CS200520B2 - Process for preparing derivatives of androstan-17-one - Google Patents

Description

(54) A method for producing androstan-17-one derivatives

The invention relates to a process for the preparation of androstan-17-one derivatives of the general formula I

in which the bond .... represents a single or double bond,

The process according to the invention is characterized in that the sterol derivative of the general formula II

RgOCHRfl

in which the binding ..,. has the above meaning,

R1 is hydrogen or C1-C1-alkyl,

R 3 represents a hydrocarbon moiety of a C 8 -C 10 sterol and R 2 is C 1 -C 6 -alkyl, optionally interrupted by an oxygen atom, is fermented with a culture of microorganisms capable of degrading the side-chain sterols, and the thusrostan-17-one derivative of formula III

the bond ...., R 1 and R 2 as defined above are cleaved in the presence of H ⁺ -ions or Lewis acids.

The process according to the invention is preferably carried out in that the sterol derivative of the general formula IIa

wherein the bond ...., R 1 and R 2 are as defined above and

R 1 represents a hydrogen atom, a methyl or ethyl group, and is fermented with a culture of microorganisms capable of degrading the sterol side chain.

Preferably, cultures of microorganisms capable of degrading the side chain of sterols of Arthrobacter, Brevibacterium, Mirobacterium, Protaminobacter, Bacillus, Nocardia, Streptomyces or, in particular, Mycobacterium species can be used for talc fermentation.

The alkyl group R 1 or R 2 is an alkyl group having 1 to 4 carbon atoms, preferably a straight chain alkyl group. Suitable alkyl groups R 1 or R 2 are, for example, propyl, butyl, isopropyl, sec-butyl and especially methyl and ethyl. An alkyl group interrupted by an oxygen atom is a group containing from 3 to 6 carbon atoms. Such groups are, for example, 2-alkoxyethylene groups such as a 2-methoxyethylene group or a 2-ethoxyethylene group.

The hydrocarbon radical R3 having 8 to 10 carbon atoms is understood to be the radical which occurs in the side chains of natural zoo- or phytosterols, for example cholesterol, stigmasterol, campesterol, brassicasterol or sitosterols.

Suitable sterol derivatives of formula (II) are, for example, those compounds which can be characterized by formula (IIa)

in which

R 1 and R 3 are as defined above, the bonds ..., represent single or double bonds and

R 1 represents a hydrogen atom, a methyl or ethyl group.

Particularly suitable sterol derivatives of general formula IIa are ^{5 5} -steroids and 5α-sterol derivatives of this general formula.

Numerous microorganisms (such as Arthrobacters, Brevibacterium, Microbacterium, Protaminobacter, Bacillus, Nocardia, Streptomyces, and especially Mycobacterium) are known to have a natural ability to degrade zoo- and phytosterols to carbon dioxide and water, and that 4-androstene- 3,17-di-one and 1,4-androstadiene-3,17-dione.

Since numerous zoo- and phytosterols (for example, cholesterol, stigmasterol, campesterol, brassicasterol or .sitosterols) are widely distributed in nature and are therefore readily available raw materials for the synthesis of pharmacology of active steroids, numerous attempts have been made to control sterol degradation to prevent further degradation of the resulting 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione.

For example, it was possible to prevent further degradation of 1,4-androstadiene-3,17-dione and 4-androstene-3,17-dione by adding inhibitors to the fermentation broths (see German Unloading Notes Nos. 1,543,269 and 1593). 327, as well as U.S. Patent No. 1,208,078j. However, the use of inhibitors renders the engineering of these reactions very expensive, not least because the inhibitors used must be removed from the fermentation cultures after the reaction to prevent the penetration of these substances into the The known reactions also have the disadvantage that they always produce 1,4-androstadiene-3,17-dione or mixtures of 1,4-androstadiene-3,17-dione and 4-androstene-3,17-dione. However, the 1,4-androstadiene-3,17-dione formed is poorly suited as a starting material for the synthesis of numerous pharmacologically active steroids.

Otherwise, further degradation of 1,4-androstadiene-3,17-dione and 4-androstene-3,17-dione could also be prevented by the use of a mutant of Mycobacterium species for the fermentation conversion of sterols (see U.S. Pat. No. 3,684,657). ). However, the mutants produced so far have the disadvantage of a very limited ability to form 1,4-androstadiene-3,17-dione or 4-androstene-3,17-dione from sterols.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for degrading the sterol side chains without the disadvantages of known methods.

