SI21368A - New derivatives of pyridilethanol(phenylethyl)amines as cholesterol biosynthesis inhibitors, procedures of their preparation and phermaceutical preparations which they contain - Google Patents

Abstract

New derivatives of pyridilethanol (phenylethyl) amines are described, having formula I, where n means an integer from 1 to 4; R1 is a hydrogen atom, hydroxyl group or a lower C1-6 alkoxy group; R2 is a hydrogen atom or a lower C1-6 alkyl group with straight or branched chain; X is a hydrogen atom, fluorine, chlorine, bromine, hydroxyl group, trifluoromethyl group, 3,4-di-Cl, 2,4-di-Cl or lower C1-6 alkoxy group, their enanthiomers, diastereoisomers or racemates or their physiologically acceptable acid addition salts, which are new ligands of sigma receptors in cholesterol biosynthesis inhibition and thus suitable for treatment of hypercholesterolemia and hyperlipidemia in humans. The greatest reduction of cholesterol has been found out in the case of 1-(3-pyridil)-2-N-(2-(3,4-dichlorophenyl)ethyl-N-propyl-amino) ethanol in the form of a dihydrobromide salt (designation BK-35.2 HBr).

Description

New pyridylethanol (phenylethyl) amine derivatives as inhibitors of cholesterol biosynthesis, processes for their preparation and pharmaceutical preparations containing them

Field of the Invention (MPK C 07 D 213/38, A 61 K 31/44)

The present invention falls within the scope of the active ingredients of the group of heterocyclic compounds and the pharmaceutical industry and relates to novel pyridylethanol (phenylethyl) amine derivatives, processes for their preparation, pharmaceutical compositions containing them and their use for inhibiting cholesterol biosynthesis. The novel pyridylethanol (phenylethyl) amine derivatives of the invention are ligands of sigma receptors, cholesterol biosynthesis inhibitors at the sterol Δ7,8 - isomerase stage, and are suitable for the treatment of hypercholesterolemia and hyperlipemia in humans.

A technical problem

There is an ongoing need for new agents as inhibitors of cholesterol biosynthesis, effective in the treatment of hypercholesterolemia and hyperlipemia, to achieve more targeted action in therapy and with fewer side effects relative to the therapeutic agents known in the art.

The state of the art

Due to the influence of high blood cholesterol on the formation of atherosclerosis, many studies have been made to find active substances that reduce mammalian blood cholesterol levels and are therefore effective in the treatment of hypercholesterolemia and hypolipemia. One such treatment method has been found to be inhibition of cholesterol biosynthesis.

Several cholesterol biosynthesis inhibitors are known in the inhibition step of 3-hydroxy-3methyl-glutaryl coenzyme A reductase (HMG-CoA reductase), as described for example in U.S. Pat. No. 4,231,938 (lovastatin), U.S. Pat. No. 4,444,784 (simvastatin) to U.S. Pat. No. 4,346,227 (pravastatin sodium) to U.S. Pat. No. 5,273,995 (atorvastatin), which are already used in therapy and are known as commercial preparations Mevacor®, Sinvacor®, Lipitor®. These HMG-CoA reductase inhibitors, also known by the common name statins, greatly reduce blood cholesterol content.

from U.S. Pat. No. 4,800,206 are known pyridine ethanolamine derivatives suitable for the treatment of obesity and / or diabetes, especially in obese adults.

Sigma ligands are known to bind to sigma receptors, which are structural homologs of the sterol A8,7-isomerase (FFMoebius et al., Brit.J.Pharmacol. (1997), 121,16), which occurs in the last stages of cholesterol biosynthesis. , however, no active substance or drug is known in medicine to inhibit cholesterol biosynthesis at this stage.

Description of solution to a technical problem with implementation examples

The invention is based on the task of finding new agents that would significantly reduce the cholesterol content of mammals by inhibiting cholesterol biosynthesis at the last stages of its biosynthetic pathway, at the sterol Δ7,8 - isomerase level, which would be more selective than the effect of known statins, which inhibit HMG-CoA reductase, which occurs during the early phase of the cholesterol biosynthetic pathway.

The novel compounds of the invention would achieve more targeted action and with fewer side effects than already established agents in therapy.

This problem has been solved by the present invention, which relates to novel pyridylethanol (phenylethyl) amine derivatives, processes for their preparation, pharmaceutical compositions containing them, and use of novel compounds of the invention for the treatment of hypercholesterolemia and hypolipemia.

The new pyridylethanol (phenylethyl) amines of the invention are compounds of general formula I

Oj- (CH ₂ ) n— ¹

Ri R ₂ where n is an integer from 1 to 4

R 1 is a hydrogen atom, a hydroxyl group or a lower C 1-4 alkoxy group

R ₂ is a hydrogen atom or a lower C _1-6 alkyl group with a straight or branched chain X is hydrogen, fluorine, chlorine, bromine, a hydroxyl group, a trifluoromethyl group, 3,4-di-CI, 2,4-di-CI or lower C 1 . ₆ alkoxy group as well as their physiologically acceptable acid addition salts.

The term lower alkyl group means a straight- or branched-chain lower alkyl group having 1 to 6, preferably 1 to 4 carbon atoms ( _C1-6 alkyl), such as methyl, ethyl, npropyl, isopropyl, n-butyl and isobutyl groups. The term lower alkoxy group means an alkoxy group of 1 to 6, preferably 1 to 4 carbon atoms (C _1-6 alkoxy) such as methoxy, ethoxy, propoxy, isopropoxy, butoxy and isobutoxy.

The compounds of formula I form salts with acids and these salts are also part of the invention. Examples of such salts are salts with physiologically acceptable mineral acids such as hydrochloric acid, hydrobromic acid, phosphoric acid; or with organic acids such as methanesulfonic acid, citric acid, oxalic acid, maleic acid, benzenesulfonic acid and others.

