Salt ethyl ester 3-(2-(4-(4-(aminoiminomethyl)phenyl)-4 - methyl-2,5-dioxoimidazolidin-1-yl)acetylamino)-3 - phenylpropionic acid, method for their production and pharmaceutical composition based on them

 

(57) Abstract:

The invention relates to compounds of the formula I, in all stereoisomeric forms and mixtures in any ratio, where NV denotes maleic acid, to a method for producing compounds of formula I, which lies in the fact that compounds of the formula II exercise anionic exchange with maleic acid and/or maleate. The invention relates to a pharmaceutical composition having inhibitory effect on platelet aggregation or overwhelming effect against thrombosis, for this reason, compounds of General formula I can be used as drugs for the prevention of diseases associated with the cardiovascular system. 3 S. and 7 C.p. f-crystals, 3 tables.

The present invention relates to salts 3-(2-(4-(4- (aminoiminomethyl)phenyl)-4-methyl-2,5-dioxoimidazolidin-1 - yl)acetylamino)-3-phenylpropionic acid of General formula I,

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where HB denotes maleic acid, and their physiologically acceptable salts, method for their production and to their use in medicines.

Hydrochloride ethyl ester 3-(2-(4-(4- (aminoiminomethyl)phenyl)-4-methyl-2,5-dioxoimidazolidin-1 - yl)acetylamino)-3-f is ako hydrochloride ethyl ester (S)-3-(2-((S)- 4-(4-(aminoiminomethyl)phenyl)-4-methyl-2,5-dioxoimidazolidin-1 - yl) acetylamino)-3-phenylpropionic acid, ethyl ester of (R) -3-(2-((R)-4-(4-(aminoiminomethyl)phenyl)-4-methyl-2,5 - dioxoimidazolidin-1-yl)acetylamino)-3-phenylpropionic acid, ethyl ester (S)-3-(2-((R)-4-(4-(aminoiminomethyl)phenyl)-4 - methyl-2,5-dioxoimidazolidin-1-yl) acetylamino)-3 - phenylpropionic acid ethyl ester (R)-3-(2-((S)-4-(4- (aminoiminomethyl)phenyl)-4-methyl-2,5-dioxoimidazolidin-1 - yl)acetylamino)-3-phenylpropionic acids have the disadvantage that they are amorphous and cannot be obtained in crystalline form. For this reason, they cannot be cleaned by means of crystallization, which makes them impracticable for use as active substances in medicinal products for which according to existing legal regulations are clearly defined requirements regarding the purity of the ingredients; they are equally unsuitable as a target product in the synthesis of pharmacologically active substances on an industrial scale, since the implementation of the methods of isolation and purification of this product, also in accordance with the statutory provisions must be complied with strictly defined conditions.

Their physical properties and because of the difficulty of their use for the preparation of pharmaceutics CLASS="ptx2">

Based on the foregoing, the invention was based on the task to obtain ethyl esters 3-(2-(4-(4-(aminoiminomethyl)phenyl)-4 - methyl-2,5-dioxoimidazolidin-1-yl)acetylamino)-3-phenylpropionic acid suitable for use in the above-mentioned purposes, non-hygroscopic form, thanks to which in a simple way provided the compliance with the requirements regarding purity, as well as the fulfillment of the requirements for the synthesis on an industrial scale, as well as compliance with the requirements of Galenika.

This task is solved in an unexpected way by getting malinovaklara salts of ethyl esters 3-(2-(4-(4-(aminoiminomethyl)phenyl)-4-methyl-2,5 - dioxoimidazolidin-1-yl)acetylamino)-3-phenylpropionic acids, especially salts containing these ethyl esters and maleic acid is in salt form (i.e., in the form of an acid additive salt) in a molar ratio of approximately 1:1.

The object of the present invention are in accordance with this salt ethyl ester 3- (2-(4-(4-(aminoiminomethyl)phenyl)-4-methyl-2,5-dioxoimidazolidin-1 - yl)acetylamino)-3-phenylpropionic acid of General formula I

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where HB denotes maleic acid, all stereoisomeric forms of the mules I are crystalline, they hygroscopic and have the unexpected advantage. They can under appropriate conditions to crystallize and purify by recrystallization and used in medicines, as well as for the preparation of medicinal products in the galenical form.

The present invention includes all stereoisomers of the compounds of General formula I, i.e., the isomer with (S)-configuration at the chiral center imidazolidinone rings and (S)-configuration at the chiral center in propionolactone fragment, isomer from (R)-configuration at the chiral center in imidazolidinone ring and (S)-configuration at the chiral center in propionolactone fragment, the isomer with (S)-configuration at the chiral center in imidazolidinone ring and (R)-configuration at the chiral center in propionolactone fragment and isomer from (R)-configuration at the chiral center in imidazolidinone ring and (R)-configuration at the chiral center in propionolactone fragment. Preferred of the above isomer and (S)-configuration at the chiral center in imidazolidinone ring and (S)-configuration at the chiral center in propionolactone fragment, i.e., the salt of the ethyl ester of (S)-3-(2- ((S)-4-(4-(aminoiminomethyl)phenyl)-4-methyl-2,5-dioxo the Yu acid, as well as its physiologically acceptable salt.

The present invention further includes all mixtures of two or more stereoisomers of General formula 1 in any proportions.

In the proposed according to the invention the compounds of General formula 1, containing ethyl esters 3-(2-(4-(4-(aminoiminomethyl)phenyl)-4-methyl-2,5-dioxoimidazolidin - 1-yl)acetylamino)-3-phenylpropionic acid and maleic acid in a molar ratio of approximately 1:1, one of the two COOH groups of maleic acid neutralized main amedieval group and submitted in salt form, i.e., in the form of a negatively charged carboxylate group (medinova group in ethyl ether after protonation of maleic acid are presented in this case in the form of positively charged amedieval group). The second of the two COOH-groups of the maleic acid can be represented in the compounds according to the invention in the acid form, i.e. in the form of COOH-groups, or it may be provided in salt form, i.e. in the form of carboxylate groups. The object of the present invention are as gidromolot, which presents one COOH-group, and which are among the preferred and their derivatives, predstavlayu.

