UA77428C2 - Peptides arginals and a method for the treatment of disseminated intravascular coagulation - Google Patents

Abstract

The invention relates to disseminated intravascular coagulation. More particularly, the invention relates to medical intervention for disseminated intravascular coagulation. The invention provides new peptides arginals, new and better compounds and methods for the treatment of DIC. The compounds and methods according to the invention have inhibitory action on clot-bound thrombin and factor Xa and are also inhibitory against plasmin and plasminogen activators.

Description

Description of the invention

The invention relates to diffuse intravascular coagulation. More precisely, the invention relates to medical 2 intervention in cases of diffuse intravascular coagulation.

Disseminated intravascular coagulation (DI) is a secondary disease and can result from any of a number of primary diseases. | See Wax, Oizzetipaieai Ipigauhazscag Soadciaiop apa Keiaiedy Zupagotevs. SKS

Rgezz, Vosa Kap, (1983). Its characteristics include the systematic activation of blood coagulation, as a result of which fibrin is produced and deposited, which leads to microvascular thrombi in various organs and contributes to the development of multiple organ failure. In the work (Visk ei ai., SiIp. Arri Tygotroviz Netozyaviv and: 3-23 (1995) it is indicated that another characteristic of IS is systemically circulating plasmin, a general proteolytic enzyme that can biologically decompose various plasma proteins (factors, hormones, etc. . etc.) and can cleave fibrinogen/fibrin, providing fibrinogen/fibrin degradation products. These products disrupt hemostasis and lead to hemorrhage. 12 The most severe clinical form of BIS is characterized by extensive consumption of coagulation proteins, significant fibrin deposition, and bleeding.

Patients with trauma are at increased risk of SIS, especially if a large area of tissue is affected (in particular, the brain), in the case of sepsis and multiple organ failure. Head trauma is a particularly common occurrence

IS in infants and children due to the high content of thromboplastin in the brain and the proportionally increased ratio of head surface area to total body surface area.

Sepsis may occur in approximately 40,905 of the total trauma patients and is a significant primary cause of BIS in all patients. The clinical condition is worsened by secondary fibrinolysis, which leads to the formation of HOR (fibrinogen/fibrin breakdown products) or "O-dimers", which interfere with normal fibrin formation and platelet function. with

Deposition of fibrin in BIS can lead to dysfunction of other organs. OIS is the main cause of acute (3) renal failure and also contributes to multiorgan system failure. | on the contrary, damaged organs contribute to OIS.

Currently, the only recognized treatment for BIS is limited to attempts to alleviate the primary disorder.

Uncontrolled IS continues despite forms of therapy aimed at correcting the bleeding or co-thrombolytic problem. In some cases where significant bleeding occurs, Ha replacement therapy using fresh frozen plasma, plasma components (eg, antithrombin I), cryoprecipitate, and/or platelet concentrates may help as long as the underlying problem is controlled, but these C therapies are extremely expensive. The use of heparin in IS is highly controversial and is not universally applicable to patients with trauma-based problems. 3o Thus, there is a need for new and improved compounds and methods for the treatment of BIS. also, for example, de dopde ei ai, Ogydve 55:767-777 (1998) apa g emi ei ai, Tygotroviv apa Naetosiavziv 82: 695 (1999)).

The invention provides new and improved compounds and methods of treating BIS. Unexpectedly, it has been discovered that anticoagulant compounds that inhibit both free and clot-bound thrombin and factor Xa, as well as inhibit plasmin and plasminogen activators, may be useful in the treatment of IS. With p 50 In the first aspect, the invention provides a composition of a substance that includes peptidyl arginal of the formula (I) z» Xaa-xLB-Ago-H (II) where Xaa represents an alpha-substituted carboxylic acid residue of the formula (II) -i o-sn(B )-Co (I) - where O represents an alkyloxycarbonylamino group with 1-3 carbon atoms, a methylamino group or their hydroxyl group, and K represents a cycloalkylmethyl group with 7-9 carbon atoms or a cycloalkyl group with 5-7 carbon 5 atoms, or 1-adamantylmethyl group, and XLB represents I-proline or I-azetidine-2-carboxylic acid residue, and its acid addition salts formed with an organic or inorganic acid. c in optimal embodiments (such compounds can have the following structures: 1 (ethoxycarbonyl-O-cycloheptylalanyl-i!-prolyl-i-arginine aldehyde, Eos-O-cHra-Pho-Aga-H), or 2 (M-methyl -O-cycloheptylalanyl-i -prolyl-i -arginine aldehyde, M-Me-O-cHra-Pho-Ago-H), or C (O-cycloheptyllactyl-i -prolyl-i -arginine aldehyde, O-cHri-Rho -Aga-H), or 4 (M-methyl-O-cyclohexylglycyl-i!-azetidine-2-carbonyl-i -arginine aldehyde, M-Me-O-Spd-Age-Aga-H), which (F. correspond to formula (I), where Xaa represents alpha-substituted alkyl carboxylic acid residues of formula (II), where Kk represents cycloheptylmethyl and cyclohexyl groups, respectively, C) represents ethoxycarbonylamino-, methylamino- and hydroxyl groups, respectively, and Xrb represents I- proline and I-azetidinyl-2-carboxylic acid residue, respectively. b5

And you.

KE nei pek - Gee! 6)

I, I know, I don't know what's going on. - still eat Sho, still eat 5 ; In the second aspect, the invention provides a pharmaceutical composition that includes the anticoagulant peptidyl 50 arginal or its pharmaceutically acceptable salt according to the first aspect of the invention and a pharmaceutically acceptable carrier, filler or diluent.

IR e) In the third aspect, the invention provides a method of treating diffuse intravascular coagulation, which includes administering to a patient with diffuse intravascular coagulation the anticoagulant peptidyl arginal, which corresponds to the formula (I) about Xa-xLB-Ago-H (II) where Xa represents an alpha-substituted carboxylic acid residue of formula (II)

О-сН(В)-Оо (И) 60 where O represents an alkyloxycarbonylamino group with 1-3 carbon atoms, a methylamino group or a hydroxyl group, and K represents a cycloalkylmethyl group with 7-9 carbon atoms, or a 1-adamantylmethyl group or a cycloalkyl group with 5 -7 carbon atoms, and XV represents I-proline or azetidine-2-carboxylic acid residue, or its pharmaceutically acceptable acid additive salt. In optimal embodiments, such compounds may have the following structures: 1 (ethoxycarbonyl-O-cycloheptylalanyl-!-prolyl-i-arginine. aldehyde,

Eos-O-cHra-Pho-Aga-H), or 2 (M-methyl-O-cycloheptilalanyl-i-prolyl-i-arginine aldehyde,

M-Me-O-cHra-Pho-Aga-H), or C (O-cycloheptyllactyl-i -prolyl-I! -arginine aldehyde, O-cHri-Pho-Ago-H), or 4 (M-methyl- O-cyclohexylglycyl-i!-azetidine-1i-carbonyl-i-arginine-aldehyde, M-Me-O-Spd-Age-Aga-H), which correspond to the formula (I), where Haa represents alpha-substituted alkyl carboxylic acid residues of formula (III), where Kk represents cycloheptylmethyl and cyclohexyl groups, respectively, C) represents ethoxycarbonylamino-, methylamino- and hydroxyl groups, respectively, and ХББББ represents ! proline and i-azetidinyl-2-carboxylic acid residue, respectively. ше Кл г Ж vidne ПЕ шо и пт мк se se я вых, ke "J І І є я. - in "and PE "and 2 in GYA m even Bash a;" The invention relates to scattered intravascular coagulation. More precisely, the invention relates to medical intervention in cases of diffuse intravascular coagulation. The invention provides new and improved compounds and methods for the treatment of OIS. Compounds according to the invention have an inhibitory effect on both free and clot-bound thrombin and factor Xa, as well as on activators of plasmin and plasminogen.

The patents and publications cited by the author reflect prior art and are hereby incorporated by reference in their entirety. Any inconsistency between these patents and other publications and this specification shall be construed in favor of this specification.

In the first aspect, the invention provides a composition of matter that includes peptidyl arginal, which has

F) formula (I) o Khaa-xLB-Ago-H (Ii) where Khaa represents an alpha-substituted carboxylic acid residue of the formula (II) o-cn(B)-Co (I) where O represents an alkyloxycarbonylamino group with 1-3 atoms carbon, a methyl group or a hydroxyl b5 group, and K represents a cycloalkylmethyl group with 7-9 carbon atoms, a 1-adamantylmethyl group or a cycloalkyl group with 5-7 carbon atoms, and Xlb represents I-proline or I-azetidinyl-2-carboxylic acid residue, and its acid additive salts formed with an organic or inorganic acid.

A particularly preferred embodiment of this aspect of the invention corresponds to structure 1 (ethoxycarbonyl-O-cycloheptylalanyl-prolyl-barginine aldehyde, Eos-O-cHra-Pho-Aga-H): 9 where o.

B PE and r and 10 and she She. In y and y

Mn and her Bi

Another particularly preferred embodiment of this aspect of the invention corresponds to 79 structure 2 (M-methyl-O-cycloheptylalanyl-!-prolyl-i-arginine aldehyde, M-Me-O-cHra-Pho-Agao-H) : tsbytnvya she I viky piy 20 0 y -en y visn -Kh iy NU i D i Their Z ve z shi. Shchy: and di ery Kei sia ; (at)

Another particularly preferred embodiment of this aspect of the invention corresponds to the structure C (O-cycloheptyllactyl-i-prolyl-i!-arginine aldehyde, O-Hri-Rho-Aga-H): :ky U shcha. I and her iy No. and what sia ooo I - pa I shi M y -

I bbeeney well, schey m.

Another particularly preferred embodiment of this aspect of the invention corresponds to structure 4 (M-methyl-O-cyclohexylglycyl-!-azetidine-2-carbonyl-i-arginine aldehyde, M-Me-O-Spd- Age-Aga-N): with ;" Ben še še і і -I Z šcheya Y goes sZ dor o, I kre » | . and

Compounds according to formulas 1, 2, C and 4 are obtained, for example, by condensation of the M or O-substituted 2-residue acid component I-arginine with a lactam protected on the guanidino group by a benzyloxycarbonyl group, and reduction of the obtained tripeptide lactam to a protected tripeptide aldehyde , with the removal of the protective group from the guanidino group of arginine, and, if C) is methylamino or 22 hydroxyl in formula (I) - also from the terminal methylamino or hydroxyl group, and the isolation of the peptide

HF) derivative of formula (I) as its addition salt formed with an organic or inorganic acid.

Compounds represented by formula (I) are obtained and used in the form of acid additive salts due to their greater stability in salt form. In the acid addition salts of the compounds of formula (I) the activity is contained in the base and the acid is less important, although for therapeutic purposes preference is given to the use of 60 pharmaceutically acceptable acid addition salts. Examples of such suitable acids are (a) mineral acids: hydrochloric, hydrobromic, phosphoric, metaphosphoric and sulfuric acids, (b) organic acids: tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, palmic and arylsulfonic acids, for example, p-toluenesulfonic acid. The optimal acid additive salt is sulfate, especially hemisulfate. because Acid additive salts are obtained in a traditional way, for example, by neutralizing the free base form of the compound of formula (I) with an acid.

