JPS638397A - Production of 17beta-hydroxy-17alpha-(3-hydroxypropyl)-androsta-4,6-dien-3-one - Google Patents

Abstract

PURPOSE:To easily produce the titled compound useful as a synthetic intermediate for antialdosterone diuretic, by reacting a specific steroid compound with an organic lithium compound and hydrolyzing the reaction product in the presence of an acid. CONSTITUTION:1mol of a compound of formula I (R<1> is alkylene) is made to react with preferably 10-20mol of an organic lithium compound of formula II (R<2> is H or lower alkyl; R<3> and R<4> are lower alkyl or together form an alkylene) preferably in a inert solvent and inert gas atmosphere at -5-+30 deg.C. The reaction product is added with an acid and hydrolyzed preferably at 0.5-3pH and 15-25 deg.C to obtain the objective compound. The starting compound of formula I is produced by reacting 7alpha-hydroxyandrost-4-ene-3,17-dione of formula III with an alkylene glycol in the presence of an acid.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing 17β-hydroxy-17α-(3-hydroxypropyl)androsta-4,6-renin-3-one.

17β-hydroxy-1 produced by the method of the present invention
7α-(3-hydroxypropyl)androsta-4,
17β-hydroxy-7α-mercapto-3 with the formula known as 6-renin-3-onha antialdosterone diuretic
It is useful as a synthetic intermediate for producing -OΦ-17α-pregnar-4-ene-21-carboxylic acid γ-lactone-7α-acetate (hereinafter referred to as spironolactone).

[Conventional technology]

Conventionally, 17β-hydroxy-17α-(3-hydroxypropyl)androst-4,6-cyen-3-one was prepared by converting androst-4-ene-3,17-dione in the presence of an alkali metal salt of a tertiary alcohol. is reacted with propargyl alcohol to obtain 17β-hydroxy-17
α-(3-Hydroxy-1-propynylfandroster 4-en-3-one was prepared by hydrogenation in the material phase in the presence of a metal complex catalyst such as chlorotris(triphenylphosphine)rhodium). 17
α-(3-hydroxypropyl)androst-4-en-3-one and then the 17β-hydroxy-17
It is known that α-(3-hydroxypropyl)androst-4-en-3-one is produced by reacting it with tetrachloro-p-benzoquinone in tert-phthyl alcohol (JP-A-52 -11155
Publication No. 2 H In addition, the above-mentioned 17β-hydroyac-17α-(
3-hydroxypropyl)androst-4-ene-3
The following methods are known as methods for producing -on.

(1) Reacting androsta-1,4-diene-3,17-dione with propargyl alcohol in the presence of an alkali metal salt of a tertiary alcohol lc! , 917β-
Hydroxy-17α-(3-hydroxy-1-propynyl)androsta-1°4-dien-3-one is obtained, and then the 17β-hydroxy-17α-(3-hydroxy-1-propynyl)androsta-1, 4-diene-3
-one in the substance phase in the presence of a metal complex catalyst such as chlorotris(triphenylphosphine)rhodium (see JP-A-50-30861).

(2) p androst-4-ene-3,17-dione
-) 3-ethoxyandrosta-3,5- obtained by reacting with ethyl orthoformate in the presence of luenesulfonic acid
Dien-17-one to 1-ethoxyethyl = 3-I
General formula (IV) by reacting with J thiopropyl ether or 2-(3-lithiopropoxy)tetrahydrobilane
A 17β-hydroxysteroid derivative represented by
A method comprising then hydrolyzing the 17β-monohydroxysteroid derivative under acidic conditions (Japanese Patent Application Laid-open No. 51-1
43654 and JP-A-52-42867).

(The person in the formula represents a 1-ethoxyethyl group or a 2-tetrahydropyranyl group) (+) 3β-hydroxyandrost-5-ene-
Grignard prepared 17-one from 2-(2-propynyloxy)tetrahydropyran.
) reagent, and the resulting 17α-(3-(2-tetrahydropyranyloxy)-1-propynyl]androstar-5-ene-3β,17β-diol is hydrogenated in the presence of a batidium-carbon catalyst. 17α-(3-(2-
Tetrahydropyranyloxy) furovir cyandrostar 5-ene-3β.