Physiologically acceptable salts are thus primarily suitable for use in the pharmaceutical industry or non-toxic salts. The cations present in such salts are derived, obtained, for example, with alkali and alkaline earth metals such as sodium, potassium, magnesium or calcium, ammonia, or a physiologically acceptable organic amines, such as triethylamine, ethanolamine or Tris-(2 - hydroxyethyl) amine. Salts of compounds of General formula I can be obtained directly according to the following method by applying it, for example, the corresponding salts of maleic acid, or adding the appropriate bases. Another possibility to obtain these salts is that the received first gidromolot General formula I, in which one COOH-group, treated with the appropriate bases, such as hydroxides, carbonates or bicarbonates of alkali or alkaline earth metals, oxides of alkaline earth metals or amines. This COOH-groups may be completely or only partially converted into the salt form. The efficiency of the process such salt formation depends, in particular, on the value of pH, which want to get for the mV - in the salt form can be carried out also in those cases when the preparation of the pharmaceutical compositions of gidromolot General formula I are used together with the basic substances.

Compounds of General formula I can be obtained due to the fact that compounds of General formula II

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where HV is any inorganic or organic acid other than maleic acid, according to methods known to a person skilled in the art realize anionic exchange with maleic acid and/or maleate. Examples of acids of General formula HV are hydrochloric acid, Hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, propionic acid or methansulfonate. Examples of compounds of General formula II are represented by the compounds of formulas IIa and llb

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where HV in the General formula II represents hydrogen chloride, respectively acetic acid. The initial compounds of General formula II can be obtained by the method described in PCT/EP94/03491 and PCT/EP96/01572 (publication WO 96/33976), for example, hydrochloride (salt with hydrogen chloride) or salt with acetic acid. Concerning this aspect, the content of PCT applications is the currency can be used for example, sodium, lithium, potassium and ammonium salts of maleic acid or salts of maleic acid with organic ammonium cations. Preferably used maleic acid.

For the exchange of anions can be chromatographically, for example, a solution of compounds of General formula II with an ion exchanger with maleic acid. Suitable for this purpose solvents are, for example, water, alcohols, such as methanol, ethanol, butanol or isopropanol, and mixtures of these solvents, in particular water-alcohol mixture. You can also use commercially available anion-exchange materials, which in the usual manner is transferred first in the form of maleic acid or, respectively, reuse, restore to this form. Anion exchange is carried out usually at temperatures in the range from -10 to 40oC, primarily from -5 to 30oC, preferably from 0 to 25oC. From the eluate ion-exchange chromatography the desired product can then, if necessary after concentration to crystallize, for example, by cooling and/or deposition, and to allocate by filtration or centrifugation.

For anionic exchange you what storiesmy this includes a mixture of two or more solvents. Suitable for these purposes are, for example, water, alcohols, in particular those with 1 to 8 C-atoms, especially those with 1-6 C-atoms, preferably those with 1-4 C-atoms, ethers such as dioxane, tetrahydrofuran, mono - and dimethyl ether of ethylene glycol and diethylene glycol, ketones, such as acetone or butanone, NITRILES, such as acetonitrile, dimethylformamide, dimethylsulfoxide, and mixtures of such solvents, for example mixtures of water and alcohols. As alcohols can be used, among others, methanol, ethanol, ISO - and n-propanol, n-, ISO-, sec - and tert-butanol, n-, ISO-, sec - and tert-pentanol, n-hexanol, 2 - ethylbutanol, 2-ethylhexanol, isooctyl alcohol, Cyclopentanol, methylcyclohexanol (mixture) or benzyl alcohol.

The molar ratio of the compound of General formula II : maleic acid, respectively maleate is this process usually 1:1 -1:10, preferably 1:1 to 1:2, most preferably about 1: 1. When you pre-load the compounds of General formula II, and then add maleic acid, respectively maleate or a mixture of maleic acid and malatov or you can work in the reverse order, but possible Tagirov, however, depending on the type of implementation anion exchange may be appropriate for one of the components of pre-load in suspension and/or add another component in the form of suspensions or solids. Anion exchange is carried out usually in the temperature range from -10 to 40oC, primarily from -5 to 30oC, preferably from 0 to 25oC.

To highlight the salts of maleic acid, if necessary after concentration of the last it is possible to crystallize, for example, by cooling and/or precipitation and separated by filtration or centrifugation. Depending on the respective requirements of the resulting product can then be further washed and, if necessary, be subjected to further purification, for example by recrystallization or leaching.

In one of the preferred variants of the method of obtaining the salts of maleic acid of General formula I according to the invention as starting compounds used salts of acetic acid of formula IIb. Particularly preferable to combine these salts in a solvent with maleic acid.

Used anionic exchange compounds of General formuch">

Compounds of General formula III

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which can be carried out according to the data in the application PCT/EP94/03491 and PCT/EP96/01572, in the presence of vodosvyatiya agents, such as dicyclohexylcarbodiimide (DCC), tetrafluoroborate O-((CYANOGEN (etoxycarbonyl)methylene)amino) -N, N, N', N' - tetramethylurea (TAUTOU) or anhydride propylphosphonic acid (agricultural and industrial financial), the usual reaction conditions may be of combination reaction with compounds of General formula IV

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which can be described in the literature methods (see , for example, E. Juaristi, D. Quintana, J. Escalante, Aldrichimica Acta, vol 27, No. 1, 1994, page 3-11; D. C. Cole, Tetrahedron, vol 50, 1994, pp. 9517-9582), results in compounds of General formula V

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In the compounds of General formulas III, IV and V of the chiral centers in imidazolidinone ring and propionolactone fragment can be represented, respectively, (R)-configuration or (S)-configuration. Compounds of General formulas III and IV can be applied in the form of pure stereoisomers (enantiomers or as mixtures of stereoisomers, i.e., for example, in racemic form or optically active form.