The two-residue acid component can be shown as U-Xaa-XBb, where Xaa represents an o-substituted carboxylic acid residue of the formula O-CH(K)-CO, where O is an alkoxycarbonylamino group with 1-3 carbon atoms, K is as defined above, and XLBb represents I-proline or I-azetidine-2-carboxylic acid residue. If Xaa, an o-substituted alkyl acid, is a 5-methylamino or o-hydroxy acid, i.e., C) represents a methylamino or hydroxyl group, the diresidue acid component can be shown as P-O-Xaa-ChrBB, where P represents an M-protecting group, such as a benzyloxycarbonyl (7) or tert-butoxycarbonyl (Boc) group or an 0-protecting group, preferably a tetrahydropyranyl (THP) group. 70 An acyl dipeptide, which is used as a starting material for compounds containing an o-amino or o-methylamino acid residue, is obtained by acylation of an o-amino acid with the appropriate ester of chloroformic acid to obtain an 1-3 carbon alkoxycarbonylamino acid and a benzyloxycarbonylamino acid, which are then combine with I-proline or I-azetidine-2-carboxylic acid to obtain O-Xaa-Xb and 7-aminoacyl Xbb, which is M-methylated to obtain the desired P-O-Xa-Xbb.

О-Хаа, which is required for connection with ХЬ, is optimally obtained by acetylation of racemic ОІ-Хаа compound, conversion of 0-acetylamino acid to its methyl ester and enzymatic decomposition of acetyl-ОІ-Хаа-ОМе racemic ester. Acetyl-O-Haa-Ote obtained in this way is then saponified and deacetylated, and then converted into the required M-protected ХО-amino acid.

The required ХО-о-hydroxy acid is optimally obtained from the corresponding О-y-amino acid. It is then converted to its O-protected form and coupled with XHO to give the desired P-XO-Xa-xXrB.

In a second aspect, the invention provides a pharmaceutical composition comprising peptidyl arginal according to the first aspect of the invention and a pharmaceutically acceptable carrier, excipient or diluent.

Pharmaceutical compositions include an effective amount of a compound of general formula (I) or a pharmaceutically acceptable salt thereof and known pharmaceutically acceptable carriers, excipients, diluents and/or other pharmaceutical excipients. at

The aforementioned carriers, diluents or fillers can be water, alcohols, gelatin, lactose, sucrose, starch, pectin, magnesium stearate, stearic acid, talc, various oils of animal or vegetable origin, as well as glycols, for example, propylene glycol or polyethylene glycol. co

Pharmaceutical excipients can be preservatives, various natural or synthetic emulsifiers, dispersants or wetting agents, dyes, flavorings, buffers, materials that promote disintegration, and other materials that improve the bioavailability of the active ingredient. what-

Pharmaceutical compositions of the invention are prepared as conventional compositions, such as oral compositions (which are administered through the mouth, such as tablets, capsules, powders, pills, dragees or granules), as well as parenteral and - compositions (medicines that are administered by bypassing the gastrointestinal system , such as injections, infusions, suppositories, patches or ointments).

In a third aspect, the invention provides a method of treating a patient with disseminated intravascular coagulation, comprising administering to a patient with disseminated intravascular coagulation a peptidyl arginal having the formula Xa-XbB-Ago-H (I), wherein Xa and XbBb are as defined above, or its pharmaceutically acceptable acid additive salt. Peptidyl arginals are also called derivatives of peptidylarginine aldehydes. According to this aspect of the invention, the invention provides a method of treating diffuse intravascular coagulation, which includes administering to an animal, including a human, peptidyl arginals according to the invention. In the method according to this aspect of the invention, a therapeutically effective amount of peptidyl arginal according to the invention is administered for a therapeutically effective period of time to an animal, including a human, having disseminated vascular coagulation in its body. In the best case, such administration is better to be carried out intravenously or - subcutaneously, in the best case - intravenously. Administration of therapeutic compositions is carried out using known procedures, in doses and for periods of time, effective for alleviating the symptoms or equivalent signs of BIS. When administered systemically, the therapeutic composition is optimally administered in a dose of 50 μM sufficient to achieve a blood level of peptidyl arginals from approximately 10 μM to approximately 100 μM. In the optimal version, the total dose is from about 0.1 mg to about 50 mg peptidyl arginal per kg i42) of body weight per day. It may be desirable to simultaneously or sequentially administer to a subject a therapeutically effective amount of one or more therapeutic compositions of the invention as a single therapeutic measure.

The following examples are presented for a more detailed explanation of some optimal embodiments of the invention and are not intended to limit the scope of the invention. Unless specifically stated otherwise, for

Gee! for the experiments presented below, human thrombin (3,000 MIN O/mg), human albumin, and human fibrinogen were obtained from bZidt Aiaghisn KA. (Budapest, Hungary), and the human factor Xa (8μg/)) - from Epgute de Kezeagsi I arogajugies (Swansea, Great Britain). ARTT reagent was obtained from KEAMAG. (Budapest, Hungary), and RT reagent, Zitriazip O, was purchased from OKOAMOM, TEKMIKA (Eppelheim, Germany). 60 Abbreviations for amino acids, peptides, substituents and reagents are used according to the IUPAC-IUB conventions. The following abbreviations are found in this application: Agud - I-arginine, Bos - tert-butoxy-carbonyl, B! - benzyl, Spd -

Y-cyclohexylglycine, OSNA - dicyclohexylamine, ONR - dihydropyran, Eos - ethoxycarbonyl, su - glycine, Me - methyl, MeRpe - M-methyl-I -phenylalanine, Mos - methoxycarbonyl, Pho - I-proline, PMA - p-nitroanilino, TRA - trifluoroacetic acid, TNR - tetrahydropyranyl, To - p-toluenesulfonyl, 7 - benzyloxycarbonyl, CT - 65 room temperature. Abbreviations of rare acids used in this application are as follows: Ada - adamantyl-i -alanine,

But - I-azetidine-2-carboxylic acid, M-Me-O-cHra - M-methyl-O-cycloheptylalanine, O-cHra -

p-cycloheptylalanine or (K)-2-amino-3-dicloheptylpropionic acid, O-Niia - ХО-cycloheptyllactic acid or (K)-2-hydroxy-3-cycloheptylpropionic acid.

The NO values specified in the examples were determined by thin-layer chromatography, using silica gel as an adsorbent (OS-AliYiIshoiep KieseYide! 60 P254, Megsk, Darmstadt), in the development systems presented below.

The numbers of the applied systems are given in parentheses after the GI abbreviation. 1. Ethyl acetate 2. Ethyl acetate - p-hexane (1:4)

C. Ethyl acetate - p-hexane (1:1) 70 4. Ethyl acetate - cyclohexane (15:85) 5. Chloroform - acetone (95:5) 6. Ethyl acetate - pyridine - acetic acid - water (960:20:6: 11) 7. Ethyl acetate - pyridine - acetic acid - water (480:20:6:11) 8. Ethyl acetate - pyridine - acetic acid - water (240:20:6:11) 9. Ethyl acetate - pyridine - acetic acid - water (120:20:6:11) 10. Ethyl acetate - pyridine - acetic acid - water (90:20:6:11) 11. Ethyl acetate - pyridine - acetic acid - water (60:20:6:11) 12. Ethyl acetate - pyridine - acetic acid - water (45:20:6:11) 13. Ethyl acetate - pyridine - acetic acid - water (30:20:6:11) The loading coefficients (K) specified in the examples were determined using the device " Rpagtasia I. KV Apaiuiisa! NRI-S Zuziet. To" as follows:

Column: GISpgozrpeg KR-18:12μm 240khiAmMM

Column temperature: ambient

Eluents: Solvent A, 0.195 TEA/water, Solvent B, 0.190 TEA/acetonitrile

Gradient profile: O-»15 min. 30- 26095 V, then isocratic 60905 V. p

Solvent flow rate: ml/min.

Detector: UV monitor I KV 2141; wavelength: 214 nm. and)

Injector: Kpeodupe 7125. Loop: 10Omkl.

Pumps: type 2 I KV 2148. Regulation system: I KV NRI S Mapadeg.

Sample concentration: 1mg/ml in Solvent A, injection volume 25μl. with

Analysis time 4 Okhv.

Acyl-arginine aldehydes are present in balanced structures, i.e. in the form of aldehyde, aldehyde hydrate and with two forms of aminocyclol. During NRI C-analysis, aldehyde hydrate and one or both forms of aminocyclol «fr are detected as two or three separate peaks. Acyl-arginine aldehydes, described in the examples, are characterized by two or three indicators of Kk. -

Mass spectrometry. RAV measurements of positive ionization were carried out in the Rippidap MAT 8430 device. The samples were dissolved in t-nitrobenzyl alcohol (MVA) and introduced directly into the ion source. In the spectrum of peptidyl-arginine aldehydes, an additional molecular ion of an additional compound formed from MVA was detected: (MANI and (MANAMVA/)". In the examples, data on EAV spectra were indicated accordingly. EI measurements of positive ionization were carried out in the device of MO Otsaigo (Rizopv). Samples was dissolved in a mixture of acetonitrile-water (1:1), which contained 190 (v/v) of formic acid and was introduced with a sample loop of 1 Oml into the ion source with a flow rate of 15-25 ml/min. and Example 1 "" Synthesis of hemisulphatuethoxycarbonyl-O-cycloheptylalanyl-i-prolyl-i-arginine aldehyde

Stage 1: Ethoxycarbonyl-O-cycloheptylalanyl-i-prolyl-M-benzyloxycarbonyl-i-arginine lactam 7.85g (20.1mM) tert-butyloxycarbonyl-O-benzyloxycarbonyl-i! -arginine lactam MVa|ie72 ei ai., 9. Med. - And Speth. 33.1729 (1990) was suspended in 20 ml of chloroform, then 20 ml of ethyl acetate saturated with NH gas (0.11-0.15 g/ml) was added while stirring and cooling with ice. Cleavage of the Bos group was monitored by thin-layer chromatography (CL (11) - 0.5 (free compound); 1.0 (Boc-compound)). Before the completion of the reaction, the suspension was diluted with 40 ml of diethyl ether, the formed crystalline mass was filtered, washed with 1 ml of acetone 7 50 and 1 ml of diethyl ether and dried under reduced pressure over KOH. Obtained as a result

Mo. benzyloxycarbonyl-I-arginine lactam hydrochloride was dissolved in 20 ml of dimethylformamide, cooled to -2090°C and added to the anhydride with further stirring. 7.12 g (20.1 mM) of ethoxycarbonyl-O-cycloheptylalanyl-I-proline (Example 1, Step U) was dissolved in 20 Oml of dimethylformamide, cooled to -159C7, then 2.2 ml (20.1 mM) of 29 M-methyl -morpholine and 2.65 ml (20.1 mM) of isobutyl chloroformate. After 10 minutes of stirring, it was added

HF) the aforementioned solution in dimethylformamide of M-benzyloxycarbonyl-I-arginine lactam, then triethylamine in an amount sufficient to bring the pH of the reaction mixture to 8 (approximately 2.8 ml). The reaction mixture was stirred at -109C for 30 minutes, then at 09 for one hour. After that, the salts were filtered off and the filtrate was diluted with 100 ml of ethyl acetate. The resulting solution was washed with 33 x 25 ml of water, 1 ml of 1M 60 KNZO, and 3 x 10 ml of water, dried over anhydrous Ma»5O, and evaporated at 2.0-2.5 kPa. The obtained product was subjected to chromatography on a column with silica gel, using 200 g of KiezeYde! 60 (0.040-0.063 mm) as an adsorbent and ethyl acetate as an eluent. Fractions that contained only the pure MK product; (1) - 0.60), combined and evaporated at 2.0-2.5 kPa. The residue after evaporation was crystallized from diisopropyl ether.

Yield 10.84g (86.1905), K, (1) - 0.55-0.65. 65 EAV mass spectrum (627 MANI") confirmed the expected structure.

