HU184896B - Process for producing new 3-alpha-amino-substituted-steroide derivatives - Google Patents

Description

The present invention relates to novel 3 'amino-substituted steroid derivatives and their acid addition salts with mineral or organic acids. U.S. Pat. No. 3,600,945; and the antimicrobial activity of certain 3 '-acylamido-5' -pregnan-20-ones and 3a-acylamido-5a-pregnan-20-ol; and pregnenamides have been reported to have anti-inflammatory and anticholesterol effects. The present invention provides novel compounds of formula I and acid addition salts thereof, wherein:

W is hydrogen or hydroxy and X is ethyl or formula (1) or (2), R 1 is hydrogen or methyl, R ₂ is hydrogen or C 1-5 alkyl,

R ₃ is C 1 -C 5 alkyl or C 2 -C 5 hydroxyalkyl, C 2 -C 5 alkoxycarbonyl, nicotinoyl, or an aminoacyl group derived from a natural α-amino acid or glycylglycine, or an N, N-bis - (lower alkyl) glycyl, with the proviso that

(i) when R _t is methyl, W and R ₂ are both hydrogen and X is (1) then R ₃ cannot be methyl, nicotinoyl or aminoacyl derived from a natural aminocarboxylic acid; (ii) when R j is methyl, W is hydrogen, R ₂ is methyl, and X is X at the same time, then R ₃ cannot be methyl; iii) when R 1 is methyl, R ₂ and W are both hydrogen, X is wherein hydroxy group having the (S) configuration, then R ₃ cannot be methyl or glycyl; (iv) if W is hydrogen, X is a group of formula (2) then one of R 1, R ₂ , and R ₃ is different from methyl;

v) when R ₂ and W are hydrogen, R is methyl and X is at the same time (2) wherein, a hydroxyl group (R) configuration, R ₃ does not represent a methyl group or ethoxycarbonyl.

In the compound of formula (I) and below, C 1-5 alkyl is, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl or pentyl; a C2 -C5 hydroxyalkyl group such as hydroxyethyl or hydroxypropyl; an alkoxycarbonyl group containing from 2 to 5 carbon atoms may be, for example, methoxy, ethoxy, or propoxycarbonyl, and the residue derived from aminocarboxylic acid may be selected from the group consisting of Al, Val, Ival, Leu, He, Asp, Asn, Glu, Gin, Ser, Thr, Cys, Met, Lys, Arg, Phe, Tyr, Trp, His and Pro, Nva, Nle, Hyp, Orn; these acids occur in the L-form as well as Gly.

Α-aminocarboxylic acids can have the L-configuration, for example, the term Ala means alinine in L-form.

The present application uses the IUPAC nomenclature, the rules of which are in particular those of Biochem. J. (1972) 126, 773-780. literary site.

Addition salts with mineral or organic acids include, for example, hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, acetic acid, formic acid, propionic acid, maleic acid, fumaric acid, succinic acid, citric acid, tartaric acid, tartaric acid, salts with, for example, methane or ethane sulfonic acid, aryl sulfonic acids such as benzene or paratoluenesulfonic acid and aryl carboxylic acid.

Among the compounds of formula (I) according to the invention and their acid addition salts with mineral or organic acids, those compounds wherein X is a group of formula (2) are preferred.

Particularly preferred compounds are those wherein X is a group of formula (3) and R ₂ is hydrogen or methyl, and acid addition salts thereof with mineral or organic acids. In particular, the following compounds may be mentioned:

- (2S) -2-amino-N - [(20S) -20-hydroxy- (5a) -pregnan-3-yl] -propionamide, - (2S) -2-amino-N - [(20S) -20-hydroxy- (5a) -pregnan-3a-yl] -3- (1H-indol-3-yl) -propionamide, 2-amino-N - [(20S) -20-hydroxy-19-nor (5a) -pregnan-3a-yl] -acetamide, 2-amino-N - [(20S) -20-hydroxy- (5a) -pregnan-3a-yl] -N-methylacetamide, and its salts other derivatives are described in the examples.

According to the invention, the derivatives of the formula I and their salts are prepared by reacting an amine of the formula II, wherein W, X and R j are as defined above, with <i) a halide of the formula III, wherein Hal represents chlorine. , bromine or iodine, and R ₃ is C 2-5 alkoxycarbonyl or nicotinoyl, to give the product of formula (I) wherein R ₂ is hydrogen, R ₃ is C 2-5 alkoxy. a-rbanyl group, or a nicotinoyl group, W, X and Rj are as defined above, and the product of formula (I) thus obtained is isolated and optionally converted to a salt; obsession

(b) reacting with a natural α-amino acid or glycine-glycine (the amine group of which may be protected by a group which is particularly readily cleaved by acid hydrolysis) or an N, N-bis (lower alkyl) glycine, followed by deprotection of the product; There is thus obtained a compound of formula (I) wherein R ₂ is hydrogen, R ₃ is a natural α-amino group or an N, N-bis (lower alkyl) glycyl or glycylglycyl group and W, X and R 1 are as defined above and the product is isolated and optionally converted to a salt, or

c) Reaction with a C 1-5 alkyl halide or an optionally protected C 2-5 hydroxyalkyl halide containing chlorine, bromine or iodine followed by cleavage of the optional hydroxy protecting group to give the product of formula I wherein R ₂ is a hydrogen atom _and R represents C1-5 alkyl or C2-5 hydroxyalkyl group and W, X and R are as defined above, is isolated and optionally converted into a salt, or, when R ₂ is hydrogen and R ₃ is C 1 -C 5 alkyl, optionally a product is reacted further with a halide of formula IV where R 1 'is C 1 -C 5 alkyl or C 2 -C 5 hydroxyalkyl or 2 to 5 carbon atoms

