NO140679B - PROCEDURE FOR THE PREPARATION OF ALFA-1,6-GLUCOSIDASES - Google Patents

Description

Process for the production of 2a-cyan-3-oxo-A <5->steroids.

The invention relates to the production of 2"-cyan-3-oxo-A5 steroids where the basic skeleton of the steroid residue contains from 17 to 23 carbon atoms, as well as possibly the steroid's

3-enol esters and the salts of the above-mentioned compounds. These substances block the thymolytic action of ACTH in immature, possibly hypophysectomized individuals. The production takes place using the corresponding [2,3-d]-isoxazole steroid with a strong base and subsequent acidification, and the characteristic feature is that a [2,3-d]-isoxazole-4 is used as starting material ,4-dialkyl-A5-steroid and that after acidification it is optionally treated with an acid anhydride to form the 3-enol ester.

The ring structure of the compounds that can be produced according to the invention is as follows:

The exact nature of the steroid residue is not critical and it may be obtained from any steroid of the general type known to have hormonal properties or

other pharmacological or endocrinological

properties. Such steroid residues contain from 17 to 23 carbon atoms in the basic skeleton. The production of esterified 3-enol ester steroids is also within the scope of the invention, but the carbon atoms that are in the acid part of the ester are not included among the carbon atoms of the steroid skeleton.

The steroid residue can be of the estrane, 18-norestrane, androstane, eticholan, pregnan or allopregnan series. The ones mentioned above can have varying degrees of unsaturation and many different kinds of substituents in the form of hydrocarbon radicals or functional groups which are commonly used in steroid technology. Representative of steroid residues, which are contained in the compounds whose preparation the invention concerns, are those which have in the 17-position a hydroxy or a hydroxy and a lower alkyl radical, which are characteristic of androgenic and anabolic steroids, or lower alkenyl -, lower alkynyl, acetyl, hydroxyacetyl, 1,2-dihydroxyethyl, 1-hydroxy ethyl and similar radicals, which are characteristic of progestational and adrenal cortical steroids. The steroid residue can also have one or more substituents in other positions in the core, e.g. hydroxy, acyloxy or oxo radicals in positions 6, 7, 11, 12, 14 or 16 halogen atoms, preferably fluorine, chlorine or bromine, e.g. in positions 4, 6, 7, 9, 12, 16, 17 or 21, and lower alkyl groups, e.g. in positions 6, 7, 11 or 16. The steroid residue may also contain one or more double bonds, especially in the 4,5 and/or 1,2 and/or 6,7 positions. The steroid residue usually contains methyl groups at C10 and C,,,, but also 18- and 19-norsteroids and 18, 19-bisnorsteroids that lack one or both methyl groups at C,., resp. C10, are representative steroids.

The basic skeleton of 18,19-bisnorsteroid, 18,19-norsteroid and normal steroid residues in compounds the invention relates to the production of contains seventeen, resp. eighteen and nineteen carbon atoms up to a total of twenty-three carbon atoms, including radicals not included.

If acyloxy radicals are present in the steroid residue, the acyl radicals are preferably derived from carboxylic acids containing from one to about ten carbon atoms, which are commonly used in the steroid technique; and which has a molecular weight of less than approx. 200. Among representative acyl radicals that may be present are lower alkanoyl radicals, e.g. formyl, acetyl, propionyl, butyryl, isobutyryl, caproyl, heptanoyl, octanoyl, trimethylacetyl and the like, carboxy-lower alkanoyl radicals, e.g. succinyl-(|3-carboxypropionyl), cycloalkyl-lower-alkanoyl radicals, e.g. (3-cyclopentylpropionyl, p-cyclohexylpropionyl and the like, mono-carbocyclic aroyl radicals, e.g. benzoyl, p-toluyl, p-nitrobenzoyl, 3,4,5-trimethoxy-benzoyl and the like, monocarbocyclic aryl-lower-alkanoyl or - alkenoyl radicals such as phenylacetyl, [3-phenylpropionyl, cinnamoyl and the like, and monocarbocyclic aryloxy-lower alkanoyl radicals such as p-chlorophenoxyacetyl and the like Esters of inorganic acids such as eg: phosphoric acid can also be found!.