Step 2: Ethoxycarbonyl-O-cycloheptylalanyl-I-prolyl-MU-benzyloxycarbonyl-I-arginine. 8.02 g (12.8 mM) ethoxycarbonyl-O-cycloheptylalanyl-I-prolyl-MU-benzyloxycarbonyl-I-arginine lactam aldehyde (Example 1, Step 1) was dissolved in 15 ml of tetrahydrofuran, and then with stirring and at a temperature that did not exceed -502С, a solution of 3,6MM HyAIN, dissolved in tetrahydrofuran, was added. The recovery process was monitored by thin-layer chromatography (solvent 7 as a developing solvent) and, if necessary, another portion of HyAIN was added. 0.5M KNBZO) was added dropwise to this reaction mixture with constant stirring and cooling until pH 3 was reached, then Zbml of water. The resulting solution was extracted with 2x15ml of hexane, then with 3xX2O0ml of dichloromethane. The extracts in dichloromethane were combined, washed with 70 ml of water, 15 ml of cold 595 sodium bicarbonate solution and again with 15 ml of water, dried over anhydrous

Ma»5zO, and evaporated at 2.0-2.5 kPa. The residue after evaporation was treated with isopropyl ether, filtered and dried under reduced pressure.

Yield 7.08g (8890), K., (8) - 0.40-0.50.

EAV mass spectrum (629 (M.-NI", 782 (MAN-MVAG)) confirmed the expected structure.

Step 3: Hemisulfate ethoxycarbonyl-O-cycloheptylalanyl-i! - spilled-and! -arginine aldehyde 6.91 g (11.0 mM) ethoxycarbonyl-O-cycloheptylalanyl-! -prolyl-y -benzyloxycarbonyl-y! -arginine aldehyde (Example 1, Stage 2) was dissolved in 85 ml of ethanol and 11.25 ml of 0.5 M sulfuric acid was added, then 0.7 g of Ra-s catalyst suspended in 14 ml of water and the mixture was hydrogenated at approximately 102С. The reaction process was monitored by thin-layer chromatography. After completion of the reaction (approximately 15 minutes), the catalyst was filtered and the filtrate was concentrated to approximately 7-9 ml at 2.0-2.5 xPa. The residue was diluted with 80 ml of water, extracted with 4 x 15 ml of dichloromethane and the aqueous solution was left to stand at 20-222C for 24 hours.

The solution was extracted again with 33 x 15 ml of dichloromethane and the pH was adjusted to 3.5 with the ion exchange resin Juozeh As 1-X8 (HO), then the solution was subjected to lyophilization.

Output 4.90g (8296). Bh (11) - 0.35-0.45. (91079 2 -77.6o (s-1.018; water). see

NRIS: K - 1.695 and 2.328. at

The EAV mass spectrum (495 MANI", 648 |M-N-MVAI") confirmed the expected structure.

Synthesis of raw materials:

Ethoxycarbonyl-O-cycloheptylalanyl-! -proline co

Stage A; 1-cycloheptylacetyl-2,5-dimethylpyrazole 58.6 mg (3/5 mM) of cycloheptylacetic acid | Slesp., Spet., Sottyp., 55: 1278-1289 s (1990)) was dissolved in 375 ml of tetrahydrofuran, cooled to -152C, then, with stirring, 41.3 ml (375 mM) of M-methylmorpholine, 49.50 ml (375 mM ) of isobutylchloroformate and after 10 minutes of stirring at -10907, a solution of 37.85g (393.75MM) of 3,5-dimethylpyrazole in 300ml of tetrahydrofuran at -1096 was added. -

Stirring was continued at -102C for 30 minutes, at 02C for an hour and at room temperature for 3 hours. After that, the salts were filtered off, the filtrate was evaporated under reduced pressure, and the residue was dissolved in 900 ml of ethyl acetate. The resulting solution was successively washed with 100 ml of 1 M Mahon, 9 ml of water, 100 ml of 1 M CARRIER and water until neutral. (The main washes were combined and acidified to obtain "-5.8g, 37.1MM, cycloheptylacetic acid). The ethyl acetate solution was dried over anhydrous Ma»5O, then evaporated at 2.0-2.5 kPa. The resulting oily product was treated as 340 mM - s 1-cycloheptylacetyl-2,5-dimethylpyrazole and used for the next step without further purification. "with K(2) - 0.8-0.9 (acidity: 0.3-0.4). i Stage B: 1-cycloheptylacetaldehyde

12.83g (338mMM) of GiIAINul was added to ZB5BOml of tetrahydrofuran, cooled to -309С7. After that, while stirring, a solution of the oily product (Example 1, Stage A) in 250 ml of cold tetrahydrofuran at -252C was added dropwise. The reaction was monitored by TI C. If necessary, 30-40 ml of a solution of -1 PAN in tetrahydrofuran (Zg/1O0Oml) was added. After completion of the recovery, the cold mixture was acidified with 6M NS! and diluted with ethyl acetate (50O0ml). The aqueous phase was extracted with ethyl acetate, the organic solutions were combined, - washed with water until neutral, dried over anhydrous Ma»5O), and then evaporated at 2.0-2.5 kPa. d) 20 The resulting oily product was crude 1-cycloheptylacetaldehyde contaminated with 2,5-dimethylpyrazole (OMP). K, (2) - 0.7-0.8 (OT. PL.: 0.25-0.35). so To the resulting oily product (62.6g) dissolved in 3500ml of methanol, a solution of 36.4g of ManbSO3 in 70ml of water was added with stirring. The reaction mixture was kept in the refrigerator until the next day.

The precipitated material was filtered off, washed with a cold mixture of 10 ml of methanol and 7 ml of water, then with 255 ml of diethyl ether, and dried. The solid material is sodium hydrosulfite adduct (73.87g, 302.38MM),

GF! K, (14) - 0.55-0.65), which was added to a mixture of 45O0ml of methylene chloride and 450ml of water, which contained 47.7g (450MM)

Ma»SO». The reaction mixture was stirred until the next day. The two phases were separated, the organic phase was washed with methylene chloride (2x1O0Oml). The combined organic layers were washed with water, dried over anhydrous

Ma»5zO,, was then evaporated at 2.0-2.5 kPa. The resulting oily product (36.97 g, 263.64 mmol) was 60 pure 1-cycloheptyl-acetaldehyde, which was used directly for the next reaction. K; (2) - 0.7-0.8.

Step C: 5-cycloheptylmethylhydantoin

To a solution of aldehyde (Example 1, Stage B) in 5095 aqueous ethanol (857ml; 3.25ml/mM) at 50-55905 with stirring was added 15.5g (290mM; 1, Tequiv.) of ammonium chloride, 27.87g (659mM; 2.5 equiv.) of ammonium carbonate and 18.9 g (290 mM; 1.Eq.) of potassium cyanide, and stirring was continued at 502C for 48 hours. The precipitated material was filtered, washed with 5095 ethanol (10 Oml) and dried in a vacuum desiccator. In the first batch, 4,226 bg (206.97 mm) of material were obtained. Ethanol was distilled off from the mother liquor, the residue was extracted with ethyl acetate (1x25Oml and 2x100ml), the organic solutions were combined, washed with water (3x5Oml), dried over anhydrous Ma»5O), then evaporated at 2.0-2.5 kPa. The residue was triturated with isopropyl ether, filtered and dried. In the second batch, 3.6 bg (17.12 mm) of material were obtained; the total yield was 45.86 g (218 mM, 82.590) of 5-cycloheptylmethylhydantoin. Total area: 240,990. MK, (6) - 0.73-0.78.

Analysis for C.44Ni8M2O" (210,28). Calculated: So - 62.83; H 90 - 8.63; Mo - 13.32. Found: Soo - 63.09; N

Pho - 8.67; M 90 - 13.25. 70 Step 0: 0-cycloheptylalanine hydrochloride 87g (1.55M) of KOH was dissolved in 435ml of p-butanol with stirring and heating and 45.71g (217 4mMM) of 5-cycloheptylmethylhydantoin (Example 1, Step C) was added. The solution thus obtained was dephlegmated for 72 hours, then diluted with water and evaporated under reduced pressure. The residue was dissolved in 220 ml of water, acidified to pH 2 using 130 ml of HCl and kept in the refrigerator until the next day. 75 The precipitated material was filtered, washed with 50 ml of water and dried in a vacuum desiccator. The thus obtained product (106.5 g) was considered as 217 mM bi-cycloheptylalanine and used for further reactions. K, (11) - 0.2-0.3.

Step E: 0-dcycloheptylalanine methyl ester hydrochloride 23.65 ml (325.25 mM) of ZOSI" was added dropwise with stirring to 217 ml of methanol at a temperature from des to -59205. After that, 0-cycloheptylalanine (217 mM from Example 1, Step 2) was added and stirred for 24 hours. After the transformation, TI C CC was carried out, (11) - 0.75-0.85 (ester), (0.3-0.4 (acid). If an unreacted amino acid was detected, the reaction mixture was cooled to -52C, 11 was added dropwise ,8 ml of ZOSI"; and the reaction mixture was stirred for another 24 hours. After that, the undissolved salts were filtered off, washed with methanol (2 x 50 ml) and the combined methanol solutions were evaporated. The residue was re-dissolved in methanol and He was evaporated again. | finally, the residue was triturated with diisopropyl ether, filtered , washed (5) with isopropyl ether and dried in a vacuum desiccator over KOH and PoO5. 33.68g (142.85MM) of 0-cycloheptylalanine methyl ester hydrochloride were obtained. K, (11) - 0.5-0.6. T Square 95.4-96.5960.

Step E: Acetyl-OiI-dcycloheptylalanine methyl ester

Acetic anhydride (162 ml, 171.43 mM) was added dropwise to a solution of 33.68 g (142.85 mM) of 0-cycloheptylalanine methyl ester hydrochloride (Example 1, Step E) in 0.140 ml of pyridine over the course of an hour with stirring and cooling in an ice bath. water The reaction mixture was stirred for 24 hours at ambient temperature, then evaporated under reduced pressure. The residue was dissolved in 300 ml of ethyl acetate, washed with 1 M KNSO, (3x500 ml) and water (3x500 ml), dried over anhydrous Ma»5O), and then evaporated at a temperature of 2.0-2.5 kPa. The resulting oily product was triturated with diisopropyl ether to a solid material, which was filtered off, washed with diisopropyl ether, then water, and dried in a desiccator. and -

24.3 bg (100.94 mM, 70.495) of methyl acetyl-Ol-dicloheptylalanine (acetyl-Ol-ester) were obtained. EC, (1) - 0.55-0.65. T. pl.: 69-7196.

Analysis for C43NozMO»z (241,332). Calculated: C bo - 64.70; H 95 - 9.61; MO - 5.80. It was found: C bo - 64.75; "

N bo - 9.76; M 9 - 5.85.

Step o: Acetyl-O-cycloheptylalanine methyl ester (enzymatic dissolution of o) with acetyl-OI-dicloheptylalanine methyl ester) "» To a solution of 8.69 g (ZbmM) of acetyl-OiI-dicloheptylalanine methyl ester, acetyl-OI -ester (Example 1 , " Stage R), in Zbml of toluene, added 72 ml of water and Zbmg Zibiyvip Sagizregd (Protease, type MI, Zidta).

Enzymatic hydrolysis of the I-enantiomer, acetyl-I-ester, which occurred at pH 7.0, was supported by an autotitrator filled with 3M MaonH. After stopping consumption of Mahon (5.vml, 17.4MM), the reaction mixture was diluted with Zbml toluene and the two layers were separated. The aqueous phase was washed with 2 x 30 ml of toluene. The combined -I solutions in toluene contained acetyl-O-ester, and the combined aqueous solutions contained the sodium salt of acetyl-II -acid.

After drying over anhydrous Ma 250, the combined solutions in toluene were evaporated under reduced pressure to obtain 3.8 g (15.75 mM) of acetyl-O-ester, K (1) - 0.55-0.65, which was used directly

Mo 70 at the next stage.

The combined aqueous solutions were acidified and extracted with 300 ml of ethyl acetate. The combined solutions in ethyl acetate were washed with water, dried over anhydrous NaCl, and evaporated under reduced pressure to obtain 4.0 g (17.6 mM) of acetyl-! - acids. K, (7) - 0.38-0.42.

