Derivatives analyoung of dihydropyridines or their salts with physiologically tolerated acids, and means blocking non-selective cationic channels

 

(57) Abstract:

The object of the invention are derived analyoung of dihydropyridines of General formula I,

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where a is benzo - or citograph;

R2is hydroxyl, alkoxyl, benzyloxy, halogen; alkyl;

m is 0, if a - citograph and 2, And if - anthropo;

R1-- 4-6cycloalkyl or a group of formula II,

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where R5- alkyl, asiagraph, halogen, alkoxyl;

n = 1, 2, or 3, n = 0, if a - citograph;

R3and R4is hydrogen, C3-6alkenyl, C3-6- quinil,1-12-alkyl which can be substituted by tanila or phenyl, or R3is hydrogen, R4- cyclohexyl, phenyl, forfinal, pyridyl or N-benzylpiperidine or R3and R4together with the nitrogen atom form pyrrolidinyl, piperidinyl, a group of the formula III

,

or piperazinil, unsubstituted or substituted, for example by a group of the formula IV

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or their salts with physiologically tolerated acids. Proposed also a means of blocking non-selective cation channels, where the active substance is used as a compound of formula I or its salt with a physiologically tolerated acid. 2 S. p. f-crystals, 15 PL.

The invention autoresponer analyoung of dihydropyridines and means, blocking the non-selective reabsorption of cations.

Known derivatives analyoung of dihydropyridines, which can be used as a tool with cardiotoxin action (see application EP 0251194 A1, MKI: C 07 D 217/18, A 61 K 31/47, 1988).

The objective of the invention is to develop a derivative analyoung of dihydropyridines, which can be used as a means of selective blocking the reabsorption of cations.

The problem is solved derived analyoung of dihydropyridines of General formula (I)

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where A is the mean benzo - or ceanography, the substituents R2, independently of one another represent hydroxyl, alkoxy with 1 to 4 carbon atoms, benzyloxy group, a halogen, alkyl with 1 to 4 carbon atoms,

m - O if A is ceanography, and 2, if A denotes anthropou,

R1- cycloalkyl with 4 to 6 carbon atoms, cycloalkenyl with 4 to 6 carbon atoms in cycloalkyl part and 1 to 5 carbon atoms in the alkyl part, or a group of the formula

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in which R5means alkyl with 1 to 4 carbon atoms, asiagraph, halogen, alkoxy with 1 to 4 carbon atoms, and n is 1, 2 or 3, and n can be 0 in that case, if A denotes t the config of alkenyl 3 6 carbon atoms, unbranched or branched quinil with 3 to 6 carbon atoms, unbranched or branched alkyl with 1 to 12 carbon atoms, and the alkyl may be substituted by tanila or phenyl, whereby phenyl may be mono-, di - or triamese by alkyl with 1-4 carbon atoms, sidegroups, alkoxyl with 1 to 4 carbon atoms, benzyloxycarbonyl, halogen, trifluoromethyl, adamantium, group-SO2NH2or a bridge-O-CH2-O-, pyridium, pyrrolidinium, N-methylpyrrolidinium, morpholinopropan, adamantium, nitrosopropane, or R3means hydrogen, a R4- cyclohexyl, phenyl, forfinal, pyridyl or N-benzylpiperidine, or R3and R4together with the nitrogen atom to which they are connected, means pyrrolidinyl, piperidinyl, a group of the formula

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or piperazinil, unsubstituted or substituted atom has the stands, unsubstituted phenyl, mono - or dialkoxybenzene with 1 to 4 carbon atoms in each CNS part, pyrimidinium, phenylalkyl with 1 to 4 carbon atoms in the alkyl part, or a group of the formula

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or their salts with physiologically tolerated acids.

Due to its ability to block non-selective channel cations new derivatives of d is s, ulcerative colitis and Crohn's disease, as well as to protect the heart and brain and inhibition of cell growth.

Due to the said biological activity of the compounds of formula (I) represent the active substance, selective blocking the reabsorption of cations tool, which is an additional object of the present invention.

Compounds of General formula (I) are the tautomers of the formula (Ia)

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The tautomers can be divided by the known methods, for example by column chromatography or selective restore (borane, sodium, or by catalytic reduction).

The compounds of formula (Ia) may exist as CIS - or TRANS-isomers:

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In that case, if the connection structure is not specified, the compound of formula (I) stands for the compound of formula (Ia).

Compounds of General formula (I) can be obtained with known methods, for example, by cyclization of diamide malonic acid of General formula (III)

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in which R1, R2, R3, R4and m have the above meanings;

Ar denotes phenyl, indolyl or thienyl,

in the presence of standard condensing agent and optionally, in inert restructure within 50oC to the boiling point of the reaction mixture.

The duration of the reaction depends on the initial compounds III and is 2 to 15 hours.

Compounds of General formula I are bases, and they can known method to translate in any physiologically acceptable adducts (salt), using inorganic or organic acids.

The proposed connection can be given orally, parenterally, or by local application. The required dosage depends on the specific situation and type of drug, it can be determined by experiments. Suitable types of drugs are, for example, tablets, capsules, suppositories, solutions, juices, emulsions, aerosols or dispersible powders, which in addition to the active substance, also contain conventional additives target. The following are examples of pharmaceutical preparations.

