Derived dipeptide p-amidino-benzylamino with n - terminal sulfanilimide residues and their salts with physiologically acceptable acids

 

(57) Abstract:

Describes the new dipeptide derivatives p-amidinopropane with N-terminal residues of the General formula I, in which R is alkyl with 1-20 carbon atoms, foralkyl with 1-3 carbon atoms, arylalkyl with 1-10 carbon atoms in the alkyl part, aryl, heteroaryl, which represents a five - or six-membered aromatic cycle containing one heteroatom of nitrogen and sulfur, hydroxyl, or R2R3N, where R2and R3are identical or different and denote hydrogen, alkyl with 1-10 carbon atoms, a represents a residue of amino acid of formula II, where R4means hydrogen, cycloalkyl with 3-7 carbon atoms or aryl, R5means hydrogen, cycloalkyl with 3-7 carbon atoms in cycloalkyl part and 1 to 3 carbon atoms in the alkyl part, arylalkyl with 1-3 carbon atoms in the alkyl part, means the residue of a cyclic-amino acids of formula III, where m represents the number 2, 3 or 4, if m means 3 or 4, two adjacent hydrogen atom may be replaced by a double bond, or their salts with physiologically acceptable acids. The compounds of formula I possess biological activity, in particular inhibiting thrombin activity. 1 C.p. f-l the biological activity, more specifically to derived dipeptide p-amidinopropane with N-terminal sulfanilimide residues and their salts with physiologically acceptable acids.

Known peptide compounds with biological activity, in particular inhibiting thrombin activity (see application WO 93/11152, C 07 K 5/06 A 61 K 37/64, 10 06.1993 year).

Object of the invention is the expansion of the range of peptide compounds with biological activity, in particular inhibiting thrombin activity.

The problem is solved, we offer dipeptide derivatives p-amidinopropane with N-terminal sulfanilimide residues of General formula (I)

< / BR>
in which R1alkyl with 1-20 carbon atoms, foralkyl with 1-3 carbon atoms, arylalkyl with 1-10 carbon atoms in the alkyl part, aryl, heteroaryl, which represents a five or six-membered aromatic cycle containing one heteroatom of nitrogen and sulfur, hydroxyl, or R2R3N, where R2and R3are identical or different and denote hydrogen, alkyl with 1-10 carbon atoms,

A denotes the residue of amino acid of formula (II):

< / BR>
where R4means hydrogen, cycloalkyl with 3-7 carbon atoms or aryl is of Lerida in the alkyl part, arylalkyl with 1-3 carbon atoms in the alkyl part,

B means the residue of a cyclic-amino acids of formula (III):

< / BR>
where m represents the number 2, 3 or 4, if m means 3 or 4, two adjacent hydrogen atom may be replaced by a double bond,

or their salts with physiologically acceptable acids.

The term "physiologically acceptable acids" are understood to include such acids as hydrochloric acid, citric acid, tartaric acid, lactic acid, phosphoric acid, methanesulfonate, acetic acid, formic acid, maleic acid, fumaric acid, succinic acid, hydroxyestra acid, sulfuric acid, glutaric acid, aspartic acid, pyruvic acid, benzoic acid, glucuronic acid, oxalic acid, ascorbic acid and acetylglycine.

The proposed compounds of General formula (I) belong to the category of low-toxic substances, they can be obtained by known methods. For example, they can be derived from amino acids of formula H-A-OH, respectively, of the protected nitrogen cyclic amino acids of formula B-OH, reaction scheme I, respectively, II (see below).

In the given reaction the carbon preferably methyl or tert-butyl;

R8means cyano or a group

< / BR>
(X means chlorine, bromide, acetyl),

P is a protective group, preferably tert-butoxycarbonyl (Boc) or benzyloxycarbonyl (Cbz).

Alternatively, a protected amino acids P-A-OH and H-B-OR7you can enter in the reaction mix with the formation of the dipeptide P-A-B-OR7and then, after removal of P with R1SO2Cl, respectively, of the radical R7with the help of p-cyano - or p-amidinopropane, to enter into interaction, and the sequence of reactions can be any.

