1

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A) Receive Boc-D-Phe-Pro-OBn.

To a solution of Boc-D-Phe-OH (89,1 g, 336 mmol), Pro-OBn-hydrochloride (81,2 g, 336 mmol), HOBT (50 g, 370 mmol) and N,N-diisopropylethylamine (176 ml, 1,008 mmol) in dichloromethane (600 ml) at 0oC add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (71 g, 370 mmol). After stirring for 18 h the mixture was diluted with diethyl ether (1 l) and washed successively three times with 1N citric acid (250 ml), once with water (250 ml), three times with saturated aqueous sodium bicarbonate solution (250 ml) and once with saturated aqueous sodium chloride (250 ml). The organic phase is dried (Na2SO4), filtered and concentrated in vacuo to obtain 140 g (92,5%) of a pale yellow foam. FD/MS m/e 452 (M+);1H-NMR

B) Obtaining p-Phe-Pro-OBnTFA.

To a stirred solution of Boc-D-Phe-Pro-OBn (68 g, 150 mmol) in dichloromethane (50 ml) at 0oC added anisole (20 ml), then triperoxonane acid (400 ml). After stirring for 3 h the solvent is evaporated in vacuum and the viscous oily residue is dissolved in diethyl ether (1.5 l) and cool (72 h). White precipitate is filtered off, washed with diethyl ether (300 ml) and dried with getting to 59.4 g (85%) of white powder. 1H-NMR.

oC add acanaloniidae (2.65 ml, 28.3 mmol) dropwise via an addition funnel. The reaction vessel is heated to 0oC and after stirring for 4 h, add water (10 ml). The organic phase is washed three times with 1N hydrochloric acid (100 ml), once with a saturated solution of sodium chloride (100 ml) and then the solvent is removed in vacuum. The product was then purified by flash chromatography on silica gel, elwira a mixture of ethyl acetate with hexane (6:4). Containing the product fraction (evaluated by TLC) are combined and concentrated to obtain 6,62 g (58%) of yellow oil, which solidifies.1H-NMR; FD-MS, m/e 445 (M+); Analysis for C23H28N2O5S:

calculated, %: C 62,14; H 6,35; N 6,30.

found, %: C 61,87; H 6,37; N 6,18.

G) Receiving EtSO2-D-Phe-Pro-OH.

To mix the solution EtSO2-D-Phe-Pro-OBn (4.5 g, 10.1 mmol) in p-dioxane (150 ml) add a solution of the monohydrate of lithium hydroxide (2.1 g, a 50.5 mmol) in water (75 ml). After stirring for 16 h the solution volume reduced in vacuo half and the solution was diluted with water (300 ml) and 0.1 n NaOH solution (100 ml).

The aqueous phase is then washed twice with diethyl ether (250 ml), elitnye extracts washed with saturated aqueous sodium chloride (200 ml), dried (MgSO4), filtered and concentrated to obtain 3.6 g (90%) of a white solid.

FD-MS, m/e 355 (M+).

Analysis for C16H22N2O5S:

calculated, %: C 54,22; H of 6.26; N of 7.90.

found, %: C 54,40; H 6.42 per; N a 7.85.

D) Obtaining N,N-di-Boc-S-methylisothiazoline

To a stirred solution of di-tert-butyl-dicarbonate (100 g, 458 mmol) in tert-butanol (300 ml) add a solution of sulphate bis-S-methylisothiazoline (32.7 g, 117 mmol) in water (150 ml), then sodium hydroxide solution (19.2 g, 480 ml) in water (150 ml). After stirring for 48 h the mixture is concentrated to about 1/3 the original volume in vacuo and diluted with diethyl ether (500 ml). The organic phase is washed once with water (250 ml), three times with 1N citric acid solution (250 ml) and once again with water (250 ml). The organic phase is then dried (MgSO4), filtered and concentrated in vacuo to obtain 42 g (62%) of a white solid.1H-NMR.

E) obtaining the NgNg'di-Boc-agmatine

To a stirred solution of 1,4-butanediamine (23 g, 258 mmol) in a mixture 2: 1 of dimethylformamide with water (300 ml) via addition funnel is added a solution of N,N'-di-Boc-S-methylisothiazoline (15 g, 52 mmol) in dimethylformamid acid (250 ml), diluted with water (250 ml) and washed with ethyl acetate (250 ml). An ethyl acetate phase is again extracted with 1N citric acid solution (100 ml) and the combined aqueous phase is alkalinized with sodium carbonate, saturated with solid sodium chloride and extracted twice with ethyl acetate (250 ml). United an ethyl acetate extracts are washed with saturated aqueous sodium chloride (200 ml), dried (MgSO4), filtered and concentrated to obtain 12.5 g (73%) of a viscous syrup.1H-NMR.

F) obtaining the EtSO2-D-Phe-Pro-Agm(Boc)2.

To a stirred solution of NgNg'di-Boc-agmatine (2 g, 6 mmol) in dichloromethane (30 ml) is added EtSO2-D-Phe-Pro-OH (2.1 g, 6 mmol), HOBT (810 mg, 6 mmol) and N,N-diisopropylethylamine (1.6 g, 12 mmol) followed by addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide-hydrochloride (1.4 g, 73 mmol). After stirring for 20 h the solution was diluted with ethyl acetate (300 ml) and washed three times with 1N citric acid solution (150 ml), once with water (150 ml) and twice with saturated aqueous sodium bicarbonate solution. The organic phase is then dried (MgSO4), filtered and concentrated in vacuo. The remainder chromatographic on silica gel, elwira increasing gradient mixture ethylacetate obtaining 2.4 g (60%) of a viscous oil.1H-NMR FD-MS, m/e: 668 (MH+).

C) Obtaining EtSO2-D-Phe-Pro-AgmHCl.

Stir the suspension EtSO2-D-Phe-Pro-Agm(Boc)2(1.6 g, 2.4 mmol) in anisole (1 ml) dissolved in triperoxonane acid (20 ml) and continue stirring for 1 h at room temperature. The solvent is then removed in vacuo and the residue partitioned between water (100 ml) and diethyl ether (50 ml). The aqueous phase is washed again with diethyl ether (50 ml) and then partially concentrated and lyophilizer to obtain 1.4 g of crude triptoreline salt. Half of this substance is then dissolved in water and purified using R P HPLC (method 1; 98/2 /A/B/); the ratio of the change up to 50/50 /A,/B/, 60 minutes) to give 490 mg (81%) of white powder.1H-NMR; FD-MS, m/e 467 (M+).

Analysis for C21H34N6O4SHCl H2O:

calculated, %: C 48,41; H 7,16; N 16,13; Cl 6,80.

found, %: C 48,01; H for 6.81; N 16,15; Cl 6,97.

Example 2

< / BR>
A) Teaching Boc-D-Cha-Pro-OBn.

By the way, is essentially equivalent to the one described in example 1-a, Boc-D-Cha-Pro-AFP obtained from Boc-D-Cha-OH and Pro-OBn. HCl to yield 91% (109 g), FD-MS, m/e 458 (M+).

B) Obtaining D-Cha-Pro-OBnTFA.

By the way, is essentially equivalent to the one described in example 1-B, D-Cha-Poluchenie EtSO2-D-Cha-Pro-OBn.

By the way, is essentially equivalent to the one described in example 1, EtSO2-D-Cha-Pro-OBn get with the release of 2.3 g (20%).

1H-NMR; FD-MS. m/e 450 (M+)

Analysis for C23H34N2O5S:

calculated, %: C 61,31; H to 7.61; N 6,22.

found, %: C 61,55; H to 7.59; N 6,28.

G) Receiving EtSO2-D-Cha-Pro-OH.

By the way, is essentially equivalent to the one described in example 1-G, EtSO2-D-Cha-Pro-HE get out 0,78 g (48%).

1H-NMR FD-MS, m/e 361 (M+).

D) EtSO2-D-Cha-Pro-Agm(Boc)2.

By the way, is essentially equivalent to the one described in example 1-G, 400 mg (40%) EtSO2-D-Cha-Pro-Agm(Boc)2receive from EtSO2-D-Cha-Pro-OH and NgNg'di-Boc-Agm.1H-NMR; FD-MS, m/e. 674 (MH+).

E) Receiving EtSO2-D-Cha-Pro-AgmHCl.

By the way, is essentially equivalent to the one described in example 1-C, EtSO2-D-Cha-Pro-AgmHCl get with the release of 100 mg (45%). The product was then purified via RPHPLC (method 1; 98/2 /A/B/, the ratio of the change to 50/50 /A,/B/, 60 min).1H-NMR; FD-MS, m/e 473 (M+)

Analysis for C21H40N6O4S1,2HCl H2O

calculated, %: C 47,20; H 8,15; N 15,73; Cl of 7.96.

found, %: C 47,47; H to 7.84; N 16,10; C 7,80.

Example 3

1H-NMR; FD-MS, m/e 335 (M+);

Analysis for C17H22N2O5:

calculated, %: C 61,07; H 6,63; N scored 8.38.

found, %: C 60,88; H 6,72; N 8,14.

B) Receiving EtOCO-D-Phe-Pro-AgmHCl.

By the way, is essentially equivalent to the one described in example 1-G, get 2.1 g (54%) EtOCO-D-Phe-Pro-Agm(Boc)2of EtOCO-D-Phe-Pro-OH and Ng-Ng'-di-Boc-Agm. Then the method is essentially equivalent to the one described in example 1-C, you get 390 mg (77%) EtOCO-D-Phe-Pro-AgmHCl. The product was then purified by RPHPLC (method 1; 98/2 /A/B/, changing to 50/50 /A,/B/, 60 min).1H-NMR; FD-MS m/e 447 (M+).

Analysis for C22H34N6O40,9 HCl 0,2 TTCN2O

calculated, %: C 51,70; H 7,22; N 16,15; Cl 6,13.

found, % C 51,73; H 7,20; N 16,54; Cl 6,36.

Example 4

< / BR>
(N-[(1-Methyl-1H-indol-2-yl)carbonyl]-D-i.e. phenylalanyl-N- [4-[(aminoiminomethyl)amino]butyl]-L-prolinamide-monohydrochloride).

A). Getting NMI-D-Phe-Pro-OH.

To a solution of N-methyl-indole-2-carboxylic acid (2.6 g, 14.9 mmol) in anhydrous tetrahydrofuran (45 ml) add pentafluorophenol (3 g, 16.5 mmol), then 1-(3-dimethylaminopropyl)- 3-ethylcarbodiimide (3.2 g, 16.5 mmol). The mixture is boiled OBR add a solution of D-Phe-Pro-OBnTFA (7 g, 14.9 mmol) and N,N-diisopropylethylamine (4 g, 30 mmol) in tetrahydrofuran (25 ml). After stirring for 2 h, the solvents are removed in vacuo and the residue is dissolved in ethyl acetate (500 ml), after which the resulting solution was washed three times with 0.1 n aqueous solution of sodium bisulfate (250 ml) and three times with 1N aqueous solution of potassium carbonate (250 ml). The organic phase is dried (Na2SO4), filtered and concentrated in vacuo to obtain 6.5 g of amorphous solids (a mixture of the desired product contaminated with pentafluorophenol). This crude product is then hydrolized by the way, is essentially equivalent to the one described in example 1-G, to obtain 3.8 g (62%) not quite white solid.1H-NMR; FD-MS, m/e 419 (M+).

B) Receiving NMI-D-Phe-Pro-AgmHCl.

By the way, is essentially equivalent to the one described in example 1-G, get 900 mg (20%) NMI-D-Phe-Pro-Agm(Boc)2.

Then the method is essentially equivalent to the one described in example 1-C, get 144 mg (31%) NMI-D-Phe-Pro-AgmHCl.

The crude product is dissolved in glacial acetic acid and purified by RPHPLC (method 1, (A/B) is from 90/10 to 40/60, 80 minutes).

1H-NMR. FD-MS, m/e 532 (M+)

Analysis for C29H37N7O30,9 HCl 0,TFK,5H2O:

races Boc-D-Phe-Pro-OH.

To a solution of Boc-D-Phe-Pro-OBn (145 g, 320 mmol) in p-dioxane (660 ml) add a solution of the monohydrate of lithium hydroxide (54 g, 1,280 mmol) in water (330 ml) under vigorous stirring. After 4 h, the solution was concentrated in vacuo to approximately 1/4 of the original volume and diluted with water (350 ml) and 0.1 n sodium hydroxide solution (100 ml). The aqueous phase is washed three times with diethyl ether (250 ml) and then acidified to pH 3 solid citric acid, resulting in precipitation. The solid is filtered, washed twice with water and then dried in a high vacuum, receiving 91 g (78%) of a white solid.1H-NMR; FD-MS m/e 363 (M+).

B) obtaining the NgNg'di-Boc-6-aminoguanidine

By the way, is essentially equivalent to the one described in example 1-E, obtain 4.7 g (66%) NgNg'di-Boc-6-amino hexylaniline of 1,6-hexanediamine.

C) Receive Boc-D-Phe-Pro-NH(CH2)6NHC(NBoc)NH(Boc).

By the way, is essentially equivalent to the one described in example 1-G, get 1.3 g (62%) of Boc-D-Phe-Pro-6-NH(CH2)6NHC(NBoc)NH (Boc) Boc-D-Phe-Pro-OH and NgNg'di-Boc-aminohexyl - guanidine.1H-NMR; FD-MS, m/e: 703 (M+).

G) obtaining D-Phe-Pro-NH(CH2)6NHC(NH)NH2HCl.

By the way, SUB>0,9 HCl 0,TFK,5H2O:

calculated, %: C 49,97; H 6,95; N 15,34;

found, %: C 49,60; H 7,13; N 15,23.

Example 6

< / BR>
A) Obtaining NgNg'di-Boc-5-aminomethylpyridine

By the way, is essentially equivalent to the one described in example 1-E, obtain 1.73 g (72%) NgNg'di-BOC-5 - aminomethylpyridine of the 1.5-pentanediamine. FD-MS, m/e 345 (M+);1H-NMR;

B) Receive Boc-D-Phe-Pro-NH(CH2)5NHC(NBoc)NH(Boc)

By the way, is essentially equivalent to the one described in example 1-G, get 1,9 g (92%) of Boc-D-Phe-Pro-NH(CH2)5NHC(NBoc)NH(Boc) Boc-D-Phe-Pro-OH and NgNg'di-Boc-5-aminomethylpyridine.

1H-NMR; FD-MS, m/e 689 (M+).

C) Getting D-Phe-Pro-NH(CH2)5NHC(NH)NH2HCl. The product was then purified via RPHPLC (method 1; (A/B) of from 98/2 to 40/60, 40 min). FD-MS, m/e 389 (M+).

Analysis for C20H32N6O20,9 HCl 0,TFK,7H2O:

calculated, %: C 48,71; H 6,79; N 15,63;

found, %: C 48,34; H of 6.68; N 16,01.

Example 7

< / BR>
A) Receive Boc-D-Phe-Pro-NH(CH2)3NHC(NBoc)NH(Boc).

To a solution of 1,3-diaminopropane (2.2 g, 30 mmol) in dimethylformamide (25 ml) add a solution of N,N'-di-Boc-S-methylisothiazoline (2.9 g, 10 mmol) in dimethylformamide (25 ml). After stirring for nutria and saturated aqueous sodium chloride (200 ml) and once with saturated aqueous sodium chloride (250 ml). The organic phase is dried (MgSO4), filtered and partially concentrated in vacuo to a volume of about 200 ml.

To this solution is then added Boc-D-Phe-Pro-OH (3.6 g, 10 mmol), HOBT (1.3 g, 10 mmol) and N,N-aminobutiramida ethylamine (1.3 g, 10 mmol), then 1-(3-dimethylaminopropyl)-3-apoHCl (2.1 g, 11 mmol). After stirring for 16 h the solvent is removed in vacuo and the residue treated with ethyl acetate (250 ml). The organic phase is washed three times with 1N citric acid solution (200 ml), once with water (100 ml), twice with saturated aqueous sodium bicarbonate solution (200 ml) and once with a saturated aqueous solution of sodium chloride. The organic phase is then dried (MgSO4), filtered and concentrated in vacuo. The residue after this chromatographic on silica gel, elwira speed gradient mixture of ethyl acetate hexane (1:4) to ethyl acetate. Containing the product fraction (measured by TLC) concentrate to obtain 2.6 g (40%) of a viscous colorless oil.1H-NMR; FD-MS, m/e 661 (M+).

B) Obtaining D-Phe-Pro-NH(CH2)3NHC(NH)NH2HCl.

By the way, is essentially equivalent to the one described in example 1-C, get 460 mg (71%) D-Phe-Pro-NH(CH2)3NHC(NH)NH2HCl. The product was then purified by RPHRLC (Metov>O2HCl 1,TFK,1H2O:

calculated, %: C 44,66; H 6,20; N 15,47;

found, %: C 44,69; H 6,10; N 15,19.

Example 8

< / BR>
A) Obtaining NgNg'di-Boc-4-amino-TRANS-2 - butenylamine

By the way, is essentially equivalent to the one described in example 1-E, obtain 2.4 g (42%) NgNg'di-Boc-4-amino-TRANS-2 - butenylamine of 1,4-diamino-TRANS-2-butene.

B) Receive Boc-D-Phe-Pro-NHCH2-trans-CH=CHCH2NHC (NBoc)NHBoc.

By the way, is essentially equivalent to the one described in example 1-G, get 2.7 g (55%) of Boc-D-Phe-Pro-NHCH2-trans - CH=CHCH2NHC(NBoc)NHBoc of Boc-D-Phe-Pro-OH and NgNg'di-Boc-4-amino-TRANS-2-butenylamine. 1H-NMR. FD-MS, m/e: 673 (M+).

C) Getting D-Phe-Pro-NHCH2-trans - CH=CHCH2NHC(NH)NH2HCl.

By the way, is essentially equivalent to the one described in example 1-C, receive approximately 100 mg of D-Phe-Pro-NHCH2-trans - CH=CHCH2NHC(NH)NH2HCl. The product was then purified by RPHRLC (method 1; (A/B from 98/2 to 40/60, 40 minutes).1H-NMR. FD-MS m/e 373 (M+)

Analysis for C19H28N6O2HCl 0,TFK,5H2O:

calculated, %: C 47,01; H for 6.81; N 16,45;

found, %: C 47,36; H 6,53; N 16,70.

Example 9

< / BR>
A) Obtaining p-H2NCH2C6H4CH2NHC(NBoc)NHBoc
CH2NHC(NBoc)NHBoc of p-xylocaine.1H-NMR.

B) Receive Boc-D-Phe-Pro-p-NHCH2-C6H4CH2NHC(NBoc)NHBoc.

By the way, is essentially equivalent to the one described in example 1-G, get 2.8 g (63%) of Boc-D-Phe-Pro-p-NHCH2-C6H4CH2NHC(NBoc)NHBoc of Boc-D-Phe-Pro-OH and p-H2NCH2C6H4CH2NHC (NBoc) NHBoc.

1H-NMR. FD-MS, m/e: 723 (M+)

C) Getting D-Phe-Pro-p-NHCH2-C6H4CH2NHC(NH)NH22TFA.

By the way, is essentially equivalent to the one described in example 1-C, get 725 mg (81%) of the target bis-TFA-salt and further purified by RPHRLC. 1H-NMR; FD-MS, m/e 423 (M+).

Analysis for C23H30N6O22,1 TFA H2O:

calculated, %: C 48,05; H of 5.05; N 12,36;

found, %: C 48,06; H is 4.85; N to 12.28.

Example 10

< / BR>
A) Obtaining N-Boc-n-(aminomethyl)benzylamine

To a stirred solution of p-cilostamide (10 g, 73 mmol) in a mixture of dimethylformamide with water (1:1, 100 ml) is added di-tert - butyl-dicarbonate (8 g, 37 mmol). After stirring for 20 h the mixture was concentrated in vacuo and the residue partitioned between diethyl ether (200 ml) and 1N citric acid solution (200 ml). The aqueous phase is washed again with diethyl, afirmou phase is then extracted 4 times with ethyl acetate (200 ml). United an ethyl acetate extracts are dried (MgSO4), filtered and concentrated to obtain 2.1 g (24%) of a viscous oil.1H-NMR; FD-MS, m/e 237 (MH+).

Analysis for C13H20N2O2:

calculated, %: C 66,07; H 8,53; N 11,85;

found, %: C 66,33; H 8,44; N 12,11.

B) Receive Boc-D-Phe-Pro-p-NHCH2C6H4CH2NHBoc.

By the way, is essentially equivalent to the one described in example 1-F, receive: 1.1 g (63%) of Boc-D-Phe-Pro-p-NHCH2-C6H4CH2NHBoc of Boc-D-Phe-Pro-OH and N-BOC-n-(aminomethyl) benzylamine.

1H-NMR; FD-MS, m/e 581 (M+)

Analysis for C32H44N4O6:

calculated, %: C 66,19; H of 7.64; N 9,65;

found, %: C 65,99; H 7,63; N 9,42.

C) Getting D-Phe-Pro-p-NHCH2C6H4CH2NH2HCl.

By the way, is essentially equivalent to the one described in example 1-C, get about 100 mg D-Phe-Pro-p-NHCH2-C6H4CH2NH2HCl. The product was then purified by RPHRLC (method 1, (A/B) of from 98/2 to 40/60 40 min).1H-NMR; FD-MS, m/e 381 (M+).

Analysis for C22H28N4O2HCl 1,1 TTCN2O:

calculated, %: C 51,87; H 5,77; N 10,00;

found, % C 51,78; H 5,88; N 10,28.

Example 11

< / BR>
A) Obtaining N-Boc-m-(aminomethyl)be the l)benzylamine of m-xylocaine. 1H-NMR; FD-MS, m/e 237 (MH+).

Analysis for C13H20N2O2:

calculated, %: C 66,07; H 8,53; N 11,85;

found, %: 65,81; H 8,48; N 11,98.

B) Receive Boc-D-Phe-Pro-m-NHCH2C6H4CH2NHBoc.

By the way, is essentially equivalent to the one described in example 1-G, get 1.6 g (95%) of Boc-D-Phe-Pro-m-NHCH2-C6H4CH2NHBoc of Boc-D-Phe-Pro-OH and N-Boc-m-(aminomethyl) benzylamine.

1H-NMR; FD-MS, m/e 581 (M+)

C) Getting D-Phe-Pro-m-NHCH2C6H4CH2NH2HCl

By the way, is essentially equivalent to the one described in example 1-C, get about 100 mg D-Phe-Pro-m-NHCH2-C6H4CH2NH2HCl.

1H-NMR; FD-MS, m/e 381 (M+).

Analysis for C22H28N4O2HCl TTCN2O:

calculated, %: C 52,51; H by 5.87; N OF 10.21;

found, %: C 52,13; H 6,21; N 10,48.

Example 12

< / BR>
A) Obtaining N-Boc-TRANS-4-(aminomethyl)cyclohexanecarboxylic acid

To a solution of TRANS-4-(aminomethyl) cyclohexanecarboxylic acid (50 g, 318 mmol) in 1N solution of sodium hydroxide (334 ml, 334 mmol) and tert-butanol (400 ml) add a solution of di-tert-butyl-dicarbonate (73 g, 334 mmol) in tetrahydrofuran (50 ml). After stirring for evadney phase is washed again with diethyl ether (250 ml) and then acidified with solid citric acid, resulting in precipitation of a white color. The solid is filtered, washed twice with water (100 ml) and dried in vacuum to obtain 48 g (59%) of white powder.1H-NMR.

B) Receiving HOCH2-TRANS-4-(N-Boc-aminomethyl)cyclohexane

To a stirred solution of N-Boc-TRANS-4-(aminomethyl) cyclohexanecarboxylic acid (15 g, 58 mmol) in tetrahydrofuran (150 ml) at 0oC add N-methylmorpholine (5.9 g, 58 mmol), and then ethylchloride (6.3 g, 58 mmol). After stirring for 30 min, add sodium borohydride (6.5 g, 175 mmol), and then through an addition funnel over 5 minutes add methanol (300 ml). The mixture is stirred for 1 h and then the solvents removed in vacuo. The residue is dissolved in ethyl acetate (500 ml) and washed twice 1N citric acid solution (250 ml), once with water (100 ml), twice with saturated aqueous sodium bicarbonate solution (250 ml) and once with saturated aqueous sodium chloride (250 ml). The organic phase is dried over magnesium sulfate, filtered and concentrated to obtain 13 g (91%) of target compound.1H-NMR.

