JPH0359915B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention is based on the formula (In the formula, the wavy line ~ indicates that the hydroxyl group or cyano group is in either the α-configuration or the β-configuration.) 17-cyano-12,17
-dihydroxyandrosta-1,4-diene-
Regarding 3-on. 17-cyano-12,17- provided by the present invention
Dihydroxyandrosta-1,4-diene-3
-one is a new compound not described in known literature,
It is useful as a synthetic intermediate for progesterone or corticoids such as brednisone and brednisolone. 17-cyano-12,17-dihydroxyandrosta-1,4-dien-3-one is 12-hydroxyandrosta-1,4-diene-3,17-dione (hereinafter referred to as 12-hydroxyADD). )
It is produced by reacting hydrogen cyanide with hydrogen cyanide in the presence of a lower alcohol and a cyano compound.
Examples of lower alcohols used as solvents include methanol, ethanol, and propanol. Examples of the cyano compound include potassium cyanide and sodium cyanide. The amount of the cyano compound to be used is preferably about 1 to 10 times the molar amount of 12-hydroxy ADD. Hydrogen cyanide may be added directly to the reaction system, or it may be generated within the reaction system by reacting an organic acid such as acetic acid or propionic acid or an inorganic acid such as hydrochloric acid or sulfuric acid to the cyano compound present in the system. good. Hydrogen cyanide is preferably used in an amount of about 1 to 8 moles relative to 12-hydroxy ADD. This reaction is carried out at a temperature of about 0-30°C. In addition, 17-cyano-12,17-dihydroxyandroster-1,4-dien-3-one can be obtained by reacting 12-hydroxyADD with acetone cyanohydrin in the presence of a small amount of pyridine, or by reacting 12-hydroxyADD with acetone cyanohydrin in the presence of a small amount of pyridine. It can also be produced by directly adding hydrogen cyanide to After the reaction is complete, the reaction mixture is poured into water, extracted with methylene chloride, etc., the extract is washed with water, sodium bicarbonate, etc., dried, and the lower boiling point substances are distilled off to obtain 17-cyano-12. , 17-dihydroxyandstar-1,4-dien-3-one crude product is obtained. Highly purified 17-cyano-12,17-dihydroxyandrosta-1,4 can be obtained by recrystallizing this crude product from, for example, ethanol.
-dien-3-one can be obtained. The raw material compound 12-hydroxy ADD is a known compound, but according to a method previously discovered by some of the present inventors, deoxycholic acid and/or
It can be obtained by culturing bacteria belonging to Pseudomonas putida or Pseudomonas ovalis that produce 12-hydroxy ADD using a salt thereof as a substrate in a medium containing deoxycholic acid and/or a salt thereof. Bacteria belonging to the above Pseudomonas putida include Pseudomonas putida D4014-A1099.
(Pseudomonas putida D4014-A1099) strain (Feikoken Joyori No. 207). This strain is Pseudomonas putida D4014, which was collected from Tuyongzhong.
(Pseudomonas Putida D4014) strain (Feikoken Joyori No. 205) was subjected to mutation treatment. In addition, as a bacterium belonging to Pseudomonas ovalis, Pseudomonas ovalis D
-40 (Pseudomonas ovalis D-40) strain (Feikoken Bacteria No. 6147). Tables 1 and 2 list the mycological properties of Pseudomonas putida D4014 strain, Pseudomonas putida D4014-A1099 strain, and Pseudomonas ovalis D-40 strain.

