JPH0696592B2 - Method for producing 17β-hydroxy-17α- (3-hydroxypropyl) androsta-4,6-dien-3-one - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] And a method for producing 17β-hydroxy-17α- (3-hydroxypropyl) androsta-4,6-dien-3-one represented by

17β-hydroxy-17α produced by the method of the present invention
-(3-Hydroxypropyl) androsta-4,6-dien-3-one is a formula known as an anti-aldosterone diuretic 17β-hydroxy-7α-mercapto-3-
Oxo-17α-pregna-4-ene-21-carboxylic acid =
It is useful as a synthetic intermediate for producing γ-lactone = 7α-acetate (hereinafter referred to as spironolactone).

[Conventional technology]

Conventionally, 17β-hydroxy-17α- (3-hydroxypropyl) androsta-4,6-dien-3-one has been prepared from androsta-4-ene-3,17-dione in the presence of an alkali metal salt of a tertiary alcohol. 17β-hydroxy-17α- (3-hydroxy-1-propynyl) androsta-4-ene-3 obtained by reacting with propargyl alcohol below
17-hydroxy-17α- (3-hydroxypropyl) androsta-4-en-3-one by hydrogenating the -one in the homogeneous phase in the presence of a metal complex catalyst such as chlorotris (triphenylphosphine) rhodium. age,
It is then known to be prepared by reacting the 17β-hydroxy-17α- (3-hydroxypropyl) androsta-4-en-3-one with tetrachloro-p-benzoquinone in tert-butyl alcohol. (See Japanese Laid-Open Patent Publication No. 52-111552).

In addition, the aforementioned 17β-hydroxy-17α- (3-hydroxypropyl) androsta-4-, which is a precursor of 17β-hydroxy-17α- (3-hydroxypropyl) androsta-4,6-dien-3-one. The following method is known as a method for producing en-3-one.

(1) By reacting androsta-1,4-diene-3,17-dione with purpargyl alcohol in the presence of an alkali metal salt of a tertiary alcohol, 17β-hydroxy-
17α- (3-Hydroxy-1-propynyl) androsta-1,4-dien-3-one is obtained, then the 17β-hydroxy-17α- (3-hydroxy-1-propynyl) androsta-1,4- A method comprising hydrogenating dien-3-one in a homogeneous phase in the presence of a metal complex catalyst such as chlorotris (triphenylphosphine) rhodium (see JP-A-50-30861).

(2) Androsta-4-en-3,17-dione is reacted with ethyl orthoformate in the presence of p-toluenesulfonic acid to give 3-ethoxyandrosta-3,5-dien-17-one obtained. 17β represented by the general formula (IV) by reacting with 1-ethoxyethyl = 3-lithyliopropyl = ether or 2- (3-thyriopropoxy) tetrahydropyran
-Hydroxysteroid derivative, and then hydrolyzing the 17β-hydroxysteroid derivative under acidic conditions (JP-A-51-143654 and JP-A-51-143654).
52-42867).

(In the formula, A represents a 1-ethoxyethyl group or a 2-tetrahydropyranyl group) (3) 3β-hydroxyandrost-5-ene-17-
The 17α- [3- (2-tetrahydropyranyloxy) -1-propynyl] androsta-5-ene obtained by reacting the one with a Grignard reagent prepared from 2- (2-propynyloxy) tetrahydropyran. −
The 3β, 17β-diol was hydrogenated in the presence of a palladium-carbon catalyst to give 17α- [3- (2-tetrahydropyranyloxy) propyl] androsta-5-ene-3β, 17.
.beta.-diol, and the .alpha .- [3- (2-tetrahydropyranyloxy) propyl] androst-5-ene-
By subjecting 3β, 17β-diol to Oppenauer oxidation reaction, 17β-hydroxy-17α-
[3- (2-tetrahydropyranyloxy) propyl]
Androsta-4-en-3-one is obtained, and then the 17β
-Hydroxy-17α- [3- (2-tetrahydropyranyloxy) propyl] androsta-4-en-3-one in ethanol in the presence of p-toluenesulfonic acid under reflux.・ Journal of Medicinal Che
mistry), Vol. 6, pp. 617-618 (1963)].