C) Obtaining NH2CH2-TRANS-4-(N-Boc-aminomethyl) cyclohexane

To mix the solution HOCH2-TRANS-4-(N-Boc-aminomethyl)boxill (13,9 g, 80 mmol) followed by a solution of diphenylphosphinite (22 g, 80 mmol) in tetrahydrofuran (100 ml). After stirring for 16 h the solvent is removed in vacuo and the residue chromatographic on silica gel, elwira speed gradient mixture of ethyl acetate hexane (1:3) to a mixture of ethyl acetate with hexane (3:1). Containing the product fraction (measured by TLC) are combined and concentrated with getting to 17.4 g of crude product (contaminated connection with a higher Rf). The crude azide was dissolved in methanol (200 ml) and this solution added to a stirred suspension of finely pulverized Na2S9H2O (51 g, 212 mmol) and triethylamine (1 g, 11 mmol) in methanol (100 ml). The resulting mixture was refluxed for 16 h, then cooled to room temperature and the solvents removed in vacuo. The residue was diluted with water (250 ml) and acidified with solid citric acid. The aqueous phase is washed twice with ethyl acetate (250 ml), alkalinized with solid sodium bicarbonate and saturated with solid sodium chloride. The aqueous phase is then extracted three times with ethyl acetate (200 ml) and the combined extracts dried (MgSO4), filtered and concentrated to obtain 6.4 g (45%) of a viscous oil.1H-NMR.2-TRANS-4-(N-Boc - aminomethyl)cyclohexane from Boc-D-Phe-Pro-OH and NH2CH2-TRANS-4-(N-Boc-aminomethyl)cyclohexane. 1H-NMR; FD-MS, m/e 587 (M+).

D) D-Phe-Pro-NHCH2-TRANS-4-(aminomethyl) HCl

By the way, is essentially equivalent to the one described in example 1-C, receive 588 mg (75%) D-Phe-Pro-NHCH2-TRANS-4-(aminomethyl)-HCl TTCN2O:

calculated, %: C 50,30; H? 7.04 baby mortality; N 9,78;

found, %: C 50,44; H 7,20; N 9,62.

Example 13

< / BR>
A) Receive Boc-D-Phe-Pro-p-NHCH2C6H4NH2< / BR>
By the way, is essentially equivalent to the one described in example 1-G, get 8 g of Boc-D-Phe-Pro-p-NHCH2C6H4NO2from Boc-D-Phe-Pro-OH and p-NO2-benzylamino-HCl. The intermediate product is dissolved in ethanol (250 ml) and heated to the boiling temperature under reflux. To this stirred solution was added a solution of Na2S2O4(12.3 g, 70 mmol) in water (125 ml). After stirring at the boiling point under reflux for 2 h the solvent is removed in vacuo and the residue distributed between ethyl acetate (250 ml) and water (250 ml). The aqueous phase is extracted again with ethyl acetate (250 ml), the combined organic phase is TBA.

1H-NMR; FD-MS, m/e 466 (M+).

Analysis for C26H34N4O4:

calculated, %: C 66,93; H 7,34; N 12,01;

found, %: C 66,69; H 7,32; N to 12.28.

B) Obtaining D-Phe-Pro-p-NHCH2C6H4NH2HCl.

By the way, is essentially equivalent to the one described in example 1-H, obtain 180 mg (60%) D-Phe-Pro-p-NHCH2-C6H4NH2HCl. The product was then purified by RPHRLC (method 1; (A/B) from 98/2 up to 60/40, 60 min).1H-NMR; FD-MS, m/e 366 (M+).

Analysis for C21H26N4O2HCl 0,TFK,5H2O:

calculated, %: C 55,51; H 6,00; N 11,66;

found, %: C 55,16; H 6,14; N 11,57.

Example 14

< / BR>
A) Receive Boc-D-Phe-Pro-p-NHCH2CH2C6H4NH2.

By the way, is essentially equivalent to the one described in example 13 And receive 3 g (23%) of Boc-D-Phe-Pro-p-NHCH2CH2- C6H4NH2from Boc-D-Phe-Pro-OH and p-NO2-penicillin-hydrochloride.

1H-NMR; FD-MS, m/e 480 (M+).

Analysis for C27H36N4O4:

calculated, %: C 67,48; H of 7.55; N 11,66;

found, %: C 67,30; H rate of 7.54; N 12,34.

B) Obtaining D-Phe-Pro-p-NHCH2CH2C6H4NH2HCl.

By the way, is essentially equivalent to the one described in example 1-is (B) the gradient of from 98/2 up to 60/40, 60 min).1H-NMR; FD-MS, m/e 380 (M+).

Analysis for C22H28N4O2HCl 0,TFK,7H2O:

calculated, %: C 55,18; H x 6.15; N 11,00;

found, %: C 55,12; H 6,18; N 10,99.

Example 15

< / BR>
(D-i.e. phenylalanyl-N-[[(4-aminoiminomethyl)phenyl] methyl] -L-Proline-amide-hydrochloride)

A) Obtaining n-(aminomethyl)benzonitrile

To a stirred suspension of sodium hydride (2.2 g, 56 mmol, 60% dispersion in oil) in tetrahydrofuran (100 ml) is added 4-(methyl bromide)benzonitrile (10 g, 51 mmol). To this mixture is added slowly through an addition funnel a solution of di-tert-butyl-imino - in primary forms (12.2 g, 56 mmol). After stirring for 16 h the mixture was diluted with diethyl ether (300 ml) and washed twice with water (150 ml). The organic phase is then dried (MgSO4), filtered and concentrated. The obtained solid is then dissolved in a minimum amount of dichloromethane. Added anisole (10 ml) and the solution cooled to 0oC. the Solution is then diluted with triperoxonane acid (200 ml) and further stirred for 1 hour. The solvent is then removed in vacuum and the oily residue is intensively stirred with diethyl ether (100 ml) and after 5 min the product hardens. The precipitate is filtered, industrial/SUP>).

B) Receive Boc-D-Phe-Pro-p-NHCH2C6H4CN.

By the way, is essentially equivalent to the one described in example 1-G, get 7,4 g (78%) of Boc-D-Phe-Pro-p-NHCH2C6H4CN from Boc-D-Phe-Pro-OH and p-(aminomethyl)benzonitrile. TPA. In this case, the product is purified by recrystallization from diethyl ether. IR;1H-NMR; FD-MS, m/e 476 (M+).

C) Receive Boc-D-Phe-Pro-p-NHCH2C6H4C(NH)NH2.

Gaseous hydrogen sulfide is bubbled through a solution of Boc-D - Phe-Pro-p-NHCH2C6H4CN (2 g, 4.2 mmol) in pyridine (25 ml) and triethylamine (2.5 ml) for 30 minutes, the Reaction vessel is then closed and allowed to stand at room temperature for 2 days. The solution is then diluted with water (100 ml) and extracted twice with ethyl acetate (200 ml). The combined organic phase is washed twice with a saturated aqueous solution of sodium chloride, dried over magnesium sulfate, filtered and concentrated in vacuo.

The residue is dissolved in acetone (50 ml), add methyliodide (10 ml) and the solution refluxed for 2 hours the Solvent is removed in vacuum, the residue is dissolved in methanol (20 ml), add ammonium acetate (712 mg, 9.2 mmol) and the solution is boiled with reverse cold the slots (100 ml) and the aqueous phase is washed twice with ethyl acetate (200 ml), then alkalinized with solid sodium bicarbonate, saturated with solid sodium chloride and extracted twice with ethyl acetate (200 ml). United an ethyl acetate extracts are dried (MgSO4), filtered and concentrated, gaining 1.4 g (67%) of a viscous oil.1H-NMR; FD-MS, m/e 494 (M+).

G) Obtaining D-Phe-Pro-P-NHCH2C6H4C(NH)NH2HCl 1,TFK,5H2O:

calculated, %: C 49,76; H 5,12; N 11,70;

found, %: C 49,75; H 5,19; N 11,58.

Example 16

< / BR>
A) Obtaining m-(aminomethyl)-benzonitrile

By the way, is essentially equivalent to the one described in example 15-a, get 10.8 g (86%) m-(aminomethyl)-benzonitrile of m- (methyl bromide)benzonitrile. IR;1H-NMR; FD-MS, m/e: 132 (M+)

B) Receive Boc-D-Phe-Pro-m-NHCH2C6H4CN.

By the way, is essentially equivalent to the one described in example 1-G, get 7.5 g (79%) of Boc-D-Phe-Pro-m-NHCH2C6H4CN from Boc-D-Phe-Pro-OH and m-(aminomethyl)benzonitrile. In this case, the product is purified by recrystallization from diethyl ether. IR;1H-NMR; FD-MS, m/e 476 (M+).

Analysis for C27H32N4O4:

calculated, %: C 68,05; H 6,77; N 11,76;

found, %: C 68,27; H 6,82; N 11,96.

C) Receive Boc-D-Phe-Pro-m-NHCH2C6H4C(N-NHCH2C6H4C (NH)NH2. FD-MS, m/e 494 (M+)

G) Obtaining D-Phe-Pro-m-NHCH2C6H4C(NH)NH20,75 HCl.

By the way, is essentially equivalent to the one described in example 1-G, get 0.65 g (63%) D-Phe-Pro-m-NHCH2C6H4C (NH)NH20,75 HCl. The product was then purified by RPHRLC (method 2; gradient (A/B) of from 98/2 to 75/25, 120 minutes). FD/MS, m/e 394 (M+).

Analysis for C22H27N5O20,75 HCl 1,TFK,5H2O:

calculated, %: C 51,72; H 5,33; N 12,36; Cl 4,69;

found, %: C 51,79; H is 4.93; N 11,96; Cl 4,82.

Example 17

< / BR>
(D-Homopolar-N-[[4-(aminoiminomethyl)phenyl] methyl] -L-Proline - amide-hydrochloride)

A) Receiving Cbz-D-hPro-HE.

D-hPro-OH (5.0 g, of 38.7 mmol) was dissolved in tetrahydrofuran (100 ml) and water (30 ml). the pH Value of the solution set equal to 9.5 with 2n sodium hydroxide solution and added dropwise to benzylchloride (5.5 ml, of 38.7 mmol) and pH support equal to 9.5 with 2n sodium hydroxide solution. The reaction mixture was stirred for 1 h at room temperature. The organic solvent is evaporated in vacuo, add diethyl ether (100 ml) and water (50 ml) to the resulting residue. The aqueous layer was separated, the pH of the solution was adjusted to 2.8 using 3n hydrochloric acid is e to obtain 9.6 g (95%) of a clear oil.1H-NMR;

FD-MS, m/e 264 (MH+).

B). Receiving Cbz-D-hPro-Pro-OH.

Cbz-D-hPro-OH (9.5 g, 36 mmol) is dissolved in ethyl acetate (100 ml) and the solution cooled to 0oC. To the resulting solution was added 2,4,5-trichlorophenol (7,1 g, 36 mmol) and 1,3-dicyclohexylcarbodiimide (7,4 g, 36 mmol). The reaction mixture was stirred for 1 h at 0oC and for 1 h at room temperature. The precipitate is filtered and the filtrate concentrated in vacuo to obtain oil. The oil is dissolved in pyridine (100 ml), add Pro-OH (4,2 g, 36 mmol) and triethylamine (5.0 ml, 36 mmol). The reaction mixture was stirred at room temperature (24 h). The solvent is removed from the reaction mixture in vacuo to obtain an oil. The residue is dissolved in water (100 ml), add diethyl ether (50 ml) and the pH adjusted to 9.5 with 1N solution of sodium hydroxide. The aqueous layer was extracted twice with diethyl ether. The aqueous layer was separated, the pH adjusted to 2.8 with 3n hydrochloric acid and add ethyl acetate (150 ml). The organic layer is separated, dried (MgSO4) and the filtrate evaporated in vacuum to obtain an amorphous solid (11.4 g, 88%). FD-MS 361 (M+).

Analysis for C19H24N4O5:

calculated, %: C 63,32; H of 6.71; N TO 7.77;

found, %: C 63,42; H is alentum described in example 1-G, obtain 2.2 g (84%) of Cbz-D-hPro-Pro-p-NHCH2C6H4CN from Cbz-D-hPro-Pro-OH and p-NH2CH2C6H4CN0,9HCl0,9HBr0,5H2O.

1H-NMR; FD-MS, m/e 357 (M+)

Analysis for C19H27N5O20,9HCl0,9HBr0,5H2O:

calculated, %: C 48,34; H 6,36; N 14,83; Cl 6,76; Br 15,23;

found, %: C 48,66; H 6,36; N 14,62; Cl 7,14; Br of 14.90.

Example 18

< / BR>
(N-[[4-(Aminoiminomethyl)phenyl] methyl] -1-[[(4aS, 8aS)-decahydro-1 (R)-ethenolysis]carbonyl]-L-prolinamide-dihydrochloride).

A) Receiving Cbz-D-1-Piq-Pro-OH.

A solution of 1-ethinlestradiol acid (50 g, in 0.288. mol) in ethanol (150 ml) and 60 ml of 5N HCl restore in the presence of 5% Rh/Al2O3(14 g) and at a hydrogen pressure of 52 bar (750 psi) in the apparatus for high pressure at 50oC for 17 h, the Reaction mixture was filtered through a layer of diatomaceous earth and the filtrate concentrated in vacuo. The solid is triturated with water, filtered and dried to obtain DL-perhydro-1-ethinlestradiol acid (DL-1-Piq-OH) (30 g, 48%); FD/MS 184 (MH+).

DL-1-Piq-OH (30,2 g, 137 mmol) was dissolved in tetrahydrofuran (150 ml) with water (150 ml). the pH of the solution set equal to 9.8 using 5N NaOH solution and added dropwise to benzylchloride (to 21.6 ml, 151 mmol), maintaining the pH 9.5 the ski solvent evaporated in vacuo and to the residue is added diethyl ether (150 ml) and water (50 ml). The aqueous layer was separated, the pH of the solution was adjusted to 2.5 using 5N HCl and add ethyl acetate (200 ml). The organic layer is separated and dried (MgSO4) and the filtrate was concentrated in vacuo, obtaining a clear oil. The oil was dissolved in diethyl ether (150 ml) and the solution allowed to stand at room temperature for 24 hours the Precipitate is filtered and dried to obtain 2-Cbz-DL-perhydro-1 - ethinlestradiol acid (Cbz-DL-1-Piq-OH) (32 g, 75%); FD-MS m/e 318 (MH+).

Cbz-DL-1-Piq-OH (31.8 g, 100 mmol) dissolved in DMF (100 ml) and cooled to 0oC. To the reaction mixture are added tert-butyl ester of Proline (17.1 g, 100 mmol), 1-hydroxybenzotriazole (13.5 g, 100 mmol) and DCC (20.6 g, 100 mmol).

The reaction mixture was stirred for 3 h at 0oC and 24 h at room temperature. The precipitate is filtered and the filtrate concentrated in vacuo to obtain an oil. The oil is dissolved in ethyl acetate (200 ml) and water (100 ml). The organic layer is separated and washed successively 1N solution of sodium bicarbonate, 1.5 n citric acid solution and water. The organic layer is dried (MgSO4) and the filtrate evaporated to obtain an oil, which was dried to obtain 2-Cbz-DL-perhydro-1-isoquinoline - carbonyl-L-prolyl-tert-butyl ether complex (Cbz-DL-1-Piq-P is, containing triperoxonane acid (100 ml), CH2Cl2(35 ml), anisole (5 ml), and stirred at room temperature for 1 h, the Reaction mixture was concentrated in vacuo without heating and the residue is added diethyl ether (100 ml) and water (100 ml). the pH of the solution was adjusted to 9.8 using 5N NaOH solution. The aqueous layer was separated, the pH of the solution was adjusted to 2.5 using 5N HCl and add ethyl acetate (200 ml). The organic layer is separated and dried over magnesium sulfate, the filtrate was concentrated in vacuo, obtaining a clear oil. The oil was dissolved in diethyl ether (700 ml) and to the solution was added (L)-(-) - methylbenzylamine. The solution is left to stand at room temperature for 5 days. The obtained solid is filtered and washed with diethyl ether. The filtrate was washed with 1.5 n citric acid solution and water. The organic layer is dried (MgSO4) and the filtrate evaporated to obtain oil. The oil was dissolved in diethyl ether (400 ml) and left to stand at room temperature for 48 hours resulting solid substance was filtered, washed with diethyl ether and dried, obtaining 2-Cbz-D-perhydro-1-athinaikon-L-Proline (Cbz-D-1-Piq-Pro-OH) (5,86 g, 36%); FAB-MS; 415 (MH+);

[]D= -34,2o(c=0.5, methanol).

B) Received the Oia in oil) in tetrahydrofuran (150 ml) is added 4-(methyl bromide)-benzonitrile (20.5 g, 105 mmol). To this mixture add (slowly via an addition funnel) solution of di-tert - butyl-iminodicarboxylate (25 g, 115 mmol). After stirring for 16 h the mixture was diluted with diethyl ether (500 ml) and washed three times with water (250 ml). The organic phase is then dried (MgSO4), filtered and concentrated, obtaining with 40.2 g of crude solid.

The obtained solid (28,3 g, 85 mmol) is then dissolved in tetrahydrofuran (150 ml) and add sodium hydroxide solution (3.4 g, 85 mmol) in methanol (300 ml). After stirring overnight the solution is concentrated to about 1/2 volume and add water to cause precipitation of the product. The precipitate is filtered and dried in vacuum, obtaining of 18.5 g (94%) of white solids. IR;1H-NMR; FD-MS, m/e 232 (M+).

Analysis for C13H16N2O2:

calculated, %: C 67,22; H 6,94; N 12,06;

found, %: C 67,19; H 7,16; N 11,82.

C) Obtaining p-(BocNHCH2)C6H4C(NH)NHCbz.

By the way, is essentially equivalent to the one described in example 15, N-Boc-p-(aminomethyl)benzonitrile (32.7 g, 140 mmol) is transformed into p-(BocNHCH2)C6H4C(NH)NH2. The remainder of this transformation is dissolved in dimethylformamide (700 ml) and to the solution was added N, is (48 g, 280 mmol). After stirring for 16 h, add water (100 ml) and then the solvents removed in vacuo. The residue is partitioned between water (250 ml) and ethyl acetate (500 ml). The phases are separated and the organic phase is washed three times with saturated aqueous solution of ammonium chloride (250 ml), once with water (200 ml) and twice with saturated aqueous sodium bicarbonate solution (250 ml). The organic phase is then dried (MgSO4), filtered and concentrated, and the product is recrystallized from diethyl ether, receiving 14 g (26%) of a white solid.1H-NMR; FD-MS, m/e 384 (M+).

G) Obtaining p-H2NCH2C6H4C(NH)NHCbz 2HCl.

To a solution of n-(BocNHCH2)C6H4C(NH)NHCbz (11 g, 28.7 mmol) in dichloromethane (125 ml) at 0oC added anisole (10 ml), then triperoxonane acid (125 ml). After stirring for 2 h the solvent is removed in vacuum and the residue is dissolved in 1N HCl (50 ml) and the solution washed twice with diethyl ether (50 ml). the pH was adjusted to 3 using ion-exchange resin (Bio Rad AG1-X8, in the main form) and the solution lyophilized, receiving of 9.2 g (90%) of white powder. 1H-NMR; FD-MS, m/e 284 (M+).

D) Receiving Cbz-1-Piq-Pro-p-NHCH2C6H4C (NH)NHCbz.

By the way, sumisho-OH and p-H2NCH2C6H4C(NH)NHCbz1,9HCl 2,5 H2O:

calculated, %: C 52,53; H 7,65; N 13,32; Cl 12,81;

found, %: C 52,63; H of 7.36; N 13,47; Cl 12,95.

Example 19

< / BR>
A) Receiving Cbz-D-3-Piq-Pro-OH.

D)-Phenylalanine (50 g, 302 mmol) is subjected to interaction with a 37% formaldehyde solution (120 ml) and concentrated HCl (380 ml) by boiling under reflux. After 30 minutes add a further 50 ml of formaldehyde and the reaction continued for 3 hours. The reaction mixture is cooled to -10oC and the precipitate filtered. The solid is dried in vacuum, obtaining D-1,2,3,4-tetrahydro-3-ethanolammonium acid (24.2 g, 45%); FD-MS 178 (MH+).

A solution of D-1,2,3,4-tetrahydro-3-ethinlestradiol acid (17 g, 96 mmol) in water (200 ml) and 20 ml of 5N HCl hydronaut in the presence of 5% Rh/Al2O3(8.5 g) at a pressure of 138 bar (2000 psi) of hydrogen in high-pressure apparatus at 120oC for 16 h, the Reaction mixture was filtered through a gasket (layer) of diatomaceous earth and the filtrate is subjected to drying by freezing, getting D-perhydro-3-ethanolammonium acid (D-3-Piq-OH) (21 g, 100%); FD-MS: 184 (MH+).

D-3-Piq-OH (21,0 g, the 95.8 mmol) dissolved in tetrahydrofuran (75 ml) and water (50 ml). the pH of the solution was adjusted to 10.0 using 5N RA the aOH. The reaction mixture was stirred for additional 1 h at room temperature. The organic solvent is evaporated in vacuo and to the residue is added diethyl ether (100 ml) and water (50 ml). The aqueous layer was separated, the pH of the solution set equal to 3.0 with 3n HCl and add ethyl acetate (250 ml). The organic layer is separated and dried (MgSO4). The filtrate was concentrated in vacuo, obtaining a clear oil 2-Cbz-D-perhydro-3-ethanolammonium acid (Cbz-D-3-Piq-OH) (25,8 g, 85%); FD-MS: 318 (MH+).

Cbz-D-3-Piq-OH (17,2 g, 54 mmol) dissolved in

DMF (50 ml) and cooled to 0oC. To the resulting solution was added tert-butyl ester of Proline (9,2 g, 54 mmol), 1-hydroxybenzotriazole (7,3 g, 54 mmol) and DNA (11.1 g, 54 mmol). The reaction mixture was stirred for 3 h at 0oC and 24 h at room temperature. The precipitate is filtered and the filtrate concentrated in vacuo to obtain an oil. The oil is dissolved in ethyl acetate (200 ml) with water (100 ml). The organic layer is separated and washed successively 1N solution of sodium bicarbonate, water, 1.5 n citric acid solution and water. The organic layer is dried (MgSO4) and filtered. The filtrate is evaporated to obtain an oil, which was dried, obtaining 2-Cbz-D-perhydro-3-isoquinoline mmol) was placed in a round bottom flask, containing triperoxonane acid (100 ml), anisole (5 ml), and stirred at room temperature (1 h). The reaction mixture was concentrated in vacuo without heating and add to the residue in diethyl ether (150 ml) and water (100 ml). the pH of the solution was adjusted to 9.8 using 5N NaOH solution. The aqueous layer was separated, the pH of the solution was adjusted to 2.8 with 3n HCl and add ethyl acetate (200 ml). The organic layer is separated, dried (MgSO4) and filtered. The oil was dissolved in diethyl ether (300 ml) and the solution allowed to stand at room temperature for 24 hours of the Obtained solid is filtered, washed with diethyl ether and dried, obtaining 2-Cbz-perhydro-3-athinaikon-L-Proline (Cbz-D-3-Piq-Pro-OH) (13.5 g, 49%); FAB-MS: 415 (MH+).

Analysis for C23H30N2O5:

calculated, %: C 66,65; H 7,29; N 6,76;

found, %: C 66,90; H 7,33; N for 6.81.

B) Receiving Cbz-D-3-Piq-Pro-p-NHCH2C6H4C(NH)NHCbz.

By the way, is essentially equivalent to the one described in example 18-D, get 1.6 g (49%) of Cbz-D-3-Piq-Pro-p-NHCH2C6H4C(NH)NHCbz from Cbz-D-3-Piq-Pro-OH and p-H2NCH2C6H4C(NH)NHCbz3HCl.

By the way, is essentially equivalent to the one described in example 17 In, receive 150 mg of D-3-Piq-Pro-p-NHCH2C6H4C(NH)NH2 ((S-CIS)-N-[[4-(Aminoiminomethyl)phenyl] methyl] -octahydro-1-D - homopolar-1H-indole-2-carboxamide-trihydrochloride)

A) Receiving Cbz-D-hPro-Ohi-OH.