A similar method of preparation, starting from 9.65 g (40 mM) of acetyl-O-ester (Example 1, Step E) gave as an output the A,bg (19.06 mM) of acetyl-O-ester and 4.44 g (19.55 mM) acetyl-II -acids.

GF! Stage H: 7.24 g of O-cycloheptylalanine hydrochloride (SOMM) of acetyl-O-ester (Example 1, Stage b) was suspended in 120 ml of 6M NCI and subjected to dephlegmation for 3 hours. The free amino acid was separated in the form of crystals. The reaction mixture was cooled, kept in the refrigerator until the next day, filtered, washed with cold water and ether, then dried in a vacuum desiccator. 5.9 g (26.69 mM, 8995) of O-cycloheptylalanine hydrochloride was obtained. EC, (12) - 0.10-0.15. (91029 - -119 (s-0.4; 1M NS).

Analysis for С 10Н419МО». NSII (221,728). Calculated: C 95 - 54.17; H 95 - 9.09; M 95 - 6.32; SI Fo - 15.99.

It was revealed: C bo - 54.27; H Fo - 9.27; M 90 - 6.30; SI Fo - 16.2.

Stage I: Ethoxycarbonyl-O-cycloheptylalanine bo To a solution of 4.43 g (20 mM) O-cycloheptylalanine hydrochloride (Example 1, Step H) in 20 ml of dimethylformamide was added 5.bml (40 mM) of triethylamine and 3.95 g (21 mM) (M-hydroxysuccinimidyl )-ethyl carbonate." After stirring at room temperature for 3 hours, the reaction mixture was evaporated, the residue, dissolved in 40 ml of ethyl acetate, was washed with 2 x 30 ml of 1 M KNBSO, and with water until neutral. After that, the organic layer was dried over anhydrous Ma»5O), then evaporated at 2.0-2.5 kPa. The resulting oily product (4.47 g, 717 mM) was ethoxycarbonyl-O-cycloheptylalanine |K, (7) - 0.85-0.90), which was used directly in the next step. (91929 - 6.32 (c-1, methanol). "Preparation of (M-hydroxysuccinimidyl)-ethyl carbonate 11.5 g (100 mM) of M-hydroxysuccinimide was dissolved in 100 ml of tetrahydrofuran, cooled to -109C, then 70 with stirring, 15 .4 ml (110 mM) of triethylamine and 10.45 ml (110 mM) of ethyl chlorocarbonate. After 2 hours, stir after heating at room temperature, the mixture was filtered and the filtrate was evaporated under reduced pressure. The oily residue crystallized after cooling. The crystalline material was suspended in petroleum ether, filtered and dried in a vacuum desiccator. Output 12.78g (68.390). T. pl. 39.4-39.726.

Analysis for SUNoMO" (187,15). Calculated: C, 44.92; N, 4.85; M, 7.48.

It was revealed: C Fo - 44.67; NOyu - 4.81; MO - 7.27.

Step y: Ethoxycarbonyl-O-cycloheptylalanyl-! -proline

A solution of ethoxycarbonyl-O-cycloheptylalanine (717 mM, Example 1, Step I) in 17 ml of TNE was combined with 374 g (20 mM) of M-hydroxysuccinimide, cooled to 1092 and combined with a solution of 4.12 g (20 mol) of 1,8-dicyclohexylcarbodiimide in approximately 20 ml of TNE . The mixture was stirred for about B5h at 229C, after which the formation of the active ester was considered complete according to the TI C data.

II-proline (1.95 g, 17 mM) was added to the stirred reaction mixture followed by the addition of 2.3 mL (17 mM) of triethylamine. The reaction mixture was stirred at 222C for about 15 hours, after which consumption of the active ester was considered complete according to the data of TI C. The reaction mixture was filtered, the sediment on the filter was washed with 29 1 bml of TNE and the filtrate was evaporated. The residue was dissolved in 30 ml of ethyl acetate and 30 ml of water. The aqueous phase was washed (9 2 x 20 ml of ethyl acetate, acidified with 20 ml of 1 M KNZBO4 and extracted with 3 x 20 ml of ethyl acetate. The combined solutions in ethyl acetate were washed with water until neutral, dried over anhydrous NaCl), then evaporated at 2.0-2.5 kPa. After trituration with diisopropyl ether, the residue crystallized. This suspension of crystals was cooled in a refrigerator, filtered with petroleum ether and dried in a vacuum desiccator. so 3o 4.2 g (11.85 MM, 70905) of ethoxycarbonyl-O-cycloheptylalanyl-I-proline were obtained. K, (7) - 0.45-0.55. Ge

Analysis for C 8HzoM2O) (354.45). Calculated: C, 60.99; N, 8.53; M, 7.90. It was revealed: C bo - 60.14; H 95 - 8.555 MU - 7.38. WITH

EAV mass spectrum (355 MANI") confirmed the expected structure. -

Example 2 M

Synthesis of M-methyl-O-cycloheptylalanyl-! -prolyl-and -arginine aldehyde sulfate

Stage 1: Benzyloxycarbonyl-M-methyl-O-cycloheptylalant-I -prolt-MU-benzyl-oxycarbonyl-I! -arginine lactam 3.93g (10MM) tert-butyloxycarbonyl-M-benzyloxycarbonyl-I -arginine lactam ((Va|iv2 ei ai, U. Med.

Spent 33, 1729 (1990) was suspended in 1 Oml of chloroform, then 1 Oml of ethyl acetate saturated with NCI gas (0.11-0.15 g/ml) was added while stirring and cooling with ice. Cleavage of the Bos group was monitored for 8 s by thin-layer IR chromatography; (11) - 0.5 (free compound); 1.0 (Vos compound)). Before the completion of the reaction, the suspension was diluted with 20 ml of diethyl ether, the formed crystalline mass was filtered, washed with 1 ml of acetone and 5 ml of diethyl ether and dried under reduced pressure over KOH. to -2090 | was added to the anhydride with further stirring. - and 4.32 g (10 mM) of benzyloxycarbonyl-M-methyl-O-cycloheptylalanyl-!-proline (Example 2, Step C) was dissolved in -1 10 ml of dimethylformamide, cooled to -159С , then with stirring, 1.12 ml (10.1 mm) was added

of M-methyl-morpholine and 1.3 ml (10.1 mM) of isobutyl chloroformate. After 10 minutes of stirring, the above-mentioned solution in dimethylformamide of MO-benzyloxycarbonyl-I-arginine lactam was added, then triethylamine in an amount sufficient to bring the pH of the reaction mixture to 8 (about 1.4 ml was required). The reaction mixture was stirred at -1092C7 for 30 minutes, then at 09C for one hour. After that, the salt was filtered off and the filtrate was diluted with 100 ml of ethyl acetate. The resulting solution was washed with 3x15 ml of water, 1 ml of 1M KNBO, and 3x5 ml of water, dried over anhydrous Ma»35O, and evaporated at 2.0-2.5 kPa. The obtained product was subjected to chromatography on a column with silica gel, using 100 g of KiezeiYide! 60 (0.040-0.063 mm) as 29 adsorbent and ethyl acetate as eluent. The fractions that contained only the pure product ((K ; (1) - 0.70) were combined and (FI evaporated at 2.0-2.5 kPa. The residue after evaporation was crystallized from diisopropyl ether. yuyu Yield b,Og ( 85965), K (1) - 0.65-0.75.

EAV mass spectrum (703 M--NI") confirmed the expected structure. in Step 2: Benzyloxycarbonyl-M-methyl-O-cycloheptylalanyl-iI-prolyl-M O-benzyloxycarbonyl-i-arginine aldehyde 5.2g (8MM) benzyloxycarbonyl -M-methyl-O-cycloheptylalanyl-iI-prolyl-MU-benzyloxycarbonyl-i-arginine lactam (Example 2, Stage 1) was dissolved in 1 Oml of tetrahydrofuran, and then, with stirring and at a temperature that did not exceed -50 C, the solution was added 2.25 mM HyAIN; dissolved in tetrahydrofuran. Process 65 The recovery was monitored by thin-layer chromatography (solvent 7 as the developing solvent) and, if necessary, another portion of HyAIN was added. To this reaction mixture, 0.5 M KNBO was added dropwise, with a constant with stirring and cooling until pH 3 is reached, then 25 ml of water. The resulting solution was extracted with 2 x 10 ml of hexane, then with 3 x 5 ml of dichloromethane. The extracts from dichloromethane were combined, washed with 3 x 15 ml of water, 15 ml of cold 595 solution of ManHsSoO" and again with 15 ml of water, dried over water

Ma»5zO, and evaporated at 2.0-2.5 kPa. The residue after evaporation was treated with isopropyl ether, filtered and dried under reduced pressure.

Yield 4.95 g (8890), K, (1) - 0.20-0.25.

The EAV mass spectrum (705 MANI", 858 |IMANAMVA/") confirmed the expected structure.

Step 3: M-methyl-O-cycloheptylalanyl-i! -spill-! -arginine aldehyde sulfate 70 4.6g (6.5MM) of benzyloxycarbonyl-M-methyl-O-cycloheptylalanyl-I-prolyl-MU-benzyloxycarbonyl-i-arginine aldehyde (Example 2, Stage 2) was dissolved in b5ml of ethanol and added 13, 5 ml of 0.5 M sulfuric acid, then 0.4 g

Ra-C of the catalyst suspended in 100 ml of water, and the mixture was hydrogenated at approximately 102. The reaction process was monitored by thin-layer chromatography. After completion of the reaction (approximately 15 minutes), the catalyst was filtered and the filtrate was concentrated to approximately 5-7 ml at 2.0-2.5 kPa. The residue was diluted with 5Oml of 75% water, extracted with 4x10ml of dichloromethane and the aqueous solution was left to stand at 20-222C for 24 hours.

The solution was extracted again with 3x1.0ml of dichloromethane and the pH was adjusted to 3.5 with the ion-exchange resin Juoueh AC 1-X8 (HO), then the solution was subjected to lyophilization.

Output 2.85g (8296). Bh (9) - 0.35-0.45. (41079 - -79.6e (s-1; water).

EAV mass spectrum (437 MANI", 590 IMANAMVAIT) confirmed the expected structure.

Synthesis of raw materials:

Benzyloxycarbonyl-M-methyl-O-cycloheptylalanyl-! -proline

Stage A: Synthesis of M-benzyloxycarbonyl-O-cycloheptylalanine

O-Cycloheptylalanine hydrochloride (Example 1, Step H) (11.09g, 5OMM) was combined with 40ml of TNE and 40ml of water at 02. The stirred mixture was adjusted to approximately pH 10 by adding 5M Mahon solution. Benzyl chloroformate (8.22 mL, 55.4 mM) was added to the reaction mixture c, while maintaining a temperature of about 32 and a pH (o) of about 10, adding 5M Mahon if necessary. After the addition of benzyl chloroformate was completed, the reaction mixture was stirred for 1 hour at 02C and the pH was maintained at approximately 10. Stirring was continued until the next day at RT and the completion of the reaction was checked by TIS (7). If necessary, a certain amount of benzyl chloroformate (1-2 x 07 bml, 5 mM) was added to the reaction mixture, while maintaining a temperature of about 32C and a pH of about 10, adding 5M of Mahon. I-butyl methyl ether (25 ml) was added and the stirred mixture was heated to 222C. The aqueous phase was separated and washed with a second 25 ml portion of -«f

I-butyl methyl ether. The content of the organic phase was checked using TI C and, if necessary, the organic phase was subjected to reverse extraction with 25 ml of water. The aqueous phases and 25 ml of ethyl acetate were combined and adjusted to a pH of 2 with concentrated HCl. The phases were separated and the aqueous phase was extracted with a second 25 ml portion of ethyl acetate.