The object was solved by a process characterized in that the sterol derivative of the formula II is fermented with a culture of microorganisms capable of degrading the sterol side chains.

It is very surprising to the person skilled in the art that this fermentative conversion results in high yields of androstan-17-one derivatives of formula I. This is because it is known that degradation of the sterol side chain carries out a very complex enzyme isystem and could not be expected all enzymes involved in the degradation of natural sterol side chains also have the ability to perform non-natural sterol side chain degradation of formula II. Moreover, it could not be predicted that the enzyme systems involved in the degradation of 1,4-androstanediene-3,17-dione and 4-androstene-3,17-dione would degrade the androstan-17-one derivatives of the general formula I.

Apart from the use of other starting compounds and the fact that this reaction can be carried out in the absence of inhibitors, the process of the invention is carried out under the same fermentation conditions as also used in the known microbiological degradation reactions of sterol side chains.

According to the invention, the fermentation is carried out using cultures of microorganisms, which are usually used to break down the sterol side chain. Suitable cultures are, for example, bacteria cultures capable of degrading the side chains of sterols of Arthrobacter, Brevibacterium, Microbacterium, Protaminobacter, Streptomyces or, in particular, Mycobacterium species. Suitable microorganisms include, for example, Microbacterium lactum IAM-1640, Protaminobacter alboflavus IAM-1040, Bacillus roseus IAM-1257, Bacillus sphaericus ATCC-7055, Nocardia gardneri IAM-105, Nocardia minima IAM-374, Nocardia corallina IDO-3338, Streptomyces IAM-74 or, in particular, Mycobacterium avium IFO-3082, Mycobacterium phlei IFO-3158, Mycobacterium phlei [Institute of Health, Budapest No. 29], Mycobacterium phlei ATCC-354, Mycobacterium smegmatis ATCC-20, Mycobacterium smegmatis IFO-3082 [Institute of Health, Budapest No. 27], Mycobacterium smegmatis ATCC-19979, Mycobacterium fortuitum CBS-49566, Mycobacterium spec. NRRL-B ^ 3805 and Mycobacterium spec. NRRL-B-3683.

Submerged cultures are pre-cultured under aeration in a suitable culture medium under the usual conditions used for these microorganisms. Subsequently, the substrate (dissolved in a suitable solvent or preferably in an emulsified form) is added to the cultures and fermented until maximum conversion of the substrate is achieved.

Suitable substrate solvents are, for example, methanol, ethanol, glycol monomethyl ether, dimethylformamide or dimethylsulfoxide. The emulsification of the substrate can be carried out, for example, by adding it in micronized form or dissolved in a water-miscible solvent (such as methanol, ethanol, acetone, glycol monomethyl ether, dimethylformamide or dimethylsulfoxide) under intense turbulence into water (preferably de-calcium) containing conventional emulsifying agents. Suitable emulsifiers are nonionic emulsifiers such as ethylene oxide adducts or polyglycol fatty acid esters, such as commercially available surfactants such as Tegin ^(R) , Tagat ^(R) , Tween ^(R) and Span ^(R) .

Emulsifying the substrate often allows for increased throughput of the substrate, thereby increasing substrate concentration. Of course, it is also possible to use other methods of increasing substrate throughput in the method of the invention, as is well known to those skilled in the art.

The optimum substrate concentration, substrate addition time and fermentation duration depend on the structure of the substrate used and the type of microorganism used. These quantities must be determined, as is generally required for microbial transformations of steroids, in preliminary cases in individual cases.

The cleavage of the compounds of formula III is carried out under such conditions as are usually used for the hydrolysis or alcoholysis of acetals. For example, the compounds can be resolved by reacting in a lower alcohol such as methanol or ethanol or an organic solvent containing water such as glycol monomethyl ether, tetrahydrofuran, dioxane, dimethyiformamide, dimethylsulfoxide, hexamethylphosphoric triamide, acetone with a mineral acid such as hydrochloric acid. , sulfuric, phosphoric, perchloric, sulfonic acids such as p-toluenesulfonic acid, with strongly acidic carboxylic acids such as formic, acetic, trifluoroacetic acid, acidic ion exchangers or Lewis acids such as boron trifluoride, zinc chloride or zinc bromide.