Gaseous HCl bubbled through a stirred suspension of (S)-indoline-2-carboxylic acid (20 g, 110 mmol) in ethanol (500 ml). When the acid is completely dissolved, the solution is brought to the boiling temperature under reflux. After 16 h, the solution is cooled and the solvent is removed in vacuum. The residue is triturated with diethyl ether and obtained not quite white solid is separated by filtration, washed with hexane and dried overnight in vacuum at 30oC to obtain hydrochloride of the ethyl ester of (S)-indoline-2-carboxylic acid (25,7 g, 78%).

The solid is dissolved in ethanol (800 ml), add 5% Pd/C (25 g) and the resulting suspension hydronaut in Parr apparatus for 8 h (4.1 bar, 60 psi). The solution is filtered and the solvent is removed in vacuum. The residue is dissolved, triturated with diethyl ether and filtered by receive 18,8 g (73%) not quite white solid (CIS-Ohi-OEtHCl).

By the way, is essentially equivalent to the one described in example 1, get 13.5 g (93%) of Cbz-D-hPro-cis-Ohi-OEt from Cbz-D-hPro-HE and CIS-Ohi-OEtHCl. 1H-NMR; FD-MS, m/e 442 (M+)

P CLASS="ptx2">

By the way, is essentially equivalent to the one described in example 1-G, get 12.5 g (102%) of Cbz-D-hPro-cis-Ohi-OH.

1H-NMR; FD-MS, m/e 414 (M+).

Analysis for C23H30N2O5:

calculated, %: C 66,65; H 7,29; N 6,76;

found, %: C to 66.4; H 7,30; N 6,86.

B) Receiving Cbz-D-hPro-Ohi-p-NHCH2C6H4C(NH)NHCbz.

By the way, is essentially equivalent to the one described in example 18-D, get a 3.3 g (67%) of Cbz-D-hPro-Ohi-p-NHCH2C6H4C(NH)NHCbz from Cbz-D-hPro-Ohi-OH and p-H2NCH2C6H4C(NH)NHCbz3HCl.

By the way, is essentially equivalent to the one described in example 18-E, get 2.2 g (66%) of D-hPro-Ohi-p-NHCH2C6H4C(NH)NH23HCl 0.5 H2ABOUT:

calculated, %: C 52,13; H? 7.04 baby mortality; N 13,22;

found, %: C 51,98; H? 7.04 baby mortality; N 13,35.

Example 21

< / BR>
(D-Homopolar-N ()- (2-phenylethyl)-N-[[4-(aminoiminomethyl)phenyl]- methyl] -glycinamide-dihydrochloride)

A) Receiving Cbz-D-hPro-N(PhCH2CH2)Gly-OH.

To a solution of phenethylamine (58 ml, 461 mmol) and triethylamine (21 ml, 154 mmol) in ethanol (200 ml) at 0oC add a solution of tert-butylbromide (30 g, 154 mmol) in ethanol (50 ml) for 1 h the Cooling bath and leave the solution allowed to warm to room temperature. After paramasivan actor washed twice with diethyl ether, alkalinized with solid sodium bicarbonate and then extracted three times with ethyl acetate (20 ml). United an ethyl acetate extracts are dried (MgSO4), filtered and left to stand for 24 hours the precipitate is filtered, washed with diethyl ether and dried to obtain 10.5 g of a white solid. The mother liquor is concentrated to a volume of about 100 ml and then diluted with diethyl ether (400 ml). After standing for 30 minutes the solution is filtered, getting more of 23.5 g of a white solid. A total of 34 g (94%) N(PhCH2CH2)Gly-O-t-Bu.1H-NMR; FD-MS, m/e 235 (M+)

By the way, is essentially equivalent to the one described in example 1, get 10.8 g (56%) of Cbz-D-hPro-N(PhCH2CH2)Gly-O-t-Bu from Cbz-D-hPro-OH and N(PhCH2CH2)Gly-O-t-Bu.

1H-NMR. FD-MS, m/e 480 (M+).

Analysis for C28H36N2O5:

calculated, %: C 69,98; H of 7.55; N OF 5.83;

found, %: C 69,68; H 7,56; N 5,77.

By the way, is essentially equivalent to the one described in example 18, to remove protection from Cbz-DL-1-Piq-Pro-O-t-Bu, gain of 9.2 g (100%) of Cbz-D-hPro-N(PhCH2CH2)Gly-OH.

1H-NMR; FD-MS, m/e 425 (M+).

Analysis for C24H28N2O5:

Russ2- C6H4C(NH)NHCbz.

By the way, is essentially equivalent to the one described in example 18-D, get 3.2 g (55%) of Cbz-D-hPro-N(PhCH2CH2)Gly-p - NHCH2C6H4C(NH)NHCbz from Cbz-D-hPro-N(PhCH2CH2)Gly-OH and p-H2NCH2C6H4C(NH)NHCbz2HCl.

By the way, is essentially equivalent to the one described in example 19-E, get the 770 mg (54%) of D-hPro-N(PhCH2CH2)Gly-p - NHCH2C6H4C(NH)NH22HCl:

calculated, %: C 58,30; H of 6.73; N 14,16;

found, %: C 58,05; H 6,60; N to 12.28.

Example 22

< / BR>
D-hPro-Pro(4-cis-PhO)-p-NHCH2C6H4C(NH)NH22HCl

(Cys-D-Homopolar-N-[[4-(aminoiminomethyl)-phenyl] methyl] -4-phenoxy - L-prolinamide-dihydrochloride).

A) Receiving Cbz-D-hPro-Pro(4-cis-PhO)-OH.

To a solution of Cbz-Pro(4-TRANS-OH)-Et (58,8 g, 200 mmol), triphenylphosphine (65,6 g, 250 mmol) and phenol (23,5 g, 250 mmol) in tetrahydrofuran (500 ml) at 0oC add (adding dropwise within 1 h) solution of diethylazodicarboxylate (40 ml, 250 mmol) in THF (50 ml). The cooling bath is then removed and the solution allowed to warm to room temperature for 16 h then the solvent is removed in vacuum and the remaining amber syrup is triturated with diethyl ether. White solid deletion is openitem gradient from hexane to a mixture of 1:1 ethyl acetate with hexane. Containing pure product fractions (assessed by TLC) are combined and concentrated in vacuo obtaining of 36.3 g (50%) of Cbz-Pro(4-CIS-phenoxy)OEt in the form of a colorless syrup. 1H-NMR; FD-MS, m/e 369 (M+).

Analysis for C21H23NO5:

calculated, %: C 68,28; H 6,28; N 3,79;

found, %: C 68,38; H 6,30; N 3,89.

To a solution of Cbz-Pro(4-CIS-phenoxy)-OEt (25 g, + 67.7 mmol) in ethanol (400 ml) is added 5% Pd/C (5 g). After ozonation of hydrogen through the solution for 3 h, the solution was filtered through a gasket (layer) diatom earth, add 3 ml of concentrated HCl and the solution was concentrated in vacuo. The residue is suspended in diethyl ether with vigorous stirring and then filtered and dried, obtaining 14.2 g (77%) Pro(4-CIS-phenoxy)-OEtHCl in the form of a white solid.1H-NMR; FD-MS, m/e 235 (M+).

Analysis for C13H18NO3Cl:

calculated, %: C 57,46; H of 6.68; N 5,15;

found, %: C 57,68; H is 6.78; N 5,18.

By the way, is essentially equivalent to the one described in example 1-a, get to 19.4 g (100%) of Cbz-D-hPro-Pro(4-CIS-phenoxy)-OEt from Cbz-D-hPro-IT Pro(4-CIS-phenoxy)-OEtHCl.

1H-NMR; FS-MS, m/e 480 (M+).

Analysis for C27H32N2O6:

calculated, %: C 67,48; H of 6.71; N OF 5.83;

Nai is Cbz-D-hPro-(4-CIS-phenoxy)-OH.

1H-NMR; FD-MS, m/e 452 (M+).

Analysis for C25H28N2O6:

calculated, %: C collected 66.36; H 6,24; N IS 6.19;

found, %: C 66,22; H 6,18; N 6,17.

B) Receiving Cbz-D-hPro-Pro(4-cis-PhO)-p-NHCH2- C6H4C(NH)NHCbz.

By the way, is essentially equivalent to the one described in example 18-D, get 4,55 g (75%) of Cbz-D-hPro-Pro(4-cis-PhO)-p - NHCH2C6H4C(NH)NHCbz from Cbz-D-hPro-Pro(4-cis-PhO)-OH and p-H2NCH2C6H4C(NH)NHCbz2HCl.

By the way, is essentially equivalent to the one described in example 18, receive 873 mg (40%) of D-hPro-Pro(4-cis-PhO)-p - NHCH2C6H4C(NH)NH22HCl:

calculated, %: C 57,47; H 6,37; N 13,40;

found, %: C 57,22; H 6,29; N 13,47.

Example 23

< / BR>
(N-(Ethylsulfonyl)-D-i.e. phenylalanyl-N-[[4-(aminoiminomethyl)phenyl] methyl] - L-prolinamide hydrochloride)

A) Obtaining p-NH2CH2-C6H4CNHCl.

To a solution of EtSO2-D-Phe-Pro-p-NHCH2-C6H4CN (1 g, 2.1 mmol) in absolute ethanol (35 ml) is added N, N-diisopropylethylamine (of 0.47 ml, 2.7 mmol), then gidroxinimesoulid (185 mg, 2.7 mmol) and the solution brought to a boil under reflux. After 16 h the solution is cooled and the solvents removed in vacuo. 250 mg of this substance is used in the next stage, and /e 501 (M+).

Analysis for C24H31N5O51,2 HCl H2O:

calculated, %: C 51,17; H 6,12; N 12,42; Cl 7,55;

found, %: C 51,04; H of 5.81; N KZT 12.39; Cl 7,18.

G) Receiving EtSO2-D-Phe-Pro-p-NHCH2- C6H4C(=NH)NH2HCl.

To a solution of EtSO2-D-Phe-Pro-p-NHCH2- C6H4C(= NOH)NH2HCl (250 mg, 0.52 mmol) in ethanol (40 ml) and water (19 ml) is added 1N HCl (1 ml), then 250 mg of 5% palladium-on-charcoal grill. Stir the suspension is placed in an atmosphere of hydrogen for 18 hours and then filtered, concentrated and purified by RPHRLC (method 1; the gradient (A/B) is from 90/10 to 60/40, within 200 min) to give 140 mg (52%) EtSO2-D-Phe-Pro-p - NHCH2-C6H4C(=NH)NH2HCl for 1.5 H2ABOUT:

calculated, %: C 52,50; H 6.42 per; N WAS 12.75;

found, %: C 52,56; H is 6.19; N 12,59.

Another 5 g of the product obtained by the method described in example 15, and purified by RPHPLC (method 3; gradient (A/B): 98/2 for 60 min, before 60/40 - for 300 min).

Example 24

< / BR>
A) Obtaining EtSO2-D-Phe-Pro-m-NHCH2-C6H4C(=NOH)NH2.

By the way, is essentially equivalent to the one described in example 23, EtSO2-D-Phe-Pro-m-NHCH2-C6H4C(= NOH)NH2receive, using m-Br-CH2-C6H4C instead of p-Br-CH2-C1
H-NMR; FD-MS, m/e 502 (M+)

Analysis for C24H31N5O5:

calculated, %: C 57,47; H 6,23; N 13,96;

found, %: C 57,28; H 6,21; N 13,66.

B) Receiving EtSO2-D-Phe-Pro-m-NHCH2- C6H4C(=NH)NH2HCl.

By the way, essentially equivalent described in examples 23 and 23-G) obtain 0.27 g (28%, stage 2) EtSO2-D-Phe-Pro-m - NHCH2-C6H4C(=NH)NH21,1 HCl 2H2O:

calculated, %: C 51,32; H 6,48; N 12,47; Cl 6,94;

found, %: C 51,33; H 6,09; N 12,20; Cl 6,66.

Example 25

< / BR>
By the way, is essentially equivalent to the one described in example 23, get 0,86 g D-1-Piq-Pro-m-NHCH2- C6H4C(=NH)NH2HCl of Cbz-D-1-Piq-Pro-OH and m-NH2CH2-C6H4CN2,5HCl 0.5 H2O:

calculated, %: C 53,99; H 7,19; N 13,69;

found, %: C to 54.19; H 7,02; N 13,81.

Example 26

< / BR>
A) Obtaining methyl-n-cyano-TRANS-cinnamate

To a stirred suspension of NaH (6,1 g of 60% oil suspension 153 mmol) and p-cyanobenzaldehyde (20 g, 153 mmol) in tetrahydrofuran (250 ml) at 0oC added through an addition funnel a solution of trimethylphosphate (28 g, 153 mmol) in tetrahydrofuran (50 ml). After stirring for 48 h the solvent is removed in vacuo and the crude residue is dissolved in ethyl) - Rev. B> and once the salt solution. The organic phase is then dried (MgSO4), filtered and concentrated in vacuo, obtaining 28 g (98%) of a white solid. IR;1H-NMR; FD-MS, m/e 187 (M+).

B) Obtaining methyl-n-cyano-digitalkamera

To a solution of methyl n-cyano-TRANS-cinnamate (13,6 g, 73 mmol) in toluene (485 ml) is added 5% Pd/BaSO4(2.7 g). After exposure to hydrogen gas under pressure 4 bar (60 psi) for 9 h, the solution is filtered, concentrated in vacuo and chromatographic on silica gel, elwira stepped gradient from hexane to hexane to 30% ethyl acetate. Containing the product fractions are combined and concentrated to obtain 10.6 g (77%) of colorless oil. IR;1H-NMR; FD-MS, m/e 189 (M+)

C) Obtaining p-canadahydrochloride acid

By the way, is essentially equivalent to the one described in example 1-G, using 1.1 equivalent LiOHH2O get a 5.1 g (58%) of n-cyano-dihydrocortisol acid from methyl-n-cyano-digitalkamera. IR;1H-NMR; FD-MS, m/e 175 (M+).

G) Receive Boc-p-NHCH2CH2-C6H4CN.

To a solution of n-cyano-dihydrocortisol acid (6.7 g, is 38.2 mmol) and triethylamine (of 5.9 ml, 42 mmol) in tert-butanol (150 ml) added definito the deposits during the night, the solution is cooled and the solvent is removed in vacuum. The residue is dissolved in ethyl acetate and the resulting solution was washed three times with 1N citric acid solution, once with saturated salt solution, twice with saturated aqueous sodium bicarbonate solution and then dried (MgSO4), filtered and concentrated in vacuo. The residue is then chromatographic on silica gel, elwira using a mixture of hexane to 10% ethyl acetate hexane to 50% ethyl acetate. Containing the product fractions, as assessed by TLC, are combined and concentrated to obtain 5.4 g (57%) of white solids. IR;1H-NMR; FD-MS, m/e 246 (M+)

Analysis for C14H18N2O2:

calculated, %: C 68,27; H 7,37; N 11,37;

found, %: C 68,39; H 7,50; N 11,40.

D) p-NH2CH2CH2- C6H4CNHCl.

By the way, is essentially equivalent to the one described in example 23-A, obtain 3.6 g (98%) p-NH2CH2CH2- C6H4CNHCl. 1H-NMR; FD-MS, m/e: 147 (MH+)

Analysis for C9H11N2Cl:

calculated, %: C 59,18; H 6,07; N 15,34; Cl 19,41;

found, %: C 58,90; H 6,16; N 15,20; Cl 19,30.

E) Receiving EtSO2-D-Phe-Pro-p-NHCH2CH2- C6H4CN.

By the way, CN of EtSO2-D-Phe-Pro-OH and p-NH2CH2CH2-C6H4CNHCl.

To mix the solution EtSO2-D-Phe-Pro-p - NHCH2CH2-C6H4CN (1 g, 2,07 mmol) and N, N-diisopropylethylamine (0.45 ml, at 2.59 mmol) added gidroxinimesoulid (180 mg, at 2.59 mmol) and the solution is brought to the boiling temperature under reflux. After 18 h (at this temperature), the solution is cooled, the solvent is removed in vacuum and the residue is dissolved in acetic acid (15 ml) and purified by RPHRLC (method 2; gradient (A/B) is from 90/10 to 60/40, within 200 minutes). The fractions containing pure EtSO2-D-Phe-Pro-p-NHCH2CH2- C6H4C(= NOH)NH2HCl, as determined by analytical RPHRLC, unite and pH set as described above and lyophilizers, receiving 0.35 g (31%) EtSO2-D-Phe-Pro-p-NHCH2CH2- C6H4C(= NOH)NH2HCl.

1H-NMR; FD-MS, m/e 516 (M+)

Analysis for C25H33N5O5SHClH2O:

calculated, %: C 52,67; H 6,36; N To 12.28; Cl 6,22;

found, %: C 52,40; H 6,10; N 12,35; Cl 6,51.

C) Obtaining EtSO2-D-Phe-Pro-p-NHCH2CH2- C6H4C(=NH)NH2HCl.

By the way, is essentially equivalent to the one described in example 23-G, get 0,98 g (50%) EtSO2-/SUB>C(=NOH)NH2HCl.

1H-NMR; FD-MS, m/e 500 (M+).

Analysis for C25H33N5O4S2,6HCl H2O:

calculated, %: C 49,03; H is 6.19; N 11,44;

found, %: C 48,87; H 5,79; N of 11.15.

Example 27

< / BR>
A) Obtaining EtSO2-D-Phe-Pro-m-NHCH2CH2- C6H4C(=NOH)NH2.

By the way, essentially equivalent described in examples 26: 26-D and 24-And receive 0.15 g EtSO2-D-Phe-Pro-m-NHCH2CH2- C6H4C(=NOH)NH2from m-cyanobenzaldehyde.1H-NMR; FD-MS, m/e 516 (M+).

Analysis for C25H33N5O5S:

calculated, %: C 58,23; H 6,45; N 13,50;

found, %: C 57,99; H to 6.57; N 13,28.

B) Receiving EtSO2-D-Phe-Pro-m-NHCH2CH2- C6H4C(=NH)NH2HCl.

By the way, is essentially equivalent to the one described in example 24-B, obtain 0.21 g (20%) EtSO2-D-Phe-Pro-m-NHCH2CH2- C6H4C(=NH)NH2HCl of EtSO2-D-Phe-Pro-m-NHCH2CH2- C6H4C(=NOH)NH2.

1H-NMR; FD-MS, m/e 500 (M+).

Analysis for C25H33N5O4S2,1HCl0,7H2O:

calculated, %: C 51,00; H 6,25; N 11,89;

found, %: C 50,79; H 5,86; N 11,54.

Example 28

< / BR>
By the way,) is 2HCl from Cbz-D-1-Piq-Pro-OH and p-NH2CH2CH2-C6H4CN2HCl 2H2O:

calculated, %: C 53,93; H 7,73; N 13,10;

found, %: C 53,94; H 7,60; N 13,06.

Example 29

< / BR>
By the way, is essentially equivalent to the one described in example 23, to obtain 0.8 g of 1-Piq-Pro-m-NHCH2CH2- C6H4C(=NH)NH22HCl from Cbz-D-1-Piq-Pro-OH and m-NH2CH2CH2-C6H4CN2HCl 2H2O:

calculated, %: C 53,93; H 7,73; N 13,10;

found, %: C 53,62; H EUR 7.57; N of 13.18.

Example 30

< / BR>
< / BR>
A) Obtaining p-HOCH2CH2CH2-C6H4CN

To a stirred solution of methyl n-cyano-digitalkamera (10 g, 53 mmol) in tetrahydrofuran (150 ml) is added LiBH4(1,15 g, 53 mmol) and the solution refluxed. After 2 h the solution is cooled and added dropwise buffer phosphate (pH 7). After the evolution of gas, add ethyl acetate and water and the layers separated. The aqueous phase is extracted once with ethyl acetate and the combined an ethyl acetate phases are washed with brine, then dried (MgSO4), filtered and concentrated to obtain 8.1 g (95%) of a viscous colorless oil. IR;1H-NMR; FD-MS, m/e: 161 (M+).

B) Obtaining p-Br-CH2-CH2CH2-C6H4CN.

To a stirred solution of p-HOCH1H-NMR; FD-MS, m/e 223 (M+).

Analysis for C10H10BrN:

calculated, %: C 53,60; H 4,50; N 6,25;

found, %: C 53,90; H of 4.67; N 6,24.

C) Obtaining n-Boc2CH2CH2CH2- C6H4CN

To a stirred suspension of NaH (1.4 g of 60% oil dispersion, 34 mmol) in DMF (100 ml) added dropwise with an addition funnel to a solution of di-tert-butyl-iminodicarboxylate (7,4 g, 34 mmol) in DMF (20 ml). After gas evolution add a solution of p-Br-CH2CH2-CH2-C6H4CN (7 g, 31 mmol) in DMF via addition funnel and the solution is heated to 70oC. After stirring for 12 h at this temperature, the solution is cooled and the solvent is removed in vacuum. The residue is dissolved in diethyl ether and washed 3 times with water. The organic phase is dried over magnesium sulfate, filter and concentrate the and. Containing the product fractions are combined and concentrated, obtaining 9,38 g (84%) of white solids. IR;1H-NMR; FD-MS, m/e 361 (M+).

Analysis: for C20H28N2O4:

calculated, %: C 66,64; H 7,83; N TO 7.77;

found, %: C 66,40; H 7,81; N EUR 7.57.

G) Obtaining p-NH2CH2CH2CH2- C6H4CNHCl

By the way, is essentially equivalent to the one described in example 23-A, obtain 4.3 g (84%) p-NH2CH2CH2CH2- C6H4CNHCl. IR;1H-NMR; FD-MS, m/e: 160 (M+).

D) EtSO2-D-Phe-Pro-p-NHCH2CH2CH2- C6H4C(=NOH)NH2HCl.

By the way, essentially equivalent described in examples 1-W and 26 W, get 0.32g EtSO2-D-Phe-Pro-p-NHCH2CH2CH2- C6H4C(= NOH)NH2HCl of EtSO2-D-Phe-Pro-OH and p-NHCH2CH2CH2- C6H4CNHCl.

1H-NMR; FD-MS, m/e 530 (M+).

Analysis for C26H35N5O5S1,2HCl H2O:

calculated, %: C 52,88; H 6,51; N 11,84;

found, %: C 52,71; H of 6.26; N 11,76.

E) Receiving EtSO2-D-Phe-Pro-p-NHCH2CH2CH2- C6H4C(=NH)NH2HCl.

By the way, essentially equivalent described in section the UB>2-D-Phe-Pro-p-NHCH2CH2CH2- C6H4C(= NOH)NH2HCl. 1H-NMR; FD-MS, m/e 514 (M+). Analysis for C26H35N5O4S1,5HCl 2H2O:

calculated, %: C 51,67; H of 6.75; N 11,59;

found, %: C 51,36; H 6,46; N 11,28.

Example 31

< / BR>
A) Obtaining EtSO2-D-Phe-Pro-m-NHCH2CH2CH2- C6H4C(=NOH)NH2HCl.

By the way, essentially equivalent described in examples 1-W and 26 W, get 0.32g EtSO2-D-Phe-Pro-m-NHCH2CH2CH2- C6H4C(= NOH)NH2HCl of m-canadahydrochloride acid.1H-NMR; FD-MS, m/e 530 (M+).

Analysis for C26H35N5O5SHCl 1,1 H2O:

calculated, %: C 53,30; H to 6.57; N 11,95; Cl 6,05;

found, %: C 52,97; H is 6.19; N 11,96; Cl 6,13.

B) Receiving EtSO2-D-Phe-Pro-m-NHCH2CH2CH2- C6H4C(=NH)NH2HCl.

By the way, is essentially equivalent to the one described in example 23-G, get 0.12 g (62%) EtSO2-D-Phe-Pro-m-NHCH2CH2CH2- C6H4C(=NH)NH2HCl of EtSO2-D-Phe-Pro-m-NHCH2CH2CH2- C6H4C(=NOH)NH2HCl.

1H-NMR; FD-MS, m/e 514 (M+).

Analysis for C26H35N5O4S1,5HCl H2O:

TSS is equivalent to the one described in example 23, obtain 0.66 g (48%) of 1-Piq-Pro-p-NHCH2CH2CH2- C6H4C(= NH)NH2HCl of 1-Piq-Pro-OH and p-NH2CH2CH2CH2-C6H4CN2,1HCl H2O:

calculated, %: C 56,21; H of 7.75; N 13,11;

found, %: C 56,36; H 7,44; N 12,79.