17β-diol, the 17α-1-3-(2-tetrahydropyranyloxy)propyl]androstar 5
-ene-3β,17β-diol by Otzpenauer (0
chef-β-hydroxy-17α-[3-(2-tetrahydropyranyloxy)propylfandroster 4-ene-3 by subjecting it to an oxidation reaction
-one, and then the 17β-hydroxy-17a-(
:3-(2-tetrahydropyranyloxy)propylphandroster 4-en-3-one t-1)-) A process consisting of heating to reflux in ethanol in the presence of luenesulfonic acid [Journal on Medicinal]・Chemistry (Journal of Medicinal)
Chemistry, Vol. 6, pp. 617-618 (1963)] 0 (In the formula, THP represents a 2-tetrahydropyranyl group) [Problems to be solved by the invention] According to the above conventional method, , androstar-4-en-3,
17-dione, androsta-1,4-diene-3,1
Using 7-dione or 3β-hydroxyandrost-5-en-17-one as a raw material, 17β-hydroxy-17α-(3-hydroxypropyl)androster
17β-hydroxy-1 via 4-en-3-one
7α-(3-hydroxypropyl)androsta-4,
6-renin-3-one is produced. In producing the 17β-hydroxy-17α-(3-hydroxypropyl)androsta-4,6-renin-3-one,
It is preferable if the raw material that can be used can be selected from among many compounds, since it is possible to appropriately change the manufacturing process depending on the raw material situation.

Therefore, the object of the present invention is to
α-(3-hydroxypropyl)androsta-4,6
- To provide a new method by which renin-3-one can be easily produced.

[Means for solving problems]

According to the present invention, the above object is achieved by the following:
4,6-renine-1-fuon is represented by the general formula (wherein R2 represents a hydrogen atom or a lower alkyl group, and R3 and R4 each represent a lower alkyl group or together represent an alkylene group) 17β-hydroxy-17α-(3-
This is achieved by providing a method for producing hydroxypropyl)androsp-4,6-cyen-3-one, and also 3,3-alkylenedioxy7androster 4,6- represented by the general formula (1). 7α-Hydroxyandrost-4-ene- as renin-1-huon, with the formula
17β-hydroxy prepared by reacting a 3,17-dione with an alkylene glycol having the general formula %() (wherein R1 is as defined above) in the presence of an acid. -17α-(3-Hydroxypropyl)androsta-4,6-renin-3-one is achieved by providing an entire method for producing it.

The alkylene group represented by R1 in the above general formula is,
For example, ethylene group, propylene group (-CH2CH(CH
3) -3,1,2-dimethylethylene group, ethylethylene group, trimethylene group, 1-methyltrimethylene group, 2
-methyltrimethylene group, tetramethylene group, etc. Examples of the lower alkyl group represented by R2 in the general formula (I[) include a methyl group, an ethyl group, a propyl group,
Examples include isopropyl group. Examples of lower alkyl groups represented by R3 and R4 in general formula (n) include methyl, ethyl, propyl, and isopropyl groups, and alkylene groups represented by R3 and R4 together include For example, trimethylene group, 1-methyltrimethylene group, 2-methyltrimethylene group, tetramethylene group, 1-methyltetramethylene group,
Examples include 2-methyltetramethylene group.

Examples of the 1J thium compound represented by the general formula (■) include 3-lithiopropyl-1-methoxyethyl-
Ether, 1-ethoxyethyl 3-IJ thiopropyl ether, 3-lithiopropyl-1-glopoxyether ether, 1-impropoxyethyl-3-lithiopropyl ether, 3-lithiopropyl 1-methoxypropyl Neefle, 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-')thiopropyl 1-, 71 Tyl-1-propoxyethyl ether, 2-(3-lithiopropoxy)tetrahydrofuran, 2-(3-lithiopropoxy)tetrahydropyran, 2-(3-lithiopropoxy)
-4-methyltetrahydropyran and the like are used. The organolithium compound may be prepared, for example, by the corresponding general formula (wherein R
1.R3 and R4 are as defined above, and X represents a halogen atom) by reacting the halide represented by the following with metallic lithium according to a commonly known method for converting an alkyl halide into an alkyllithium. [For example, The Journal on Organic Chemistry]
Organic Chemistry), Volume 37,
Page 1947 (1972); Journal on the
American Chemical Society (Journal)
of the American Chemical
5ociety), Volume 99, Page 2751 (1977
): Japanese Patent Application Laid-open No. 143654 (1982); Japanese Patent Application Publication No. 52-
42867, etc.].

