NO812944L - PROCEDURE FOR THE PREPARATION OF NEW AMINOTIOL DERIVATIVES - Google Patents

Description

1

The present invention relates to derivatives of aminothiols with the general formula (I):

.where: j

- R, means hydrogen or a straight or branched C1-C10 alkyl residue;

- R, and R2 together with the adjacent carbon atom can form

a C^Cq cycloalkyl radical,

- R^means hydrogen, a straight-line or branched C-^-C^' , alkyl residue, a straight-line or branched C^-C^alkahoyl thio residue, a straight-line or branched lower C^-C^alkyloxycarbonylthio residue, a straight-line or branched C -^-C^q alkylthio residue or a residue . - (S) ^CR^^CH^N^Rg where' n

is 1 or 2;

1

- R^ and R,- which may be the same or different mean: j

- hydrogen, ,

- a straight or branched C^C^ alkyl residue,

a straight or branched C2-C8 alkyl residue substituted with: - hydroxyl, carboxyl or a straight or branched C1-C4 alkoxycarbonyl group,

- a naphthyloxy group,

- a phenyl, phenoxy, phenylthio or benzyloxy group which is optionally substituted with one, two or more halogen atoms such as chlorine, with a,

two or more straight-chain or branched C1-C4 alkyl residues, one, two or more close-chain or branched C1-C4 alkyloxy residues,

a straight-chain or branched C 1 -C 4 alkoxy or alkylthio group,

- a C 1 -C 4 alkylamino group optionally substituted with a phenyl group,'!

I!

to the erythro and threo configurations respectively, and these two

in racemates can be dissolved by traditional methods, e.g. by the formation of diastereoisomeric salts by the action of optically active acids such as tartaric acid, diacetyltartaric acid, vinanilic acid, dibenzoyltartaric acid, ditoliyltartaric acid and . separation of the mixture of diastereoisomers through crystallisation, in distillation, chromatography, thereby releasing the optically active bases from these salts.

For the products with three asymmetry centers, it is possible to obtain 8 optical isomers. The same methods can be used for

in solution of these mixtures. IN

: I

Thus, the derivatives can be used either in the form of mixtures containing several diastereoisomers, regardless of their mutual ratio, or in the form of enantiomer pairs in equal proportions (racemic mixture), or not, or as optically pure compounds.

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The derivatives are hypolipidemic, antithrombotic, antiarthritic; tic, antimitotic, antihypertensive, vasodilatory and • ; immunomodulating properties. These properties make it possible to use the products in the treatment of cardiovascular \< diseases such as hypercholesterolaemia, thrombotic disorders and arteriosclerosis, for the treatment of rheumatoid arthritis, for the treatment of cancerous disorders and for the treatment of hypertension and especially for the prevention of vascular lesions that follow hypertension.

The present invention also requires pharmaceutical mixtures containing at least one compound of the general formula (I) and/or a salt thereof as active ingredient with a pharmaceutical diluent. These mixtures are formulated on such a way that they can be given orally, rectally, parenterally or topically.

Thus, e.g.' the compositions for oral administration may be liquid or solid and be available in the form of tablets, pastilles, coated tablets, cardboard capsules, granules, pul^- \

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vere, syrups or suspensions. The dry oral forms contain the commonly used additives and excipients in galenic pharmacy, inert diluents, disintegrants, binders and lubricants such as lactose, starch, talc, gelatin, stearic acid, cellulose and derivatives, silicon-! acid, magnesium stearate, polyvinylpyrrolidone calcium phosphate, in calcium carbonate, etc.

Such preparations can be prepared to prolong bursting and consequently the duration of the activity of the active ingredient. in .

Aqueous suspensions, emulsions and oily solutions j are prepared in the presence of sweeteners such as dextrose or I glycerol, flavoring agents, for example vanillin, and can also

i contain thickeners, wetting agents, preservatives | laughing..

The emulsions and oily solutions are prepared in an oil of vegetable or animal origin and may contain flavourings. emulsifiers, dispersants, sweeteners and antioxidants. Sterile water, an aqueous solution of polyvinylpyrrolidone, ground nut oil, ethyl oleate, .etc. used as a carrier for parenteral administration. These aqueous or oily injectable solutions may contain thickening, wetting, dispersing and gelling agents.

Preparations intended for topical administration may be semi-solid or liquid and formulated in the form of hydrophobic lubricating bases, hydrophilic lubricating bases, emulsified bases, pastes, gels and creams. They may contain additives and diluents that are generally used in galenic pharmacy, surfactants, solvents, thickeners, emollients, antioxidants, preservatives such as sorbitan derivatives, cetyl alcohol ethers, alginic acid, propylene glycol, glycerin, sorbitol, cetyl alcohol, stearyl alcohol, cetyl stearyl alcohol, propyl gallate, benzyl alcohol, p-hydroxybenzoic acid esters. '■j

i Several methods can be used for the manufacture of the products. As stated above, these methods also form a

. part of the invention. When these methods give new intermediate products, these new products and the methods for their production are also the subject of the present invention, in

In i

i A first method consists in the reductive transfer of! an amide, an imine or a corresponding iminium salt in an amino-r thiol derivative of formula (I). This method consists, in particular, in subjecting an aldehyde, a ketone or, a corresponding acid, to a sulphurisation, a condensation with an amine and a reduction, which gives the said derivative. in

According to a first variant of this method, an aldehyde is treated with a sulphurizing agent, then condensed with a primary amine and the imine (II) obtained thereby is reduced to ;

amine. The following reaction scheme illustrates this process

Late.