Examples of pharmaceutical preparations

a) Bean.

1 core tablet contains:

The active substance of General formula I - 30.0 mg

Milk sugar - 100.0 mg

Corn starch is 75.0 mg

Gelatin - 3.0 mg

Magnesium stearate - 2.0 mg

Total: - 210,0 mg

Getting

The mixture of the active substance, lactose and corn rajmalai 40oC and again forced through a sieve. Thus obtained granules are mixed with magnesium stearate and pressed. The obtained core covered with a shell, caused by aqueous suspension of sugar, titanium dioxide, talc and gum Arabic. The finished tablets are polished with beeswax.

b) Tablets

The active substance of General formula I - 30.0 mg

Milk sugar - 100.0 mg

Corn starch - 70.0 mg

Soluble starch - 7,0 mg

Magnesium stearate - 3.0 mg

Only 210 mg

Getting

The active ingredient and magnesium stearate granularit with an aqueous solution of the soluble starch, the granules are dried and thoroughly mixed with milk sugar and cornstarch. Then the mixture is pressed into tablets weighing 210 mg.

) Capsules

The active substance of General formula - 20.0 mg

Milk sugar 230,0 mg

Corn starch - 40,0 mg

Talc - 10.0 mg

All of 300.0 mg

Getting

The active ingredient, lactose and corn starch are mixed first in the mixer, and then grinding machine. The mixture is again fed into the mixer and thoroughly mixed with talc, then through exercise machine filling the mixture into capsules of hard gelatin.

e) Pills

The active substance of General formula (I) - 20.0 mg

Milk sugar - 100.0 mg

Corn starch - 65,0 mg

Colloidal silicic acid - 2,0 mg

Soluble starch - 5.0 mg

Magnesium stearate - 3.0 mg

All 195,0 mg

Getting

The active substance and excipients is pressed analogously to example 1 in the kernel that are using sugar, talc and gum Arabic dragonaut usual method.

e) Suppositories

The active substance of General formula (I) - 50.0 mg

Milk sugar - 250.0 mg

The material for the manufacture of suppositories To 1.7 grams

Getting

The active ingredient and the lactose are mixed with each other, and the mixture evenly suspended in the molten mass for the manufacture of suppositories. The suspension is poured into a cooled form. So get suppositories weight of 1.7 g

C) Capsules

The active substance of General formula (I) - 10.0 mg

Sodium chloride - 7,0 mg

Bi-distilled water to 1.0 ml

and Sperm

The active substance of General formula (I) to 0.70 g

Methyl ester p.-hydroxybenzoic acid, 0.07 g

Complex propyl ether p.-hydroxybenzoic acid 0.03 g

Demineralized water Up to 100.00 ml

Getting

The active substance and the preservatives are dissolved in demineralized water, the solution is filtered and bottled, each with a capacity of 100 ml.

The proposed connection can be applied interline or parenteral. Primarily for oral use are from 0.1 to 500 mg of active substance per dose, while intravenous use from 0.05 to 150 mg per dose. In the case of tablets, pills, capsules and juices individual dose of 1 to 200 mg, preferably 20 to 50 mg per 75 kg of body weight. Depending on the severity of the disease give in General 1 to 3 separate doses per day.

The following examples serve to illustrate obtain the derivative di is a 4-trimethoxyphenyl)ethyl]aminocarbonyl-isoquinoline

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a) N, N-di-[2-(2,3,4-trimethoxyphenyl)-ethyl] amide of 2-(3,4 - acid)ethyl-aminocarbonyl-phenyl-acetic acid

To a solution of 18.0 g (a 52.4 mmol) of complex monoethylene ether 2- (3,4-acid)ethylamide phenylmalonate acid in 150 ml of anhydrous dimethylformamide at room temperature under stirring portions added 9.0 g (of 55.5 mmol) of N,N'- carbonyldiimidazole. After 30 minutes add 18.0 g (44,3 mmol) of di-[2-(2,3,4-trimethoxyphenyl)ethyl]amine and stirred for 30 minutes. The solvent is then distilled off in vacuo, the residue is absorbed in 1.5 l of methylene chloride and successively shaken out twice with 250 ml of water and twice with 200 ml of 1 N. hydrochloric acid. The organic phase is dried over sodium sulfate, concentrated and the residue purified by chromatography on containing silica gel column using as eluent a mixture of methylene chloride and methanol in the ratio of 100 : 2, and then crystallized from a mixture of ester of acetic acid and simple ether.

Output: 35,5,

b) Hydrochloride 3,4-dihydro-1-benzyl-6,7-dimethoxy -- [di-2- (2,3,4-trimethoxyphenyl)ethyl]aminocarbonyl-isoquinoline

35,0 g (47,5 mmol) obtained in stage a) amide and 15 ml (164 mmol) of phosphorus oxychloride in 150 ml of anhydrous acetone and unreacted phosphorus oxychloride is distilled off in vacuum. To the residue add ice water, alkalinized by addition of soda solution and portions extracted with about 1 l of methylene chloride. The organic phase is washed with water, dried over sodium sulfate and concentrated. The residue is twice purified by chromatography on filled with silica gel column using as the first elution solvent a mixture of methylene chloride and methanol in the ratio of 100: 2 to 100: 4, and the second elution solvent a mixture of methylene chloride and a complex ester of acetic acid in the ratio of 1 : 1.

