The tripeptide or its pharmaceutically acceptable salt or solvate, the pharmaceutical composition having inhibitory activity against thrombin amidase

 

(57) Abstract:

Usage: the invention provides (1R, 4aR, 8aR)-1,2,3,4,5,6,7,8-perhydrosqualene-1-carbonyl-(L)-prolinol-(L)-arginine aldehyde and pharmaceutically acceptable salt and solvate, pharmaceutical formulation containing the above compounds and methods of their use as thrombin inhibitors, inhibitors of coagulation and drugs for the treatment of thrombosis embolism. 2 S. and 2 C.p. f-crystals, 1 table.

The invention relates to inhibitors of thrombin, which are anticoagulants in humans and animals. In particular, it concerns a derivative of the dipeptide L-Proline-L-arginine-aldehyde with high antithrombotic activity.

Inhibition of thrombin currently achieved by the introduction of heparins and coumarins. The mechanism of action of these drugs is studied. Heparins are only introduced parenterally, and their levels must be controlled accurately. Coumarins are blocking or inhibition of the formation of prothrombin and require some time to achieve maximum efficiency.

Although the heparins and coumarins are effective anticoagulants, there is a need in antithrombolytic existing clots.

The present invention discovers that the connection of the present invention is much stronger inhibitor of thrombin than the corresponding 4aS, 8aS enantiomer.

Accordingly, the main purpose of the present invention is (1R, 4aR, 8aR)-1,2,3,4,5,6,7,8-perhydrosqualene-1-carbonyl-(L)-prolinol-(L)-arginine-aldehyde, its pharmaceutically acceptable salt and solvate, which are suddenly more powerful inhibitors of thrombin and suitable as anticoagulants and drugs against thrombosis embolism.

Proposed by this invention a compound is expressed by the following formula I.

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where A is a

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and its pharmaceutically acceptable non-toxic salts and solvate.

The peptide expressed by the formula I, suitable antithrombotic drug and can be used as an auxiliary drug tissue plasminogen activator (tPA), streptokinase, or urokinase therapy.

Connection receive conventional methods of obtaining [1-3]. For example, in the interaction of Cbz-1,2,3,4,5,6,7,8-perhydro-hydro-1-ethinlestradiol acid ester of L-Proline is formed ether Cbz-1,2,3,4,5,6,7,8-perhydrosqualene-1-carbonyl-Pro. The ether group is Cbz-1,2,3,4,5,6,7,8-perhydrosqualene-1-carbonyl-Pro-Arg of lactam protected on the amino group form. Arg laktamovogo ring open recovery and remove a protective group from arginine amino and perhydrosqualene nitrogen and receive aldehyde 1,2,3,4,5,6,7,8-perhydrosqualene-1-carbonyl-Pro-Arg. The peptide is converted into a suitable salt such as the acetate and sulfate.

1,2,3,4,5,6,7,8-perhydro-1-ethanolammonium acid is easily obtained by hydrogenation of 1-ethinlestradiol acid in the standard or other suitable alcohol in the presence of 5N hydrochloric acid or other suitable strong inorganic acid over 5% Rh/A12O3or other appropriate catalyst at a pressure of from about 500 to 1000 pounds/inch2and at a temperature of from about 30oC to approximately 80oC.

This technique allows to obtain (1R, 4aS, 8aS)-1,2,3,4,5,6,7,8-perhydro-1-ethanolammonium acid and (1S, 4aR, 8aR)-1,2,3,4,5,6,7,8-perhydro-1-ethanolammonium acid as a racemic mixture. Individual enantiomers may be obtained by separation of the racemate classical methods. Such methods include the formation of salts with optically active acids and separation using liquid chromatography high pressure of the racemate by chiral column.

Thermodynamic from the OIC acid, allocate obtaining esters of racemic mixtures using the above methods and reaction of these esters with sodium ethylate in the pattern and then deesterification the resulting racemic mixture thermodynamic isomers.

Otherwise, the racemic mixture of isomers and racemic mixture thermodynamic isomers, as acids protected at the amino group interacts with the Proline-protected carboxyl, with the formation of the dipeptide. Diastereoisomer formed by the interaction with L-Proline, separated by crystallization.

Ester group, protecting carboxypropyl prolinnova part of the dipeptide, then remove (release or deesterification) and dipeptide in the form of the free acid interacts with the arginine lactam. This product (arginine lactam product) is reacted with a hydride reducing agent, preferably alumoweld lithium or three-tert-butoxylated lithium, in an inert solvent or mixture of solvents, to restore laktamovogo rings and receive Tripeptide in orginaltitel form. Then remove the protective group by known methods, such as hydrogenation metal catalyst.

The invention also provides m is of use in this method.

As shown in the formula I, asymmetric center prolinnova and arginine parts is L.