The novel compounds according to the invention have at least one asymmetric carbon atom and can therefore exist as optically active enantiomers, as diastereomers or as racemates.

Compounds of formula I wherein n = 2 and wherein R 1 is a hydroxyl group, R ₂ is a methyl or n-propyl group and X is a hydrogen atom or two chlorine atoms at the sites 3 and 4 of the phenyl nucleus are novel pyridylethanol derivatives (phenylethyl ) amines and are preferred compounds of the invention.

Of the above compounds, the following compounds are preferred:

1- (3-pyridyl) -2- (N- (2-phenylethyl) -N-propylamino) ethanol and its dihydrobromide salt of formula II (hereinafter referred to as BK-31)

N.

II

CH ₃

1- (3-pyridyl) -2- (N- (2- (3,4-dichlorophenyl) ethyl) -N-methylamino) ethanol and its dihydrobromide salt of formula ffl (hereinafter referred to as BK33)

- (3-pyridyl) -2- (N- (2- (3,4-dichlorophenyl) ethyl) -N-propylamino) ethanol and its dihydrobromide salt of formula IV (hereinafter referred to as BK35)

and 1- (4-pyridyl) -2- (N- (2- (3,4-dichlorophenyl) ethyl) -N-methylamino) ethanol and its dihydrobromide salt of formula V (hereinafter referred to as BK-38) )

CI

CI

Of the above compounds of the invention, particularly preferred is 1- (3-pyridyl) -2- (N- (2- (3,4-dichlorophenyl) ethyl) -N-propylamino) ethanol and its dihydrobromide salt (BK-35.2HBr) as an inhibitor of cholesterol biosynthesis and thus suitable for the treatment of hypercholesterolemia and hyperipemia.

The compounds of the invention can be prepared in two different ways, which are illustrated in the following scheme as variant (a) and variant (b), as follows:

variant a):

alkylation of secondary amines of formula VI

NHR ₂ CH ₂ CH ₂ Z

VI in which R _{2 has the} above meaning and Z means a group in which X has the above meaning, with pyridyl oxiran (pyridine ethylene oxide) of formula VII

VII to the desired title pyridylethanol (phenylethyl) amines of formula I, which are optionally converted to their physiologically acceptable acid addition salts.

The secondary amines of formula VI can be prepared by alkylation of primary amines of formula XII h ₂ n - ch ₂ ch ₂ zz alkyl iodides of formula XIII

R ₂ J according to the following reaction scheme:

H ₂ N - CH ₂ CH ₂ Z + R ₂ J

XII

XIII

HNR ₂ - CH ₂ CH ₂ Z wherein the substituents R ₂ and Z have the above meaning.

The primary amines of formula XII and alkyl iodides of formula XIII are known and commercially available chemicals.

At sites 2, 3 or 4, substituted pyridyloxides of formula VII in the alkylation process of secondary amines of formula VI are prepared in situ by reaction at sites 2, 3 or 4 of substituted bromoacetylpyridine hydrobromide with complex metal hydrides, such as sodium borohydride in an inert solvent as a lower inert solvent alkanol, such as ethanol, at room temperature.

At sites 2, 3, or 4, substituted bromoacetylpyridine hydrobromide is prepared by metabolizing the starting material at sites 2, 3, or 4 of substituted acetylpyridine, which are known and commercially available chemicals, by bromination with bromine and hydrochloric acid.

The alkylation process of secondary amines of formula VI with pyridyloxylated of formula VII is carried out at temperatures from about room temperature to reflux temperature of the reaction mixture, in an inert solvent such as lower aliphatic alkanol, for example ethanol. The obtained crude pyridylethanol (phenylethyl) amines of formula I are isolated and purified in known manner, preferably by column chromatography.

Variant (b):

alkylation of primary amines of formula VI11

R ₂ NH ₂ in which R _{2 has the} above meaning, with pyridyl oxirane of formula VII

VII to the intermediates of formula IX

IX in which R2 has the above meaning, which are condensed with phenyl acetic acid of formula X

HOOCCH2Z X in which Z has the above meaning, to novel intermediates of formula XI

N. R ₂ O

T1-ch-ch ₂ n-cch ₂ with XI in which the substituents R ₂ and Z have the above meanings, which are reduced to the desired title pyridyethanol (phenylethyl) amines of formula I, which are then optionally converted to their physiologically acceptable acid addition salts.

Primary aliphatic amines of formula Vlyl, such as methylamine or n-propylamine, are known and commercially available chemicals that are alkylated with pyridyl oxirane of formula VII in an inert solvent, such as lower aliphatic alkanol, for example ethanol, to intermediates of formula IX. The latter intermediates are condensed with phenyl acetic acid derivatives of formula X, wherein the substituent Z has the above meaning, in an inert solvent and at room temperature. Known condensing agents can be used as a condensing agent,

-1010 as dicyclohexylcarbodiimide (DCC), such as an inert solvent such as methylene chloride (dichloromethane).

In the final synthesis step, the carbonyl group in the new intermediate compounds XI is reduced to the alcohol group. The reaction may be carried out by conventional reducing agents, preferably those suitable for the reduction of the carbonyl group in the group -R ₂ HN-CO-. Particularly preferred is a complex metal hydride such as UAIH4 in an inert solvent, preferably an ether such as tetrahydrofuran (THF), diethyl ether, dioxane and the like. The desired title pyridylethanol (phenylethyl) amines of the formula I obtained are isolated and purified by conventional methods, preferably by silica gel column chromatography and then optionally converted into their physiologically acceptable acid addition salts.

The procedures for the production of novel pyridylethanol (phenylethyl) amine derivatives of the formula I according to variants (a) and (b) are illustrated in Figure 5.