According to the invention, 2q-cyan-3-oxo-A5 steroids can be prepared by cleaving a steroid-[2,3-d]isoxazole with a strong base according to the following scheme

in which Q represents the remainder of the steroid molecule. The 2-cyan group assumes the α-configuration in steroids of the androstane series and in A4 and A5 steroids, while it assumes the (^-configuration in steroids of the etiocholan series.

Any strong base can be used to convert the isoxazole to the cyano ketone, but it is preferred to use alkali metal alkoxides, and the reaction is best carried out in an anhydrous medium.

It has been shown that the 2-cyan-3-oxo-A5 steroids have endocrinological activity, especially hormonal activity, e.g. anabolic and adrenal-inhibiting properties. A particularly preferred class of 2-cyano-3-oxy-A5 steroids, which have adrenal blocking properties, are those having two lower alkyl radicals in the 4-position and a double bond in the 5,6-position of the steroid nucleus, and which is represented by the following par-tiaMormel:

The above-mentioned structural features are decisive, consequently the invention relates to the production of 2a-cyan-3-oxo-A5 steroids, where the basic skeleton of the steroid residue contains from seventeen to twenty-three carbon atoms, not including ester radicals, and which also have two lower alkyl radicals in the 4-position and a double bond in the 5,6-position. The lower alkyl radicals preferably contain from one to four carbon atoms.

To prepare the above-mentioned steroid-[2,3-d]isoxazoles, a 2-(1-ny-hydroxyalkylidene)-3-oxo-steroid is reacted with hydroxylamine or with an acid addition salt thereof according to the following equation:

In the foregoing general formulas, Q represents the rest of the steroid molecule.

The condensation of hydroxylamine with a 2-hydroxyalkylidene-3-oxo-steroid takes place by heating the steroid together with at least one molar equivalent of hydroxylamine or an acid addition salt thereof in an inert solvent at approx. 50—150°C. The inert solvent is preferably a lower alkanol, e.g. methanol or ethanol, or a lower alkanoic acid, e.g. acetic acid or propionic acid, or a mixture of an alcohol and an acid. The reaction is catalyzed by the presence of a weak or medium strong acid, e.g. acetic acid, but if a strong acid is present this can cause dehydration of a hydroxy group in the 17-position followed by a profound transformation in the steroid nucleus. If an acid addition salt of hydroxylamine is used, e.g. the hydrochloride, to prevent dehydration and rearrangement, becomes an approximately equivalent amount of a salt of a strong base and a weak acid, e.g. sodium acetate, added to convert addition acid from a strong to a weak acid. 17-hydroxy groups can also be protected by esterification before isoxazole formation. The above-mentioned compounds can be modified in various ways before they are reacted by the method according to the invention. For example, a steroid [2,3-d]isoxazole having the hydroxy group in the 17-position of the steroid nucleus (V, VI or VII, R' is H, Z is OH) can be oxidized to the corresponding 17-oxo compound . Or a steroid [2,3-d]isoxazole which has a 1-hydroxyethyl radical in the 17-position can be oxidized to the corresponding 17-acetyl compound.

The intermediate 2-hydroxyalkylidene-3-oxo-A5-steroids are prepared by condensing a 3-oxo-A5-steroid, with a lower-alkyl-lower-alkanoate, RCOOR'", in which R is lower-alkyl and R '" is lower alkyl or with HCOOR'" where R'" has the same meaning as above, in the presence of a strong base under anhydrous conditions. The strong base is preferably an alkali metal lower alkoxide or amide (cf. Ruzicka, U.S. Patent No. 2,281,622), or

-hydride. An acyl group enters the 2-position during the elimination of a molecule of an alcohol, as follows:

A particularly preferred group of compounds, which are derived from readily available starting materials, are those having the following structural formula:

in which R' represents hydrogen or lower alkyl, lower alkenyl, lower alkynyl, acetyl, hydroxyacetyl, 1,2-dihydroxyethyl or 1-hydroxyethyl, X represents H2,

(H) (OH) or O, Y and Y' represent hydrogen or methyl and Z represents hy-

the drug or, hydroxyl, and wherein Z is limited to be hydroxy when R' represents hydrogen or lower alkyl, lower alkenyl or lower alkynyl, and wherein, if desired, R' and Z together form an oxo group, and wherein the steroid optionally contains other substituents, which are inert for the reaction used in the preparation of the aforementioned compounds. A particularly preferred class of compounds are those having two alkyl substituents, e.g. methyl groups, in the 4-position and a double bond in the 5,6-position, as indicated above.