General formula (II)
), the amount of the organolithium compound used is about 10 to 20 moles per mole of 3,3-alkylenedioxyandrosta-4,6-renine-1-fuone. It is preferable that

The reaction is preferably carried out in an organic solvent, and examples of organic solvents include chain formulas such as diethyl ether, dipropyl ether, dibutyl ether, 1,2-dimethoxyethane, diethylene glycol dimethyl ether, tetrahydrofuran, and tetrahydropyran. Alternatively, solvents that may adversely affect the reaction, such as cyclic ethers, are used alone or in a mixture of two or more.

The reaction is preferably carried out at a temperature in the range of about -5 DEG to 30 DEG C., and good results are obtained when carried out in an atmosphere of a gas such as argon, helium, nitrogen, etc. which does not adversely affect the reaction.

3,3 represented by the general formula ([) obtained in this way
-alkylenedioxyandrosta-4,6-renin-
The reaction product of 1 phoon and the organolithium compound represented by the general formula (n) is hydrolyzed in the presence of an acid to prevent isolation from the reaction mixture. In this case, 3,3-alkylenedioxyandrosta-4, represented by general formula (1),
A reaction mixture containing a reaction product of 6-renine-1-fuon and an organolithium compound represented by the general formula (■), a concentrate thereof, and an organic layer obtained by contacting the reaction mixture with saline, water, etc. etc. may be subjected to a hydrolysis reaction in the presence of an acid. Although water alone may be used as the solvent for the hydrolysis reaction, it is preferable to use an organic solvent that is at least partially soluble in water and does not adversely affect the hydrolysis reaction.

Examples of organic solvents include cyclic ethers such as tetrahydrofuran and dioxane; methanol, ethanol,
Lower alcohols such as isopropyl alcohol are used alone or as a mixture of two or more. The amount of water used is preferably in the range of about 5 to 20 times the volume of 3,3-alkylenedioxyandrosta-4,6-renine-1-fuone represented by the general formula (1). Examples of acids present in the hydrolysis reaction system include hydrochloric acid, sulfuric acid, phosphoric acid,
Inorganic acids such as perchloric acid; I)-) various acids such as luenesulfonic acid and trifluoroacetic acid are used.

The amount of acid used is not particularly limited, but it is preferable to use an amount such that the temperature of the reaction solution is within the range of about 0.5 to 3, especially an amount that makes the temperature of the reaction solution 18 degrees. She seems like a good wife. The reaction is usually carried out at a temperature in the range of about 0 to 60°C;
In particular, it is preferred to carry out the reaction at a temperature in the range of about 15 to 25°C.

17 from the hydrolysis reaction mixture thus obtained.
β-hydroxy-17α-(3-hydroxypropyl)
Separation of androsta-4,6-renin-3-one・'f
The pI can be produced, for example, by the following method. After neutralizing the reaction mixture by mixing it with a basic aqueous solution such as aqueous sodium bicarbonate, it is extracted with a solvent such as ethyl acetate, chloroform, or methylene chloride. 17β-hydroxy-17α-(3-hydroquinopropyl)androsta-4,6-diene-3- You can get it on.

The 3,3-alkylenedioxyandrosta-4,6-diene-1 huone represented by the general formula (1) is, for example,
It can be manufactured by the method described above.

The amount of alkylene glycol represented by the general formula (I[[) is α-hydroxyandrost-4-ene-
Preferably, the amount is about 15 to 30 moles per mole of 3,17-dione. As the acid present in the reaction system, organic acids such as fumaric acid and oxalic acid are preferably used. Use of acid fi:ij 1 of 7α-hydroxyandrost-4-ene-3,17-dione
Preferably, it is about 0.1 to 0.5 mole K per mole. The reaction is preferably carried out in an organic solvent, and examples of the organic solvent include aromatic hydrocarbons such as evaporate, benzene, toluene, and xylene; and halogenated hydrocarbons such as 1,2-dichloroethane and carbon tetrachloride. Organic solvents can be used that have no adverse effects.

The reaction temperature is preferably within the range of about 50-100°C.

From the viewpoint of promoting this reaction, it is preferable to carry out the reaction after removing generated water from the reaction system. As a method for removing the produced water from the reaction system, the method normally used for distilling the water produced in an organic reaction out of the reaction system can be adopted as is. It is preferable to adopt a method of distilling it out of the system.

