CS200518B2 - Process for preparing derivatives of androstanone - Google Patents

Description

(54) A method for producing androslan derivatives

The invention relates to a process for the preparation of androstanone derivatives of the general formula I

in which

X is carbonyl or hydroxy, methylene, n is 1 or 2,

R 1 represents an atom, a hydrogen atom or a C 1 -C 4 -alkyl group and. R 2 denotes a C 1 -C 6 -alkyl group optionally interrupted by an oxygen atom or, in the case n denotes 2, also denotes a hydrogen atom.

The principle of the process according to the invention consists in that the sterol derivative of the general formula

II

wherein n, R 1 and R a are as defined above and

R 3 represents a hydrocarbon residue of a C 8 -C 10 sterol, is fermented with a culture of microorganisms capable of degrading the sterol side chain and the resulting compound of formula III below is hydrogenated.

The process according to the invention can advantageously be carried out by providing a sterol derivative of the formula IIa

bond .... means a single or double bond,

R1 and R2 are as defined above and R4 is hydrogen, methyl or ethyl, is fermented with a culture of microorganisms capable of degrading the sterol side chain, and the 5-androsten-17-one derivative of formula III thus produced

R 1, R 2 and n have the same meanings as above and are hydrogenated.

The process of the invention may also be carried out by providing a sterol derivative of formula (IIb)

wherein R 2, R 2 and R 2 are as defined above. a .....,

R4 represents a hydrogen atom, a methyl or ethyl group, is fermented with a culture of microorganisms capable of side chain degradation and the resulting compound of formula III is hydrogenated.

The microorganisms capable of degrading the sterol side chain are preferably microorganisms of the species Arthrobacter, -Brevibacterium, Microbacterium, Protaminobacter, Bacillus, Nocardia, Streptomyces, and in particular of the species Mycobacterium.

By alkyl ^1, 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, isopro-propyl, sec-butyl, and in particular methyl and ethyl. Oxygen interrupted by an oxygen atom means a group containing 3 to 6 carbon atoms. Such groups are, for example, 2-alkoxyethylene groups, such as a 2-methoxyethylene group or a 2-ethoxyethylene group.

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

Suitable sterol derivatives of formula II are, for example, those compounds which are characterized by the formulas

Ha or lib

where

R1 and R2 are as defined above, the bonds are single bonds or double bonds,

R4 represents a hydrogen atom, a methyl group or an ethyl group.

Particularly suitable sterol derivatives of general formulas IIa and IIb are -isteroidy A ^5-5a and derivatives of sterols such Formulas.

Numerous microorganisms (e.g., Arthrobacters, Brevibacterium, Microbacterium, Protaminobacter, Bacillus, Nocardia, Streptomyces, and in particular Mycobacteriumj) are known to have a natural ability to degrade zoo- and phytosterols to carbon dioxide and water, and that 4-androstene-3 17-dione α1,4-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 pharmacologically 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.

Thus, 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 DE 1 543 269 et al.). No. 593,327 as well as U.S. Patent No. 1,208,078). However, the use of inhibitors makes the engineering of such reactions very expensive; not least because the used inhibitors have to be removed from the fermentation cultures after the reaction to prevent the penetration of these substances into the waste water. Furthermore, the known reactions have the disadvantage that they always produce 1,4-androstadiene-3,17-dione or a mixture of 1,4-androstadiene-3,17-dione and 4-androstene-3,17-dione. However, the 1,4-androstadiene-3,17-dione formed is not suitable 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 using a mutant of Mycobacterium species for fermentation conversion of sterols (see U.S. Patent 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 reductive conversion results in high yields of androstan-17-one derivatives of the general formula I. This is because it is known that degradation of the sterol side chain carries out a very complex enzyme system and could not be expected all enzymes involved in the degradation of the side chains of natural sterols also have the ability to carry out non-natural sterol side chain degradation of the general formula II. Furthermore, it could not be predicted that the enzyme systems involved in the degradation of 1,4-androstadiene-3,17-dione and 4-aridrostene-3,17-dione did not degrade 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, Protaminobacterium, Streptomyces or, in particular, Mycobacterium species. Suitable microorganisms include, for example:

Miorobacterium lactum 1 AM-1B40, Protaminobaicter alboflavus IAM-1040, Bacillus roseus IAM-1'257,

Bacillus sphaericus ATCC-7055

Night Gardneri IAM-105,

Nocardia minima IAM-374

Nocallia corallina IFO-3338,

Streptomyces rubescens IAM-74, or particularly microorganisms

Mycobacterium avium IFO-3082

Mycobacterium phlei IFO-3158

Mycobacterium phlei (Health Institute, Budapest No. 29),

Mycobacterium phlei ATCC-354, Mycobacterium smegmatis ATCC-20, Mycobacterium smegmatis IFO-3Q84, Mycobacterium smegmatis (Institute of Health, Budapest No. 27),