Rl'R2°^R4^are ^e above^or stated meanings, X means hydrogen or an easily cleavable group such as chlorine, bromine, tosyl or mesyl groups, Rg means hydrogen or the SO^Na group.

The carbon-sulphur bond is formed by using sulphurizing agents such as sodium hydrosulfite (Na2S203).., sodium hydrosulfid j

i (NaSH) or sulfur monochloride (S„C19).■■.■■•]

In the latter case, a symmetrical disulfide is obtained with: for-• Il mel (III): j j

I 1 The sulphurisation reaction is carried out in an inert organic solvent such as a chlorinated solvent, an aliphatic or aromatic hydrocarbon, an alcohol, an ether or a nitrile. in

i i The temperature is between the room temperature and the reflux temperature for the selected solvent. in

The transfer to the imine takes place readily in an inert organic solvent selected for example from alcohols, chlorinated solvents, aromatic or aliphatic hydrocarbons, and generally under acid catalysis, by e.g. introduce into the reaction mixture a catalytic amount of p-toluenesulfonic acid or an organic acid or mineral acid.

The resulting imine is then reduced according to methods that are well described in the literature.

This reduction can be carried out in the usual way, e.g. by hydrogenation in the presence of a catalyst such as palladium on carbon, Raney nickel or platinum, in the presence of a solvent such as methanol or ethanol at normal or high pressure, or by the action of alkali metal hydrides such as sodium borohydride in a solvent such as methanol or ethanol, preferably at low temperature, or as lithium aluminum hydride in ether or tetrahydrofuran, preferably at low temperature, or by the action of an aluminium-

in an alcoholate such as aluminum isopropylate in a solvent such as isopropanol, preferably under reflux of the latter,

or by the action of diborane in the same solvents.

1

i When a disulfide is formed in the first step, it can be transferred i

thiol, e.g. under the action of a reducing agent such as phosphine, especially tributylphosphine or lithium or aluminum hydride. This reaction takes place in an inert organic solvent such as a chlorinated solvent, an alcohol, an ether or a hydrocarbon and at a temperature for the solvent.. I

A second variant of this method consists in condensing an aldehyde of formula (IV). with a secondary amine under !

in the formation of an iminium salt and reduce the latter to an amine.<1>The following scheme illustrates this variant.

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R^, in R3'R5°9Rg have the same meanings as given above.

The condensation of aldehyde (IV) with the secondary amine readily takes place in an inert organic solvent selected for example from alcohols, chlorinated solvents, aromatic or! aliphatic hydrocarbons,, and advantageously under acid catalytic conditions, e.g. by introducing a catalytic amount of. p.toluenesulfonic acid or an organic acid or raineral acid in the reaction mixture. It is likewise an advantage to optionally introduce the secondary amine in the form of a salt, e.g. !in the form of the hydrochloride. in

The iminium salt is then reduced to amine under the influence of a reducing agent such as e.g. hydride of lithium and aluminum in ether or tetrahydrofuran. |

According to a third variant, an amide (V), which is easily produced from acid and amine through the acid chloride, is first treated with a sulfurizing agent and then reduced to aminothiol according to the reaction scheme: j-j

R1' R2'R4 °^ R5^ are previously indicated meanings, X means hydrogen or an easily cleavable group such as chlorine, bromine, tosyl or methyl groups, R^ means hydrogen or the NaSO^ group. The carbon-sulphur bond is formed by the use of sulphurizing agents such as e.g. sodium hyposulphite (Na2S20^)., sodium hydrosulphide (NaSH). or sulfur monochloride (S2C12)... In the latter case, a symmetrical disulfide of formula (VII) is obtained.. ■■ ' ; 'in

The sulfurization reaction is carried out in an inert organic solvent such as a chlorinated solvent, an aliphatic or aromatic hydrocarbon, an alcohol, ether or a nitrile. The temperature is between normal temperature and reflux

the temperature of the selected solvent. i i<!>i i 1 ! ! The amide (VI) is then reduced in the usual way, e.g. by hydrogenation in the presence of a catalyst, such as palladium/carbon, ' Raney nickel or platinum, in the presence of a solvent 1 ir such as methanol or ethanol at normal or i.higher pressure, |

1 or by the action of metal hydrides such as the hydride of aluminum lithium in ether or tetrahydrofuran, or by the action of diborane in the same solvents.

i When a disulfide is formed:- in the first step, it can be transferred i i thiol by the influence of e.g. a reducing agent such as a phosphine, especially tributylphosphine or lithium aluminum hydrides. This reaction takes place in an inert organic solvent such as a chlorinated solvent, an alcohol, in an ether or a hydrocarbon and at a temperature between normal temperature and the reflux temperature of the solvent.