When water is distilled off as an azeotrope, an organic solvent that does not adversely affect the reaction and can form an azeotrope with water is used. As such an organic solvent,
-Has Aromatic hydrocarbons such as benzene, toluene and xylene; halogenated hydrocarbons such as 1,2-dichloroethane and carbon tetrachloride, and the like, but it is preferable to use aromatic hydrocarbons.

3,3 represented by the general formula (1) obtained in this way
-alkylenedioxyandrosta-4,6-renin-
Separation and purification of 1 phoon from the reaction mixture can be carried out, for example, by the following method.

After neutralizing the reaction mixture by mixing it with a basic aqueous solution such as aqueous sodium bicarbonate, benzene, toluene, xylene, 1
．． By extracting with an organic solvent such as 2-dichloroethane or carbon tetrachloride, distilling off the solvent from the extract, and then subjecting the residue to purification operations such as recrystallization and chromatography, a product represented by general formula (1) is obtained. 3,3-alkylenedioxyandrosta-4,6-renine-1-fuone can be obtained.

7α-hydroxyandrosta-4 used as raw material
-ene-3,17-dione can be produced, for example, by using 3α,7α-dihydroxy-5β-co27 acid (hereinafter referred to as CDCA) and/or its salt as a substrate to produce 7α-hydroxyandrost-4-ene-3°. Pseudomonas with the ability to produce 17-dione
Genus Putida g or Pseudomonas (
Pseudomonas)
ri) G bacteria are cultured in a medium containing CDCA and/or its salts to produce 7α-hydroxyandrost-4-ene-3,17-dione, which is collected with high selectivity. and produced with good yield.

Examples of bacteria belonging to the genus Pseudomonas species that have the ability to produce 7α-hydroxyandrost-4-ene-3,17-dione using CDCA and/or its salt as a substrate include Pseudomonas putida p 4 Q.
l 4-A l 5 l 5 (Pseudomona
sputida D4014-A1615) strain (Feikoken Bibori No. 6572), and also 7α-hydroxyandrosta-4 using CDCA and/or its salt as a substrate.
-Ene-3,17-dione-producing bacteria belonging to the Pseudomonas sutcheri species include, for example, Pseudomonas sutcheri M-1492 (Pseudomonas
domonas 5tutzeri M-1492)
There is a strain (Feikoken Bibori No. 8771). Pseudomonas putida D4014-A1615 strain was collected from soil.
These are mutant strains obtained by subjecting No. 205) to mutation treatment, and the mycological properties of these strains are described in Japanese Patent Application Laid-open No. 1496-1985.
This is as described in Japanese Patent Application Laid-open No. 98 or Japanese Patent Application Laid-Open No. 59-39298. In addition, Pseudomonas stucchei IJM-1492 strain was isolated from Pseudomonas sutscheri C-37-2 (Pseudomonas sutscheri C-37-2), which was collected from the soil.
The following table shows the mycological properties of these strains.

(Note 1): In physiological properties, +, - and - mean the following.

＋・・・・・・The property or its generation±・・・・・・
・It is not possible to determine its properties or the presence or absence of its generation.
...its properties or its production Note 2): The carbon source of Hugh & Lifeson medium is saccharide 1 to 15, respectively.
The results show observations of acid production and gas generation from sugars by bacteria in a different medium.

＋・・・・・・Acid generation or gas generation±・・・・・・
・The determination is whether or not acid or gas is generated.
...No production of alcohol or gas Based on the mycological properties shown in the table above, Pseudomonas stuccheri C-3
7-2 strain and Pseudomonas stutcheri M-149
Two bacterial strains were identified. Pseudomonas sutuche I
The JC-37=2 strain has microscopic findings such as being a bacillus, having a polar monoflagellate, and negative Durham staining, as well as positive oxidase and catalase reactions. Purge Aids Manual of Determinative Bacteriology (B
manual of determination
Active Bacteriology) 8th edition, it was identified as a bacterium belonging to the genus Pseudomonas. Furthermore, Pseudomonas stutcheri strain C-37-2 was identified as belonging to the Pseudomonas stutcheri species because it does not produce soluble pigments, performs denitrification reactions, does not decompose gelatin, and decomposes starch. did. Furthermore, it is generally believed that a mutant strain belongs to the same species as its parent strain, and it was determined that the Pseudomonas stutcheri strain M-1492 belongs to the species of Pseudomonas stutcheri.