PermitRootLogin, including D - 1,2,3,4,4 and,5,6,7,8,8 and-DECA - hydroisoquinoline-1-carbonyl(perhydrosqualene-1-carbonyl or I-Pig) and D-1,2,3,4,4 a, 5,6,7,8,8 and-decahydroquinoline-3-carbonyl (perhydrosqualene-3-carbonyl or 3-Pig) derived Pro-Arg-H below.

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PermitRootLogin can exist as CIS - or transcutaneously. For example, the following pair of stereoisomers that are identified as CIS-, can be formed for D-perhydrosqualene-1-carbonyl-L-prolyl-L-arginine-aldehyde:

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Pharmaceutically acceptable salts of the peptides of the invention include salts formed with inorganic or carboxylic acids. Examples of inorganic acids, forming salts include halogen acids such as hydrochloric, Hydrobromic acid, phosphoric acid, sulfuric acid. Salts of carboxylic acids are formed with such acids as acetic, propionic, malonic, maleic, citric, succinic, malic, benzoic, fumaric, and other carboxylic acids. Salts with acids receive conventional methods, for example, through the neutralization of the compound I in the form of NWO is but higher the present invention includes a solvate of the compounds of this invention and their pharmaceutically acceptable salts. The compound of the present invention or its pharmaceutically acceptable salt may form a solvate with water or common organic solvents. Such a solvate is included in the scope of the present invention.

The compound represented by formula I, get known methods of peptide synthesis. According to one such method, acid A-COOH, where A has the same meaning given for formula I and the nitrogen atom is protected with suitable protective group for the amino group interacts with the Proline-protected carboxy, with the formation of the dipeptide. Ether group, protecting carboxypropyl prolinnova part of the product, is removed, and the dipeptide in the form of the free acid interacts with the lactam form of arginine. The above sequence of reactions is illustrated in the following diagram.

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where P represents a protective group for the amino group.

Obtained Arg(P)lactam, the product (c) restored with lithium aluminum hydride in an inert solvent to the cleavage lactam ring and get Tripeptide in orginaltitel form formula:

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first converted into an active ester form, such as a mixed anhydride, with chloroformiate, such as ethylchloride to isobutylacetate. The formation of the ester is performed in the presence of a tertiary amine, such as N-methylmorpholine. Adding a strong tertiary amine as the base, such as triethylamine, carries out internal acylation to obtain a lactam form diaminohexane arginine, as shown below:

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Before the interaction with A(C=O)-Pro-OH as shown in the diagram above, Boc protective group is selectively removed from triperoxonane acid to obtain the desired free amino group.

Linking ACOOH connection with prolinnova ether is performed after protection of the amino group of amino acids. For temporary protection or blocking of amino groups typically use standard protective groups for amino groups. Examples of such protective groups are alkoxy, alkenylacyl, cycloalkane and aryloxyalkyl groups, such as etoxycarbonyl, tert-butyloxycarbonyl (Boc), cyclohexyloxycarbonyl, adamantanecarbonyl, trichlorocyanuric, benzyloxycarbonyl (Cbz), diphenylmethylene and other podobn is advised of the commonly used easily removable groups such as tert-butyl, benzyl, n-nitrobenzyl, n-methoxybenzyl, diphenylmethyl, trichlorethyl, pencil or trialkylsilyl esters. When performing a reaction between use such ester group for Proline, which is removed in the conditions under which the protective group of the amino group remains intact. The protective group of amino group alleluya acid ACOOH, thus, remains in place to protect the amino group in the subsequent transformation with arginylation connection with the formation of form C.

Peridomicile group obtained by hydrogenation or partially restored or unsaturated acids by conventional methods. For example, 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid hereroense over platinum oxide in a solvent such as ethanol or acetic acid, before the formation of perhydro(decahydro)isoquinoline-1 - carboxylic acid. Targetability then used, as described above, when the acylation prolinnova ether. Examples of such PermitRootLogin represented by formula I are N-(D-decahydroquinoline-1-carbonyl)-L-prolyl-L-arginine-aldehyde and N-(D-decahydroquinoline-3-carbonyl)-L-prolyl-L-arginine.

The process described above, gidrirovanii are formed in larger quantities. For example, the compounds include D-I-(4aS, 8aS)-Pig-(L)-Pro-(L)-Arg-H, D-I-(4aR, 8aR)-Pig-(L)-Pro-(L)-Arg-H, D-I-(4aS, 8aR)-Pig-(L)-Pro-(L)-Arg-H, D-1-(4aR, 8aS)-Pig(L)-Pro-(L)-Arg-H, D-3-(4aS, 8aS)-Pig-(L)-Pro-(L)-Arg-H, D-3-(4aR, 8aR)-Pig-(L)-Pro-(L)-Arg-H, D-3-(4aS, 8aR)-Pig-(L)-Pro-(L)-Arh-H, and D-3-(4aR, 8aS)-Pig-(L)-Arg-H.