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[Table] Based on the mycological properties shown in the table above, Pseudomonas putida strain D4014, Pseudomonas putida
putida D4014-A1099 strain and Pseudomonas
The Ovaris D-40 strain was identified. Pseudomonas putida strain D4014 is a bacillus;
Microscopic findings such as having polar flagella, negative Gram staining, and positive oxidase and catalase reactions;
Based on the physiological properties such as being aerobic and the O-F test result being oxidative, Pseudomonas It was identified as a bacterium belonging to the genus. Furthermore, Pseudomonas putida
Strain D4014 was identified as a bacterium belonging to the putida species of the genus Pseudomonas based on the fact that the culture solution takes on a fluorescent color, that it does not liquefy gelatin, that it grows at 37°C, and that it produces arginine dihydrolase. . Generally, mutant strains are considered to belong to the same species as their parent strain, and Pseudomonas
The putida strain D4014-A1099 was determined to be a bacterium belonging to the putida species of the genus Pseudomonas. Also,
Pseudomonas obalis D-40 strain is a bacillus, has polar flagella, has negative Gram staining, is aerobic, and has a negative O-F test result. It was identified as a bacterium belonging to the genus Pseudomonas based on the physiological properties such as the fact that Furthermore, Pseudomonas ovalis strain D-40 is a short bacillus with rounded ends, the colonies formed on the cold plate are circular and shiny, it does not produce indole, and it does not produce acids and gases from sugars. Since no production was observed, it was identified as a bacterium belonging to the genus Pseudomonas spp. 12-hydroxy ADD using the above microorganisms
In the production method, deoxycholic acid and/or its salt is used as a substrate. Specifically, the salt of deoxycholic acid is a salt of an alkali metal such as sodium or potassium, or a salt of an alkaline earth metal such as calcium or magnesium. The concentration of deoxycholic acid and/or its salt may normally be in the range of about 1 to 200 g/, but from an economic standpoint such as the yield of 12-hydroxy ADD to be produced, culture conditions, and operability, the concentration of deoxycholic acid and/or its salt is about 2 to 50 g/. A range is preferred. The culture method is basically the same as that used for aerobic culture of general microorganisms, but
Usually, a shaking culture method or an aeration culture method using a liquid medium is used. The medium uses the above deoxycholic acid and/or its salt as a substrate and 12-hydroxy
It is sufficient that it contains a nutrient source that can be used as an assimilation agent by bacteria belonging to Pseudomonas putida or Pseudomonas ovalis that produce ADD. As the carbon source, deoxycholic acid and/or its salt may be used as the sole carbon source, but preferably glucose, glycerin, peptone, meat extract, malt extract, yeast extract, etc. are used in combination with deoxycholic acid and/or its salt. It is better. Further, as the nitrogen source, for example, an inorganic nitrogen source such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium nitrate, sodium nitrate, potassium nitrate, or an organic nitrogen source such as boripeptone, peptone, meat extract, etc. is used. In addition, hydrogen phosphate 2
Inorganic salts such as potassium, potassium dihydrogen phosphate, and magnesium sulfate are added. Although there are no specific culture conditions, shaking culture or aerated agitation culture is usually carried out at 25 to 35°C for 10 hours to 7 days. In order to separate and collect the 12-hydroxy ADD accumulated in the culture solution in this way, the culture solution is made alkaline as desired, and then the 12-hydroxy
An extraction method is used that uses an organic solvent that dissolves ADD and undergoes phase separation from water, such as ethyl acetate, chloroform, or a mixture of chloroform and methanol. This extraction with a solvent may be performed on the culture fluid containing the bacterial cells, or the bacterial cells and other insoluble components in the culture fluid may be separated and removed by filtration or centrifugation, and the resulting culture The test may be carried out on the filtrate or supernatant. When using the latter extraction method, the insoluble components that are separated and removed by filtration or centrifugation, if necessary, can be extracted with the organic solvent described above, so that they can be precipitated or precipitated in the culture solution. The 12-hydroxy ADD that is present can be recovered. By collecting the extract obtained in this way and distilling off the solvent, it is possible to recover almost all of the deoxycholic acid oxides including 12-hydroxyADD and unconverted deoxycholic acid. can.
12-hydroxy ADD can be obtained by recrystallizing the obtained extract from ethyl acetate, dichloromethane/methanol, etc., or by adsorbing it on a silica gel column and eluting it with a mixture of chloroform and ethanol. 17-cyano-12,17- provided by the present invention
Dihydroxyandroster 1,4-diene-3
-one is obtained by (i) subjecting it to a dehydration reaction, followed by partial hydrogenation, protecting the ketone moiety at position 3 of the obtained product, reacting it with methylmagnesium halide, and further converting the product into If necessary, progesterone can be derived by subjecting it to a partial hydrogenation reaction, and (ii) 17-cyano-
The hydroxyl group at the 12th position of 12,17-dihydroxyandroster-1,4-dien-3-one is converted to the β-hydroxyl group at the 11th position by a conventional method, and the cyano group at the 17th position is used to induce a side chain. This can lead to corticoids such as prednisone and prednisolone. The present invention will be specifically explained below using Examples and Reference Examples. Reference example 1 Method for obtaining Pseudomonas putida D4014-A1099 strain Medium 1 (composition: deoxycholic acid 0.5%, sodium hydroxide 0.05%, peptone 0.5%, yeast extract
Pseudomonas putida grown on slants of 0.5% sodium chloride, 0.5% sodium chloride and 1.5% agar).