The definition given above also includes 2-cyan-3-enol ester steroids in which free hydroxy groups are present. The esters are derived from carboxylic acids that have from one to ten carbon atoms and are usually used in the steroid technique, and the esters are produced by usual working methods, e.g. by treating the steroid alcohol with the appropriate acid halide or anhydride. Included are thus enol esters which have the following partial formula:

where Ac represents an acyl radical. The enol esters. is prepared by heating the 2-cyano-3-oxo-A<5> compound together with the appropriate acid anhydride in the presence of pyridine. The invention also relates to salts of the aforementioned compounds, where R', when it represents a lower alkyl, a lower alkenyl or a lower alkynyl radical, contains from one to approx. four carbon atoms and may be straight or branched, thus including such groups as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary-butyl, vinyl, 1-propenyl, 2-propenyl, ethynyl, propargyl and the like.

The 2-cyan-3-oxo-A5 steroids are acidic in nature in that they have an active hydrogen atom in the 2-position, and therefore form salts with strong bases such as alkali metal hydroxides or alkoxides. Thus, during the cleavage of the isoxazole, a salt of the 2-cyano-3-oxo-steroid is first formed, and this salt is converted into the free 2-cyano-3-oxo-steroid by making the solution acidic.

The compounds produced according to the invention can be prepared for parenteral use by dispersing them as an aqueous suspension or by dissolving them in a pharmacologically acceptable oil or oil-water emulsion, or for oral use they can be incorporated into tablets together with suitable substances.

The following examples illustrate the invention in more detail, without limiting it.

Example 1.

(a) 17$- hydroxy- 4, 4, 17 a- trimethyl-5- androstene [ 2, 3- d] isoxazole

can be prepared from 4.5 g of 2-hydroxymethylene-4,4-17α-trimethyl-5-androstene-17fS-ol-3-one, 0.91 g of hydroxylamine hydrochloride, 1.03 g of sodium acetate and 150 ml of acetic acid by the working method described in ex. 1, part (a) of Belgian Pat. 580 902. The crude product was chromatographed on silica gel in benzene solution, eluted with benzene containing 25% ether and recrystallized from ethanol, whereby 17p-hydroxy-4,4,17a-trimethyl-5-androstene [2,3- d] isoxazole, m.p. 177.6—180.8°C (corr.), [a] V = —66.9 ± 0.1° (1 per cent in 95 per cent ethanol), ultraviolet maximum at 229 m\ in (E = 6.200).

Analysis:

calcd for C28H.SoN02: C 77.70; H 9.36; 0 9.00. found: C 77.71; H 9.17; O 9.30. (b) 2a-4,4,17a-trimethyl-5-androstene-17fiol-3-one. 1 a 250 ml Erlenmeyer flask, which was equipped with a calcium chloride tube and a stirring rod, was placed a solution of 11.7 g (0.0330 mol) 17(3-hydroxy-4,4,17a-trimethyl-5- androsten [2,3-d]isoxazole in 50 ml of tetrahydrofuran. The solution was stirred and 3.6 g (0.0667 mol) of sodium methoxide was added. The solution became clear, but after a few minutes the sodium salt of the product began to precipitate out. An additional 20 mL of tetrahydrofuran was added, and stirring was continued for 1 hour. Ether (200 mL) was added to the mixture, which was then extracted with water in portions of 250 mL and 100 mL. The aqueous layers were acidified with a small excess hydrochloric acid at room temperature.The resulting solid product was filtered and dried at 60°C to give 11.5 g of 2α-cyano-4,4,17α-trimethyl-5-androstene-176-ol-3- on, m.p. 225-230°C (uncorrected). The compound was further purified as follows: 187 g of product was dissolved in 1 liter of hot tetrahydrofuran, the solution was filtered and 1 liter of hot ethyl cetate was added slowly to the filtrate. The solution was concentrated to a volume of 1200 ml and allowed to stand in a refrigerator for approx. 17 hours. The product that had separated was filtered off, and the mother liquor was evaporated to 400 ml, cooled and a new portion of precipitated product was collected. In this way, 119° with m.p. 222.8—228.2°C (corr.), [a] y = —34.9° (1 per cent in chloroform), ultraviolet maximum at 238 mji (E = 7.600), and 44 g with m.p. 221.8—224.2°C (corr.), [a]2D5 = —35.5°, ultraviolet maximum at 238 imi (E = 7,300)..