Using CDCA and/or its salt as a substrate, bacteria belonging to Pseudomonas putida sp. By culturing in a medium containing Schiff α-hydroxyandrost-4-ene-3,17-dione. Specific examples of the salt of CDCA include salts of alkali metals such as sodium and potassium, or salts of alkaline earth metals such as calcium and magnesium. The concentration of CDCA and/or its salt in the medium is usually about 1 to 100%. /l, but from the viewpoint of easily obtaining 7α-hydroxyandrost-4-ene-3,17-dione in a high yield under mild conditions, the range is about 10 to
50? A range of /l is preferred. The culture method is basically the same as that used for aerobic culture of general microorganisms, but usually a shaking culture method using a liquid medium or an aerated agitation culture method is used. The medium used was 7α-hydroxyandroster using the above CDCA and/or its salt as a substrate.
It is sufficient that it contains a nutrient source that can be assimilated and utilized by bacteria belonging to the Pseudomonas putida species or Pseudomonas stutcheri species that have the ability to produce 4-ene-3,17-thion.

As a carbon source, CDCA and/or its salt may be used as a single carbon source, or CDCA and/or its salt may be combined with pentoses such as arabinose; hequinoses such as glucose, mannose, fructose, and galactose; sucrose, maltose, etc. disaccharides; starch decomposition products: sugar alcohols, polyhydric alcohols such as glycerin; or polypeptone, peptone, meat extract, malt extract, cornstarch liquor, yeast extract, various amino acids, organic acids, etc. good. Examples of nitrogen sources include inorganic nitrogen sources such as ammonium nitrate, sodium nitrate, and potassium nitrate, or polypeptone, peptone,
Organic nitrogen sources such as meat extracts are used. In addition, inorganic salts such as dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and magnesium sulfate are added. Although there are no specific culture conditions, shaking culture or aerated agitation culture is usually performed at 25 to 35°C for 10 hours to 7 days.

Most of the 7α-hydroxyandrost-4-ene-3,17-dione thus accumulated in the culture solution is usually obtained as a precipitate. In order to separate and collect the accumulated 7α-hydroxyandrost-4-ene-3,17-dione, 7α-hydroxyandrost-4-ene
An extraction method can be adopted using an organic solvent that dissolves -ene-3,17-dione and undergoes phase separation from water, such as ethyl acetate, chloroform, or a mixture of chloroform and methanol. Extraction with an organic solvent may be performed on a culture containing precipitates and bacterial cells, or may be performed on insoluble components separated from the culture by filtration or centrifugation. When the former extraction method is used, it is preferable to make the culture solution alkaline by adding an aqueous alkaline solution such as an aqueous solution of sodium hydroxide, and then perform the extraction operation using the organic solvent described above. When the latter extraction method is adopted, an alkaline aqueous solution such as an aqueous solution of sodium hydroxide is preferably added to the culture filtrate or supernatant, which is separated and removed by filtration or centrifugation as necessary. After making it alkaline,
By performing the extraction operation with the above organic solvent, 7α-hydroxyandrosta-4 dissolved in the culture solution is extracted.
-ene-3,17-dione can be recovered. The extracts obtained in this way were collected and the solvent was distilled off to obtain 7α-hydroxyandrosta-4-
Almost all of the ene-3,17-dione can be recovered. The recovered 7α-hydroxyandrost-4-ene-3,17-dione is purified by recrystallization from methanol, an aqueous methanol solution, or the like.

17β-hydroxy produced by the method of the present invention
17α-(3-hydroxypropyl)androsta-4
, 6-renin-3-one is disclosed in, for example, JP-A-52-1999.
17β-hydroxy-17α-(3-
Spironolactone is derived by oxidizing 7α-acetate (hydroxypropyl)-7α-mercaptoandroster-4-en/-3-one with Jones reagent.