R1-SO2-A-OH you can also directly enter in the reaction mix with a pair of H-B-NHCH2C6H4R9order to obtain the desired product of formula (I), respectively, of the intermediate product of formula (IV)

< / BR>
where R1A and B have the specified value.

Required of combination reaction is carried out in standard conditions of peptide chemistry.

Tert-butoxycarbonyl protective group otscheplaut using hydrogen chloride in dioxane or triperoxonane acid in methylene chloride; benzyloxycarbonyl protective group otscheplaut by hydrogenolysis or lithium hydroxide in alcohol, such as methanol or ethanol. Tert-butyl esters omelet using acids, such as, for example, triperoxonane acid.

Interaction with sulphonylchloride R1-SO2Cl in the presence of organic bases such as triethylamine, pyridine or N,N-diisopropylethylamine carried out in organic solvents, such as, for example, methylene chloride, tetrahydrofuran or dimethylformamide. In the case of free carbonyl groups interaction is carried out in the presence of aqueous solutions of hydroxide or carbonate of an alkali metal.

Amidine obtained from NITRILES as precursors by classical synthesis of Silver or preferably by a modified synthesis of Pinner, which proceeds through the formation of complex salts of aminothiophenol as an intermediate stage. Catalytic hydrogenation of N-hydroxyamides, which is obtained by joining the hydroxylamine to the cyano in the presence of Raney Nickel, respectively, palladium-on-charcoal, in alcohols as solvents also leads to amidines.

As mentioned above, the proposed compounds of General formula (I) possess inhibiting thrombin activity, and especially of thromboembolic disease, such as myocardial infarction, peripheral occlusive disease, deep venous thrombosis, amboli pulmonary artery and stroke. In addition, they can be used to prevent re-occlusion after opening the blood vessels by mechanical methods or by lysis.

The proposed compounds of General formula (I) can be entered as usual oral or parenteral (subcutaneously, intravenously, intramuscularly, administered intraperitoneally, rectally). They can also enter through vapors or aerosols through the nasopharynx.

The dosage depends on age, health and weight of the patient, as well as the form of application. As a rule, the daily dose of active substance per person is about 10 to 2000 mg by oral administration and from about 1 to 200 mg of parenteral introduction. This dose can be administered in the form of two to four single doses or once daily in the form of prolonged action.

New connections can be used in conventional, solid or liquid, ready medicinal forms, such as, for example, tablets, capsules, powders, granules, pills, suppositories, solutions, ointments, creams and aerosols. Get them in the usual way. Active basestack binder for tablets, fillers, preservatives, leavening agents for tablets, the fluidity regulators, softeners, wetting, dispersing agents, emulsifiers, solvents, helps to slow down the excretion of the active substance means, antioxidants and/or working gases. Thus, the resulting finished dosage forms containing an active ingredient generally in amounts of from 0.1 to 99 wt.%.

Receipt of the proposed compounds of General formula (I) is illustrated by the following examples that use the following abbreviations: Boc = tert-butoxycarbonyl, Phe = i.e. phenylalanyl, Pro = Proline, MC = mass spectrum.

Example 1

Acetate N-isopropylphenyl-D-phenylalaninol-(p-lidinopril)amide

a) BOC-0-phenylalaninol-(p-cyanobenzyl)amide.

To a solution of 5.1 g (of 14.2 mmol) of Boc-D-Phe-Pro-OH and 1.53 g (15,2 mmol) N-methyl-research in 15 ml of dimethylformamide at a temperature of -15oC for two minutes, add 2.0 g (14.6 mmol) of isobutyl ether of Harborview acid, optionally stirred for ten minutes and then add a solution of 1.9 g (of 14.2 mmol) of p-cyanoaniline (and 1.53 g of N-methylmorpholine in 3 ml of dimethylformamide. After stirring additionally for three hours at a temperature of -15oC according to obnarujivayut no parent compounds.