The reaction described above is preferably performed at low temperatures from -20oC to 15oC. the Reaction is performed in an inert organic solvent such as dimethylformamide, dimethylacetamide, tetrahydrofuran, methylene chloride, chloroform, and other such conventional solvents. Mainly used anhydrous conditions, when the reaction between the active ester alleluya acid is used.

Compounds of the invention are better allocated in the form of a salt with the acid. Salts of compounds of formula I formed with acids, as mentioned above, suitable pharmaceutically acceptable for administration antithrombotic drugs and to obtain preparative forms of these agents. Other salts with acids can be obtained and used for the separation and purification of peptides. For example, salts formed with sulfonic acids, such as methanesulfonate, n-butanesulfonate, n - toluensulfonate and naphthalenesulfonate, can be used.

Preferred is an in Pat. USA 5250660 includes preparative purification of C18reversed-phase chromatography. The aqueous phase comprises sulfuric acid or hydrochloric acid in concentrations between about 0.01 and about 0.05%; the organic phase is acetonitrile, THF, methanol or other suitable solvents. The acidic pH of the eluate is adjusted between about pH 4 and about pH 6, the exact pH, which is a function of individual peptide, with the main resin, such as Bio-Rad AG-IX8 resin in the hydroxyl form. After adjusting the pH of a solution of salts of Tripeptide, such as sulfate or hydrochloride, spend lyophilization to obtain a purified salt in the form of a dry powder. In the example of crude 1-(1R, 4aR, 8aR)-Pig-L-Pro-L-Arg-H sulfate contaminated epimeres D-Arg-H sulfate, dissolved in water and the solution is injected on column 5 x 50 cm Vydac C18RPHPLC. Use the gradient of 2-20% B (A = 0,01% H2SO4; B = acetonitrile) for 10 hours. Fractions are collected and the fractions containing the desired product, as determined by analytical RP-HPLC (reversed-phase liquid chromatography high pressure), unite. The value of pH of the combined fractions is adjusted approximately from 4.0 to 4.5 with resin Bio-Rad AG-IX8 in hydroxyl form. The solution polagayut for the selective inhibition of thrombin in comparison with other proteases and non-enzymatic proteins in blood coagulation without significant obstacles ability to dissolve natural clots in the blood (compounds have a low inhibitory effect on the dissolution of blood clots). In addition, this selectivity allows use with drugs that dissolve blood clots, without significant intervention in the processes of dissolution of blood clots and blood clots.

The connection proposed by the invention (formula I) selectively inhibits the action of thrombin in humans and animals (mammals). Inhibition of thrombin is shown by inhibiting the activity of amidase thrombin. The following table I lists the apparent equilibrium constant (Kass) for the interaction between the test compound (inhibitor) and thrombin. The data in the table obtained by the analysis, in which thrombin hydrolyzes the chromogenic substrate, N-benzoyl-D-i.e. phenylalanyl-L-poured-L-arginine-n-nitroanilide.

The analysis performed in 50 μl buffer (0.03 M Tris-buffer, 0.15 M NaC1, pH of 7.4) with 25 μl of a solution of human thrombin (purified human thrombin, Enzyme Research Laboratories, South Bend, Indiana, at 8 NIH units/ml) and 25 μl of test compound in the solvent (50% aqueous methanol (about:). Then make 150 μl of an aqueous solution of a chromogenic substrate (0.25 mg/ml) and measure the rate of hydrolysis of the substrate by monitoring the reaction at 405 nm for the release of n-nitroaniline. Standard curves get build dependencies free com is converted to the amount of free thrombin in the corresponding analysis using standard curves. The amount of bound thrombin (associated with the test compound) is calculated by subtracting the amount of free thrombin observed in each analysis, from a known initial amount of thrombin observed in each analysis. The amount of free inhibitor in each assay is calculated by subtracting the number of moles of bound thrombin from the number of moles of added inhibitor (test connection).

Value Kass is a constant hypothetical equilibrium for the reaction between thrombin and the tested compound (1).

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Kass is calculated for a range of concentrations of the test compounds and the average value is given in units of liter per mole.

Using mainly the following techniques described above for human thrombin using other serine proteases of the coagulation system of the blood and uses serine proteases fibrinolytic system, with the appropriate chromogenic substrates, identified below, the selectivity of the compounds of the present invention in accordance with serine proteases of blood coagulation and fibrinoliticakimi serine proteases appreciate as well as the absence of the intervention is South Bend, Indiana. Chromogenic substrate N - benzoyl - Ile - Glu - Gly - Arg - n-nitroanilide (for factor Xa) buy from Kabi Vitrum, Stockholm, Sweden, or the Midwest Biotech, Fishers, Indiana. Bovine trypsin buy from Worthington Biochemicals, Freehold, New Jersey. Chromogenic substrate, N - benzoyl-Phe - Val - Arg - n - nitroanilide, a substrate for thrombin person and trypsin, are synthesized according to the methods described above for compounds of the present invention using well-known methods of peptide synthesis from commercially available reagents or buy from Midwest Biotech, Fishers, Indiana.