It is likewise possible to introduce, in a derivative with formula (I), substituents R^ and R,- which are different from hydrogen on the nitrogen atom, and the substituent R^ on the sulfur atom by | j alkylation or by acylation. ■ '' '\

a) alkylation on the nitrogen atom

R1'R2°^R3^are ^e previously stated meanings and Q means the substituent R^ or a protecting group for the thiol function,

X means an easily cleavable group such as a halogen atom which

chlorine, bromine and iodine or tosyl and methyl groups, A list of protecting groups for the thiol function can be found in ! special works such as e.g. "Protective Groups in Organic Chemistry", Ed. J. F. W. McOmie, Plenum Press (1973)... i I j!

in

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The alkylation of the amine is normally carried out in an inert organic solvent such as a chlorinated solvent, an aromatic or aliphatic hydrocarbon, an alcohol or an ether, and advantageously by refluxing the solvent. The presence of an organic or mineral base such as sodium hydroxide or triethylamine in the reaction mixture aids the reaction and increases the yield. j-i

b) acylation on the nitrogen atom

R1 °^ R2 ^are ^e meanings indicated above, Q means the group or protecting group for the thiol function, .Y means an easily cleavable group such as e.g. chlorine, bromine, or the R'^COO J group. R<1>^means the substituent R^whose chain is one carbon atom shorter. A list of protecting groups for the thiol function can e.g. can be found in the book referred to above.

i The acylation is normally carried out in an inert organic solution

agent such as a chlorinated solvent, an aromatic or, aliphatic hydrocarbon or an ether, and advantageously by solu- | the reflux temperature of the liquid. The presence of organic or mineral base in the reaction mixture aids the reaction and increases the yield.

The reduction of the amide (VIII) can be carried out in the usual way, for example by hydrogenation in the presence of a catalyst such as palladium on carbon, Raney nickel or platinum in the presence of a solvent such as methanol or ethanol at normal or elevated pressure, or by impact of metal- '

in hydrides such as hydride of aluminum and lithium in ether or tetrahydrofuran, or by the action of diborane in the same solvents. j

c) alkylation on the sulfur atom i

R^, R2, R^, R^ and R^, have the meanings indicated above and

: X means an easily cleavable group such as e.g. a halogen atom such as chlorine, bromine or iodine, or a tosyl or mesyl group.

The alkylation is normally carried out in an inert organic solvent such as a chlorinated solvent, an aromatic or aliphatic hydrocarbon, an alcohol or an ether and advantageously at the reflux temperature of the solvent. The presence of organic or mineral base in the reaction mixture aids the reaction and increases the yield.

According to another method, an episulphide of formula (IX) is reacted with an amine according to the general scheme:

R^, R^/R^ and R^, have the meanings indicated above. Depending on the reagents used, the opening of the episulphide can out-; carried out at atmospheric pressure or at a pressure higher than atmospheric pressure, at normal temperature or at a higher temperature, or also by catalysis with Ag ions. The reaction can be carried out with or without a solvent. If a solvent is used, it may be water or an inert organic solvent such as an aromatic or aliphatic hydrocarbon, a chlorinated solvent or an ether. A tertiary amine favorably affects the course of the reaction, which in this case usually takes place at a temperature between 25 and; o * ' • 100 c. 1 i i i Yet another variant is illustrated by the following scheme:

R 1 , R 2 , R 4 and R 1 - have the meanings given above and R 1 means a lower C 1 -C 2 alkyl group.

A thiol carbonate of formula (X) is treated with an acid in an inert organic solvent such as an aliphatic or aro-j

1 matic hydrocarbon or a chlorinated solvent or a | ether. The acid is preferably organic such as p.toluenesulfonic acid (TsOH) or methanesulfonic acid. The reaction temperature is higher than the normal temperature and preferably close to the boiling point of the chosen solvent. The thus obtained derivative (XI) is then treated with a . amine under, reflux in an inert . j. organic solvent and gives the products of formula (I)..;. |

According to a final variant, a derivative ay 2-mercapto-ethanol (XII) is esterified with an acid and then treated with an amine according to the scheme: ; R^, R2, R^ and R,, have the previously indicated meanings and Rg means tyr a group so that the acyloxy group can be easily removed. ] F. e.g. Rg can be a trihalomethyl group such as trifluoro-; methyl or the trichloromethyl group. The esterification is normally carried out in an inert organic solvent selected e.g. among 'the aromatic solvents, chlorinated solvents or the ethers and preferably under reflux of the solvent.

It is advantageous to eliminate water as it is formed, e.g. by azeotropic distillation. The ester (XIII) is then heated in an inert organic solvent as mentioned above in the presence of an excess of amine and forms the products with. formula (I).

i Detailed examples of the preparation of several derivatives of aminothiols according to the invention are given below.

These examples are particularly intended to further illustrate the special characteristics of the methods according to the invention. in !

EXAMPLE 1

2-methyl-1-(2-phenoxyethylamino)-2-propanethiol, hydrochloride ' ; (derivative no. 3 in table 1).<;>

, a. - 817 ml (9 mol; 648.9 g) of isobutylaldehyde is placed under

. stirring to 650 ml of carbon tetrachloride. The mixture is heated! to 50°G and 360 ml (4.5 mol; 135.0 g) of sulfur monochloride are added slowly. The reaction is exothermic. At the end of the reaction, the temperature in the medium is kept at 40°C for 48 hours. The solvent is then evaporated under reduced pressure and the residue is distilled. The main fraction is collected between 94 and 100°C

at a pressure of 0.8 Torr. A further distillation gives 365 g (1.8 mol; 40%) of α,α'-dithiodiisobutyraldehyde. j i

b. - A mixture consisting of 20.6 g (0.1 mol) of the above-mentioned product, 27.4 g (0.2 mol) 2-pheoxyethylamine and 130 ml of isopropanol is heated in 1, 5 hours at reflux temperature.<1>The resulting medium is evaporated to dryness under reduced pressure. The remainder is taken up in hexane. An insoluble part is then filtered, the filtrate is then evaporated. This gives diimino sulphide, which is used as such in the following step. c. - Place .7.6 g (0.2 mol) of lithium and aluminum hydride in suspension in 250 ml of anhydrous ether in a 1 liter reactor. A solution of the above is then added while stirring;

product in 250 ml of the same solvent at such a rate that a slight reflux is achieved and persists. On ! at the end of the reaction, reflux is maintained by heating for a further 2 hours. The product is allowed to cool, and then the excess of hydride is split off. in

i The dried organic phase is placed in contact with. gaseous dry hydrochloric acid until precipitation is complete. The hydrochloric acid is filtered off, washed well with ether and then dried under reduced pressure.