For separation of the target product, the reaction mixture was poured into 200 ml of water, and is allocated oil, which after a short time, hardens, and after grinding the product is filtered under vacumm. Still wet the residue is dissolved in a mixture of 250 ml of ethyl acetate and 50 ml of diethyl ether and the resulting solution was washed sequentially with a five percent aqueous solution of citric acid, sodium hydrogen carbonate solution and saturated solution of sodium chloride. After drying over sodium sulfate the solvent is distilled off in vacuum, the residue is mixed with n-hexane and then filtered under vacuum. After recrystallization from 50 ml of ethyl acetate to obtain 5.6 g of pure according to thin-layer chromatography of the product.

The melting point of 156 to 157oC.

b) D-phenylalaninol-(p-cyanobenzyl)amidogidrolaza.

Visaelectron compound is dissolved in 100 ml of 5 N. hydrochloric acid and stirred for three hours at room temperature, and the precipitated hydrochloride. It is filtered under vacuum, washed not containing hydrogen chloride in diethyl ether and dried in vacuo over potassium hydroxide. Obtain 4.6 g (95% of theoretically Russ is

in) N-isopropylphenyl-D-phenylalaninol-(p-cyanobenzyl)amide.

2,05 g (6.05 mmol) videolounge hydrochloride suspended in 50 ml of methylene chloride. After addition of 1.35 g (13.5 mmol) of triethylamine solution is formed, in which at a temperature of from 0 to 5oC was added dropwise a solution of 0.9 g (6.1 mmol) of acid chloride of propane-2-sulfonic acids in 10 ml of methylene chloride. The reaction mixture is additionally stirred for five hours at room temperature and then shaken out with water, a five per cent solution of citric acid and a five per cent solution of sodium bicarbonate. Obtained after drying over sodium sulfate and removal of the solvent viscous oily residue vykristallizovyvalas from a mixture of ethyl acetate with diethyl ether in a ratio of 1:1.

g) N-isopropylphenyl-D-phenylalaninol-(p-diamidines)amide.

4.1 g videolooking connection and 4 ml of triethylamine dissolved in 40 ml of pyridine, at a temperature of 0oC saturate the resulting solution with hydrogen sulfide and incubated over night at room temperature. According to the control by means of thin layer chromatography (mixture of methylene chloride with methanol in the ratio 9:1) turning in thioamide occurs on the TA and the resulting solution washed with sodium chloride solution, a five per cent solution of citric acid and sodium hydrogen carbonate solution. After drying and removal of the solvent obtain 4.1 g of pure crystalline thioamide.

e) Acetate N-isopropylphenyl-D-phenylalaninol-(p-lidinopril)amide.

Tioned dissolved in 150 ml of acetone and, after addition of 7 ml under the conditions incubated over night at room temperature. Obtained after removal of solvent, the amorphous residue is stirred with anhydrous diethyl ether and then dried. Hydroiodide methyl ester S-methyl-thioamide-acid are dissolved in 50 ml of ethanol, mixed with 15 ml of a ten percent solution of ammonium acetate and heated for three hours at 60oC. For separation of the target product, the solvent is removed, the residue is dissolved in 100 ml of methylene chloride, the insoluble component parts is filtered and then distilled methylene chloride. By infusion, with a mixture of ethyl acetate with diethyl ether to separate the soluble impurities in this mixture. The obtained iodide acetate mixed salt dissolved in a mixture of acetone with water in the ratio 3:2 and using ion-exchange resin brand IRA in acetate form was transferred to a clean acetate, which after heating in acetonitril the/SUP>C (decomposition); MS (fast atom bombardment): (M+ H+) = 500.

Example 2

Acetate N-(thienylmethyl)-D-phenylalaninol-(p-lidinopril)amide

a) BOC-D-phenylalaninol-(p-lidinopril)amide.