(In the formula, THP represents a 2-tetrahydropyranyl group) [Problems to be Solved by the Invention] According to the above conventional method, androsta-4-ene-3,17
-Dione, androsta-1,4-dien-3,17-dione or 3β-hydroxyandrosta-5-en-17-one was used as a starting material, and 17β-hydroxy-17α- (3-
17β-Hydroxy-17α- (3-hydroxypropyl) androsta-4,6-dien-3-one has been prepared via hydroxypropyl) androsta-4-en-3-one. The 17β-hydroxy-17α- (3-hydroxypropyl) androsta-4,6-diene-3-
It is preferable that the raw material that can be used in the production of ON is selected from many compounds, because the production process can be appropriately changed depending on the raw material circumstances.

Therefore, an object of the present invention is 17β-hydroxy-17α-
An object of the present invention is to provide a new method capable of easily producing (3-hydroxypropyl) androsta-4,6-dien-3-one.

[Means for Solving Problems] According to the present invention, the above-mentioned object is represented by the general formula (In the formula, R ¹ represents an alkylene group) 3,3-alkylenedioxyandrosta-4,6
-Diene-17-one can be represented by the general formula (In the formula, R ² represents a hydrogen atom or a lower alkyl group, R ³ and R ⁴ each represent a lower alkyl group or, together, represents an alkylene group). Characterized by hydrolyzing a reaction product in the presence of an acid
17β-hydroxy-17α- (3-hydroxypropyl)
3,3-Alkylenedioxyandrosta-4,6-, which has been achieved by providing a process for the preparation of androsta-4,6-dien-3-one and which has the general formula (I) above.
As the dien-17-one, the formula 7α-hydroxyandrost-4-ene-
A compound produced by reacting 3,17-dione with an alkylene glycol represented by the general formula HO-R ¹ -OH (III) (wherein R ¹ is as defined above) in the presence of an acid is used. Used 17β-hydroxy-17α-
This is accomplished by providing a process for making (3-hydroxypropyl) androsta-4,6-dien-3-one.

Examples of the alkylene group represented by R ¹ in the above general formula include an ethylene group, a propylene group [-CH ₂ CH (CH ₃ )-], 1,
Examples thereof include a 2-dimethylethylene group, an ethylethylene group, a trimethylene group, a 1-methyltrimethylene group, a 2-methyltrimethylene group and a tetramethylene group. Examples of the lower alkyl group represented by R ² in the general formula (II) include a methyl group, an ethyl group, a propyl group and an isopropyl group. Examples of the lower alkyl group represented by R ³ and R ⁴ in the general formula (II) include, for example, a methyl group, an ethyl group, a propyl group, an isopropyl group,
Examples of the alkylene group represented by R ³ and R ⁴ together include trimethylene group, 1-methyltrimethylene group, 2-methyltrimethylene group, tetramethylene group, 1
Examples include -methyltetramethylene group and 2-methyltetramethylene group.

Examples of the organic lithium compound represented by the general formula (II) include
For example, 3-lithiopropyl = 1-methoxyethyl = ether, 1-ethoxyethyl = 3-lithiopropyl = ether, 3-lithiopropyl = 1-propoxyethyl = ether, 1-isopropoxyethyl = 3-lithiopropyl = ether, 3-lithiopropyl = 1-methoxypropyl = ether, 1-ethoxypropyl = 3-lithiopropyl = ether, 3-lithiopropyl = 1-propoxypropyl = ether, 3-lithiopropyl = 1-methoxy-1-methylethyl = Ether, 1-ethoxy-1
-Methylethyl = 3-lithiopropyl = ether, 3-
Lithiopropyl = 1-methyl-1-propoxyethyl =
Ether, 2- (3-lithiopropoxy) tetrahydrofuran, 2- (3-lithiopropoxy) tetrahydropyran, 2- (3-lithiopropoxy) -4-methyltetrahydropyran and the like are used. Organolithium compounds have, for example, the corresponding general formula (Wherein R ² , R ³ and R ⁴ are as defined above, and X represents a halogen atom) according to a commonly known method for converting an alkyl halide to an alkyl lithium. It can be easily produced by reacting with metallic lithium [for example, The Journal of Organic Chemist
ry), vol. 37, p. 1947 (1972); Journal of the American Chemical Society (Journal)
of the American Chemical Society), Vol. 99, Vol. 2751
Page (1977); JP-A-51-143654; JP-A-52-42
867, etc.].