Compounds of General formula V, for example, by conventional methods used for periodlast hydroxylamine, accordingly hydroxylammonium salts and bases, for example tertiary amine or hydroxide, carbonate or bicarbonate of an alkali metal, can be converted into compounds of General formula VI

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then restore in normal conditions, for example, by hydrogenation with hydrogen in the presence of metal catalysts, and by adding an acid of General formula HV primarily by hydrogenation in the presence of an acid of General formula HV, can be converted into compounds of General formula II. As catalysts for hydrogenation, which can be conducted at atmospheric pressure or preferably at elevated pressures can be used, among other catalysts based on noble metals, such as palladium on coal. To obtain compounds of formula IIb in the preferred method, the hydrogenation can be carried out, for example, in an appropriate solvent in the presence of acetic acid or the acetic acid as a solvent.

Pharmacological properties of the compounds of General formula I does not depend on the fact that we are talking about maleic acid. Like, for example, corresponding hydrochloride offered with the result of the interaction of Arg-Gly-Asp-containing proteins, such as fibronectin, fibrinogen or factor a background of Villebranda with the so-called integrins. Integrins are transmembrane glycoproteins, receptors Arg-Gly-Asp-containing extracellular proteins (see E. Ruoslahti and M. D. Pierschbacher, Science 238 (1987) 491-497; D. R. Phillips, I. F. Charo, L. V. Parise and L. A. Fitzgerald, Blood 71 (1988) 831-843). In addition, they inhibit the binding of other adhesive proteins such as vitronectin, collagen and laminin, to the corresponding receptors on the surface of cells of different types. First of all compounds of General formula I according to the invention inhibit platelet aggregation and are suitable for the prevention of thrombosis. Compounds of General formula I also inhibit the metastasis of cancer cells. The object of the present invention in accordance with this is the use of compounds of General formula I and/or their physiologically acceptable salts for these purposes, as well as their use for the preparation of drugs designed to inhibit platelet aggregation, prevent thrombosis, or for inhibiting metastasis of cancer cells.

Compounds of General formula I and their physiologically acceptable salts can be administered to animals, preferably mammals, and before compelling compositions, which are suitable for enteral or parenteral use and which contain as active ingredient an effective dose of at least one of the compounds of General formula I or one of its physiologically acceptable salts along with conventional, pharmaceutically perfect fillers and additives. The object of the present invention in accordance with this are also compounds of General formula I and their physiologically acceptable salts, intended for use as medicines and pharmaceutical compositions containing as active substance one or more compounds of General formula I and/or one or more of their physiologically acceptable salts along with pharmaceutically acceptable excipients and additives and optionally one or more other pharmacologically active substances. Such pharmaceutical compositions usually contain in its composition from 0.5 to 90 wt.% compounds of General formula I or their physiologically acceptable salts.

Drugs can be administered orally, for example in the form of pills, tablets, film-coated tablets, pills, granules, terdoslavich and megkozeliteni capsules, raseev, or parenterally, e.g. in the form of injection solutions, infusion solutions, microcapsules or rods, percutaneous, for example, in the form of ointments or tinctures, or in any other way, for example, in the form of nasal sprays or aerosol mixtures.

Preparation of pharmaceutical compositions is carried out by known methods using pharmaceutically inert inorganic and/or organic fillers and additives, which then together with the active substances receive required, ready to use form. For the manufacture of pills, tablets, pills and terdoslavich capsules can be used, for example, lactose, corn starch or its derivatives, talc, stearic acid or its derivatives, etc. as fillers for megkozeliteni capsules and suppositories are, for example, fats, waxes, semisolid and liquid polyols, natural or hardened oils, etc. as fillers for the preparation of solutions and syrups are suitable, for example, water, sucrose, invert sugar, alcohols, vegetable oils, etc. as fillers for microcapsules, implants or rods are suitable copolymers composition: glycolic acid-dairy colatosti also other added substances, as, for example, fillers, substances that promote disintegration, binders, softeners, wetting, stabilizers, emulsifiers, preservatives, sweet substances, dyes, korrigentami taste or flavoring agents, thickeners, diluents, buffer substances, and also solvents or agents dissolved or means of achieving depot effect, and also salts for modifying the osmotic pressure, means for coating or antioxidants. They can also contain two or more compounds of General formula I and/or their physiologically acceptable salts and optionally one or more other therapeutically effective agents.

Examples of such therapeutically effective substances are among the other facilities that promote circulation, such as dihydroergocristine, nicergoline, nicotinic acid and its esters, benzilan, naftidrofuryl, derived prostacyclin, PGE1-derivatives, antigenemia compounds such as isosorbidedinitrate, isosorbidemononitrate, glyceroltrinitrate molsidomin, nifedipine and verapamil; -blockers, such as propranolol, oxprenolol, atenolol, metoprolol and penbutolol. In addition, the compounds according to the invention can be applied to the Mgr, enalapril, lisinopril and trandolapril, inhibitors of platelet function, such as acetylsalicylic acid, ticlopidine and clopidogrel, and anticoagulants, such as heparin and low molecular weight heparins.