BOC-D-phenylalaninol-(p-cyanobenzyl)amide (receipt see example 1 (a)) according to the method of example 1 by means of hydrogen sulfide is transferred to thioamide and then according to the method of example 1 g) turn in amidon. Amidin receive in the form of white crystals.

The melting point of 237 to 239oC;

MS (fast atom bombardment): (M-H+) = 347.

b) D-phenylalaninol-(p-lidinopril)amidohydrolase.

According to the method of example 1 (b), from videolooking connections using 5 n solution of hydrogen chloride in dioxane otscheplaut the BOC-protective group. The dihydrochloride obtained as a very hygroscopic powder.

The melting point of 130 to 140oC;

MS (fast atom bombardment): (M-H+) = 247.

in) Acetate N-(thienylmethyl)-D-phenylalaninol-(p-lidinopril)amide.

A solution of 3.9 g (12.6 mmol) of N-phenylsulfonyl-D-phenylalanine in 40 ml of tetrahydrofuran, after addition of 1.9 g (12.6 mmol) of 1-HYDR is Noah temperature. Precipitated precipitated urea is filtered off under vacuum and optionally washed with a small amount of tetrahydrofuran.

At a temperature of 5oC to this filtrate is added a solution of 4.1 g (12.6 mmol) of N-(p-lidinopril)polyamideacid and 1.6 g of sodium bicarbonate in 6 ml of water. After stirring for forty-eight hours at room temperature the solvent is distilled off, the residue is treated with ethanol, filtered off from the insoluble parts and again concentrated.

The residue is purified on a column of silica gel using a mixture of methylene chloride with methanol and fifty percent acetic acid in the ratio of 45:5:1,5. From the eluate homogeneous fractions distilled solvents, to end with toluene as an additive, and the remainder will recrystallized from 50 ml of acetone with the addition of a small amount of water. Allot of 3.3 g (48% of theoretically calculated amount) of midinotate in the form of white crystals.

The melting point of 95 to 98oC;

MS (fast atom bombardment): (M-H+) = to 540.5.

Example 3

Acetate N-(2-naphthylmethyl)-D-phenylalaninol-(p-lidinopril)amide

a) Boc-Proline-(p-cyanobenzyl)amide.

Am add 163,9 g (0.97 mol) of 4-cyano-benzylaminopurine and 230 ml of 1.34 mol) of diisopropylethylamine. The suspension is stirred for forty-eight hours in a bath with melting ice and then filtered. The filtrate is extracted four times with a twenty-percent solution of sodium hydrosulphate, three times with saturated sodium hydrogen carbonate solution and twice with saturated sodium chloride solution, dried and the solvent is removed in a rotary evaporator. After recrystallization of the residue from methyl tert-butyl ether get 261 g (90% of theoretically calculated amount) of white crystals.

The melting point of 124 to 125oC.

b) N-(4-cyanobenzyl)polyamideacid.

260 g (0,79 mol) videolounge connection with the Boc-protective group is dissolved in one liter of diethyl ether and, after the addition of excess ether solution of hydrogen chloride is stirred overnight. Precipitated precipitated hydrochloride is filtered off, washed not containing hydrogen chloride in diethyl ether and then recrystallized from ethanol. Receive 200 g (95% of theoretically calculated amount) of white crystals.

The melting point of 209 to 211oC.

C) N-(2-naphthylmethyl)-D-phenylalaninol-(p-cyanobenzyl)nasledovatela mixed with 7.8 g (of 21.3 mmol) of N-(2-naphthylmethyl)-D-phenylalanine, 2.15 g (of 21.3 mmol) of triethylamine and 3.25 g (of 21.3 mmol) of 1-hydroxybenzotriazole. At a temperature of from 0 to 5oC and with stirring a solution of 4.4 g (21,3 mmol) dicyclohexylcarbodiimide in 30 ml of ethyl acetate and then stirred for forty-eight hours at room temperature. After filtration under vacuum drawn in the precipitated urea solvent is distilled off in vacuum, the residue is dissolved in 200 ml of ethyl acetate and sequentially washed with the solution obtained using a five per cent solution of sodium bicarbonate, chetyrekhvalentnogo citric acid solution and water. Obtained after drying and distillation of the ethyl acetate oily residue is dissolved in 30 ml of methylene chloride and vykristallizovyvalas by adding 50 ml of diethyl ether. Get 9,1 g (76% of theoretically calculated amount) of the target compound.

g) Acetate N-(2-naphthylmethyl)-D-phenylalaninol-(p-lidinopril)amide.