The combined organic phase was washed with 20 ml of 1 M NaCl and 2 x 30 ml of water, dried over anhydrous NaCl, and evaporated at 2.0-2.5 kPa. The residue after evaporation was dissolved in 45 ml of TNE and this solution of benzyloxycarbonyl-O-cycloheptylalanine was kept for use in the next step without further purification. "

Step B: Benzyloxycarbonyl-O-cycloheptylalanyl-! -proline A solution in TEA of benzyloxycarbonyl-O-cycloheptylalanine obtained in Example 2, Step A, was combined with 5.9 g (5124 mM) and M-hydroxysuccinimide, cooled to 1096; and combined with a solution of 11 g (53.3 MM) of 1,3-dicyclohexylcarbodiimide in approximately 25 ml of TNE. The mixture was stirred for approximately 4.5 hours at 229C, after which the formation of the active ester was considered complete according to TI C data.

II-proline (5.9 g, 51.24 mM) was added to the stirred reaction mixture, followed by the addition of 7.2 ml (51.24 mM) of triethylamine. The reaction mixture was stirred at 222C for about 15 hours, after which consumption of the -I active ester was considered complete according to the data of TI C. The reaction mixture was filtered, the sediment on the filter was washed with 25 ml of TNE and the filtrate was evaporated. The residue was dissolved in 500 ml of ethyl acetate and 500 ml of water. The aqueous phase was washed with 2 x 20 ml of ethyl acetate, acidified with 200 ml of 1 M KHBO,) and extracted with 3 x 40 ml of ethyl acetate. d) 250 The combined solutions in ethyl acetate were washed with water until neutral, dried over anhydrous Ma»5O, then evaporated at 2.0-2.5 kPa. The residue, benzyloxycarbonyl-O-cycloheptylalanyl-I-proline, was dissolved. in so bOml TNE and used in the next step without further purification.

Step C: Benzyloxycarbonyl-M-methyl-O-cycloheptylalanyl-i-proline

Iodomethane (17.95 mL, 288 mM) was added to a solution in TNE of benzyloxycarbonyl-O-cycloheptylalanyl-I-proline 2o from Example 2, Step B. This solution was cooled to 82C and transferred together with 20 mL of TNE for washing to

HF) of a stirred aqueous mixture of 4.75 g (119 mM) of sodium hydride 6095 in 35 ml of TNE, while simultaneously maintaining the temperature below 132C. The reaction mixture was stirred at 112 for 24 hours. Excess sodium hydride was decomposed by carefully adding 1.bml of water to the reaction mixture, while maintaining the temperature below 139 and controlling foaming. The quenched reaction mixture was stirred for about 20 min at 22 2C, and then 60 was concentrated to about 40 ml under reduced pressure at a temperature below 302. Water (70 ml) was added to the residue, followed by the addition of 30 ml of I-butyl methyl ether. The phases were separated and the aqueous phase was washed again with 30 ml of i-butyl methyl ether. The aqueous phase was combined with 40 ml of ethyl acetate and adjusted to pH 2.2 with 3M sulfuric acid solution. The phases were separated and the aqueous phase was back-extracted with 40 ml of ethyl acetate. The combined organic phase was washed with 7 Oml of 595 sodium thiosulfate solution. Phases 65 were separated and the organic phase was concentrated to a small volume under vacuum conditions (33-45kPa), while maintaining the temperature below 5020. The residue was combined with 18.bml of TNE and 100ml of water and adjusted to pH 85 with cyclohexylamine. The resulting aqueous mixture was concentrated to 100 ml under reduced pressure (9-33 kPa) at 25-559C, brought to 259C, diluted with 71.5 ml of water and stirred for approximately 10.5 hours. The aqueous mixture was filtered, washed with water and dried in air at 4597 to obtain 16.72 g of benzyloxycarbonyl-M-methyl-O-cycloheptylalanyl-y-proline cyclohexylamine salt (6390 yield from p-cycloheptylalanine). EC (8) - 0.55-0.65.

Example C

Synthesis of Y-cycloheptyllactyl-i-prolyl-i-arginine aldehyde hemisulfate

Stage 1: Tetrahydropyranyl-O-cycloheptyllactyl-i-prolyl-MU-benzyloxycarbonyl-i-arginine lactam 5.08 g (13 mM) tert-butyloxycarbonyl-MU-benzyloxycarbonyl-i-arginine lactam (Va|shv2 ei ai, u. Mei Snet . 33, 1729 (1990)| suspended in 13ml of chloroform, then 13ml of ethyl acetate, saturated with HCI gas (0.11-0.15g/ml) was added while stirring and cooling with ice. Cleavage of the Vos group was monitored by thin-layer IR chromatography; (11) - 0.5 (free compound); 1.0 (Voc-compound)). Until the end of the reaction, suspension 75 was diluted with 25 ml of diethyl ether, the formed crystalline mass was filtered, washed with 7 ml of acetone and 7 ml of diethyl ether and dried under reduced pressure over potassium hydroxide. Obtained as a result

Mo. benzyloxycarbonyl-arginine lactam hydrochloride was dissolved in 133 ml of dimethylformamide, cooled to -2090 and added to the anhydride with further stirring.

A solution of tetrahydropyranyl-Y-cycloheptyllactyl-I-proline triethylammonium salt obtained in Step 1

Example C (12 mM) was cooled to -202C, then 1.bml (12 mM) of isobutyl chloroformate was added with stirring. After 10 minutes of stirring, the above solution in dimethylformamide was added

Mo. benzyloxycarbonyl- and -arginine lactam, then triethylamine in an amount sufficient to bring the pH of the reaction mixture to 8 (about 1.8 ml was required). The reaction mixture was stirred at -1092C for 30 minutes, then at 02 for one hour. After that, the salts were filtered off and the filtrate was diluted with 5 ml of ethyl acetate. The resulting solution was washed with 3x25ml of water, 7ml of 1M potassium hydrosulfate and 3x/7ml of i) water, dried over anhydrous Ma»5O, and evaporated at 2.0-2.5 kPa. The obtained product was subjected to chromatography on a column with silica gel, using 130 g of KiezeYde! 60 (0.040-0.063 mm) as an adsorbent and ethyl acetate as an eluent. Fractions containing only the pure product (CC; (1) - 0.60) were combined and evaporated at Fu with 2.0-2.5 kPa. The residue after evaporation was crystallized from isopropyl ether.

Output 5.0 g (7.8 mm, 6496), K. (1) - 0.6 s

EAV mass spectrum (640 (М--Н") confirmed the expected structure. "I

Stage 2: Tetrahydropyranyl-O-cycloheptyllactyl-i-prolyl-MU-benzyloxycarbonyl-i-arginine-aldehyde m 4.8g (7.51MM) tetrahydropyranil-O-cycloheptyllactyl-i! -prolyl-MU-benzyloxycarbonyl-Barginin lactam

Zo (Example C, Stage 1) was dissolved in 15 ml of tetrahydrofuran, then with stirring at a temperature that is not exceeded -502С, a solution of 3,bmMM of lithium aluminum hydride dissolved in tetrahydrofuran was added. The recovery process was monitored by thin-layer chromatography with Mo8 developing solvent, and if necessary, another portion of lithium aluminum hydride was added. 0.5 M sulfuric acid was added dropwise to this reaction mixture with constant stirring and cooling until pH 3 was reached, then 35 ml of water was added.

The resulting solution was extracted with 2 x 15 ml of hexane, then with 3 x 200 ml of dichloromethane. The extracts in c) with dichloromethane were combined, washed with 3x15 ml of water, 15 ml of cold 595 sodium bicarbonate solution and again with 15 ml of water, dried over anhydrous sodium sulfate and evaporated at 2.0-2.5 kPa. The residue after evaporation was treated with diisopropyl ether , filtered and dried under reduced pressure.

Yield 3.9g (6.07mM, 81965). B (8) - 0.40. (93579 - 16.09(s-1, tetrahydrofuran). -1 but the EAV mass spectrum (642 MAN, 795 |MAN MVA) confirmed the expected structure.

Step 3: Y-cycloheptyllactyl-i-prolyl-i-arginine aldehyde hemisulfate and 3.21 g (BmMM) of tetrahydropyranyl-O-cycloheptyllactyl-i-prolyl-M-benzyloxycarbonyl-i-arginine aldehyde (Example C, Step 2) were dissolved in 40 ml of ethanol and bml of 0.5M sulfuric acid was added, then 0.3 g of Ra-c 20 catalyst suspended in bml of water, and the mixture was hydrogenated at approximately 109C. The reaction process was monitored by thin-layer chromatography. After completion of the reaction (approximately 15 minutes), the catalyst was filtered off and the filtrate was concentrated to approximately 4-bml at 2.0-2.5 kPa. The residue was diluted with 40 ml of water, extracted with 4 x 7 ml of dichloromethane and the aqueous solution was left to stand at 20-229C for 24 hours. The solution was extracted again with 5 ml of dichloromethane and the pH was adjusted to 3.5 with the ion exchange resin Yuozheh AS 1-XV (HO), then the solution was subjected to lyophilization. o Yield 1.65 g (3.5 mmMM, 7096) (oShcho-9U - -94.72 (s-1, water)

NRIS:K - 2.702 and 3.010. and EAV mass spectrum (424 (M.-NI", 577 (MANAMVAG)) confirmed the expected structure.

Synthesis of starting materials: 60 Triethylammonium salttetrahydropyranyl-O-cycloheptyllactyl-! -proline

Stage A: Chloroacetyl-OiI-dicloheptylalanine methyl ester

To a solution of 15.2g (b65mM) of 0-cycloheptylalanine methyl ester hydrochloride (Example 1, Step E) in bbml of dichloromethane was added 9.1ml (b65mM) of triethylamine and 14.9g (78mM) of (M-hydroxysuccinimidyl)-chloroacetate."

After stirring at room temperature for 3 hours, the reaction mixture was diluted with 100 ml of dichloromethane and successively washed with 300 ml of water, 1 M KNBSO, water, 595 Manso, and finally with water until neutral. After that, the organic layer was dried over anhydrous Ma 250.4, then evaporated at 2.0-2.5 kPa. The resulting oily product was triturated with petroleum ether. The solid material was filtered off, washed with petroleum ether and dried in a vacuum desiccator. 17.54g (63.6MM, 79895) of chloroacetyl-OI-dicloheptylalanine methyl ester was obtained, which was used directly for the next reaction. K, (7) - 0.73-0.83. T. pl.: 78-80260.

Analysis for С43Н22МозСИ (275,777). Calculated C 95 - 56.62; H 95 - 8.04; M 95 - 5.08; SI 95 - 12.86.

It was revealed: C bo - 55.65; H Fo - 7.93; M 90 - 5.06; SI Fo - 12.72. Preparation of (M-hydroxysuccinimidyl) chloroacetate 70 32 ml (450 mM) of chloroacetyl chloride was added to 23 g (200 mM) of M-hydroxysuccinimide and the mixture was refluxed for 10 minutes, then poured onto crushed ice, filtered, washed with cold water and dried in a vacuum desiccator.

Yield 20.23g (105.9mM, 5390). T. pl.: 113.3-113.720,

Analysis for S eNhMO)sSI SUNoMO»B (191.55). Calculated: So - 37.62; НОб - 3.16; Mine - 7.31; SiIFo - 18.51. 75 Found: C 95 - 37.37; NOyu - 3.16; M 90 - 7.23; SiIFo - 18.35.

Stage B: Chloroacetyl-Y-cycloheptylalanine methyl ester (enzymatic dissolution of chloroacetyl-Y-dicloheptylalanine methyl ester)

To a solution of 8.71 g (31.bmol) of chloroacetyl-OH -dicloheptylalanine methyl ester, 0 -ester, (Example 3,

Stage A) 7/Oml of water and 5Omg BiBbiyvip Sagizregu (Protease, type MIM, 5idta) were added to ZOml of toluene. Enzymatic hydrolysis of the I-enantiomer, the I-ester, which occurred at pH 7.0, was supported using an autotitrator filled with ZM Mahon. After stopping the consumption of Mahon (5.522 ml, 16.57 mM), the reaction mixture was diluted with 3 ml of toluene and the two layers were separated. The aqueous phase was washed with 2 x 20 ml of toluene. The combined solutions in toluene contained the SO-ester, and the combined aqueous solutions contained the sodium salt of the I-acid.