The plasmin person buy from Boehringer Mannheim, Indianapolis, Indiana; nt-PA buy as a single purchase from American Diagnostica, Greenwich, Connecticut. Chromogenic plasmin substrate H-D-Val-Leu-Lys-n-nitroanilide and the substrate of tissue plasminogen activator (t-PA), H-D-Ile-Pro-Arg-n-nitroanilide, buy from Kabi Vitrum, Stockholm, Sweden.

In chromogenic substrates described above, use the three-letter symbols - Ile, Glu, Gly, Pro, Arg, Phe, Val, Leu and Lys, they show a group of the corresponding amino acids, isoleucine, glutamic acid, glycine, Proline, arginine, phenylalanine, valine, leucine, and lysine, respectively.

The table represents the list of values Kass obtained with these compounds.

The compound of the invention selectively ironer compounds have low inhibitory dissolution of fibrin ability.

The invention in one of its aspects provides a method for inhibiting the formation of blood clots in humans and animals (mammals), which includes the introduction of man or animals, the compounds of formula I at a dose of effectively inhibiting the formation of clots and non-toxic. Connection-anticoagulant is administered orally, parenterally, e.g. by intravenous infusion (in/in), intramuscular (I/m) or subcutaneously (s/C).

Dose, effectively inhibiting the formation of clots, is between about 5 mg and about 1000 mg Mode of doses may vary, for example, for prophylactic use can be entered or a single daily dose, or multiple doses of 3 or 5 times a day, respectively. In critical situations the compound of the invention is introduced into/in infusion at a dose between about 0.1 mg/kg/hour and up to about 1.0 mg/kg/hour.

In oral introduction the dose of a compound of the present invention is from about 0.1 mg/kg to about 20 mg/kg and may be administered one or more times within 24 hours. Preferably, the dose is about 0.5 mg/kg to about 5 mg/kg and placed 4 times per 24 hours. These connections can the ptx2">

The method of this invention may also be used in combination with agents that dissolve clots, for example, tissue plasminogen activator (tPA), a modified tPA, streptokinase or urokinase. When is the formation of clots and blocked artery or vein, or partially, or fully, is commonly used agent, dissolving clots. The compound of the invention can be administered together with a dissolving agent, or after use to prevent re-formation of clots.

This invention also provides a pharmaceutical formulation for use in the above-described therapeutic methods. Pharmaceutical formulation include effective for the inhibition of thrombin amount of the compounds of formula I in combination with a pharmaceutically acceptable carrier, excipient or diluent. For oral administration antithrombin drugs formulated in gelatin capsules or tablets, which may include excipients, such as binders, lubricating substance, disintegrity agent and the like. For parenteral administration antithrombosis substance is formulated in a pharmaceutically acceptable diluent, such as is subramania can be formulated in preparative form including a unit dose comprising between about 0.1 mg and about 1000 mg, Preferably, the compound used in the form of pharmaceutically acceptable salts, such as, for example, sulfate, acetate or phosphate. For example, preparative form in a unit dose includes 5 mg of the compound of the present invention as a pharmaceutically acceptable salt in 10 ml sterile glass vials. Another example of the preparative form a unit dose form comprising 10 mg of the compound of the present invention as a pharmaceutically acceptable salt in 20 ml of isotonic saline, placed in a sterile vial.

The compounds can be administered in various ways, including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular and inside of the nose. Compounds of the present invention are preferably formulated prior to their introduction. Another embodiment of the present invention is a pharmaceutical form of application that includes 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 forms castlestone with other ingredients of the formulation and not having a harmful effect on patients.

These preparative pharmaceutical form get by known methods from readily available ingredients. The compositions of this invention may be formulated using known methods to provide after administration to the patient quickly, supported or delayed release of the active ingredient. When preparing the compositions of the present invention the active ingredient is usually mixed with a carrier, or diluted by a carrier, or include inside the carrier, which may be in the form of capsules, bags, paper or other container. When the carrier serves as a diluent, it may be solid, semi-solid, or liquid material which acts as a binder, experient or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pellet, powder, pellets, bags, those capsules, 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 following examples preparative forms illustrate, but not limit the scope of the invention. "Active ingredient" is, of course, Osnach is 1

Hard gelatin capsules are prepared using the following ingredients (mg/capsule):

Active ingredient: 250

Starch, dried 200

Magnesium stearate - 10

The total number of 460

Preparative form 2

Tablets obtained using the ingredients below (mg/capsule):

Active ingredient: 250

Cellulose, microcrystalline - 400

Silicon dioxide, dust - 10

Stearic acid - 5

Total 665

The components are mixed and pressed into tablets, each weighing 665 mg

Preparative form 3

Spray solution derived from the following components:

Active ingredient - 0,25

Ethanol - 25,75

The propellant 22 (Chlorodifluoromethane) - 70,00

Total 100,00

The active compound is mixed with ethanol and the mixture added to a portion of the propellant 22, cooled to -30oC and transferred to a filling device. The required number then bring in a stainless steel container, dilute the remaining part of the propellant. Then to secure the container valve.