Example 33

< / BR>
By the way, is essentially equivalent to the one described in example 23, to obtain 0.64 g (46%) of 1-Piq-Pro-m-NHCH2CH2CH2- C6H4C(= NH)NH2HCl of 1-Piq-Pro-OH and

1H-NMR; FD-MS, m/e 440 (M+).

Analysis for C25H37N5O22HCl H2O:

calculated, %: C 56,60; H 7,79; N 13,20;

found, %: C 56,92; H of 7.55; N 13,26.

Example 34

< / BR>
A) Receive Boc-p-NHCH2C6H4NO2< / BR>
To a stirred solution of 4-nitrobenzylamine-hydrochloride (15 g, 79 mmol) and N,N-diisopropylethylamine (14 ml, 79 mmol) in dichloromethane (200 ml) is added di-tert-BUTYLCARBAMATE (17 g, 79 mmol). After 48 h the solvent is removed in vacuum and the residue is dissolved in ethyl acetate (500 ml) and washed twice 1M citric acid solution, once with water and once with saturated aqueous sodium bicarbonate. The organic phase is dried (MgSO4), filtered and concentrated in vacuo to obtain not quite white solid, which Perek is the Aquum, receiving of 11.5 g (58%) of white solids. IR;1H-NMR; FD-MS, m/e 252 (M+).

Analysis for C12H16N2O4:

calculated, %: C 57,13; H to 6.39; N 11,10;

found, %: C 57,27; H 6,60; N 11,13.

B) Obtaining p-BocNHCH2C6H4NH2< / BR>
To a stirred solution of p-BocNHCH2C6H4NO2(7.5 g, 29.7 mmol) and NiCl26H2O (17,7 g of 74.3 mmol) in methanol (150 ml) at 0oC add NaBH4(5.6 g, 149 mmol) in small portions during 30 minutes after the addition of NaBH4and after another 15 minutes the solvent is evaporated in vacuum and the residue is dissolved in concentrated ammonium hydroxide solution and extracted twice with dichloromethane. The combined organic extracts washed with brine, dried (MgSO4), filtered and concentrated in vacuo to obtain 6.4 g (97%) of a white solid.

IR; 1H-NMR: FD-MS, m/e 222 (M+).

Analysis for C12H18N2O2:

calculated, %: C 64,84; H 8,16; N 12,60;

found, %: C 65,10; H 8,42; N 12,76.

C) Obtaining N,N-di-Cbz-S-methylisothiazoline

To a stirred suspension of sulfate bis-S-methylisothiazoline (20 g, 144 mmol) in dichloromethane (200 ml) is added 5N solution g is. the same time add the 2n sodium hydroxide solution in such a quantity to maintain the pH around 11. The cooling bath is then removed and after heating to room temperature, the phases are separated and the aqueous phase extracted with dichloromethane (250 ml). The combined organic phases are then washed twice with 0.1 n HCl (250 ml) and once with brine (250 ml). The organic phase is then dried (MgSO4), filtered and concentrated in vacuo to obtain 41 g (79%) of a viscous colorless syrup.1H-NMR.

G) Obtaining p-BocNHCH2C6H4NHC(NCbz)NHCbz

To a stirred solution of p-BocNHCH2C6H4NH2(5 g, to 22.5 mmol) in tetrahydrofuran (50 ml) is added N,N'-di-Cbz-S-methylisothiazoline (8,9 g of 24.7 mmol). After 48 h the solvent is removed in vacuum and the residue is dissolved in chloroform. Add silica gel and the solvent is removed in vacuum to obtain not quite white powder, which is then in a dry condition make silicagel column. The contents of the column then elute stepped gradient from a mixture of hexane with 5% ethyl acetate to a mixture of hexane to 30% ethyl acetate. Containing the product fraction (determined by TLC) are combined and concentrated in vacuo to obtain 7.6 g (63%B>O6:

calculated, %: C 65,40; H the 6.06; N 10,52;

found, %: C 65,66; H 6,35; N 10,59.

D) Obtaining HClp-NH2CH2C6H4NHC(NCbz)NHCbz

By the way, is essentially equivalent to the one described in example 23-A, obtain 4.7 g (89%) HClp-NH2CH2C6H4NHC(NCbz)NHCbz, in the form of a white solid of p-BocNHCH2C6H4NHC(NCbz)NHCbz. IR;1H-NMR; FD-MS, m/e 433 (M+).

E) Receiving EtSO2-D-Phe-Pro-p - NHCH2C6H4NHC(NH)NH2Is HCl.

By the way, is essentially equivalent to the one described in example 1-G and in example 18-E, get 1.1 g EtSO2-D-Phe-Pro-p - NHCH2C6H4NHC(NH)NH2p-NH2CH2C6H4NHC (NCbz)NHCbz.

Analysis for C24H32N6O4SHCl H2O:

calculated, %: C 51,73; H 6,36; N 15,14;

found, %: C 52,32; H of 5.99; N 14,79.

Example 35

< / BR>
Ways, essentially equivalent to the one described in example 34, obtain 1.8 g EtSO2-D-Phe-Pro-p-NHCH2CH2C6H4- CNH(NH)NH2HCl of 4-nitrophenylamino. HCl. IR;1H-NMR; FD-MS, m/e 515 (M+).

HRMS (FAB), m/e, C25H35N6O4S: calculated: 515,2441;

found: 515,2483.

Example 36

< / BR>
Ways, essentially R>
The methods are essentially equivalent described in examples 26-G 26-B (using 5% Pd/C instead of Pd/BaSO4and ethyl acetate instead of toluene) and 34 G-34-E, get 0,85 g EtSO2-D-Phe-Pro-m - NHCH2CH2C6H4NHC(NH)NH22,2 HCl 0.5 H2O:

calculated, %: C 49,73; H 6,21; N 13,92;

found, %: C 49,45; H of 5.82; N 13,55.

Example 38

< / BR>
Ways, essentially equivalent to the one described in example 34, receive 0,94 g D-1-Piq-Pro-p-NHCH2C6H4NHC(NH)NH22HCl:

calculated, %: C 55,31; H 7,26; N 16,82; Cl 14,20;

found, %: C 55,05; H of 7.23; N 16,55; Cl 14.24 from.

Example 39

< / BR>
Ways, essentially equivalent to the one described in example 35, the gain of 1.03 g D-1-Piq-Pro-p-NHCH2CH2C6H4NHC(NH)NH22HCl. The target product was then purified by RPHRLC (method 2; gradient (A/B) of from 98/2 to 70/30, 180 min).1H-NMR; FD-MS, m/e 441 (M+).

Analysis for C24H36N6O22HCl 1,5 H2O:

calculated, %: C 53,33; H 7,65; N 15,55; Cl 13,12;

found, %: C 53,41; H 7,45; N Shed 15.37; Cl 13,48.

Example 40

< / BR>
Ways, essentially equivalent to the one described in example 36, obtain 1.04 g D-1-Piq-Pro-m-NHCH2C6H4NHC(NH)NH22HClH2O:

calculated, %: C 53,38; H 7,40; N 16,24; Cl 13,70;

found, %: C 53,25; H 7,50; N 16,23; Cl 13,8 D-1-Piq-Pro-m-NHCH2CH2C6H4NHC(NH)NH22HCl. The target product was then purified by RPHPLC (method 2, the gradient (A/B) of from 98/2 to 70/30, 180 min).1H-NMR; FD-MS, m/e 441 (M+).

Analysis for C24H36N6O22,1 HCl for 1.5 H2O:

calculated, %: C 52,97; H to 7.61; N 15,44; Cl 13,68;

found, %: C 52,80; H EUR 7.57; N 15,46; Cl 13,35.

Example 42

< / BR>
A) Receiving Cbz-DL-1-Piq-cis-Ohi-OEt.

By the way, is essentially equivalent to the one described in example 1, receive 16.6 g (100%) of Cbz-DL-1-Piq-cis-Ohi-OEt of Cbz-DL-1-Piq-OH and CIS-Ohi-OEtHCl. 1H-NMR; FD-MS, m/e 496 (M+).

Analysis for C29H40N2O5:

calculated, %: C 70,13; H 8,12; N 5,64;

found, %: C 69,96; H 8,23; N 5,73.

B) Receiving Cbz-D-1-Piq-cis-Ohi-p - NHCH2C6H4C(NH)NHCbz.

The methods are essentially equivalent described in examples 1-D and 18 D, Cbz-D-1-Piq-cis-Ohi-p-NHCH2C6H4C(NH)NHCbz and Cbz-L-1-Piq-cis-Ohi-p-NHCH2C6H4C(NH)NHCbz get from Cbz-D,L-1-Piq-Pro-OH and p-H2NCH2C6H4C(NH)NHCbz2HCl 1H2O:

calculated, %: C 58,27; H 7,79; N 12,58;

found, %: C 58,66; H 7,56; N 12,78.

Example 43

< / BR>
A) Obtaining D-3-Piq-cis-Ohi-p-NHCH2C6H4C(NH)NH22HCl.

calculated, %: C 60,22; H to 7.67; N 13,00;

found, %: C 59,95; H 7,73; N 12,89.

camid hydrochloride).

A) Receive Boc-D-Phg-cis-Ohi-OEt

By the way, is essentially equivalent to the one described in example 1, receive 14.9 g (58%) of Boc-D-Phg-cis-Ohi-OEt from Boc-D-Phg-OH and the acid chloride ethyl ester of (S)-CIS-octahedron-2-carboxylic acid.1H-NMR; FD-MS, m/e 430 (M+).

Analysis for C24H34N2O5:

calculated, %: C 66,95; H of 7.96; N 6,51;

found, %: C 66,69; H 8,02; N 6,40.

B) Obtaining D-Phg-cis-Ohi-OEtHCl.

To a cooled (0oC), stirred solution of Boc-D-Phg-CIS-Ohi-OEt in ethyl acetate, passed through ozonation within 10 min of gaseous HCl. After stirring for 2 h, allowed to warm to room temperature, the solvent is removed in vacuum. The obtained solid is suspended in diethyl ether and then allocate by filtering, receiving 10.7 g (97%) D-Phg-CIS-Ohi-OEtHCl. 1H-NMR; FD-MS, m/e 331 (M+).

Analysis for C19H27N2O3Cl:

calculated, %: C 62,20; H 7,41; N OF 7.64;

found, %: C 62,42; H of 7.36; N a 7.85.

C) Obtaining EtSO2-D-Phg-cis-Ohi-OEt.

To a solution of D-Phg-cis-Ohi-OEtHCl HCl (10 g, 27 mmol) and N,N-diisopropylethylamine (10,7 ml, 61 mmol) in THF (200 ml) at -78oC was added dropwise from a dropping funnel a solution of acanaloniidae (3.9 g, 30 mmol) in THF (20 ml). Ohver concentrated in vacuo. The residue is dissolved in ethyl acetate (20 ml), washed twice 1N citric acid solution (200 ml), saturated aqueous sodium bicarbonate (200 ml) and brine (200 ml). The organic phase is then dried over magnesium sulfate, filtered and concentrated in vacuo to obtain 11.2 g (97%) of a yellow foam. 1H-NMR; FD-MS, m/e 422 (M+).

Analysis for C21H30N2O5S:

calculated, %: C 59,69; H 7,16; H 6,63;

found, %: C 59,94; H 7,08; N is 6.78.

G) Receiving EtSO2-Phg-cis-Ohi-p - NHCH2C6H4C(NH)NH2.

The methods are essentially equivalent described in examples 1-D, 18 D and 18 E, get 0,62 g EtSO2-Phg-CIS-Ohi-p - NHCH2C6H4C(NH)NH2HCl:

calculated, %: C 57,69; H 6,45; N 12,46;

found, %: C 57,47; H 6,48; N 12,20.

Example 45

< / BR>
((S-CIS)-N-[[4-(Aminoiminomethyl)phenyl] methyl] -1-[N-(ethylsulfonyl)- D-i.e. phenylalanyl]-1H-indole-2-carboxamide hydrochloride)

A) Obtaining EtSO2-Phe-cis-Ohi-p - NHCH2C6H4C(NH)NH2HCl of Boc-D-Phe-OH.

Analysis for C28H37N5O4SHClH2O:

calculated, %: C 56,51; H 6,94; N 11,77;

found, %: C 56,24; H 6,55; N 11,72.

Example 46

< / BR>
(N-(Carboxymethyl)-D-i.e. phenylalanyl-N-[[4-(aminoiminomethyl D-Phe-Pro-OBnHCl (20 g, 51 mmol) in DMF (100 ml) is added tert-butyl-bromoacetate (9,9 g, 56 mmol) at once, and within 30 minutes added dropwise N,N-diisopropylethylamine a (17.4 ml, 101 mmol). This mixture is allowed to mix at room temperature for 18 hours and Then at once add di-tert-BUTYLCARBAMATE (16.6 g, 76 mmol) and N,N-diisopropylethylamine (13,2 ml, 76 mmol) and the reaction mixture is additionally stirred for 24 hours the Solvent is removed in vacuo and the residue distributed between ethyl acetate (1 l) and 1 M aqueous citric acid solution (500 ml). The layers are separated and the organic phase is washed once with 1M aqueous citric acid solution, twice with saturated aqueous sodium bicarbonate solution and once with brine (500 ml each of wash funds). The organic phase is dried (MgSO4), filtered and concentrated in vacuo. Amber oil purified by chromatography on silica gel, elwira gradient ethyl acetate/hexane (hexane to a mixture of hexane to 30% ethyl acetate). Containing the product fractions are combined and concentrated to obtain 19,0 g (66%) of colorless oil that slowly crystallized upon standing.1H-NMR; FD-MS, m/e 566 (M+).

Analysis for C32H42N2O7:

Rasch">

To a solution of N-(t-BuOOCCH2)-N-Boc-D-Phe-Pro-OBn (18.5 g, 33 mmol) in ethyl acetate (250 ml) is added 5% Pd/C as catalyst (5 g). This solution for several hours Tegaserod in vacuum and placed in an atmosphere of hydrogen for 2 h under stirring. The hydrogen source is removed, add diatomaceous earth and the suspension is filtered through a layer of diatomaceous earth. The filtrate was concentrated in vacuo obtaining of 13.2 g (84%) of a white foam. 1H-NMR; FD-MS, m/e 476 (M+).

Analysis for C25H36N2O7:

calculated, %: C 63,01; H to 7.61; N 5,88; found, %: C 63,23; H 7,73; N 5,59;

C) Obtaining N-(t-BuOOCCH2)-N-Boc-D-Phe-Pro-p - NHCH2C6H4C(NH)NHCbz.

By the way, essentially equivalent to example 18-D, get 2.7 g (90%) of N-(t-BuOOCCH2)-N-Boc-D-Phe-Pro-p - NHCH2C6H4C(NH)NHCbz N-(t-BuOOCCH2)-N-Boc-D-Phe-Pro-OH-OH and p-H2NCH2C6H4C(NH)NHCbz2HCl:

calculated, %: C 54,97; H 5,96; N 13,35;

found, %: C 55,21; H 6,11; N 13,39.

Example 47

< / BR>
A) Obtaining tert-BuOOCCH2-D-Phe-cis-Ohi-OEt.

To a solution of D-Phe-cis-Ohi-OEtHCl (30 g, 79 mmol) in acetonitrile (400 ml) is added N,N-diisopropylethylamine (41 ml, 236 mmol) and tert-butyl-bromoacetate (14 ml, 87 mmol). This solution is heated to 50oC and kept at this pace ethyl acetate (300 ml) and this solution is washed twice with a saturated aqueous solution of ammonium chloride (200 ml), double-saturated aqueous sodium bicarbonate (200 ml) and twice with saline solution (200 ml). The organic layer is dried over magnesium sulfate, filtered and concentrated in vacuo to obtain an orange oil, which was purified by chromatography on silica gel, elwira gradient from hexane to 1: 1 hexane with ethyl acetate. Containing the product fractions (identified by TLC) are combined and concentrated to obtain a 33.2 g (92%) of colorless oil.1H-NMR; FD-MS, m/e 458 (M+).

Analysis for C26H38N2O5:

calculated, %: C 68,10; H 8,35; N 6,11;

found, %: C 68,37; H of 8.47; N 5,90.

B) Obtaining N-(t-BuOOCCH2)-N-Boc-D-Phe-cis-Ohi-OH.

To a solution of tert-BuOOCCH2-D-Phe-cis-Ohi-OEt (30 g, 65 mmol) in THF (200 ml) is added N, N-diisopropylethylamine (17 ml, 98 mmol) and di-tert-butyl-dicarbonate (15.7 g, 72 mmol). This solution is brought to a light boil under reflux and maintained at this temperature for 16 hours the Heating is stopped, and after cooling, the solution was concentrated in vacuo. The residue is dissolved in ethyl acetate (400 ml) and washed twice 1M citric acid solution (200 ml), twice with saturated aqueous sodium bicarbonate solution (200 ml) and twice with saline solution (200 ml). The organic is La (24.8 g, 44 mmol) is dissolved in 300 ml of dioxane. There is added a solution containing 2,05 g LiOHH2O (49 mmol) in 150 ml of water. The mixture is stirred for 5 h at room temperature, then add 100 ml of a saturated aqueous solution of ammonium chloride. The solvents are removed in vacuo and the residue partitioned between saturated aqueous sodium bicarbonate and diethyl ether. The layers are separated and the aqueous layer was acidified to pH 3 with citric acid. The acidified aqueous solution is extracted 3 times with diethyl ether (200 ml) and the extracts combined, dried over magnesium sulfate, filtered and concentrated to obtain 24.3 g of N-(t-BuOOCCH2)-N-Boc-D-Phe-cis-Ohi-OH as a white foam.1H-NMR; FD-MS, m/e 530 (M+).

Analysis for C29H42N2O7:

calculated, %: C 65,64; H 7,98; N 5,28;

found, %: C 65,39; H 8,04; N 5,39.

C) Obtaining HOOCCH2-D-Phe-cis-Ohi-p - NHCH2C6H4C(NH)NH22HClH2O:

calculated, %: C 57,24; H of 6.52; N 11,92;

found, %: C 57,40; H 6,30; N of 11.69.

Example 48

< / BR>
(N-Carboxymethyl)-D-cyclohexylethyl-N-[[4-(aminoiminomethyl) phenyl] methyl]-L-prolinamide)

A) Obtaining HOOCCH2-D-Cha-Pro-p - NHCH2C6H4C(NH)NH2HCl 0.5 H2O:

designed ethyl)phenyl] methyl] -1-[N-(carboxymethyl)- D-cyclohexylethyl]-1H-indole-2-carboxamide hydrochloride).

A) Obtaining HOOCCH2-D-Phe-cis-Ohi-p - NHCH2C6H4C(NH)NH2HCl. To clean this substance is used HPLC, method 2, using a gradient (A/B) of from 98/2 to 70/30 for 3 h1H-NMR; FAB-MS, m/e: 512,3 (MH+).

Analysis for C28H41N5O4HCl:

calculated, %: C 61,36; H 7,72; N 12,78;

found, %: C 61,08; H 7,47; N 12,53.

Example 50

< / BR>
A) Receiving Cbz-Pro(4-TRANS-OH)-OEt

To a solution of Cbz-Pro(4-TRANS-OH)-OH (33 g, 124 mmol) in ethanol (500 ml) is added p-toluensulfonate (1 g) and the solution refluxed. After 16 h the solution is cooled to room temperature and the solvent is removed in vacuum. The residue is dissolved in ethyl acetate (400 ml) and washed twice with saturated aqueous sodium bicarbonate solution and twice with a saturated aqueous solution of sodium chloride. An ethyl acetate solution is dried (MgSO4), filtered and concentrated in vacuo to obtain 34,5 g (95%) of a colorless oil.1H-NMR; FD-MS, m/e 293 (M+).

Analysis for C15H19NO5:

calculated, %: C 61,42; H 6,53; N 4,77;

found, %: C 61,20; H of 6.65; N 4,73.

B) Receiving Cbz-Pro(4-oxo)-OEt

Cbz-Pro(4-TRANS-OH)-OEt (32.7 g, 111 mmol) dissolved in dichloromethane (500 ml) under mechanical stirring at cropton is quite small portions while maintaining the intensive mixing. After stirring for 12 h at room temperature, add diethyl ether (200 ml) and dark color, the suspension is decanted from the tarry residue and passed through a column of silica gel (200 g). The residue is washed twice with dichloromethane (200 ml) and the combined washing fluid is passed through the layer of silicon dioxide. The filtrate is washed by passing through a column of silica gel with a mixture of 1:1 ethyl acetate with hexane (4 l) and collecting fractions of 500 ml containing All the product fractions as determined by TLC, combined and concentrated in vacuo to obtain a 23.8 g (74%) of colorless oil.1H-NMR; FD-MS, m/e 291 (M+).

Analysis for C15H17NO5:

calculated, %: C 61,85; H 5,88; N 4,81;

found, %: C 61,57; H of 5.82; N 4,71.

C) Receiving Cbz-Pro(4-isobutylamides)-OEt

Tert-piperonyl potassium (34 g, 288 mmol) suspended in tetrahydrofuran (800 ml) dried in a drying Cabinet two-neck round bottom flask equipped with a tube for input of nitrogen, rod magnetic stirrer and addition funnel. To this suspension parts add italytraveltraininfov (120 g, 288 mmol). After stirring for 30 min via the addition funnel are added dropwise within 1 h of rest the command solution of NH4Cl (100 ml). This solution is diluted with ethyl acetate (750 ml) and the layers separated. The organic layer is washed twice with 1N citric acid solution, twice with saturated aqueous sodium bicarbonate solution and twice with a saturated aqueous solution of sodium chloride. The organic solution is dried over magnesium sulfate, filtered and concentrated to obtain yellow oil. This oil is purified by flash chromatography on silica gel, elwira a mixture of 2:1 hexane with ethyl acetate. Containing the product fraction (determined by TLC) are combined and concentrated in vacuo to obtain 37 g (45%) of colorless oil.1H-NMR; FD-MS, m/e 345 (M+).

Analysis for C20H27NO4:

calculated, %: C 69,54; H 7,88; N 4,05;

found, %: C 69,74; H a 7.85; N 3,99.

G) Obtaining Pro(4-CIS-isoamyl)-OEtHCl

To a solution of Cbz-Pro(4-isobutylamides)-OEt (37 g, 107 mmol) in ethanol (500 ml) is added 5% Pd/C (5 g). Through this solution bubbled nitrogen for 5 min and then for 3 h bubbled hydrogen gas. The solution is filtered through a layer of diatomaceous earth. Then through the solution until saturation bubbled gaseous hydrogen chloride and the solution was concentrated in vacuo to obtain 26 g (97%) of an amber color m is read, %: C 57,70; H 9,68; N 5,61;

found, %: C 57,46; H 9,50; N of 5.82.

D) HOOCCH2-D-Phe-Pro(4-CIS-isoamyl)-p - NHCH2C6H4C(NH)-NH2HCl. Purification of this compound using HPLC method 2, using a gradient (A/B) of from 98/2 to 50/50 for 3 h1H-NMR; FAB-MS, m/e: 528,4 (MH+).

Analysis for C29H45N5O41,9 HCl:

calculated, %: C 58,34; H a 7.92; N 11,73; Cl 11,28;

found, %: C 58,30; H a 7.85; N 11,83; Cl 11,27.

Example 51

< / BR>
(D-Cyclohexylethyl-N-[[4-(aminoiminomethyl)phenyl] methyl] -L - prolinamide-dihydrochloride)

A) Receive Boc-D-Cha-Pro-p-NHCH2C6H4C(NH)NHCbz

The methods are essentially equivalent described in examples 1-A, 46-D and 18-D, get 32,5 g (94%) of Boc-D-Cha-Pro-p - NHCH2C6H4C(NH)NHCbz, on the basis of Boc-D-Cha-Pro-OH.1H-NMR; FD-MS, m/e 634 (M+).

Analysis for C35H47N5O6:

calculated, %: C 66,33; H 7,47; N 11,05;

found, %: C to 66.30; H 7,47; N of 11.26.

B) Obtaining D-Cha-Pro-p-NHCH2C6H4C(NH)NHCbz2HCl

By the way, is essentially equivalent to the one described in example 18, receive 0.74 g (62%) D-Cha-Pro-p-NHCH2C6H4C(NH)NH22,1 HCl:

calculated, %: C 55,50; H 7,43; N 14,71; Cl 15,64;

found, %: C of 55.64; H 7,50; N 14,65; Cl 15,81.