In this way, for example, 3β-hydroxy-5-androsten-17-one, whose esters are known pharmacologically active substances (Chem. Abstr. 65, 1966, 1266f) and DE-A-1 643 046 J.

The starting compounds of the process of the invention are known or can be prepared by known methods [J. Pharm. Soc. 54, 514 (1965), Can. J. Chem. 49, 2418 (1971), Synthesis

1975, 276, and 1976, 244, Tetrahedron Letters

1976, 809, Can. J. Chem. 50, 2788 (1972) and Bull. Soc. Chim. Prance 1960, 297).

The following examples serve to illustrate the invention.

A) Examples of embodiments relating to microbiological side chain degradation

Example 1

(a) A 750 ml Erlenmeyer flask is charged with 200 ml of a sterile nutrient solution containing 1% yeast extract, 0,45% disodium hydrogen phosphate, 0,34% potassium dihydrogen phosphate and 0,2% Tagat ^(R) 02, adjusted to pH 6.7, inoculated with a washed dry culture of Mycobacterium spec. NRLL-B-3805 and shaken for 3 days at 30 ^° C at 190 rpm.

(b) Inoculate a 501 liter fenmentor with 40 liters of sterile nutrient solution containing 1,23 o / _o yeast extract (65%), 0,68% potassium dihydrogenphosphate and 0,2% Tagat ^(R) 02, adjusted to pH 6,0, 200 ml of Mycobacterium spec. and the preculture is incubated for 48 hours at 30 ° C under aeration (2 m ³ per hour).

(c) 400 g of cholesterol are dissolved in 6 liters of formaldehyde dimethyl methylacetal, 400 g of diatomaceous earth and 200 g of phosphorus pentoxide are added in portions with stirring, and the reaction mixture is stirred for 2 hours at room temperature. Insoluble materials were filtered off, washed with formaldehyde dimethyl acetal and the solvent was distilled off in vacuo. After addition of sodium bicarbonate solution, the solid crude product is filtered off with suction, washed with water and dried to give 440 g of 3/3-methoxymethoxy-5-cholestene. The compound recrystallized from acetone has a melting point of 79-80 ° C.

400 g of the thus prepared 3,3-methoxymethoxy-S-cholestene are emulsified with 120 g of Tegin ^(R) , 10 liters of deionized water and 40 ml.

N sodium hydroxide at 95 ° C for 30 minutes. The emulsion is sterilized for 20 minutes at 120 ° C.

d) 40 liters of sterile nutrient solution containing 2.0% corn steep liquor, 0.3% diammonium hydrogen phosphate and 0.25% Tagat ^(R) 02, adjusted to pH 6.5, are introduced into a 501 liter fermenter, seeded with liters of preculture Mycobacterium speč. and incubated for 24 hours at 30 ° C under aeration (0.5 m ³ per hour) and stirring (250 rpm). Then the emulsion of 3, 3-methoxymethoxy-5-cholestene prepared according to c) is added to the culture and fermented for a further 120 hours.

After completion of the fehmentation, the culture is extracted 3 times with 5 liters of ethylene chloride, the ethylene chloride extract is filtered and evaporated in vacuo.

The residue (156 g j is chromatographed on a silica gel column, recrystallized from ethyl acetate to give 86 g 3/3 ^1-methyl hoxy.m'ethoxy- ^| 5 Andros 17'Onu-one mp 129/131 DEG-132 DEG C. .

Example 2

(a) A 21-liter Erlenmeyer flask with 500 ml of sterile culture medium is not grown under the conditions of Example 1a) Mycobacterium spec. NRRL-3805.

b) 10 g of sitosterol was converted by the reaction as described in Example 1c) to give 11 g of 24-ethyl-3- (3 ' -methoxymethoxy-5-cholestene), a compound recrystallized from acetone having a melting point of 70-71 ° C.

g of the 24-ethyl-3,3-methoxymethoxy-5-cholestene thus obtained is emulsified with 4 g of Tegin ^(R) and 300 ml of water at 95 DEG C. for 10 minutes. The emulsion was sterilized for 20 minutes at 120 ° C ^Q

(c) The contents of 20 Erlenmeyer flasks each containing 85 ml of sterile broth containing 2,0 o / ₀ maize extract, 0,3% dlammonium hydrogen phosphate and 0,25% Tagat ^(R) , set at a p'H value of 6,5, inoculate 5 ml of Mycobacterium spec. and shake for 24 hours at 220 rpm at 30 ° C.