2α-cyano-4,4,17α-trimethyl-5-androsten-17|3-ol-3-one showed no marked effect on normal animals, but was very effective in reversing effects due to strong adrenal cortex action due to ACTH. Laboratory tests with rats showed that this compound inhibits the synthesis of corticosterone, and these results show that the compound has a beneficial effect under pathological conditions which have been produced by overactivity of the adrenal glands. 2a-cyano-4,4,17a-trimethyl-5-androsten-17|3-ol-3-one also lowered blood pressure (by 20-28 mm Hg) in rats with renal hypertension when given in

individual oral doses of 2.5-10 mg/kg. Daily subcutaneous injection of the compound (5 mg/kg/day) for 4 days resulted in a sustained hypotensive effect (10-40 mm Hg). 2α-cyano-4,4,17α-trimethyl-5-androstene-17|3-ol-3-one is very mildly toxic. No LDr>() could be determined, as doses of up to 8000 mg/kg have been tolerated. Five-day experiments with monkeys showed no adverse effects of doses of 100 mg/kg.

When the compound 2-cyano-17a-methyl-4-androstene-17p-ol-3-one according to ex. 1(c) in Australian Patent 242,477 was subjected to the same tests as above, no inhibitory effect could be observed.

Example 2.

2- cyano- 3-($- cyclohexylpropionoxy)- 4, 4, 17a- trimethyl- 2, 5- andro-stadien- 17fiol

((3-Cyclohexylpropionate enol ester of 2α-cyano-4,4,17α-trimethyl-5-androstene-17(3-ol-3-one).

A mixture of 8.6 g (0.0242 mol) 2a-cyano-4,4,17a-trimethyl-5-androstene-17p-01-3-one, 21.4 g (0.726 mol) p-cyclohexylpropionic anhydride and 50 ml of pyridine was boiled for 3 hours under reflux. The reaction mixture was poured into ice water, allowed to stand for approx. 17 hours, and was then extracted with ether. The ether extracts were washed with 5% sodium carbonate solution, dried over anhydrous sodium sulfate and evaporated in vacuo. The residue was chromatographed on neutral alumina using pentane as solvent. The column was eluted with pentane containing gradually increasing amounts of ether, and finally with pure ether. The crystalline product thus obtained was recrystallized from a mixture of ether and pentane, whereby 4.2 g of 2-cyano-3-(p-cyclohexylpropionoxy)-

4,4,17a-trimethyl-2,5-androstadien-17p-

etc., m.p. 104—110°C (uncorrected). A sample of the compound that was recrystallized once more had m.p. 106.2—109.4°C (corr.), [a]2D5= —49.8° (1 per cent in chloroform), ultraviolet maximum at 219 m^i (E = 9.940), infrared maxima at 2, 88, 3.43, 4.50, 5.65, 6.01 and 6.89 µ, suggesting the presence of a hydroxy group and the absence of a carbonyl group.

2-Cyano-3-(p-cyclohexylpropionoxy)-4,4,17a-trimethyl-2,5-androstadien-17p-ol was found to have a pharmacological activity similar to that of 2a-eyan-4,4,17a -trimethyl-5-androstene-17p-ol-3-one.

Example 3.