In the reaction of the 3,3-alkylenedioxyandrosta-4,6-dien-17-one represented by the general formula (I) with the organolithium compound represented by the general formula (II), the amount of the organolithium compound used Is about 10 to 1 mol of 3,3-alkylenedioxyandrosta-4,6-dien-17-one.
It is preferably in an amount of -20 mol. The reaction is preferably carried out in an organic solvent, and examples of the organic solvent include chain ethers or cyclic ethers such as diethyl ether, dipropyl ether, dibutyl ether, 1,2-dimethoxyethane, diethylene glycol dimethyl ether, tetrahydrofuran and tetrahydropyran. Solvents which do not adversely influence the reaction such as ether are used alone or as a mixture of two or more kinds. The reaction is preferably carried out at a temperature in the range of about -5 to 30 ° C, and good results are obtained if the reaction is carried out in an atmosphere of a gas such as argon, helium or nitrogen which does not adversely influence the reaction.

The thus obtained 3,3− represented by the general formula (I)
Alkylenedioxyandrosta-4,6-diene-17-
The reaction product of the on and the organolithium compound of general formula (II) is hydrolyzed in the presence of an acid without isolation from the reaction mixture. In this case, it is represented by the general formula (I).
3,3-Alkylenedioxyandrosta-4,6-diene-
A reaction mixture containing a reaction product of a 17-one and an organolithium compound represented by the general formula (II), a concentrate thereof, an organic layer obtained by contacting the reaction mixture with saline, water, etc. is acidified. It may be subjected to a hydrolysis reaction in the presence of.
Although only water may be used as a solvent for the hydrolysis reaction, it is preferable to use an organic solvent which is at least partially soluble in water and which does not adversely affect the hydrolysis reaction. As the organic solvent, for example, cyclic ethers such as tetrahydrofuran and dioxane; lower alcohols such as methanol, ethanol and isopropyl alcohol are used alone or as a mixture of two or more kinds. The amount of water used is preferably in the range of about 5 to 20 times the volume of 3,3-alkylenedioxyandrosta-4,6-dien-17-one represented by the general formula (I). As the acid to be present in the hydrolysis reaction system, inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid and perchloric acid; organic acids such as p-toluenesulfonic acid and trifluoroacetic acid are used.
The amount of acid used is not particularly limited, but an amount such that the pH of the reaction solution is within the range of about 0.5 to 3 is preferable.
Particularly, an amount such that the pH of the reaction solution becomes about 1 is preferable.
The reaction is usually carried out at a temperature in the range of about 0 to 60 ° C, particularly preferably at a temperature in the range of about 15 to 25 ° C.

Separation and purification of 17β-hydroxy-17α- (3-hydroxypropyl) androsta-4,6-dien-3-one from the thus obtained hydrolysis mixture can be carried out, for example, by the following method. After the reaction mixture is neutralized by mixing with a basic aqueous solution such as sodium bicarbonate water,
Extract with a solvent such as ethyl acetate, chloroform, methylene chloride, evaporate the solvent from the extract, and then subject the residue to the same purification operation as that used for the purification of the product obtained by a general organic synthesis reaction. By doing so, 17β-hydroxy-17α- (3-hydroxypropyl) androsta-4,6-dien-3-one can be obtained.

The 3,3-alkylenedioxyandrosta-4,6-dien-17-one represented by the general formula (I) can be produced, for example, by the above-mentioned method. The amount of the alkylene glycol represented by the general formula (III) used is preferably about 15 to 30 mol per 1 mol of 7α-hydroxyandrosta-4-ene-3,17-dione. As the acid to be present in the reaction system, it is preferable to use an organic acid such as fumaric acid or oxalic acid. The amount of the acid used is preferably about 0.1 to 0.5 mol based on 1 mol of 7α-hydroxyandrost-4-ene-3,17-dione. The reaction is preferably carried out in an organic solvent, and as the organic solvent, for example, aromatic hydrocarbons such as benzene, toluene and xylene; halogenated hydrocarbons such as 1,2-dichloroethane and carbon tetrachloride are adversely affected. It is possible to use an organic solvent which does not exceed the above range. The reaction temperature is preferably within the range of about 50 to 100 ° C. From the viewpoint of accelerating this reaction, it is preferable to carry out the reaction while removing generated water out of the reaction system. As a method of removing the produced water to the outside of the reaction system, a method usually used for distilling the water produced in an organic reaction to the outside of the reaction system can be adopted as it is, and in particular, water is used as an azeotropic mixture to react. It is preferable to employ a method of distilling out of the system. When distilling out water as an azeotrope, an organic solvent which does not adversely influence the reaction and can form an azeotrope with water is used. Examples of such an organic solvent include aromatic hydrocarbons such as benzene, toluene and xylene; halogenated hydrocarbons such as 1,2-dichloroethane and carbon tetrachloride.However, aromatic hydrocarbons are used. preferable.