After drying over anhydrous Ma 250, the combined solutions in toluene were evaporated under reduced pressure to obtain 4.18 g (15.16 mM) of O-ester. K, (7) - 0.73-0.83, which was used directly to obtain ХО-cycloheptylalanine. and)

The combined aqueous solutions were acidified and extracted with 300 ml of ethyl acetate. The combined solutions in ethyl acetate were washed with water, dried over anhydrous Ma»b5O, and evaporated under reduced pressure to obtain 3.46 g (13.22 mM) of I-acid IR, (7) - 0.45-0.50), which was used directly to obtain ee

I-cycloheptylalanine.

A similar method of preparation, starting from 8.25 g (30 mM) of 0-ester (Example 2, Step A) yielded 4.08 g (14.79 mM) of O-ester and 3.23 g (12.34 mM) of I-acid. "AND

Stage C: O-cycloheptylalanine hydrochloride 8.27 g (ZOMM) O-ester (Example 3, Stage B) was suspended in 120 ml of 6M NI and subjected to dephlegmation for 3 - hours. The free amino acid was separated in the form of crystals. The reaction mixture was cooled, kept in a refrigerator until the next day, filtered, washed with cold water and ether, then dried in a vacuum desiccator. 5.9 g (26.69 mM, 89965) of O-cycloheptylalanine hydrochloride was obtained. P, (12) - 0.10-0.15. |sdr2O - -119 (c-0.4; 1 MNOI). "

Analysis for С 10Н419МО». NSII (221,728). Calculated: C 95 - 54.17; H 95 - 9.09; M 95 - 6.32; SI Fo - 15.99.

Revealed: C 95 - 54.27; H 905. - 9.27; M 90 - 6.30; Sibo - 16.2. - c Step Or: Dicyclohexylammonium salt of O-cycloheptyllactic acid m 5.78 g (26.15 mM) of O-cycloheptylalanine hydrochloride (Example 3, Step C) was dissolved in 2 bml of water, diluted with 105 ml of water and 52.5 l of glacial acetic acid and cooled to 520. A solution of 18.0 g (261 mM) of Mamo" in ZOml of water was added dropwise to this mixture while stirring and cooling. Stirring was continued at 52C for one hour and at room temperature until the next day. The next day, the reaction mixture was acidified with 25 ml of sodium chloride under stirring. After that, the mixture was evaporated to dryness at 5023 under reduced pressure conditions. The residue was dissolved in 100 ml of water and evaporated in the same way, triturated with toluene and evaporated again. The final residue was dissolved in 50 ml of ethyl acetate and 20 ml of water. The aqueous phase was washed with ethyl acetate, and the combined solutions in ethyl acetate were washed with water until neutral, dried over anhydrous MAO, and evaporated under reduced pressure. The resulting solid was dissolved in 20 ml of isopropyl ether. 5 ml (25 mM) of dicyclohexylamine was added to this solution, after which the crystalline salt was separated. After cooling, the crystals were filtered, washed with cold ether and dried in a vacuum desiccator to obtain 5.5 g (14.96 mM, 57.295) of dicyclohexylamine salt of O-cycloheptyllactic acid, O-cNia.OSNA. EV, (7) - 0.53-0.60. (oШ079 - -19.59 (s-1; methanol). T. pl.: 147-15026.

Analysis for SiONivOz (Co2NAMOz). Calculated: C 90 - 71.89; H 90. 11.24; M 90 - 3.81.

GF) Revealed: By himself - 71.57; H Fo - 11.34; M 95 - 3.83.

GF The conversion of 0.35 g (1 mM) O-cNia.OSNA to free o-hydroxy acid yielded 0.15 g (0.8 mM) O-cNia,

IFCh09 - -10.12 (s-1; methanol.); t. pl.: 125-12726. in Analysis for SoNivOz (186.52). Calculated: C 9o - 64.48; H 95 - 9.74. Revealed: C 95 - 64.54; BUT - 9.86.

Step E: Benzyl ester of O-cycloheptyllactic acid

To a solution of 11.21 g (30.5 mM) of the dicyclohexylammonium salt of O-cycloheptyllactic acid (Example C, Step 0) in 30 mL of dimethylformamide, 3.57 mL (30 mL) of benzyl bromide was added. The mixture was stirred at room temperature for 24 hours, then filtered and evaporated at 2.0-2.5 kPa. The residue was dissolved in 20 ml of 0.5 M 65 potassium bicarbonate and 1 ml of diethyl ether. The organic phase was successively washed with 20 ml of water, 0.5 M KNZO,) and water, then dried over anhydrous sodium sulfate and evaporated under reduced pressure.

The oily residue was 8.3 g (700 g) of benzyl ester of O-cycloheptyllactic acid |K; (3) - 0.2-0.3), which was used directly at Stage R.

Step E: Benzyl ester of tetrahydropyranyl-O-cycloheptyllactic acid

To a solution of 8.29 g (300 g) of benzyl ester of O-cycloheptyllactic acid (Example 3, Step E) in 30 ml of dichloromethane was added 3.01 ml (333 mM) of 3,4-dihydro-2H-pyran and 0.3 ml of 3M NOI in ethyl acetate and the mixture left to stand at room temperature for 16 hours. After that, the reaction mixture was diluted with 40 ml of dichloromethane, washed with 3x2O0 ml of water, dried over anhydrous sodium sulfate and evaporated at 2.0-2.5 kPa. The residue was subjected to chromatography on a column with silica gel, using 200 g of KiezeiYide! 60 7/0 (0.040-0.063 mm) as adsorbent and 85:15 mixture of cyclohexane and ethyl acetate as eluent. The fractions containing only the pure IR product, (4) - 0.60-0.701, were combined and evaporated at 2.0-2.5 kPa. The oily residue was 7.9 g (21.9 mM, 7395) of tetrahydropyranyl-Y-cycloheptyllactic acid benzyl ester, which was used directly in the next step.

Step c: Triethylammonium salt of tetrahydropyranyl-O-dcycloheptyllactic acid 7.21 g (20 mM) of benzyl ester of tetrahydropyranyl-Y-cycloheptyllactic acid (Example C, Step B) was dissolved in 20 ml of dimethylformamide, 2.8 ml (20 mM) of triethylamine was added and hydrogenated in the presence of 0 , 1 g of Ra/C catalyst. The reaction process was monitored by thin-layer IR chromatography; (1) - 0.30 (ester), 0.00 (acid)). After completion of the reaction, the catalyst was filtered and washed with 2x5 ml of dimethylformamide.

The filtrate and washings were combined and used in the next step as a solution of 20 mM triethylammonium salt of 2O tetrahydropyranyl-O-cycloheptyllactic acid.

Step H: Benzyl ester tetrahydropyranyl-Y-cycloheptyllactyl-i! - proline

A solution of the triethylammonium salt of tetrahydropyranyl-Y-cycloheptyllactic acid obtained in Step o

Example C (20 mM) was cooled to 52 and with stirring, 2.7 g (20 mM) of 1-hydroxybenzotriazole, 4.83 g (20 mM) of I-proline benzyl ester hydrochloride and 4.12 g (20 mM) of dicyclohexylcarbodiimide were added. The reaction mixture was kept at room temperature until the next day, then filtered and evaporated at 2.0-2.5 kPa.

The residue was dissolved in 50 ml of ethyl acetate and washed with 20 ml of water and 59 g of sodium bicarbonate, dried over anhydrous NaCl, and evaporated under reduced pressure. The residue was subjected to chromatography on a silica gel column using 200 g of Kieseide! 60 (0.040-0.063 mm) as an adsorbent and a 1:1 mixture of n-hexane and ethyl acetate as an eluent. The fractions containing only the pure product (K; (3) - 0.3-0.4) were combined and evaporated at 2.0-2.5 kPa. The oily residue was 5.95 g (13MM, 6590) of tetrahydropyranyl-O-cycloheptyllactyl-! -proline, which was used directly at the next stage. with

Stage I: Triethylammonium salt of tetrahydropyranyl-O-cycloheptyllactyl! -proline «I 5.5g (12mM) of tetrahydropyranyl-O-cycloheptyllactyl-I-proline benzyl ester (Example 3, Step H) was dissolved in 12ml of dimethylformamide, 1.68ml (12mM) of triethylamine was added and hydrogenated in the presence of -bO, 1g of catalyst Ra/S. The reaction process was monitored by thin-layer chromatography (TLC; (1) - 030 Kk (ester), 0.00 (acid)! After the completion of the reaction, the catalyst was filtered and washed with 2x2 ml of dimethylformamide. The filtrate and washings were combined and used as a solution containing 12 mM of triethylammonium salttetrahydropyranyl -O-cycloheptyllactyl!-proline.

Example 4 "

Synthesis of hemisulfate M-methyl-O-cyclohexylglycyl-! -azetidine-2-carbonyl-i-arginine aldehyde shshc Step 1: c Benzyloxycarbonyl-M-methyl-O-cyclohexylglycyl-I -azetidine-2-carbonyl-M O-benzyloxycarbonyl-i -arginine "» lactam 1.97g ( 5mMM) tert-butyloxycarbonyl-MU-benzyloxycarbonyl-I -arginine lactam (Ba): ей ай., .. Med.

Spent 33.1729 (1990) was suspended in bml of chloroform, then with stirring and cooling with ice - and bml of ethyl acetate saturated with NH gas (0.11-0.15g/ml) was added. Cleavage of the Bos group was monitored -1 by thin-layer chromatography |K; (11) - 0.5 (free compound); 1.0 (Voc-compound)) until the completion of the reaction, the suspension was diluted with 10 ml of diethyl ether, the formed crystalline mass was filtered, washed with 3 ml of acetone and 3 ml of diethyl ether and dried under reduced pressure conditions over KOH. Obtained as a result g 50 Me. benzyloxycarbonyl- and -arginine lactam hydrochloride was dissolved in 5 ml of dimethylformamide, cooled to -2090 and added to the anhydride with further stirring. with 1.95 g (5 mM) of benzyloxycarbonyl-M-methyl-O-cyclohexylglycyl-I-azetidine-2-carboxylic acid (Example 4,

Stage B) was dissolved in 5 ml of dimethylformamide, cooled to -152C, then 0.5 ml (5005 mM) of M-methyl-morpholine and 0.6 ml (5.05 mM) of isobutyl chloroformate were added with stirring. After 10 minutes of stirring, the above-mentioned solution in dimethylformamide of MO-benzyloxycarbonyl-I-arginine lactam was added, then

HF) triethylamine in an amount sufficient to bring the pH of the reaction mixture to 8 (approximately 0.7 ml was required). c The reaction mixture was stirred at -109C7 for 30 minutes, then at 092 for one hour. After that, the salts were filtered off and the filtrate was diluted with 500 ml of ethyl acetate. The resulting solution was washed in 3x7ml of water, 3ml of 1M KNBO, and 3x3ml of water, dried over anhydrous Ma»5O, and evaporated at 2.0-2.5 kPa.

The obtained product was subjected to chromatography on a column with silica gel, using 5Og KiezeiYide! 60 (0.040-0.063 mm) as an adsorbent and ethyl acetate as an eluent. Fractions that contained only the pure MK product; (1) - 0.70), combined and evaporated at 2.0-2.5 kPa. The residue after evaporation was crystallized from diisopropyl ether. 65 Yield 2.35g (7190), K; (1) - 0.45-0.55

The EAV mass spectrum (661 IMNANI) confirmed the predicted structure.

