FI77871B - FOERFARANDE FOER FRAMSTAELLNING AV NYA TERAPEUTISKT ANVAENDBARA 3-AMINOSUBSTITUERADE STEROIDDERIVAT. - Google Patents

Description

! 77871

Process for the preparation of new therapeutically useful 3-amino-substituted steroid derivatives

The invention relates to a process for the preparation of novel 3-amino-substituted steroid derivatives and to their salts formed by the addition of mineral or organic acids of general formula I: wherein W is a hydrogen atom or a hydroxyl group or together with X an ethylidene group, X is an ethyl group or a group CH, or CH0 / CN / C \

20 7 X0 '' OH

or together with W an ethylidene group, the wavy lines indicate that the corresponding radical has either the «or <? configuration, has a hydrogen atom or a methyl group, R 2 is a hydrogen atom or an alkyl group having 1 to 5 carbon atoms or 2 to 5 a hydroxyalkyl group having 1 to 5 carbon atoms and R 1 is a hydrogen atom, an alkyl group having 1 to 5 carbon atoms or a hydroxyalkyl group having 2 to 5 carbon atoms, an acyl group or an alkoxycarbonyl group having 2 to 8 carbon atoms, or a peptide derived from a β-amino acid or 2 to 30 amino acids a group in which: - on the one hand at least one of R2 and R1 is not a hydrogen atom, - on the other hand: 2 77871 i) when at the same time R1 is a methyl group, W and R2 are hydrogen atoms and X is a CH8 / CN) group, in which case R8 is not may be a methyl, acyl group or a residue derived from an aminocarboxylic acid, ii) when R 1 is simultaneously a methyl group, W is a hydrogen atom, R 2 is a methyl group and X is a group CH 8 10, C.

In which case Rg cannot be a methyl or acetyl group, iii) when at the same time R1 is a methyl group, R2 and W are hydrogen atoms, X is a group CH8 whose hydroxyl is in the (S) configuration and the amino group is in the position 34, in which case Rg cannot be a methyl, 2q acetyl or glycyl group, iii) when W is a hydrogen atom, X is a group CH-.

/ ^ OH

and the amino group is in the 3-position, in which case the substituents R 1, R 2 and R 8 are not simultaneously methyl groups, iiiii) when Rg and W are simultaneously hydrogen atoms, R 1 is a methyl group, X is a group CH 8

/ ^ 0H

whose hydroxyl is in the configuration (R) and the amino group 30 is in position 3 (C in which case Rg is not an ethoxycarbonyl, methyl or acetyl group, iiiiii) when R1, R2 and R8 are simultaneously methyl groups and the amino group is in position 3, in which case in which W and X cannot together be an ethylidene group.

In the general formula I and hereinafter, the term 1-5 alkyl group having 1 to 5 carbon atoms means, for example, 3,77871 methyl, ethyl, isopropyl, butyl, isobutyl or pentyl groups; the term hydroxyalkyl group having 2 to 5 carbon atoms means, for example, hydroxyethyl or hydroxypropyl group; the term acyl group having 2 to 8 carbon atoms means, for example, an acetyl, propionyl, n-butyryl, isobutyryl, benzoyl or nicotinoyl group; An alkoxycarbonyl group having 2 to 8 carbon atoms means, for example, a methoxy, ethoxy or propoxycarbonyl group; residues derived from aminocarboxylic acids can be selected from the group consisting of Ala, Val, Ival, Leu, Ile, Asp, Asn, Glu, Gln, Ser, Thr, Cys, Met, Lys, Arg, Phe, Tyr, Trp, His and Pro, Nva, Nle, Hyp, Orn, these acids may be in D or L form, and Sar and Gly, all of the above acids may be N-alkylated when there is a group derived from a peptide of 2-3 amino acids, selected from the group consisting of the above-mentioned amino acids.

It should be noted from the convention that the symbols for α4-aminocarboxylic acids refer to these acids in the D or L configuration (for example, the term Ala means alanine in the D or L form).

Unless otherwise stated, the nomenclature used in this requirement follows the IUPAC nomenclature, the regulations of which are published in particular by Biochem. J. (1972), 126, 773-780.

Salts formed by the addition of inorganic or organic acids may be, for example, salts formed with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, acetic acid, formic acid, propionic acid, benzoic acid, maleic acid, fumaric acid, fumaric acid, , citric acid, oxalic acid, glyoxylic acid, aspartic acid, alkanesulfonic acids such as methane or ethanesulfonic acids, arylsulfonic acids such as benzene or paratoluenesulfonic acids and aryl carboxylic acids.

4 77871

Among the products obtained according to the process to which the invention relates, mention may be made in particular of the derivatives of the above-mentioned formula I and their salts formed by the addition of mineral or organic acids, in which said formula X is a group CH1.

I 3, CH.

/ \

OH

Among these, there may be mentioned in particular derivatives in which said formula IX has a group CH-, 10 * x 1 and 1 * 2 is a hydrogen atom or a methyl group, and their salts formed by the addition of inorganic or organic acids, and in particular: ^ - (2S) 2-amino-N - [(20S) -20-hydroxy (5S) pregnan-3oC-yl] -propanamide, - (2S) 2-amino-N - [(20S) -20-hydroxy (5S) pregnen-3 «C-yl] -acetamide, -2-amino-N - [(20S) -20-hydroxy (5o-pregnen-3O-yl) -N-2q-methyl-acetamide, and their salts; derivatives described in the examples.

The process for the preparation of the derivatives of the formula I and their salts, according to the invention, is characterized in that the amine of the formula II is CH.