The thus obtained 3,3− represented by the general formula (I)
Alkylenedioxyandrosta-4,6-diene-17-
Separation and purification from the ON reaction mixture can be carried out, for example, by the following method. The reaction mixture is neutralized by mixing with a basic aqueous solution such as sodium bicarbonate solution, and then extracted with an organic solvent such as benzene, toluene, xylene, 1,2-dichloroethane, carbon tetrachloride, etc., and the solvent is distilled from the extract. The residue was removed, and the residue was subjected to purification operations such as recrystallization and chromatography to give the 3,3-alkylenedioxyandrosta-4,6-diene-17- of the general formula (I). You can get on.

7α-Hydroxyandrosta-4 used as raw material
-Ene-3,17-dione is, for example, 3α, 7α-dihydroxy-5β-cholanic acid (hereinafter, referred to as CDCA) and / or its salt as a substrate, 7α-hydroxyandrosta-.
4 Pseudomonas having the ability to produce ene-3,17-dione (Pseudomonas) genus putida (putida) species or Shiyudomonasu (Pseudomonas) Sutsuchieri (stutzeri)
Bacteria belonging to the species are cultured in a medium containing CDCA and / or a salt thereof to obtain 7α-hydroxyandrosta-4-en-3,
By producing 17-dione and collecting it, it can be produced with high selectivity and in good yield.

Examples of the bacterium belonging to the genus Pseudomonas putida having the ability to produce 7α-hydroxyandrosta-4-ene-3,17-dione using CDCA and / or a salt thereof as a substrate include, for example, C. petitidae D4014-A1615 ( Pseu
domonas Putida D4014-A1615) strain
No. 2), and a bacterium belonging to the genus Stutieri of the genus Studieri of the genus Cichudomonas, which has the ability to produce 7α-hydroxyandrosta-4-ene-3,17-dione using CDCA and / or a salt thereof as a substrate, for example, There is a strain of Pseudomonas stutzeri M-1492 ( Pseudomonas stutzeri M-1492) (Microtechnology Research Institute, No. 8771). C. sutidomonas putida D4014-A1615 strain is a mutant strain obtained by subjecting Pseudomonas putida D4014 ( Pseudomonas putida D4014) strain (Microtechnology Research Institute No. 205) collected from soil to mutation treatment. The mycological properties of the strain are disclosed in JP-A-58
As described in JP-A-149698 or JP-A-59-39298. In addition, C. suttieri M-1492 strain is a strain of C.
Studio C-37-2 ( Pseudomons stutzeri C-37-
2) Mutant strains obtained by subjecting the bacterial strains (Microtechnology Research Institute No. 8770) to mutation treatment, and the mycological properties of these strains are shown in the following table.

Based on the mycological properties shown in the above table, the strains of C. scuttieri C-37-2 and C. scuttieri M-1492 were identified. C. 37-2 strain of C. suttieri is a bacillus, has polar flagella, microscopic findings such as negative Gram stain, and positive for both oxidase reaction and catalase reaction. , Aerobic, O
-Because of the physiological properties such as the result of the F test being oxidative, it is possible to use the Bergey's Manual of Determinative Bacteriology (Bergey ').
s Manual of Determinative Bacteriology) 8th edition, it was identified as a bacterium belonging to the genus C. pseudomonas. Furthermore, the strain of C. 37-2 was identified as a bacterium belonging to the genus Studieri of the genus C. steinae because it does not produce soluble pigments, undergoes denitrification, does not degrade gelatin, and degrades starch. did. In addition, it is generally considered that the mutant strain belongs to the same species as its parent strain, and it was determined that the C. scuttieri M-1492 strain is a bacterium belonging to the genus St.