Stage 2:

Benzyloxycarbonyl-M-methyl-O-cyclohexylglycyl-I -azetidine-2-carbonyl-M C-benzyloxycarbonyl-i -arginine aldehyde 2.15g (3.25MM) benzyloxycarbonyl-M-methyl-O-cyclohexylglycyl-i -azetidine-2 -carbonyl-M 3-benzyloxycarbonyl-and -arginine lactam (Example 4, Stage 1) was dissolved in 5 ml of tetrahydrofuran, and then, with stirring and at a temperature that did not exceed -50 °C, a solution of 2.25 mM HyAIN; dissolved in tetrahydrofurans. The recovery process was monitored by thin-layer chromatography (solvent 7 as a developing solvent) and, if necessary, another portion of HyAIN was added. 0.5M KNBZO was added dropwise to this reaction mixture, with constant stirring and cooling until pH 3 was reached, then 13 ml of water. The resulting solution was extracted with 2x5ml of hexane, then with 3x/ml of dichloromethane. The extracts in dichloromethane were combined, washed with 3x/ml of water, 7 ml of cold 596 solution of ManHsSO»z and again with 7 ml of water, dried over anhydrous

Ma»5zO, and evaporated at 2.0-2.5 kPa. The residue after evaporation was treated with isopropyl ether, filtered and dried under reduced pressure.

Output 1.6bg (74965), K (7) - 0.33-0.43

The EAV mass spectrum (663 MANI") confirmed the expected structure.

Step 3: M-methyl-O-cyclohexylglycyl-! -azetidine-2-carbonyl-I-arginine aldehyde sulfate 1.53g (2.3MM) benzyloxycarbonyl-M-methyl-O-cyclohexylglycyl-azetidine-2-carbonyl-M-benzyloxycarbonyl-i-arginine aldehyde (Example 4, Step 2) was dissolved in 233 ml of ethanol, 4.8 ml of 0.5 M sulfuric acid was added, then 0.15 g of the Ra-C catalyst suspended in 3.5 ml of water and the mixture was hydrogenated at approximately 102. The reaction process was monitored by thin-layer chromatography. After completion of the reaction (approximately 15 minutes), the catalyst was filtered and the filtrate was concentrated to approximately 2-Zml at 2.0-2.5 kPa. The residue was diluted with 20 ml of 29% water, extracted with 4 x 4 ml of dichloromethane and the aqueous solution was left to stand at 20-222C for 24 hours. Gee)

The solution was again extracted with Zhiml dichloromethane and the pH was adjusted to 3.5 with the ion-exchange resin Juomzheh AC 1-X8 (HO), then the solution was subjected to lyophilization.

Output 1.02g (9090). K, (12) - 0.40. so zo EAV mass spectrum (395 MANI", 548 |M--Н-МВАЙ") confirmed the expected structure.

Synthesis of source materials: p

Benzyloxycarbonyl-M-methyl-O-cyclohexylglycyl-! -azetidine-2-carboxylic acid "E

Stage A: Synthesis of 2, 4, 5-trichlorophenyl ester of benzyloxycarbonyl-O-cyclohexylglycine

8.8 g (22 mM) of benzyl pentachlorophenyl carbonate was added to a stirred suspension of 5.42 g (20 mM) of the trifluoroacetate salt of 10-cyclohexylglycine and 5.0 mL (40 mM) of triethylamine in 1 mL of dimethylformamide (Apieshpiv ei cha a!: Vii. os. Spit. Ved. 96, 775 (1987)) and b.1 bml (44 mM) of triethylamine. After 3 hours of stirring, the reaction mixture was evaporated under reduced pressure, the residue was dissolved in bOml diethyl ether and 60 water. The phases were separated, the organic phase was washed with water, and the combined aqueous phases were washed with diethyl ether, acidified with 1M NaOH to pH 3, then extracted with 300 ml of ethyl acetate. The organic phase was washed with water to neutrality, dried over anhydrous Ma»5O), and evaporated at 2.0-2.5 kPa. The residue after evaporation was benzyloxycarbonyl-O-cyclohexylglycine, which was dissolved in 20 ml of tetrahydrofuran and combined with 4.54 g (22 mM) of 2,4,5-richloro-phenol and 4.54 g (22 mM) of dicyclohexylcarbodiimide After three hours the reaction mixture was filtered, the filtrate and washings were combined and evaporated under reduced pressure. acetone as an eluent. The fractions containing only the pure product (KK; (5) - 0.7-0.8) were combined and evaporated at 2.0-2.5 kPa. The oily residue was triturated with diethyl ether, filtered, washed diethyl ether and

Sh- dried. Yield: 8.34 g (88.596) of pure 2,4,5-trichlorophenyl ester of benzyloxycarbonyl-O-cyclohexylglycine.

Stage B: Synthesis of benzyloxycarbonyl-O-methyl-O-cyclohexylglycyl-I! -azetidine-2-carboxylic acid and 1-azetidine-2-carboxylic acid (1.01 g, 1 mM) and triethylamine (1.4 ml, 71 mM) were added to a solution of 2,4,5-trichlorophenyl ester of benzyloxycarbonyl-O-cyclohexylglycine (5, 18g, 11mMM, from Example 4, Step A) in 1Oml of pyridine. After stirring until the next day, the reaction mixture was evaporated under reduced pressure and the residue was dissolved in 50 ml of 595 Manso with and 500 ml of diethyl ether. The organic phase was washed with water and the 5B combined aqueous phases were washed with diethyl ether, acidified with 1M KHBO to pH 3, then extracted

3x5Oml of ethyl acetate. The extracts in ethyl acetate were combined, washed with water until neutral, dried over (F; anhydrous Ma»zoO, and evaporated at 2.0-2.5 kPa. ka The residue after evaporation is benzyloxycarbonyl-O-cyclohexylglycyl-I-azetidine-2-carboxylic acid , which was dissolved in 1 Oml of tetrahydrofuran and combined with 5.Oml (8BOMM) of iodomethane and cooled to 02. To this solution was added 1.2g (ZOMM) of 6090 sodium hydride and the reaction mixture was stirred at RT until the next day. The excess sodium hydride was carefully decomposed by adding 0.4 ml of water. The quenched reaction mixture was concentrated to approximately 10 ml under reduced pressure at a temperature below 302C. The residue was diluted with 15 ml of water and 100 ml of i-butyl methyl ether. The phases were separated and the aqueous phase was washed again with 10 ml

Ibutyl methyl ether. The aqueous phase of the product was combined with 1.5 ml of ethyl acetate and adjusted to pH 22 using 3M sulfuric acid solution. The phases were separated and the aqueous phase was subjected to reverse extraction with 10 ml of ethyl acetate. The combined organic phase was washed with 595 mL of sodium thiosulfate solution. The phases were separated and the organic phase was evaporated under pressure at a temperature below 402. The oily residue is 3.75 g of benzyloxycarbonyl-M-methyl-O-cyclohexylglycyl-! -azetidine-2-carboxylic acid (9.65 mM,

E(7) - 0.85-0.95), which was dissolved in 9.65 ml of tetrahydrofuran and kept for use in Step 1 of Example 4.

The EAV mass spectrum (403 MANI") confirmed the expected structure.

Example 5

Inhibition of coagulation by peptidyl arginals inhibitory effect on plasma coagulation was evaluated by assays of thrombin time (TT), activated partial thromboplastin time (ARTT) and prothrombin time (PT) using purified human 70 plasma as a substrate |Vadau, O. ei ai: Tygotr. Naetozvziav 67325 (1992)). In the TT analysis, inhibition of one stage, coagulation of fibrinogen under the action of exogenous thrombin (final concentration of 2.5 MIN SHO/ml) is measured.

Plasma coagulation in ARTT and RT assays was caused by recalcification. The produced endogenous thrombin can theoretically be present at a final concentration of 50 MIN IU/ml (ARTT, RT). These assays reveal a combined inhibitory effect on fibrin formation and thrombin generation that involves several proteolytic reactions 75 mediated by thrombin or other enzymes, such as IXa. Anticoagulant activity is expressed in

St", which is the concentration (nM) required to double the clotting time. (C1, C2) for plasma coagulation (column A), clot-bound thrombin and factor Xa (column B) and fibrinolytic enzymes (column C) Spd - cyclohexylglycyl. ; thromboplastin time) and RT (prothrombin time). and - tissue plasminogen activator, OK - urokinase.

Some of these compounds were superior to EGedigap (C, O-MeRPNe-Pho-Ag9-H, parent peptide arginal) in their ability to prolong clotting time (see Table 1). Column A of Table 1 presents the anticoagulant effect of the compounds of formula (I) according to the invention (1-4) compared to the activity of Eiedayap (C), a known anticoagulant |Va|y572, 5. ei. a: 9). May Spent 33,1729 (1990); 05. Rai, Mo. 4,703,036 (1982);

From" Wadau, O. ek a: Tygotr. Naetoviaz. 67, 357 apa 68,125 (1992); Chaskwop, OM. ей ай.: Siip. Arri.

Tpgotroviv/Naetozviaziv 2, 258 (1996)| and other related peptidyl arginals (for example, C1 and C2)

IVainv2, 5. ei ai!.: Vioogd. 5 Med. Spent Z, 1079 (1995); 05. Paradise. Mo. 6,121,241 (2000); PCT Ryb. Mo. MUO97/465761. 49 Analysis of TT shows new analogues almost as effective as eidejap in inhibiting the reaction and thrombin-fibrinogen. ARTT, on the other hand, indicates that the new peptides are more effective than egedaiyigap in inhibiting the earlier, thrombin-generating steps of coagulation.

Example 6 Shk Inhibition of Thrombin and Factor Xa 20 Inhibition of the enzyme was studied in platelet-enriched plasma clots using chromogenic substrates, i.e. Tov-c1u-Pho-Agd-PMA (51) for thrombin and Mos-O-Spd-SiIu-Agao-pRMA (52) for the Xa factor, as was briefly described in the publication (Va|iv2, 5. es a: PCT Rybr. Mo. UUO97/46576). Analyzes were carried out at room temperature in glass tubes and 96-well microtiter plates.

Solutions, () Buffer A: 0.1 M sodium phosphate/0.05 M Mass (pH 8.5). (ii) Inhibitors: 0.1, 1.0, and 1Omg/ml 25 solutions in buffer A containing 0.0295 human albumin, (ii) Substrates: 1 mM 51 and 2 mM 52 in distilled

GF) water. (b) Obtaining plasma clots. Platelet-enriched plasma samples (200 μl), placed in glass tubes, were incubated at room temperature with 30 μl of 40 mM CaCl for 2 min. Clots were washed with 2 ml aliquots of saline (0.995 Mass) with gentle agitation to remove unbound enzymes. In 60 cases of successful washing, the optical density of the reaction mixture of the washed substance and 51 is less than 595 from the control. The plasma clot obtained in this way was kept under saline in a test tube until use. (c) Evaluation of enzyme inhibition in clots. After removal of the saline, the plasma clot was incubated at 37 C with 40 μl of inhibitor (or buffer A as a negative control) for 2 h and with 10 μl of substrate 51 or 53 for 30 h, then the reaction was stopped with 10 μl of 5090 acetic acid. Portions of 1500 μl of the reaction mixtures were placed in the wells of the microtitration 62 tablet and read at 405 nm (EOBA KEAOEK 800, Vio-Tech

Ipvigitepiv Ips. M/hypoxic. MT. AA). Values of IS5o were displayed graphically according to attenuation data.

The results are shown in column B of Table 1. Compounds 71, 2, C, and 4 are the only analogs that can outperform Etedaigap in inhibiting clot-bound thrombin, but each analog outperforms Eggedaigap in inhibiting clot-bound factor Xa. Thus, 1, 2, C and 4 are the best inhibitors for both clot-bound enzymes.