: * in which the wavy line, W, X and Re have the same meaning as above, may react 35 "with either the halide of formula III: 5 77871

Hal-R'2 III

wherein Hal is a chlorine, bromine or iodine atom and R'3 is an acyl or alkoxycarbonyl group having 2 to 8 carbon atoms to give a product of formula I wherein R2 is a hydrogen atom, R3 is an acyl or alkoxycarbonyl group having 2 to 8 carbon atoms. the alkoxycarbonyl group and W, X, R 1 and the wavy line represent, as already indicated above, a compound of formula I which is isolated and, if desired, formed into a salt, a 10- or N-amino acid or a peptide comprising 2-3 N-amino acids with an amino function protected by a readily leaving group which is further cleaved by acid hydrolysis, then treated to cleave the protecting group to give a product of formula I wherein R 2 is a hydrogen atom, R 2 is an α-amino acid or 2 A group derived from a peptide comprising 3-amino acid, and W, X, R 2 and the wavy line have the same meaning as already indicated above, and which is separated and, if desired, formed into a salt with a C 20 -C 3 alkyl halide or a 2- 5 carbon atom mia with a hydroxyalkyl halide comprising a chlorine, bromine or iodine atom to give a product of formula I wherein R 2 and R 2 are hydrogen atoms or an alkyl group having 1 to 5 carbon atoms or a hydroxyalkyl group having 2 to 5 carbon atoms and W, X , R 1 and the wavy line have the same meaning as already indicated above, the product of formula I is either separated and, if desired, made a salt or, in the case where R 3 is a hydrogen atom, reacted with a halide of formula IV:

Hal-R "3 IV

wherein R "3 is an alkyl group having 1 to 5 carbon atoms or a hydroxyalkyl group having 2 to 5 carbon atoms or an alkoxycarbonyl group having 2 to 8 carbon atoms and Hal is as defined above, 6,77871 to give a product of formula I having a C1-C5 or a hydroxyalkyl group having 2 to 5 carbon atoms, an alkyl group having 1 to 5 carbon atoms or an acyl or acyl-oxy group having 2 to 8 carbon atoms and W, X, and the wavy line have the same meaning as indicated above, the product is isolated and if desired, it is salted, or even in the case where R 1 is a hydrogen atom, said product of formula I is reacted with a «C-amino acid or a peptide of 2-3 amino-10 acids, the amino function of which is protected by a readily leaving group, in particular by acid hydrolysis , is then treated to remove the protecting group to give a product of formula I wherein R 2 is an alkyl group of 1 to 5 carbon atoms or 2 to 5 carbon atoms a hydroxyalkyl group, R 1 is a group derived from a β-amino acid or a peptide of 2 to 3 amino acids, and W, X, R 1 and the wavy line have the same meaning as already indicated above, the product is isolated and, if desired, salted.

In best embodiments of the invention, the manufacturing method described above is carried out as described herein below.

1) The reaction of an amine of formula II with a halide of formula (III) 25 is carried out in the presence of an acid scavenger, in particular an alkali metal hydroxide, alkali metal carbonate (e.g. potassium carbonate, alkali metal bicarbonate, alkali acetate, alkaline earth metal carbonate, tertiary amine or e.g. pyridine) or an alkali metal alcoholate (e.g. sodium ethylate).

The reaction may take place, for example, in an inert solvent or suspension such as dioxane, dimethylformamide, benzene, toluene or methylene chloride. In the case of acylation, it is also possible to use a free acid or a functional derivative of the acid other than a halide, such as an anhydride.

When using the free acid, it is necessary to carry out the amidation with an activating agent such as carbodiimide. Other techniques are useful, such as in "The Peptides" Vol. 1,

Academic Press, 1979.

2) The reaction of an amine of formula II or a product of formula I) having a hydrogen atom and having an alkyl group or a hydroxyalkyl group with an αC amino acid or a peptide whose amino function is protected by a readily leaving protecting group takes place in the presence of a condensing agent. In this case, the purpose of the condensing agent is to activate the acid function of the amino acid.

The following can be used as a condensing agent

A-N = C = N-B

wherein A and B are alkyl groups having 1 to 8 carbon atoms, which are optionally carriers of a dialkylamino group or are cycloacyl groups.

Mention may be made, for example, of dicyclohexylcarbodiimide or 1-ethyl-3- (3-dimethylaminopropyl) -25carbodiimide, in particular the latter.

2-Chloro-N-methylpyridine halide such as iodine can also be used.

An alkyl chloroformate such as methyl, ethyl or isobutyl chloroformate may also be used; an alkyl pyrophosphite such as ethyl pyrophosphite may also be used.

As the easily leaving protecting group, for example, a benzyloxycarbonyl group or a (Z) terbuthyloxycarbonyl group (BOC) can be used, as the case may be. 4 β 77871

In particular, the invention relates to the above-mentioned method, wherein the protecting group is a terbuthyloxycarbonyl group.

An acid such as hydrochloric acid is preferably used as a cleaving agent to remove the above-mentioned easily leaving protecting group 5; used as an aid e.g.

an alkanolic solution of hydrochloric acid or anhydrous hydrochloric acid mixed with nitromethane. Acids such as paratoluenesulfonic acid, formic acid or trifluoroacetic acid can also be used. Hydrogen can also be used in the presence of palladium as a group protector (Z) for example.

3) The reaction of an amine of formula II with an alkyl or hydroxyalkyl halide is carried out in the presence of the same acid scavengers and solvents as in the reaction with a halide of formula III.

It is advantageous to protect the hydroxy function of the hydroxyalkyl group with a leaving group, in particular by acid hydrolysis with a leaving group. This protecting group is preferably a tetrahydropyranyl group. It can be cleaved under the same conditions as indicated above for amino acid protecting groups.

4) Reaction of a product of formula I having a hydrogen atom, R 2 is a C 1 -C 5 alkyl group or a hydroxyalkyl group having 2 to 5 carbon atoms and W, X, R 1 and the wavy line have the same meaning as indicated above with a halide of formula IV is made according to the nature of the R "3 substituent under the conditions described above for the reaction between an amine of formula II and a halide or alkyl halide of formula III.