-2184896 alkoxycarbonyl or nicotinoyl group and Hal is as defined above, to give a product of formula (I) in which _R2 is 1-5C-alkyl, alkyl of 1-5 carbon atoms, R ₃ is 2-5 carbon atoms a hydroxyalkyl group or a C 2 -C 5 alkoxycarbonyl or nicotinoyl group and W, X and R _t are as defined above and the product is isolated and optionally converted into a salt, or eb) with a natural α-amino acid or glycylglycine or derivative thereof, or an N, N-bis (lower alkyl) glycine derivative containing easily cleavable by acid hydrolysis protected amino group, is further processed, then the optionally present protective group is removed and the product of formula (I) obtained wherein R ₂ represents 1-5 alkyl, R ₃ is a natural amino acid or κ-glycine-glycine or an N, N-bis (rövidsz an aminoacyl group derived from a (C1 -C4 alkyl) glycine and W, X and R 1 are as defined above, isolated and optionally converted to a salt.

In a preferred embodiment of the process of the invention:

The reaction of an amine of formula II with a halide of formula III according to process a), in particular an alkali metal hydroxide, an alkali metal carbonate such as potassium carbonate, an alkali metal bicarbonate, and an alkali metal acetate or alkaline earth metal. carbonate or a tertiary amine such as trialkylamine or pyridine or an alkali metal alcoholate such as sodium ethylate.

The reaction may be carried out, for example, in an inert solvent or suspension medium such as dioxane, dimethylformamide, benzene, toluene, or methylene chloride. Acylation may also involve the use of a free acid or a functional derivative other than a halide, such as an acid anhydride.

When a free acid is used, the acid must be converted to an amide with an activating agent such as carbodiimide. Other technologies may be used, such as those described in "The Peptides"

Volume 1, Academic Press 1979.

2. An amine of formula (II) or a compound of formula (I) wherein R ₂ is hydrogen and R ₃ is alkyl and a natural amino acid or glycylglycine or derivatives thereof which are readily deprotected. or reacting a N, N-bis (lower alkyl) glycine derivative according to process b) or eb) in the presence of a condensing agent. The role of the condensing agent in this case is to activate the acid group of the amino acid.

The condensing agent used is the carbodiimide of formula (VIII), wherein A and B are C 1-8 alkyl optionally containing dialkylamino or cycloalkyl.

Carbodiimides include, for example, dicyclohexylcarbodiimide or 1-ethyl-3- (3-dimethylylaminopropyl) carbodiimide, the latter being the more preferred.

In addition, 2-chloro-N-methylpyridinium halide such as iodide may also be used.

It is also possible to use an alkyl ester of chloroformate, such as methyl, ethyl or isobutyl ether of chloroformate, and an alkyl pyrophosphite such as ethyl pyrophosphite.

Examples of easy cleavable protecting groups include, for example, benzyloxycarbonyl or (Z) tert. butyloxycarbonyl (BOC).

According to the invention, it is particularly advantageous to provide a protecting group. butyloxycarbonyl.

An acid such as hydrochloric acid is preferably used as the cleaving agent to remove the readily cleavable protecting group, and the cleavage can be effected, for example, with the alkanolic side of hydrochloric acid or with anhydrous hydrochloric acid by bubbling the latter into nitronethane. Acids such as p-toluenesulfonic acid, formic acid or trifluoroacetic acid may also be used. In the presence of palladium, hydrogen may also be used to cleave the (Z) protecting group.

3. The reaction of the amine of formula II with an alkyl or hydroxyalkyl halide of process c) may be carried out in the presence of similar acid scavengers and solvents as the reaction of the compounds of formula II with the halides of formula III according to process a) .

The hydroxy group of the hydroxyalkyl group is preferably protected by a protecting group which is easily cleaved by particularly acidic hydrolysis. Such a protecting group is preferably tetrahydropyranyl. The protecting group may be cleaved under similar conditions as the amino group protecting group of the amino acid.

4. The reaction of the product of formula I wherein R ₂ is hydrogen, R ₃ is C 1 -C 5 alkyl, W, X and R j is as defined above, and a halide of formula IV) according to process ca) Depending on the nature of the substituent, it is carried out under the conditions described above for the reaction of the amine of formula II with the halide or alkyl halide of formula III according to process a).

5. The preparation of the N-alkylated products of formula (I) may, in certain cases, require special treatment:

For the preparation of the N-monomethylated product of formula (I), the carbamate of the amine of formula (II) may be prepared, for example, from an alkyl ester of halogenoformate, in particular ethyl ester, according to process (a) and subsequently reduced with lithium aluminum hydride ;

- first preparing the corresponding N-acetylated derivative to produce the N-monoethylated product of formula (1) and then reducing it;

It is sufficient to repeat the above procedure to obtain the N-diethylated derivative.

SHE

II

In these three cases, when X is a -C-CH ₃ group, a ketal at position 20, for example, with ethylene glycol, should be temporarily prepared and then hydrolyzed, for example, with a mineral acid, after reduction.

The N-mono- or diisopropylated product of formula (I) may also be reacted with acetone in the presence of a reducing agent such as sodium cyanoborohydride to form the N-mono- or diisopropylated product.

It will be appreciated that in the process of the invention, the amine groups need not be protected in cases where these amine groups are alkylated.