When CDCA and / or a salt thereof is used as a substrate, a bacterium belonging to the genus Cichudomonas putida or the genus Studieri of the genus Studieri of the genus Streptomyces having the ability to produce 7α-hydroxyandrosta-4-ene-3,17-dione The 7α-hydroxyandrosta-4-ene-3,17-dione is produced by culturing in a medium containing the same. Specific examples of the CDCA salt include salts of CDCA with alkali metals such as sodium and potassium and salts of alkaline earth metals such as calcium and magnesium. The concentration of CDCA and / or its salt in the medium may be usually in the range of about 1 to 100 g / l, but 7α-hydroxyandrosta-4-ene-3,17-
The range of about 10 to 50 g / l is preferable from the viewpoint of obtaining dione easily and in high yield under mild conditions. The culture method is, in principle, the same as the method employed in the aerobic culture of general microorganisms, but usually the shaking culture method using a liquid medium or the aeration stirring culture method is used. As the medium, the above CDCA and / or its salt is used as a substrate for 7α-hydroxyandrosta-
Any bacterium containing a nutrient source that can be assimilated and utilized by a bacterium belonging to the genus Pseudomonas putida or the genus Studieri of the genus Studieri, which has the ability to produce 4-en-3,17-dione, can be used. The carbon source may be CDCA and / or a salt thereof as a single carbon source, or pentoses such as arabinose may be added to CDCA and / or a salt thereof, hexoses such as glucose, mannose, fructose and galactose; sucrose, maltose and the like. Disaccharides; starch degradation products; polyhydric alcohols such as sugar alcohols and glycerin; or polypeptone, peptone, meat extract, malt extract, corn steep liquor, yeast extract, various amino acids, organic acids, etc. may be used in combination. . As the nitrogen source, for example, an inorganic nitrogen source such as ammonium nitrate, sodium nitrate or potassium nitrate, or an organic nitrogen source such as polypeptone, peptone or meat extract is used. Also,
In addition to this, inorganic salts such as dipotassium hydrogen phosphate, potassium dihydrogen phosphate and magnesium sulfate are added. Although the culture conditions are not characteristic, shaking culture or aeration stirring culture is usually performed at 25 to 35 ° C. for 10 hours to 7 days.

Most of the 7α-hydroxyandrosta-4-en-3,17-dione thus accumulated in the culture medium is obtained as a precipitate. To separate and collect the accumulated 7α-hydroxyandrosta-4-ene-3,17-dione, 7α-hydroxyandrosta-4-ene-
A method of extraction is carried out using an organic solvent which dissolves 3,17-dione and phase-separates from water, such as ethyl acetate, chloroform, a mixed solution of chloroform and methanol. The extraction with an organic solvent may be performed on the culture containing the precipitate and the cells, or may be performed on the insoluble component separated from the culture by filtration or centrifugation. When the former extraction method is adopted, it is preferable that the culture solution is made alkaline by adding an alkaline aqueous solution such as an aqueous solution of sodium hydroxide thereto, and then the extraction operation with the above-mentioned organic solvent is carried out. When using the latter extraction method,
To the culture filtrate or supernatant that is separated and removed by filtration or centrifugation as necessary, preferably an alkaline aqueous solution such as an aqueous solution of sodium hydroxide is added to make the culture filtrate or supernatant alkaline, and then the above organic By performing the extraction operation with a solvent, 7α-hydroxyandrosta-4-ene-3,17-dione dissolved in the culture medium can be recovered. The extracts thus obtained were collected, and the solvent was distilled off therefrom to give 7α-
It is possible to recover almost all of hydroxyandrosta-4-en-3,17-dione. Purification of the recovered 7α-hydroxyandrost-4-ene-3,17-dione is carried out by recrystallization from, for example, methanol or aqueous methanol solution.

17β-hydroxy-17 produced by the method of the present invention
α- (3-hydroxypropyl) androsta-4,6-
Dien-3-one is obtained by reacting it with thioacetic acid according to the method described in, for example, JP-A-52-111552, to obtain 17β-hydroxy-17α- (3-hydroxypropyl) -7α-mercapto. Androsta-4-en-3-
It is derivatized to spironolactone by oxidizing on = 7α-acetate with the Jones reagent.

Alternatively, 17β-hydroxy-17α- (3-hydroxypropyl) androsta-4,6-dien-3-one is oxidized with 17D-Yones reagent to give 17β-hydroxy-3-oxo-17α-pregna. −4,6-Diene-21−
Carboxylic acid = γ-lactone (hereinafter referred to as “canrenone”), and the canrenone is also reacted with thioacetic acid to induce spironolactone.

[Examples] Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited to these Examples.