After a recrystallization in a mixture of ethyl acetate and benzene, 3 8.5 g (0.15 mol; 7 5 %) of the expected compound, melting point 13 9.9°C are obtained.

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IR, NMR and the mass spectrum are consistent with the structure. - I 1Elementary analysis: j

EXAMPLE 2 I 2-Methyl-1-(2-p.tolyloxyethylamino)-2-propanethiol, hydrochloride (derivative No. 11 in Table 1). i i ii i This compound is prepared according to the method described in example 1 by replacing 2-phenoxyethylamine with i 2-p.tolyloxyethylamine. ' • I Yield: 56%, m.p. 115.2°C (AcOEt). ; j Elementary analysis:

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. EXAMPLE 3

1-(2-p.chlorophenoxyethylamine)-2-methyl-2---propanethiol hydrochloride.

(derivative no. 13 in table 1)..

This compound is produced according to the operating method described! example 1, by replacing 2-phenoxyethylamine with 2-p.chlorophenoxyethylamine.

Yield: 54% - m.p. ; 141.5°C (AcOEt).

Elemental analysis:

' i

■ : i

EXAMPLE 4 I ;<!>i bis-[2-methyl-1-(2-phenoxyethylamino).-2-propyl] disulfide, dihydrochloride (derivative no.. 14 in table 11.

, i

i This disulphide is produced at the expense of the product which j results from the method described under b. in example no. 1.

A solution of 16.1 g (35 mmol) of rat diiminodisulfide in 90 ml of ethanol is heated to 60°C, then slowly a ! solution of 4.0 g (105 mmol). sodium borohydride in 10.5 ml of: the same solvent. After the addition, the solution is heated at reflux for 2 hours. The mixture is then cooled to room temperature and poured over 350 g of crushed ice. The aqueous phase is extracted using chloroform. The organic extract] is evaporated to dryness under reduced pressure. The evaporation residue is dissolved in ether and the solution is brought into contact with gaseous dry hydrogen chloride. The salt that precipitates out is filtered and recrystallized in propanol. Weight: 11.8 g (23 mmol; 66%L m.p.; 181.8°C. ;

in I

The IR, NMR and mass spectra are consistent with that | expected disulfide structure. i i Elementary analysis:<!>

EXAMPLE 5

3-(2-raethyl-2-mercapto-butylamino).propanoic acid (derivative no. 5

in table 1).. " j

a. - 36.9 g (150 mmol) of 1-amino-2-benzylthio-2-methylbutane hydrochloride are mixed with 200 ral of isopropanol and 62 ml of triethylamine. Stir while 23.0 g (150 mmol). 3-bromopropanoic acid is added slowly. After the addition is complete, it is heated for 2 hours at the reflux temperature. The mixture is cooled to room temperature and evaporated to dryness under reduced pressure. The remainder is distributed between water and chloroform. The ori<ga>-;

The lower phase is dried and evaporated. The rest is taken up in a bit'

, I

acetone which gives a white solid. Weight: 19.0 g (68 mmol; 45%). m.p.; 140.9°C. -in

in

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The NMR spectrum is consistent with the structure for 3-(2-methyl-2-benzylthiobutylamino)propanonic acid. j

! 1 b. - 8.4 g (30 mmol) of the above product is added at a temperature between -50 and -60°C to 200 ml of liquid ammonia, then sufficient metallic sodium is added in small successive portions so that the blue color remains. On this ■. time, the amount of ammonium chloride which discolours the medium is added, and the ammonia is then evaporated once the cooling bath has been removed. in 30 ml of water is added and the mixture is acidified progressively, until

pH 2 under a nitrogen atmosphere. Evaporation is carried out carefully to dryness under reduced pressure, and then the residue is taken up in acetonitrile. The addition of ether causes the precipitation of a white solid which is filtered, washed carefully with 1

in ether and dried.

.Weight: 2.4 g (11 mmol; 37%).. mp: 117.2°C. Examination of the IR, NMR and mass spectra confirms the structure of the expected product. '

Elemental analysis:

EXAMPLE 6

1-(4-benzyl-1-piperazinyl)..-2-propanethiol, dihydrochloride (derivative no. 32 in Table 1).