Derivatives of formula (I) are basic in nature, except those derivatives wherein R ₃ is alkoxycarbonyl. The acid addition salts of the products of formula (I) may advantageously be prepared by:

-3184896 when reacting a derivative of formula (I) with a stoichiometric amount of a mineral or organic acid. The salts may be prepared without isolation of the appropriate bases.

The derivatives of the present invention have interesting pharmacological properties, in particular immunotherapeutic properties. They are particularly suitable for inducing immune responses.

These properties are described in the experimental section below. These properties warrant the use of the 3- (amino-substituted) steroid derivatives of formula (I) and their pharmaceutically acceptable salts as medicaments.

Preferably, the novel 3- (amino-substituted) -steroid derivatives of formula (I) wherein X is a group of formula (2) and addition salts thereof with pharmaceutically acceptable acids are preferred as a medicament.

Particularly preferred are the compounds of formula I as pharmaceutically active compounds wherein X is a group of formula 3 and R ₂ is hydrogen or methyl, and addition salts thereof with pharmaceutically acceptable acids.

The following derivatives are particularly preferred: a

2-amino-N - [(20S) -20-hydroxy (5a) pregnan-3a-yl] -propionamide;

(2S) -2-amino-N - [(20S) -20-hydroxy (5a) pregnan-3a-yl] -3- (lH-indol-3-yl) -propionamide;

2-Amino-N - [(20S) -20-hydroxy-19-nor- (5a) -pregnan-3a-yl] -acetamide and the

2-Amino-N - [(20S) -20-hydroxy- (5a) -pregnan-3a-yl] -N-methylacetamide, and pharmaceutically acceptable acid addition salts thereof.

The pharmaceutical compositions of the present invention may be used, for example, to treat autoimmune diseases resulting from the deficiency of certain lymphocytes, such as non-specific connective tissue diseases of an organ, e.g.

The pharmaceutical compositions of the present invention may be used in antibiotic therapy as adjunctive therapy or in anticancer chemotherapy.

Depending on the derivative, the usual dosage may vary from 10 mg to 1 g per day, depending on the patient being treated and the complaint, for oral administration in man, for the derivative used in Example 14 when used as an adjuvant in antibiotic therapy.

Derivatives of formula (I) and pharmaceutically acceptable acid addition salts thereof may be used to prepare pharmaceutical compositions containing at least one of the aforementioned derivatives or salts thereof as active ingredients. Pharmaceutical compositions containing the compounds of the formula I or their acid addition salts may be administered to the body via the gastrointestinal tract or parenterally.

These pharmaceutical compositions may be in solid or liquid form, such as in the form of pharmaceutical forms customary in human medicine, such as plain or sugar-coated tablets, gelatin capsules, granules; suppositories or injectable preparations which are prepared by conventional means. The active ingredients are mixed with excipients. Examples of such excipients are talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous solvents, animal or vegetable fats, paraffin derivatives, glycols, various wetting, dispersing or emulsifying agents. and preservatives.

The starting materials of formula (II), if not known, may be prepared by reacting a compound of formula (V) wherein R is as defined above with an excess of ethyl triphenylphosphonium bromide and an excess of potassium tert-butylate. The product of formula (VI), wherein R ₁ is as defined above, is converted, for example, to the tosylate, and then reacted with sodium azide or directly with diphenyl azidophosphate in the presence of ethyl azodicarboxylate and triphenylphosphine to give the azide of formula VII. R 1 is as defined above - and the configuration at the 3-position is reversed with respect to the compound of formula (V) or (VI) and the compound is then reduced with, for example, lithium aluminum hydride to give _the amine of formula IIA ferti-, this compound is either isolated or, for example, a cathysizer, is an advantage sen rhodium, platinum or a palladium; is reduced by hydrogenation in the presence of catalyst can give _(IIB) to give a compound of formula - wherein R is as defined above - by using diborane or hydrated form (II _C) of the product of formula are obtained - aho R | are as defined above - and the product isolated or oxidized for example with chromic acid to give (II _D) to give the product of formula - wherein R is as defined above - cut; ' subjecting cis-dihydroxylation conditions, for example with osmium anbidriddel trimetiloxamin presence after protect which optionally readily hydrolyzable group, such as trifluoroacetic anhydride to give (II _E), ^said: product ÁNOS formula obtained - wherein R is as defined above, protecting group or hydrogen; and R 1 is as defined above, and the resulting compound is isolated when R is hydrogen, or when R is a protecting group, it is reacted with a deprotecting agent, for example, in the case of trifluoroacetyl, subjected to alkaline hydrolysis and the resulting free amine is isolated, or to oxidation is subjected to, for example, chromic acid and then (II _F) of the product of formula are obtained - wherein R and R are as defined above - a product where R is hydrogen, is isolated or, if R is a protecting group, is reacted with a suitable deprotecting agent place ongoing, and the free amine is isolated or, for example sodium borohydride reducing Jk and (II _G) of the product of formula are obtained - aho: R and R are as defined above - which, when R is hydrogen, is isolated or, if R is a protecting group, the protecting group and the free amine is isolated.

The preparation of the products of formula (II) is illustrated in the Examples.

Further details of the invention are illustrated by the following examples.