Example 52

>A) Obtaining HOOCCH2CH2-D-Cha-Pro-p - NHCH2C6H4C(NH)NH21,9 HCl:

calculated, %: C 55,50; H 7,25; N 12,94; Cl 12,45;

found, %: C 55,26; H 7,26; N 13,21; Cl 12,85.

Example 53

< / BR>
A) Receive Boc-4-(aminomethyl)pyridine

By the way, is essentially equivalent to the one described in example 34-A, get a 19 g (87%) of Boc-4-(aminomethyl)-pyridine 4-(aminomethyl)pyridine. 1H-NMR.

B) Obtaining 4-BocNHCH2-N-Cbz-piperidine

4-BocNHCH2-pyridine (10 g, 48 mmol) dissolved in ethanol (280 ml) and add 5% Pd/C (10 g). The suspension is shaken in an atmosphere of hydrogen (4.1 bar, 60 psi) overnight at 60oC. Then, the catalyst was filtered and the solution concentrated in vacuo to obtain 9.0 g of gray solid. 3.2 g of a solid substance dissolved in THF (75 ml) and added dropwise an aqueous solution (75 ml), potassium carbonate (4,2 g, 30 mmol). To this mixed solution add benzylchloride (2.3 ml, 16 mmol). After 15 min the solution was concentrated in vacuo to about 1/2 the original volume and then diluted with ethyl acetate. The organic phase is separated and washed with brine, then dried (MgSO4), filtered and concentrated in vacuo to obtain 4.6 g (76%) as a white solid.1H-NMR; FD-MS, m/e: 3 the figures described in example 23-A, get 3 g (84%) 4-NH2CH2-N-Cbz-HCl of 4-BocNHCH2-N-Cbz-piperidine. IR;1H-NMR; FD-MS, m/e: 249 (MH+).

G) Obtaining N-(tert-BuO2CCH2)-N-Boc-D-Cha-Pro-OH.

N-(t-BuO2CCH2)-N-Boc-D-Phe-Pro-OH (13 g, 27 mmol) dissolved in ethanol (750 ml) and add PtO2(13 g). The suspension is shaken in a hydrogen atmosphere at a pressure of 4.1 bar (60 psi) at 40oC for 16 hours, the Catalyst is filtered and the filtrate concentrated in vacuo to obtain 11.7 g (90%) of a white foam. IR;1H-NMR; FD-MS, m/e 483 (M+).

Analysis for C25H42N2O7:

calculated, %: C 62,22; H 8,77; N 5,80;

found, %: C 62,99; H 8,96; N 5,48.

D) HO2CCH2-D-Cha-Pro-4-NHCH2- piperidine-hydrochloride

The methods are essentially equivalent described in examples 1-G and 46 G, obtain 1.1 g HO2CCH2-D-Cha-Pro-4-NHCH2- piperidine-hydrochloride of N-(t-BuO2CCH2)-N-Boc-D-Cha-Pro-OH and HCl4-NH2CH2-N-Cbz-piperidine. The product was then purified by RPHRLC, method 2, using a gradient (A/B) of from 98/2 to 70/30 for 2 h IR; 1H-NMR; FD-MS, m/e 423 (M+).

Analysis for C22H38N4O42HCl 1,5 H2O:

calculated, %: C 50,57; H 8,29; N OF 10.72;

found, %: C 50,31; H 8,46; N of 10.93.


Ways, essentially equivalent to the one described in example 52, receive 0,59 g HO2CCH2-D-Cha-Pro-4-NHCH2CH2- piperidine-hydrochloride of 4-aminomethylpyridine. The product was then purified by RPHRLC, method 2, using a gradient (A/B) of from 98/2 to 70/30 for 2 h IR;1H-NMR; FD-MS, m/e 437 (M+).

Analysis for C23H40N4O42,5 HCl for 1.5 H2O:

calculated, %: C 49, 80mm; H of 8.27; N 10,10;

found, %: C 49,95; H 8,08; N 10,34.

Example 55

< / BR>
A) Obtaining 4-hydroxypropyl-N-Cbz-piperidine

Ways, essentially equivalent to the one described in example 53-B, receive 28 g (67%) 4-hydroxypropyl-N-Cbz-piperidine from 4-hydroxypropylamino. 1H-NMR.

B) Obtaining 4-(NH2CH2CH2CH2)-N-Cbz - piperidine-hydrochloride

The methods are essentially equivalent described in examples 30-B, 30-b and 30-G, get to 7.3 g of 4-(NH2CH2CH2CH2)- N-Cbz-piperidine-hydrochloride of 4-hydroxypropyl-1-N-Cbz-piperidine.1H-NMR; FD-MS, m/e 276 (M+).

C) Obtaining HO2CCH2-D-Cha-Pro-4 - NHCH2CH2CH2-HCl

The methods are essentially equivalent described in examples 53-D 53-D, obtain 0.39 g HO2CCH2-D-Cha-Pro-4 - NHCH2CH2CH THE RPHPLC, method 2, using a gradient (A/B) of from 98/2 to 70/30 for 2 h IR;1H-NMR; IS-MS, m/e: 451,4 (MH+).

Analysis for C24H42N4O42HCl H2O:

calculated, %: C 53,23; H 7,56; N 10,35;

found, %: C 53,43; H 8,63; N 10,19.

Example 56

< / BR>
HO2CCH2-D-Cha-Pro-4-NHCH2-1-HCl

The methods are essentially equivalent described in examples 34-G, 23-A, 1-G (using N-(tert-BuO2CCH2)-N-Boc-D - Cha-Pro-OH), 18-D, 1-3 and obtain 0.35 g HO2CCH2-D-Cha - Pro-4-NHCH2-1-amidinopropane-hydrochloride of 4-BocNHCH2of piperidine. The target product was then purified by RPHRLC, method 2 (the gradient (A/B) of from 98/2 to 75/25 within 150 minutes). IR;1H-NMR; FAB-MS, m/e 465 (MH+).

Analysis for C23H40N6O42HCl:

calculated, %: C 51,39; H 7,88; N 15,63; Cl 13,19;

found, %: C 51,66; H 7,98; N 15,80; Cl 13,48.

Example 57

< / BR>
HO2CCH2-D-Cha-Pro-4-NHCH2CH2-1 - HCl

Getting HO2CCH2-D-Cha-Pro-4-NHCH2CH2- 1-HCl

The methods are essentially equivalent described in examples 34-G, 23-A, 1-G (using N-(tert-BuO2CCH2)-N-Boc-D - Cha-Pro-OH), 18-D, 1-3 and obtain 0.34 g HO2CCH2-D-Cha - Pro-4-NHH2CH2-1-amidinopropane (A/B) of from 98/2 to 75/25, 150 min, IR;1H-NMR; FAB-MS, m/e: 479,4 (MH+).

Analysis for C24H42N6O42HCl:

calculated, %: C 52,26; H 8,07; N 15,24; Cl 12,86;

found, %: C 52,56; H 8,15; N Shed 15.37; Cl 13,07.

Example 58

< / BR>
HO2CCH2-D-Cha-Pro-4-NHCH2-3,4-degidro - HCl

A) Obtaining 4-BocNHCH2-N-methyl-pyridine-iodide

To a stirred solution of 4-BocNHCH2-pyridine (20 g, 96 mmol) in acetonitrile (200 ml) add itmean in (8.9 ml, 144 mmol).

After 16 h, the solution was concentrated in vacuo obtaining of 33.8 g (96%) of a viscous light yellow oil. FD-MS, m/e: 223,1 (M+).

B) Obtaining 4-BocNHCH2-N-Fmoc-3,4-degidro-piperidine

To a stirred solution of 4-BocNHCH2-methylpyridinium (7.7 g, 34 mmol) in 1,2-dichloroethane (100 ml) was added 1,8-bis(dimethylamino)naphthalene (1.5 g, 6.8 mmol), then 2-chloroethylphosphonic (5.3g, 37 ml). The solution is refluxed and after 2 h, cooled to room temperature and the solvent is removed in vacuo, and the residue quickly chromatographic through a column of silica gel, elwira mixture of hexane to 20% ethyl acetate. Organic solvents are removed in vacuo and the residue is dissolved in methanol (300 ml) and refluxed for 20 mi is. the STATCOM is dissolved in water (200 ml) and washed with this solution twice with hexane, and then saturated with solid NaCl and extracted several times with ethyl acetate.

United an ethyl acetate extracts are dried (MgSO4), filtered and concentrated in vacuo to obtain a slightly yellowish oil, which was dissolved in dichloromethane (75 ml). To this stirred solution was then added N, N-diisopropylethylamine (2.1 ml, 12.2 mmol), then - 9-fluorenylmethoxycarbonyl (3.2 g, 12.2 mmol). After 2 h the solvent is removed in vacuum and the residue is dissolved in ethyl acetate (250 ml) and washed twice 1N citric acid solution, once with brine, twice with saturated aqueous sodium bicarbonate solution and finally once with brine. The organic phase is then dried over magnesium sulfate, filtered and concentrated in vacuo and the residue chromatographic through a column of silica gel, elwira stepped gradient from hexane to 5% ethyl acetate hexane to 50% ethyl acetate. Containing the product fraction (determined by TLC) are combined and concentrated to obtain 4 g (27%) of white solids. IR;1H-NMR; FD-MS, m/e 435 (M+).

C) Obtaining N-(t-BuO2CCH2)-N-Boc-D-Cha-Pro-4 - NHCH2-N-Fmoc-3,4-is UB>2
CCH2)-N-Boc-D-Cha-Pro-OH), obtain 2.5 g of N-(t-BuO2CCH2)-N-Boc-D-Cha-Pro-4 - NHCH2-N-Fmoc-3,4-degidro-piperidine from 4-BocNHCH2-N-Fmoc - 3,4-degidro-piperidine. IR;1H-NMR; FD-MS, m/e: 799 (M+).

G) Obtaining N-(t-BuO2CCH2)-N-Boc-D-Cha-Pro-4 - NHCH2-3,4-degidro-piperidine

N-(t-BuO2CCH2)-N-Boc-D-Cha-Pro-4-NHCH2-N - Fmoc-3,4-degidro-piperidine (1.5 g, 1.9 mmol) was dissolved in morpholine (25 ml) and after stirring for 5 h the solvent is evaporated in vacuum. The residue is dissolved in ethyl acetate and the resulting solution was washed twice with saturated aqueous sodium bicarbonate solution, dried (MgSO4), filtered and concentrated in vacuo. The residue is then dissolved in a small volume of chloroform and chromatographic on silica gel, elwira gradient (A/B) from 5% to 10% A/B (A=9:1 methanol/concentrated NH4OH; In = chloroform). Containing the product fractions according to TLC, are combined and concentrated in vacuo to obtain 890 mg (82%) of a white solid.1H-NMR; FD-MS, m/e 576 (MH+).

D)

Through a solution of N-(tert-BuO2CCH2)-N-Boc-D-Cha-Pro - 4-NHCH2-3,4-degidro-piperidine (820 mg, 1.4 mmol) and anisole (1 ml) in dioxane (25 ml) at 0oC for 10 min bubbled gaseous HCl. Last solution is washed twice with diethyl ether. The aqueous phase is then concentrated to a volume of about 20 ml in vacuo and the residue purified by RPHRLC (method 2; gradient (A/B) of from 98/2 to 70/30, 2 h). Containing the product fractions as determined by analytical RPHRLC, unite, partially concentrated in vacuo and lyophilized with getting 442 mg (68%) of white solids. IR;1H-NMR; FD-MS, m/e 423 (MH+).

Analysis for C22H36N4O42HCl 1,5 H2O:

calculated, %: C 50,57; H 8,29; N OF 10.72;

found, %: C 50,31; H 8,46; N of 10.93.

Example 59

< / BR>
Getting HO2CCH2-D-Cha-Pro-4-NHCH2CH2- 3,4-degidro-HCl

Ways, essentially equivalent to the one described in example 58, obtain 73 mg of HO2CCH2-D-Cha-Pro-4-NHCH2CH2- 3,4-degidro-piperidine-hydrochloride of 4-BocNHCH2CH2-pyridine. The target product was then purified by RPHRLC method 2 (the gradient (A/B) of from 98/2 to 70/30, 2 h). IR;1H-NMR; IS-MS, m/e: 435,2 (MH+).

Analysis for C23H38N4O42,3 HCl 3H2O:

calculated, %: C 48,26; H 8,15; N 9,79; Cl 14.24 from;

found, %: C 48,31; H of 7.93; N 9,66; Cl 14,56.

Example 60

< / BR>
Getting HO2CCH2-D-Cha-Pro-4 - NHCH2CH2CH2-3,4-degidro-piperidin,3HCl H2O:

calculated, %: C 52,37; H 3,11; N 10,1 R,8AR)-decahydro-1- (R)-ethenolysis]carbonyl]-1-propylamide-trihydrochloride)

Rfin this example, is determined by thin-layer chromatography on silica gel (silica gel 60 F-254) in the following systems (by volume):

(A) chloroform : methanol : acetic acid = 135:15:1

(B) ethyl acetate : acetic acid : absolute ethanol = 90:10:10

(C) ethyl acetate : hexane = 70:30

(G) chloroform

A) N-Methoxycarbonylmethylene

To a stirred solution of phenethylamine (75,2 ml, 0.6 mol) and triethylamine (83 ml, 0.6 mol) in THF (500 ml) slowly add methylchloroform (46.2 ml, 0.6 mol), dissolved in THF (50 ml). After the reaction stirred for further 1 h at room temperature, add diethyl ether (2 l) and 1 n HCl (800 ml). The organic layer was washed with water, dried over magnesium sulfate, filtered and the filtrate concentrated in vacuo to produce in the form of a clear oil pure target compound (102 g, 95%).

B) 2-Methoxycarbonyl-DL-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid

To a solution of N-methoxycarbonyl-phenethylamine (102 g of 0.57 mol) in triperoxonane acid (300 ml) is added Glyoxylic acid (63 g of 0.68 mol) and the mixture is heated to the boiling temperature under reflux. After boiling for 4 h under reflux, the reaction mixture is cooled to room the mixture was adjusted to pH 12 with 5N NaOH and the aqueous layer was separated. To the aqueous layer add diethyl ether (500 ml) and the solution is acidified to pH of 2.5 using 5N HCl. The organic layer is separated, dried over magnesium sulfate, filtered and the filtrate was concentrated in vacuo, receiving in the form of oil the pure target compound (107 g; 80%). FAB-MS: 236 (MH+).

C) Tert-butyl ether 2-methoxycarbonyl-DL-1,2,3,4 - tetrahydroisoquinoline-1-carboxylic acid

To a stirred cooled (0oC) solution of 2-methoxycarbonyl-DL-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid (2) (105 g, 0.45 mol) in CH2Cl2(200 ml) is added tert-butanol (52 ml, 0.54 mol) and DCC (92 g, 0.45 mol). After 2 h at 0oC and 24 h at room temperature, the solvent is removed in vacuo and to the residue is added ethyl acetate (800 ml) with 1N solution of sodium bicarbonate (300 ml). The organic layer was separated, washed with water, 1.5 n citric acid solution and water. The organic layer is dried over magnesium sulfate, filtered and the filtrate concentrated in vacuo to produce in the form of oil the pure target compound (106 g; 81%); FAB-MS: 292 (MH+) TLC: Rf(A)= 0,61; elemental analysis for C16H21NO4:

calculated, %: C 65,96; H 7,27; N 4,81;

found, %: C 66,24; H 7,28; N 4,73.

G) Tert-butyl ether 2-methoxycarbonyl-(1RS,4a is hydroisoquinoline-1-carboxylic acid (105 g, 0.36 mol) in tert-butanol (800 ml) restore in the presence of 5% Rh/Al2O3(52,5 g) under hydrogen pressure of 55 bar (800 psi) in the equipment of high pressure at 50oC and within 24 hours the Reaction mixture was filtered through a layer of diatomaceous earth and the filtrate concentrated in vacuo. The oil obtained is dried to obtain the pure target compound (96,5 g, 90%); FD-MS: 298 (MH+); TLC: Rf(B)=0,63.

D) Ethyl ester 2-methoxycarbonyl-(1RS, 4aRS,8aRS)- perhydrosqualene-1-carboxylic acid

To a solution of tert-butyl methyl ether 2-methoxycarbonyl-(1RS,4aSR,8aSR)- perhydrosqualene-1-carboxylic acid (81,2 g, 273 mmol) in ethanol (500 ml) add ethoxide sodium (21% in ethanol) (88,4 ml, 273 mmol) and the reaction mixture is refluxed for 24 hours of the Organic solvent is evaporated in vacuo, add ethyl acetate (400 ml) and water (100 ml) to the resulting residue. The organic layer is separated, washed twice with water, dried over magnesium sulfate, filtered and the filtrate concentrated in vacuo to produce in the form of oil the pure target compound (70 g, 95%); FAB-MS: 270 (MH+); TCX: Rf(A)=0,61.

E) 2-Methoxycarbonyl-(1RS, 4aRS, 8aRS)-perhydrosqualene-1-carboxylic acid

To a solution of the product of stage D) (themperature (30 hours). The organic solvent is evaporated in vacuo, to the residue is added diethyl ether (400 ml) and water (100 ml). The aqueous layer was separated and add ethyl acetate (400 ml). the pH of the solution was adjusted to 2.0 with 5N HCl. The organic layer is dried (MgSO4), filtered and the filtrate concentrated in vacuo to obtain a clear oil. The oil is crystallized from hexane (200 ml) to give the pure target compound (46.4 g, 74%); FAB-MS: 242 (MH+); TCX: Rf(A)=0,36; elemental analysis for C12H19NO4:

calculated, %: C 59,74; H 7,94; N 5,81;

found, %: C 59,95; H 7,88; N, 5,54.

Values obtained by NMR experiment homonuclear rassheplenia, COSY, HMQC and DEPT.

W) 2-Cbz-(1RS,4aRS,8aRS)-perhydrosqualene-1-carboxylic acid

To a stirred solution of the product of stage E) (46 g, 191 mmol) in anhydrous CH3CN (200 ml) at room temperature and in an inert atmosphere, add a solution of attributively (of 62.4 ml, 440 mmol) in CH3CN (60 ml). The reaction mixture was stirred at 55oC for 30 min and cooled to room temperature. The reaction is suppressed by adding water (100 ml), then add sodium metabisulfite (1 g). the pH of the reaction medium is set to 10,0 using 5N NaOH solution and is added and stirred for an additional 30 minutes at room temperature, the organic solvent is evaporated in vacuum and add diethyl ether (200 ml). The reaction mixture was then allowed to stand at room temperature for 2 h and added ethyl acetate (200 ml). The aqueous solution is acidified to pH of 2.5 using 5N HCl; the organic layer is separated, dried (MgSO4), filtered and the filtrate concentrated in vacuo to obtain pure target compound in the form of oil (39.5 g, 65%); FAB-MS: 318 (MH+).

Elemental analysis for C18H23NO4:

calculated, %: C 68,12; H 7,30; N TO 4.41;

found, %: C 66,37; H 7,52; N 4,37.

C) 2-Cbz-(1RS,4aRS,8aRS)-perhydrosqualene-1-carbonyl-Pro-O-tert-Bu

To a stirred, cooled to 0oC to a solution of the product of stage G) (39 g, 123 mmol) in DMF (200 ml) is added tert-butyl ester of Proline (21.1 g, 123 mmol), 1-hydroxybenzotriazole (16.6 g, 123 mmol) and DCC (25,3 g, 123 mmol). The reaction mixture was stirred for 2 h at 0oC and 24 h at room temperature. The precipitate is filtered and the filtrate concentrated in vacuo to obtain an oil. The oil is dissolved in ethyl acetate (200 ml) with water (100 ml). The organic layer is washed successively 1N solution of sodium bicarbonate, water, 1.5 n citric acid solution and water. The organic layer from the target compound in the form of a mixture of diastereomers (52.7 g, 91%). FAB-MS: 471 (MH+).

And 2Cbz-(4AR,8aR)-perhydrosqualene-1(R)-carbonyl-Pro-OH

To a stirred solution of the product of stage C) (52,4 g, 111 mmol) in CH2Cl2(20 ml) is added triperoxonane acid (70 ml) and anisole (5 ml). The reaction mixture was stirred at room temperature for 1 h and concentrated in vacuo without heating. The residue was diluted with diethyl ether (400 ml), water (100 ml) and the pH of the solution was adjusted to 10.0 using 5N NaOH solution. The aqueous layer was separated and added ethyl acetate (100 ml). the pH of the solution was adjusted to 2.5 using 5N HCl; the organic layer is separated, dried over magnesium sulfate, filtered and the filtrate concentrated in vacuo to obtain a clear oil. The oil was dissolved in diethyl ether (500 ml) and to the solution was added (L)-(-)-alpha-methylbenzylamine. The solution is incubated at room temperature for 24 hours resulting solid substance was filtered, washed with diethyl ether and dried. The solid is suspended in ethyl acetate, washed obtained a suspension of 1.5 n citric acid solution and water. The organic layer is dried over magnesium sulfate, filtered and the filtrate is evaporated to obtain the target compound in the form of oil (20.2 g, 44%) FAB-MS: 415 (MH+); []D= 3,2o(c = 0.5, methanol); elem,36; N 6,63.

K) 2-Cbz-(4aR,8aR)-perhydrosqualene-1(R)-carbonyl-Pro - NH-(CH2)4-NH-Boc

In flask 1 product stage) (1.06 g, 2.55 mmol) was dissolved in DMF (10 ml), cooled to -15oC and add N-methylmorpholine (of 0.28 ml, 2.55 mmol), then add isobutylparaben (0.33 ml, 2.55 mmol). The reaction mixture is stirred at -15oC for 2 min.

In flask 2 N-Boc-1,4-diamino-butane (0,48 g, 2.55 mmol) was dissolved in DMF (10 ml), cooled to 0oC and to the solution was added N-methylmorpholine (of 0.28 ml, 2.55 mmol). The reaction mixture was stirred for 2 min at 0oC.

The contents of the flask 2 is added to the flask 1 and the reaction mixture stirred for 4 h at -15oC and for 24 h at room temperature. To the reaction mixture is added 1N solution of sodium bicarbonate (1 ml) and the solvent is removed in vacuum to obtain oil. The residue is dissolved in ethyl acetate (200 ml) and washed sequentially with 1.5 n citric acid solution, water, 1N solution of sodium bicarbonate (100 ml) and water. The organic solution is dried (MgSO4), filtered and concentrated to dryness in vacuum to give crude target compound as a solid (1.47 g, 99%) FAB-MS: 585 (MH+); TLC: Rf(A)=0,70.

L) (4aR,8aR)-peligroso is (2 ml) is added triperoxonane acid (25 ml) and anisole (2.5 ml). The reaction mixture was stirred at room temperature for 30 min and concentrated in vacuo without heating. The reaction mixture was diluted with diethyl ether (100 ml) and the suspension is decanted. The oil obtained proscout twice in diethyl ether and dried. Dried oil was dissolved in THF (20 ml), add triethylamine (0,66 ml, 4.8 mmol) and the mixture of bis-Cbz-S-methylthymidine (0,859 g, 2.4 mmol). The reaction mixture was stirred at room temperature for 48 hours the Organic solvent is evaporated in vacuum and the residue is dissolved in ethyl acetate (200 ml) and washed successively 1N solution of sodium bicarbonate (100 ml) and water. The organic solution is dried (MgSO4), filter and concentrate the filtrate to dryness in vacuo to obtain crude solid (1.5 g, 79%); TLC: Rf(G)=0,33. The crude solid (1.5 g, of 1.93 mmol) dissolved in ethanol (50 ml) with water (10 ml) and 1N HCl (5.8 ml, 5.8 mmol), hydronaut in the presence of 5% Pd/C as catalyst (2.5 g) at room temperature and under pressure. The catalyst is removed by filtration and the filtrate was concentrated in vacuo to obtain an oil. The oil is dissolved in triperoxonane acid (10 ml), add thioanisole (1.0 ml) and to this mixture trsut ethyl ether (100 ml). Separated in the settling liquid is decanted and the oil obtained twice triturated with diethyl ether and dried in vacuum to obtain crude solid (1.3 g). Solid (1.3 g) was dissolved in a 0.05% solution of HCl and applied to a column of size 5 x 25 cm with a fixed phase Vydac C18-resin. For elution of the peptide from the column using a gradient of increasing concentrations of CH3CN (from 2% to 25%). Fractions are collected and unite on the basis of relevant analytical RPHRLC and lyophilizers obtaining pure target compound (0,139 g, 15%); FAB-MS 393 (MH+).