5) In certain cases it may be of interest to prepare N-alkylated products of formula I in a specific way: 35 - In order to prepare an N-monomethylated product of formula (I), an amine carbamate of the product of formula (II) can be prepared, for example alkyl and especially by ethyl haloformate, then by reducing the carbamate with, for example, aluminum lithium hydride; 5 - to prepare an N-aminoethylated product of formula (I), the corresponding N-acetylated derivative can be prepared and then reduced; - in order to prepare the N-diethylated derivative, all that is required is to repeat the previous step; In these three cases, when X is a group -C-CH 2, it is necessary to prepare a ketal at the 20-position, for example with ethylene glycol, and then, after reduction, to hydrolyze this ketal, for example with an inorganic acid.

To prepare an N-mono or diisopropylated product of formula I, the product of formula II may be reacted with acetone in the presence of a reducing agent, e.g. sodium cyanoborohydride.

It is quite obvious that in the process according to the invention the protection of amino functions does not apply in cases where these amino groups have been alkylated.

The derivatives of the formula I, with the exception of those having an acyl or acyloxy group, are basic in nature. Salts can be readily prepared from the derivatives of formula I by reacting an inorganic or organic acid with precisely stoichiometric proportions of said derivative of formula I. The salts can be prepared without isolating the corresponding bases.

The derivatives prepared by this method, which is the subject of the present invention, have very interesting pharmacological properties; in particular, they have remarkable immunotherapeutic properties. In particular, they are able to stimulate immunoreactions.

10 77871 These properties are described in the experimental section below.

These properties justify the use of the 3-amino-substituted steroid derivatives of formula (I) and their pharmaceutically acceptable salts as medicaments.

Among the medicaments according to the invention, in particular the medicaments formed from the novel 3-amino-substituted steroid derivatives according to the formula 10 I in which X is a group CH 2

'M) H

as well as their salts formed by the addition of acceptable pharmaceutical acids.

15 Of these, it is worth recalling in particular formula I

compounds wherein X is a group CH 2 CH.

/ '' OH

and R 2 is a hydrogen atom or a methyl group, and salts thereof formed by the addition of pharmaceutically acceptable acids.

Among the latter, mention may be made in particular of the derivatives whose names are: - 2-amino-N - ((20S) -20-hydroxy (5O) -pregnan-3 * E-yl] -propanamide, - (2S) 2-amino-N- [1 (20S) -20-hydroxy (5et) pregnan-3'-yl] -1H-indole-3-propanamide, -2-amino-N - [(2S) -20-hydroxy-19-nor (5o, pregnan-3 * Cyl / acetamide 30-2-amino-N- [120S) -20-hydroxy (5 (t) pregnan-3-yl] -N-methylacetamide and their salts formed by the addition of pharmaceutically acceptable acids.

The medicaments according to the invention are used e.g.

35 in the treatment of autoimmune diseases due to a deficiency of certain lymphocytes, such as non-specific connective tissue disease of an organ, for example rheumatoid arthritis, systemic lupus erythematosus or a specific disease of an organ such as thyroiditis, soft tissue.

The medicaments according to the invention can also be used as adjunctive therapy in antibiotic therapy and cancer chemotherapy.

The usual dose depending on the derivative used, the subject to be treated and the disease in question may be, for example, 10 mg to 1 g per day orally in humans for the derivative of Example 14 used as an adjunct to antibiotic therapy.

The derivatives of the formula I and their salts formed by the addition of pharmaceutically acceptable acids can be used for the preparation of pharmaceutical preparations in which at least one of said derivatives or at least one of said salts is active ingredient.

The derivatives of the formula I and their salts formed by the addition of pharmaceutically acceptable acids can be mixed as medicaments with pharmaceutical preparations intended for oral or parenteral administration.

The pharmaceutical preparations may be, for example, solid or liquid and in pharmaceutical forms commonly used in human medicine, such as tablets, simple or granules, gels, granules, suppositories, injectables; they are prepared by standard methods. The active ingredient (s) may be mixed with excipients commonly used in pharmaceutical preparations, such as talc, acacia, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous vehicles, animal or vegetable i2 77871 fats / paraffin derivatives, glycols, miscellaneous.foaming, dispersing or emulsifying agents, preservatives.

The starting materials of formula II, when not known, can be prepared as follows:

Reacting a product of formula V wherein R 1 and the wavy line have the same meaning as indicated above with an excess of ethyl triphenylphosphonium bromide or potassium terbutylate to give a product of formula VI: wherein R 1 and the wavy line have the same meaning as indicated above, which is converted, for example, by introduction into a tosylate solution and then treatment with sodium azide or by direct reaction with diphenylazidophosphate in the presence of ethyl azodicarboxylate and triphenylphosphine to give the azide of formula VII:

VII

wherein R 1 and the wavy line have the same meaning as already indicated above and the configuration at position 3 is the inverse i3 77871 for compounds V and VI, which is reduced, for example, with aluminum-lithium hydride to give the amine of formula IIA: - wherein R 1 and the wavy line have the same meaning as indicated above, which - either isolated, - or reduced, for example, by hydrogenation in the presence of a catalyst based on rhodium,

15 palladium or platinum to give formula IID

D

- wherein and the wavy line represent the same as indicated above, - or hydrogenated, for example with diborane, to give the product of formula IIC: wherein R 1 and the wavy lines have the same meaning as already indicated, which is either isolated or oxidized, e.g. with chromic acid to give the product of formula IID: 14 77871 CH3 5 H2N4 wherein and the wavy line denote the same as already indicated, - or cis-dihydroxylation is carried out, for example with osmium anhydride, in the presence of trimethyloxamine after the amino group is protected or not readily with a hydrolyzable group such as a trifluoroacetyl group to give a product of formula IIE: qj

R

wherein R is a protecting group or a hydrogen atom as defined above, and the R 2 wavy line has the same meaning as already indicated, which is either isolated when R is a hydrogen atom or when R is a protecting group, removal of said protecting group is carried out, for example by alkali hydrolysis. when the group is trifluoroacetyl, then the free amine obtained is isolated, or oxidation is carried out, for example, with chromic acid to give the product of formula ΙΙρ 778 71 CH3

11F

5?