-4184896

Example 1 (2S) -2-Amino-N - [(20S) -20-hydroxy-5a-pregnan-3a-yl] -propionamide (hydrochloride and base)

Step A: 2- [1,1-Dimethyl-ethoxycarbonylamino] -N - [(20S) -20-hydroxy- (5a) -pregnan-3'-yl] -propionamide

1.92 g of (20S) -3a-amino-5a-pregnan-20-ol and 2.27 g of tert-butyloxycarbonyl-L-alanine (BOC-L-alanine) are dissolved in 60 cm ^{3 of} chloroform and 12 cm ^{3 of} pyridine. The solution is stirred at 0-5 ° C under an inert atmosphere and 1.14 g of 15-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride are added. After 1 hour and 15 minutes, 1.15 g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride are added again and the mixture is stirred for 50 minutes at 0-5 ° C and then evaporated to dryness with aqueous It is taken up in 20 sodium bicarbonate solution, extracted with ethyl acetate, washed with aqueous sodium chloride solution and then with aq. washed, dried at 60 ° C under reduced pressure to give the desired product. Melting point: 171 ° C.

Step B: (2S) -2-Amino-N - [(20S) -20-hydroxy-5a-pregnan-3a-yl] -propionamide hydrochloride and base

Alternative 1: Under an inert atmosphere, 1.55 g of the product of Step A is suspended in 100 cm <^{3> of} nitromethane, and the hydrochloric acid is bubbled through the suspension for 10 minutes and stirred at 20-25 [deg.] C. for 35 minutes to remove excess hydrochloric acid. separated, washed with ethyl ether, dried at 60 ° C under reduced pressure and recrystallized from methanol.

990 mg of the desired hydrochloride are obtained. Mp: about 220 ° C. t

Alternative 2: Under inert atmosphere, 2.7 g of the product obtained in Step A are dissolved in 10 cm ^{3 of} ethanol, cm ^{3 of} 3.5N hydrochloric acid ethanol are added, and the mixture is stirred ^{for one} hour, evaporated to dryness, taken up in ethyl acetate, cooled, , wash with ethyl acetate,

It was dried at <RTIgt; C0 </RTI> under reduced pressure and recrystallized from methanol. 2.2 g of the desired hydrochloride are obtained.

Mp: about 220 ° C.

Preparation of the base: 1.74 g of hydrochloride is dissolved in 100 cm ^{3 of} 30% water in tetrahydrofuran, 4 cm ^{3 of} 2N sodium hydroxide are added and the mixture is dried, taken up in 100 cm ^{3 of} chloroform, washed with water, evaporated and recrystallized from ethyl acetate. 1.27 g of the desired base are obtained. Mp 224 ° C.

Following the above procedure, the following compounds of formula (S) were prepared:

In compounds of formula I

Example dry Version Starting amino acid rj Op. Second 1+ comment BOC-L-glutam insav Ο CH —CH ₂ —CH ₂ —COOH (S) r nil 220 ° C (hydrochloride) Third 1 Boc-L-serine O CH -CH ₂ OH (S) 1 NH _; 210 ° C (hydrochloride) 4th 1 BOC-L-phenylalanine Group 4 (S) 240 ° C (hydrochloride) 5th 1 BOC-L-tryptophan Group 5 (S) 135 ° C (base) 6th 2 BOC-L-proline Group 6 (S) 275 ° C (hydrochloride) 7th CF ₃ COOH BOC-glycylglycine O CH ₂ —NH — CO — CH ₂ NH ₂ 240 ° C (hydrochloride) (200, then 252 ° C base)

Note to Example 2: The second acidic group of L-glutamic acid is protected as the benzyl ester and liberated by catalytic hydrogenation in acetic acid in the presence of palladium prior to cleavage of BOC.

Example 8

N - [(20S) -20-hydroxy-5a-pregnan-3 "-yl] pyridine-3-carboxamide

Under inert atmosphere, 960 mg of (20S) -3a-amino-5a-pregnan-20-ol and 813 mg of nicotinic acid were dissolved in 30 cm ^{3 of} chloroform in 6 and 6 cm ^{3 of} pyridine and 582 mg of 1-ethyl-3- (3-dimethyl55 aminopropyl). ) -carbodiimide hydrochloride. After 3 hours 30 minutes, while stirring, 291 mg of 1-ethyl-3- (3-methylmethylaminopropyl) carbodiimide hydrochloride was added and the whole was stirred for 16 hours, evaporated to dryness, taken up in 30 cm ^{3 of} water, cooled and separated. The reaction mixture was washed with water, dried at 80 ° C under reduced pressure and recrystallized from methanol. 920 mg of the desired product are obtained. M.p. 258 ° C. [α] _D = + 14.5 ° ± Γ (c = 1% pyridine).

-511

Example 9

2-Amino-N - [(20S) -17a, 20-dihydroxy-5a-pregnan-3a-yl] -acetamide hydrochloride

The procedure described in Example 1, Variant 2 was followed, starting from BOC-glycine and (20S) -3'-amino-5α-pregnan-17α, 20-diol to give the desired product. Mp: about 200 ° C. [α] _η = + 4 ° ± Γ (c = 1.5% ethanol 95 °).

The starting (20S) -3a-amino-5? -pregnan-17?, 20-diol can be prepared as follows:

Step 1: (Z) - (5z) -pregn-17 (20) -en-3 [J-ol

Triphenethylphosphonium bromide (59.4 g) was added to a solution of 16.1 g of potassium tert-butylate in 160 cm ^{3 of} tetrahydrofuran, the mixture was stirred for 30 minutes, 23.2 g of epiandrosterone was added, and the mixture was poured into ice water. The mixture was extracted with ethyl acetate, washed with water, dried, evaporated to dryness, purified by silica gel column chromatography (eluent: cyclohexane: ethyl acetate = 7: 3), and then crystallized from methanol, cooled, isolated and dried. 23.1 g of the expected product are obtained. Mp: 160 ° C.