In the absence of moisture and at a temperature of 0oC 80 ml of methanol saturated with gaseous hydrogen chloride, then dissolve 5.6 g (10 mmol) videolounge connection and maintained at a temperature of 0oC within forty-eight hours. After that, the solvent is removed under cembalet to 80 ml of a saturated methanolic ammonia solution. After boiling under reflux for three hours, the cooled solution is filtered, the solvent is distilled off and the residue using ion-exchange resin in the acetate form is transferred to emediatead. After recrystallization from acetone with the addition of a small amount of water gain of 5.1 g (80%) of target compound as white crystals.

The melting point of 221 to 225oC;

MS (fast atom bombardment): (M+H+) = 584,5.

Example 4

Acetate N-(pyridylsulfonyl)-D-phenylalaninol-p-lidinopril)amide

a) N-(3-pyridylsulfonyl)-D-phenylalaninol-(p-cyanobenzyl)amide.

According to the method of example 1), D-phenylalaninol-(p-cyanobenzyl)amidogidrolaza (receipt in accordance with the method of example 1 (a) and 1 (b)) enter into interaction with the acid chloride pyridine-3-sulfonic acids. Rfis 0.57 (a mixture of methylene chloride with methanol in the ratio 9:1).

b) Acetate N-(3-pyridylsulfonyl)-D-phenylalaninol-(p-gidroksietilirovanny)amide.

2 g videolooking connection, 0.74 g of hydroxylaminopurine and 2.2 g of triethylamine dissolved in 30 ml of ethanol and stirred in nitrogen atmosphere for two hours at a temperature of from 60 to x the starting materials. The reaction mixture is mixed with water and using glacial acetic acid to set the pH of 3-4. The aqueous phase is repeatedly extracted with methylene chloride, the combined methylenechloride extracts are dried and the methylene chloride is distilled off. The residue, which still contains acetic acid, directly used in the subsequent reaction.

Rf0.1 (a mixture of methylene chloride with methanol in the ratio 9:1).

in) Acetate N-(3-pyridylsulfonyl)-D-phenylalaninol-(p-lidinopril)amide

A solution of 2.4 g (5 mmol) videolounge compound in 40 ml of methanol hydronaut in the presence of 0.4 g of 10% palladium-on-coal for seven hours at a temperature of 50oC. Then the catalyst is filtered off under vacuum, the solvent is distilled off and the residue is mixed with ethyl acetate and heated. Due to the addition of acetone and a small amount of water forms a clear solution, from which crystallizes upon cooling amidated. Obtain 1.3 g (49,5% of theoretically calculated amount) of white powder.

The melting point of 201 to 202oC;

Rfis 0,28 (a mixture of methylene chloride with methanol and fifty percent acetic acid in the ratio Ispolin-(p-cyanobenzyl)amide

To a solution of 7.0 g (40 mmol) of Boc-glycine in 240 ml of methylene chloride at a temperature of 0oC successively added 30 ml of diisopropylethylamine, 10.6 g (40 mmol) of N-(p-cyanobenzyl)polyamideacid and 32 ml (44 mmol) of anhydride papapostolou acid (50% solution in ethyl acetate). After stirring additionally for two hours at a temperature of 0oC the organic phase is washed with 1 n sodium hydroxide solution, water and saturated sodium chloride solution, dried and the solvent is distilled off. Obtain 14.8 g (96% of theoretically calculated amount) of white powder.

Rfis 0.57 (a mixture of methylene chloride with methanol in the ratio 9:1).