Preparative form 4

Tablets, each containing 60 mg of active ingredient, are prepared as follows (mg):

Carboximetilkrahmal sodium - 4,5

Magnesium stearate and 0.5

Talc - 1

Total - 150

The active ingredient, starch and cellulose are passed through sieves with openings N 45 U.S. and thoroughly mix. An aqueous solution containing polyvinyl pyrrolidone is added to the obtained powder, the mixture is passed through sieve No. 14. The granules thus obtained, dried at 50oC and passed through sieve No. 18. Carboximetilkrahmal sodium, magnesium stearate and talc, previously passed through sieve # 60 is added to the granules which, after mixing is pressed on a special device to obtain tablets, and get the pills, each of which weighs 150 mg.

Preparative form 5

Capsules, each containing 80 mg of the active ingredient, was prepared as follows (mg):

Active ingredient - 80

Starch - 59

Microcrystalline cellulose - 59

Magnesium stearate - 2

Total - 200

The active ingredient, cellulose, starch and magnesium stearate are blended, passed through sieve No. 45 and the mixture is filled capsules hard gelatin amount of 200 mg.

Preparative form 6

Suppositories, each containing 225 mg of accesat - 2000

Total - 2225

The active ingredient is passed through sieve # 60, suspended in glyceride saturated fatty acids, previously melted using the minimum heat. The mixture is then poured into the form of suppositories with a nominal capacity of 2 g and cool.

Preparative form 7

The suspension containing 50 mg of active ingredient per 5 ml dose, was obtained as follows (mg):

Active ingredient - 50

Carboxymethylcellulose sodium - 50

Syrup - 1,25

A solution of benzoic acid - 0,10

Odorant - mind

Dye - mind

Purified water to total 5

The active ingredient is passed through a sieve No. 45, mixed with carboxymethylcellulose sodium and syrup until smooth paste, add with stirring a solution of benzoic acid, fragrance and dye, diluted part water, and then make the necessary amount of water to obtain the desired volume.

Preparative form 8

Preparative form for intravenous obtained as follows (mg):

Active ingredient - 100

Isotonic saline solution - 1000

The solution of the above ingredient dose includes 5 mg sulfate (1R, 4aR, 8aR)-perhydrosqualene-1-carbonyl-L-prolyl-L-arginine-aldehyde in 10 ml sterile glass vials. Another example of a formulation with a single dose is 10 mg sulfate (1R, 4aR, 8aR)-perhydrosqualene-1-carbonyl-L-prolyl-L-arginine-aldehyde in 20 ml of isotonic saline, placed in a sterile vial.

The preferred preparative form is the form of a unit dose comprising between 5 mg and 50 mg sulfate (1R, 4aR, 8aR)-perhydrosqualene-1-carbonyl-L-prolyl-L-arginine-aldehyde in a sterile vial.

The following examples offer a further description of the invention and do not limit the scope of the invention.

The value of Rfin the following examples is determined by thin-layer chromatography on silica gel using kieselgel 60F-254 (Merck, Darmstradt) in the following solvent system:

(A) chloroform-methanol-acetic acid, 135:15:1, about:about:about

(B) ethyl acetate - acetic acid, absolute ethanol, 90:10:10, about:about: about

(C) chloroform : methanol : acetic acid, 90:30:5, about:about:about

(D) ethyl acetate : hexane, 30:70, about:about

In the examples, the following methods analytical HPLC (liquid chromatography high pressure).

Method 1. Waters 600 E with spawanie gradient A = 0.01 M ammonium acetate and B = acetonitrile.

Method 2. Pharmacia FPLC using Pep RPC 0.5 x 5.0, see the Monitoring is performed on a Pharmacia UV-M at 214 nm using a gradient or A = 0.01 M ammonium acetate, or B = acetonitrile.

The symbols used here have the following meanings:

Amino acids: Arg = arginine, Pro = Proline, Phg = phenylglycine

Boc = tert-butyloxycarbonyl

Bz1 = benzyl

Cbz (or z)=benzyloxycarbonyl

DCC = dicyclohexylcarbodiimide

DMF = dimethylformamide

DMSO = dimethylsulfoxide

FAB-MS = mass spectrum at the bombardment of stable atoms

FD-MS = mass spectrum (field desorption

THF = tetrahydrofuran

TLC = thin layer chromatography

Example 1. Obtaining (1R, 4aR, 8aR)-perhydrosqualene-1-carbonyl-L-prolyl-L-arginine-aldehyde

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Methylcarbamate-phenethylamine (I)