Mycobacterium smegmatis ATCC-19979, Mycobacterium fortuitum SBS-49566, Mycobacterium spec. NRRL-B-3805 and Mycobacterium spec. NRRL-B-36 & 3

Submerged cultures are grown under aeration in a suitable culture medium under the usual conditions used with 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) with vigorous turbulence into water (preferably devoid of calcium) conventional emulsifying agents. Suitable emulsifying agents are nonionic emulsifiers, for example ethylene oxide adducts or polyglycol fatty acid esters. Suitable emulsifiers are, for example, commercially available surfactants such as Tegln ^(R) , Tagat ^(R) , Tween ^(R) and Span ^(R) .

Emulsifying the substrate often allows increased substrate throughput and 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 ensured, as is generally required for microbial transformations of steroids, in individual cases by preliminary experiments.

The 5-androsten-17-one derivatives of formula (III) are hydrogenated to the corresponding 5α-androstan-17-one derivatives, according to reaction conditions the 17-keto group is retained or reduced to the corresponding 17β-hydroxyl group.

For example, 5α-androstan-17-one derivatives of formula I can be converted after reduction of the 17-keto group to the 17-hydroxy group, esterification thereof, cleavage of the acetal bond and oxidation of the 3-hydroxyl group in the corresponding 17β-acyloxy-5α-androstane-3- those known to be anabolic agents (E, ndocrinology 66, 1960, 13).

The starting compounds of the process of the invention are known or can be prepared by known methods [J. Pharm. Soc. 54, 414 (1965); J. Chem. 49, 2418 (1971), Synthesis 1975, 276 & 1976, 244, Tetrahedron Letters 1976, 80Θ, Can. J. Chem. 50, 2788 (1972), and Bull. Soc. Chim. France, 1960, 297).

The following examples serve to illustrate the invention.

A) Exemplary embodiments relating to microbiological side chain degradation

Example 1

(a) 200 ml of sterile nutrient solution containing 1% yeast extract, 0,45% dibasic sodium phosphate, 0,34% potassium dihydrogenphosphate and 0,2% Tagat ^(R) 02, adjusted to a pH of 6,7 shall be introduced into a 750-ml Erlenmeyer flask. , inoculated with a washed dry culture of Mycobacterium spec. NRLL-B-3805, and shake for 3 days at 30 ° C at 190 rpm.

(b) A 40 liter sterile nutrient fermenter containing 1,23% yeast extract (65%), 0,68% potassium dihydrogen phosphate and 0,2% Tagat ^(R) 02, adjusted to pH 6,0, is seeded. 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 acetal, 400 g of diatomaceous earth and 200 g of phosphorus pentoxide are added under stirring at room temperature, and the reaction mixture is stirred for 2 hours at room temperature. Insoluble materials were filtered off, washed with dimethyl acetal formaldehyde and the solvent was distilled off in vacuo. After addition of sodium bicarbonate solution, the solid dry product is filtered off with suction, washed with water and dried to give 440 g of 3/1-methoxymethoxy-5-cholestene. The compound recrystallized from acetone has a melting point of 79-80 ° C.

400 g of the thus prepared 3'-methoxymethoxy-5-cholestene are emulsified with 120 g of Tegine ⁽ 10 ^R) , 10 liters of deionized water and 40 ml of 1 N sodium hydroxide at 95 ° C for 30 minutes. The emulsion is sterilized for 20 minutes at 120 ° C.

dj 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 and inoculated with 2 liters of Mycobacterium spec preculture . and incubated for 24 hours at 30 ° C under aeration (0.5 m ³ per hour) and stirring (250 rpm). Emulsion 3 (S-methoxymethoxy-5-cholestene, prepared according to c) was then added to the culture and fermented for an additional 120 hours.

After the fermentation was complete, the culture was extracted with 3X5 liters of ethylene chloride, the ethylene chloride extract was filtered and evaporated in vacuo.

The residue, (158 g) is chromatographed on silica gel, recrystallized from ethyl acetate to give 86 g of 3, 3-methoxymethoxy-5-androsten-17-one, m.p. 129/131-132 ° C.

Example 2

(a) In a 21-liter Erlenmeyer flask with 50 ml of sterile broth, 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-p-methoxymethoxy-5-cholestene. The compound recrystallized from acetone has a melting point of 70-71 ° C.

g of the thus obtained 2'-4-ethyl-3- (3-methoxymethoxy-5-cholestene) is emulsified with 4 g of Tegin ^(R) and 300 ml of water at 95 DEG C. for 10 minutes, and the emulsion is sterilized for 20 minutes at 120

(c) The contents of 20 Erlehmeyer flasks each containing 85 ml of sterile broth containing 2,0% corn extract, 0,3% diammonium hydrogen phosphate and 0,25% Tagat ^(R) , adjusted to a pH of 6,5, shall be inoculated with: 5 ml of Mycobacterium spec. and shake for 24 hours at 220 rpm at 30 ° C. Celsius.