Alternatively, 17β-hydroxy-17α-(3-
hydroxypropyl) androsta-4゜6-renin-
3-one is oxidized with Jones reagent to form 17β-
Hydroxy-3-oxo-17α-pregna-4,6-
Renin-21-carboxylic acid = γ-lactone (hereinafter referred to as canrenone), which can also be induced into spironolactone by reacting with thioacetic acid. However, the present invention is not limited to these examples.Reference Example 1 Obtaining medium 1 of Pseudomonas stucchelli M-1492 strain (composition: peptone 0.5%, meat extract 0.5%) Pseudomonas succeri C-37-2 grown on a slant of 0.5% salt and 1.5% agar;
Place a loopful of the bacterial strain in a test tube (inner diameter: 21 m; length:
Prepare 9 medium 2 (m composition: CDCA 3%) in 200sm).
, nitric acid 7y molybum 0.2%, potassium dihydrogen phosphate 0.1%, dipotassium hydrogen phosphate 0.6%, magnesium sulfate heptahydrate 0.02%, yeast extract 0.02%
, glucose O, osl, peptone O, 0.05% and sodium hydroxide 0.3%; Tania, 5) OloWLl was inoculated and cultured with shaking at 30°C for 244 hours. Q, 4w, l of this culture solution was prepared in a test tube (inner diameter: 211EIl;
length? 200 eyes)・Medium 3 prepared within (composition: CD
CAo, 5%, ammonium nitrate 0.2%, potassium dihydrogen phosphate 0.1%, dipotassium hydrogen phosphate 0.6%, magnesium sulfate heptahydrate 0.02%, yeast extract 0.0
2% and glucose 0.1%: 1 of F4' (:8.5)
The cells were added to Otnl and cultured with shaking at 30°C for 6 hours. Next, the bacterial cells in the logarithmic growth phase were collected by aseptic filtration using a membrane filter with a pore size of 0.45μ, and after washing with 20rnl of 0.1M phosphate buffer (F4-1?7.0), the same Suspended in 25 dK buffer.

Put 4 d of this bacterial suspension into a test tube (inner diameter: 21 m, length: 200 m), and add N-methyl-N'-nitro-N-nitrosoguanidine to a final concentration of 50 μ2/co. and after 10 minutes, the 0. l rLi! t
- Culture i4 [composition near α-hydroxyandrosta-4-
0.1% ene-3,17-dione, ammothi nitrate, 0.05% yeast extract and 0.05% polyoxyethylene (20) sorbitan monooleate (Tween 80);
)' + 1 near, 53, and penicillin G was added to a concentration of 1 ootl/σ, and then 1
The culture was shaken for 88 hours. The obtained culture solution was diluted and applied to a plate of medium 1 so that about 100 to 500 colonies appeared on the plate, and then cultured overnight at 30°C. After the colonies that appeared were isolated on a slant in medium 1, each isolate was cultured in regression culture in 10 mJ of medium 2 for 2 days. The products in each of the obtained culture solutions were assayed by dilute chromatography, and one strain was found that selectively accumulated 7α-hydroxyandrost-4-ene-3,17-dione.

It was named Pseudomonas cholera IJM-1492.

Reference Example 2 Pseudomonas putida strain D4014-A1615 was cultured by the method shown below. CDCA10F.

Ammonium nitrate 2.05', potassium dihydrogen phosphate 1.
0t1 dipotassium hydrogen phosphate 6, Or, magnesium sulfate heptahydrate 0-2', yeast extract 0.2 P
, glucose o, sr, peptone 0.5F and sodium hydroxide 1.0? Add tap water to the mixture to make a volume of 1f!
The μ was adjusted to 1t (:8.5) and used as a culture medium. This medium was placed in a 26 volume fermenter and steam sterilized at 120°C for 15 minutes. 50 m of inoculum grown in advance in the same medium as the above medium in a test tube shaker for 20 hours was added to the above medium, stirring speed 300 rpm, aeration volume o, 5 v.
Aerated agitation culture was carried out for 48 hours under conditions of vm and 30°C. After culturing, the culture solution was thoroughly mixed and a portion thereof was analyzed by thin layer chromatography, and as a result, it was confirmed that no CDCA remained. After adjusting the size to 10 by adding a 1N aqueous solution of sodium hydroxide to the culture solution, the culture solution was extracted with 5 tons of a mixed solvent of chloroform/ethanol (volume ratio: 4/1). 57. Insoluble materials such as denatured bacterial strains are filtered from the extract, and the solvent is distilled off from the 4th solution under reduced pressure. Q? The dry matter was obtained.

The dried product was analyzed by high performance liquid chromatography and was found to be 7α-hydroxyand-a-ster-4-ene-3,17-dione with a purity of 86. 4. Recrystallizing the 7α-hydroxyandrost-4-ene-3,17-dione using 80 volumes of aqueous methanol solution; A purified product of Of was obtained.

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

Melting point = 245-247°C Mass spectrum m/e: 302 (M) + 284 (
M-H20]'' The presence of 3-keto-4-ene was also confirmed from m/13=124.