1H-NMR (deuterated dimethyl sulfoxide), d M. D. : 1,4 (, N, (CH3)3); 1.7 to 2.2 (m, 4H, CH2- CH2); 3,3 - 3,6 (m, 2H, N-CH2Proline); and 3.8 (m, 2H, N-CH2glycine); a 4.3 to 4.5 (m, 3H, CH and N-CH2-Ar); to 6.8 (m, 1H, Boc-); of 7.4 - 7.5 (m (apparent triplet due to the second geometric isomer), 2H, Ar - H); 7,8 (d, 2H, Ar - H); and 8.5 and 8.8 (in each case, multiplet, form together 1H (two geometric isomers), NH).

b) Hydrochloride of N-gillbryan-(p-cyanobenzyl)amide.

According to the method of example 1 (b), from videolooking tx2">

1H-NMR (deuterated dimethyl sulfoxide), d M. D.: 1.7 to 2.2 (m, 4H, CH2- CH2); 3,4 - 4,0 m, 4H, N-CH2Proline and glycine); of 4.2 to 4.5 (m, 3H, CH and N-CH2-Ar); 7.5 (d, 2H, Ar); 7,8 (d, 2H, Ar); 8.3 (the ush. s, 3H, NH3+); 8,9 - 9,2 (in each case m, form together 1H (two geometric isomers), NH).

C) N-(2-naphthylmethyl)gillbryan-(p-cyanobenzyl)amide.

According to the method of example 1), from videolooking connection due to its interaction with 2-attilalongoria gain of 3.3 g of white powder.

Rfis 0.59 (a mixture of methylene chloride with methanol in the ratio 9:1).

1H-NMR (deuterated dimethyl sulfoxide), d M. D.: 1,6-2,0 (m, 4H, CH2- CH2); 3,3 - 3,5 m, 2H, N-CH2Proline); 3.7 m, 2H, N-CH2glycine); 4,1 - 4,4 (m, 3H, CH and N-CH2-Ar); 7.4 to 8.5 range (13H, aromatic protons and 2NH).

g) N-(2-naphthylmethyl)gillbryan-(p-diaminobenzene)amide.

Visaelectron connection according to the method of example 1 g) was transferred to tioned. Obtain 3.0 g (85% of theoretically calculated amount) of yellowish powder.

1H-NMR (deuterated dimethyl sulfoxide), d M. D.: 1,5-2,0 (m, 4H, CH2- CH2); 3,3 - 3,5 (m, 2H, N-CH2Proline); 3,7 (m, 2H, is sulfonyl)gillbryan-(p-lidinopril)amide.

The target connection receive according to the method of example 1 d). Allocate 2.5 g (68% of theoretically calculated amount) of hydroiodide [Rfis 0.09 mixture of methylene chloride with methanol in the ratio 9:1)] and then using ion-exchange resin in the acetate form (amberlite) transferred to acetate. Purity according to HPLC, 99%.

MS (fast atom bombardment): (M-H+) = 493,5.

1H-NMR (deuterated dimethyl sulfoxide), d M. D.: 1,6 - 2,0 (m, 4H, CH2- CH2); about 3.5 (m, 2H, N-CH2Proline, partially overlapped with a signal of H2O); 3,7 (m, 2H, N-CH2glycine); 4,1 - 4,4 (m, 3H, CH and N-CH2-Ar); 7,3 - 8,5 (13H, aromatic protons and NH); about 8.4 to 9.2 (4H, amidon).

Example 6

N-(1-naphthylmethyl)gillbryan-(p-lidinopril)amide

The target connection receive according to the method of example 5 using 1-attilalongoria.

MS (fast atom bombardment): (M-H+) = 493.

Example 7

Acetate N-(n-hexadecacarbonyl)-D-phenylalaninol-p-lidinopril)amide

The target connection receive according to the method of example 1 in the form of white powder.

Temperature R> Acetate N-(n-butylsulfonyl)-D-phenylalaninol-(p-lidinopril)amide

The target connection receive according to the method of example 1 in the form of white powder.