Subsequently, 14 ml of a suspension of 24-ethyl-3I / 3-methoxymethoxy-5-cholestene (corresponding to 0.5 g of 24-ethyl-3I / 3 ^, -) methoxymethoxy-5-cholestene is added to each culture and further fermented. 120 hours at 30 ° C on a shaker.

After processing as in the example. 1d) 2.1 g of 3'-methoxymethoxy-5-androsten-17-one of melting point 130 DEG-132 DEG C. are obtained.

Example 1 a d 3

a) 10.5 g of stigmasterol were converted by the reaction as in Example 1c) to give 10.7) of 5 g of 24-ethyl 1-3 / 3-methoxymethoxy-5,22-chloro -stadien. The compound recrystallized from acetone has a melting point of 103-104 ° C.

b) 10 g of 24-ethyl-3 / 3'-methoxymethoxy-5,22-cholestadiene were emulsified under the conditions described in Example 2b).

(c) Under conditions described in Example 2 (a) and (c), 85 ml of a culture of Mycobacterium spec. NRRL-B-3805 and 14 µl of a suspension of 24-ethyl-3-p-methoxymethoxy-5,2,12-cholestadiene (corresponding to 0.5 g of 24-ethyl-S / S-methoxymethoxy-S 2 Z-cholestadiene) was added.

Incubation for an additional 120 hours at 30 ° C on a shaker is followed by treatment as described in Example 1d).

2.3 g of 3/3-methoxymethoxy-5-androsten-17-one of melting point 130 DEG-132 DEG C. are obtained.

He attaches

(a) Dissolve 20 g of cholesterol in 300 ml of formaldehyde diethyl acetal, add 30 g of diatomaceous earth at room temperature and in portions of 15 g of phosphorus pentoxide with stirring and stir for 4 hours. The reaction mixture was freed of insoluble matter by filtration, the filter was washed with diethyl acetaldehyde and the solvent was distilled off in vacuo. The residue crystallized at 0 ° C. After addition of sodium bicarbonate solution, the crude product is filtered off with suction, washed with water and, after drying, 22.5 g of 3/3-ethoxy, methoxy-5-cholestene is obtained. The compound recrystallized from ethyl acetate had a melting point of 64-66 ° C.

b) 10 g of 3/3-ethoxymethoxy-5-cholestene are emulsified under the conditions described in Example 2b).

c) To 85 ml of Mycobacterium spec. N-RRL-B-3805, prepared under the conditions of Example 2a, ac), is added 14 ml of a suspension of 3/3-ethoxymethoxy-5-cholestene (this corresponds to 0.5 g of 3/3-ethoxymethoxy-5-ol). cholestene).

Incubation for an additional 120 hours at 30 ° C on a shaker followed by treatment as in Example 1d).

1.5 g of 3/3-ethoxymethoxy-5-androsten-17-one with a melting point of 121 to 123 ° C are obtained.

Example 1 and -d 5

(a) 38.67 g of cholesterol are dissolved in 250 ml of .alpha.-methylene chloride and 164.2 g of formaldehyde-bis-glycolmonomethylether acetal (obtainable, for example, according to DE 2 405 633), 60 g of diatomaceous earth are added under stirring at room temperature; 30 g of phosphorus pentoxide and stirred for 1 hour at room temperature. Insoluble products are filtered off, washed with methylene chloride and neutralized with methanolic potassium hydroxide solution. After distilling off the solvent in vacuo, the product is taken up in methanol, filtered and the compound is slowly evaporated from methanol, crystallizing. 25 g of 2 ^: [3- (2,5-dioxahexyloxy) -5-cholestene are obtained, m.p. 41-42 ° C.

b) 10 g of 30- (2,5-dioxahexyloxy) -5-cholestene are emulsified under the conditions of Example 2'b).

c) 25 ml of a culture of Mycobacterium spec. NRRL-B-3'805 and 14 ml of a suspension of 3 / 3- (2,5-dioxahexyloxy) -5-cholestene (corresponding to 0.5 g of 3 / 3- (2,5-dio.xahexyloxy) -5) is added. -cholesten).