Synthesis of new derivatives of pyridylethyl (phenylethyl) amines of formula I, wherein R1 is a hydrogen atom, can be carried out such that the new compounds of the invention of formula I, wherein R1 is a hydroxyl group, are first acetylated in the usual manner as with acetanhydride and then obtained The O-acetyl compound is catalytically hydrogenated by known methods, such as palladium on a carrier, such as barium sulfate, according to the following variant c)

-1111

acetanhydride

-chch ₂ nr ₂ ch ₂ ch ₂ with ococh ₃ h ₂

CH ₂ CH ₂ NR ₂ CH ₂ CH ₂ Z

Pd / BaSO ₄

Alternatively, new pyridylethanol (phenylethyl) amine derivatives of formula I can be prepared, wherein R1 represents a hydrogen atom, according to the following variant d)

LIAIH4 .N.

((3 ^ ch ₂ cooh - * (Q ^ ch ₂ cooch ₂ ch ₃ [^ CH ₂ CH ₂ OH

SOCI2 / CHCI3 β2 # ΟΗ ₂ ΟΗ ₂ ΟΙ

H ₂ NCH ₂ CH ₂ Z [T ^ -ch ₂ ch ₂ nhch ₂ ch ₂ z

-1212

Initially at sites 2, 3 or 4 - the substituted pyridyl acetic acid is esterified in the usual manner known in the art, for example by metabolizing to its ethyl ester pyridyl acetic acid, which is then reduced by conventional reducing agents, preferably those for reducing the ester group to an alcoholic group. Particularly preferred is a complex metal hydride such as lithium aluminum hydride (LiAIH ₄ ) in an inert solvent, preferably ether, such as diethyl ether, tetrahydrofuran, dioxane and the like. The 2, 3 or 4-substituted pyridyl ethanol thus obtained is reacted into the 2, 3 or 4-substituted pyridyl ethylene chloride with conventional chlorinating agents, for example thionyl chloride in an inert solvent such as chloroform. With the resulting substituted pyridylethylene chloride, the primary amines of formula VI are alkylated to the title derivatives of the pyridylethanol (phenethyl) amines of formula I, wherein R1 is a hydrogen atom.

In accordance with the invention, the influence of novel derivatives of pyridylethanol (phenylethyl) amines as sigma receptor ligands in the inhibition of cholesterol biosynthesis was evaluated. We used the ex vivo method of metabolic labeling of human immortal hepatocyte cell lines. Cells were added with a radiolabelled cholesterol precursor [ ³ H] acetate, with or without the addition of sigma ligands. Two mutually independent experiments on metabolic labeling and sterol analysis were performed. The results obtained in both experiments are reproducible and indicate that the test substances significantly inhibit cholesterol synthesis.

Among the new sigma receptor ligands of the invention, the greatest inhibition of cholesterol synthesis was demonstrated by the compound 1- (3-pyridyl) -2- (N- (2- (3,4-dichlorophenyl) ethyl) -Npropylamino) ethanol, in the form of a dihydrobromide salt ( code BK 35.2 HBr).

Namely, sigma ligands are known to bind to sigma receptors, which are structural homologues of the Δ8,7 - isomerase because they belong to the same gene family. Sterol on A8,7-isomerase occurs in the late stages of cholesterol biosynthesis, as illustrated in Fig. 1. It can be seen from Fig. 1 that the most common substrates are Δδ-cholestenol and cymosterol, which differ from each other in the saturation of the side chain at the position

-1313

Δ24,25. Figure 2 illustrates cholesterol biosynthesis with designated cholesterol inhibitor sites.

Thus, the influence of novel pyridylethanol (phenylethyl) amines as sigma ligands of the invention is more selective than the effect of statins such as lovastatin or pravastatin that inhibit HMG CoA reductase, which occurs at the early stage of the cholesterol biosynthetic pathway. By inhibiting cholesterol synthesis at the last steps of the biosynthetic pathway, the novel pyridylethanol (phenylethyl) amines of the invention can achieve a more targeted action and with fewer side effects, making these compounds particularly suitable for the treatment of hypercholesterolemia and hyperlipemia. Such action of the new pyridylethanol (phenylethyl) amines of the invention was truly unexpected, since no active substances that would lower cholesterol levels in the last stages of the cholesterol biosynthetic pathway have been used in medicine and therapy.

By using the new pyridylethanol (phenylethyl) amines of the formula I of the invention in patients with pathologically elevated cholesterol biosynthesis, the blood cholesterol level of these patients is significantly reduced. The dose and frequency of administration depend both on the properties of each compound, its bioavailability, its pharmacokinetic properties and the patient's condition.

Pharmaceutical preparations contain the active substance together with a physiologically acceptable organic or inorganic carrier such as water, lactose, starch and starch derivatives, magnesium stearate, talc, vegetable oils and the like. The pharmaceutical preparations are preferably used orally, for example in the form of tablets, capsules, pills, powders, granules, solutions, syrups, suspensions, elixirs and the like. The use may also be parenteral, for example in the form of sterile solutions, suspensions or emulsions. The pharmaceutical preparations may be sterilized and / or contain ingredients such as condoms, stabilizers, emulsifiers, buffering agents, and other additives.

The invention is described, but in no way limited by the following examples:

-1414

EXAMPLE 1

1- (3-pyridyl) -2- (N- (2-phenylethyl) -N-propylamino) ethanol (BK 31)

Preparation of starting compounds:

N-propyl- (p-phenylethyl) amine

Pour 1.2 ml (9.5 mmol) of phenylethylamine, 0.93 ml (9.5 mmol) of propyliodide, 5 ml of triethylamine and 5 ml of THF (tetrahydrofuran) into the flask, and heat the reaction mixture at reflux temperature for 3.5 hours. The reaction mixture was then cooled, the salt recovered filtered off, the solution evaporated and the desired compound purified by silica gel column chromatography (silica gel 60, mobile phase: CHCI ₃ : CH ₃ OH = 10: 3). 0.62 g (40%) of N-propyl- (phenylethyl) amine are obtained in the form of an oil (molar mass: 163.264, formula: C11H17N).