The melting point of 203 to 211oC;

MS (fast atom bombardment): (M-H+) = 526,5.

Example 9

Acetate N-(isopropylaminocarbonyl)-D-phenylalaninol-(p-lidinopril)amide

a) N-(isopropylaminocarbonyl)-D-phenylalaninol-(p-cyanobenzyl)amide.

According to the method of example 1), D-phenylalaninol-(p-cyanobenzyl)amide hydrochloride enter into interaction with the acid chloride of isopropylaminocarbonyl.

b) N-(isopropylaminocarbonyl)-D-phenylalaninol-(p-gidroksietilirovanny)amide.

The target connection receive according to the method of example 4 (b) by reacting the compound of example 9 (a) hydroxylaminopurine.

in) Acetate N-(isopropylaminocarbonyl)-D-phenylpyrrole-(p-lidinopril)amide.

Hydrogenation of the compound of example 9 (b) is carried out in a mixture of methanol with glacial acetic acid and tetrahydrofuran as a solvent in the presence of palladium-on-coal at a temperature of 40oC for thirteen hours. Then canola, the residue is treated with water, the aqueous phase is extracted three times with ethyl acetate and then the aqueous phase, which contains the target product, lyophilizer. Get a solid white color.

The melting point of 199 to 205oC;

MS (fast atom bombardment): (M+H+)= 515.

Example 10

Acetate N-(dimethylaminomethyl)-D-phenylalaninol-(p-lidinopril)amide

The target connection receive according to the method of example 9 in a solid white color, which decomposes starting temperature 90oC.

MS (fast atom bombardment): (M+H+) = 501.

Example 11

N-hydroxysulfonic-D-phenylalaninol-(p-lidinopril)amide

To a solution of 1.8 g (4,36 mmol) of the hydrochloride of D-phenylalaninol-(p-cyanobenzyl)amide and 1.68 g (13,0 mmol) diisopropylethylamine in 20 ml of methylene chloride slowly at a temperature of 20oC and upon cooling, was added dropwise a solution of 0.58 g (0.33 ml, 5 mmol) of chlorosulfonic acid in 10 ml of methylene chloride. After stirring for thirty minutes at room temperature, dilute to volume of 100 ml with methylene chloride, then extracted first with 2 N. hydrochloric acid, then Dugdale. Obtain 2.0 g of N-hydroxysulfonic-D-phenylalaninol-(p-cyanobenzyl)amide in the form of a crude product without further purification injected in the subsequent reaction.

2.0 g of the specified crude product together with 0.9 g (13 mmol) of hydroxylaminopurine and 2.5 ml of Diisopropylamine in 50 ml of ethanol is stirred over night at room temperature, then concentrated and volatile component parts removed in a high vacuum for one hour at a temperature of 50oC. based On the high water solubility of the product of the extractive processing is not possible. The crude product (1.8 g) is directly used in the subsequent hydrogenation.

Hydrogenation of the crude product is carried out at slightly elevated pressure of hydrogen in a mixture of 40 ml of methanol and 5 ml of glacial acetic acid in the presence of added at the tip of the spatula 10% palladium-on-coal at a temperature of 50oC. After five and a half hours the catalyst is filtered off, the solution was concentrated in vacuo in a rotary evaporator and re-distilled with methanol and toluene. After repeated mixing the resulting product with methylene chloride receive 1.5 g (73% of theoretically calculated amount through three stages) chistina. The white powder.

The melting temperature of from 220 to 224oC;

MS (fast atom bombardment): (M-H+) = 474.

Analogously to examples 1-11 receive the following connections.

Example 12

Acetate N-trifloromethyl-D-phenylalaninol-(p-lidinopril)amide

Crystals white.

The melting point of 240 to 242oC (decomposition);

MS (fast atom bombardment): (M - H+) = 526.

Example 13

Acetate N-(,, -cryptonemiales)-D-phenylalaninol-(p-lidinopril)amide

Crystals white.