The obtained purified product (6.5 g) by dissolving about 50 ml of ethanol and adding alcoholic hydrochloric acid is converted into the hydrochloride. In the drying and thickening in high vacuum at a temperature of 50oC gain of 11.5 g of the target product.

Melting point: 56 - 64oC, amorphous.

Example 2

N-(2-(3,4-acid)ethyl)-N', N'-di-(2-(2 - forfinal)ethyl)-diamid 2-phenylmalonate acid

To a solution of 17.2 g (0.05 mol) of N-(2-(3,4-acid)ethyl)- monoamide phenylmalonate acid in 200 ml of anhydrous methylene chloride at room temperature and under stirring added in several portions of 8.1 g (0.05 mol) of N,N'-carbonyldiimidazole. After about 30 minutes into the reaction mixture under stirring on the anhydrous methylene chloride. Continue to stir for 15 hours, then add 200 ml of water, acidified by adding diluted hydrochloric acid, the organic phase is separated and the aqueous phase is extracted three times, each time using 100 ml of methylene chloride. The combined organic phases are dried over sodium sulfate and concentrated.

Oxalate R, S-(3,4-dihydro-6,7-dimethoxyisoquinoline-1-yl)-2-phenyl - N,N-di-(2-(2-forfinal)ethyl)-ndimethylacetamide

The mixture 29,8 g (0,049 mol) obtained in the previous phase amide, 150 ml of anhydrous methylene chloride and 17.5 g (= 9.4 ml; 0.10 mol) of phosphorus oxychloride is heated under reflux for 10 hours. Upon completion of the reaction (monitoring by TLC) the reaction mixture is poured into a mixture of 400 ml of ice water and 200 ml of methylene chloride. After addition of saturated soda solution is brought to neutrality, the organic phase is separated, and the aqueous phase is extracted three times, each time using 100 ml of methylene chloride. The combined phases are dried over sodium sulfate and concentrated in vacuum. The residue is purified by chromatography on filled with silica gel column using as eluent a mixture of methylene chloride and methanol in the ratio of 100 : 2, then dissolved in a small amount of this is, the which is brought to crystallization by adding a simple ether.

Melting point: 144 -146oC.

Example 3

Oxalate (R, S)-(3,4-dihydro-6,7 - dimethoxyisoquinoline-1-yl)-2-(4-methoxyphenyl)-N,N-di-(2-(2,3,4 - trimethoxyphenyl)ethyl)-ndimethylacetamide

Source connection:

5 g (6,57 mol) of N-(2-(3,4-acid)ethyl)-N'-(2-(2,3,4-trimethoxyphenyl)-ethyl-diamide 4-methoxyphenylalanine acid (hereinafter: "diamed")

18 ml of acetonitrile

to 3.02 g (19,7 mmol) of phosphorus oxychloride

600 mg (6,66 mmol) of anhydrous oxalic acid

200 ml of a simple ester

The implementation of the response

The reaction mixture is from "diamide", acetonitrile, and phosphorus oxychloride in a nitrogen atmosphere heated at a temperature of phlegmy within hours. After cooling, ice water was diluted by adding 100 ml of ester of acetic acid and successively washed twice with ice water, 50 ml saturated sodium bicarbonate solution, water and saturated sodium chloride solution. The organic phase is dried over magnesium sulfate and the mixture is poured into a solution of 706 mg (7,84 mmol) of anhydrous oxalic acid in 200 ml of absolute simple ether.

The product separates as a salt of oxalic acid in the form of oil, which Chris is the significance of the night, washed with simple ether and dried.

Melting point: 107 - 110oC

The structure is confirmed by NMR analysis.

The value of Rf: 0,53 (ester acetic acid)

The value of Rf: 0.6 (mixture of acetonitrile and water in the ratio 9 : 1)

Analogously to examples 1 to 3 obtain compounds of General formula (I) shown in tables 1 - 6.

The following examples illustrate how to obtain the original compounds.

Example 4

A. Complex diethyl ether 4-methoxyphenylalanine acid

Source connection:

150 ml of absolute ethanol

7.5 g (0.33 mol) of sodium

300 ml of diethylmalonate

62.5 g (0.3 mol) of a compound ethyl ester 4-methoxyphenylacetic acid

The implementation of the response

Sodium is dissolved in absolute ethanol and evaporated in vacuum to dryness. To the residue while cooling with ice water and while stirring add diethylcarbamyl and complex ethyl ester 4 - methoxyphenylacetic acid. The ethanol is then slowly (over 2-3 hours) is distilled in a high vacuum at a temperature of 40 - 70oC and 200 mm RT.article on a column of 40 cm in diameter, equipped with rings of Rashit. After cooling, acidified by addition of 30 ml of glacial acetic acid and add 150 ml what Ulfat magnesium.