Culture medium 2 (composition: 2% deoxycholic acid, 0.2% sodium hydroxide, 0.2% ammonium nitrate, 01% potassium dihydrogen phosphate, 0.6% dipotassium hydrogen phosphate,
The cells were inoculated into 10 ml of 0.02% magnesium sulfate heptahydrate and 0.02% yeast extract, and cultured with shaking at 30°C for 14 to 15 hours. Medium 3 (composition: deoxycholic acid 0.5%,
Sodium hydroxide 0.05%, glucose 0.1%, ammonium nitrate 0.2%, potassium dihydrogen phosphate 0.1
%, dipotassium hydrogen phosphate 0.6%, magnesium sulfate heptahydrate 0.02% and yeast extract 0.02%).
ml and cultured at 30°C for 8 to 9 hours. Then,
The cells in the logarithmic growth phase were collected by aseptic filtration using a 0.45 μ membrane filter, washed with 20 ml of 0.1 M phosphate buffer (PH: 7.0), and washed with 25 ml of the same buffer.
ml. Mutation treatment was carried out by adding N-methyl-N'-nitro-N-nitrosoguanidine to the mixture at a final concentration of 50 μg/ml and allowing it to stand for 3 to 4 minutes. The cells subjected to mutation treatment were collected by filtration using a 0.45μ membrane filter, washed with 20ml of 0.1M phosphate buffer (PH: 7.0), and then suspended in 20ml of the same buffer. The obtained bacterial suspension was diluted with sterile physiological saline and added to medium 4.
(Composition: Deoxycholic acid 0.5%, sodium hydroxide 0.05%, ammonium nitrate 0.2%, potassium dihydrogen phosphate 0.1%, dipotassium hydrogen phosphate 0.6%, magnesium sulfate heptahydrate 0.02%, yeast extract 0.02
% and agar 1.5%) on an agar plate so that 500 to 1000 colonies appear, and then incubated at 30°C.
The cells were cultured for 3 to 4 days. After isolating the tiny colonies among the colonies that appeared on the slant of medium 1,
Culture medium 5 (composition: deoxycholic acid 0.2%, sodium hydroxide
0.02%, glucose 0.1%, ammonium nitrate 0.2
%, potassium dihydrogen phosphate 0.1%, dipotassium hydrogen phosphate 0.6%, magnesium sulfate heptahydrate 0.02%
and yeast extract 0.02%) and inoculated at 30℃.
It was cultured with shaking for 24 hours. The products in each culture solution obtained were assayed by thin layer chromatography, and the target 12α-hydroxyandroster 1,4-diene-3,17-dione (hereinafter referred to as
We found a strain that selectively accumulates 12α-hydroxyADD) and named it Pseudomonas putida D4014-A1099. Reference Example 2 Pseudomonas putida D4014-A1099 strain (Feikoken Bibori No. 6327) was cultured by the following method. Deoxycholic acid 0.5g, glucose 0.1g,
Ammonium nitrate 0.2g, potassium dihydrogen phosphate 0.1
g, dipotassium hydrogen phosphate 0.6 g, magnesium sulfate heptahydrate 0.02 g, yeast extract 0.02 g, and sodium hydroxide 0.05 g, add tap water to make the volume 100.
ml (PH: 8.4) and used as a medium. Pour this medium into a 500ml Sakaguchi flask and heat at 120℃ for 15 minutes.
Steam sterilization was performed for 1 minute. Add 10 ml of the inoculum grown in advance in the same medium as the above medium in a test tube shaker for 15 hours to the above 500 ml Sakaguchi flask,
Shaking culture was performed at 30°C for 28 hours. After culturing, collect the culture fluid and remove the bacterial cells using a centrifuge. Add a 1N aqueous sodium hydroxide solution to the resulting culture supernatant to bring the pH of the culture supernatant to around 12. It was adjusted. Then, the culture supernatant was diluted with 300% ethyl acetate.
ml, and 12α-
360 mg of hydroxy ADD was obtained. Take a part of the obtained 12α-hydroxy ADD,
Methanol was added to this to make a 2% solution, and 25 μl of this solution was injected into a high performance liquid chromatography system (manufactured by Waters, USA, model HLC-GPC-244) equipped with a Micro Bondapak C-18 column. 30% water/methanol adjusted to PH4.0 as mobile phase
A mixed solution having a volume ratio of 70 was flowed at a flow rate of 1 ml/min, and detection was performed using a refractive index method. The area ratio of each peak in the obtained liquid chromatograph was determined using an integrator (Shimadzu Chromato Pack C-R1A, manufactured by Shimadzu Corporation),
The purity of the above 12α-hydroxy ADD was determined from this area ratio and was 99.5%. Furthermore, the 12μ-hydroxy ADD obtained above was confirmed by the following method. Mass spectrum m/z: 300 [M] ⁺

Claims (1)

[Claims] 1 17-cyano-12,17-dihydroxyandrosta-1,4-dien-3-one.