R

wherein R 1 and the wavy line denote the same as already indicated, which are either isolated when R is a hydrogen atom or when R is a protecting group, said protecting group is removed, then the free amine obtained is isolated, or reduced with, for example, sodium borohydride, to give the product of formula II_: wherein R, R 1 and the wavy lines have the same meaning as already indicated, which is either isolated when R is a hydrogen atom or when R is a protecting group, is carried out on said protecting group cleavage, then isolating the resulting free amine.

Examples of the preparation of products of formula (II): · are described below in the experimental section.

The following examples illustrate the invention without, however, limiting it.

ie 77871

Example 1: (2S) 2-Amino-N - [(20S) -20-hydroxy- (50L) -pregnan-3 '-yl] propanamide (chlorohydrate and base)

Step A: 2- (1,1-Dimethylethoxycarbonylamino) -5 N - [(20S) -20-hydroxy- (5o () pregnan-3-yl] propanamide

Dissolve 1.92 g of (20S) 3ε-amino- (5o {) pregnan-20-ol and 2.27 g of tert-butyloxycarbonyl-L-alanine 3 3 (BOC-L-Alanine) in 60 cm of chloroform and 12 cm of pyrrole. -diiniä. The solution is stirred at 0 ° C / + 5 ° C under an inert atmosphere and 1.14 g of 1-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, stirred for 50 minutes at 0 ° C / + 5 ° C, evaporated to dryness, dissolved in aqueous sodium bicarbonate solution, extracted with ethyl acetate. , washed with saturated sodium chloride solution and then 1N aqueous hydrochloric acid and again with saturated sodium chloride solution, dried, evaporated to dryness, crystallized from isopropyl ether, air dried, washed with isopropyl ether, dried at 60 ° C under reduced pressure to give the desired product.

Sp. = 171 ° C.

Step B: (2S) 2-Amino-N - [(20S) -20-hydroxy- (5ol) -25 pregnan-30-yl] propanamide hydrochloride and base.

Variation 1: Suspend 1.55 g of the product obtained in step A in 100 cm of nitromethane under an inert atmosphere, allow to mix for 10 minutes with 30 hydrochloric acid, stir at 20-25 ° C for 35 minutes, remove excess hydrochloric acid, air dry, washed with ethyl ether, dried at 50 ° C under reduced pressure, recrystallized from methanol to give 990 mg of the expected product. Sp. ^ 220 ° C.

17 77871

Variation 2: Dissolve 2.7 g of the product obtained in step A in 10 cm of ethanol under an inert atmosphere, add 40 cm of ethanolic hydrochloric acid (3.5N), stir for 8 hours, evaporate to dryness, dissolve in ethyl acetate, cool, air-dried, washed with ethyl acetate, dried at 60 ° C under reduced pressure, recrystallized from methanol to give 2.2 g of the desired chlorohydrate. Sp. ^ 220 ° C.

Preparation of base: Dissolve 1.74 g of chlorohydrate in 100 cm of 30% tetrahydrofuran in water, add 4 cm2 of 2N soda, evaporate to dryness, dissolve in 100 cm of chloroform, wash with water, dry, evaporate to dryness, recrystallize from ethyl acetate to give 1.27 g of the desired base. Sp. = 224 ° C.

By proceeding analogously to the above, the derivatives in the table below were prepared.

ie 77 8 71 i i i i i “in UOUO Ό Ό Ό g ad ° o | a ° o§ <p | 0U ~ ou§ Jl (xj CM-H-H rH-H- ~ 0-i-1 ~ -.inW IT) -H .—. O -H -p (M +) tN M -H ^ -rH m: gl γ- ρ -h p -h -h 8 3 8 ± i ^ 8 S ^ i fs8tifN8ti “ou

r-j ra iH ftf rH (tf ^ r — I (tf H (Ö O (N

Mu ^ g Mg ^ Jtf ^ rooin ^ / -N, fN / co co to co 0 5

V / J V.> V. ^ CO

l o 15 'f

W _ Cti Γ Ä-Ä V

^ ^ u a \ - ^ · a pi I o I \ g <3 N N CS n I \ -2 - / a a a a I z-a ', \ u υ υ υ k ^ \ _ I OI | CN I CM | <S * \ SS SIS KEii 5333 ^ ^ \ u-E u-E u-E o- £ - ^ _U _S Λ -i _ V-O ia I -H Λ ä

tl i! If s II I I H

v_ / || ^, 5 ¥ | l r | ¥ · Is, V_ «r | .s f ΐ f” fl f pa / il 8 S 8 8 88 a a! -p m m a m_ a a__: .3 a • d + 3 §

n - * (rt f— (<—I ΓΊ U

g (rt g n 3 e 8 p * ä s__ ° 5 M oi m <t ir \ Ό r * »

JS

m M____ is 778 71

Note to example no. 2: the second acid function of L-glutamic acid is blocked in the form of a benzyl ester; it is liberated by catalytic hydrogenation in acetic acid in the presence of palladium before cleavage of BOC.

Example 8: N - [(20S) -20-hydroxy (5® O pregnan-3'-yl] -3-pyridinecarboxamide

Dissolve 960 mg of (20S) 3-aminoamino (pregnan-20-ol and 813 mg of nicotinic acid-3 3 po in 30 cm of chloroform and 6 cm of pyridine in an inert atmosphere, add 582 mg of 1-ethyl-3- (3-dimethylaminopropyl After 3 hours and 30 minutes, 291 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride are added with stirring, the mixture is stirred for 16 hours, evaporated to dryness and dissolved in 30 cm of water. , cooled, evaporated to dryness, washed with water, dried at 80 ° C under reduced pressure, recrystallised from methanol to give 290 mg of the expected product, mp = 258 ° C = 14.5 ° + 20 1 ° (c = 1% pyridine).