The residue is distilled in a vacuum oil pump.

Boiling point at 0.5 mm RT.article: 150 - 155oC.

B. Complex monotropy ether 4-methoxyphenylalanine acid

The connection you use:

of 52.2 g (0,196 mol) obtained at the stage And diethyl ether complex

120 ml ethanol

120 ml of water

12.3 g (0.22 mol) of potassium hydroxide in 60 ml water and 60 ml ethanol

The implementation of the response

To the ethanol solution of complex diethyl ether 4-methoxyphenylalanine acid with stirring and ice cooling type water-alcohol solution of potassium hydroxide and stirred for 75 minutes. Then acidified with saturated citric acid solution and extracted three times with methylene chloride. The organic phase is washed successively with water and saturated saline, dried over magnesium sulfate and the solvent is distilled off in vacuum. The crystalline residue is recrystallized from a mixture of methylene chloride and simple petroleum ether in a ratio of 40 : 800.

Output: 34.4 g (73,6% of theory)

The structure is confirmed by NMR analysis.

Century Amide complex monoethylene ether N-(2-3,4 - acid)ethyl)- 4-methoxy-phenyl-malonic KIS is th acid

150 ml of anhydrous tetrahydrofuran (THF)

23.3 g (0.144 mol) of N,N'-carbonyldiimidazole

of 26.1 g (0.144 mol) of 2-(3,4-acid)ethylamine in 50 ml of anhydrous tetrahydrofuran (THF)

The implementation of the response

To a solution of complex Palmyra 4-methoxy - phenylmalonate acid in tetrahydrofuran with stirring at a temperature of 50oC portions add carbonyldiimidazole. Stirred at room temperature for 30 minutes, followed by cooling with ice add Amin and continue to stir at room temperature for 16 hours. The reaction mixture was concentrated and the residue is absorbed in methylene chloride. Washed twice with water, 10% solution of acid potassium sulfate, saturated sodium bicarbonate solution, water and saturated salt solution. After drying over magnesium sulfate the organic phase is concentrated and the oily residue (7.7 g) is crystallized from ether complex of acetic acid.

G N-(2-(3,4-acid)ethyl)-amide 4-methoxy - phenylmalonate acid

The connection you use:

44,16 g (0.11 mol) amide complex Palmyra 4-methoxy-phenylmalonate acid

300 ml of methanol

120 ml (0.12 mol) 1H. sodium liquor

The implementation of the response

To methanol Aut sodium lye. Stirred for 3 hours at room temperature, then concentrated, diluted with water and extracted twice with chloroform. The aqueous phase by the addition of concentrated hydrochloric acid adjusted to pH 1, and extracted twice with chloroform. Washed with a saturated solution of sodium chloride, after which the organic phase is dried over magnesium sulfate and concentrated. The crystalline residue is crystallized from chloroform.

Example 5

A. 2-(2,3,4-trimethoxyphenyl)-1-nitro-ether

The connection you use:

400 g (2.04 mol) of 2,3,4-trimethoxybenzaldehyde

1740 ml of glacial acetic acid

172,6 g (2,24 mol) of anhydrous ammonium acetate

656 ml of nitromethane

The implementation of the response

A mixture of benzaldehyde and ammonium acetate, nitromethane and glacial acetic acid is stirred under nitrogen atmosphere for 30 minutes at boiling temperature. Then cooled to a temperature of -5oC and with stirring, poured into 5 l of ice water. The viscous residue icerays extracted with methylene chloride. The organic phase is washed with water, dried over magnesium sulfate and concentrated. Get 510 g of a brown oily residue, which is left to stand for crystallization. It is dissolved in 470 ml of complex EPE,4-trimethoxyphenyl)ethylamine

The connection you use:

144,5 g (of 0.60 mol) obtained at the stage of A connection

1.9 l of water

232 ml of concentrated hydrochloric acid (abs.)

88 g of 10% palladium on coal

The implementation of the response

The above reaction mixture was subjected to hydrogenation at a temperature of 60oC and a pressure of 35 bar for 2.25 hours. Then concentrated in vacuo, the residue is absorbed in ethanol and evaporated to dryness. After this was dissolved in ethanol and by adding a simple ester of the reaction product is brought to crystallization.

Century 2,3,4-trimethoxybenzyl alcohol

The connection you use:

500 g (2.55 mol) of 2,3,4-trimethoxybenzaldehyde

5.0 l of methanol

13,0 g of platinum oxide

The implementation of the response

The restoration is carried out at a temperature of 20oC and a pressure of 5 bar, and it ends after 30 minutes. After evaporation of the solvent the residue (501,5 g) is distilled in a high vacuum (boiling point: 116oC, 0.5 bar).

, 2,3,4-trimethoxybenzoic

The connection you use:

50.0 g (0.25 mol) of 2,3,4-trimethoxybenzyl alcohol

800 ml of anhydrous methylene chloride

to 59.4 g (0.5 mol) of thionyl chloride

The implementation of the response

To a solution of the alcohol to be illore. Continue to stir with cooling for 15 minutes, then stirred at room temperature for 2 hours. The solvent and excess thionyl chloride removed in vacuo, the residue is absorbed in methylene chloride and successively shaken with saturated sodium bicarbonate solution, water and saturated salt solution. Dried over magnesium sulfate, then the solvent is removed in vacuum and the residue is subjected to distillation in a vacuum oil pump (boiling point 118oC, 0.1 mbar).