Example 7

Antifibrinolytic activity

The inhibitory effect of the compounds of the invention on plasmin (PI) and plasmin production by plasminogen activators 7/0 (progen), such as tissue plasminogen activator (IPA) and urokinase (SK), was investigated by fibrin plate assay |see, for example, Wadau, O. , Vagaraz, E., Va|iv2, 5., apa 52eiI, E., Tygotr. Naetozviazv., 67:325-330 (1992); Vagaraz, E. 52eiy, E. apa Va|iv7, 5, Viood Soadshayop apa Ribgipoiuviv, 4:243 (1993)).

The results are shown in Table 1, column C. In some cases, the analogs have a slightly greater inhibitory effect than EtTedaigap on the three fibrinolytic enzymes. The exceptions are C1 against RI and C1, C2 against SHK, while Z/5 has almost the same activity as EtTedaigap against OK.

Example 8

A rabbit model of disseminated intravascular coagulation

Compounds 1 and 3 according to the invention and other peptidyl arginases from Table 1, as well as C3, were tested for their ability to inhibit BIS in rabbits injected with endotoxin (lipopolysaccharide, I P5) as described |CsNegeg,

M. U. eyi a: Har. Appetite zZsi. 45,538 (1995)). The analysis procedure lasted for 4 hours. Endotoxin was reduced in doses of 80 and 40 (μg/kg by intravenous bolus injection, at 0-1 and 120 minutes, respectively, while peptidyl arginals 1, C and C-C3C (0.25 and/or 0, 5mg/kg/h) was infused throughout the experiment. The control group of animals was injected with 0.995 saline. Hemostatic parameters were determined at 0, 120 and 24 Ohv.

Despite careful treatment, mortality was 3195, most likely due to the high sensitivity of the rabbits to endotoxin. U. Siip. Hebrew Kev. 21., 214 (1992). of other arginals (C1 and C2) and heparin (H) on the mortality of rabbits injected with endotoxin

Vaveezhmsi 00000100000060010090010000110 vdoeyun 011111101000003010000001000m80001 M zvooevmyyud 111010950010101151 shchi zavmyyutod 111 17111011116le1111

Еге МИ ПИ ЗИ ПА ТА ПО ПО « shuochovmachyud 0000000100000960000100006001000088600 Z is povi 1111010000092000100000100089000010v. pozovmiyud 10000001000000y8000010000800001000000800001000056000 » yanyuyutd 11010100000901009019000113 znyyued 00170100 60001008 5 te ulvatsi suri tt pit. 0115 05 legend li soy ben pen let, -I adiv. in Table 1, the structures of peptidyl arginals 1, 3, C, C1 and C2, C3 - pRIa-Pho-Ago-H, where HRia - 2-hydroxy-4-phenyl-butyric acid. - As shown in Table 2, compounds 1 and C significantly reduce the mortality of rabbits injected with endotoxin, while other anticoagulants either did not affect mortality (C1) or caused a certain increase in mortality, and the highest rate, "1.8 times, gave C3. From the data in Table 1, it can be seen that compounds 1 and 3, which reduced mortality, most inhibited clot-bound thrombin, as well as factor Xa, and effectively inhibited both plasma coagulation and fibrinolytic enzymes, plasmin and activators plasminogen.

Example 9

A rat model of disseminated intravascular coagulation

The most serious consequences of IS include deposition of fibrin in various organs, changes in blood cells, for example, a decrease in the number of platelets and changes in fibrin breakdown products. The effect of compound 1 according to (F.'s invention) and two control anticoagulants, Etedaigap (C) and heparin (H), on these phenomena was studied in rats injected with endotoxin (Roga, A. u. apa I opdgidde, O.9U. : Vg. 9). Ripagtasoi. 110, Zirri. 131R (1993);

Nazedaula, M.; her a)I.: At. 9. Caesar. Stia Sage Med. 153.1831 (1996); Oisppeye, 0. ei aI.: Tygotr. Kezv. 77,357 (1995)). Intravenous bolus injections of 1Omg/kg of endotoxin were administered to male rats. They were followed by an intravenous infusion of saline or test compounds for four hours. Compounds 1 and C were administered at 0.25 mg/kg as an initial bolus injection followed by an IV infusion of 0.25 mg/kg/h over four hours. Heparin (H) was administered in a similar manner, 50 IU/kg as an initial bolus injection followed by an IV infusion of 50 IU/kg/h over four hours. The control group of animals was injected with 65 0.990 saline.

Deposition of 725I-fibrin was studied in selected organs (liver and kidneys). 722I-fibrinogen was administered by injection for ZOkhv before endotoxin injection. Radioactivity in tissue samples was measured in a gamma counter (M/ayas Muigaga 1470). The formation of microthrombi from organs was evaluated using the ratio of 725) activity of the organ to the total 7292| injection activity, defined as microthrombi index.

Changes in this parameter are expressed as a percentage compared to the group treated with saline.

The number of platelets was determined using an automatic device (Zuztech E-800) and correlated with control indicators.

Determination of BOR (fibrin breakdown products) was carried out by analyzing the precipitation of Addysiip (ristocetin). Animals were killed four hours after endotoxin administration. The results are summarized in 70 Table 3. on the deposition of 125|-fibrin and on changes in the number of platelets and fibrin breakdown products (FRP) in rats injected with endotoxin e

The data in Table C indicate that the OiIS inhibitory potential of 1 was more pronounced than that of heparin or EGedigap. everything

Example 10 Study of survival in the rat model of diffuse intravascular coagulation o

Male rats were given an intravenous bolus injection of ZOmg/kg of I P5 (endotoxin). Peptides in doses of 0.5 and/or 0.75, 1.0 and 1.5 mg/kg were administered as a primary bolus injection with subsequent infusion within eight hours immediately after administration of I RB5. Mortality was recorded 4, 5, 6, 7, 8 hours after the introduction of IR.

The data in Table 4 indicate that the introduction of Y/P5 reduced the survival rate of rats to 2095 by the end of the experiment (8 hours). All studied new peptidyl arginals prolonged the survival time and reduced the mortality compared to group I RB5. Compounds 1, 2, C and 4 were more effective than the standard C. 15 oBoiv 15 ovo 15. blo zo zoo|loo oo 100 oo (105109 109| 100 100 100 109100. « 74717609 |va zo lob o 309, ve 109 10900100. 109 98 | 100. 100. z -; s 61 vk| e6ozoo lov oo oo, v, 10910903 109 109 tu | 100100. khz 71172 za |v ovo vo loo tu v loo/too loo/too vu v 100 " voi |v 92 to volos vo vo )cho0) vo) 922 vo vo 100)

Male rats were given an intravenous bolus injection of ZOmg/kg and RB5. The test compounds were administered as an initial bolus injection followed by an intravenous infusion within eight hours immediately after the administration of I P5. - and I

Mortality was recorded 4, 5, 6, 7 and 8 hours after the introduction of I P5.

Cheka" at 7

Claims (24)

The formula of the invention IR e) 1. A compound having the formula (I) Xa-Chrrf-Aga-H, (I) where Xa represents an alpha-substituted carboxylic acid residue of the formula (II) O-CH(K)-CO,, (II) i) where O represents an alkyloxycarbonylamino group with 1-3 carbon atoms, a methylamino group or a hydroxyl group, and K represents a cycloalkylmethyl group with 7-9 carbon atoms, a 1-adamantylmethyl group or a cycloalkyl group with 5-7 carbon atoms, and Xb represents I -proline or I - azetidine-2-carboxylic acid 60p residue, and its acid additive salts formed with an organic or inorganic acid. 2. A compound that has the structure 1: b5 shto ANNA, 1 shet shu NE and yah NEO Mt, where - M zho 70 more: SHY and its acid additive salts. Q. A compound having the structure 2: 75 I is not and its acid addition salts. 4. A compound that has structure 3: o ss dz so vena dev De lo i yoOooyto v s some and "I ph otv Me: What is it - Om pd i- and its acid additive salts. 5. A compound that has structure 4: "A YN, 4 ey : Tv ПЧ shsh ts oo, what 7 sh t as AKED SE J sere - 2 Esh KE DN? -iI and pokopi and its acid additive salts. to 20 6. A pharmaceutical composition containing the compound specified in claim 1. 7. A pharmaceutical composition containing the compound specified in claim 2 8. A pharmaceutical composition containing the compound specified in claim 3. 9. Pharmaceutical composition containing the compound specified in claim 4. 10. A pharmaceutical composition containing the compound specified in claim 5. 11. Compound ethoxycarbonyl-O-cycloheptylalanylprolyl-MO-benzyloxycarbonyl-i-arginine aldehyde. GF) 12. The compound tetrahydropyranyl-O-cycloheptyllactyl-I-prolyl-MO-benzyloxycarbonyl-I-arginine. aldehyde. GI 13. Compound benzyloxycarbonyl-M-methyl-O-cycloheptilalanyl-i-prolyl-M-benzyloxycarbonyl-i-arginine aldehyde. in 14. Compound M-methyl-O-cyclohexylglycyl-! -azetidine-2-carbonyl-and -arginine aldehyde. 15. A pharmaceutical composition according to any one of claims 6-10, which is characterized in that the aforementioned composition can be in the form of a tablet, capsule, powder, pill, dragee, granule, solution, infusion, suppository, patch or ointment. 16. A method of treating a patient with disseminated intravascular coagulation, in which the patient is administered de Peptidyl arginal, which has an inhibitory effect on clot-bound thrombin, factor Xa, plasmin, and plasminogen activators. 17. A method of treating a patient with disseminated intravascular coagulation, in which the patient is administered peptidyl arginal, which has the formula (I) Xa-Chrrf-Aga-H, (I) where Xa is an alpha-substituted carboxylic acid residue of the formula (II) O-CH( K)-CO,, (Ii) where O represents an alkyloxycarbonylamino group with 1-3 carbon atoms, a methylamino group or a hydroxyl group, and K represents a cycloalkylmethyl group with 6-9 carbon atoms, a 1-adamantylmethyl group or a cycloalkyl group with 5-7 carbon atoms , and ХЬ represents I-proline or I-azetidine-2-carboxylic acid residue, or its pharmaceutically acceptable acid addition salts. 18. A method of treating a patient with disseminated intravascular coagulation, in which the patient is administered peptidyl arginal, which has the structure 1 theo ZI, 1 or its pharmaceutically acceptable acid addition salts. 19. A method of treating a patient with disseminated intravascular coagulation, in which peptidyl arginal, which has the structure 2 o DY Vela, that TE s tires - with Shei - or its pharmaceutically acceptable acid addition salts, is administered to the patient. 20. A method of treating a patient with diffuse intravascular coagulation, in which peptidyl arginal is administered to the patient, which has the structure Z « santnt VINY, Z Sheeey z neds - s Ta te moya ch Ek inya sho y - pe She ME sl ky - v she SHY iz ZHI or its pharmaceutically acceptable acid additive salts. yes 21. A method of treating a patient with disseminated intravascular coagulation, in which the patient is administered peptidyl arginal, which has the structure 4 Ne DYN, 4 t zay iy ESHEE in p: NEK VE VEN or its pharmaceutically acceptable acid additive salts. 22. The method according to any one of claims 16-21, characterized in that the patient is administered from about 0.1 mg to about 50 mg/kg peptidyl arginal, depending on the body weight of the patient. 23. The method according to any one of claims 16-21, characterized in that peptidyl arginal is administered to the patient in a dose sufficient to achieve a peptidyl arginal blood level of from about 6 μm to about 100 μm. 24. The method according to any one of claims 16-23, which is characterized by the fact that peptidyl arginal is administered simultaneously or sequentially. Official bulletin "Industrial Property". Book 1 "Inventions, useful models, topographies of integrated microcircuits", 2006, M 12, 15.12.2006. State Department of Intellectual Property of the Ministry of Education and Science of Ukraine. se that o (ee) se «I cha - - with . a -I -I t» with 50 so (F) ko bo b5