3-Bromoacetylpyridine hydrobromide

Add 30 ml of 48% hydrochloric acid to 10 g (82.5 mmol) of 3-acetylpyridine. The reaction mixture was warmed to 70 ° C and 4.2 ml of bromine was slowly added dropwise while stirring. After complete addition of bromine, the reaction mixture was stirred for 15 minutes at the same temperature, then the reaction mixture was cooled on ice, filtered off the crystalline compound thus obtained and washed well with acetone. 21 g (90%) of 3-bromoacetylpyridine hydrobromide are obtained, which melts at 195-200 ° C. Preparation of the title 1- (3-pyridyl) -2- (N- (2-phenylethyl) -N-propylamino) ethanol

20 ml of abs were added to 1.01 g (3.6 mmol) of 3-bromoacetylpyridine hydrobromide. ethanol and 0.5 g (13.2 mmol) of sodium borohydride were added. The reaction mixture was stirred at 20 ° C for two hours. The reaction mixture was then filtered, and 0.96 g (5.9 mmol) of N-propyl- (p-phenylethyl) amine was added to the filtrate containing 3-pyridyloxiran. The reaction mixture was heated at reflux temperature for 4 hours. Then

-1515 the reaction mixture is evaporated to a dry residue, to which is added 20 ml of chloroform, the solid is filtered off, the filtrate is evaporated and the oily residue obtained is purified by silica gel column chromatography (silica gel 60, mobile phase: CHCI ₃ : CH ₃ OH = 10 : 3). 0.56 g (55%) of the title compound are obtained as an oil.

0.56 g (2 mmol) of the purified oily base of 1- (3-pyridyl) -2- (N- (2-phenylethyl) -N-propylamino) ethanol was dissolved in 5 ml of acetone. The resulting solution was cooled on ice and 2.5 ml of ethanolic hydrochloric acid solution (0.35 g (4.3 mmol HBr)) was added while stirring. This eliminates the precipitate, which is topped up with 3 ml of diethyl ether. After stirring on ice for 2 hours, the crystalline product was filtered off and washed with diethyl ether. 0.7 g (80%) of 1- (3-pyridyl) -2- (N- (2-phenylethyl) -N-propylamino) ethanol dihydrobromide are obtained, which melts at 113-120 ° C (molar mass: 446.238, gross formula : C18H24N2O.2HBr).

¹ H NMR spectrum, D ₂ O, ppm relative to DSS (Oppm): 8.89. 8.80 (2H), 8.65, 8.57 (1H), 8.10 (1H), 7.38 (5H), 5.47 (1H), 3.7-3.1 (8H), 1.80 (2H), 0.97 (3H).

The IR (infra-red) spectrum (KBr plate) is illustrated in Figure 6.

EXAMPLE 2

1- (3-pyridyl) -2- (N- (2- (3,4-dichlorophenyl) ethyl) -N-methylamino) ethanol (BK 33)

Preparation of 1- (3-pyridyl) -2-methylaminoethanol

Add 20 ml of abs to 1.01 g (3.6 mmol) of 3-bromoacetylpyridine hydrobromide prepared according to Example 1. ethanol and 0.5 g (13.2 mmol) of sodium borohydride were added and the reaction mixture was stirred at 20 ° C for two hours. The reaction mixture was then filtered and to the filtrate containing 3-pyridyloxidane was added 1.3 ml of 33% ethanol methylamine solution. The reaction mixture was heated at reflux temperature for 5 hours. The reaction mixture is then evaporated to a dry residue, to which is added 20 ml of chloroform, the solid is filtered off, the filtrate is filtered off.

The -1616 was evaporated and the resulting oily residue was purified by silica gel column chromatography (mobile phase: CHCI ₃ : CH ₃ OH = 10: 3). 0.33 g (60%) of the title compound are obtained as an oily base (molar mass: 152.196, gross formula: C ₈ H ₁₂ N ₂ O).

Preparation of 1- (3-pyridyl) -2- (N- (2- (3,4-dichlorophenyl) acetyl-N-methylamino) ethanol

Weigh 542 mg (2.6 mmol) of DCC (dicyclohexylcarbodiimide) into the flask and make up with 2 ml of methylene chloride (CH ₂ Cl ₂ ) and then dropwise a solution of 538 mg (2.6 mmol) of 3,4-dichlorophenyl acetic acid in 3 ml of methylene chloride, while stirring. eliminating the precipitate. The reaction mixture was then stirred for 5 minutes and 400 mg (2.6 mmol) of 1- (3-pyridyl) -2-methylaminoethanol was added thereto. The reaction mixture was then stirred for another 1 hour at 20 ° C. The precipitate was filtered off and the resulting solution was evaporated. The evaporated filtrate was purified by silica gel column chromatography (silica gel 60, mobile phase: CHCl ₃ : CH ₃ OH = 10: 0.5). 715 mg (80%) of 1 (3-pyridyl) -2- (N- (2- (3,4-dichlorophenyl) acetyl-N-methylamino) ethanol was obtained (molar mass: 339.224, gross formula: Ci ₆ H ₁₆ N ₂ O ₂ Cl2)

Preparation of the title 1- (3-pyridyl) -2- (N- (2- (3,4-dichlorophenyl) ethyl-N-methylamino) ethanol (BK-33)

Weigh 0.53 g (13.9 mmol) of lithium aluminum hydride (L1AIH4) into the flask and make up to 6 ml of anhydrous tetrahydrofuran (THF) and cool the mixture on ice. A solution of 1.1 g (3.2 mmol) of 1- (3-pyridyl) -2- (N '(2- (3,4-dichlorophenyl) acetyl) -N-ethylamino) ethanol in 10 ml of anhydrous was then added dropwise to the reaction mixture. tetrahydrofuran (THF). After the addition is complete, the reaction mixture is stirred for another 1 hour at 20 ° C. The reaction mixture was then cooled on ice and then 6.5 mL of 15% NaOH was gradually added under vigorous stirring and 16 mL of methylene chloride (CH ₂ Cl ₂ ) was added. The organic phase was separated, dried with anhydrous Na ₂ SO ₄ and evaporated on a rotary evaporator to an oily residue which

-1717 was purified by silica gel column chromatography (silica gel 60, mobile phase: CH ₃ OH: ethyl acetate = 10: 2). 0.63 g (60%) of the title compound are obtained as an oily base.