(a) 17fi- hydroxy- 4, 4- dimethyl- 5- androstene [ 2, 3- dl isoxazole

was prepared from hydroxylamine and 2-hydroxymethylene-4,4-dimethyl-4-androstene-17p-ol-3-one (m.p. 148-154°C, from 4,4-dimethyl-4-androstene-17p- ol-3-one and ethyl formate in the presence of sodium methoxide in pyridine) and it had m.p. 192.6—195.4

(corr.) after recrystallization from ether-pentane, [a]D = —39.6° (1 percent in chloroform).

(b) 2a- cyano- 4, 4- dimethyl- 5- androsten-17$- ol- 3- one

was prepared by basic cleavage of 17(5-hydroxy-4,4-dimethyl-5-androstene [2,3-d] isoxazole, and had m.p. 215.0-218.2°C (corr.) after recrystallization from isopropyl alcohol, [a]D = -23.5° (1% in pyridine). 2a-cyano-4,4-dimethyl-5-androstene-17p-ol-3-one was shown to have pharmacological activity in similarity to that of 2α-cyano-4,4,17α-trimethyl-5-androstene-17β-ol-3-one.

Example 4.

(a) 17$-(phy- cyclohexylpropionoxy)-4, 4, 17a- trimethyl- 5-androstene [ 2, 3- d]-isoxazole

was prepared by refluxing a mixture of 17β-hydroxy-4,4,17α-trimethyl-5-androstene [2,3-d]isoxazole and an excess of β-cyclohexylpropionic anhydride in pyridine for 7 hours. The product had m.p. 121—124°C (uncorrected).

(b) 2α-cyano-4,4,17α-trimethyl-17β-(P-cyclohexylpropionoxy)-5-androstene-3-one

was prepared by basic cleavage of 17(5-(p-cyclohexylpropionoxy)-4,4,17a-trimethyl-5-androstene [2,3-d]isoxazole, and had a m.p. 168-171°C (uncorrected) after recrystallization from acetone.

Example 5.

(a) 17fi- hydroxy- 4, 4- dimethyl- 17a- ethynyl-5-androstene [2,3-d]isoxazole was prepared from hydroxylamine and 2-hydroxymethylene-4,4-dimethyl-17α-ethynyl-4-androstene-17β-ol-3-one, and had m.p. 211—221°C (uncorrected) after recrystallization from methanol, ultraviolet maximum at 229 m(i (E = 5.840). (b) 2a- cyano- 17a- ethynyl- 4, 4- dimethyl-5- androstene- 17fi- ol- 3-on was prepared by basic cleavage of 17(3-hydroxy-4,4-dimethyl-17a-ethynyl-5-androstene [2,3-d]-isoxazole.

Example 6. (a) 17,20:20,21-bismethylenedioxy derivative of 17a,21-dihydroxy-11,20-dioxo-4-pregnene [2,3-d]isoxazole was prepared from hydroxylamine and 17,20: The 20,21-bis-methylenedioxy derivative of 2-hydroxymethylene-4-pregnene-17a,21-diol-3,11,20-trione, and had m.p. 273-278°C (uncorrected) after recrystallization from a toluene-ethyl acetate mixture. (b) The 17,20:20,21-bismethylenedioxy derivative of 2α-cyano-4-pregnene-17α,21-diol-3,11,20-trione was prepared by basic cleavage of 17,20:20,21- the bismethylenedioxy derivative of 17α,21-dihydroxy-11,20-dioxo-4-pregnene [2,3-d]isoxazole, and had m.p. 228—236°C (uncorrected).

Claims (2)

1. Process for the production of a 2a-cyan-3-oxo-A5-steroid, optionally its 3-enol ester, as well as their salts, which block the thymolytic action of ACTH in immature, possibly hypophysectomized individuals, by converting the corresponding [2 ,3-d] isoxazole steroid with a strong base, after which it is acidified, characterized by the fact that a [2,3-d] isoxazole-4,4-dialkyl-A5 steroid is used as starting material and that after acidification it is optionally treated with an acid anhydride to form the 3-enol ester. 2. Process as stated in claim 1, characterized in that 17|3-hydroxy-4,4,17a-trimethyl-5-androsteno-[2,3-d]-isoxazole is used as starting material.