JPH0614874B2 - Steroid conversion process - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] There are 19 examples of the first use of corticosteroids for treatment.
In the fifties, he introduced cortisone acetate treatment for rheumatoid arthritis.

In subsequent research, Hydrocortisone and Cortisone 1,
It has been demonstrated that the insertion of unsaturation at the 2-position increases the efficacy of the resulting steroids prednisolone and prednisone and reduces drug-induced salt retention.
Most other steroids used for the treatment of diseases that respond to corticosteroids have since been synthesized with a double bond in the 1,2-position of the steroid molecule. In 1977, two US patents were issued showing a new method for the synthesis of corticosteroids from sterol precursors. US Patent No. 4.035,236 describes a method of making 9α-hydroxyandrostenedione through fermentation of sitosterol, stigmasterol or cholesterol. US Patent No. 4,041,055
The issue reveals a general method for synthesizing medically useful corticosteroids from this androstene.
The intermediates dealt with in this chemistry may have the 3-keto-Δ ^{4,9 (11)} configuration.

[Problems to be Solved by Conventional Techniques and Inventions]

The following is a prior art method that revealed the biological conversion of 1,2-saturated steroids to the corresponding 1,2-dehydrosteroids.

US Patent No. 2,837,464 "Corynebacterium (Cory
Method for producing dienes by nebacterium) "Description of 1-dehydrogenation of steroids in fermentation broth by Arthrobacter (Corynebacterium) simplex.

-U.S. Patent No. 3,360,439 entitled "Process for Making 1-Dehydrosteroids".

Description of the 1-dehydrogenation of steroids by the use of A. simplex cells pretreated with a lower alkanol or a lower alkanone such as acetone before mixing with a substrate and a hydrogen carrier.

・ Charney W. and Harzog
Herzog.H., 1967, microbial conversion of steroids. Academic Press, New York, 4-9
Pp. 236-261.

Historical background on the biotransformation of steroids and a taxonomic list of microorganisms known to perform 1-dehydrogenation.

・ T. Yamane, H. Nakatani, E. Sada, T. Omata, A. Tanaka and S. Fukui, Biotechnology and Bioengineering 21
Volume 1979.

The authors studied the 1-dehydrogenation of 4-androstene-3,17-dione by Nocardia rhodocrous in a mixture consisting of 50% benzene-heptane. The previously reported advantage of adding this solvent to the bioconversion mixture was to increase the reaction rate by a factor of 180. However, benzene is a known carcinogen. The preferred electron acceptor for the activity of Nocardia rhodoclass was phenazine methosulfate. This electron acceptor is too expensive and unstable to render this microbial activity practical or useful in bioconversion manufacturing processes. Nocardia rhodoclassic enzymes are inactive in the presence of the electron acceptor menadione selected for the Altrobacter simplex. The enzymes in N. rhodochlas must be different from those made in A. simplex. The reaction proceeded with organic solvent levels that were only 20% saturated with steroids. It was shown that the reaction rate decreased with increasing steroid concentration. The enzyme is an essential reagent for the 1-dehydrogenation reaction. The solvent (50% benzene-heptane) is flammable in the presence of the enzyme. How to introduce oxygen into the reactor without creating an explosive environment was not discussed.

・ K. Sonomoto, I. Jin, A. Tanaka and S. Fukui, Agric.
Biol. Chem. 44: 119-1126, 1980.

The same laboratory studied 1-dehydrogenation of hydrocortisone by A. simplex. Two water-immiscible solvents, n-butanol and n-amyl alcohol, were tested, but the bioconversion reaction did not proceed in the presence of these solvents.
A water-miscible solvent such as 10% methanol-water or 10-propylene glycol-water was chosen as the preferred solvent.

The prior art did not disclose or suggest this improved method.

[Means for solving the problem]

The best known prior art process using air-dried or heat-dried microbial cells capable of catalyzing the 1-dehydrogenation of steroids in an aqueous environment, especially in the presence of exogenous electron acceptors. Efficient conversion of 1,2-saturated steroids to the corresponding 1,2-dehydro derivatives is obtained than that obtained in.

Further, the invention of the divisional application of the present application involves adding an aromatic hydrocarbon as a solvent immiscible with water to an aqueous slurry comprising A. simplex cells, an exogenous electron acceptor and a steroid to be dehydrogenated. A method is disclosed and claimed. The slurry is stirred to supply oxygen to the extent that the oxygen content of the gas cavity becomes lower than the minimum combustion amount of oxygen, or the reaction is performed at a temperature lower than the flash point of the organic solvent.

These procedures are superior to microbial prior art steroid dehydrogenation techniques for the following reasons.

a) This bioconversion reduces the level of unconverted steroid substrate.

b) This method can be performed at higher steroid concentrations.

c) For converting a predetermined amount of steroid, microbial cells,
For example, the amount of A. simplex is small.

d) The present method is highly resistant to organic soluble impurities present in steroids, impurities present in microbial cells, or impurities made from steroids by microbial cells.

e) Higher yields of the desired product are obtained due to the reduction or total elimination of steroid-degrading enzymes present in the microbial cells.