Reference Example 1 Acquisition of C. suttieri M-1492 Strain Medium 1 (composition: 0.5% peptone, 0.5% meat extract, 0.1% salt).
5% and agar 1.5%; pH: 7.8) one platinum loop of C. 37-2 strain of S. suttieri grown in slant was prepared in a test tube (internal diameter: 21 mm; length: 200 mm) 2 (composition: CDCA 3%, ammonium nitrate 0.2%, potassium dihydrogen phosphate 0.1%, dipotassium hydrogen phosphate 0.6%, magnesium sulfate heptahydrate 0.02%, yeast extract 0.02%, glucose 0.05%, peptone 0.05% and hydroxylation sodium
0.3%; pH: 7.5) was inoculated into 10 ml and shake-cultured at 30 ° C. for 24 hours. 0.4 ml of this culture solution was previously tested in a test tube (internal diameter: 21 mm;
Prepared medium 3 (composition: CDCA 0.5%, ammonium nitrate 0.2%, potassium dihydrogen phosphate 0.1%, dipotassium hydrogen phosphate 0.6%, magnesium sulfate heptahydrate 0.0) prepared in length: 200 mm)
2%, yeast extract 0.02% and glucose 0.1%; pH: 8.5)
Was added to 10 ml of the above and cultured at 30 ° C. for 6 hours with shaking. Then
The bacterial cells in the logarithmic growth phase were aseptically collected by filtration with a membrane filter having a pore size of 0.45 μm, and then 0.1 M phosphate buffer (p
After washing with 20 ml of H: 7.0), the cells were suspended in 25 ml of the same buffer.
4 ml of this bacterial suspension was placed in a test tube (internal diameter: 21 mm; length: 200 mm), and N-methyl-N'-nitro-N-nitrosoguanidine was added to this so that the final concentration would be 50 μg / ml. ,Ten
After a minute, 0.1 ml of the medium 4 [composition: 7α-hydroxyandrost-4-en-3,17-dione 0.1%, ammonium nitrate 0.2%, potassium dihydrogen phosphate 0.1%, dihydrogen phosphate 0.6%, magnesium sulfate. Heptahydrate 0.02%, yeast extract 0.05% and polyoxyethylene (20) sorbitan monooleate (Tween 80) 0.05%; pH: 7.5], and then penicillin G to a concentration of 100 U / ml After that, shaking culture was performed at 30 ° C. for 18 hours. The obtained culture solution was diluted and applied to the plate of medium 1 so that about 100 to 500 colonies appeared, and then the plate was cultured overnight at 30 ° C. After the emerged colonies were isolated on slants in medium 1, each isolate was added to 10 of medium 2.
Culture was carried out in ml for 2 days with shaking. The products in each of the obtained culture solutions were assayed by thin layer chromatography, and 7α
One strain that selectively accumulated -hydroxyandrosta-4-en-3,17-dione was found, and this strain was designated as C. suttieri M-1492 strain.

Reference Example 2 Production of 7α-Hydroxyandrosta-4-ene-3,17-dione A strain of Cydudomonas putida D4014-A1615 was cultured by the following method. A mixture of 10 g of CDCA, 2.0 g of ammonium nitrate, 1.0 g of potassium dihydrogen phosphate, 6.0 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate heptahydrate, 0.2 g of yeast extract, 0.5 g of glucose, 0.5 g of peptone and 1.0 g of sodium hydroxide. Tap water was added to the mixture to adjust the volume to 1 (pH: 8.5), which was used as a medium. Place this medium in a 2 liter fermentor and mix at 120 ° C for 1 hour.
Steam sterilization was performed for 5 minutes. 50 ml of the inoculum that had been grown for 20 hours on a test tube shaker in the same medium as the above medium was added to the above medium, the stirring speed was 300 rpm, and the aeration rate was 0.5 vvm and 30.
The culture was performed under aeration and agitation for 48 hours under the condition of ° C. After culturing,
As a result of thoroughly mixing the culture solution and analyzing a part thereof by thin layer chromatography, it was confirmed that CDCA did not remain. After adjusting the pH to 10 by adding 1N sodium hydroxide aqueous solution to the culture solution, the culture solution was mixed with 5 l of chloroform / ethanol mixed solvent (volume ratio: 4/1).
It was extracted with. Insoluble substances such as denatured cells were filtered off from the extract, and the solvent was distilled off from the filtrate under reduced pressure to obtain 7.0 g of a dried solid product. The dry solid was analyzed by high performance liquid chromatography, and it was found that the dry solid was 7α-hydroxyandrosta-4-en-3,17-dione having a purity of 86%. This 7α-hydroxy androstar
The 4-ene-3,17-dione was recrystallized using an 80% by volume aqueous methanol solution to obtain 4.0 g of a purified product.

The analysis results of the purified product of 7α-hydroxyandrost-4-ene-3,17-dione are shown below.

Melting point: 245 to 247 ° C. Mass spectrum m / e: 302 [M] ⁺ : 284 [MH ₂ O] ^{+ and} m / e = 124 confirmed the presence of 3-keto-4-ene.