Step 2: (Z) -3a-Azido (5R) -pregn-17 (20) -en

1.92 g of ethyl azodicarboxylate and 3.02 g of diphenyl azidofoszfátot added in 30 cm ³ of benzene and 5 cm ³ of tetrahydrofuran was added 1.66 g of the above product and the mixture was stirred in an ice bath for 20 minutes and 2.88 g of triphenylphosphine in 30 cm A solution of ³ benzene was added and the whole was stirred for 40 minutes at 10 ° C. The mixture was washed with 2N hydrochloric acid, water, dried, evaporated to dryness and purified by silica gel column chromatography eluting with heptane followed by 1: 1 heptane / ethyl ether. 1.67 g of the expected product are obtained in the form of crystals. 114 DEG C., after recrystallization from methanol.

Step 3: (Z) -5 '-pregn-17 (20) -en-3a-amine (and hydrochloride)

14.5 g of (Z) -3a-azido (5 ') - pregn-17 (20) -ene are dissolved in 290 cm ^{3 of} tetrahydrofuran, and the mixture is stirred at 25-27 ° C and 800 mg for 1 hour. lithium aluminum hydride is added. The mixture was stirred again for 1 hour, the excess hydride was removed with methanol, the residue was filtered and the filtrate was washed with aqueous Seignette salt followed by brine, dried, evaporated to dryness to give 13.1 g of the desired amine crystals. M.p. about 90 ° C.

The base was dissolved in 150 cm ^{3 of} ethyl acetate and 30 cm ^{3 of} methylene chloride and 27 cm ^{3 of} 1.7 N hydrochloric acid in ethyl acetate were added. The whole was isolated, washed with ethyl acetate and the resulting crystals were dried under reduced pressure. 13.2 g of hydrochloride crystals are obtained. Mp: above 300 ° C. [ _α ] _{D at} 1% in pyridine containing 10% water = + 38.5 ° ± 1.5 °.

Step 4: N - [(Z, 5a) -pregn-17 (20) -en-3a-yl] -trifluoroacetamide

Under an inert atmosphere, 16.5 g of the hydrochloride obtained in Step ^{3 are} suspended in 165 cm ^{3 of} methylene chloride and 16.5 cm ^{3 of} pyridine, cooled to about 5 ° C and treated with 16.5 cm ^{3 of} trifluoroacetic anhydride over 5 minutes. into the suspension, which was stirred at room temperature for 15 min, evaporated to dryness under reduced pressure, 200 cm ³ of water are added and the product was isolated, washed with water and dried under reduced pressure. 18.1 g of crystals are obtained. Mp 204 ° C.

Step 5: N - [(20S) -17a, 20-Dihydroxy- (5a) -pregnan-3a-yl] trifluoroacetamide

Under an inert atmosphere, 18.1 g of the product obtained above are dissolved in 100 cm ^{3 of} methylethylketone and then 9 g of dihydrate trimethylamine N-oxide are added together with a solution of 360 mg of osmium tetroxide in 71 cm ^{3 of} methylethylketone. The solution was heated under reflux for 2 hours, allowed to cool, and a 10% aqueous solution of sodium thiosulfate (200 cm ³ ) was added. The solution is stirred for 30 minutes at room temperature, decanted, washed with water, dried over magnesium sulfate, filtered and evaporated to dryness under reduced pressure. The resulting oil was purified by chromatography on a silica column. The eluent was a 7: 3 mixture of benzene and ethyl acetate. 14 g of product are obtained. 172 ° C then 192 ° C.

Step 6: (20S) -3a-Amino-5a-pregnan-17a, 20-diol

Under inert atmosphere, 4 g of the product obtained above are dissolved in 20 cm <^{3> of} methanol, then 8 cm <^{3> of} sodium hydroxide solution are added and the whole is stirred for 1.5 hours. After stirring for 10 minutes with 50 cm ^{3 of} water, the mixture was separated, washed with water, dried under reduced pressure at 40 ° C to give 3 g of the desired product. Mp 210 ° C.

Example 10

2-Amino-N - [(20S) -20-hydroxy-19-nor- (5a) -pregnan-3a-yl] -acetamide hydrochloride

Example 1, variant 1, starting with BOC-glycine and (20S) -3a-amino-19-nor- (5a) -pregnan-20-ol. The desired product is formed which melts at about 270 ° C during sublimation. [α] _D = + 39.5 ° ± 1.5 ° (c = 1% methanol).

The starting (20S) -3a-amino-19-nor- (5a) -pregnan-20-ol can be prepared as follows:

The method used to prepare (Z) -5α-pregn-17 (20) -en-3β-amine in Steps 1, 2 and 3 of Example 9 and using (Z) -5α-19 from 19-norepiandrosterone -nor-pregn-17 (20) -en-3a-amine.