D. 2,3,4-trimethoxybenzaldehyde

The connection you use:

of 75.8 g (0.35 mol) of 2,3,4-trimethoxybenzaldehyde

700 ml of anhydrous acetone

of 3.45 g (is 0.023 mol) of sodium iodide

of 25.7 g (of 0.53 mol) of dry sodium cyanide in powder form

The implementation of the response

The reaction mixture consisting of the above benzylchloride, sodium iodide and sodium cyanide in anhydrous acetone, stirred at boiling temperature for 20 hours. After cooling is sucked off and the solvent is removed in vacuum. The residue is dissolved in a complex ester of acetic acid, then shaken first with water, then with saturated sodium chloride solution and dried over magnesium sulfate. After removal dissolve the Naya acid

The connection you use:

138,5 g (0.67 mol) of 2,3,4-trimethoxybenzaldehyde

of 53.5 g (of 1.34 mol) of sodium hydroxide dissolved in 215 ml of water

The implementation of the response

A mixture of the above benzylcyanide and aqueous sodium liquor is stirred at a temperature of phlegmy for 7 hours. After cooling, acidified by adding 6N. sulfuric acid and extracted three times with methylene chloride. The organic phase is washed with water and saturated saline, then dried over magnesium sulfate. After removal of solvent the residue is dissolved in 200 ml of methylene chloride and adding 1500 ml of cyclohexane is brought to crystallization.

J. 2,3,4-trimethoxyphenyl-N-(2-(2,3,4-trimethoxyphenyl)ethyl)-amide acetic acid

The connection you use:

72.4 g (0.32 mol) of 2,3,4-trimethoxyphenylacetic acid

400 ml of anhydrous tetrahydrofuran (THF)

51.8 g (0.32 mol) of N,N'-carbonyldiimidazole

to 79.3 g (0.32 mol) of the hydrochloride of 2-(2,3,4-trimethoxyphenyl)ethylamine

500 ml of anhydrous tetrahydrofuran (THF)

32,4 ml (0.32 mol) of triethylamine

The implementation of the response

Phenylacetic acid, dissolved in anhydrous tetrahydrofuran by adding portions of N, N'- carbonyldiimidazole when temperature is ri stirring suspension of hydrochloride of the above amine and triethylamine in anhydrous tetrahydrofuran. After 16 hours, concentrated in vacuo and the residue is distributed between methylene chloride and 2 N. hydrochloric acid. Then sequentially washed with water, saturated sodium bicarbonate solution, water and saturated solution of sodium chloride layers. Dried over magnesium sulfate, after which the organic phase is concentrated, the residue is dissolved in 200 ml of ester of acetic acid and the resulting product is brought to crystallization by adding 700 ml of cyclohexane.

3. Hydrochloride, di-2-(2,3,4-trimethoxyphenyl)ethylamine

The connection you use:

to 113.4 g (0.27 mol) obtained at the previous stage of phenylacetic acid amide

470 ml of anhydrous tetrahydrofuran (THF)

270 ml (0.54 mol) BH3S(CH3)2in tetrahydrofuran (2 mol per l)

The implementation of the response

To a solution of the amide acid in anhydrous tetrahydrofuran at a temperature of 65oC in nitrogen atmosphere drops add a mixture of the specified complex of borane and tetrahydrofuran. After the addition, continue to stir at a temperature of 65oC for 15 minutes. Then the reaction mixture is cooled to a temperature of 5oC. Carefully acidified by the addition of methanolic hydrochloric acid, concentrated in vacuo and the residue CRIS3,4 - trimethoxyphenyl)ethyl) di-amide-4-methoxy-phenylmalonate acid

The connection you use:

of 3.73 g (10 mmol) polyamide 4-methoxy - phenylmalonate acid

25 ml of anhydrous tetrahydrofuran (THF)

of 1.62 g (10 mmol) of N,N'-carbonyldiimidazole

4.1 g (10 mmol) of the hydrochloride di-2-(2,3,4-trimethoxyphenyl)ethylamine

40 ml of anhydrous tetrahydrofuran (THF)

1.01 g (10 mmol) of triethylamine (= 1.39 ml)

The implementation of the response

To a solution of the above monoamide in tetrahydrofuran at a temperature of 5oC under stirring portions add carbonyldiimidazole. The reaction mixture was stirred at room temperature for 30 minutes. Then, with ice cooling is subjected to interaction with the suspension of the amine hydrochloride in tetrahydrofuran and triethylamine. Stir at room temperature for 16 hours, then concentrated and the residue is dissolved in a complex ester of acetic acid. The organic phase is successively washed with water, 5% solution of acid potassium sulfate, saturated sodium bicarbonate solution, water and saturated salt solution. After drying over magnesium sulfate concentrated and the residue (7.2 g) purified by chromatography on 210 g of silica gel using as eluent a mixture of ester of acetic acid and n-hexane in ennoe application of known derivatives of dihydropyridines in the new order.