The dosage may vary within wide limits and depends on the individual patient. As a rule, in oral introduction to achieve the desired effect, the daily dose is administered in amounts of from 0.1 to 5 mg/kg body weight, preferably from 0.3 to 3 mg/kg, especially from 0.5 to 2 mg/kg intravenously daily dose is generally from 0.01 to 0.6 mg/kg body weight, preferably from 0.05 to 0.3 mg/kg, especially from 0.05 to 0.1 mg/kg Daily dose primarily in the appointment of increased quantities can be divided in several doses, for example 2, 3 or 4. Under certain conditions, depending on the specific condition of the patient may be necessary the daily dose to appoint above respectively below the specified limits. Pharmaceutical compositions usually contain from 0.2 to 500 mg, preferably from 1 to 200 mg, especially from 10 to 100 mg of active substance of General formula I, respectively, of its salts per dose.

Proposed according to the invention of the cardiovascular system, system diseases of the coronary vessels, vascular system, brain, or peripheral arterial disease. They can find application in risk of acute thrombosis and to prevent chronic arteriosclerosis and thrombosis, for example, in the treatment and prevention of diseases of the blood vessels, such as acute myocardial infarction, secondary prevention of myocardial infarction, prevention of reocclusion after lysis and dilatation (RTSA), unstable angina, transient ischemic attacks, stroke, coronary bypass surgery, including the prevention of reocclusion during shunting, pulmonary embolism, peripheral occlusive endarterite, dissecting aneurysm; next, in the treatment of venous diseases and microcirculation vessels, as, for example, deep phlebothrombosis, disseminated intravascular koagulirovannogo, post-operative or post-partum trauma, surgical or infectious shock, septicemia or diseases associated with hypersensitivity of platelets in thrombocytopenic thromboembolitic purple, preeclampsia, premenstrual syndrome or dialysis or extracorporeal circulation; plaguemix according to the invention compounds of General formula I and/or their physiologically acceptable salts for the treatment or prevention of these diseases is also an object of the present invention, as well as their use for the manufacture of drugs for the treatment and prevention of the above diseases. The subject invention is primarily the application of the proposed compounds for the manufacture of medicinal products intended for the treatment and prevention of diseases of the coronary vessels or vascular system of the brain, peripheral arterial diseases, venous and microcirculation vessels, as well as the use for the manufacture of drugs used in dialysis or extracorporeal circulation.

Compounds of General formula I were pilot-tested for their inhibitory efficacy in platelet aggregation and adhesion of fibrinogen to platelets (used gel-filtered platelets donated human blood, activated ATP [adenosine-5'- diphosphate] or chrominum), as well as their in vivo efficacy in the inhibition of platelet aggregation and inhibition of thrombosis.

Examples

All products are identified using mass spectroscopy and NMR spectra.

Example 1

Gidromolot ethyl ester (S)-3-(2- ((S)-4-(4-(aminoiminomethyl)phenyl)-4-methyl-tion, the company Fluka) was stirred for 10 min at room temperature with 1000 ml of 2 M solution of maleic acid. Then a solution of maleic acid was filtered and ion-exchange re-double-handled similarly 2 M solution of maleic acid. Then the ion exchange resin is washed with water until neutral state. 1.51 g (3 mmole) of the hydrochloride of the ethyl ester of (S)-3-(2-((S)-4-(4- (aminoiminomethyl)phenyl)-4-methyl-2,5-dioxoimidazolidin-1 - yl)acetylamino)-3-phenylpropionic acid was dissolved in 15 ml of water.

The solution was passed through a column of ion exchanger with maleic acid, as described above, filled with 80 ml of Amberlite IRA93 . Then the column was suirable water. Next, the collected fractions of 25 ml each. From fractions 2 and 3 at room temperature, precipitated in the form of crystals of pure gidromolot ethyl ester (S)-3-(2-((S)-4-(4- (aminoiminomethyl)phenyl)-4-methyl-2,5-dioxoimidazolidin-1-yl) acetylamino)-3-phenylpropionic acid. The crystals were isolated by filtration.

Output: 771 mg of a white crystalline solid (44%). TPL228oC. []D=-54,4o(C=1; methanol; 22oC). MS (FAB): 466 (M+N)+.

X-ray analysis of kristalliset confirmed that the obtained product Prestel)acetyl-amino)-3 - phenylpropionic acid (a molar ratio of ethyl ether:maleic acid 1: 1), which may be represented by the following ionic formula is equivalent to the above formula.

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From mother solutions of fractions 2 and 3 and from fractions 4-12 freeze-drying the aqueous solution has allocated a further 740 mg of pure gidromolot ethyl ester (S)-3-(2-((S)-4-(4- (aminoiminomethyl)phenyl)-4-methyl-2,5-dioxoimidazolidin-1 - yl)acetylamino)-3-phenylpropionic acid.

Total yield: 1,511 g (87%).

Example 2

Gidromolot ethyl ester (S)-3-(2-((S)-4-(4-(aminoiminomethyl)phenyl)-4 - methyl-2,5 - dioxoimidazolidin-1-yl)acetylamino)-3-phenylpropionic acid

2a. Ethyl ester of (S)-3-(2-((S)-4-(4-tianfeng)-4-methyl-2,5 - dioxoimidazolidin-1-yl)acetylamino)-3-phenylpropionic acid

7,7 kg (33,5 mole) of the hydrochloride of the ethyl ester of (S)-3-amino-3 - phenylpropionic acid pre-loaded into the boiler and mixed with a solution of 9,14 kg (33,5 mole) of 2-((S)-4-(4 - tianfeng)-4-methyl-2,5-dioxoimidazolidin-1-yl) acetic acid in 50 l of ethyl acetate. When the internal temperature of 20-23oC pump filed 7,72 kg (8,54 l; 67 moles) N-ethylmorpholine. The mixture was stirred for 15 min, then at 20-23oC through loading the gateway to one portion was added 11 kg (33,5 mole) of tetrafluoroborate O-((CYANOGEN (etoxycarbonyl)matelasse which was added 50 l of water and the mixture was stirred for 30 min at a temperature of 20-23oC. was Then added 50 l of methyl tert-butyl ether, after which the mixture was stirred for 2 hours at a temperature of 8-12oC. the White suspension was transferred into a centrifuge and centrifuged. The filter cake was washed first with 28 l of water, then 28 l of ethanol and finally re-56 l of water. The product was dried in a vacuum drying Cabinet at 40oC in a stream of nitrogen.