A mixture prepared from 4.5 g (60 mmol) of propylene sulphide and 10.5 g (60 mmol) of N-benzylpiperazine is stirred for 20 hours at 60°C. The temperature of the mixture is brought to 100°C over the course of 3 hours and is kept at this temperature for a further hour. The reactor is then regulated under reduced pressure (4.10^ Torr) overnight. To the residue is added 150 ml of ether, then hydrochloric acid ether until the precipitation caused thereby is complete. Then filtered and recrystallized from ethanol to constant ! melting point (215 o C).. This gives 12.8 g (40 mmol; 67.%). of the expected hydrochloride whose structure is confirmed through, IR, NMR and mass spectra.

in Elementary Analysis:

EXAMPLE 7 j' 5,5-dimethyl-3-(2-phenoxyethyl).-2-thiazolidinone (derivative no. 23 in Table 1). , 1 10.5 g (40 mmol). 2-methyl-1-^(2^phenoxy'ethylaminol-2-propanethiol hydrochloride is dissolved in 100 ml of methanol which is further added r 11.0 ml (8.0 g; 80 mmol) triethylamine. The mixture is stirred for 15 minutes and then evaporated to dryness under reduced pressure. The residue is taken up in ether and filtered and the filtrate is evaporated again to dryness under reduced pressure. The product obtained is dissolved after drying in 250 ml of toluene, which then: a further 8.0 ml of triethylamine is added before the product is mixed with 250 ml of an aqueous solution of sodium bicarbonate. Vigorous stirring is then carried out while 3 4 ml (65 mmol). of a 20% phosgene solution is added slowly. When the addition is finished,; continue to stir for 2 hours. The organic phase is decanted, dried and then evaporated to dryness under reduced pressure. The rest consists of a dark oil that crystallizes spontaneously. The recrystallization in n-heptane then gives a white solid weighing 5.2 g (21 mmol: 53%). M.p.: 51f3 o C. I ( ';

The structure of the cyclization product is confirmed through IR, NMR and the mass spectrum. il Elementary analysis: ,

EXAMPLE 8

2-Methoxycarbonyldithio-2-methyl-1-(2-phenoxyethylamino)-propane hydrochloride (derivative No. 24 in Table 1).

in i

9.8 g (77 mmol) of methoxycarbonylsulfenyl are dissolved in 600 ml of methanol in an inert atmosphere (oxygen-free nitrogen).. Solution- ■ | in which is stirred and cooled to 0°C, a solution of 15.7 g (60 mmol) of the product from example 1 in 300 ml of the aforementioned solvent is added over the course of half an hour;

After the addition is complete, continue stirring in; half an hour before the mixture is allowed to return to room temperature.

i I !<!>Evaporation is carried out under reduced pressure. The remaining i' syrup crystallizes after being stored in the refrigerator. One crystallizes from benzene and isopropanol in sequence. ! In this way, 10.6 g (30 mmol; 50%L) of the expected disulphide is obtained, the structure of which is confirmed from IR, NMR and the mass spectra. Mp.: 104.1° C.,

Elementary analysis: j i

The melting points and recrystallization solvents for the derivatives produced according to the invention are given in the following table I:

The products have been subjected to a number of biochemical and pharmaceutical tests, the methodology of which is described below. The results of the experiments are listed in Table TI where. the figures in column 1 correspond to the figures in column 1 of Table I.

Information about the test results is listed in Table II.

1. ACUTE TOXICITY IN MICE

The substances in suspension in a 1% gum tragacanth mucilage are given orally by means of intragastric tubes to groups of five male mice that were fasted for 18 hours. Mortality is recorded over a 14-day period after treatment. The DL50 values are calculated according to Litchfield and Wilcoxon's method (J. Pharmacol. Exp. Ther. 96, 99, 1949). and is expressed in mg/kg.

2. INHIBITING PLATELET AGGREGATION

Platelet aggregation is measured according to the turbidimetric method described by G.V.R. Born and M.J. Cross (J. Physiol., 168, 178-195, 1973).

Citrated human platelet-rich plasma is pre-incubated for 3 minutes before introducing the inducer. The variations in the optimal transmission are recorded over a period of 10 minutes in a "Chronolog" aggregometer. The experiment is carried out in duplicate in the presence of the product or the corresponding solvent.

. The results are given in % inhibition of the maximum aggregation amplitude compared to controls. The product concentration [expressed in micromoles (µM or 10 - 6M)J which causes a 50% inhibition of platelet aggregation under the experimental conditions (CIj-q) is calculated from the results obtained at 6

different concentrations."

3. VASODILATORY ACTIVITY

The peripheral vasodilatory activity of the products was measured on anesthetized dogs at the level of femoral artery circulation. Consequently, the femoral artery became whose collaterals

was ligated perfused with a constant flow rate of blood taken from the aorta. Thus, the perfusion pressure measured at the level of the femoral artery varied as a function of the resistance in the perfused area. The investigated products and the corresponding solvents were directly injected into the system in a dose of 30 µg/kg. The blood flow rate was kept constant and a vessel dilation was measured through a reduction of the perfusion pressure. Latter

is stated in comparison with the effect of papaverine as a standard and. injected once per group of 4 products. When of interest, the products were examined at other doses under the same conditions.

The vasodilatory activity was indicated as follows:

0 : inactive reduction <10 mm Hg

1 : 1/3 of papaverine activity

2 : 2/3 of the papaverine activity

3: activity similar to that of papaverine.

4 : activity higher than that of papaverine.

4. INHIBITION OF CHOLESTEROL BIOSYNTHESIS.

a) Rat liver homogenate.

Rat liver homogenates are prepared and incubated in a PAS medium. in 14 send medium. Aliquots are incubated for 60 minutes with 1-C acetate and the compound to be studied at a concentration of 10<_4>M.

After saponification of the medium, the sterols are extracted with petroleum ether and the dry residue is precipitated with digitonin in an alcohol-14

acetone solution. The C content of the precipitate dissolved in pyridine is measured by liquid scintillation counting according to the method described by N. Bucher (J. Biol. Chem. 222, 1-15, 1956): the results are given in % inhibition.

b) Culture of fibroblasts.