To a solution of phenethylamine (75,22 ml, 0.6 mol) and triethylamine (83 ml, 0.6 mol) in THF (500 ml) with stirring, slowly add methylchloroform (46.2 ml, 0.6 mol) in THF (50 ml), stirred for 1 hour at room temperature and contribute diethyl ether (2 l) and I N HCl (800 ml). The organic layer was washed with water, dried (MgSO4), filtered, the filtrate evaporated in vacuum and get a clear oil of pure titled compound is Arbalet-phenethylamine (1) (102 g, of 0.57 moles) in triperoxonane acid (300 ml) was added glyoxalase acid (63 g of 0.68 moles), boiled for 4 hours, cooled to room temperature, the solvent evaporated in vacuum. The residue is treated with diethyl ether (800 ml) / water (100 ml), the pH of the reaction mixture was adjusted to 12 by addition of 5N NaOH. The aqueous layer was separated, added thereto diethyl ether (500 ml), acidified to pH 2.5 to 5N HCl. The organic layer is separated, dried (MgSO4), filtered, evaporated in vacuum and get pure titled product as an oil (107 g, 80%); FAB-MS 236 (MH+).

Tert-butyl ether methylcarbamate-DL-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid (3)

To a cooled (0oC) to a solution methylcarbamate-DL-1,2,3,4 - tetrahydroisoquinoline-1-carboxylic acid (2) (105 g, 0.45 mol) in CH2Cl2(200 ml) under stirring add tertbutanol (52 ml of 0.54 moles), 4-dimethylamino-pyridine (10 g, of 0.08 mol) and DCC (92 g, 0.45 mol), stirred for 2 hours at 0oC, 24 hours at room temperature, evaporated the solvent in vacuo and add to the residue ethyl acetate (800 ml)/IN NaHCO3(300 ml). The organic layer was separated, sequentially washed with water, 1.5 N citric acid and water. The organic layer is dried (MgSO4), filtered, the filtrate evaporated in elementry analysis for C16H21NO4:

Calculated: C 65,96 H 7,27 N 4,81

Found: C 66,24 H 7,28 N 4,73

Tert-butyl ether methylcarbamate-DL-1,2,3,4,6,7,8-perhydrosqualene-I-carboxylic acid (4)

A solution of tert-butyl methyl ether of methylcarbamate-DL-1,2,3,4-tetrahydroisoquinoline-I-carboxylic acid (3) (105 g of 0.36 moles) in tertbutanol (800 ml) hydronaut 5% Rh/Al2O3(52,5 g) at 800 pounds/inch2equipment high pressure at 50oC for 24 hours. The reaction mixture was filtered through a pad of celitethe filtrate is evaporated in vacuum. The oil obtained is dried and receive net named connection points (96.5 g, 90%); FD-MS 298 (MN+); TLC Rf(C) 0,63.

Ethyl ether methylcarbamate-1,2,3,4,4 aS,6,7,8,8 aS perhydrosqualene-S-I-carboxylic acid + Ethyl ether methylcarbamate-1,2,3,4,4 aR, 6,7,8,8 aR-perhydrosqualene-R-I-carboxylic acid (5)

To a solution of tert-butyl methyl ether methylcarbamate-DL-I-2,3,4,6,7,8-perhydrosqualene-I-carboxylic acid (4) of them (81.2 g, 273 mmol) in EtOH (500 ml) is added sodium ethylate (21% in ethanol) (88,4 ml, 273 mmol), the reaction is boiled for 24 hours. The organic solvent is evaporated in vacuum, the residue is treated with ethyl acetate (400 ml) and water (100 ml). The organic layer was separated, washed with water, dried (MgSO+); TLC Rf(A) 0,61.

Methylcarbamate-1,2,3,4,4 aS, 6,7,8,8 aS perhydrosqualene-S-I-carboxylic acid + Methylcarbamate-1,2,3,4,4 aR,6,7,8,8 aR-perhydrosqualene-R-I-carboxylic acid (6)

To a solution of 5 (50 g, 260 mmol) in THF (250 ml) was added 2 N NaOH (156 ml, 312 mmol), the reaction is stirred for 30 hours at room temperature. The organic layer is evaporated in vacuo, the residue treated with diethyl ether (400 ml) and water (100 ml). The aqueous layer was separated, add ethyl acetate (400 ml), the solution acidified with 5N HCl to pH of 2.0. The organic layer is dried (MgSO4), filtered, the filtrate evaporated in vacuum and get a clear oil, which crystallized from hexane (200 ml) and obtain pure titled compound (46.4 g, 74%); FAB-MS 242 (MH+); TLC 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

Cbz-1,2,3,4,4 aS, 6,7,8,8 aS perhydrosqualene-S-I-carboxylic acid + Cbz-1,2,3,4,4 aR,6,7,8,8 aR-perhydrosqualene-R-I-carboxylic acid (7)