0.60 g (1.84 mmol) of the purified oily base was dissolved in 3.5 ml of acetone. The solution was cooled on ice and 2.4 ml of ethanolic hydrochloric acid solution (0.328 g HBr; 4.1 mmol) were added while stirring. This eliminates the precipitate, to which is added 2 ml of diethyl ether. After stirring the reaction mixture for 2 hours on ice, the resulting crystalline product was filtered off and washed with diethyl ether. 0.72 g (80%) of 1- (3-pyridyl) -2- (N- (2- (3,4-dichlorophenyl) ethyl) -N-methylamino) ethanol dihydrobromide are obtained, which melts at 157-161 ° C ( molar mass: 487.074, gross formula: Ci ₇ H ₁₈ N ₂ OCl2.2HBr).

¹ H NMR spectrum; D ₂ O, ppm relative to DSS (Oppm): 8.90 (1H), 8.78 (1H), 8.64 (1H), 8.10 (1H), 7.50 (2H), 7.24 (1H), 5.50 (1H), 3.52 ( 4H). 3.08 (5H).

The IR spectrum (KBr plate) is illustrated in Figure 9.

EXAMPLE 3

- (3-pyridyl) -2- (N- (2- (3,4-dichlorophenyl) ethyl) -N-propylamino) ethanol (BK-35)

Preparation of 1- (3-pyridyl) -2-propylaminoethanol

To 1.01 g (3.6 mmol) of 3-bromoacetylpyridine hydrobromide prepared according to Example 1, add 20 ml of absolute ethanol and add 0.5 g (13.2 mmol) of sodium borohydride (NaBH ₄ ). The reaction mixture was stirred for 2 hours at 20 ° C. The reaction mixture was then filtered, and 0.7 ml (8.5 mmol) of n-propylamine was added to the filtrate containing 3-pyridyloxiran. The reaction mixture was then heated at reflux temperature for 5 hours. The reaction mixture was then evaporated to

-1818 of the dry residue, to which is added 20 ml of chloroform, the solid is filtered off, the filtrate is evaporated and the oily residue is purified by silica gel column chromatography (silica gel 60, mobile phase: CH ₃ OH: ethyl acetate = 10: 2). 0.33 g (50%) of 1- (3-pyridyl) -2-propylaminoethanol in the form of an oil base are obtained (molar mass: 180.25, gross formula: C 10 H ₆ N ₂ O)

Preparation of 1- (3-pyridyl) -2- (N- (2- (3,4-dichlorophenyl) acetyl) -N-propylamino) ethanol

Weigh 630 mg (3.1 mmol) of DCC (dicyclohexylcarbodiimide) into the flask and make up to volume with 3 ml of methylene chloride and dropwise with stirring a solution of 625 mg (3.1 mmol) of 3,4-dichlorophenyl acetic acid in 5 ml of methylene chloride, removing the precipitate. The reaction mixture was then stirred for 5 minutes and a solution of 550 mg (3.05 mmol) of 1- (3-pyridyl) -2-propylaminoethanol in 6 ml of methylene chloride was added. The reaction mixture was then stirred for another 1 hour at 20 ° C, the precipitated precipitate filtered off and the resulting solution evaporated. The evaporated filtrate was purified by silica gel column chromatography (silica gel 60, mobile phase: CHCl3: CH3OH = 10.0.5). 0.56 g (50%) of 1- (3-pyridyl) -2- (N- (2- (3,4-dichlorophenyl) acetyl) -propylamino) ethanol is obtained in the form of an oil (molar mass: 367.278, gross formula: C18H20N2O2Cl2).

Preparation of the title 1- (3-pyridyl) -2- [N- (2- (3,4-dichlorophenyl) ethyl) -propylaminoethanol (BK-35)

Weigh 0.43 mg (11.3 mmol) of lithiumaluminum hydride (LiAIH ₄ ) into the flask and make up to 6 ml of anhydrous tetrahydrofuran (THF) and cool the mixture on ice. A solution of 1 g (2.7 mmol) of 1- (3-pyridyl) -2- (N2- (3,4-dichlorophenyl) acetyl) -N-propylamino) ethanol in 10 ml of anhydrous THF was then added dropwise. After the addition is complete, the reaction mixture is stirred for another 1 hour at 20 ° C. The reaction mixture was then cooled on ice and 6.4 mL of 15% NaOH was gradually added under vigorous stirring and 16 mL of methylene chloride was added.

-1919

The organic phase was separated, dried with anhydrous Na ₂ SO ₄ and evaporated on a rotary evaporator. The evaporated residue was purified by silica gel column chromatography (silica gel 60, first mobile phase: CHCI ₃ : CH ₃ OH = 10: 0.5; second mobile phase: ethyl acetate: CH ₃ OH = 10: 1.5). 0.58 g (60%) of the title compound are obtained as an oily base.