Example 9: 2-Amino-N - [(20S) 17β, 20-dihydroxy (5jJ preg-nan-3β / 5-yl] acetamide hydrochloride By analogy to the procedure described in Example 1 (Variation 2) using 25 BOC-glycine as starting material and (20S) 3β-amino-Seβ-pregnan-1,2,20-diol gives the desired product, mp -200 ° C.

Mp = + 4 ° + 1 ° (c = 1.5% ethanol 95%).

The (20S) 3 *? -Amino-5β-pregnan-17β-diol starting material can be prepared as follows: Step 1: (Z) (5γ0 pregnan-17 (20) en-3-ol)

59.4 g of triphenylethylphosphonium bromide are added to a solution of 16.1 g of tertiary potassium 3-butylate in 160 cm of tetrahydrofuran, stirred for 30 minutes, 23.2 g of epiandrosterone are added, stirred for 15 hours, poured into ice-cold water, extracted 2o 7 7871 in ethyl acetate, washed with water, dried, distilled to dryness, purified by silica gel chromatography (eluent: cyclohexane-ethyl acetate 7-3), crystallized from methanol, cooled, dried in air, dried to give 23.1 g of the desired product. Sp. = 160 ° C.

Step 2: (Z) 3 {{-Azido (5'-pregnan-17 (20) -ene) Add 1.92 g of ethyl azodicarboxylate and 3.02 g of diphenylazidophosphate to a solution of 3.66 g of 1.66 g of the above product in 30 cm in benzene 3 and 5 cm in tetrahydrofuran, stirred in an ice bath, a solution of 2.88 g of 3 triphenylphosphine in 30 cm of benzene is added over 20 minutes, stirred for a further 40 minutes at 10 ° C, washed with 2N hydrochloric acid 15 poly solution, then water, dried and distilled to dryness, chromatographed on silica gel (eluent: heptane, then heptane-ethyl ether 1-1) to give 1.67 g of the desired product as crystals, mp = 114 ° C after recrystallization from methanol.

20 Step 3; (Z) 5 * k-Pregnan-17 (20) -ene-30α-amine (and chlorohydrate) 3

Dissolve in 290 cm of tetrahydrofuran 14.5 g of (Z) 3 o4-azido (5e (O pregnan-17 (20) -ene as obtained above), stir with heating at 25-27 ° C, add for one hour 800 mg of aluminum-lithium hydride, stirred for a further 1 hour, the excess hydride is removed with methanol, filtered, the filtrate is washed with aqueous Seignette salt and then with saturated sodium chloride solution, dried, distilled to dryness to give 13.1 g of crystals of the desired amine. ° C.

• 3

Dissolve the base in 150 cm of ethyl acetate • 3 3 and 30 cm of methylene chloride, add 27 cm of ethyl acetate relative to 1.7 N hydrochloric acid, dry in air, wash with ethyl acetate and dry the crystals obtained under reduced pressure to give 13 , 2 g of the hydrochloride crystals of the desired product. Sp. > 300 ° C.

1% in pyridine, 10% in water = 38.5 ° + 1.5 °.

Step 4: N - [(Z, 5E) -Pregnan-17 (20) -ene-3 ° -yl] -5-trifluoroacetamide

Suspend in an inert atmosphere 16.5 g of the hydrochloride obtained in step 3 above in 165 cm3 of methylene chloride and 16.5 cm of pyridine, cool to about 5 [deg.] C., add 16.5 cm @ 3 of trifluoroacetic anhydride over 5 minutes, stirred for 15 minutes at room temperature, distilled to dryness under reduced pressure, added 200 cm -1 of water, dried in air, washed with water, dried under reduced pressure to give 18.1 g of crystals. Sp. = 204 ° C.

Step 5: N - / (20S) 17o6 20-Dihydroxy (5 (i)) pregnan-3O-yl / trifluoroacetamide.

Dissolve 18.1 g of the product obtained above in 100 cm of methylethyl acetone under an inert atmosphere, add 9 g of trimethylamine N-oxide dihydrate 3 and stir a solution of 360 mg of osmium tetroxide in 71 cm of methylethyl acetone, allow to stand for 2 hours under a condenser, give cool, add 200 cm of 10% aqueous sodium thiosulphate solution, stir for 30 minutes at room temperature, decant, wash with water, dry over magnesium sulphate, filter, distill off to dryness under reduced pressure, purify the oil obtained by silica gel chromatography (eluent: benzene-ethyl acetate) and give 7- 14 g of the desired product; mp. = 172 ° C then 192 ° C.

Step 6: (20S) 3e (-amino-5o-pregnan-17o6 20-diol

Dissolve 4 g of the product obtained above 3 in 20 cm of methanol in an inert atmosphere, add 8 cm of soda solution, stir for 1 hour 30 minutes, add 50 cm of water, stir for 10 minutes, dry in air 35, wash with water, dry under reduced pressure. ° C and 3 g of the desired product are obtained. Sp. = 210 ° C.

22 778 71

Example 10; 2-Amino-N - [(20S) -20-hydroxy-19-nor (50O-pregnan-3-yl] -acetamide hydrochloride By analogy to the procedure described in Example 1 (Variation 1) using 5 BOC-glycine as starting material and (20S ) 3α-Amino-19-nor (50 ° pregnan-30-ol is obtained in the desired product. M.p. - 270 DEG C. with stirring. (M.p. + 39.5 DEG C. + 1.5 DEG C. (c = 1% methanol)) 20S) 3 * E-amino-19-nor (5U.) Pregnan-20-ol can be prepared as follows: By proceeding in the same manner as in Example 9 (steps 1,2,3) to prepare (Z) 5? -Pregnan-17 ( 20) -ene-3w (ramine) was prepared from (Z) 5 * E-19-nor-pregnan-17- (20) -ene-3,4-amine using 19 nor-epiandrosterone as starting material.