When the cells run together, the culture medium is removed and replaced

14

of 10 ml of new medium containing 100 ul C acetate and the product to be examined in a concentration • p■ å o 5.10 -5M. The platelets are incubated for 2 hours at 37°C. After incubation, the synthesis of cholesterol is measured by measuring the radioactivity of the purified "^C cholesterol from the culture medium (Stoffel and Klotzbucher, Hoppe-Seyler's Z. Ph6siol. Chemie 359, 199-209, 1978).

The amount of proteins in the cells is determined according to Folin's method. The results are expressed in cpm cholesterol per gram of protein.

5. INHIBITING COLLAGEN DEGRADATION.

After incubation of the collagen at 37°C in the presence of collagenase in a buffered medium at pH 7.2 in the presence of a drug in a concentration of 10 M or without a drug, the released amino acids are retained by colorimetric reaction with ninhydrin and measuring the absorbance at 570 nm.

. 6. INHIBITION OF LIPOLYSIS.

Bituminous fat is taken from fasted rats. Fragments of adipose at (+ 150 mg) are incubated in Krebs-Ringer buffer containing 3% bovine serum albumin and the substance to be examined

—4

in the concentration of 10 M.

A comparison sample is taken after an incubation of 30 minutes at 37°C. Norepinephrine is used to induce lipolysis. The amount of free amino acids is measured after 20 minutes of incubation (W.G. Duncombe, Clin. Chim. Acta, 8 , 122 , 1964).; the results are given as % inhibition.

In this experiment, products 7,13,16,17,18 and 19 are active.

7.. ANTISPASMODIC ACTIVITY.

The antispasmodic activity of the investigated substances was tested against contractions in the last section of the guinea pig small intestine induced by histamine and acetylcholine.

These tests enable the registration of an antihistamine, anti-cholinergic or musculotropic activity. The reaction to the contraction agent (submaximal concentration) was repeated every 5 minutes before and after injection of increasing doses

-7 -4

of the investigated products (10 -10 M)..

The percentage of inhibition under the influence of the investigated products is calculated and the theoretical concentration which gives 50% inhibition is determined graphically for each experiment. These values are expressed in -log CI50(M)... In this sample, the products 9,14,15 and 21 are particularly noteworthy with a CIj_q value higher than 6. 8. INHIBITION OF LDL UPTAKE BY HUMAN FIBROBLASTS.

Skin fibroblasts from humans are cultured according to the Ross method

(J. Cell. Biol. 50,. 170-186, 1971).. The low-density lipoproteins (LDL) are prepared from human plasma by preparation 125

ultracentrifugation. They are isolated with iodine according to the method of Bilheimer et al (Biochémica Biophysica Acta 260., 212, 1972).

The measurement of LDL binding is carried out according to Chait et al.'s method (Proced. Nat. Acad. Sei., in press).."

For the experiment, the culture medium is removed and replaced with 2 ml of a new medium containing 5 µg/ml 125iodine. LDL. After incubation for 24 hours at 37°C, cells and medium are separated. The cells are washed and then incubated in the presence of 4 mg/ml dextran*^sulfate.

The radioactivity of the supernatant material makes it possible to measure the binding. Cells are then dissolved in 0.1 N NaOH, and an aliquot is counted (uptake)...

Finally, the breakdown of 125 iodine LDL is determined by measuring the radioactivity of <125> iodine associated with free tyrosine in the culture medium.

In this experiment, the products 3,7,13,18,25,2.7,34,44 and 48 proved to be particularly active.

9. ' IMMUNOMODULATING ACTIVITY.

The immunomodulating activity is measured in mice C 57BL/10, sensitized by intrapedial injection of pertussis vaccine. After 15 days, an intramuscular injection was made in the lumbar region.

After 4 days, the animals are euthanized and the knee lymph nodes are dissected and weighed. Treatments are carried out by "gavage" from day 13 to day 18.

The products are tested in a dose equivalent to 50 mg/kg D. penicillamine. Under these conditions, penicillamine inhibits the weight gain of the knee lymph nodes on the sensitized side (2.53 g + 0.12 mg for treated animals compared to 4.16 + 0.24 mg for controls). In this experiment, the products 2,3,13,14,18,19,27,30 and 35 showed an equivalent effect with D.penicillamine.

In all these experiments, it turns out that the compounds 3, 14, 18 and 33 show beneficial effects in most experiments. Moreover, it has also been shown that the compound 24 has an anticancer activity. The latter can be detected in animals that have been inoculated with cancer cells and treated orally with the manufactured product.

It must also be noted that many products produced according to the invention have negligible vasodilatory activity. Compound 28 and especially compound 24 has a particularly high activity (from a dose of 0.1 µg/kgL and especially an activity over a long period of time (up to 30 minutes after intra-arterial injection).

A more extensive provision was made with connection 3.

It has thus been shown that this compound has an anti-hypertensive activity in spontaneously hypertensive rats when given in a dose of 60 mg/kg to rats whose systolic blood pressure is compressed between 180 and 220 mm Hg, as it causes a pressure reduction of approx. 30 to 50 mm Hg.

It has also been shown that when this product is given to rats orally in doses of 50 to 10 mg/kg it causes an increase in prostacyclin release by the blood vessels. The experiment was carried out according to the method described by Villa et al in British J. Hematol. 42, 425-431, 19-79.