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

After processing as described in Example ld), 2.1 g 3 | / 3-methoxymethoxy-5-androsten-17-one with a melting point of 130-132 C. Example 3 ^and

a) 10.5 g of stigmasterol were converted by the reaction of Example 1c) to give 10.75 g of 24-ethyl-3? -mothoxymethoxy-5,22-eholestadiene. The compound recrystallized from acetone has a melting point of ^103-104 ° C.

b) 10 g of 24-ethyl-3 (3 ^: -methoxymethoxy-5,22-choestadiene) was 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 24-ethyl-3β / 3-methoxymethoxy-5,22-cholestadiene (this corresponds to 0.5 g of 24-ethyl-3; p-methoxymethoxy-5,22-cholestadiene) .

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-p-methoxymethoxy-5-androsten-17-one with a melting point of 130 DEG-132 DEG C. are obtained. Celsius.

Example 1 a d 4

(a) 20 g of cholesterol are dissolved in 300 ml of formaldehyde diethyl acetal, while stirring, 30 g of diatomaceous earth are added at room temperature and in portions of 15 g of phosphorus pentoxide and stirred for 4 hours. The reaction mixture was freed of insoluble materials by filtration, washed with formaldehyde diethyl acetal and the solvent was distilled off in vacuo. The residue was crystallized at 0 ^C C. After addition of sodium carbonate, the crude product is filtered off, washed with water and after drying, 22.5 g 3--ethoxymethoxy-'5'-cholě'stenu. The compound recrystallized from ethyl acetate had a melting point of 64-66 ° C.

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

c) To 85 ml of Mycobacterium spec. NRRL-B-3805, prepared under the conditions of Example 2a) and c), 14 ml of a suspension of 3,3-ethoxymethoxy-5-cholestene (corresponding to 0.5 g of 3α-ethoxymethoxy-5-eholestene) is added,

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

1.5 g of 3'-ethoxymethoxy-5-androsten-17-one are obtained, m.p. 121-1.23 ^and C. Example 5

a) 38.67 g of cholesterol are dissolved in 250 ml of methylene chloride and 164.2 g of formaldehyde-bis-glycolmonomethylether acetal (for example according to Dé No. 2 405 633), while stirring at room temperature, 60 g of diatomaceous earth are added. and 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 was taken up in methanol, filtered and the compound crystallized by slow evaporation. 25 g of 2 / 3- (2,5-dioxahexyloxy) -5-cholestene are obtained, m.p. 41-42 ° C.

b) Under the conditions of Example 2b) 10 g of 3 ') 3 -' (2,5-, dioxahexyloxy) -5-cholestene are emulsified.

cj Under the conditions of Example 2a) and c), 25 ml of a culture of Mycobacteirium spec. NlRRL-B-3805 and add 15 ml of a suspension of 3/3 - (2,5-dioxahexyloxy) -5-cholestene (corresponding to 0.5 g of 3 / 3- (2,5-dioxahexyloxy) -5- cholestene).

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

1.75 g of 3- [2,5-dioxahexyloxy] -5-androsten-17-one of melting point 65 DEG-67 DEG C. are obtained. Celsius.

Example 6

(a) 12 g of cholesterol are suspended in 100 ml of acetaldehyde dimethylacetal, 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- (1-methoxyethoxy) -5-cholestene are obtained, m.p. 94-95 ° C.

b) 10 g of 3 / 3- (1-methoxyethoxy) -5-cholestene were 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 14 ml of a suspension of 3 / 3- (1-methoxyethoxy) -5-cholestene (corresponding to 0.5 g of 3 / 3- (1-methoxyethoxy) -5-cholestene) is added. After an additional 120 hours of incubation at 30 ° C on a shaker, processing is carried out as described in Example 1d).

There were obtained 1.89 g of 3/3 ((1-methoxyethoxy) -5-androsten-17-one), m.p.

Example 7

(a) To 19,33 g of cholesterol in 200 ml of meth.

Ethylene chloride was added with 23.6 ml of vinyl ether and 86 mg of p-toluenesulfonic acid (anhydrous) and stirred for 1.5 h. After neutralization with sodium bicarbonate solution, the reaction mixture was extracted with brine, dried with sodium sulfate and the solvent was distilled 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 waxy 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 of Mycobacterium spec. NRRL-B-3805 and 14 ml of a suspension of 3 / 3- (1-ethoxyethoxy) -5-cholestene is added. [This corresponds to 0.5 g of 3 / 3- (1-ethoxyethoxy) -5-cholestene). 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 9e 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-ol-17-one, m.p. 138/148-149 ° C.