Under a stream of nitrogen, 156 mg of sodium borohydride was suspended in 5 cm ^{3 of} tetrahydrofuran and a solution of 0.5 cm ^{3 of} boron trifluoride etherate in 2.5 cm ^{3 of} tetrahydrofuran was added dropwise at 5 ° C. the mixture was stirred for 1 hour in an ice bath. 296 mg of (Z) -5a-19-nof-pregi-17 (20) -en-3a-amine in 3 cm ^{3 of} tetrahydrofuran

-6184896 was added and the mixture was stirred for 1 hour 30 minutes at room temperature and then cooled in an ice bath. Sodium hydroxide (2 cm ^{3) was} added slowly and stirred at room temperature for 5 minutes. After decantation, the aqueous phase was extracted with tetrahydrofuran and the organic phase was washed with water (4 cm ³ ) and 5 cm ^{3 of} hydrogen peroxide (110 ml). The mixture was stirred for 45 minutes, extracted with ethyl acetate, washed with water, dried and evaporated to dryness under reduced pressure. The dry extract was taken up in 10 cm ^{3 of} methanol with 5 cm ^{3 of} hydrochloric acid, heated for 30 minutes in a water bath at 50 ° C, poured into saturated sodium bicarbonate solution, extracted with methylene chloride, washed with water, dried and concentrated under reduced pressure. mg of crystalline product is obtained. M.p. 190 ° C.

Example 11

2-Amino-N- [17-hydroxy-20-oxo-5a-pregnan-3a-yl] -acetamide hydrochloride

Example 1, variant 2, starting with BOC-glycine and 3oc-amino-17α-hydroxy-5α-pregnan-20-one. The desired product is obtained, m.p. [α] _D = + 50 ° ± Γ (c = 1% ethanol 95 °).

The starting material 3a-amino-17oe-hydroxy-5a-pregnan-20-one can be prepared as follows:

N- (17α-hydroxy-20-oxo-5α-pregnan-3a-yl) -trifluoroacetamide (m.p. 178 ° C and 186 ° C) was prepared according to the method of Example 9.

The product of Step 5 is subjected to percutaneous oxidation. The product was treated according to Example 9, Step 6 to give 3α-amino-17α-hydroxy-5α-pregnan-20-one. 216 DEG C. (after crystallization from water).

Example 12

2-amino-N - [(20R) -20-hydroxy-5a-pregnan-3a-yl-acetamide hydrochloride,

Example 1, variant 1, using BOC-glycine and (20R) -3z-amino-5α-pregnan-20-ol as starting material. The desired product is obtained, m.p. 210 ° C and 260 ° C. [ _α] D = + 22 ° ± 1 ° (c = 0.8% in pyridine containing 10% water).

Example 13

Ethyl N - [(20S) -20-hydroxy- (5a) -pregnan-3a-yl] -carbamate 5 g (20S) -3a-amino-5a-pregnan-20-ol 500 cm ³ dropwise over 1 min. methylene chloride solution was added to 15 cm ^{3 of} ethyl chloroformate and 45 cm ^{3 of} methylene chloride, the mixture was stirred for 30 minutes, 20 cm ^{3 of} sodium hydroxide was added and the whole was stirred for 30 minutes, decanted and extracted with methylene chloride, washed with water, dried, filtered, evaporated to dryness under reduced pressure, taken up in ethyl ether and isolated. 5.7 g of the expected product are obtained. M.p. 198 ° C. [.Alpha.] D @ 20 = + 25 DEG ± 1.5 DEG (c = 1% methylene chloride).

Example 14

2-Amino-N - [(20S) -20-hydroxy-5a-pregnan-3a-yl] -N-methyl-acetamide hydrochloride

Reduction of the product of Example 13 gave (20S) -3a-methylamino-5a-pregnan-20-ol (m.p. 170 ° C) as described by Vetter et al., Bull. Soc. (1963) 1324 and the compound was reacted with BOC-glycine according to Example 1, Variant 2 to give the desired product, which was prepared as its hydrochloride. Mp: about 250 ° C.

Example 15

2 (S) -2-Amino-N-methyl-N - [(20S) -20-hydroxy-5a-pregnan-3o-yl] -propionamide hydrochloride (20S) -3a-methylamino-5a-pregnan-20- Example 1

Reaction with BOC-L-alanine as described in Variant 1 gives the desired product, which is prepared in the hydrochloride, m.p. 270 ° C.

Example 16

2 (R) -2-Amino-N-methyl-N - [(20S) -20-hydroxy-5a-pregnan-3a-yl] -propionamide hydrochloride (20S) -3a-methylamino-5a-pregnan-20- Example 1

Alternatively, it is reacted with BOC-L-alanine to give the desired product, whose hydrochloride is prepared. The product sublimates at about 260 ° C.

Example 17 (20S) -3a-Ethylamino-5a-pregnan-20-ol hydrochloride (20S) -3a-amino-5a-pregnan-20-ol is reacted with ethyl iodide in the presence of sodium carbonate to give the product as the base. After recrystallization from ethyl acetate, m.p .: 129 C ^3rd

Addition of hydrochloric acid ethyl acetate gives the hydrochloride, m.p. 270 ° C.

Example 18

2-amino-N-ethyl-N - [(20S) -20-hydroxy-5a-pregnan-3a-yl-acetamide hydrochloride,

The product of Example 17 is reacted with BOC-L-glycine as the base in the same manner as in Example 1, Variant 1 to give the desired product, which is prepared as its hydrochloride. Mp: about 230 ° C.

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EXAMPLE 19 ^ OS3 -? -? - Kl -hydroxyethyl-O-amino-α-pregnan-O-ol hydrochloride g (20S) -3oc-amino-5? -Pregnan-20-ol and 7 sodium carbonate in 100 cm? ^{It is} suspended in ³ anhydrous dioxane and 6.6 cm ^{3 of} 2- (2-bromoethoxy) tetrahydropyran are added. The mixture was heated at reflux for 24 hours. It is then cooled to room temperature, diluted with water, extracted with ether, dried, evaporated to dryness under reduced pressure and purified by column chromatography on silica. Chloroform: methanol: acetic acid (85: 5: 10) was used as eluent. 7.7 g of a yellow oil are obtained.