Compound No. 3 also has a very beneficial effect in preventing some reactions in the arterial wall when hypertension is developing in rats made hypertensive by Goldblatt's method (constriction of renal arteries,

and it is found that:

- in the case of preventive administration from the day of the surgical operation, the product has a significant protective effect on the aortic area; it reduces hypertrophy and ADN, collagen and cholesterol contents and it reduces synthesis of proteins and collagen. It has no similar effect on normal rats. - in the case of curative administration to rats that have been made hypertensive one month before, compound no. 3 significantly reduces the blood pressure and the amount of cholesterol in the aorta.

Due to all these experimental results, the properties of the product manufactured according to the invention enable use in the therapy of: 1) cardiovascular disorders such as hypertension and atherosclerosis,

2) rheumatic disorders,

3) cancer

.4) auto-immune disorders.

The active compounds can be given in association with various pharmaceutical diluents orally, parenterally or rectally.

For oral administration, lozenges, granules, tablets, capsules, pills and drops with controlled release of the active ingredients, sub-lingual tablets, solutions, syrups, emulsions containing common additives or diluents within galenic pharmacy will be used. For parenteral administration, sterile water or an oil such as ground nut oil or ethyl oleate will be used. For rectal administration, suppositories or rectal capsules will be used.

These active compounds can be used alone and in combination with other active products with a similar or different activity.

The products according to the invention can be used in different forms. _ The following examples are not limiting and concern galenic formulations which as active product contain one of the following compounds indicated by

"A" :

2-methyl-1-(2-phenoxyethylamino)-2-propanethiol 2-methyl-1-(2. p. tolyloxyethylamino).-2-propanethiol 2-methyl-1-(2-p. chlorophenoxyethylaminol.-2- propanethiol 2-methyl-1-(2-benz<y>lox<y>et<y>lamino).-2-propanethiol bis-[2-methyl-1-(2-phenoxyethylamino).-2-propyl ] disulfide 3- (2-merca<p>to-2-met<y>l<p>rop<y>laminoJ.propanonic acid bis- [2-methyl-l-(2-m. chlorophenoxyethylamino).-2 ^-propyl.] disulfide 2-methyl-1-n.octylamino-2-propanethiol 2-methyl-1-n.dodecylamino-2-propanethiol 2-methyl-1-(2-phenylethylaminoL-2-propanethiol 2-methoxycarbonyldithio-2 -methyl-1-(2-phenoxyethylamino).-propane - Whitepsol H15 : mixture of partially or fully esterified glycerides.

Depending on the cases, the route of administration, the intended activity and the individual compound used, the derivatives produced according to the invention are given in daily doses of 100 to 2500 mg, and in intravenous injection the derivatives are given

. in doses of 5 to 100 mg.

Claims (14)