Output: 9,84 kg (85%). MS (FAB): 449 (M+N)+.

2B. Ethyl ester of (S)-3-(2-((S)-4-(4- (aminohydrocinnamic)phenyl)-4-methyl-2,5-dioxoimidazolidin-1 - yl)acetylamino)-3-phenylpropionic acid

120 l of ethanol was pre-loaded into the boiler. At room temperature in the boiler under stirring was added 12 kg (26,786 mmol) ethyl ester of (S)-3-(2-((S)-4-(4-tianfeng)-4-methyl-2,5 - dioxoimidazolidin-1-yl)acetylamino)-3-phenylpropionic acid. 3 Quiroga added 3,74 kg (53,813 mole) of hydroxylamine hydrochloride and 5,44 kg (7,45 l; 53,86 mole) of triethylamine. The reaction mixture was heated to the temperature of reflux distilled and was stirred for 2 h at 80oC. When the bath temperature 50oC drove under vacuum to 90 l of ethanol. The residue was dissolved in 220 l of ethyl acetate and was extracted three times with portions of 50 l of water, respectively. An ethyl acetate phase was evaporated under vacuum at the and was evaporated to dryness.

Output: 11,1 kg (85%). MS (FAB): 482 (M+N)+.

2B. Acetic acid salt of the ethyl ester of (S)-3-(2-((S)-4-(4- (aminoiminomethyl) phenyl)-4-methyl-2,5-dioxoimidazolidin-1 - yl)acetyl-amino)-3-phenylpropionic acid

11 kg (22,869 mole) of ethyl ester of (S)-3-(2-((S)-4-(4-(aminohydrocinnamic)phenyl) -4-methyl-2,5-dioxoimidazolidin-1-yl)acetylamino)-3 - phenylpropionic acid was dissolved in 70 l of acetic ether and placed in the autoclave with a volume of 125 liters of this solution was added a suspension of 1.0 kg of palladium on coal (10%; 50% water) and 5 l of acetic ether. Then was first made for 15 h at 50oC and a hydrogen pressure of 10 bar. The catalyst was filtered at suction pressure, in a nitrogen atmosphere and washed with 5 l of acetic ether. The filtrate is completely evaporated at a bath temperature of 60oC under vacuum using a rotary evaporator. The residue was dissolved in 30 l of acetone and the solution for 30 min at 20oC was added to 200 l of methyl tert-butyl ether. Then for 1 h and was stirred at room temperature and precipitated precipitated product was centrifuged. The residue on the filter is washed with 10 l of methyl tert-butyl ether, unloaded and dried under vacuum. In this way got a 10.1 kg (85%) of product. MS (FAB): 466 (M+N)+.

Output: 7,2 kg (60%). MS (FAB): 466 (M+N)+.

G Gidromolot ethyl ester (S)-3-(2- ((S)-4-(4-(aminoiminomethyl)phenyl)-4-methyl-2,5-dioxoimidazolidin - 1-yl)acetylamino)-3-phenylpropionic acid

75,68 g (144 mmole) acetic acid salt of ethyl ether (S)-3-(2-((S)-4-(4- (aminoiminomethyl)phenyl)-4-methyl-2,5-dioxoimidazolidin-1 - yl)acetylamino)-3-phenylpropionic acid was dissolved in 340 ml of water. The solution was filtered and stirring was mixed with a solution of 16,87 g (144 mmole) of maleic acid (99%) in 48 ml of water. Stirring was continued for in the form of a crystalline solid. Then was filtered and dried under high vacuum over pjatiokisi phosphorus.

Output: 80,7 g of a white crystalline solid (96%). TPL228oC. [] =-54,4o(C=1; methanol; 22oC). MS (FAB): 466 (M+N)+.

Getting the original compounds used in stage 2A

I. the Hydrochloride of the ethyl ester of (S)-3-amino-3 - phenylpropionic acid

Ia. (R)-2-amino-2-phenylethanol

20 g (920 mmol) of lithium borohydride was dissolved in 420 ml of absolute tetrahydrofuran. Then under stirring was added dropwise 233,5 ml (1,84 mole) of trimethylchlorosilane, then for 4 h portions were added for 69.5 g (0,46 mol) of (R)-phenylglycine. The reaction mixture was stirred over night at room temperature. Then added 690 ml of methanol was stirred for 2 h at room temperature and concentrated under vacuum. The residue was dissolved with stirring in 690 ml of 20% aqueous solution of potassium hydroxide. The aqueous phase was extracted three times with ethyl ether, acetic acid. The combined organic phases are washed with water, dried over magnesium sulfate and concentrated under vacuum.

Output: 41,2 g (65,3%). MS (FAB): 138 (M+N)+.

IB. (R)-2-benzyloxycarbonylamino-2-phenylethane whenoC added is 73.5 g of N-(benzyloxycarbonyloxy)succinimide (295 mmol) and was stirred for 1 h at 0oC. Then the ice bath was removed and the mixture was left to stand for 48 h at room temperature. The reaction solution was concentrated under vacuum, then the residue was dissolved in 500 ml ethyl ester of acetic acid. The organic phase is twice washed with 10% aqueous citric acid solution and once with water. Then was dried over anhydrous sodium sulfate and concentrated. The obtained crude crystalline product (82.3 g) was re-dissolved in ethyl ether, acetic acid. The organic phase is twice washed with 10% aqueous citric acid solution and once with water. Then recrystallized from ethyl ether/petroleum ether.