Hydrolysis of the protecting group The above product is dissolved in 30 cm ^{3 of} ethanol and 15 cm ^{3 of} 2N hydrochloric acid, and the mixture is refluxed for 1.5 hours and then cooled; It was poured into 100 cm ^{3 of} aqueous sodium bicarbonate solution, cooled for 15 minutes, isolated, washed with water, dried at 40 ° C under reduced pressure and then dissolved in 350 cm ^{3 of} 10% methanol in methylene chloride. The mixture was filtered, ca. The reaction mixture was concentrated to a volume of 50 cm ³ , ethyl acetate (100 cm ^{3) was} added and the whole was concentrated in half. The precipitate was collected. The precipitate was dissolved in 100 cm <^{3> of} methanol, filtered and concentrated to 30 cm <^3> . Ethyl acetate (70 cm ^{3) was} added and the mixture was concentrated to half, isolated, dried at 40 ° C under reduced pressure to give the desired product as a base (1.65 g). M.p. 215 ° C.

Preparation of hydrochloride

1.5 g of the above base was dissolved in 40 cm ^{3 of} methanol and 1.6 N hydrochloric acid in ethyl acetate was added until an acidic pH was obtained. After addition of 60 cm ^{3 of} ethyl acetate, the mixture was concentrated under reduced pressure, It was dried at <RTIgt; C </RTI> under reduced pressure. 1.5 g of the expected product are obtained, m.p.

Example 20 α-Dimethylamino-N - [(20S) -20-hydroxy-5 '-pregnan-3' -yl] -acetamide

In the same manner as in Example 1, Step A, 2.552 g of (20S) -3-amino-5α-pregnan-20-ol and 3.350 g of N, N-dimethylglycine hydrochloride are used. 4 g of crude product are obtained containing the desired derivative and the Ο, Ν-disubstituted derivative. Saponification with sodium hydroxide in methanol gives the desired product. M.p. 206 ° C. [ _α ] _D = + 29 ° ± 1 ° (c = 1% CHCl ₃ ).

Example 21

Tablets are prepared containing the active ingredient (2S) -2-amino-N - [(20S) -20-hydroxy-5a-pregnan-3a-yl] -propionamide hydrochloride in 20 mg of excipient in 1 tablet up to 100 mg.

(The other ingredients are: lactose, starch, talc magnesium stearate.)

Example 22

A tablet having the following active ingredient is prepared:

2-Amino N - [(20S) -hydroxy-5a-pregnan-3a-yl] -N-methylacetamide hydrochloride 15 mg of excipient in 1 tablet up to 100 mg.

(Excipient composition: lactose, starch, talc, magnesium stearate.)

Farm; .cological examination

1. Anaphylactic shock adjuvant

Principle: Animals are administered a product which is capable of stimulating the activity of the immune system, and this effect is expressed by an increase in the shock to the antigen-induced reaction to which the animal is sensitized.

Male males weighing 30-35 g were sensitized by intra-plantar route with bovine serum albumin. After 8 days, the animals receive the same antigen intravenously. Under minimal sensitization conditions, control animals do not suffer a fatal shock at the time of the last administration.

The product to be tested was injected intra-plantar, mixed with antigen: if this product is adjuvant, it will increase sensitization and result in a lethal shock when administered by intravenous administration.

An active dose is one that causes 50% or greater mortality in animals.

Results:

Pcldaszám Dose / animal mg First 0.5 Second sl 4th 5 5th 5 6th 2 7th 1 8th 5 9th 1 10th 0.5 13th 5 14th 0.5

2. Rosette test with lamb's erythrocytes

Principle: Animals are dosed with a product that stimulates the activity of the immune system and the effect is expressed as an increase in the capacity of the reaction induced by injection of the immunogenic product.

Male 3-month-old rats are sensitized with lamb erythrocytes by the intraperitoneal route (day 0). Seven days later (day 7), the spleen of the animals was removed and the spleen cells were contacted with the lamb's erythrocytes: the percentage of leukocytes around which the erythrocytes formed rosettes was calculated.

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The product to be tested was orally administered as described in FIG. day to day 1 daily.

An immuno-stimulating dose is defined as a dose which gives a percentage of rosettes observed in control animals of about 10%. doubles.

Results;

example Number Dose / animal in mg / kb per os First 1 5th 2 6th 5 8th 5 10th 5 11th 5 14th 10

3. Acute toxicity test

The various compounds lethal doses LD ₀ was evaluated in mice when administered by the oral route.

The maximum dose that does not cause mortality at the end of day 8 is called LD ₀ .