To a solution of 6 (46 g, 191 mmol) in anhydrous CH3CN (200 ml) under stirring at room temperature in an inert atmosphere was added a solution of attributively (of 62.4 ml, 440 mmol) in CH3CN (60 ml). The reaction mixture is stirred for 30 minutes at 554), filtered, the filtrate evaporated in vacuum and get the pure titled compound as oil (39.5 g, 65%); FAB-MS 318 (MN+);

Elemental analysis for C18H23NO4:

Calculated: C 68,12 H 7,30 to 4.41 N

Found: C 66,37 H 7,52 N 4,37

Cbz-1,2,3,4,4 aS, 6,7,8,8 aS perhydrosqualene-S-I-carbonyl-Pro-tert-Bu + Cbz-1,2,3,4,4 aR,6,7,8,8 aR-perhydrosqualene-R-I-carbonyl-Pro-tert-Bu (8)

To a solution of 7 (39 g, 123 mmol) in DMF (200 ml) under stirring and cooling (0oC) was added tert-butyl ether Proline (21.1 g, 123 mmol), I-hydroxybenzoate-azole (16.6 g, 123 mmol) and DCC (25,3 g, 123 mmol), stirred for 2 hours at 0oC and 24 hours at room temperature. The precipitation is filtered off, the filtrate evaporated in vacuo to an oil. The oil was dissolved in EtOAc (200 ml) and water (100 ml). Organic elaut, dried (MgSO4), filtered, the filtrate evaporated to an amorphous solid and get a named connection (52.7 g, 91%), FAB-MS 471 (MN+).

Cbz-1,2,3,4,4 aR,6,7,8,8 aR-perhydrosqualene-(IR)-carbonyl - Pro-OH (9)

To a solution of 8 (52,4 g, III mmol) in CH2Cl2(20 ml) was added triperoxonane acid (70 ml) and anisole (5 ml), stirred for 1 hour at room temperature, evaporated in vacuo without heating, diluted with diethyl ether (400 ml), water (100 ml) and the pH of the solution adjusted to a pH of 10.0 with 5N NaOH. The aqueous layer was separated, add ethyl acetate (300 ml), acidified with 5N HCl to pH of 2.5. The organic layer is separated, dried (MgSO4), filtered, the filtrate evaporated in vacuum and get a clear oil. The oil was dissolved in diethyl ether (500 ml) and add L(-)-[]D-methylbenzylamine, stand 24 hours at room temperature, the precipitate is filtered off, washed with diethyl ether and dried. The solid residue is suspended in ethyl acetate, washed with 1.5 N citric acid and water. The organic layer is dried (MgSO4), filtered, the filtrate evaporated and get a named connection in the form of oil (20.2 g, 44%); FAB-MS 415 (MN+); []D=3,2o(C, 0.5 in MeOH);

Elemental analysis for C23H30N2O5rg-OH x Hcl) (82,1 g, 250 mmol) is dissolved in 240 ml of 5 N NaOH in a three-neck round-bottom flask, cooled to -5oC and added dropwise within 56 minutes benzylchloride (143 ml, 1.0 mol, 4 equiv.) support by adding 5 N NaOH (250 ml) pH at 13,2-13,5. The reaction mass is stirred for 1 hour at a temperature of -5oC, diluted with 100 ml of water and 500 ml of diethyl ether. The aqueous layer was separated, extracted twice with 40 ml portions of diethyl ether. The aqueous layer was acidified with 3 N H2SO4(560 ml) to pH 3.0 and extracted with 550 ml of ethyl acetate. The separated aqueous layer is extracted only by ethyl acetate, the extract combined with the previous an ethyl acetate extract. The combined extracts washed with water, dried over MgSO4, evaporated in vacuum to dryness. The remainder RUB clean with ether, the precipitated product is filtered off and dried, yielding of 66.1 g (3) Boc-Arg(Cbz)-OH (65% of theory): TLC Rf(C) 0,43; FD-MS 408(M+).

1H NMR (CDCl3), to 1.42 (s, 9H), 1,61 is 1.91 (m, 4H), 3,23-to 3.41 (m, 2H), 4,17 (d, IH), a total of 5.21 (s, 2H), 5,62 (d, IH), 7,30-7,42 (m, 6H), of 8.37 (m, 1H).

(B) Boc-Arg(Cbz)-lactam

A solution of Boc-Arg(Cbz)-OH (A) obtained by the method described previously (66,0 g rate £ 0.162 moles) in 230 ml of dry THF cooled in a bath of ice-acetone to -10oC, to the cooled solution was added N-methylmorpholine (18.7 ml of 1.05 is dobavlaut the triethylamine (23,5 ml, 1,05 EQ. ), stirred for 1 hour at -10oC and 1 hour at room temperature. The reaction mixture is poured into 1 l of a mixture of ice-water and drops the named compound, which is filtered off, washed with cold water, dried under vacuum, and crystallized from ethyl acetate and get 38,05 g (60% of theory) of the named compound, Boc-Arg(Cbz)-lactam; TLC Rf(A) 0,77; FD-MS 291 (MH+).