1. Procedure for the preparation of aminothiol derivatives with the general formula (I): where: - R^ means hydrogen or a straight-chain or branched C^-C^0 alkyl residue; - R 2 means a straight or branched C 1 -C 4 alkyl residue; - and R^ can, together with the adjacent carbon atom, form a C-^-Cg cycloalkyl residue; - R^ means hydrogen, a straight-chain or branched lower C^- alkyl residue, a straight-chain or branched C-^-C^ alkanoylthio residue, a straight-chain or branched lower C-^ -C^ alkyloxycarbonylthio residue, a straight-chain or branched C1~ C10 alkylthio residue or a residue - (S). n-CR]LR2-CH2-NR4R5 where n is 1 or 2; - R^ and R^, which may be the same or different, mean: - hydrogen, - a straight-chain or branched C^^ C^ g alkyl residue, - a straight-chain or branched C2-C <g> alkyl residue substituted with: - a hydroxyl group, a carboxyl or a linear or branched C 1 -C 2 alkoxycarbonyl group, - a naphthyloxy group, - a phenyl, phenoxy, phenylthio or benzyloxy group which is optionally substituted with one, two or more halogen atoms such as chlorine, with one, two or more straight-chain or branched C-^-C. alkyl residues, with one, two or more straight-chain or branched C-^-C^ alkyloxy acid residues, - a straight-chain or branched C₁-C₂ alkoxy or alkylthio group, - a C-^-C^ alkylamino group which is optionally substituted with a phenyl ring, - a straight-chain or branched C-^-Cg alkylcarbox-i I samido group which is optionally substituted with a phenyl or phenoxy group, - a C 1 -C 8 cycloalkylcarboxamido group, - a C 1 -C 8 cycloalkyl radical, - a straight-chain or branched C^ -C^ g alkenyl residue, - a straight-chain or branched C^Cg alkynyl residue, - and R,, together with the adjacent nitrogen atom can form a heterocyclic ring such as piperidine, pyrrolidine, morpholine, piperazine, optionally substituted in the 4-position with a benzyl group, a straight-chain or branched C-^-C^ alkyl group or a phenyl group as in itself may be substituted with 1,2 or more halogen atoms such as fluorine and chlorine, straight-chain or branched C-^-C^ alkyl residues, straight-chain or branched C^-C^ olefin residues, - R_ and R^ can form a thiazolidine ring which is optionally substituted in the 2-position with one or two straight-chain or branched C1-C-3 alkyl groups or a thiazolidinone ring; R^, R^, R^ in R4°9 R5 cannot simultaneously be hydrogen, and not toxic and pharmaceutically usable salts of these derivatives, characterized in that a) an amide, an imine or a corresponding iminium salt is transferred into an aminothiol derivative of formula I, or b) a derivative with formula: is reacted with a compound R^ X or R^ COY, where R^ , R 2 and R^ have the meanings given above, Q means the substituent R^ or a protecting group for the thiol group, R^ means the substituent R^ whose chain is one carbon atom shorter, X means an easily cleavable group such as halogen, especially chlorine, bromine, iodine 'or tosyl or mesyl groups, Y means an easily cleavable group such as f .ex. chlorine, bromine or also the group R^COO, which after possible reduction gives a compound with the formula: orc) a derivative with formula: is reacted with a compound FUX, where R^, R^, R^, R^ and R,- have the meanings given above and X means an easily cleavable group such as halogen, especially chlorine, bromine, iodine or tosyl or mesyl groups, which gives an aminothiol of formula (I); or an episulfide of formula: a 2-oxathiolanone of formula: an ester of formula: is reacted with an amine of the type HNR^ R^ , to the corresponding aminothiol derivative. 2. Method according to claim 1 for the preparation of compounds of formula I where R 1 means hydrogen or a branched C 1 -C 1 alkyl residue, characterized in that correspondingly substituted starting materials are used. 3. Method according to claim 1 in the preparation of compounds of formula I where R-^ means a cg^ c^ g alkyl residue, c a r a_k teris t by using correspondingly substituted starting materials. 4. Process according to claim 1 for the preparation of compounds of formula I where R-^ denotes a C^-Cg alkyl group, characterized in that correspondingly substituted starting materials are used. 5. Method according to claim 1 in the production of. compounds of formula I where R-^ means hydrogen or a methyl residue, characterized by using correspondingly substituted starting materials. 6. Method according to one of claims 1-5 for the preparation of compounds of formula I where R2 means hydrogen or a methyl residue, characterized in that using correspondingly substituted starting materials. 7. Process according to claims 1-6 in the preparation of compounds of formula I where R 1 means an ethanoylthio, methoxycarbonylthio or a straight-chain or branched c 2 c alkyl thiorest, characterized by using correspondingly substituted starting materials. 8. Method according to claims 1-7 for the preparation of compounds of formula I where R4 . and R 5 , which may be the same or different, means hydrogen, a straight-chain or branched C 1 -C 8 alkyl radical, a C 1 -C 8 cycloalkyl radical, a straight or branched C 1 -C 3 alkenyl radical or a straight or branched C 2 -C 8 alkenyl residue, characterized by using correspondingly substituted starting materials. 9. Method according to claims 1-7 for the preparation of compounds of formula I where R^ and R^ which may be the same or different mean a straight-chain or branched C^^ C^ alkyl residue substituted with: - a hydroxyl, a carboxyl or a straight-chain or branched C₁ -C₁ alkoxycarbonyl group, - a phenyl, phenoxy, phenylthio or benzyloxy group which may optionally be substituted with one or two chlorine atoms, with one, two or more straight-chain or branched C1-C2 alkyl groups, with one or two straight-chain or branched C1-C3 alkoxy or with a or two C-,-C 3 alkylthio residues, characterized by using correspondingly substituted starting materials. 10. : : Process according to claims 1-7 in the preparation of compounds of formula I in which R. and Rj. which may be the same or different, a straight-chain or branched C₁-C₂ alkylamino group optionally substituted with a phenyl group, or a straight-chain or branched C₁-C₂ alkylamino group. alkylcarboxamido group which is optionally substituted with a phenyl or phenoxy group, characterized by using correspondingly substituted starting materials. 11. Process according to claims 1-7 for the preparation of compounds of formula I where R^ and Rj. together with the adjacent nitrogen atom forms a heterocyclic ring such as piperidine, pyrrolidine, morpholine, piperazine optionally substituted in the 4-position with a benzyl group or a phenyl group which itself may optionally be substituted with a chlorine atom, characterized by using correspondingly substituted starting materials. 12. Method according to claims 1-7 in the production of pre- .compounds of formula I where R^ means hydrogen and R,- means a phenoxyethyl group whose phenyl ring is optionally substituted with one or two chlorine atoms or with one or two methyl or methoxy acid residues, characterized by using correspondingly substituted starting materials. 13. Process according to claims 1-7 in the preparation of compounds of formula I where R 1 means hydrogen or a methyl residue, characterized in that correspondingly substituted starting materials are used. 14. Procedure according to claims 1-14 for the production of in 2-methyl-1-(2-phenoxyethylamino)-2-propane thiol 2-methyl-1-(2.p.tolyloxyethylamino)-2-propanethiol 2-methyl-1-(2-p.chlorophenoxyethylamino).-2-propanethiol 2-methyl-1-(2-benzyloxyethylamino)_-2 -propanethiol bis-[2-methyl-1-(2-phenoxyethylamino)-2-propyl J disulfide 3-(2-mercapto-2-methylpropylaminolpropanoic acid bis-[2-methyl-1-(2-m. chlorophenoxyethylamino ).-2-propyl]disulfide 2-methyl-1-n.octylamino-2-propanethiol 2-methyl-1-n-dodecylamino-2-propanethiol 2-methyl-1-(2-phenylethylamino)-2-propanethiol or 2-methoxycarbonyldithio-2-methyl-1-(2-phenoxyethylamino)-L-propane, characterized in that correspondingly substituted starting materials are used.