Elemental analysis for C20H36N6O25HCl3H2O:

calculated, %: C 38,28;

found, %: C 38,34.

Example 62

< / BR>
Getting HO2CCH2-D-Cha-Pro-4 - NHCH2C6H4NH22,2 HCl for 1.5 H2O:

calculated, %: C 51,37; H 7,35; N 10,42; Cl 14,50;

found, %: C 50,87; H 6,72; N 10,41; Cl 14,18.

Example 63

< / BR>
Getting HO2CCH2-D-Cha-Pro-4 - NHCH2CH2C6H4NH22,2 HCl 0.5 H2ABOUT:

calculated, %: C 54,00; H 7,40; N 10,50; Cl 14,61;

found, %: C 53,65; H to 7.59; N 10,24; Cl 14,33.

Example 64

< / BR>
A) Obtaining 4-Boc2NCH2-3-F-C6H3NO2< / BR>
To move the ID (5.7 g, 32 mmol) followed by benzoyl peroxide (0,78 g, 3.2 mmol) and the solution refluxed. After 12 h the heat is removed and the mixture is diluted with carbon tetrachloride (100 ml) and washed with water. The organic phase is then diluted with ethyl acetate (300 ml), dried (MgSO4), filtered and concentrated in vacuo. The residue is dissolved in tetrahydrofuran (50 ml) and added dropwise to a stirred solution of NaH (60% dispersion in oil; 1.3 g, 32 mmol) in di-tert-butylimidazolium (6,9 g, 32 mmol) in THF (100 ml). After stirring overnight the solvent is removed in vacuo and the residue chromatographic on silica gel, elwira stepped gradient from hexane to a mixture of hexane to 20% ethyl acetate. Containing the product fraction (determined by TLC) are combined and concentrated in vacuo to obtain 39 g (33%) of white solids. IR;1H-NMR; FD-MS, m/e 370 (M+).

Analysis for C17H23N2O6:

calculated, %: C 55,13; H of 6.26; N 7,56;

found, %: C 55,27; H 6,23; N 7,44.

B) Obtaining HO2CCH2-D-Cha-Pro-4-NHCH2-3 - F-C6H3NH2HCl

The methods are essentially equivalent described in examples 23-A, 1-G (using N-(tert-BuO2CCH2)-N-Boc-D-Cha-Pro-OH), 23-D and 58-C, gain of 0.44 g of HO21H-NMR; FAB-MS, m/e: 449,3 (MH+).

Analysis for C23H33N4O4F1,3HCl:

calculated, %: C 55,70; H 6,97; N 11,30; Cl 9,29;

found, %: C 55,38; H 6,97; N 11,05; Cl 9,31.

Example 65

< / BR>
(N-(Carboxymethyl)-D-cyclohexylethyl-N-[[5-(aminoiminomethyl)thiophene - 2-yl]methyl]-L-prolinamide hydrochloride)

A) Obtaining 2-cyano-5-formylthiophene

In dried in a drying Cabinet three-neck flask with a capacity of 1 l make Diisopropylamine (9 ml, 66 mmol) and THF (150 ml) under nitrogen atmosphere. The flask was cooled to an internal temperature of -78oC (dry ice with acetone). To this mixed solution with a syringe and add n-utility (1.6 ml in hexane, to 41.3 ml of 66.1 mmol) and the mixture is left under stirring for 5 minutes To this solution add a solution of 2-thiophenecarbonitrile (6,55 g, 60 mmol) in THF (30 ml) for 10 minutes Received a bright red solution was stirred at -78oC for 45 min, then add the syringe dimethylformamide (23,3 ml, 300 mmol). This mixture is stirred for 2 h at -78oC, then add citric acid (about 10 g) and then water (60 ml). Volatile solvents are removed in vacuo and the residue partitioned between diethyl ether and brine (200 ml each). Layers razdelim solution dried over magnesium sulfate, filtered and concentrated in vacuo to obtain a yellow solid, which is purified by chromatography on silica gel using a gradient of ethyl acetate/hexane (hexane to hexane to 50% ethyl acetate). The fractions containing pure product are combined and concentrated in vacuo, obtaining of 6.9 g (84%) of 2-cyano-5-formyl-thiophene. 1H-NMR.

B) Obtain 2-cyano-5-(hydroxymethyl)thiophene

To a solution of 2-cyano-5-formyl-thiophene (6.9 g, 50 mmol) in ethanol (100 ml) is added in portions sodium borohydride (1.9 g, 50 mmol). After stirring for 5 min the solvent is removed in vacuo and the residue distributed between ethyl acetate and brine. The layers are separated and the organic phase is washed once with 1M citric acid solution and once with brine, then dried (MgSO4), filtered and concentrated in vacuo to obtain 6,1 g (88%) of 2-cyano-5-(hydroxymethyl)thiophene. 1H-NMR; FD-MS, m/e: 140 (M+).

Analysis for C6H5NOS:

calculated, %: C 51,78; H 3,62; N 10,06;

found, %: C 51,54; H 3,62; N 9,86.

C) Obtaining 2-cyano-5-(methyl bromide)thiophene

To a solution of 2-cyano-5-(hydroxymethyl)thiophene (6.0 g, 43 mmol) in THF (50 ml) is added triphenylphosphine (15.7 g, 47 mmol) and Tetra the solvent is removed in vacuum and the residue is dissolved in chloroform, then adsorb on silica gel and make silicagel column. Product elute using a gradient of ethyl acetate/hexane. The fractions containing pure product (as determined by TLC) are pooled and concentrated in vacuo to obtain 6.5 g (75%) of 2-cyano-5-(methyl bromide)thiophene.1H-NMR; FD-MS, m/e 203 (M+).

Analysis for C6H4NSBr:

calculated, %: C 35,66; H 1,99; N 6,93;

found, %: C 35,71; H 2,03; N 6,95.

G) Obtaining 2-cyano-5-(aminomethyl)HCl

To a cooled to 0oC to a solution of 2-cyano-5-(methyl bromide)-thiophene (6.0 g, 30 mmol) in THF (50 ml) under nitrogen atmosphere add portions NaH (60% dispersion in oil; 1.3 g, 33 mmol). To this stirred suspension over 30 minutes a solution of di-tert-butylmethacrylate (7,1 g, 33 mmol) in THF (50 ml). After stirring for 3 h, add saturated aqueous solution of ammonium chloride (100 ml). Volatile solvents are then removed in vacuum and the residue distributed between ethyl acetate and water. The layers are separated and the organic phase is washed twice with saline, dried (MgSO4), filtered and concentrated in vacuo to obtain 10.5 g (100%) 2-cyano-5-Boc2NCH2-thiophene, which crystallizes upon standing. This solid is dissolved in the comfort of dry gaseous HCl and the mixture is stirred for 2 h, during which time it warmed to room temperature. The solvents are removed in vacuo and the resulting solid is suspended in diethyl ether and separated by filtration. White solid is dried overnight in a vacuum, getting to 5.2 g (100%) 2-cyano-5-(aminomethyl)HCl. 1H-NMR; FD-MS, m/e: 139 (M+).

Analysis for C6H7N2SCl:

calculated:% C 41,26; H of 4.04; N 16,04;

found, %: C 41,19; H 4,12; N 15,82.

D) N-(tert-BuO2CCH2)-N-Boc-D-Cha-Pro - 4-NHCH2-2-cyanothiophene

By the way, is essentially equivalent to the one described in example 1-G (using N-(tert-BuO2CCH2)-N-Boc-D-Cha-Pro-OH), obtain 4.6 g (93%) of N-(tert-BuO2CCH2)-N-Boc-D-Cha-Pro - 4-NHCH2-2-cyanothiophene of 2-cyano-5-(aminomethyl)HCl. IR;1H-NMR; FD-MS, m/e 602 (M+).

E) Obtaining N-(tert-BuO2CCH2)-N-Boc-D-Cha-Pro - 4-NHCH2-2-C(NH)NHBoc-thiophene

Through a solution of N-(tert-BuO2CCH2)-N-Boc-D-Cha-Pro - 4-NHCH2-2-cyanothiophene (1.5 g, 2.5 mmol) and triethylamine (4.5 ml) in pyridine (45 ml) over 5 minutes pass by ozonation gaseous hydrogen sulfide and the vessel is then closed and allowed to stand over night. The next morning through the solution for 5 min bubbled ass water and once with saline, then dried (MgSO4), filtered and concentrated in vacuo. The residue is then dissolved in toluene and the resulting solution was concentrated in vacuo, repeating the operation twice.

The residue is then dissolved in acetone (100 ml) and add itmean (5 ml). After stirring over night at room temperature the solvent is removed in vacuum. Received a Golden foam is then dissolved in methanol (20 ml), add NH4OAc (0.39 g, 5 mmol) and the solution refluxed. After 1 h the solvent is removed in vacuum and the residue is dissolved in THF (10 ml). To this stirred solution was added a solution of K2CO3(1.73 g, 12.5 mmol) in water (10 ml), then add di-tert-butyl-dicarbonate (2.2 g, 10 mmol). After stirring for 1 h, the suspension is diluted with ethyl acetate (400 ml) and washed with water, then brine. The organic phase is then concentrated in vacuo and chromatographic the residue on silica gel, elwira stepwise gradient of hexane to 10% ethyl acetate hexane to 75% ethyl acetate. Containing the product fractions as determined by TLC, combined and concentrated in vacuo, obtaining 1.1 g (61%) of a white foam.1H-NMR; FD-MS, m/e: 720 (M+).

Analysis for CAluchemie HO2CCH2-D-Cha-Pro-4-NHCH2- 2-HCl

By the way, is essentially equivalent to the one described in example 58-D, receive 500 mg of HO2CCH2-D-Cha-Pro-4-NHCH2- 2-amidinohydrolase. The product was then purified by RPHRLC, method 2 (the gradient (A/B) of from 98/2 to 70/30, 2 h). IR;1H-NMR; FAB-MS, m/e: 464,2 (MH+).

Analysis for C22H33N5O4S2HCl H2O:

calculated, %: C 47,65; H of 6.73; N 12,63; Cl 12,79;

found, %: C 47,53; H to 6.57; N 12,59; Cl 12,67.

Example 66

< / BR>
(N-(Carboxymethyl)-D-cyclohexylethyl-N-[[5-(aminoiminomethyl)- pyridine-2-yl]methyl]-L-prolinamide hydrochloride).

A) Obtaining N-(tert-BuO2CCH2)-N-Boc-D-Cha-Pro-2 - NHCH2-5-cyanopyridine

Ways, essentially equivalent to the one described in example 64-A, example 23 and example 1-G (using N-(tert-BuO2CCH2)- N-Boc-D-Cha-Pro-OH), obtain 4.4 g of N-(tert-BuO2CCH2)-N - Boc-D-Cha-Pro-2-NHCH2-5-cyanopyridine 2-methyl-5-cyanopyridine. IR;1H-NMR; FD-MS, m/e 597 (M+).

B) Obtaining HO2CCH2-D-Cha-Pro-2-NHCH2-5 - HCl

The methods are essentially equivalent described in examples 65 and 65-W, get 130 mg HO2CCH2-D-Cha-Pro-2 - NHCH2-5-amidinopropane-hydrochloride of N-(tert-BuO2CCH1H-NMR; FAB-MS, m/e: 459,3 (MH+); HRMS (FAB), m/e: to C23H35N6O4:

calculated: 459,2720;

found: 459,2707.

Example 67

< / BR>
Getting HO2CCH2-D-Cha-Pro-2-NHCH2-5 - amidinopropane-hydrochloride

N-(tert-BuO2CCH2)-N-Boc-D-Cha-Pro-2-NHCH2- 5-cyanopyridine (1.2 g, 2 mmol) get methods are essentially equivalent described in examples 23-A, 23-B and 1-B. the Product was then purified by RPHRLC, method 2 (the gradient (A/B) of from 98/2 to 70/30, 2 h). The fractions containing the secondary (minor) product, as determined by analytical RPHRLC, unite, partially concentrated in vacuo and lyophilizer to obtain 93 mg (9% pale green solid. IR; 1H-NMR; IS-MS, m/e: 465,5 (MH+).

HRMS (FAB), m/e: to C23H41N6O4:

calculated: 465,3189;

found: 465,3191.

Example 68

< / BR>
(N-(Carboxymethyl)-D-cyclohexylethyl-N-[[5-(aminoiminomethyl)- 1,2,3,4-tetrahydro-pyridine-2-yl]methyl]-L-prolinamide hydrochloride).

Getting HO2CCH2-D-Cha-Pro-2-NHCH2-5-amidino-5,6 - degidro-piperidine-hydrochloride

N-(tert-BuO2CCH2)-N-Boc-D-Cha-Pro-2-NHCH2- 5-cyanopyridine (1.2 g, 2 mmol) get methods are essentially equivalent described, operasie the main product, as determined by analytical RPHRLC, unite, concentrate, and partially lyophilizer with getting 422 mg (39%) of white solids IR;1H-NMR; IS-MS, m/e: 463,3 (MH+).

Analysis for C23H38N6O42,9 HCl 2H2O:

calculated, %: C 45,71; H 7,49; N 13,91; Cl 17,01;

found, %: C 45,51; H 6,83; N 13,66; Cl equal to 16.83.

Example 69

< / BR>
HO2CCH2-D-Cha-Pro-3-NHCH2-6-amidino - 3HCl

(N-(Carboxymethyl)-D-cyclohexylethyl-N-[[6-(aminoiminomethyl)- pyridazin-3-yl]methyl]-L-prolinamide-trihydrochloride).

A) Obtaining 3-methyl-6-sinoamerican

To a stirred solution of 3-methyl-pyridazine (11 g, 118 mmol) in dichloromethane (200 ml) is added AlCl3(0.05 g), then trimethylsilylacetamide (21 g, 211 mmol). After 20 min via addition funnel is added a solution of p-toluensulfonate (38 g, 201 mmol) in dichloromethane (50 ml) and the solution is incubated under stirring overnight. The next morning, the solvent is removed in vacuo and the residue suspended in ethanol under stirring for 15 min and then filtered to obtain solid white. The solid is dissolved in THF (200 ml) and to this stirred solution gobelet between hexane and saturated aqueous ammonium chloride. The phases are separated and the aqueous phase is alkalinized with solid sodium carbonate, then extracted three times with ethyl acetate. United an ethyl acetate phase is dried (MgSO4), filtered and concentrated in vacuo to obtain 9 g (64%) of white solids. IR;1H-NMR; FD-MS, m/e: 119,1 (M+).

B) Obtaining HO2CCH2-D-Cha-Pro-3-NHCH2- 6-amidino-pyridazin-hydrochloride

Ways, essentially equivalent to the one described in example 66, receive 90 mg of HO2CCH2-D-Cha-Pro-3-NHCH2- 6-amidino-pyridazin-hydrochloride of 3-methyl-6-cyanopyridine. The product was then purified by RPHRLC, method 2 (the gradient (A/B from 98/2 to 70/30, 2 h). IR;1H-NMR; FAB-MS, m/e: 460,3 (MH+).

Analysis for C22H33N7O43HCl 2H2O:

calculated, %: C 43,68; H 6,66; N 16,21;

found, %: C 44,04; H 6,45; N 15,57.

Example 70

< / BR>
(N-(Carboxymethyl)-D-cyclohexylethyl-N-[[1-(aminoiminomethyl)- 1,2,3,6-tetrahydropyridine-4-yl]methyl]-L-prolinamide hydrochloride)

A) Obtaining N,N'-Boc2-thiourea

To a stirred suspension of NaH (60% dispersion in oil; 9.4 g, 234 mmol) in THF (500 ml) at 0oC add thiourea (4.0 g, 52 mmol). After 30 minutes the cooling bath removed and the reaction mixture peremeshivayu add a solution of di-tert-butyl-dicarbonate (25 g, 115 mmol) in THF (100 ml). After stirring for 30 min at 0oC and an additional 2 h at room temperature, add saturated aqueous sodium bicarbonate solution. The solution is then concentrated to approximately half the original volume under vacuum and add ethyl acetate. The organic phase is then washed with saturated aqueous sodium bicarbonate, then brine and then dried (MgSO4), filtered and concentrated to obtain 11.9 g (83%) of white solids. IR;1H-NMR; FD-MS, m/e 276 (M+).

Analysis for C11H20N2O4S:

calculated, %: C 47,81; H 7,30; N 10,14;

found, %: C 47,69; H 7,28; N 10,34.

B) Obtaining N-(tert-BuO2CCH2)-N-Boc-D-Cha-Pro - 4-NHCH2-1-(N, N'-Boc2-amidino)-3,4-degidro-piperidine

To a stirred solution of N-(tert-BuO2CCH2)-N-Boc - D-Cha-Pro-4-NHCH2-3,4-degidro-piperidine (0.6 g, 1 mmol) and triethylamine (0.35 g, 3.4 mmol) in dimethylformamide (10 ml) is added N,N-Boc2-thiourea (0.28 g, 1 mmol), then HgCl2(0.28 g, 1 mmol). After 4 h the solvent is removed in vacuum and the residue is dissolved in ethyl acetate and the resulting solution washed twice with saline. The organic phase is then dried over sulfate-ment from 20% ethyl acetate/hexane to 75% ethyl acetate/hexane. The fractions containing the product, as determined by TLC, combined and concentrated in vacuo to obtain 800 mg (94%) of a white foam. IR;1H-NMR; FD-MS, m/e: 820 (MH+).

Analysis for C42H70N6O10:

calculated, %: C 61,59; H 8,61; N 10,26;

found, %: C 61,81; H 8,79; N 10,45.

C) Obtaining HO2CCH2-D-Cha-Pro-4-NHCH2- 1-amidino-3,4-degidro-piperidine-hydrochloride

Ways, essentially equivalent to the one described in example 58-D, get 0,22 g (55%) HO2CCH2-D-Cha-Pro-4-NHCH2- 1-amidino-3,4-degidro-piperidine-hydrochloride of N-(tert-BuO2CCH2)-N-Boc-D-Cha-Pro-4-NHCH2-1- (N, N'-Boc2-amidino)-3,4-degidro-piperidine. The product was then purified by RPHRLC, method 2 (the gradient (A/B) of from 98/2 to 70/30, 2 h). IR;1H-NMR; FAB-MS, m/e: 463,3 (MH+).

Analysis for C23H38N6O42,2 HCl 2H2O:

calculated, %: C 47,73; H of 7.70; N 14,52; Cl 13,47;

found, %: C 47,49; H of 7.64; N 14,55; Cl 13,48,

Example 71

< / BR>
(N-(Carboxymethyl)-D-cyclohexylethyl-N-[[4-(aminoiminomethyl)- 2-forfinal]methyl]-L-prolinamide hydrochloride)

Getting HO2CCH2-D-Cha-Pro-4-NHCH2-3-F - HCl

Ways, essentially equivalent to the one described in example 66, obtain 0.27 g HO2CCH2-D-Ch is HT (A/B) of from 98/2 to 70/0, 2 h). IR;1H-NMR; FAB-MS, m/e: 476,3 (MH+).

Analysis for C24H34N5O4F2HCl 1,5 H2O:

calculated, %: C 50,09; H 6,83; N 12,17; Cl 12,32;

found, %: C 49,89; H of 6.65; N 12,17; Cl 12,42.

Example 72

< / BR>
HO2CCH2-D-Cha-Pro-4-NHCH2A 3.5-F2- HCl

(N-(Carboxymethyl)-D-cyclohexylethyl-N-[[4-(aminoiminomethyl)- 2,6-differenl]methyl]-L-prolinamide hydrochloride)

Getting HO2CCH2-D-Cha-Pro-4-NHCH2A 3.5 - F2-benzamidine-hydrochloride

Ways, essentially equivalent to the one described in example 65, obtain 0.28 g of HO2CCH2-D-Cha-Pro-4-NHCH2A 3.5 - F2-HCl of the 3.5-F2-benzonitrile. The product was then purified by RPHRLC, method 2 (the gradient (A/B) of from 98/2 to 70/30, 150 min). IR; 1H-NMR; FAB-MS, m/e: 494,2 (MH+).

Analysis for C24H33N5O4F21,5 H2O:

calculated, %: C 48,57; H 6,45; N 11,80; Cl 11,95;

found, %: C 48,26; H 6,17; N 11,89; Cl 11,90.

Example 73

< / BR>
HO2CCH2-D-Cha-Pro-4-NHCH2-2 - HCl

A) Obtaining 4-methyl-2-phthalimidopropyl

To a stirred solution of 4-methyl-2-aminopyridine (50 g, 460 mmol) in acetic acid (1 l) was added phthalic anhydride (68 g, 460 mmol) and the reaction mixture is heated is kasny anhydride (43 ml, 460 mmol) and the solution continued to stir while boiling under reflux for an additional 48 hours, the Solvent is then removed in vacuum and the solid residue suspended in toluene and the solution was concentrated in vacuo, repeating this operation twice. The solid is then suspended in ethyl acetate with vigorous stirring, and filtered. After repeating this operation of washing in ethyl acetate, the solid is dried overnight in vacuum to obtain 46.6 g (42%) of white solids. IR;1H-NMR; FD-MS, m/e 238 (M+).

Analysis for C14H10N2O2:

calculated, %: C 70,58; H to 4.23; N 11,76;

found, %: C 70,42; H 4,29; N 11,70.

B) Obtaining N-(tert-BuO2CCH2)-N-Boc-D-Cha-Pro-4 - NHCH2-2-phthalimidopropyl

The methods are essentially equivalent described in examples 64-A, 23-a and 1-G (using N-(tert-BuO2CCH2)-N-Boc - D-Cha-Pro-OH), obtain 2.4 g of N-(tert-BuO2CCH2)-N-Boc - D-Cha-Pro-4-NHCH2-2-phthalimidopropyl of 4-methyl-2-phthalimidopropyl. IR;1H-NMR; FD-MS, m/e: 717,7 (M+).

C) Obtaining HO2CCH2-D-Cha-Pro-4-NHCH2- 2-aminopyridine-hydrochloride

To a stirred solution of N-(tert-BuO2CCH2)-N-Boc - D-Cha-Pro-4-NHCH2-2-teli removed in vacuum and the residue is dissolved in ethyl acetate and the resulting solution was concentrated in vacuo, by repeating this operation twice. The remainder of the transform method is essentially equivalent to the one described in example 58-D, getting 380 mg of a white solid (37%). The product was then purified by RPHRLC, method 2 (the gradient (A/B) of from 98/2 to 70/30, 150 min). IR;1H-NMR; FAB-MS, m/e: 432,3 (MH+).

Analysis for C22H33N5O42,1 HCl H2O:

calculated, %: C 50,23; H 7,11; N 13,31;

found, %: C 50,05; H 7,08; N 13,54.

Example 74

< / BR>
Getting HO2CCH2-D-Cha-Pro-5-NHCH2-2 - aminopyridine-hydrochloride

Ways, essentially equivalent to the one described in example 73, obtain 0.88 g HO2CCH2-D-Cha-Pro-5-NHCH2-2 - aminopyridine-hydrochloride of 5-methyl-2-aminopyridine. The product was then purified by RPHRLC, method 2 (the gradient (A/B) of from 98/2 to 70/30, 150 min). IR;1H-NMR; FAB-MS, m/e: 432,3 (MH+).

Analysis for C22H33N5O42HCl H2O:

calculated, %: C 50,58; H 7,14; N 13,40;

found, %: C 50,79; H 7,20; N of 13.58.

Example 75

< / BR>
HO2CCH2-D-Cha-Pro-5-NHCH2CH2-2 - HCl

A) Receiving 5-methyl-2-Boc2N-pyridine

To a stirred solution of 5-methyl-2-aminopyridine (10.5 g, 100 mmol) in dichloromethane (200 ml) at 0oC add N,N-diisopropylethylamine (25,8 g, 200 Moscow bath removed and the solution stirred overnight. The mixture was then diluted with ethyl acetate (600 ml) and washed three times with saturated aqueous solution of NH4Cl, once with brine, twice with saturated aqueous sodium bicarbonate solution and once again with brine. The organic phase is then dried (MgSO4), filtered, concentrated in vacuo and chromatographic on silica gel, elwira gradient from 10% ethyl acetate/hexane to 75% ethyl acetate/hexane. The fractions containing the product, as determined by TLC, combined and concentrated in vacuo, receiving 12.8 g (42%) of white solids. IR;1H-NMR; FD-MS, m/e 308 (M+).

Analysis for C16H24N2O4:

calculated, %: C 62,32; H to 7.84; N REMAINING 9.08;

found, %: C 62,51; H 8,11; N 9,37.