Suspend 156 mg of sodium borohydride in 5 cm3 of tetrahydrofuran under nitrogen, add dropwise at + 5 ° C a solution of 0.5 cm3 of boron trifluoride-dietherate in 2.5 cm3 of tetrahydrofuran, stir for 1 hour in an ice bath, 296 mg of (Z) 5 * 4-19-nor-20 pregnan-17 (20) -ene-3 * E-amine dissolved in 3 cm3 of tetrahydrofuran are added, stirred for 1 hour 30 minutes at room temperature, cooled in an ice bath, 3 are slowly added 2 cm 6N soda, stir for 5 minutes at room temperature, decant, extract the aqueous phase into tetrahydrofuran, wash the organic phase with water, add 4 cm 5N soda, 2 cm oxidized water per 110 volumes, stir for 45 minutes, extract with ethyl acetate, wash with water, dried and distilled to dryness under reduced pressure. The dry extract is redissolved in 10 cm of methanol containing 5 cm of 1N 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 evaporated under reduced pressure. 357 mg of crystals of the desired product are obtained. Sp. ^ 190 ° C.

23 77871

Example 11; 2-Amino-N- [17 * (hydroxy-20-oxo-5 - {(pregnan-3-yl) / acetamide hydrochloride By analogy to the procedure described in Example 1 (Variation 2) using 5 BOC-glycine and 3α-amino- 17eL-hydroxy-5H-pregnan-20-one gives the desired product, mp> 300 ° C.

= -50 ° + 1 ° (c = 1% ethanol 95%).

The starting material 3o-amino-17-ethyl-hydroxy-5'-trans-2-one can be prepared as follows: By oxidizing the product obtained in Example 9, step 5 with perchromate, N- (17-hydroxy-20-oxo- 5α-pregnan-3α-yl) trifluoroacetamide (m.p. = 178 ° C then 186 ° C), which is treated as shown in Example 9, Step 6 to give 3N-C-amino-17 * C-15 hydroxy- 5E6-pregnan-20-one. Sp. = 216 ° C (after crystallization from water).

Example 12: 2-Amino-N - [(20R) -20-hydroxy-5,4-pregnan-30 / t-yl] acetamide hydrochloride By analogy to the method described in Example 1 (Variation 1) using BOC-glycine as starting material and ( 20R) 3eE-amino-5oE-pregnan-20-ol gives the desired product. Sp. = 210 ° C then 260 ° C.

= + 22 ° + 1 ° (c = 0.8% pyridine in 10% water). Example 13: N - [(20S) -20-hydroxy (5H) -pregnan-3-yl] -25-ethylcarbamate.

5 g of (20S) 3 -6-amino-5 '-pregnan-20-ol 3 are added dropwise over 10 minutes to a solution of 500 cm of methylene chloride, 15 cm3 of ethyl chloroformate and 45 cm of methylene chloride, stirred for 30 minutes. 20 cm-1 soda soda are added, the mixture is stirred for a further 30 minutes, decanted, extracted with methylene chloride, washed with water, dried, filtered, evaporated to dryness under reduced pressure, redissolved in ethyl acetate, air-dried.

^ O

35 sa and 5.7 g of the expected product are obtained. Sp. - 198 C.

24 7 7 8 71 + 25 ° + 1.5 ° (c = 1% methylene chloride)

Example 14: 2-Amino-N - [(20S) -20-hydroxy-5-methylpregnan-3 * t-yl] -N-methylacetamide hydrochloride By reduction of the product obtained in Example 13, (20S) 30 ° methylamino-5 was prepared. rpregnan-20-ol sp. = 170 ° C) by the method of Vetter and colleagues described in Bull. Soc. (1963) 1324, it was reacted with BOC-glycine according to the method described in Example 1 (Variation 1) to give the desired product from which the hydrochloride was prepared. Sp. 250 ° C.

Example 15: 2 (S) 2-Amino-N-methyl-N - [(20S) -20-hydroxy-5-β-pregnan-3-e-yl] propanamide hydrochloride 15 (4S) 3,4-methylamino is given. React -5-ε pregnan-20-ol with BOC-L-alanine as described in Example 1 (Variation 1) to give the desired product from which the hydrochloride was prepared. F> 270 ° C.

Example 16: 2 (R) 2-Amino-N-methyl-N - [(20S) 20-hydroxy-20-silicon-5'-pregnan-3] style / propanamide chlorate hydrate

Reacting (20S) 3α-methylamino-5H-pregnan-20-ol with BOC-L-alanine according to the procedure described in Example 1 (Variation 1) gives the desired product from which the hydrochloride was prepared. (Turbids at about 260 ° C).

Example 17: (20S) 30α-Ethylamino-5,4-pregnan-20-ol

React (20S) -3 * amino-5-pregnan-20-ol with ethyl iodide in the presence of sodium carbonate to give the base of the desired product. Sp. = 129 ° C after recrystallization from ethyl acetate.

Chlorohydrate formation: The chlorohydrate is prepared by adding ethyl acetate hydrochloride to give the desired product. Sp. > 270 ° C.

i 25 778 71

Example 18; 2-Amino-N-ethyl-N - [(20S) 20-hydroxy-5,4] pregnan-3 * 4-yl] acetamide hydrochloride

The base of the product of Example 17 is reacted with 5 BOC-L-glycine according to the procedure described in Example 1 (Variation 1) to give the desired product from which the hydrochloride is prepared. Sp. - 230 ° C. Example 19: (20S) 304 - / (2-hydroxyethyl) amino [5e (Rpreg-nan-20-ol) 7 g of (20S) 3'-amino-5 * tpregnan-20-3

Oil and 7 g of sodium carbonate in 100 cm of anhydrous dioxane, 6.6 cm of 2- [2-bromoethoxy / tetrahydropyran are added and the mixture is kept under reflux for 24 hours under an inert atmosphere. Cool to room temperature, dilute with water, extract into ether, dry, distill to dryness under reduced pressure, purify by silica gel chromatography (eluent: chloroform-methanol-acetic acid 85-5-10) to give 7.7 g of a yellow oil.