Claims (8)

A process for the preparation of novel 3a-amino-substituted steroid derivatives of formula I and acid addition salts thereof with mineral or organic acids - wherein W is hydrogen or hydroxy, X is ethyl or a group of formula (1) or (2), Rj is hydrogen or methyl, R ₂ is hydrogen or C 1-5 alkyl, R ₃ is C 1 -C 5 alkyl, C 2 -C 5 hydroxyalkyl, C 2 -C 5 alkoxycarbonyl, nicotinod or an amino group derived from natural α-amino acid or glycylglycine, or N, N-bis (lower alkyl) glycyl, with the proviso that i) when R is methyl, W and _R2 are both hydrogen and X is (1) wherein or R, is methyl, nikotinod- or other than one derivable natural aminokarbonsavból aminoacyl group, ii) when R _(is methyl , W is hydrogen 5, R ₂ is methyl, and X is at the same time R ₁ is other than methyl, iii) when R 1 is methyl, W and R ₂ are both hydrogen, X is a group of formula 2, wherein the hydroxy group is in the (S) configuration, then R 1 is other than methyl or glycyl, (iv) if W is hydrogen, X is a group of formula (2) then one of R _p R ₂ and R ₃ is not methyl, 15 v) when R is methyl, R ₂ and W is a hydrogen atom and X is a group of formula (2) wherein the hydroxyl group (R) configuration, then R₁ is ethoxycarbonyl, or a methyl group other than - preparation comprising that an amine of formula (II) of formula (II) wherein W, X and Rj are as defined above, a) (III) halide of formula - wherein Hal is chlorine, bromine or iodine, R represents a C2-5 alkoxikarbond- nikoti25 or hexanoyl group - and the resulting product of Formula (I) - wherein R ₂ is hydrogen R 1 is C 2 -C 5 alkoxycarbond or nicotinoyl, W, X and R 1 are as above - isolated and optionally converted to a salt, or B) reacting with a natural amino acid or glycylglycine or a derivative having a protected amino group that can be easily cleaved by acid hydrolysis or with an N, N-bis (lower alkyl) glycine and optionally denoting the resulting product and removing the resulting compound of formula I wherein R ₂ is hydrogen, R 1 is an aminoacyl group derived from a natural α-amino acid or glycd-glycine or an N, N-bis (lower alkyl) glycine, and W, X and R, 40 is as defined above - isolated and optionally converted to a salt, or c) reacting with a C 1 -C 5 alkyl halide or an optionally protected C 2 -C 5 hydroxyalkyl halide, where the halogen atom can be chlorine, bromine or 45 iodine, then cleaving the optional hydroxy protecting group and 11) the product of formula - wherein said C1-5 alkyl or C2-5 hydroxyalkyl group and W, X and R je50 Lentes R ₂ is hydrogen and R is - the salt thus obtained is isolated and, if desired, when R ₂ or is hydrogen and R 1 is C 1 -C 5 alkyl, if desired, a product is further reacted with a halide of formula IV where R 1 is C 1 -C 5 alkyl or C 2 -C 5 hydroxyalkyl; group or C2-5 alkoxikarbond group or r ikotinod group, and Hal are as defined above - and the resulting product of formula (I) - wherein R ₂ is C 1 -C 5 alkyl, R 1 is C 1 -C 5 alkyl, C 2 -C 5 hydroxyalkyl or C 2 -C 5 alkoxycarbonyl or nicotinoyl, and W, X and R _t are as defined above, and the desired or (eb) with a protecting group readily cleaved by a natural amino acid or glycylglycine or by 65 acid hydrolysis thereof. -9184896 with a protected amino group or with an N, N-bis (lower alkyl) glycine derivative, then deprotecting, if any, and obtaining the product of formula (I) wherein R ₂ has from 1 to 5 carbon atoms alkyl, R ₃ is an amino acyl group derived from a natural α-amino acid or glycylglycine or an N, N-bis (lower alkyl) glycine and W, X and R ₍ as defined above) are isolated and desired case. 2. A process according to claim 1 wherein the amino group is a tert-butyloxycarbonyl protecting group. 3. A process according to claim 1 or 2, wherein the starting material is a compound of formula II wherein X is (2). 4. A process according to any one of claims 1 to 4, wherein the starting material is X in place of a compound of formula (3). A process for carrying out the process according to claim 1 b) or cb) or claim 2 according to (2S) -2-amino-N - [(20S) -20-hydroxy- (5R) -pregnan-3oc-yl]. -propionamide, (2S) -2-amino-N - [(20S) -20-hydroxy- (5a) -pregnan-3a-yl] -3- (1H-indol-3-yl) -propionamide, 2-amino-N - [(20S) -20-hydroxy-19-nor- (5a) -pregnan-3a-yl] -acetamide or 2-amino-N - [(20S) -20-hydroxy- (5a) -pregnan-3a-yl] -N-methylacetamide and its addition salts with mineral or organic acids, characterized in that Suitable starting materials are used. 6. A process for the preparation of a pharmaceutical composition, which comprises as an active ingredient a derivative of the formula (I) according to claim 1, wherein W, X, R 1, R ₂ and R ₃ are as defined in claim 1) or pharmaceutically acceptable mineral or organic acid addition salts thereof, in admixture with conventional pharmaceutically acceptable carriers or excipients and / or other excipients and formulated as a pharmaceutical composition. 15 7. A process according to claim 6 wherein the active ingredient is (2S) -2-amino-N - [(20S) -20-hydroxy- (5a) -pregnan-3a-yl] -propionamide, 2S) -2-amino-N - [(20S) -20-hydroxy- (5R) -pregnan-3z-yl] -3- (1H-indol-3-yl) -propionamide, 2-amino20 -N - [(20S) -20-hydroxy-19-nor- (5a) -pregnan-3a-yl] -acetamide or 2-amino-N - [(20S) -20-hydroxy- (5 ') - pregnane- 3z-yl] -N-methylacetamide or its addition salts with pharmaceutically acceptable mineral or organic acids. 8. A process according to claim 6 wherein the active ingredient is (2S) -2-amino-N - [(20S) -20-hydroxy- (5a) -pregnan-3a-yl] -3- (1) H-Indol-3-yl) -propionamide is used.