Example

(a) 38.7 g of cholesterol are dissolved in 500 ml of methylene chloride, 25 ml of ethylene oxide and 0.5 ml of boron trifluoride etherate are added at a temperature of 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 grams) is chromatographed on silica gel in hexane-ethyl acetate mixtures.

10 g of 3 / 3- (2-hydroxyethoxy) -5-cholestene are obtained with a melting point of 91-95 ° C,

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

c) Under the conditions of Example 2a) and c), 85 ml of a culture of Mycobacterium spec. NRRL-3805 and treated with 14 ml of a 3 / 3- (2-hydroxyethoxy) -5-cholestene suspension (this corresponds to 0.5 g of 3 / 3- (2-hydroxyethoxy) -5-cholestene). After an additional 120 hours of incubation at 30%: the shaker was treated as described in Example 1d).

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

Example 9

a) 2.0 g of 5α-cholestan-3R-ol are reacted with formaldehyde dimethyl acetal as described in Example 1c) to give

21.5 g of 3/3-methoxymethoxy-5α-cholestane. The compound recrystallized from acetone has a melting point of Θ5 to 68 ^and C.

b) 10 g of 3 H -methoxymethoxy-5α-cholestane was emulsified under the conditions described in Example 2b).

cj Under conditions described in Example 2a j and cj, 85 ml of Mycobacterium spec. NRRL-3805 and treated with 14 ml of a 3/3-methoxymethoxy-5'-cholestane suspension (this corresponds to 0.5 g of 3/3-methoxymethoxy-5'-cholestane).

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

2.05 g of Si, 4-methoxymethoxy-5α-androstane-17-one are obtained, m.p. Celsius.

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

Example 10 g 17./3 -benzyloxy-3'-methoxymethoxy-5-androstene are hydrogenated in tetrahydrofuran / methanol (7: 3) in the presence of 0.8 g palladium catalyst (5% on calcium carbonate) at 50 ° C. C and a pressure of 1.5. 10 ⁶ Pa until the consumption of hydrogen was terminated. the product precipitated upon cooling was transferred with methylene chloride to the solution, was filtered from the catalyst washed with methylene chloride and the solvents removed in vacuo. to give 2 g 17 / 3- benzyloxy-3/3-methoxymethoxy-5α-androstane, m.p. 118-120 ° C.

Example 11

1.1 g of 3/3-imethoxymethoxy-5-androstan-17-one is hydrogenated as in Example 10. The product obtained is not completely uniform and contains, in addition to 3/3-methoxymethoxy-5a-androstan-17-one, 0 65 g of crystallization from methanol, still aliquots of 3, [3-methoxymethoxy-5? -Androstain-17?] 3-ol.

Further hydrogenation or reduction with sodium borohydride converts this product into the main product.

Claims (4)

Subject A process for the preparation of androstanone derivatives of the general formula I wherein: X is carbonyl or hydroxymethylene, n is 1 or 2, R 1 represents a hydrogen atom or a C 1 -C 1 -alkyl group and R2 denotes a C1-C6-alkyl group optionally interrupted by an oxygen atom or, in the case n denotes 2, also denotes a hydrogen atom, characterized in that the sterol derivative of the general formula II OF THE INVENTION with 8 to 10 carbon atoms, is fermented with a culture of microorganisms capable of degrading the sterol side chain, and the 5-androsten-17-one derivative of formula III thus produced, wherein: R 1, R 2 and n are as defined in 1, are hydrogenated. 2. The method of claim 1, wherein the sterol derivative of formula (IIa) wherein the bond. ,. . means a single or double bond, R 1 and R 2 are as defined in point 1 and R 4 is hydrogen, methyl or ethyl, n, R 1 and R 2 are as defined above, and R3 represents the hydrocarbon residue stero200518 is fermented with a culture of microorganisms capable of degrading the sterol side chain, followed by a second reaction step. 3. The process of claim 1 wherein the sterol derivative of formula (IIb) wherein the bond. . ,. and R2 are as defined in 1 a R 4 represents a hydrogen atom, a methyl or ethyl group, and is fermented with a culture of side-degrading migroorganisms, followed by a second reaction step. Method according to one of Claims 1 to 3, characterized in that cultures of microorganisms capable of degrading the side chain of sterols of the species Arthrohacter, Brevibacterium, Microbacterium, Protaminobacter, Bacillus, Nocardia, Streptomyces or in particular Mycobacterium are used for fermentation.