After an additional 120 hours of incubation at 30 ° C on a shaker, the treatment is as described in Example 1d).

1.75 g of 3./3- (2,5-dioxahexyloxy) -5-androsten-17-one of melting point 65 DEG-67 DEG C. are obtained.

Example

(a) 12 g of cholesterol are suspended in 100 ml of acetaldehyde dimethyl acetal, 25 g of diatomaceous earth are added under stirring at room temperature, and 12 g of phosphorus pentoxide are added in portions and stirred at room temperature for 45 minutes. The insoluble matter is filtered off with suction, washed with methylene chloride and the solution is neutralized with methanolic sodium hydroxide solution. After distilling off the solvents in vacuo, the crude product was recrystallized after addition of activated carbon from acetone. 10.2 g of 3- [3- (1-methoxyethoxy) -5-cholestene, m.p. 94 DEG-95 DEG C., are obtained.

bj 10 g of 3- [(1-methoxyethoxy) -5-cholestene is emulsified under the conditions described in Example 2b).

c) Under conditions described in Example 2a) and c), 85 ml of Mycobacterium spec. NRRL-B-3805 and add 14 ml of a suspension of 3 / 3- (1-methoxyethoxy) -5-cholestene (this corresponds to 0.5 g of 3 / 3- (1-methoxyethoxy) -5-cholestene). hours of incubation at 30 ° C on a shaker followed by treatment as described in Example 1d,).

There was obtained 1.80 g of 3 / 3- (1-methoxyethoxy) -5-androsten-17-one, m.p. 111-118 ° C.

Example 7

a) To 19.3.3 g of cholesterol in 200 ml of methylene chloride were added 23.6 ml of vinyl ether and 86 mg of p-toluenesulfonic acid (anhydrous) and stirred for 1.5 hours. After neutralization with sodium bicarbonate solution, the reaction mixture was extracted with brine, dried over sodium sulfate and the solvent was evaporated. distills off. The oily crude product is chromatographed on a silica gel column in hexane-ethyl acetate mixtures. 18.4 g of 3,3- (1-ethoxyethoxy) -5-cholestene are obtained in the form of a colorless viscous substance.

. b) 10 g of 3 / 3- (1-ethoxyethoxy) -5-cholestene are emulsified under the conditions described in Example 2b).

c) Under conditions described in Example 2a) and c), 85 ml Mycobacterium -spec culture was prepared. NRRL-B-3805 and a 14 m-1 suspension of 3 / 3- (1-ethoxyethoxy) -5-cholestene (corresponding to 0.5 g of 3/3 '- (1-ethoxyethoxy) -5-cholestene) is added. It is then incubated on a shaker (at 30 ° C) for a further 120 hours.

The combined cultures were extracted with ethylene chloride. The extracts are concentrated in vacuo, the residue is refluxed with 70 ml of methanol, 12 ml of water and 6 ml of concentrated hydrochloric acid for 1 hour. After cooling to 12 ° C, the reaction product is precipitated with 100 ml of water and filtered off. Recrystallization from acetone gave 0.62 g of 5-androsten-3/3-10-o-17-one, m.p. 138/148-149 ° C.

Example 8

(a) 38.7 g of cholesterol are dissolved in 500 ml of methylene chloride, 25 ml of ethylene oxide and 0.5 ml of trifluoride etherate are added at 0 ° C. The reaction mixture was allowed to stand overnight at room temperature, shaken with water, dried over sodium sulfate and the solvent was distilled off in vacuo. The crude product (50 g) was chromatographed on silica gel in hexane-ethyl acetate mixtures.

10 g of 3 / 3- (2-hydroxyethoxy) -5-cholestene are obtained, m.p. 91-95 ° C.

b) 10 g of 3- [(2-hydroxyethoxy) -5-cholestene] were emulsified under the conditions described in Example 21b).

c) 85 ml of a Mycobacterium spec. NRRL-3805 and treated with a 14 ml suspension of 3 '/ 3- (2-hydroxyethoxy) -5-cholestene [this corresponds to 0.5 g of 3 / 3- (2-hydroxyethoxy) -5-cholestene], after a further 12 hours incubation at 30 ° C on a shaker followed by treatment as described in Example 1d).