0.50 g (1.4 mmol) of the obtained purified oily base was dissolved in 4 ml of acetone. The resulting solution was cooled on ice and 1.1 ml of ethanolic hydrochloric acid solution (0.25 g HBr; 3.1 mmol) was added while stirring. A white precipitate is removed, to which is added 3 ml of diethyl ether and after 2 hours of stirring on ice, the crystalline product is filtered off and washed with diethyl ether. 0.62 g (85%) of 1- (3-pyridyl) -2- (N- (2- (3,4-dichlorophenyl) ethyl) -N-propylamino) ethanol dihydrobromide are obtained, which melts at 198-202 ° C ( molar mass: 515.124; gross formula: Ci ₈ H ₂₂ N ₂ OCI ₂ .2HBr) ¹ H NMR spectrum, D ₂ O, ppm relative to DSS (Oppm): 8.91 (1H), 8.81 (1H), 8.64 (1H) , 8.12 (1H), 7.54 (2H), 7.27 (1H), 5.50 (1H), 3.58 (2H), 3.48 (2H), 3.34 (2H), 3.16 (2H), 1.82 (2H), 1.00 (3H)

The IR spectrum (KBr plate) is illustrated in Figure 8.

EXAMPLE 4

1- (4-pyridyl) -2 (N- (2- (3,4-dichlorophenyl) ethyl) -N-methylamino) ethanol (BK-38)

Preparation of 1- (4-pyridyl) -2-methylaminoethanol

To 1.01 g (3.6 mmol) of 4-bromoacetylpyridine hydrobromide prepared according to Example 1, add 20 ml of absolute ethanol and add 0.5 g (13.2 mmol) of sodium borohydride and stir the reaction for 2 hours at 20 ° C. The reaction mixture was then filtered and to the filtrate containing 4-pyridyloxiran added 1.3 ml of a 33% ethanol methylamine solution. The reaction mixture was then heated at

-2020 the reflux temperature of the reaction mixture for 3 hours. The reaction mixture is then evaporated to a dry residue, to which is added 20 ml of chloroform, the solid is filtered off, the filtrate is evaporated, and the resulting oil residue is purified by silica gel column chromatography (silica gel 60, mobile phase: CH3OH: ethyl acetate = 10: 2) . 0.30 g (55%) of the title compound are obtained as an oily base (molar mass: 152.196; gross formula: C ₆ H ₁₂ N ₂ O).

Preparation of 1- (4-pyridyl) -2- (N- (2- (3 _t 4-dichlorophenyl) acetyl) -N-methylamino) ethanol

Weigh into the flask 0.54 g (2.6 mmol) of DCC (dicyclohexylcarbodiimide) and make up to volume with 2 ml of methylene chloride and then dropwise with a solution of 0.54 g (2.6 mmol) of 3,4-dichlorophenyl acetic acid in 4 ml of methylene chloride, while removing the precipitate. The reaction mixture was stirred for 5 minutes and then a solution of 400 mg (2.6 mmol) of 1- (4-pyridyl) -2-methylaminoethanol in 3 ml of methylene chloride was added. The reaction mixture was stirred for another 1 hour at 20 ° C. The precipitate was filtered off and the resulting solution was evaporated. The evaporated filtrate was purified by silica gel column chromatography (silica gel 60, mobile phase: CHCl ₃ : CH ₃ OH = 10: 0.5). 0.53 g (60%) of 1- (4-pyridyl) -2- (N- (2- (3,4-dichlorophenyl) acetyl) -Nmethylamino) ethanol is obtained.

Preparation of the title 1- (4-pyridyl) -2 - [(N- (2- (3,4-dichlorophenyl) ethyl) -Nmethylamino) ethanol (BK-38)

Weigh 510 mg (13.5 mmol) of lithiumaluminum hydride (LiAIH ₄ ) into the flask and make up to 6 ml of anhydrous THF and cool the mixture on ice. A solution of 1.02 g (3 mmol) of 1- (4-pyridyl) -2- (N- (2 (3,4-dichlorophenyl) acetyl) -N-methylamino) ethanol in 10 ml of anhydrous THF was then added dropwise. After the addition is complete, the reaction mixture is stirred for another hour at room temperature.

The reaction mixture was then cooled on ice and then 6.6 mL of 15% NaOH was gradually added under vigorous stirring and 16 mL of methylene chloride was added. The organic phase was separated, dried with anhydrous Na ₂ SO ₄ and evaporated on a rotary evaporator to an oily residue which was purified by column chromatography over

-2121 silica gel (silica gel 60, mobile phase: CHCI ₃ : CH ₃ OH = 10: 1). 0.54 g (55%) of the title compound are obtained as an oily base.

0.50 g (1.54 mmol) of the purified oily base was dissolved in 3 ml of acetone. The solution was cooled on ice and 1.8 ml of ethanolic hydrochloric acid solution (0.274 g HBr; 3.4 mmol) were added while stirring. This eliminates the precipitate, which is topped up with 3 ml of diethyl ether. After stirring the reaction mixture for 2 hours on ice, the resulting crystalline product was filtered off and washed with diethyl ether. 0.64 g (85%) of 1- (4-pyridyl) -2- (N-2- (3,4-dichlorophenyl) ethyl) -N-methylamino) ethanol dihydrobromide are obtained, which melts at 191-194 ° C (mol. weight: 487,074, gross formula C17H18N2OCI2. 2 HBr) ¹ H NMR spectrum, D2O, ppm relative to DSS (Oppm): 8.81 (2H), 8.14 (2H), 7.47 (2H), 7.22 (1H), 5:54 (1H); 3.50 (4H), 3.08 (5H)

The IR spectrum (KBr plate) is illustrated in Figure 7.

EXAMPLE 5

Testing of four sigma receptor ligands (BK-31.2 HBr, BK-33.2 HBr, BK-35.2 HBr and BK-38.2 HBr) from cases 1 to 4, sterol Δ7,8 - isomerase cholesterol biosynthesis inhibitors

The influence of four new sigma receptor ligands (BK-31.2 HBr, BK-33.2 HBr, BK-35.2 HBr and BK-38.2 HBr), prepared according to Examples 1 to 4, on the inhibition of cholesterol synthesis was determined. We used the ex vivo method of metabolically labeling human non-lethal hepatocyte cell lines. Cells were added with radiolabelled cholesterol precursor [ ³ H] acetate, with or without ligand supplementation. We performed two mutually independent experiments on metabolic labeling and sterol analysis.