Output: 74,6 g (93,3%). MS (FAB): 272 (M+N)+.

V ((R)-2-benzyloxycarbonylamino-2 - phenylethyl)-4-methylphenylsulfonyl

53,9 g (K)-2 - benzyloxycarbonylamino-2-phenylethanol (198,7 mmol) was dissolved in a mixture of 500 ml of methylene chloride, and 80.3 ml (993,5 mmole) of pyridine. Under stirring at 0oC was added to 45.5 g (238,4 mmol) taillored in 240 ml of methylene chloride and left to stir for 7 h at room temperature. Then he added 11,36 ulali to stand over night at room temperature, then concentrated under vacuum. The residue was dissolved in ethyl ether, acetic acid. The organic phase is washed three times 10% citric acid solution and twice with water, dried over magnesium sulfate and concentrated under vacuum. The residue is triturated with diethyl ether, filtered off, washed with diethyl ether and dried over pjatiokisi phosphorus.

Output: 60,9 g (72%). The mother liquor was concentrated, dissolved in n-heptane/ethyl ether acetic acid (6:4) and was chromatographically using silica gel. Yield: 3.5 g (4,2%).

Total yield: 64.4 g (76,2%). MS (FAB): 426 (M+N)+.

Iك. (S) -3-benzyloxycarbonylamino-3-phenylpropionitrile

of 60.5 g ((R)-2-benzyloxycarbonylamino-2-phenylethyl)-4-were - sulfonate (142,2 mmole) was dissolved in 675 ml of dimethylformamide. 3 Quiroga was added to 13.9 g of potassium cyanide (213,3 mmol), 5,64 g of 18 - crown-6 (21,33 mmole) and 520 mg of potassium iodide (3.13 mmole) and stirred for 20 h at 50oC. the Reaction solution was poured into 500 ml of ice water, after which it was stirred for 5 h at 0oC. Then was filtered and the residue was dissolved in ethyl ether, acetic acid. The organic phase is washed three times with water, dried over magnesium sulfate and concentrated under vacuously phosphorus.

Output: 25,3 g (63.5 per cent). MS (FAB): 281 (M+N)+.

Ia. Ethyl ester of (S)-3-benzyloxycarbonylamino-3 - phenylpropionic acid

15 g of (S)-3-benzyloxycarbonylamino-3 - phenylpropionitrile (53,51 mmole) suspended in a mixture of 110 ml of absolute ethanol and 30 ml of dioxane. Under stirring and cooling at 10-15oC was injected HCl-gas. After some time formed a clear solution. When cooled continued flow of HCl gas until thin-layer chromatogram showed complete consumption of starting material. Then through the reaction solution for 15 min was passed a stream of nitrogen, then concentrated under vacuum. The residue was mixed with water until turbidity. Next was stirred for 30 min at room temperature, after which the aqueous phase was extracted three times with ethyl ether, acetic acid. The combined organic phases are washed with water, dried over magnesium sulfate and concentrated under vacuum. The residue was dissolved in ethyl ester acetic acid/petroleum ether (1:1) and was chromatographically using silica gel.

Output: 10,55 g (60%). MS (FAB): 328 (M+N)+.

The Oia. The hydrochloride of the ethyl ester of (S)-3-amino - 3-phenylpropionic acid

10,29 g of ethyl ester of (S)- benzeri simultaneous addition of a solution of 2H HCl in ethanol at a pH of 4 was carried out by catalytic hydrogenation using palladium on charcoal. The catalyst was filtered through silica gel and the filtrate was concentrated. The residue is triturated with diethyl ether, filtered off, washed with diethyl ether and dried over pjatiokisi phosphorus.

Output: of 5.05 g (70%). MS (FAB): 194 (M+N)+.

II. 2-((S)-4-(4-tianfeng)-4-methyl-2,5 - dioxoimidazolidin-1-yl) acetic acid

IIa. (R,S)-4-(4 - bromophenyl)-4-methyl-2,5-dioxoimidazolidin

of 49.8 g (0.25 mol) of 4 - bromoacetophenone and 21.2 g (0,325 mole) of potassium cyanide and 211,4 g (2,2 mol) of ammonium carbonate suspended in 1.0 l of an aqueous solution of ethanol (0.5 l of distilled water and 0.5 l of ethanol). The suspension was stirred at 60oC until a thin layer chromatogram showed complete consumption of starting material (after 8 hours). Then was allowed to cool to room temperature. The pH of a solution using policecontributing hydrochloric acid was set equal to 6.3. The product precipitated as a white precipitate. The composition was left to stand overnight at 4oC. the White precipitate was filtered, washed with water and dried under vacuum over pjatiokisi phosphorus.

Output: 65,3 g white solid (97%). MS (FAB): 269 (M+N)+.

IIb. (R,S)-2-amino-2-(4-bromophenyl)propionic acid

to 5.3 g (20 mmol) of (R,S)-4-(4-bromophenyl)-4 - 1 h at 145oC with excess nitrogen pressure of 10 bar.

The cooled reaction solution was diluted with 150 ml of water and with vigorous stirring with acetic acid under ice cooling was set to a pH of 4. Then was stirred at 0oC for 2 hours the precipitation was filtered, washed with water and dried under vacuum over pjatiokisi phosphorus.

Output: the 3.65 g of a white solid (75%). MS (FAB): 244 (M+H)+.