2.1 g of 3 / 3- (2-hydroxyethoxy) -5-androsten-17-one with a melting point of 173/175 to 177 ° C are obtained.

Example 9 and 20 g of 5os-cholestan-3 / 3'-ol are reacted with formaldehyde dimethyl acetal as described in Example 1c) to give

21.5 ^{g, 3/3-methoxyme'thoxy} 5a'-cholestane. The compound recrystallized from acetone has a melting point of 65-68 ° C.

b) 10 g of 3- (3-methoxymethoxy-5α-cholestasis) were emulsified under the conditions described in Example 2b).

c] Under the conditions described in Example 2a) and c), 85 ml of Mycobacterium spec. NRRL-3805 and treated with a 14 ml suspension of 3/3-methoxy-methoxy-5α-cholestane (this corresponds to 0.5 g of N-methoxy-5α-cholestane). After an additional 120 hours of incubation at 30 ° C on a shaker, the treatment was as in Example 1d).

2.05 of 3/3-methoxymethoxy-5α-androstan-17-one is obtained, m.p. 97-98 ° C.

B) Examples of further chemical processing of the androstan-17-one derivatives of formula III

Example g of the 3/3-methoxymethoxy-5-androsten-17-one obtained in Examples 1 to 3 was heated to reflux for 1 hour with 125 ml of methanol, 25 ml of water and

12.5 ml of concentrated hydrochloric acid, the reaction mixture was cooled, precipitated with water and filtered off. 21.6 g of 5-androsten-3/3-ol-17-one are obtained, m.p. 148-149 ° C (from methanol).

Example 11

0.5 g of the 3/3-ethoxyethoxy-5-androsten-17-one obtained in Example 4 is reacted under the conditions of Example 10. 0.4 g of S-androsteh-S / S-ol- is obtained. 148 DEG-149 DEG C. (from methanol).

Example 12

0.38 g of 3 / 3- (2,5-dioxahexyloxy) -5-aryl-17-one obtained in Example 5 was reacted and worked up under conditions analogous to Example 10. After recrystallization from methanol yielded 0.2 g of 5-androstene-3/3-ol-17-one with a melting point of 148 to 149 ^Q C

EXAMPLE to 1 ad 1 g of 3 3 / 3- (l-methoxy-ethoxy) ^{-5-androsten-17-Nu} obtained in Example 6 was reacted under the conditions described in Example 10, however, for 1 hour at room temperature. 0.8 g of 5-androsten-3/3-ol-17-one is obtained, m.p. 148 ^DEG- 149 ^DEG C. (from methanol).

SUBJECT

OF THE INVENTION

Claims (3)

SUBJECT OF THE INVENTION A process for the preparation of androstan-17-one derivatives of the general formula I in which the bond ... is a single or double bond, characterized in that the sterol derivative of the general formula II in which the bond. is as defined above and R 1 represents a hydrogen atom or an alkyl of 1 to 4 carbon atoms, Ra represents a C1-C6-alkyl group optionally interrupted by an oxygen atom, R 3 is a C 6 -C 10 hydrocarbon sterol moiety, fermented with a culture of microorganisms capable of degrading the sterol side chain, and the thus prepared androstan-17-one derivative of formula III wherein R 1 and R a are as described above is cleaved in presence of H ⁺ -ions or Lewis acids. 2. A process according to claim 1, wherein the sterol derivative of the formula (IIa) Wherein R 1, R 1 and R a are as defined in point 1 and R 4 is a hydrogen atom, a methyl or ethyl group, is fermented with a culture of microorganisms capable of breaking down the sterol side chain, followed by a second reaction step. 3. The method according to claim 1, characterized in that a culture of microorganisms capable of degrading the side chain of sterols of the species Arthrobacter, Brevibacterium, Microbacterium, Protaminobacteir, Bacillus, Nordcardia, Streptomyces or in particular Mycobacterium is used for fermentation. Severografla, np _t zivod 7, Most