1H-NMR (CDCl3), to 1.48 (s, 9H), 1,78-to 1.98 (m, 2H), 2,50 (m, IH), to 3.41 (m, IH), 4,43 (m, IH), of 4.90 (m, IH), of 4.16 (s, 2H), 5,27 (m, IH), 7,28 was 7.45 (m, 6H), 9,41 (m, IH), 9,68 (m, IH).

Cbr-1,2,3,4,4 ar, 6,7,8,8 ar-perhydrosqualene-R-I-carbonyl-Pro-Arg(Cbz) lactam (II)

The bulb 1. To compound 9 (13,9 g, 33.5 mmol) in DMF (50 ml), cooled to -15oC was added N-methylmorpholine (3,7 ml, 33.5 mmol), then, contribute isobutylparaben (4,4 ml, 33.5 mmol), stirred for 1 minute at -15oC.

The bulb 2. To HCl x Arg(Cbz)-lactam (10,9 g, 33.5 mmol) dissolved in DMF (50 ml) and cooled to 0oC, add diisopropylethylamine (14.6 ml, is 83.8 mmol), stirred for 1 minute at 0oC.

The contents of the flask 2 was added to the contents of the flask 1, the reaction mixture is stirred for 2 hours at -15oC, then 2 hours at room temperature, make I N NaHCO3(10 ml) and evaporated in vacuo of the solvent to oil. the second solution is dried (MgSO4), filtered, evaporated in vacuo to a crude solid residue, which is purified chromatographically on silica gel using a stepwise gradient elution (hexane 100 to hexane-EtOAc 30:70), and allocate the net connection 10. (9.0 g, 39%); FAB-MS 687 (MH+);

Elemental analysis for C37H46N6O7:

Calculated: C 64,71 H 6,75 N 12,24

Found: C 64,23 H 6,69 N 11,88

Cbr-1,2,3,4,4 ar, 6,7,8,8 aR-perhydrosqualene-R-I-carbonyl-Pro-Arg (Cbz)-H (12)

To a solution of II (9.0 g, 13.1 mmol) in anhydrous THF (100 ml) with stirring under cooling to -70oC in nitrogen atmosphere add to alumoweld lithium I M in THF (13.1 ml, 13.1 mmol), stirred for 30 minutes at this temperature, make dropwise 5 ml of THF and 5 ml of 0.5 N H2SO4, diluted with EtOAc (175 ml) and water (100 ml). The organic layer is separated, dried (MgSO4), filtered, evaporated in vacuo of the organic solvent and receive an amorphous solid product of the above compound (8.2 g, 91%); FAB-MS 689 (MH+).

1,2,3,4,4 ar, 6,7,8,8 ar-perhydrosqualene-R-I-carbonyl-Pro-Arg-H H2SO4(12)

Compound 12 (8,2 g, 11.9 mmol), dissolved in ethanol (50 ml), water (10 ml) and the I N H2SO4(30 ml, 29.7 mmol), hydronaut in the presence of 5% Pd/c catalyst (4.0 g) at room temperal and add 50 ml of water. The pH value of the solution was adjusted to 4.2 using resin BioRad AGI-X8 (hydroxide form). The resin is removed by filtration, the solution lyophilizer and get soggy named connection (5,46). A solid residue (5,46 g) dissolved in 0.01% H2SO4and put on a column 5 x 25 cm Vydac C18the resin. For elution of the peptide from the column using a gradient of increasing c concentration of CH3CN (1% to 5%). Fractions are collected and combine according to analytical RP-HPLC (reversed-phase liquid chromatography high pressure). The pH value of the combined fractions was adjusted to 4.2 using AGI-X8 resin (Bio-Rad analytical uninominal resin with 50 - 100 holes) in the hydroxide form. The solution is filtered, the filtrate lyophilizer and get the pure titled compound (2.4 g, 39%); FAB-MS 421 (MH+; []D= -102,8o(C, 0,5/A 0.01 N H2SO4);

Elemental analysis for C21H36N6O3H2SO42H2O:

Calculated: C got 45.47 H 7,63 N 15,16

Found: C 45,05 H 7,44 N R 15,02

1. The Tripeptide of the formula

< / BR>
where And is a

or its pharmaceutically acceptable salt or solvate.

2. Sulfate compounds on p. 1.

3. Connection PP.1 and 2 having inhibitory activity in the in respect of amidase thrombin, containing as active principle a derivative of the dipeptide L-propyne-L-arginine-aldehyde and pharmaceutically acceptable carrier, excipient or diluent, characterized in that as a derivative of the dipeptide L-Proline-L-arginine-aldehyde it contains a connection on p. 1 or 2 in an amount of 0.1 to 1000 mg per unit dose.

 

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