_{NMR (CDCl 3, 90MHz) δ} : 0.92 (s, 3H, 18-CH 3), 1.22 (s, 3H, 19-CH 3), 4.09 (m,
1H, 7β-H), 5.79 (s, 1H, 4-H) ppm Reference Example 3 Production of 7α-Hydroxyandrosta-4-ene-3,17-dione In Reference Example 2, C. deputy D4014-A1615
Instead of the strain, use C. destructor M-1492 strain and change the amount of CDCA in the medium from 10g to 30g. Also, change the amount of sodium hydroxide in the medium from 1.0g.
By doing the same operation except changing to 3.0g,
21.9 g of a dry solid was obtained. As a result of analyzing a part of the obtained culture solution by thin layer chromatography, it was confirmed that CDCA did not remain in the culture solution. As a result of analyzing the dried solid by high performance liquid chromatography, it was found that the dried solid was 7α-hydroxyandrosta-4- having a purity of 85%.
It was found to be en-3,17-dione. This 7α-
The hydroxyandrosta-4-ene-3,17-dione was recrystallized using methanol to obtain 10.5 g of a purified product.

Example (A) Preparation of 3,3-ethylenedioxyandrosta-4,6-dien-17-one 7α-hydroxyandrosta-4-en-3,17-dione 5.0 g, ethylene glycol 5 ml, fumaric acid 0.5 g And a mixture of 400 ml of benzene with Dean Stark (Dean-Sta
rk) was heated to reflux with stirring in a reactor equipped with a water separation tube. 1 day after the start of heating and refluxing, ethylene glycol 15 ml
Was added, and 1 day after that, 5 ml of ethylene glycol and 0.1 g of fumaric acid were added, and the mixture was further heated and refluxed for 1 day. During the heating under reflux, the produced water was removed from the reaction system using a Dean-Stark water separation tube. The obtained reaction mixture was poured into ice-cooled saturated aqueous sodium hydrogen carbonate, the benzene layer was separated, and the aqueous layer was extracted with benzene. The benzene layer separated earlier and the benzene extract were combined, washed with saturated brine, and dried over anhydrous potassium carbonate. The dried liquid was concentrated under reduced pressure to obtain 6.21 g of crude crystals. 16m of this crude crystal
By recrystallizing using a mixed solvent of l methanol and a few drops of pyridine, 3.9 g of 3,3-ethylenedioxyandrosta-4,6-dien-17-one was obtained (yield: 72
%). Obtained 3,3-ethylenedioxyandrosta
The analysis results of 4,6-dien-17-one are shown below.

Melting point: 153-159 ° C Specific rotation: ▲ [α] ²² _D ▼ + 149 ° (c = 1.10, CHCl ₃ ) (b) 17β-hydroxy-17α- (3-hydroxypropyl) androsta-4,6-diene Preparation of -3-one Lithium wire (1.38 g) was cut into small pieces and placed in a flask. After washing twice with n-pentane under an argon atmosphere, 40 ml of anhydrous diethyl ether was added. Under stirring, 3 g of 3-bromopropyl = 1-ethoxyethyl = ether (3 g) was added to this mixture at room temperature, and when the temperature of the solution exceeded 30 ° C., it was cooled using an ice-salt bath. While maintaining the temperature of the solution at about 5 ° C., 16 g of 3-bromopropyl = 1-ethoxyethyl ether was added to 20 ml of anhydrous diethyl ether.
The solution obtained by dissolving in (1) was added dropwise over about 40 minutes, and after completion of the addition, stirring was continued at 0 ° C. for another 50 minutes. A solution obtained by dissolving 1.7 g of 3,3-ethylenedioxyandrosta-4,6-dien-17-one in 10 ml of anhydrous tetrahydrofuran was added dropwise to the obtained mixture at 0-5 ° C., and then 0- 5 ° C
The stirring was continued for 1 hour at room temperature and for a further 4 hours at room temperature. The solution in the obtained reaction mixture was poured into ice-cooled saturated saline solution so that solids were not mixed. The organic layer was separated from this, and the aqueous layer was extracted with 50 ml of tetrahydrofuran. The organic layer obtained above and the extract were combined and concentrated under reduced pressure to a volume of about 30 ml.

20 ml of 2N hydrochloric acid was added to the obtained concentrate, and the mixture was allowed to stand at room temperature for 2
Stir for time. The obtained reaction mixture was poured into saturated aqueous sodium hydrogen carbonate and extracted twice with ethyl acetate. Combine the extracts,
The extract was washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine, and dried over anhydrous magnesium sulfate. Concentrate the dried solution under reduced pressure
4.1 g of concentrate was obtained. This is 17 according to the analysis results below.
It was found to contain β-hydroxy-17α- (3-hydroxypropyl) androsta-4,6-dien-3-one.