In these experiments we investigated are shown in tables 7 - 14 connection.

As already mentioned, the compounds of General formula (I) and (II) can be used as tools for the treatment of chronic inflammatory processes, ulcerative colitis and Crohn's disease, as well as inhibiting the proliferation of cells of funds. The cause of action of these compounds is to block non-selective cation channels (hereinafter referred to as SAC).

The basis of the pathophysiology of chronic bronchial asthma are inflammatory processes that are triggered by the activation of inflammatory cells (see Barnes, 1987, Seiffert and Schultz, 1991).

Adjustable receptors activation of inflammatory cells (e.g. neutrophils and mast cells or permanent cell lines HL-60 or sensibilizing, that is loaded with gammaglobulin E RBL cells [RBL = rat basophilic lymphoma (basophilic liposome rat)]) inhibited regardless of stimulating agonist (e.g., endothelium, PHAT [platelet activating factor, leukotrienes, chemotactic peptide fMLP or antigen against sensibilizing fat cells) NCC blockers (see Rink, 1990). Through these channels extracellular calcium enters the cells, which is necessary for postanesthesia, it blocked the activation of inflammatory cells.

Classical calcium antagonists type of dihydropyridines or phenylalkylamine not inhibit either the IAC or inflammatory processes (see Wells and others, 1986).

To assess cell activation or to assess its inhibition by NCC blockers measured corometrics the kinetics of the concentration of calcium ions in the cytoplasm of cells loaded with Fura-2, according to the method described by Grynkiewicz and others (1985 ). This method has proven itself as a reliable screening method for the detection of NCC blockers in the framework of this proposal.

To determine the specific characteristics of the NCC blockers suitable so-called functional inhibition of Thapsigargin. Thapsigargin is a described Thastnip and others (see Proc.Natl.Acad.Sci. (USA), 87, pp. 2466-2470, 1990 ) the promoter of the tumor, which selectively and irreversibly inhibits Ca2+-transferring APR-ABC intracellular sensitive about IP3warehouses Ca2+. This leads to a rapid emptying warehouses Ca2+As described by J. Putney (see Calcium, 11, PP 611 - 624, 1990), the emptying of these warehouses is a physiological signal for the opening of the IAC in the cell membrane. Consequently, cells merging a large number of isimage from agonists and IP3opening the IAC.

In the framework of the present invention the stimulation of the NCC thapsigargin successfully carried out on cells HL-60 (leukemia cells), nerve cells in the hippocampus and cortex cells and RBL and thus confirmed the presence of these channels in the respective cell lines.

The concentration of Ca2+([Ca2+]iin the cytoplasm plays an important role in cell proliferation and tumor growth (for an overview, see L. R. Zacharski, Journal of Medicine, 19, pp. 145 - 177, 1988). I believe that in particular the transition of the Ca2+in the cell, encouraged by the activation of receptors with subsequent mediation triphosphate of Inositol and IP3crucial reproduction in cancer cells (see U. Kikkawa and Y. Nishizuka, Ann. Rev. Cell. BioL, 2, page 149 -178, 1986 ). This mechanism plays a role in the formation of metastasis and resistance to many drugs (for an overview, see above source L. R. Zacharski, J. Med., 19, page 145 -177, 1988).

This hypothesis is confirmed by the fact that thapsigargin as a means of indirect stimulation NCC leads to an overload of cells, calcium ions, and, in addition, it is a highly effective promoter of tumors (see V. Thastrup, and others , Proceedings of the Natl. Acad. Sci. (USA), 87, page 2466 - 2470, 1990).

Blokirovka and thus, inhibition of tumor growth, etc.

Classical calcium antagonists do not inhibit NCC. Unexpectedly found that the compounds according to the invention inhibit the passage of calcium into the cells through the IAC.

As shown by S. N. Murch and others (see Lancet, 339, pp. 381 - 385, 15 February 1992), an important pathophysiological role in inflammatory bowel diseases, e.g. ulcerative colitis and Crohn's disease, play the endothelium I. using immunohistochemical methods revealed that patients suffering from Crohn's disease in the submucosal area of the shell, and patients, patients with ulcerative colitis in the anterior lamina epithelium of a thick intestine, have significantly and strongly increased concentration of endothelin-1 in comparison with healthy people. Suppose that the local secretion of endothelin causes severe vasospasm consistent with diffuse ischemia with microinfarcts, which probably represent the actual cause of these diseases. Vasospasmen action of endothelin explain the overload of vascular myocytes calcium ions. While endothelin primarily causes intracellular allocation of Ca2+a mediator is IP3, then p is Aly (see M. S. Simonson and others, Clin. Invest. Med., 14, pp. 499 - 507, 1991; T. Masakai, J. Cardiovasc. PharmacoL, 13, Suppi. 5, S1 - S4, 1989; D. W. Hay, R. J. PharmacoL, 100, page 383 -392, 1990). These channels represent the IAC, which is also in cells mucosa colon was found recently (see Chr. Siemer and N. Gogelein, Europ. J. Physiol., 420, pages 319-328, 1992).