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Materials and methods

Cell culture with the addition of sigma receptor ligands

The permanent human HepG ₂ liver cell line was seeded into 75 cm ² vials in a 1: 2 ratio, two vials per experimental condition. Cells were grown in DMEM medium (L-arginine.HCL 0.084 g / l, L-cystine.2HCI 0.0626 g / l, Lglutamine 0.584 g / l, glycine 0.03 g / l, L-histidine.HCI.H ₂ O 0.042 g / l, L-isoleucine 0.105 g / l, L-leucine 0.105 g / l, L-lysine.HCI 0.146 g / l, L-methionine 0.03 g / l, L-phenitalanin 0.066 g / l, L-serine 0.042 g / l, L-threonine 0.095 g / l, L-tryptophan 0.016 g / l, L-tyrosine 2Na.2H ₂ O 0.10379 g / l, L-valine 0.094 g / l, choline chloride 0.004 g / l, folic acid 0.004 g / l, myoinositol 0.0072 g / l, niacinamide 0.004 g / l, D-pantothenic acid 0.004 g / l, pyridoxal.HCI 0.004 g / l, riboflavin 0.0004 g / l, thiamine.HCI 0.004 g / l, calcium chloride. 2H ₂ O 0.265 g / l, ferric nitrate.9 H ₂ O 0.0001 g / l, magnesium sulfate [anhydrous] 0.09767 g / l, potassium chloride 0.4 g / l, sodium chloride 6.4 g / l, sodium phosphate monobasic [anhydrous] 0.109 g / l, glucose 4.5 g / l and phenol red. At 0.0159 g / l) with 5% calf serum and 1% L-glutamine. After 24 hours, the medium was added to cells with 100 μΜ concentration of sigma receptor ligands (BK-31.2 HBr, BK-33.2 HBr, BK-35.2 HBr and BK-38.2 HBr). A medium with known cholesterol synthesis inhibitors was used as a positive control of cholesterol synthesis inhibition: 100 μΜ of lovastatin or pravastatin, both HMG CoA reductase inhibitors and 100 μΜ of fluconazole, which is an inhibitor of P450 cytochrome subfamily enzymes, including 14 demethylase (CYP51). As a negative control, cells were grown in basic culture medium without the addition of inhibitors. After 24 hours, the medium was replaced. After 48 hours, [ ³ H] acetate was added at a concentration of 40 μθί per 1 ml of medium (400 μθΐ per bottle). After five hours, the medium was aspirated, cells were trypsinized with 2 ml of trypsin, collected in 4 media, cells were centrifuged and the cell pellet was dissolved in distilled water (1 ml per bottle). The cells were homogenized by freezing and thawing. Sterols were extracted from the homogenates. Protein concentration was determined in the homogenate with a Bio-Rad protein determination reagent according to the instructions of this manufacturer.

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Sterol extraction

The homogenate was transferred into glass vials with an inert lid, 3 ml of extraction solution (75% n-heptane: 25% isopropanol (vol./vol.)) Was added. The bottles were shaken vigorously on a shaker in a dark place for 2 hours. After extraction, the bottles were centrifuged (2000 g, 10 minutes), the organic phase was transferred to glass tubes, dried under nitrogen flow, washed with 2 ml of n-heptane HPLC purity, centrifuged (2000 g, 5 minutes) and transferred to a fresh tube. The sample was stored in HPLC solvent until cold and dark.

HPLC analysis (High performance liquid chromatography)

The dried extract was dissolved in 250 μΙ n-heptane. Aliquots of 100 μΙ were injected on a column for normal phase (ChromSpher Si, 150 mm x 3 mm, particle size 5 pm) with a mobile phase of 99.5% n-heptane: 0.5% isopropanol at a flow rate of 1 ml / min and room temperature. temperature.

Determination of sterols

Sterols were determined using an ultraviolet detector at two wavelengths: 200 nm for lanosterol / T-MAS and cholesterol and 249 nm for 4,4-dimethyl-acholeste-8,14,24-trien-3p-ol (FF-MAS) and the internal standard ergosterol. We used flow cell detectors to determine sterols after metabolic cell labeling. The determination was made based on the retention times of the standards: lanosterol (Steraloids), cholesterol (Steraloids), ergosterol (Sigma), 4,4-dimethyl-a-cholesterol-8,14,24, -trien-3p-ol (FF- MAS) and [ ³ H] FF-MAS (AGByskov Laboratory, Rikhospitalitet, University of Copenhagen). The results were normalized by the amount of ergosterol internal standard as well as by

-2424 protein concentration in homogenate. They are shown as the average of two measurements with the associated standard deviation.

Results

Metabolic cell labeling has shown excellent results in reducing the amount of cholesterol synthesized. For cholesterol, we found a peak on the radio detector at about 6.0 min. Figure 3 illustrates the amount of radiolabelled cholesterol after metabolic labeling of cells and the addition of various inhibitors. Negative control, normal medium - medium without inhibitors, A - analysis 1, B - analysis 2. AU - random units. The cholesterol peak, as illustrated in Figure 3, decreased significantly in cells cultured with sigma ligands, most notably for the compound labeled BH-35.2HBr. In addition to the complete inhibition of cholesterol synthesis, as illustrated in Figure 3, the tested compounds also showed an effect on the accumulation of early intermediates of the squalene moiety of cholesterol biosynthesis. An increase in the concentration of sterols corresponding to lanosterol and 4,4-dimethyl-acholest-8 (9), 24, -diene-3β-ol (T-MAS) was detected, respectively. From this point of view, the BK-35.2HBr labeling compound showed the greatest influence, where a tenfold increase in the amount of the intermediate was observed, as illustrated in Figure 4. Figure 4 illustrates the amount of the sterol X radioactive intermediate eluting behind cholesterol (7dehydrocholesterol or latosterol) . The labels are the same as in Figure 3. A - analysis, B analysis 2.