The melting point of 87 to 89oC;

MS (fast atom bombardment): (M-H+) = 540.

Example 14

Acetate N-(n-octylsilane)-D-phenylalaninol-(p-lidinopril)amide

Crystals white.

MS (fast atom bombardment): (M-H+) = 570.

Example 15

Acetate N-methylsulphonyl-(D,L)-diphenylalanine-(p-lidinopril)amide

Crystals white.

MS (fast atom bombardment): (M-H+) = 548.

Example 16

Acetate N-methylsulphonyl-di(4-chlorophenyl)alanelpaso-(p-lidinopril)amide

Chris is Acetate N-methylsulphonyl-(D)-cyclohexenyl-digitalen-(p-lidinopril)amide

MS (fast atom bombardment): 476 (M-H). White amorphous crystals;

Example 18

Acetate N-methylsulphonyl-(D)-cyclohexenyl-Proline-(p-lidinopril)amide.

MS (fast atom bombardment): 478 (M-H). White amorphous crystals.

Example 19

Acetate N-methylsulphonyl-(D)-cyclohexylethyl-Proline-(p-lidinopril)amide.

MS (fast atom bombardment): 464 (M-H). White amorphous crystals.

Example 20

Acetate N-benzylmethyl-(L)-i.e. phenylalanyl-Proline-(p-lidinopril)amide

MS (fast atom bombardment): 548 (M-H). White amorphous crystals.

Example 21

Acetate (p-lidinopril)amide N-methylsulphonyl-(D)-cyclohexadienyl-pipecolinic acid.

MS (fast atom bombardment): 492 (M-H). White amorphous crystals.

Example 22

Acetate (p-lidinopril)amide N-methylsulphonyl-(D)-cyclohexadienyl-azetidin-2-carboxylic acid.

MS (fast atom bombardment): 462 (M-H). White amorphous crystals.

The biological activity of the proposed compounds of formula (I) can be confirmed by experience.

Experience

Inhibiting thrombin activity

10 μl of a solution issleduemoi/l Tris, 154 mmol/l NaCl, pH 8.0) containing thrombin isolated from human plasma, to a final concentration of 0.1% inhibitor/ml and incubated at a temperature of 20-28oC for 5 minutes. The experience, which was held in microtronix plates with 96 cavities initiated by adding 50 μl of a solution of tested compound in buffer (final concentration: 100 umol/l) and was stopped after 5-minute incubation at 20-28oC by adding 50 μl of 35% citric acid. The absorbance at 405/630 nm each recess was determined by comparison with the control experiment. According to obtained results were determined by the concentration of tested compound in mol/l, required for 50% inhibition amylolyticus activity of thrombin (the concentration of the IC50).

The results of the experiment are summarized in table.

1. Derived dipeptide p-amidino-benzylamino with N-terminal sulfanilimide residues of the General formula I

< / BR>
in which R1is alkyl with 1 to 20 carbon atoms, foralkyl with 1 to 3 carbon atoms, arylalkyl with 1 to 10 carbon atoms in the alkyl part, aryl, heteroaryl, which represents a five - or six-membered aromatic cycle containing one heteroatom of nitrogen and sulfur, hydrox is to 10 carbon atoms;

A denotes the residue of amino acid of formula II

< / BR>
where R4means hydrogen, cycloalkyl with 3 to 7 carbon atoms or aryl;

R5means hydrogen, cycloalkyl with 3 to 7 carbon atoms in cycloalkyl part and 1 to 3 carbon atoms in the alkyl part, arylalkyl with 1 to 3 carbon atoms in the alkyl part;

B means the residue of a cyclic-amino acids of formula III

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where m represents the number 2, 3 or 4, if m means 3 or 4, two adjacent hydrogen atom may be replaced by a double bond,

or their salts with physiologically acceptable acids.

2. Derived dipeptide p-amidino-benzylamino with N-terminal residues of the General formula I under item 1, with inhibiting thrombin activity.

 

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