As a suitable model of screening for the detection of functional endothelin antagonists proved itself encouraged by endothelin activation loaded with Fura-2 leukemic human cells (cells HL 60). Like G. Grynkiewicz, and others (see J. Biol. Chem., 260, pages 340 - 3450, 1985), the intracellular concentration of calcium ions in the cytoplasm of cells of HL 60 (in suspension) can be observed by spectrofluorimetry and quantified as a measure of cell activation by endothelin. Stimulation was carried out by adding 0.1 µmol endothelin, and she succumbed dependent dose-dependent inhibition by the compounds according to the invention.

Functional antagonism of the compounds according to the invention against endothelin acts through blockade of the IAC. Therefore, the proof of the functional antagonism against thapsigargin on cells RBL-hm1 is suitable as a screening method for functional antagonists of endothelin.

Khujand is the ones the incubation medium stimulated by the addition of 0.1 µmol thapsigargin. After 4 minutes add the intracellular calcium ions to the number of 1.5 mmol and using the fluorescence of Fura-2 define a strong increase in the concentration of calcium ions in the cytoplasm, due to the strong transmembrane influx of Ca2+through the IRP.

This flow is only possible and depending on the dose to inhibit using the NCC blockers. Neither classical antagonists against calcium or specific barriers agonists, stimulating the renewal of IP3not suitable for braking indirectly caused by thapsigargin transmembrane influx of calcium ions. Compounds according to the invention are distinguished by inhibition of NCC.

Fluorometrically measurement of calcium in the cytoplasm of separate interconnected cells RBL-2H3 were performed similarly described Kudo and Ogura (1986) method for nerve cells. Used fluorescently microscope AXIOVERT 35, Zeiss, combined with imaging system Hamamatsu, consisting of a system of image processing, camera control unit and the image intensifier DVS 3000.

The kinetics of the cytoplasmic concentration of Ca2+expressed in the form of the curve "concentration - time" activation of cells, called 0,1 µmol thapsigargin. Comparing the. the horse below these curves (NCP) is measured in total and is a measure of cell activation. The intensity of the inhibitory effect of the studied barriers IRP is determined by the following equation:

< / BR>
and % T inhibition in percent passage of calcium through the IRP, which is stimulated and inhibited by 10 Microm of the investigated compounds;

PNKINGthe area under the curve, which is obtained in the presence of stimulator plus 10 µmol inhibition of tested compound;

PNKCOUNTER.the area under the curve obtained only in the presence of stimulator.

The results of measurements

Determine the inhibition percentage of the IAC in cells RBL-hm1 after stimulation with the use of 0.1 µmol thapsigargin. The compounds, without exception, used at a concentration of 10-5the mole.

The compounds and their effects are shown in table 15.

Functional anti-inflammatory effect can be shown by the following experience.

Use separate cells RBL-2H3 (native fat cell line tumor cells), adherent to glass plastics.

The cultivation of cells RBL-2H3 carried out according to the method described Hide and Beaven (1991). For sensibil the trade solution gammaglobulin E (dilution 1 : 2000) in the presence of complex dinitrophenol and bovine serum albumin. The cells are then washed. The addition of 0.1 ml of a solution dinitrophenol and bovine serum albumin (10 μg/ml) caused a strong imunnologicheskie activation of cells, mediator, which is an overload of cytoplasmic calcium ions. Fluorometrically determination of calcium in the cytoplasm of the individual adhering cells RBL-2H3 carry out the above method, described Kudo and Ogura (1986) in connection with nerve cells.

As comparative compounds in this experiment is 10 µmol chromoglycate, resulting in an approximately 50% increase inhibition induced by antigen activated cells.

In this experiment, these connections are expressed in % of the braking action, comparable with the specified values.

In the experiments microculture different tumor cell lines of the human experience of tetrazole to determine retarding the propagation steps of the proposed compounds have unexpectedly revealed that the effect of the investigated compounds 5 to 100 times greater than the effect of the comparative substance verapamil.

Inhibiting the multiplication effect of the studied compounds was investigated using the MTT experience [MTT = bromide 3-(4,5-ot and other (J. Immunol. Meth. , 89, page 271 -277, 1986), and J. Eliason other (Int. J. Cancer, 46, pp. 113 - 117, 1990). This indicator only live cells with intact mitochondria metabolized with getting blue product formazan. In our experience used the following tumor cell lines human: 549 (lung adenocarcinoma), And 431 (carcinoma of the epidermis of the vulva), PC 3 (adenocarcinoma of the prostate), SK BR 3 (mammary adenocarcinoma), HT 29 (SH 1) (adenocarcinoma of the colon) and 562 (chronic maloichskoye leukemic cell). For experiment used microtitre plate. In each recess has filed 100 μl of cell suspension (0.2 to 106cells per ml). As the incubation environment was RPMI 1640 containing 10% deactivated by heat embryonic serum of calves and 50 μg/ml gentamicin. Cell suspensions were incubated for 0, 24, 48, or 72 hours when saturated with moisture the air in a mixture of 5% carbon dioxide and 95% air at a temperature of 37oC, in the presence and absence of different concentrations of inhibiting the multiplication of connections. The latter was dissolved in dimethyl sulfoxide to a final dilution of 0.1%. Then added 10 μl of MTT solution (3 mg/ml) and, after 3 hours, 100 μl containing 0,08 N. hydrochloric acid rastvor nm) in a suitable counter. Absorption of light is directly proportional to the number of living cells. Premaxillae concentration inhibition of the studied compounds was approximately 1 μg/ml.