B) Receiving 6-BrCH2-2-Boc2N-pyridine

By the way, is essentially equivalent to the one described in example 64, get approximately 11.6 g of 5-BrCH2-2-Boc2N-pyridine (which is contaminated with starting material) of 5-methyl-2-Boc2N-pyridine.1H-NMR; FD-MS, m/e: 386,3 (M+).

Analysis for C16H23N2O4Br:

calculated, %: C 49,62; H of 5.99; N, 7.23 PERCENT;

found, %: C 49,86; H 6,00; N 7,07.

C) Receiving 5-NCCH2-2-Boc2N-pyridine

To stir the solution gently is 5 mmol), then KCN (1,95 g, 30 mmol). After stirring for 6 h the solvent is removed in vacuo and the residue chromatographic on silica gel, elwira stepped gradient from hexane to a mixture of 40% ethyl acetate with hexane. The fractions containing the product, as determined by TLC, combined and concentrated in vacuo to obtain 2.6 g (31%, during the procedure in two operations) of a white solid. IR; 1H-NMR; FD-MS, m/e: 333,4 (M+).

Analysis for C17H23N3O4:

calculated, %: C 61,25; H 6,95; N 12,60;

found, %; C 61,09; H 6,92; N 12,53.

D) receiving the HO2CCH2-D-Cha-Pro-5-NHCH2CH2- 2-HCl

To a stirred solution of 5-NCCH2-2-Boc2N-pyridine (2.5 g, 7.5 mmol) in methanol (150 ml) is added CoCl2(0.97 g, 7.5 mmol) and water (0,81 g, 45 mmol). After 5 min in small portions during 15 min add NaBH4(2,84 g, 75 mmol). After an additional 5 min, the solvent is removed in vacuum and the residue is dissolved in a concentrated aqueous solution of NH4OH and extracted several times with ethyl acetate. United an ethyl acetate extracts are dried (MgSO4), filtered and concentrated in vacuo. Then, by the way, essentially equivalent described in examples 1-a and 73-b, statutum RPHRLC, method 2 (the gradient (A/B) of from 98/2 to 60/40, 150 min). IR;1H-NMR; FAB-MS, m/e: 446,3 (MH+).

HRMS (FAB): for C23H36N5O4:

calculated: 446,2767;

found: 446,2769.

Example 76

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A) Obtaining 4-BocNHCH2CH2-2-CN-pyridine

To a solution of 4-BocNHCH2CH2-pyridine (2,22 g, 10 mmol) in acetone (50 ml) is added via addition funnel over 10 min a solution of m-chloro-adventurou acid (5.2 g, 30 mmol) in acetone (50 ml). After stirring overnight, the solvent is removed in vacuo and the residue partitioned between water (100 ml) and diethyl ether (100 ml). The organic phase is separated and extracted three times with water. The combined aqueous phase is then saturated with solid NaCl and extracted three times with dichloromethane (100 ml). The combined dichloromethane extracts are washed once with saline, dried (Na2SO4), filtered and concentrated in vacuo to a small volume and then add diethyl ether. White precipitate was filtered (2.0 g) and dried in vacuum.

Half of the selected solids (4.2 mmol) dissolved in dichloromethane (10 ml) and to this stirred solution was added trimethylsilylacetamide (from 0.84 ml, 6.3 mmol), then N,N-dimethylcarbamoyl (1 ml) and the mixture is distributed between ethyl acetate and water. The organic phase is then washed three times with saline, dried (MgSO4), filtered and concentrated in vacuo to obtain 0.6 g (58%) amber oil which crystallized upon standing.

IR;1H-NMR; FD-MS, m/e 247 (M+).

Analysis for C13H17N3O2:

calculated, %: C 63,31; H 7,02; N 16,99;

found, %: C 63,31; H 7,02; N 16,71.

B) Obtaining 4-BocNHCH2CH2-2-CbzNH-pyridine

To a stirred solution of 4-BocNHCH2CH2-2-CN-pyridine (0.5 g, 2 mmol) in methanol (2.4 ml) is added 5N NaOH (1.6 ml, 8 mmol) and the solution refluxed. After 24 hours the solution is cooled to room temperature and stirred for an additional 48 hours and Then the pH adjusted to 7 using 1N HCl and the solvents removed in vacuo.

The residue is suspended in toluene (50 ml) and refluxed. To this stirred solution was added sequentially triethylamine (of 0.36 ml, 2.6 mmol), benzyl alcohol (0,27 ml, 2.6 mmol) and diphenylphosphoryl (to 0.72 g, 2.6 mmol). After stirring while boiling under reflux overnight, the solution is allowed to cool and then diluted with ethyl acetate (200 ml) and washed twice saturated concentrated in vacuo. The residue is then chromatographic on silica gel, elwira stepped gradient from hexane to a mixture of hexane to 50% ethyl acetate. Containing the product fractions as determined by TLC, combined and concentrated in vacuo to obtain 0,37 g (50%) of a white solid.

1H-NMR; FD-MS, m/e: 371,2 (M+).

Analysis for C20H25N3O4:

calculated, %: C 64,67; H is 6.78; N 11,31;

found, %: C 64,90; H 7,07; N 11,06.

C) Obtaining HO2CCH2-D-Cha-Pro-4-NHCH2CH2- 2-HCl

The methods are essentially equivalent described in examples 23-A, 1-G (using N-(tert-BuO2CCH2)-N-Boc-D-Cha-Pro-OH), 23-D and 58-D, get 48 mg HO2CCH2-D-Cha-Pro-4 - NHCH2CH2-2-aminopyridine-hydrochloride of 4-BocNHCH2CH2-2-CbzNH-pyridine. The product was then purified by RPHRLC, method 2 (the gradient (A/B) of from 98/2 to 70/30, 150 min).1H-NMR; FAB-MS, m/e: KZT 446.4 (MH+).

Analysis for C23H35N5O41,5 HCl H2O:

calculated, %: C 53,30; H 7,49; N 13,51;

found, %: C 53,64; H 7,27; N 13,80.

Example 77

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Getting HO2CCH2-D-Cha-Pro-4-NHCH2-2-F - aniline-hydrochloride

By the way, is essentially equivalent to the one described in example 64, get 0.55 g HO2CCH21H-NMR; FAB-MS, m/e: 449,3 (MH+).

Analysis for C23H33N4O4F0,9HCl H2O:

calculated, %: C 55,32; H 7,25; N 11,22; Cl 6,39;

found, %: C 55,49; H 6,93; N Of 11.15; Cl 6,23.

Example 78

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Getting HO2CCH2-D-Cha-Pro-4 - NHCH2C6H4CH2NH22,2 HCl 0.5 H2O:

calculated, %: C 54,00; H 7,40; N 10,50; Cl 14,61;

found, %: C 54,18; H rate of 7.54; N 10,31; Cl 14,86.

Example 79

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Getting HO2CCH2-D-Cha-Pro-4 - NHCH2C6H10CH2NH22,7 HCl 0.5 H2O:

calculated, %: C 51,65; H 8,25; N 10,04;

found, %: C 51,47; H 7,87; N becomes 9.97.

Example 80

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(N-(Ethylsulfonyl)-D-cyclohexylethyl-N-[[4-(aminoiminomethyl) phenyl] methyl]-L-Proline-amidogidrolaza)

Getting EtSO2-D-Cha-Pro-4 - NHCH2C6H4C(NH)NH2HCl. The product was then purified by RPHPLC, method 2 (the gradient (A/B) is from 90/10 to 50/50, 180 min). IR;1H-NMR; FAB-MS, m/e: 492,3 (MH+).

Analysis for C24H37N5O4SHCl:

calculated, %: C 54,58; H 7,25; N 13,26; Cl of 6.71;

found, %: C 54,31; H 7,31; N 13,37; Cl of 6.71.

Example 81

< / BR>
Getting EtSO2-D-Cha-Pro-4 - NHCH2C6H4CH2NH2HCl. The product was then purified by RPHPLC, method 2 (the gradient (A/B) is from 90/10 to 50/50, 180 min)./BR> calculated, %: C 54,07; H of 7.75; N 10,51; Cl 6,65;

found, %: C 54,13; H 7,44; N 10,51; Cl is 6.78.

Example 82

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Getting EtSO2-D-Cha-Pro-4-NHCH2-3-F - C6H3NH2HCl.

By the way, is essentially equivalent to the one described in example 64, using EtSO2-D-Cha-ProOH instead get 0,53 g EtSO2-D-Cha-Pro-4-NHCH2-3-F-C6H3NH20.5 H2O:

calculated, %: C 51,95; H 7,03; N 10,54; Cl 7,33;

found, %: C 52,09; H 6,94; N accounted for 10.39; Cl 7,24.

Example 83

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< / BR>
Getting EtSO2-D-Cha-Pro-4-NHCH2-2 - aminopyridine-hydrochloride

By the way, is essentially equivalent to the one described in example 73, using EtSO2-D-Cha-ProOH instead get 0,22 g EtSO2-D-Cha-Pro-4-NHCH2-2-aminopyridine,1HCl:

calculated, %: C 52,25; H 7,19; N 13,85; Cl 7,71;

found, %: C 52,49; H of 6.96; N 13,96; Cl 7,76.

Example 84

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EtSO2-D-Cha-Pro-5-NHCH2-2-aminopyridine,15HCl:

calculated, %: C 52,06; H 7,18; N 13,80; Cl 8,03;

found, %: C 52,38; H 6,97; N 14,20; Cl 8,46.

Example 85

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HOOCCH2CH2CH2-D-Cha-Pro-p - NHCH2C6H4C(NH)NH22,1 HCl:

calculated, %: C 55,15; H 7,35; N 12,19; Cl 13,24;

found, %: C 55,55; H 7,37; N 12,19; Cl of 13.58.

Example 86

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(N-(Carboxymethyl)-D-cyclohexylglycine-N-[[4-(aminoiminomethyl)is actually equivalent to the one described in example 53-B, get 16,1 g (16%) D-cyclohexylglycine on the basis of D-phenylglycine. 1H-NMR; FD-MS, m/e: 117 (M+).

Analysis for C8H15NO2:

calculated, %: C 61,12; H 9,62; N 8,91;

found, %: C 61,23; H 9,56; N 8,73.

B) Receive Boc-D-cyclohexylglycine

By the way, is essentially equivalent to the one described in example 17 (using di-tert-BUTYLCARBAMATE) obtain 22 g (90%) of Boc-D-cyclohexylglycine.1H-NMR; FD-MS, m/e 258 (M+)

Analysis for C13H23NO4:

calculated, %: C 60,68; H 9,01; N 5,44;

found, %: C 60,91; H 9,18; N 5,38.

C) Receive Boc-Pro-p-NHCH2C6H4C(NH)NHCbz

By the way, is essentially equivalent to the one described in example 1-W, get to 20.5 g (76%) of Boc-Pro-p-NHCH2C6H4C(NH)NHCbz of Boc-Pro-OH and NH2CH2C6H4C(NH)NHCbz2,5HCl:

calculated, %: C 51,67; H 6,69; N 13,10;

found, %: C 51,84; H 6,50; N 13,15.

Example 87

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(N-(Carboxymethyl)-D-homophenylalanine-N-[[4-(aminoiminomethyl) phenyl] methyl]-L-prolinamide hydrochloride)

A) Obtaining HOOCCH2-D-hPhe-Pro-p - NHCH2C6H4C(NH)NH22,1 HCl H2O:

calculated, %: C 53,61; H 6,32; N 12,50; Cl to 13.29;

found, %: C 53,58; H between 6.08; N 12,59; Cl 13,67.

Example 88

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(N-(Carboxymethyl)-D-homocyclic, essentially equivalent to the one described in example 53-G, get a 5.1 g (100%) of Boc-D-hCha-OH from Boc-D-hPhe-OH.1H-NMR; FD-MS, m/e 240 (M+).

Analysis for C15H27NO4:

calculated, %: C 63,13; H 9,54; N 4,91;

found, %: C 63,38; H 9,39; N 5,12.

B) Receiving HOOCCH2-D-hCha-Pro-p - NHCH2C6H4C(NH)NH22,2 HCl 0.5 H2O:

calculated, %: C 53,54; H 7,22; N 12,49; Cl 13,91;

found, %: C 53,29; H 7,01; N 12,46; Cl 14,30.

Example 89

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A) Obtaining HOOCCH2-Gly-N - C6H5CH2CH2Gly-p - NHCH2C6H4C(NH)NH22,2 HCl for 1.5 H2O:

calculated, %: C 49,60; H 6,09; N 13,15; Cl 14,64;

found, %: C 47,79; H 5,71; N 13,31; Cl 14,49.

Example 90

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A) Receive Boc-(-OBn)-Glu-Pro-p - NHCH2C6H4C(NH)NHCbz

By the way, is essentially equivalent to the one described in example 1-G, get 2.7 g (64%) of Boc-(-OBn)-Glu-Pro-p - NHCH2C6H4C(NH)NHCbz from Boc-(-OBn)-Glu-OH and Pro-p-NHCH2C6H4C(NH)NHCbz-OBn)-Glu-Pro-p-NHCH2C6H4C(NH)NHCbz-OBn)-Glu-Pro-p - NHCH2C6H4C(NH)NHCbz2HCl. 1H-NMR; FD-MS, m/e 600 (M+).

Analysis for C33H39N5O6Cl2:

calculated, %: C 58,93; H of 5.84; N 10,41;

found, %: C 58,64; H 6,00; N 10,63.

B) Receive (C2H5)2C(NH)NHCbzHCl (1.3 g, 2 mmol) in THF/H2O (50 ml each) add K2CO3(to 1.38 g, 10 mmol) and 2-ethylbutyrate (0.3 g, 2.2 mmol). After stirring for 10 min the volatile component parts removed in vacuum. The resulting residue partitioned between water and ethyl acetate (100 ml each). The layers are separated and the organic phase is washed twice, each time with a saturated aqueous solution of ammonium chloride and brine, dried (MgSO4), filtered and concentrated in vacuo to obtain 1.45 g (100%) connection. 1H-NMR; FD-MS, m/e: 698 (M+).

Analysis for C39H47N5O7:

calculated, %: C 67,13; H 6,79; N 10,04;

found, %: C 67,11; H 6,70; N 9,74.

G) Receiving (C2H5)2CHCO-Glu-Pro-p - NHCH2C6H4C(NH)NH21,5 HCl 1,1 H2O:

calculated, %: C 51,10; H 6,91; N 12,41; Cl 9,43;

found, %: C 51,10; H for 6.81; N 12,41; Cl 9,62.

Example 91

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A) get (C2H5)2CHCO-Met(O2)- Pro-p-NHCH2C6H4C(NH)NH21,1 HCl:

calculated, %: C 52,63; H 7,01; N 12,79; Cl 7,12;

found, %: C 52,42; H 7,03; N 12,80; Cl 6,99.

Example 92

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(N-(Methylsulfonylmethyl)-L-cyclohexylethyl-N-[[4-(aminoiminomethyl) phenyl]methyl]-L-Proline-amide-hydrochloride).

Polo 6,97; N 12,04; Cl 7,32;

found, %: C 51,58; H 6,84; N 12,18; Cl to 7.61.

Compounds of the invention may selectively inhibit thrombin relative to other proteases and non-enzymatic proteins, causing clotting of blood, without appreciable interference with the ability to lyse natural coagulation in the body (compounds have a low inhibitory effect on fibrinolysis). Further, this selectivity allows you to use them with thrombolytic agents without significant interference with thrombolism and fibrinolysis. In addition, the compounds of the present invention can be orally effective.

In one of its aspects the invention relates to a method of inhibiting thrombin in a mammal which comprises the administration to a mammal in need of treatment an effective (inhibiting thrombin) doses of the compounds of formula I.

Inhibition of thrombin, according to the present method, includes as acceptable as a medical therapeutic and/or prophylactic treatment.

Further, the invention relates to the treatment, in the case of a person or animal, conditions which require inhibition of thrombin. Compounds of the invention may be suitable in Lochboisdale state, under which compounds have potential utility for the treatment or prevention represent thrombosis and hypercoagulability in the blood and tissues. These painful conditions using the compounds for treatment and/or prevention include venous thrombosis and pulmonary embolism, arterial thrombosis, such as myocardial ischemia, myocardial infarction, unstable angina, stroke due to thrombosis and peripheral arterial thrombosis. Further, the compounds used for the prevention of atherosclerotic diseases such as coronary arterial disease, cerebral arterial disease and peripheral arterial disease. Further, the compounds can be used in conjunction with thrombolytic agents in acute myocardial infarction. Further, the compounds used for the treatment or prevention for reocclusion after thrombolyse, percutaneous transluminal angioplasty (PTCA) and operations associated with coronary bypass surgery. Further, the compounds may be useful in the prevention of rethrombosis after microsurgery. Further, the compounds may be suitable for anticoagulant treatment in connection with artificial organs, and heart valves. Next, audistas coagulation. A further possible application is in the flushing of catheters and mechanical devices used in patients in vivo, and as an anticoagulant for conservation in vitro of blood, plasma and other blood products. Further, the compounds may be useful in other diseases, where the coagulation of blood may be the fundamental concomitant process or a source of secondary pathology, such as cancer, including metastasis, and inflammatory diseases, including arthritis and diabetes. Anticoagulant compound is administered orally or parenterally, e.g. by intravenous infusion (IV), intramuscular injection (im) or subcutaneous injection (PC).

The specific dose of a compound, administered according to the present invention, in order to achieve therapeutic and/or prophylactic effects naturally should be determined by the individual circumstances of the disease, including, for example, the input connection, the rate of administration and the condition being treated.

A typical daily dose for each of the above useful applications is from about 0.01 to 1000 mg/kg Dosing regimen can for preventive purposes differ, nopriv day. In critical anxiety connection according to the invention is administered by intravenous infusion at a rate of approximately 0.01 to 20 mg/kg/hour and, preferably, about 0.1-5 mg/kg/hour.

The method according to the present invention is also carried out in combination with lyse the thrombus (blood clot) agent, such as plasminogen tissue activator (t-PA), a modified t-PA, streptokinase or urokinase. In cases when there is a blood clot and is blocked artery or vein, either partially or completely, usually used lyse the thrombus agent. The connection according to the invention can be introduced before or together with the lytic agent or after, preferably, in addition together with aspirin to prevent a resumption of clotting.

The method according to the present invention is also carried out in combination with an antagonist trombotsitnoy glycoprotein receptor (Pb/sa), which inhibits platelet aggregation. The compound of the invention can be introduced before or together with the IB/Sha-antagonist or after it to prevent the resumption of clotting.

The method according to the present invention is also carried out in combination with aspirin. Xu prevent the recurrence of clotting. As stated above, the compound of the present invention preferably is administered in combination with lyse the thrombus agent and aspirin.

The present invention also relates to pharmaceutical compositions for use in the above method of treatment. The pharmaceutical compositions according to the invention include an effective inhibitory amount of thrombin compounds of formula I in combination with a pharmaceutically acceptable carrier, excipient or diluent. For oral administration, antithrombotic compound is prepared in the form of gelatin capsules with the medicine or tablets which may contain excipients such as binders, lubricants, disintegrating agents, etc., For parenteral administration, an antithrombotic agent is prepared in a pharmaceutically acceptable diluent, for example, in the form of a physiological saline solution (0.9%), 5% dextrose, ringer's solution, etc.,

The compound of the present invention may be part of a single dosage of preparations containing a dose of about 0.1-1000 mg, Preferably, the connection is in the form of pharmaceutically acceptable salts, such as, for example, sulfate, acetate or phosphate. An example of a single dosing the ampoule 10 ml Another example of a unit dosage form contains about 10 mg of the compound of the present invention in the form of a pharmaceutically acceptable salt in 20 ml of isotonic saline solution contained in a sterile vial.

Connections can be entered in various ways, including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular and introduction through the nose. Compounds of the present invention, preferably prepared in the form of a composition prior to the introduction. Therefore, the next object of the present invention is a pharmaceutical composition comprising an effective amount of the compounds of formula I or its pharmaceutically acceptable salt or MES in combination with a pharmaceutically acceptable carrier, diluent or excipient.

The active ingredient in such compositions is 0.1 to 99.9 wt.% in the calculation of the composition. The phrase "pharmaceutically acceptable" means that the carrier, diluent or excipient must be compatible with other ingredients of the composition and not to harm the patient.

These pharmaceutical compositions are prepared by known methods using well-known and easily doauth with a carrier, or diluted by a carrier, or include in the media, which can be in the form of a capsule, packet, paper or other container. When the carrier serves as a diluent, it may be solid, semi-solid or liquid material, which acts as a diluent, excipient, or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, pellets, wafers, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), soft or hard gelatin capsules, suppositories, sterile injectable solutions, sterile packaged powders, etc., the compositions of the present invention can be designed in such a way, that was fast, continuous or delayed release of the active ingredient after administration to the patient by means well known to the specialist procedures.

The following examples of compositions are for illustration only,

but in no way limit the scope of protection of the invention. Under the "active ingredient" of course, understand the connection corresponding to the formula I, or its pharmaceutically acceptable salt or MES.

Part 1

Hard gelatin capsules produced using the following ingredients mg/capsule: the AB 2

The tablet obtained using the following ingredients mg/tablet:

Active ingredient: 250

Microcrystalline cellulose - 400

Silicon dioxide - 10

Stearic acid - 5

Just - 665

The components are mixed and compressed to obtain tablets each weighing 665 mg

Part 3

Spray the solution obtained in the form containing the following components, wt%:

Active ingredient - 0,25

Ethanol - 25,75

Propellant 22 (Chlorodifluoromethane) - 70,00

Just 100,00

The active compound is mixed with ethanol and the mixture added to part of the Propellant 22, cooled to -30oC and transferred into a bootable device. The required amount is then introduced into a stainless steel container and diluted with the remainder of the propellant. Then the supply container valve elements.

Part 4

Tablets, each containing 60 mg of active ingredient, are prepared as follows:

The active ingredient 60 mg

Starch - 45 mg

Microcrystalline cellulose 35 mg

Polyvinylpyrrolidone (as 10% aqueous solution) 4 mg

Sodium salt of carboxymethyl amylum - 4.5 mg

Magnesium stearate 0.5 mg

Talc 1 mg

150 mg
oC and passed through sieve No. 18 mesh U.S. Sodium salt of carboxymethyl amylum, magnesium stearate and talc, previously passed through sieve # 60 mesh U. S., then added to the granules which, after mixing is pressed on the machine for the manufacture of tablets to obtain tablets each weighing 150 mg

Part 5

Capsules, each containing 80 mg of the active ingredient, are prepared as follows, mg:

Active ingredient - 80

Starch - 59

Microcrystalline cellulose - 59

Magnesium stearate - 2

Only 200

The active ingredient, cellulose, starch and magnesium stearate are blended, passed through sieve No. 45 mesh U.S. and fill this mixture in hard gelatin capsules in the amount of 200 mg.

Part 6

Candles, each containing 225 mg of active ingredient, are prepared as follows, mg:

Active ingredient: 225

Glycerides of saturated fatty acids - 2000

Total - 2225

The active ingredient is passed through sieve # 60 mesh U. S. and suspended in pre-molten when used in the s candle-making a nominal capacity of 2 g and allowed to cool.

Part 7

Suspensions, each containing 50 mg of active ingredient per dose 5 ml, prepared as follows:

Active ingredient: 50 mg

Sodium carboxymethyl cellulose 50 mg

The syrup 1.25 ml

A solution of benzoic acid 0.10 ml

Odorant - Sufficient

Dye - Sufficient

Purified water to a total volume of 5 ml

The active ingredient is passed through a sieve No. 45 mesh U.S. and mix with the sodium salt of carboxymethyl cellulose and syrup to obtain a soft paste. Add with stirring a solution of benzoic acid, fragrance and dye, diluted part water. Then add sufficient water to achieve the required volume.

Part 8

Composition for intravenous infusion can be obtained in the following way:

Active ingredient: 100 mg

Isotonic saline solution - 1000 ml

The solution of the above ingredients is usually administered intravenously to a patient at a rate of 1 ml/min

The compounds of formula I according to the invention are orally active and selectively inhibit the action of thrombin in a mammal.

The ability of the compounds of the present invention the life of the test.

Inhibition of thrombin demonstrated in vitro inhibition amidase activity of thrombin, as defined in the test in which the thrombin hydrolyzes the chromogenic substrate, N-benzoyl-L-i.e. phenylalanyl-L-poured-L-arginyl-p-nitroanilide.