IIb. Ethyl ester of (R,S)-2-amino-2-(4-bromophenyl)propionic acid

27,3 g (112,3 mmol) of (R,S)-2-amino-2-(4-bromophenyl)propionic acid are suspended in 150 ml of 9.8 H of a solution of hydrogen chloride in ethanol. The suspension was heated for 18 h under reflux, was re-added 50 ml of 9.8 H of a solution of hydrogen chloride in ethanol and continued heating under reflux for 5 hours Then the solution was concentrated and the residue was distributed between ethyl ether, acetic acid and saturated sodium bicarbonate solution. The organic phase is washed with water and dried over sodium sulfate. The crude product (23,22 g) was distilled to clean under vacuum (boiling point 129-130oC at a pressure of 2 Torr).

Output: 20.7 g (68%). MS (FAB): 272 (M+N)+.

Yield: 12.5 g (28%).

[] = +52,7o(c=1; H 2,15 solution of hydrogen chloride in ethanol; 22oC). MS (FAB): 272 (M+N)+.

D. N-((S)-1-(4-bromophenyl)-1- (etoxycarbonyl)ethyl)-N'-(ethoxycarbonylmethyl) urea

12.4 g (45.6 mmol) of ethyl ester of (S)-2-amino-2-(4-bromophenyl) propionic acid was dissolved in 70 ml of methylene chloride. Then at 0oC for 15 min was added dropwise a solution of 5,11 ml (45.6 mmol) of ethyl ether isocyanurate acid in 35 ml of methylene chloride. For c=1; 2,15 H a solution of hydrogen chloride in ethanol; 22oC). MS (FAB): 401 (M+N)+.

IIe. 2-((S)-4-(4-bromophenyl)-4-methyl-2,5 - dioxoimidazolidin-1-yl) acetic acid

18 g (44,9 mmole) of N- ((S)-1-(4-bromophenyl)-1-(etoxycarbonyl)ethyl)-N'- (ethoxycarbonylmethyl) urea was mixed with 180 ml 6 H of a solution of hydrogen chloride. Then the reaction mixture for 10 h was heated under reflux to boiling. Then was allowed to cool to 0oC and precipitated precipitated product was filtered, after which it was rinsed with water and dried under vacuum over pjatiokisi phosphorus.

Yield: 11.4 g (78%).

= +32,8o(c=1; H 2,15 solution of hydrogen chloride in ethanol; 22oC). MS (FAB): 327 (M+N)+.

G. 2-((S)-4-(4-tianfeng) - 4-methyl-2,5-dioxoimidazolidin-1-yl) acetic acid

of 11.75 g (35.9 mmol) of 2-((S)-4-(4-bromophenyl)-4-methyl-2,5 - dioxoimidazolidin-1-yl) acetic acid was dissolved in 90 ml of dimethylformamide. Then added 14,15 g (158 mmol) of copper cyanide (1) and with stirring, they were heated for 20 hours under reflux.

The reaction mixture was cooled, then poured into 300 ml of water. The aqueous phase was acidified with concentrated hydrochloric acid (pH= 1-1,5), was stirred for 30 min and was filtered through filtenesta phase was washed with water, was dried over magnesium sulfate and concentrated under vacuum.

Yield: 9.3 g (95%).

=+33,4o(C=1; H 2,15 solution of hydrogen chloride in ethanol; 22oC). MS (FAB): 274 (M+N)+.

Example 3

Determining the water vapor sorption

Determination of water absorption of the substance was performed using the automatic double-microbalance used in vacuum, respectively, in an atmosphere having a specific composition, respectively, a certain pressure. Scales on one side is dry sample, on the other teruomi weight. In the weight of the camera created an atmosphere saturated with water vapor, the pressure of which is increased stepwise by. The increase in sample weight was recorded depending on the pressure of water vapor. The measurements were carried out at constant temperature of 25oC.

In the weight of the camera was first created vacuum to constant weight and were determined by the initial weight of the sample. Then in weight insulated chamber through the inlet valve gave a certain amount of water vapor. In the following the experience installed on the first stage pressure of water vapor at a temperature of 25oC corresponded to a relative humidity of 4.7%. When the hearth atmosphere of water vapor, there was observed the increase of the sample weight. Then by further supply of water vapor, which was carried out step by raising the partial pressure of water vapor (each indicator pressure of water vapor corresponds to at that specific relative humidity at a temperature of 25oC). At each step after reaching constant weight was observed the increase of the sample weight. In the study of gidromolot ethyl ester (S)-3-(2- ((S)-4-(4-(aminoiminomethyl)phenyl)-4-methyl-2,5 - dioxoimidazolidin-1-yl)acetylamino)-3-phenylpropionic acid was observed following a gradual increase in the weight of the sample in relation to its original weight (see table. 1 in the end of the description).

The results show that even at a relative humidity of about 80% is only a slight increase in weight (approximately 0.5%) of gidromolot ethyl ester (S)-3-(2- ((S)-4-(4-(aminoiminomethyl)phenyl)-4-methyl-2,5 - dioxoimidazolidin-1-yl)acetylamino)-3-phenylpropionic acid, which, consequently, indicates nephroscopes substance.