NMR (CDα3e 90 MHz) δ; 0.92 (
B, 3 1g-CH5), 1.22 (11,3H,1
9-CH3), 4.09 (AIH, 7β-H),
5.79 (4-H to 3,1) ppm Reference Example 3 In Reference Example 2, Pseudomonas putida D4014-
Pseudomonas stuccheri M instead of A1615 strain
-1492'm strain was used, and the amount of CDCA used in the medium was changed from 10r to 3O5IK, and the λ used for hydroxylated lamb in the medium was changed from 1.02 to 3.0? By performing the same operation except for changing the dry matter to 21
．． 9? Obtained. Hikoo analyzed a part of the obtained culture solution by thin layer chromatography, and found that there was CDC in the culture solution.
After confirming that A does not remain, the next step is to proceed. Analysis of the dried product by high performance liquid chromatography revealed that the dried product was 7α-hydroquinandrost-4-ene-3,17-dione with a purity of 85%. This 7α
- by recrystallizing hydroxyandrost-4-ene-3,17-dione using methanol, 10
．． 57 purified products were obtained.

(2) 7α-hydroxyandrost-4-ene-3°17-hydrochloride
Zeon 5. Of, ethylene glycol 5 rptl
A mixture of 0.5 F., 7 malic acid and 400 g of benzene was heated to reflux with stirring in a reactor equipped with a Dean-8 tark water separation tube. 91 days after the start of heating and refluxing, 15ffi of ethylene glycol was added, and one day after that, 5 ml of ethylene glycol and 0.1 ml of fumaric acid were added. was added and heated under reflux for another day. During heating and refluxing, the water produced was removed from the reaction system using a Dean-Stark water separation tube. The resulting reaction mixture was poured into ice-cooled saturated sodium bicarbonate solution, the benzene layer was separated, and the aqueous layer was extracted with benzene. The previously separated benzene layer and benzene extract were combined, washed with saturated brine, and dried over anhydrous potassium carbonate. By concentrating the dried liquid under reduced pressure, the crude crystals were obtained at 6.21? Obtained. By recrystallizing the crude crystals using a mixed solvent of 16d methanol and a few drops of pyridine, 3.3-ethylenedioxyandrost-4,6-dien-17-one was obtained.
9 was obtained (yield: 72 cm). The analysis results of the obtained 3,3-ethylenedioxyandrosta-4,6-renin-1-phoon are shown below.

Melting point = 153-159°C Specific rotation: [α), +149° (c = 1.1 o, (
Jα3) 1.38 f of lithium wire was cut into pieces and placed in a flask, washed twice with n-pentane under an argon atmosphere, and then 40 d of anhydrous diethyl ether was added. Add 3-bromopropyl- to this mixture at room temperature while stirring.
1-Ethoxyethyl=ether 3? was added, and when the temperature of the solution rose to over 30°C, it was cooled using an ice-salt bath. While maintaining the temperature of the solution at about 5°C, a solution obtained by dissolving 16'l of 3-bromopropyl-1-ethoxyethyl ether in 20ml of anhydrous diethyl ether was added dropwise over about 40 minutes. After the dropwise addition was completed, stirring was continued for an additional 50 minutes at 0°C. A solution obtained by dissolving 1.72 of 3,3-ethylenedioxyandrosta-4,6-renin-1-fuone in 10 d of anhydrous tetrahydrofuran was added dropwise to the resulting mixture at 0 to 5°C, and then O to 1 at 5℃
Stirring was continued for an additional 4 hours at room temperature. The solution in the obtained reaction mixture was poured into ice-cooled saturated brine while being careful not to mix with solids. The organic layer was separated from this, and the aqueous layer was extracted with 50ml of tetrahydrofuran. The organic layer and extract obtained earlier are combined for about 30%! The mixture was concentrated under reduced pressure to a volume of 1/2.

2Qrnl of 2N hydrochloric acid was added to the obtained concentrate, and the mixture was stirred at room temperature for 2 hours. The resulting reaction mixture was poured into saturated aqueous sodium bicarbonate and extracted twice with ethyl acetate. After combining the extracts and washing sequentially with saturated sodium bicarbonate solution and saturated saline solution,
It was dried with anhydrous magnesium sulfate. Concentrate the dried liquid under reduced pressure to obtain 4.1? Obtain the concentrate. This product was found to contain υ17β-hydroxy-17α-(3-hydroxypropyl)androsta-4,6-dien-3-one according to the following analysis results.