The test is carried out by mixing 50 μl of buffer (0.03 M TRIS, 0.15 M NaCl, pH 7,4) with 25 μl of a solution of bovine thrombin and human thrombin (0.21 mg/ml trombotto bovine thrombin, Paeke-Davis, or purified human thrombin, Enzyme Research Laboratories, South bend, India, in an amount of about 8 N1T units/ml in the same buffer) and 25 μl of the test compound in the solvent (50% aqueous methanol by volume). Add 150 μl of an aqueous solution of a chromogenic substrate (0.25 mg/ml) and rate of hydrolysis of the substrate was measured by monitoring the reactions at 405 nm for the release of p-nitroaniline. Standard curves are built by drawing a curve, the concentration of free thrombin on the rate of hydrolysis. The rate of hydrolysis observed with test compounds, and then converted into values of free thrombin in the respective tests by using standard curves. Bound thrombin (associated with the test connection) calculate putea thrombin, used in the experiment. The amount of free inhibitor in each experiment calculated by subtracting the number of moles of bound thrombin from the number of moles of the added inhibitor (test connection).

Ka- the value is a constant estimated equilibrium for the reaction between thrombin and test connection:

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Kass-value calculated for a range of concentrations of the tested compounds and the average value is expressed in units of liter per mole.

Following essentially the above-described methods for human thrombin and using other human blood coagulation system serine proteases and protease fibrinolytic system with the appropriate chromogenic substrates, identified below, estimate the selectivity of the compounds of the present invention in relation to the coagulation factor serine proteases and in respect of the fibrinolytic system serine proteases, as well as to a significant extent, the lack of interference with the serine proteases fibrinolytic system. Thrombin inhibitors, preferably, should not affect fibrinolysis, inductive urokinase, plasminogen tissue activator (t-PA) and streptocide the optical therapy due to streptokinase, t-PA or urokinase and for use of such agents as endogenous, not affecting fibrinolysis (in respect of t-PA and urokinase) antithrombotic agent. In addition to the lack of interference with amidase activity fibrinolytic proteases, such gentle fibrinolytic system can be investigated by using human plasma clots and their lysis by an appropriate fibrinolytic plasminogen activators.

Human factors X, Xa, IXa, XIa and XIIa were obtained from Enzyme Research Laboratories, South Bend, India, human urokinase obtained from Leo Pharmaceuticals, Denmark; and recombinant activated protein C (aPC) was obtained from Eli Lilly and Co. essentially, according to the U.S. patent 4981952. Chromogenic substrates - N-benzal-Ile-Glu-Gly-Aig-p-nitroanilide (for factor Xa); N-Cbz-D-Arg-Gly-Arg-p-nitroanilide (for factor test IXa as a substrate of factor Xa); pyroglutamyl-Pro-Arg-p-nitroanilide (for factor XIa and aPC); H-D-Pro-Arg-p-nitroanilide (for factor XIIa); and pyroglutamyl-Gly-Arg-p-nitroanilide (for urokinase) - purchased from Kabi Vitrum, Stockholm, Sweden, or from Midwest Biotech, Fishers, Indiana. Bovine trypsin obtained from Worthington Biocemicals, Freehold, New Jersey, and kallickrein human plasma obtained from Kabi Vitrum, Stockholm, Sweden. Chromogenic substrate H-D-P is ID, substrate for human thrombin and trypsin, synthesized according to the method described above for the compounds of the present invention using known methods combination of peptides from commercially available reagents, or received from Midwest Biotech, Fishers, Indiana.

Human plasmin obtained from Boehringer Mannheim Indianapolis, Indiana; nt-PA as the sole chain activity pattern (chain activity reference) was purchased from American Diagnostica, greenwich, Connecticut; the modified t-PA6 (mt-PA6) was obtained from Eli Lilly and Company is known in the prior art method (see Burck and other J. Biol. Chem. 265, 5120-5177 (1990)). Chromogenic plasmin substrate H-D-Val-Leu-Lys-p-nitroanilide and the substrate fabric plasminogen activator (t-PA) H-D-Ile-Pro-Arg-p-nitroanilide obtained from Kabi Vitrum, Stockholm, Sweden.

In the above chromogenic substrates of the three-letter symbols Ile, Glu, Gly, Pro, Arg, Phe, Val, Leu and Lys are used to indicate corresponding group of amino acids: isoleucine, glutamic acid, glycine, Proline, arginine, phenylalanine, valine, leucine, and lysine, respectively.

In the following table 1 shows Kass-values obtained with the specified compound corresponding to the formula (I).

It should be noted that, unexpectedly, the compounds in which X oboznachaet the show especially surprisingly high efficacy in inhibiting factor Xa, when comparing, for example, from corresponding compounds in which X denotes a D-fenilalanina fragment.

Materials

Dog plasma get from waking dogs of mixed breed (either sex, Hazelton-LRe, Kolamazoo, Michigan, USA) by venipuncture in 3.8% citrate. Fibrinogen is obtained from fresh dog plasma, human fibrinogen derived from in-date ACD human blood into fractions 1-2, in accordance with the specified methods and descriptions: Smith, Biochem. J., 185, 1-11 (1980); and Smith and others, Biochemistry, 11, 2958-2967 (1972). Human fibrinogen (98% purity and without plasmin) is obtained from Americam Diagnostica, Greenwich, Connecticut. Labeling radioactive isotope preparations of fibrinogen 1-2 is carried out, as previously reported: Smith and others, Biochemistry, 11, 2958-2967 (1972). Urokinase obtained from Leo Pharmaceuticals, Denmark, in the form of 2200 Ploug units/vial. Streptokinase obtained from Hoechst Roussel Pharmaceuticals, Somerville, new Jersey.

Methods - Impact on the lysis of clots (thrombi) in human plasma at the expense of t-PA

The human plasma clots get in microtubes by adding 50 μl of thrombin (73 N1H units/ml) to 100 µl of human plasma, which contains the labeled isotope iodine-125 fibrinogen with radioactivity 0,0229 microcurie. Lysis of the clot study by the things temperature. After incubation, the tubes centrifuged in microcentrifuge Beckmann. 25 μl of supernatant is added to 1.0 ml of 0.03 M TRIS/0.15 M NaCl buffer for gamma counting. Controls counting for 100% lysis is obtained by not adding thrombin (and replace the buffer). Thrombin inhibitors evaluated for possible interference with fibrinolysis due to the inclusion of compounds in the coating solutions at concentrations of 1.5 and 10 IU/ml approximation values IC50estimated by linear extrapolation from data points to the value, which should mean 50% lysis, for this special concentration of fibrinolytic agent.

Anticoagulant activity

Materials

The plasma of the dog and rat plasma get from waking dogs of mixed breed (either sex, Hazelton - LRe, Kalamazoo, Michigan, USA) or from shot male rats Sprague - Dawley (Harlan Sprague - Dawley, Inc., Indianapolis, Indiana, USA) by venipuncture in 3.8% citrate. Fibrinogen is obtained from in-date ACD human blood in the form of fractions 1-2, in accordance with the specified methods and descriptions: Smith, Biochem. J., 185, 1-11 (1980); and Smith and others, Biochemistry, 11, 2958-2967 (1972). Human fibrinogen obtained in 98% purity and without plasmin, from American Diagnostica, Greenwich,Miami, Florida. Bovine thrombin obtained from Parke-Davis (Ann. Deytroyt, Michigan), used for experiments on coagulation in plasma.

Methods - determination of anticoagulation

Methods of making tests of coagulation such as previously described; Smith and others, Trombosis Research, 50, 163-174 (1988). A device for determining coagulation CoAScreener (American Labor. Inc) is used for all measurements, tests for coagulation. Prothrombin time (PT) is measured by adding 0.05 ml of saline solution and 0.05 ml of reagent thromboplastin-C to 0.05 ml test plasma. Activated partial thromboplastin time (APTT) was measured by incubation 0.06 ml test plasma with 0.05 ml of reagent Actin for 120 sec followed by the addition of 0.05 ml of 0.02 M solution of CaCl2. Thrombin time (TT) is measured by adding 0.05 ml of saline solution and 0.05 ml of thrombin (10 N1H D./ml to 0.05 ml test plasma. The compounds of formula I added to human plasma and plasma of animals in the form of a large number of concentrations to determine the prolonged effects when tested against APTT, PT and TT. Linear extrapolation is carried out for evaluation of the concentration required to double the time of formation of clots for each test.

Animals

Males cu is on (120 mg/kg, subcutaneously) and contain heated water surface (37oC). Jugular vein (vein) kanyoro, in order to allow infusion (infusion).

The model of arteriovenous shunt

The left jugular vein and right carotid artery kanyoro using 20 cm long tubing made of polyethylene PE 60. In 6 cm in length of the Central segment of the broader tubing (PE 190) into the lumen of the insert cotton yarn (5 cm) between the longer pieces to the full path of arteriovenous shunt. The blood circulates through the shunt within 15 minutes before the thread carefully removed and weighed. The wet weight of the thread is subtracted from the total weight of the filament and a blood clot (see J. R. Smith, Br. J. Pharmacol., 77, 29 (1982)).

When the compound of example 48 is compared with D-MePhe-Pro-Arg-H (discussed above on pages 2), in the model of arteriovenous shunt, antithrombotic efficacy was found 9 times greater during continuous intravenous infusion. In relation to weight reduction of the thrombus to the same extent (approximately 20% of control), the compound of example 48 prolong thrombin time, plasma is about 3 times, whereas thrombin time, plasma is extended more than 20 times in the infusion process standard connections. Prothrombin time and APTT is prolonged only the hotel arterial damage

The carotid arteries are isolated through cretinously and cervical incision. For each artery place thermocouple and the temperature of the vessel is continuously recorded on a chart tape recorder. Around each carotid artery, directly above thermocouple, placed the cuff tubing (0,058 ID x 0,077 OD x 4 mm, Baxter Med. Grade Silicone), a longitudinal cut (cut longitudinally). The uranyl FeCl3dissolved in water and the concentration (20%) expressed only from the point of view of the actual weight FeCl3. Smoking damages the arteries and promote thrombosis, 2,85 µl pipette is introduced into the cuff artery above thermocouple probe. Arterial occlusion is characterized by rapid temperature drop. Time occlusion is recorded in the minutes and the mean time elapsed between the introduction of FeCl3and a rapid fall in temperature in the vessel (see K. D. Kurz, Thromb. Res., 60, 269 (1990)).

Model of spontaneous thrombolyse

In vitro data suggest that peptide inhibitors of thrombin inhibits thrombin and other serine proteases, such as plasmin and tissue plasminogen activator. For the purpose of evaluation, inhibit connections fibrinolysis in vivo, determine the rate of spontaneous thrombolyse by implantation entirely me (4 IU, Parke-Davis) and labeled125I human FibroGen (5 microcurie, ICN), immediately make elasticity tubing and incubated at 37oC for 1 h Subjected to the aging of the blood clot is removed from the tubing, cut into segments 1 cm, washed 3 times with 1N solution of salt and each segment is subjected to pulse counting in a gamma counter. The segment with the known pulse count is sucked into the catheter, to then be implanted in the jugular vein. The thin end of the catheter is injected adjacent to the right atrium and the clot push in pulmonary blood flow. An hour later after implantation, the heart and lungs removed and separately counts the number of pulses. Thrombosis expressed in percentage, where:

< / BR>
(cpm = counts per minute).

Fibrinolytic dissolution of the implanted clot occurs in a time-dependent (see J. R. Clozel, Cardiovas. Pharmacol. 12, 520 (1988)).

The parameters of coagulation

Thrombin time, plasma (TT) and activated partial thromboplastin time (APTT) was measured using vibrometra. Blood taken from the jugular catheter and collected in a syringe containing sodium citrate (3,8%; 1 part to 9 parts blood). To measure TT, rat plasma (0.1 ml) mixed with saline solution (0.1 ml) and bovine tro is irout for 5 min at 37oC and to initiate coagulation add 0.025 M solution of CaCl2(0.01 ml). Experiments repeated two times and take the average value.

Index of bioavailability

Measurement of bioactivity, thrombin time (TT) plasma is used as a replacement test classification connection with respect to the assumption that the growth in TT are the result of inhibition of thrombin only due to the classification of compounds. Time, flowing from exposure to thrombin inhibitor relative to TT, is determined after intravenous drugs shot rats after oral processing awake rats. Due to the restriction of blood volume and the number of points required to define the time elapsing from the time of processing to the time when the response returns to the values before treatment, use two populations of rats. Each sample population means alternating sequential time points. Average TT above elapsed time is used to calculate the area under the curve (AUC). Index of bioavailability calculated using the following formula and expressed as percentage relative activity.

The area under the curve (AUC) elapsed time TT of the plasma Oprah is highlighted as:

< / BR>
p.o. = oral; i.v. = intravenous).

Connection

Solutions of the compounds are prepared fresh daily in a 1N saline solution and injected in a bolus or pour, starting at 15 min before and continuing throughout the experimental perturbations, which is 15 minutes in a model of arteriovenous shunt and 60 min in FeCl3model of arterial injury and in models of spontaneous thrombosis. Injectively volume of the bolus is 1 ml/kg intravenously and 5 ml/kg oral and infusion volume is 3 ml/h

Statistics

The results are expressed as values +/-SEM. To identify statistically significant differences using the routine analysis of variance and then used the Dunnett test to determine which values are different. The level of significance of departures from the null assumption of adequate values is P < 0,05.

Animals

Dogs (hounds; in age from 18 months to 2 years; weight of 12-13 kg; Marshall Farms, North Rose, NY 14516) tied for the night and after 240 minutes after dosing fed food by prescription according to Purina (Purina Mills, St. Lous, Missouri). Water give sufficient for survival. Maintain room temperature at 24,9-28oC; otnositelnaya prepared immediately before dosing by dissolving in sterile 0,9% saline solution to obtain a preparation with a concentration of 5 mg/ml Dogs receive a single dose of 2 mg/kg of the test compounds orally. Blood samples (4.5 ml) is withdrawn from the lateral saphenous vein in the hours 0,25, 0,5, 0,75, 1, 2, 3, 4 and 6 after dosing. Samples collected in tetrathiophene Vacutainer tube and keep on ice until the plasma concentration by centrifugation. Plasma samples transform using dinitrophenylhydrazine and analyzed by HPLC (high performance liquid chromatography (HPLC) (column with Bond SB-C8), elwira methanol with 500 mmol of sodium acetate, bringing to pH 7 with phosphoric acid (60:40 by volume). The plasma concentration of the test compounds are recorded and used to calculate the pharmacokinetic parameters: the rate constant of elimination, Ke; total clearance, CIt; volume of distribution (vascularization), VD; time of maximum plasma concentration of the test compound, Tmax; the maximum concentration of the test compounds at TmaxCmax; the half-life of plasma, t0,5; area under the curve, A. U. C. and the absorbed portion of the test compounds, F.

The model of the coronary arterial thrombosis in the rabbit

Surgical preparation and instrumentation in relation to dogs such, o MI, USA) anaesthetize pentobarbital sodium (30 mg/kg intravenous, i. v. ), inkubiruut and ventilate the room air. Tidal otlivnyy volume and rate of breathing regulate by maintaining blood PO2, PO2and pH in the normal range. Subcutaneous needle electrodes inserted for recording biopotential-P on the electrocardiograph.

The left jugular vein and common carotid artery are isolated through the left mediolaterally cervical incision. Arterial blood pressure (ABP) was measured continuously using a pre-calibrated transducer (sensor) Millar (model MPC-500, Millar Instruments, Houston, TX, USA) inserted into the carotid artery. Jugular vein kanyoro blood collection during the experiment. In addition, the femoral veins of both hind paws kanyoro for the introduction of the test compounds.

A left thoracotomy is performed in the fifth intercostal cavity and the heart suspended in a pericardial frame. The size of 1-2 cm segment of the left circumflex coronary artery (LCX) isolated close to the first main diagonal ventricular branches, 26-gauge with a thin needle end of the wire anode electrode (coated with Teflon30-gauge silver-PL is AI (fix until the end of the experiment). The stimulation cycle is completed by placing the cathode in the subcutaneous (s.c.) a plot. Adjustable plastic closing device is placed near LCX, above the area of the electrode. Pre-calibrated electromagnetic flow probe (Carolina Medical Electronics, King, U. S. A.) is placed near LCX, nearest to the anode, for measuring coronary blood flow (CBF). The closing device (occluder) is adjusted to achieve a 40-50% inhibition of the response hyperemic blood flow observed after the first 10 seconds mechanical occlusion of LCX. All hemodynamic and ECG measurements are recorded and analyzed by the system data Bank (model M3000, Modular. Instruments, Malven, PA, USA).

The formation of thrombus and modes of introduction connections

Electrolytic damage to the interior is reached by application to the anode of a constant current of 100 µa (DC). The current is maintained for 60 minutes and then interrupted to check occluded vessel or not. A blood clot occurs spontaneously, up until LCX not fully occluded (define as zero CBF and increased S-t-segment). Introduction connections start after the occluding thrombus exists already within 1 hour. Infusion for 2 h with the same (for example, tissue plasminogen activator, streptokinase, AP SAC). The reperfusion carried out for 3 h after injection of the test compounds. Reocclusion coronary arteries after a successful thrombolyse defined as zero CBF, which persists for 30 minutes

Hematology and definition of the template bleeding time

Counts of cells whole blood, hemoglobin, and values hematocrit numbers determine the sample volume of 40 µl tetrathiophene (3,8%) blood (1 part citrate to 9 parts blood) using a Hematology analyzer (Cell-Dyn 900, Sequoia-Turner, Mount View, VA, USA). Template time gingival (gum) bleeding is determined using the Simplate device II for measuring bleeding time (Organon Teknika Durham, N. C., USA). The device used to make 2 horizontal incision in the gums any upper or lower left jaw of the dog. Each slit has a width of 3 mm and a depth of 2 mm Make incisions and use a stopwatch to determine what duration is bleeding. A cotton swab is used to absorb the blood as it slowly flows (leaks) from the section. Template bleeding time is the time from incision to prekrseni the processes, after the introduction of the test compound (120 min) and at the end of the experiment.

All data analyzed by routine analysis of variance (ANOVA), followed by Student - Neuman - Kuelspost - hoc t-test to determine the level of confidence. Repeated measure ANOVA used to determine significant differences between time points during the experiments. The values determined for the statistical difference was minimal at p < 0,05. All values represent the average number of SEM. All studies are carried out in accordance with the guidelines of the American Physiological Society. Further, detailed consideration of the methods described by Jackson and others, J. Cardiovasc. Pharmacol., 21, 587-599 (1993).

In table. 2 given the anticoagulation of human plasma.

1. The peptide derivative of General formula I

X - Y - NH - (CH2)r- G

or its pharmaceutically acceptable salt, or MES,

where X is a D-homopolymer,

< / BR>
T - C3- C8cycloalkyl,1- C8alkyl;

< / BR>
a = 0 or 1;

Q - NH-A;

And is hydrogen, C1- C4alkyl, R"SO2-(where R1- C4alkyl), R"OC(O)-(where R1- C4alkyl), R"C(O)-(where R" is a 9 - or 10-membered unsubstituted or samisen may be substituted WITH1- C4by alkyl), or -(CH2)g- COOH;

g = 1, 2, or 3;

In - hydrogen;

R' is hydrogen;

m = 1;

n = 1;

Z is hydrogen;

Y denotes

< / BR>
where Rg- C1- C6alkyl, C3- C8cycloalkyl or -(CH2)p-L-(CH2)q-T';

Ppis hydrogen, C1- C6alkyl, C3- C8cycloalkyl or -(CH2)p-L-(CH2)q-T'; where R = 0, 1, 2, 3, or 4; L is a bond, -0-, -S - or-NH-; and q = 0, 1, 2 or 3, and T' is hydrogen, C1- C4alkyl, C3- C8cycloalkyl or Ar, where Ar is phenyl; Ry- -CH2and Rztaken with Ryand three adjacent carbon atoms, form a saturated carbocyclic ring of 5 to 8 atoms;

r = 1 or 2;

G IS -(CH2)s-R, where s = 0-5, -CH=CH-(CH2)t-R, where t = 0-3,

< / BR>
where D and E are each CH, or one of D and E Is N and the other is CH; k = 0 or 1; b = 1; M - S; each W is CH and

< / BR>
or its pharmaceutically acceptable salt, or pharmaceutically acceptable MES specified connection, or its salt, provided that a is not hydrogen, C1- C4the alkyl or tertbutyloxycarbonyl, when G is -(CH2)s-R or-CH= CH-(CH2)t-R and R-C(=NH)NH2or-NH-C(=NH)NH2Y - not is when r = 1 and s = 0; and, in addition, provided that a does not denote hydrogen, C1- C4alkyl, methylsulphonyl or -(CH2)g-COOH, when G is -(CH2)s-R, in which R is-C(= NH)NH2or-NH-C(=NH)NH2, Y does

the unsubstituted Proline (RR- hydrogen) or 4-hydroxyproline (RR- HE), R' is hydrogen, T is cyclohexyl and Q is-NH-A; and, in addition, provided that R"SO2is not arylsulfonyl, when G is -(CH2)s-R, in which R is-NH-C(=NH)NH2Y is an unsubstituted Proline (RR- hydrogen) or 4-methylthiophenyl (RR- SCH3and Q is-NH-A; and provided that not means R"SO2-when G denotes

< / BR>
T - C1- C8alkyl or

< / BR>
and Q is-NH-A; and, in addition, the compounds of formula I are excluded compounds, where X is D-homopolymer.

< / BR>
in which T - C3- C8cycloalkyl,1- C8alkyl,

< / BR>
a = 0 or 1;

Q-NH-A;

And is hydrogen, C1- C4alkyl, R"SO2-(where R1- C4alkyl), R"OC(O)-(where R1- C4alkyl), R"C(O) - (where R1- C4alkyl) or -(CH2)g-COOH;

g = 1, 2, or 3;

In - hydrogen;

R' is hydrogen;

m = 1;

n = 1;

Z = hydrogen;

< / BR>
in which RR- What SUB>-C(=NH)-NH2(i.e. G - 4-lidinopril or 4-guanidinate); or

< / BR>
where k = 0 and R' is-C(=NH)NH2or-NH2-C(=NH)-NH2or k = 1 and R' is-C(= NH)NH2; or

< / BR>
where R' is-C(=NH)NH2; and, in addition, where the alkyl by itself or as part of another substituent is stands, ethyl, n-propylene, isopropyl, n-bootrom, tribution, isobutyl or second-bootrom;

WITH3- C8cycloalkyl is cyclopropyl, methylcyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 4-methylcyclohexyl or cyclooctyl;

9 - or 10-membered heterocyclic ring is indolium, hyalinella or izokhinolinom; and, in addition, where any heteroaromatic group, listed for the definition of R" is independently unsubstituted or substituted by one Deputy, which leads to a stable structure independently selected from C1- C4the alkyl.

2. The compound or its salt, or its MES under item 1, where

< / BR>
D-homopolymer or 1-Piq; Y - prolinal and Q is NHA in which A - hydrogen or R"SO2-(where R 'is ethyl), R' is hydrogen, Z is hydrogen and hydrogen; R - guanidino or amidinopropane.

3. The compound or its salt, or its MES under item 1 or 2, where G is a 4 - amidinophenoxy group.Aya contains an effective amount of a compound of formula I, or its pharmaceutically acceptable salt, or MES referred to in any of paragraphs. 1 to 3, and a pharmaceutically acceptable carrier, diluent or excipient.

5. The method of obtaining the compounds of formula I

X - Y - NH - (CH2)r- G

specified in any of paragraphs.1 to 3, which includes (a) removal of simultaneous or sequential protective(s) group(s) R the corresponding compounds of formula II

(P)X - Y - NH - (CH2)r- G(P),

where (P)H - radicals X, which may contain one or more protective groups R are independently selected from aminosidine groups R of the compounds of formula I, in which X comprises the main NH-part of, and carboxyterminal group R of the compounds of the formula I, in which X includes carboxyl residue, and G(P) - radical of G, which may contain one or more independently selected aminosidine groups R; or (b) for compounds of formula I, in which

< / BR>
hydrogenation of the corresponding compounds of formula I, in which

< / BR>
then, when the desired salt of the compounds of formula I, the formation of salts with a pharmaceutically acceptable acid.

6. A method of inhibiting thrombin in a mammal, comprising introducing an effective dose of the compound, and the I in the inhibition of trombino.

 

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