20 Hydrolysis of the protecting group 3

Dissolve 3 g of the product obtained above in 30 cm 3 of ethanol and 15 cm of 2N hydrochloric acid, stir under a condenser for 1 hour 30 minutes, cool, pour into 100 cm3 of aqueous sodium bicarbonate solution, cool for 15 minutes, air dry, wash with water , dried at 40 ° C under reduced pressure, dissolved in 350 enamels of 10% methylene chloride in methanol, filtered, concentrated to about 50 cm, added 100 cm of ethyl acetate, concentrated in half and dried in air. Dissolve in 100 cm 3 of methanol, filter, concentrate to about 30 cm, add 70 cm 3 of ethyl acetate, concentrate in half, dry in air and at 40 ° C under reduced pressure to give 1.65 g of the base of the desired product.

35 Sp. = 215 ° C.

3 26 7 7 8 71

Chlorohydrate formation

Dissolve 1.5 g of the base obtained above in 40 cm of methanol, add an ethyl acetate solution 3 acidified with 1.6N hydrochloric acid, add 60 cm 5 of ethyl acetate, concentrate under reduced pressure, dry in air and at 40 ° C under reduced pressure to give 1, 5 g of the desired product. Sp. > 260 ° C.

Example 20: oi-dimethylamino-N - [(20S) 20-hydroxy-5 * 4-pregnan-3'-yl] acetamide.

By operating as in Example 1, Step A, using 2.552 g of (20S) 3 ', 4-amino-5'-impregnan-20-ol and 3,350 g of Ν, Ν-dimethylglycine hydrochloride. 4 g of crude product containing the desired derivative and an O, N-disubstituted derivative are obtained. Saponification of soda-15a in methanol gives the desired product. Sp. 206 ° C.

(U = + 29 ° + 1 ° (C = 1% CHCl 3).

Example 21;

Tablets of the following formula were prepared: (2S) 2-Amino-N - [(20S) -20-hydroxy-5H-pregnan-3'-yl] -20-propanamide hydrochloride 20 mg of excipient ad. 100 mg (Specification of excipient: lactose, starch, talc, magnesium stearate).

Example 22: Tablets of the following formula were prepared: 2-amino-N - [(20S) -20-hydroxy-5β-pregnan-3H-yl] -N-methylacetamide hydrochloride 15 mg of excipient ad. 100 mg (Specification of excipient: lactose, starch, talc, magnesium stearate).

Example 23 (o <S, 20S) -o (.-Amino-N - ["20-hydroxy-5 oC-pregnan-3 / S-yl] -1H-indole-3-propanamide. Proceed as in Example 1 (embodiment 2) by stirring for 6 hours in the presence of 5N-ethanol hydrochloride to give the title compound, mp 140. The starting material is (20S) -3 / 5-amino-5 c * -pregnan-20-ol.

27 7 7 8 71

Pharmacological study 1. Anaphylactic shock adjuvant

Principle:

When a product is administered to an animal that is capable of stimulating the function of the immune system, it manifests as an increased shock response to the administration of the antigen to which the animal is sensitized.

Male mice weighing 30-35 g are sensitized by injecting bovine albumin into the soles of the feet. 8 days later, they are given the same antigen intravenously. At this minimum sensitization situation, control animals do not receive a fatal shock after this last dose.

The test product is injected into the soles of the animal's feet mixed with the antigen: if this product is an adjuvant, it increases sensitization and causes a fatal shock when administered intravenously.

An effective dose is considered to be a dose that causes death in 50% or more of the animals.

20 Results: Product of the example Dose per animal in _mg 1 0.5 2 - 1 4 5 25 5 5 6 2 7 1 8 5 9 1 30 10 0,5 13 5 14 0,5 2. Percentage of red blood cells

Principle: When a product is administered to an animal that is capable of stimulating the immune systems, it manifests itself in an increase in their ability to react when injected with the immunogenic product.

28 7 7 8 71 3-month-old male rats were sensitized through the peritoneal cavity with sheep erythrocytes (day 0), 7 days later (day 7) their spleen was removed and the splenocytes were allowed to come into contact with sheep 5 erythrocytes: the percentage of leukocytes around which the erythrocytes were formed collections (rosettes).

The test product was administered orally every day from day -1 to day 1.

An immunostimulant dose of a product is considered to be a dose that approximately doubles the amount of rosin detected compared to control animals.

Results: Product of the example Dose per animal __mg / kg_ nc oral 15 1 1 5 2 6> 5 8 5 10 5 20 11 5 14 10 3. Acute toxicity study Lethal doses of LD for various test compounds were determined when mice were administered the compound orally.

The LDQ value is the dose that does not cause death after 8 days.

29 7 7 8 71

Results: Product of the example LD0 mg / kg 1,200 2> 400 3> 400 5 4> 400 5> 1000 6> 800 7 £ 400 9> .600 10 10> 400 11> 1000 12> 400 13> 400 14> 200 15

Pharmacological Comparative Experiments Using the anaphylactic shock test, the new compounds from Examples 2, 4, 5, 6, 7, 9, 10 and 14 were compared with known funtumins and N-glycylfuntumins (CR Acad. Sc. Paris t. 260 and Bull. Soc. Chim. Fr. Paris, 1967, No. 10).