All analyzes confirm that the new pyridylethanol (phenylethyl) amine derivatives as sigma ligands of the invention block the synthesis of cholesterol most likely at the sterol level of A8,7-isomerase. In the presence of a compound labeled BK-35.2HBr, the amount of cholesterol is reduced and the amount of intermediates present in the biosynthesis pathway prior to sterol A8,7-isomerase is increased.

Tests in cells cultured with well-known HMG-CoA reductase inhibitors (lovastatin and pravastatin) and Ianosterol 14a-demethylase (fluconazole) served as a positive control of the assay reliability. She was on lovastatin and pravastatin

-2525 biosynthesis is completely inhibited, as illustrated in Figure 3. In fluconazole, inhibition of cholesterol synthesis was worse, which is expected since fluconazole is not a specific inhibitor of human lanosterol-14a demethylase. The amount of lanosterol and 4,4-dimethyl-a-cholest-8 (9) and 24, -dien-3p-ol (T-MAS), however, did not increase in statins, since these compounds inhibit biosynthesis at the HMG-CoA reductase step, which is at the beginning of the cholesterol biosynthesis sequence, ie before lanosterol and T-MAS.

Conclusions

We found that in cells cultured in culture medium with the addition of the tested compounds labeled BK-31. 2 HBr, BK-33.2 HBr, BK-35.2 HBr, and BK-38.2 HBr, significantly reduced the amount of cholesterol formed. On this basis, it is concluded that all tested compounds or novel derivatives of the pyridylethanol (phenylethyl) amines of the invention are inhibitors of cholesterol biosynthesis, most likely at the level of the sterol A7,8-isomerase. The greatest reduction in cholesterol was observed with the compound labeled BK-35.2 HBr, i.e. 1- (3-pyridyl) -2- (N- (2- (3,4-dichlorophenyl) ethyl) -Npropylamino) ethanol dihydrobromide. The results obtained in two independent experiments are consistent and indicate that, among all sigma ligands tested, the BK-35.2 HBr labeled compound is the best cholesterol biosynthesis inhibitor of the invention and is therefore particularly suitable for the treatment of hypercholesterolemia and hyperlipemia.

LEK pharmaceutical company d.d.

Claims (11)

PATENT APPLICATIONS Compounds of formula I -X Ri R ₂ where n is an integer from 1 to 4 R 1 is a hydrogen atom, a hydroxyl group or a lower C 1-6 alkoxy group R ₂ is a hydrogen atom, a lower C _1-6 alkyl group with a straight or branched chain X is a hydrogen atom, fluorine, chlorine, bromine, hydroxyl group, trifluoromethyl group, 3,4-di-CI, 2,4-di-CI or lower A C _1-6 alkoxy group, their enantiomers, diastereoisomers or racemates or their physiologically acceptable acid addition salts. Compounds according to claim 1, wherein n is an integer 2, Rt is a hydroxyl group, R _{2 is} methyl, ethyl, n-propyl, isopropyl, n-butyl or isobutyl group and X is a hydrogen atom or with two chlorine atoms substituted phenyl at sites 3 and 4 or at positions 2 and 4. Compounds according to claims 1 and 2, wherein R 1 is a hydroxyl group in the RS configuration. -2727 4. 1- (3-Pyridyl) -2- (N- (2- (3,4-dichlorophenyl) ethyl-N-propylamino) ethanol and its dihydrobromide salt. 5. 1- (3-Pyridyl) -2- (N- (2-phenylethyl) ~ N-propylamino) ethanol and its dihydrobromide salt. 6. 1- (3-Pyridyl) -2- (N- (2- (3,4-dichlorophenyl) ethyl-N-methylamino) ethanol and its dihydrobromide salt. 7. 1- (4-Pyridyl) -2- (N- (2- (3,4-dichlorophenyl) -N-methylamino) ethanol and its dihydrobromide salt. Compounds of formula I according to any one of claims 1 to 7 and their physiologically acceptable acid addition salts as sigma receptor ligands in the inhibition of cholesterol biosynthesis for the treatment of hypercholesterolemia and hyperlipidemia in humans. Pharmaceutical preparations comprising a compound of formula I according to any one of claims 1 to 7 or a physiologically acceptable acid addition salt thereof. Use of the compounds of formula I according to any one of claims 1 to 7 or their physiologically acceptable acid addition salts as sigma receptor ligands in the inhibition of cholesterol biosynthesis for the preparation of pharmaceutical compositions for the treatment of hypercholesterolemia and hyperlipemia in humans. A process for the preparation of compounds of formula I according to any one of claims 1 to 7, characterized in that a) alkylation of the secondary amines of formula VI NHR2CH2CH2Z VI -2828 in which R ₂ in Formula I has the indicated meaning and Z represents a group in which X in Formula I has the indicated meaning, with pyridyl oxinated by Formula VII VII and optionally convert the resulting compounds of formula I into a salt or b) alkylation of primary amines of formula Vlil R ₂ NH ₂ VIII in which R _{2 is as defined} in Formula I, with pyridyl oxinated by Formula Vil -2929 νπ to the intermediates of formula IX IX in which R _{2 is as defined} in Formula I, which is fused with phenyl acetic acid derivatives of Formula X HOOCCH ₂ Z in which Z has the above meaning, to the intermediates of formula XI R ₂ O Γ II ch-ch ₂ n — cch ₂ z XI -3030 which are reduced to the title compounds of formula I, which are optionally converted into a salt.