Vazospasticescoe the above-mentioned functional antagonists of endothelin or thapsigargin confirmed on a stand-alone vessel. On retrograde perpendicularly, spontaneously beating hearts of rats on Langendorff continuously measured coronary perfusion using electromagnetic measurement device flow (device company Hugo Sachs Elektronik, , Mapx, DE). This experience allows high accuracy to determine the size, duration and type of spasms in the blood vessels. If you perpendiculat at a concentration of endothelin 100 nmol, coronary flow perfusion is reduced from 11 to 5 ml/min Reduction in perfusion can be removed by the addition compounds according to the invention. The force of action of the compounds according to the invention relative to the braking thapsigargin on loaded with Fura-2 cells RBL-hm1 or braking effect of endothelin on loaded with Fura-2 cells HL 60 unambiguously correlated with confirmed on the drug Langendorff vazospasticescoy effect of the studied compounds. From this we can conclude that the basis in the and information

Barnes P. J. , 1. W. Rodger and N. C. Thomson, Pathogenesis of asthma, Asthma, basic mechanisms and clinical management, edited by P. J. Barnes, publisher Academic Press, London, 1988

Grynkiwicz, G., M. Poenie, and R. Y. Tsien, A new generation of Ca2+-indicators with greatly improved fluorescence properties, J. Biol. Chem., 260 p. 3440 - 3450, 1985

Hide, M., and M. A. Beaven, Calcium influx in a rat mast cell (RBL - 2H3) line, J. Biol. Chem., 266 p. 15221 -15229, 1991

Kudo, Y. and A. Ogura, Glutamate-induced increase in intracellular Ca2+-concentration in isolated hippocampal neurones, Br. J. Pharmacol., 89, pages 191 - 198, 1986

Putney, J. W. Jr, Capacitative Calcium entry revised, Cell Calcium, 11, PP 611 - 624,1990,

Rink, T. J., Receptormediated calcium entry, Fes. Lett., 2686, page 381 -385,1990,

Seifert, R. and G. Shultz, The superoxide forming NADPH oxidase of phagocytes: An enzym system regulated by multiple mechanism, Rev. Physiol. Biochem. Pharmacol., volume 117, Springer Verlag, 1991

Wells, E. , C. G. Jackson, S. T. Harper, J. Mann, and R. P. Eaoy, Characterization of primate bronchalveolar mast cells II, inhibition of histamine, LTC4and PGF2alfarelease from primate bronchoalveolar mast cells and a comparison with rat peritoneal mast cells, J. Immunol., 137, page 3941 - 3945, 1986

1. Derivatives analyoung of dihydropyridines of General formula I

< / BR>
where A is the mean benzo - or lianoglou;

the substituents R2, independently of one another represent hydroxyl, alkoxy with 1 to 4 carbon atoms, benzyloxy, halogen, alkyl with 1 to 4 carbon atoms;

m is 0, if Eoda, cycloalkenyl with 4 to 6 carbon atoms in cycloalkyl part and 1 to 5 carbon atoms in the alkyl part, or a group of the formula

< / BR>
where R5means alkyl with 1 to 4 carbon atoms, asiagraph, halogen, alkoxy with 1 to 4 carbon atoms;

n is 1, 2 or 3, and n can be 0 in that case, if A denotes lianoglou;

R3and R4independently of one another denote hydrogen, unbranched or branched alkenyl with 3 to 6 carbon atoms, unbranched or branched quinil with 3 to 6 carbon atoms, unbranched or branched alkyl with 1 to 12 carbon atoms, and the alkyl may be substituted by tanila or phenyl, whereby phenyl may be mono-, di - or triamese by alkyl with 1 to 4 carbon atoms, sidegroups, alkoxyl with 1 to 4 carbon atoms, benzyloxycarbonyl, halogen, trifluoromethyl, adamantium, group-SO2NH2or-O-CH2-O-, pyridium, pyrrolidinium, N - methylpyrrolidinium, morpholinopropan, adamantium, nitrosopropane; or R3is hydrogen, and R4- cyclohexyl, phenyl, forfinal, pyridyl or N-benzylpiperidine; or R3and R4together with the nitrogen atom to which they are connected, means pyrrolidinyl, piperidinyl, a group of the formula

what alkoxyphenyl 1 4 carbon atoms in each CNS part, pyrimidinium, phenylalkyl with 1 to 4 carbon atoms in the alkyl part, or a group of the formula

< / BR>
or their salts with physiologically tolerated acids.

2. Means blocking non-selective cationic channels containing the active substance, characterized in that the active substance contains compounds of the formula I under item 1 or its salt with a physiological tolerable acid.

 

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