Example 4

Pharmacological studies on the model of Cyclical fluctuations in coronary blood flow in the dog" (model unstable angina)

or to determine hemodynamic parameters (peripheral blood pressure, pressure in the left ventricle contractility of the heart muscle, heart rate) and to record the ECG. The gases of the blood and the hematocrit was maintained constant by the introduction of electrolytes. After opening the left thoracic cavity was exposed heart. Ramus circumflexus sin. the coronary artery was equipped with an electromagnetic flow measuring cylinder to determine the average coronary circulation. Distal regards to this measuring head Ramus circumflexus briefly pinched terminal to damage of the vascular endothelium. On the damaged area using a small plastic cylinder (inner diameter depending on the size of the vessels was 1.0-1.7 mm, length 4 mm) was created concentric compression. This measure contributes to the suppression of reactive hyperemia due to a 10-second occlusion of the Ramus circumflexus and leads to cyclic fluctuations of blood (approximately 10 hours), due to the re-formation of platelet-rich thrombi in the area of the stenosis. After 60 min following the first occurrence of the regular oscillations of blood flow subjects the substance was injected intravenously or vnutriaortalina bolus. Then the cyclical fluctuations in the blood was observed on the edge of what I thrombotic coronary vessels was determined on 5-8 experimental animals by comparing the average number of oscillations of blood circulation in the hours before and after administration of the tested substances. The animals observed number of cyclical fluctuations circulation per hour remained for several hours constant (literature: Folts, J. D. and others (1976), Circulation 54: 365-370; Just, M. and others (1989), Journ. Cardiovasc. Pharmacol. 14 (Suppl. 11): pp. 129-136).

In the study of gidromolot ethyl ester (S)-3-(2-((S)-4-(4- (aminoiminomethyl) phenyl)-4-methyl-2,5-dioxoimidazolidin-1 - yl)acetylamino)-3-phenylpropionic acid were obtained the following results. Below are stated in terms of percentage decrease in the number of induced stenosis of cyclic changes in coronary blood flow per hour after the first, second and third hours after administration of the substance compared with the period before the introduction of the latter. The specified dosage in mg per kg of body weight (see table. 2 at the end of the description).

Obtained in this experiment, the in vivo results confirm the high antithrombotic activity of the tested substances, primarily coronary antithrombotic efficacy. All revealed in the course of experimental verification of the hemodynamic parameters did not show any appreciable changes.

Example 5

Pharmacological studies on the model of the Suppression of ex vivo platelet aggregation in the dog"

Dogs, osoba is ncii was administered orally in gelatin capsules or intravenously. Before the introduction of the tested substances, and several times within 24 hours after administration took 20 ml of blood from the Vena cephalica and anticoagulatory sour citratecitrate (volume 9+1). Centrifugation was received platelet-rich plasma (BTP). Platelet aggregation in BTP was determined after addition of ATP (3-30 mcmole/ml) or collagen (0.3 to 10 μg/ml) + epinephrine (10 μm) at 37oC using aggregometer (BioData). The comparison was carried out by expressed in percent of the maximum platelet aggregation before and after administration of the test substance. Below are presented in terms of percentage inhibition of platelet aggregation at the lowest concentration of the agonist, which has caused irreversible maximum aggregation. Further, when sampling blood from the inner side of the front paw of the animal, which had previously been removed the hair, using the Simplate device 1 measured the duration of cutaneous bleeding. On the basis of the obtained data was calculated in percentage terms, the increase in the duration of bleeding after administration of the test substance compared to the original figure.

In the study of gidromolot ethyl ester (S)-3-(2-((S)-4-(4-(aminoiminomethyl) phenyl)-4-methyl-2,5 - dioxido in terms of percentage inhibition of platelet aggregation after 1 h, 4 h, 8 h and 24 h after administration of the test substance.

The specified dosage in mg per kg of body weight, n means the number of animals tested (see table. 3).

The maximum change in the duration of bleeding was: increase duration by 68% (p<0.05) after 4 h after intravenous dose of 0.5 mg/kg, respectively, at 122% (p<0.05) after 2 h after an oral dose of 2 mg/kg. In oral introduction of lower doses of significant changes in the duration of bleeding was not observed. The results of this experience confirms the pronounced efficacy of the test substance compared with in vivo inhibitor of platelet aggregation.

1. Salt ethyl ester 3-(2-(4-(4-(aminoiminomethyl)phenyl)-4-methyl-2,5-dioxoimidazolidin-1-yl)acetylamino)-3-phenylpropionic acid of General formula I

< / BR>
where HB denotes maleic acid,

all stereoisomeric forms and mixtures in any ratio, and their physiologically acceptable salts.

2. Compounds of General formula I on p. 1, characterized in that the chiral center in imidazolidinone ring represented by (S)-configuration at the chiral center in propionolactone fragment represented by (S)-configuration is gidromolot ethyl ester (S)-3-(2-((S)-4-(4-(aminoiminomethyl)phenyl)-4-methyl-2,5-dioxoimidazolidin-1-yl)acetylamino)-3-phenylpropionic acid.

4. Compounds of General formula I on PP.1 to 3 or of their physiologically acceptable salts with overwhelming effect on platelet aggregation or preventive effect against thrombosis.

5. The method of obtaining compounds of General formula I according to one or more paragraphs.1 to 3, characterized in that compounds of General formula II

< / BR>
where HV is any inorganic or organic acid other than maleic acid,

perform anion exchange with maleic acid and/or maleate.

6. The method according to p. 4, characterized in that for the anion exchange compounds of General formula II chromatographic using an ion exchanger with maleic acid.

7. The method according to p. 4, characterized in that for the anion exchange compounds of General formula II are combined in a solvent with maleic acid or maleate.

8. The method according to one or more paragraphs.4 to 6, characterized in that the anionic exchange carried out with salts of acetic acid of formula IIb

< / BR>
9. The method according to one or more paragraphs.4 to 7, characterized in that to obtain the compounds of General formula II compounds of General formula III

< / BR>
and connections about is that using hydroxylamine transferred to compounds of General formula VI

< / BR>
which by hydrogenation and the addition of an acid of General formula HV transferred to compounds of General formula II preferably by hydrogenation in the presence of acetic acid in the compounds of formula IIb.

10. Pharmaceutical composition having inhibitory effect on platelet aggregation or overwhelming effect against thrombosis, containing the compound of General formula I or its physiologically acceptable salt, together with pharmaceutically acceptable excipients and additives.

 

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