IR (film) ^ν max: 3350 (s, hydroxyl group), 1665 (vs, conjugated carbonyl group), 1620
(M, double bond), 1585 (w, double bond) cm ^-1 NMR (CDCl ₃ , 60 MHz) δ: 5.6 (s, 1H, 4-H), 6.04 (s, 2H, 6-H and 7 -H) pp
m Reference example 4 (A) Production of canrenone 17β-hydroxy-17α- (3-hydroxypropyl) androsta-4,6-diene-3-obtained in Example
4.1 g of the crude product containing corn was dissolved in a mixed solvent of 50 ml of acetone and 60 ml of tert-butyl alcohol under heating. 10 ml of Jones reagent was added dropwise to this solution under ice cooling. After completion of dropping, continue stirring for 20 minutes under ice cooling,
Further, 5 ml of Djones reagent was added, and the mixture was stirred at room temperature for 20 minutes. Isopropyl alcohol was added to the obtained reaction mixture.
After adding 10 ml, the mixture was concentrated under reduced pressure. The obtained concentrate was diluted with water, extracted with ethyl acetate, the extract was washed with saturated aqueous sodium hydrogen carbonate, and dried over anhydrous magnesium sulfate. The dried liquid was concentrated under reduced pressure to obtain 2.0 g of a concentrate.

The obtained concentrate was dissolved in 10 ml of acetone, 2 ml of the di-Jones reagent was added, and the mixture was stirred at room temperature for 20 minutes. 1 ml of isopropyl alcohol was added to the obtained reaction mixture, which was then concentrated under reduced pressure, and the concentrate was subjected to the same treatment operation as above to obtain 2.0 g of an oily substance.

By purifying the obtained oily substance by column chromatography [filler: silica gel (20 g); eluent: n-hexane / ethyl acetate mixed solvent (volume ratio: 2/1 to 1/1)] Thus, 0.98 g of an oily substance containing canrenone was obtained. The oily substance was recrystallized using a mixed solvent of 1.5 ml of n-hexane and 1 ml of acetone to obtain 0.56 g of canrenone. The yield of canrenone was 32% based on the 3,3-ethylenedioxyandrosta-4,6-dien-17-one used in Example (b). The analysis results of the obtained canrenone are shown below.

Melting point: 163-168 ° C Specific rotation: ▲ [α] ²² _D ▼ + 22 ° (c = 1.05, CHCl ₃ ) (b) Production of spironolactone 0.400 g of canrenone was dissolved in 1.6 ml of methanol, and thioacetic acid 0.24 was added to this solution. ml was added under heating under reflux, and heating under reflux was continued for another 30 minutes. Water at 60 ° C was added to the obtained reaction mixture.
After adding 6 ml to the room temperature, the mixture was diluted with 20 ml of ethyl acetate. The obtained solution was washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine, and dried over anhydrous magnesium sulfate. The dried liquid was concentrated under reduced pressure to obtain 0.607 g of crude crystals. The crude crystals were recrystallized from methanol to obtain 0.408 g of spironolactone (yield: 83%). The analysis results of the obtained spironolactone are shown below.

Melting point: 130-135 ° C. Specific optical rotation: ▲ [α] ²² _D ▼ -36.6 ° (c = 1.13, CHCl ₃ ) [Effect of the invention] According to the present invention, as apparent from the above-mentioned examples, 7
From α-hydroxyandrosta-4-en-3,17-dione to 3,7-alkylenedioxyandrosta-4,6-dien-17-one represented by the general formula (I), 17β
-Hydroxy-17α- (3-hydroxypropyl) androsta-4,6-dien-3-one can be easily produced.

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Claims (2)

[Claims] 1. A general formula (In the formula, R ¹ represents an alkylene group) 3,3-alkylenedioxyandrosta-4,6
-Diene-17-one can be represented by the general formula (Wherein R ² represents a hydrogen atom or a lower alkyl group, R ³ and R ⁴ each represent a lower alkyl group or together represent an alkylene group) 17β-Hydroxy-17α-characterized in that the reaction product is hydrolyzed in the presence of an acid.
A method for producing (3-hydroxypropyl) androsta-4,6-dien-3-one. 2. A 3,3-alkylenedioxyandrosta-4,6-dien-17-one represented by the general formula (I) is 7α.
-Hydroxyandrosta-4-ene-3,17-dione and an alkylene glycol represented by the general formula HO-R ¹ -OH (III) (wherein R ¹ is as defined above) in the presence of an acid; The production method according to claim 1, which is produced by reacting.