IR (film) νmax: 3350 (s, hydroxyl group) t 1665 (vs, conjugated carbonyl group), 1620 (m, double bond), 1585 (
W, double bond) cIM-1 NMR (CDαs, 60 MHz) δ: 5.6 (8, I
H, 4-H), 6.04(s, 2H, 6-H and 7-
H) ppm 5COCH3 (a) 17β-hydroxy-17α-(3-
4.12 of the crude product containing (nitroxypropyl) androsta-4,6-renin-3-one was added to 50 mJ of acetone.
and tert-p'J- were dissolved in a mixed solvent of 60 d of alcohol under heating. Jones reagent IQtrtl was added dropwise to this solution under water cooling. After the dropwise addition was completed, stirring was continued for 20 minutes while cooling with water, and then 5 ytl of Jones reagent was added, followed by stirring at room temperature for 20 minutes. One part of isopropyl alcohol was added to the resulting reaction mixture.
After adding 0 m/, the mixture was concentrated under reduced pressure. The resulting concentrate was diluted with water, extracted with ethyl acetate, and the extract was washed with saturated aqueous sodium bicarbonate and dried over anhydrous magnesium sulfate.

By concentrating the dry liquid under reduced pressure, 2. A concentrate of Qf was obtained.

The obtained Nenkamono was dissolved in 10xl of acetone, 2ml of Jones reagent was added, and the mixture was stirred at room temperature for 20 minutes. One portion of isopropyl alcohol was added to the resulting reaction mixture. After adding g of the mixture, it was concentrated under reduced pressure, and the fine product was treated in the same manner as above to obtain an oily substance of 2.02.

The obtained oily substance was purified using column chromatography [Packing material: Nisilica gel (20 t); Eluent: n-hexane/ethyl acetate mixture (volume ratio: 2/1 to 1/1)]. 0 oily substances containing canrenone
．． Obtained 98P. This oily substance was mixed with 1°5 of n-hexane.
By recrystallizing using a mixed solvent of rul and acetone lrnl, 0.56f' of canrenone was obtained. The yield of canrenone was 32% based on the 3,3-ethylenedioxyandrosta-4,6-renine-1-fuon used in Example (b). The analysis results of the obtained canrenone are shown below.

Melting point = 163-168°C Specific rotation: [α'l, +22° (c = 1.05, CH
Q! 3)(b) Production of spironolactone Canrenone 0.4005' was dissolved in 5 m/m of methanol, Q thioacetate, 24 rne was added to this solution under heating under reflux, and heating under reflux was continued for an additional 30 minutes.

1.6 rne of water at 60°C was added to the resulting reaction mixture, cooled to room temperature, and then diluted with ethyl acetate. The resulting solution was washed with saturated aqueous sodium bicarbonate and saturated brine, and then dried over anhydrous magnesium sulfate. The dried liquid was concentrated under reduced pressure to obtain 0.6072 crude crystals. This crude crystal □ was recrystallized using methanol to obtain 0.4089 spironolactone (yield: 83φ). The analysis results of the obtained spironolactone are shown below.

Melting point: 130-135°C Specific rotation: [α cod-36.6' (C=1.13, CHα
3) [Effect of the invention] According to the present invention, as is clear from the above examples, 7
From α-hydroxyandrost-4-ene-3,17-dione, 17β-hydroxy- 17α-(3-hydroxypropyl)androsta-4,6-renin-3-one can be easily produced.

Claims (1)

[Claims] 1. 3,3-alkylene dioxyandroster represented by the general formula ▲ Numerical formula, chemical formula, table, etc. ▼ (I) (in the formula, R^1 represents an alkylene group)
4,6-dien-17-one with the general formula ▲ mathematical formula, chemical formula,
There are tables etc.▼(II) (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 represent an alkylene group), and the resulting reaction product is hydrolyzed in the presence of an acid. A method for producing 17β-hydroxy-17α-(3-hydroxypropyl)androsta-4,6-dien-3-one, characterized by: 2. 3,3-alkylenedioxyandrost-4,6-dien-17-one represented by general formula (I) is 7α
-Hydroxyandrost-4-ene-3,17-dione in the presence of an acid to form an alkylene compound of the general formula HO-R^1-OH(III) (wherein R^1 is as defined above) The manufacturing method according to claim 1, which is manufactured by reacting with glycol.