Example 2 i 25 p "" 'OH, HCl h-n-ch-co-hn ^' 'v ^^ 3 ^ /

2 I

CH 2 CH 2 OH

30

Example 4 f Γ '' OH, HCl 35 H2N-CH-CO-HN ° 'ch2-c6h5 30 77871

Example 5 ι

- "YOU

H2N-CH-CO-HNv'v ° CU'1

B

Example 6 and HCl

B

Example 7 Γ -OH, HCl? C-CH2-NH-CO-CH2-NH2

Example 9

r-y% H

HCl:.: H2N-CH2-CO-Hlf '3i 77871

Example 10 ^ y '' ΌΗ, HC1 o \ / Η2Ν-0Η2-00-ΗΝ ""

Example 14 Ö I ÖH, HCl ch3 \ N * / CO-CH2HN2

Compound A: Η2 * funtumin

Compound B:

- | ^ O

.CC

H2N-CH2-CO-NH "N-glycylfuntumin 32 77871 RESULTS;

TABLE

ADPA

Example No. mg / kg 2 4i k 5 5 5 6 2 7 1 9 1 10 0.5 14 -__ 0.5

Compound A ^ 5, _ ineffective

Compound B ____, ,, _ ______ at a dose of 2

The results show that the immune-stimulating activity of the new compounds is excellent. The required AD5Q for funtumin is higher than the maximum dose tested for the new compounds: 5 mg / kg and N-glycylfuntumin is completely ineffective at 2 mg / kg.

Claims (2)

33 77871 A process for the preparation of therapeutically useful novel 3-amino-substituted steroid derivatives of the general formula (I) R 1 - f (i) R 3 wherein W is a hydrogen atom or a hydroxyl group, X is a group CH, or CH, 1 s · The CH 2 O 2 OH dashed lines indicate that the corresponding group is in either the γ or γ configuration, is a hydrogen atom or a methyl group, R 2 is a hydrogen atom or an alkyl group having 1 to 5 carbon atoms or a hydroxyalkyl group having 2 to 5 carbon atoms, and R 1 is a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, a hydroxyalkyl group having 2 to 5 carbon atoms, or a QC amino acid Ala, Val, Ival, Leu, Ile, Asp, Asn, Glu, Gln, Ser, 25 a group derived from Thr, Cys, Met, Lys, Arg, Phe, Tyr, Trp, His or Pro, Nva, Nle, Hyp, Orn in D or L form or Sar or Gly, provided that - at least one of the substituents R 2 and R 2 are not a hydrogen atom, - i) when at the same time R 1 is a methyl group, W and R 2 represent a hydrogen atom and X is a group CH 2, then R 1 cannot be a J 3 0¾. Ia is a methyl group, ^ * - 0 - ii) when R1 is simultaneously a methyl group, W is a hydrogen atom, R2 is a methyl group and X is a group CH3, then R3 cannot be a methyl group, 34 7 7 8 71 - iii) when at the same time is a methyl group, R 2 and W represent a hydrogen atom, X is a group CH 2 having a hydroxy-OH OH in the (S) configuration, and an amino group at the 3 ° C position, so that R 1 cannot be a methyl or glycyl group, iiii) when W is a hydrogen atom, X is a group CH 2, and the amino group - "CH 2, u 2 OH is in the 3: X- position, then the substituents R 1, R 2 and R 8 cannot simultaneously mean a methyl group, ^ - iiiii) when at the same time R 2 and W represent a hydrogen atom, R 1 is a methyl group, X is a group CH 8 t whose hydroxyl-__-CH 1 L 1 is in the (R) configuration, and the amino group is in the 3-position, so R 8 cannot be a methyl group, and for the preparation of their salts with inorganic or organic acids, characterized in that a) an amine of formula (II) CH 3 ·; 25 wherein the wavy line, W, X and R 2 denote s is reacted with an amino acid whose amino group is protected by a readily cleavable group, especially one which is cleaved by acid hydrolysis, then deprotected to give a compound of formula I wherein R 2 is a hydrogen atom, R 9 is a group derived from o (-amino acid) and W, X, R 1 and the wavy line have the same meaning as above and are isolated and, if desired, converted into a salt, or b) the amine of formula (II) is reacted with 35 C 1 -C 4 alkyl or 2 to 5 carbon atoms hydroxyalkyl halides. with a halogen atom of chlorine, bromine or iodine to give a compound of formula I in which R 2 and R 3 represent a hydrogen atom or an alkyl or hydroxyalkyl group having 1 to 5 carbon atoms and W , X, R 1 and the wavy line have the same meaning as above, and which is isolated and, if desired, converted into a salt, or c) in the case where R 1 is a hydrogen atom, the compound obtained in b) reacted with a halide of formula (IV) Hal-R "3 (IV) wherein R" 3 is an alkyl group having 1 to 5 carbon atoms or a hydroxyalkyl group having 2 to 5 carbon atoms and Hal represents the same as above to give a compound of formula I wherein R 2 is C 1 -C 4 -alkyl or hydroxyalkyl group having 2 to 5 carbon atoms, R 3 is alkyl or hydroxyalkyl group having 2 to 5 carbon atoms and W, X, R 1 and the wavy line have the same meaning as above and are isolated and, if desired, converted into a salt, or D) in the case where R3 is a hydrogen atom, the compound obtained in b) is reacted with an o (-amino acid whose o (-amino group) is protected by an easily cleavable group, in particular by acid hydrolysis, then the protecting group is removed to give a compound of formula I a compound wherein R 2 is an alkyl group having 1 to 5 carbon atoms or a hydroxyalkyl group having 2 to 5 carbon atoms, R 3 is a group derived from an amino acid, and W, X, R 1 and the wavy line have the same meaning as above, and which isolates distant and, if desired, converted to salt. Process according to Claim 1, characterized in that one of the following derivatives of the formula I or their salts formed by the addition of inorganic or organic acids is prepared: - (2S) -2-amino-N- (J (20S) -20- hydroxy- (δ'Χ) -pregnan-30 ((yl) -7-propanamide, - (2S) -2-amino-N- [720S) -20-hydroxy- (50'-pregnan-30 (-yl) -7-propanamide); 1H-indole-3-propanamide, 36 7 7 8 71 - 2-amino-N-Z120S) -20-hydroxy-19-nor- (5o () -pregnan-39 (-yl) acetamide, -2-amino -N - [(20S) -20-hydroxy- (5o (') -pregnan-3α (-yl) -N-methylacetamide. 57 77871