In addition, solvent procedures are superior to the prior art for dehydrogenating steroids with other microbial activity such as Nocardia rhodochras. The reaction catalyzed by A. simplex can be carried out with stable and cheap electron acceptors such as menadione.

Microorganisms The microorganisms that can be used in the present method are any of the many known microorganisms known as 1-dehydrogenase. Such microorganisms are named by Charney W. and Herzog H., 1967, "Microbial Transformation of Steroids" (Academic Press, New York). is there.

Bacteria that perform 1-dehydrogenation of steroids are described in "Barkeys Manual of Determining Biology", No. 8
It is divided into two groups, which are decided by the edition. The procedure described herein has been successfully demonstrated for species from the genera Arthrobacter and Corynebacterium included in "Part 17, Actinomycetes and Related Organisms". 17th such as Nocardia, Mycobacterium, Streptomyces and Bacteria
1-dehydrogenated species of several other genera of the part are also probably useful.

Two commonly available microorganisms known to perform 1-dehydrogenation of steroids have been shown to be useful in this type of process. Bacterium included as 1-dehydrogenase-ATCC No. 12673 in US Pat. No. 3,065,146
Cyclooxidans was tested as a useful microorganism. A more widely used bacterium for 1-dehydrogenation of steroids is Arthrobacter simplex, ATCC 6946, which is disclosed in US Pat. No. 2,837,464. As such, many of the following use this microorganism to exemplify the method. However, this method
-It should be understood to include the use of dehydrogenating microorganisms.

Microbial growth Microorganisms grow in an aqueous nutrient medium containing:

(a) Inorganic compounds or organic nitrogen compounds such as nitrates or ammonium salts that supply nitrogen necessary for growth (yeast extract, pefton, corn steep liquor, etc.).

(b) carbon / energy sources such as carbohydrates and sugar derivatives,
Oils, fatty acids and their methyl esters, alcohols, amino acids or organic acids.

(c) Ions and trace elements such as tap water or levels of sodium, potassium, magnesium, phosphate, sulphate, manganese, copper, cobalt, supplied by less purified medium components (such as corn steep liquor). Molybdenum, etc.

Living organisms require oxygen present in the atmosphere for growth.
The growth temperature range is 10-45 ℃, and 28-37 ℃ is optimal for A. simplex. The optimum pH for growth is near neutral.

Production of Steroid-1-Dehydrogenase A 1,2-saturated 3-keto-steroid compound such as androsta-4-ene-3,17-dione or cortisone acetate was added to 0.005% (w / v) of the medium or it Addition at the above levels induces the steroid-1-dehydrogenase activity of bacterial cells. Cultivation is continued for at least 6 hours in the presence of the inducer before the cells are taken up for drying.

The inducer can be added at any point in the growth cycle. Culture media grown on nutrients such as lard oil rapidly initiate the synthesis of steroid-1-dehydrogenase, whereas culture media grown on glucose require substrate depletion before enzymatic synthesis occurs. Cultivation is continued for 6 hours or more after the addition of the inducer, then the cells are taken up for the solvent procedure or for drying.

Bacterial cell recovery procedure for drying The bacterial cells are separated from the nutrient medium and concentrated by conventional means such as centrifugation or flocculation, filtration, and ultrafiltration. The isolated cells have a water content in the range of about 1 to about 10%, 1
It is dried under reduced pressure at ~ 85 ° C (preferably 55-75 °), or by air drying with heating, or spray drying or tumble drying. A water content of about 5% is preferred. The cells are stored at 5 ° C until used for bioconversion. The dried active cells were trapped in polyacrylamide gel and collagen, or polymerized as described in "Methods of Enzymology", Volume XLIV, 1976 (Academic Press, New York), pages 11 to 317. It can also be prepared by immobilizing dry cells by covalent attachment of the cells to the electrolyte carrier.

Bioconversion Method Bioconversion is achieved by exposing the prepared cells to steroid substrates. Typically a pH of 6-10
Suspend cells and steroids in a weakly buffered aqueous solution (optimum pH 7.25 to 8.5). The amount of bacteria is 0.1-50g per 1
And the steroid is added at a weight ratio of 0.05 to 5.0 (steroid: fungus body). When bioconversion is performed in an aqueous environment, 8-10 per 5-10 g of steroid
A bacterial load of g is preferred. A catalytic amount (eg 5 × 10 ⁻⁴ M menadione) of exogenous electron carrier is added to stimulate the reaction. A useful compound is menadione (2-methyl-
1,4-naphthoquinone), phenazine methosulfate,
Includes dichlorophenol-indophenol, 1,4-naphthoquinone, menadione bisulfite, ubiquinones (coenzyme Q) and vitamin K type compounds. 5 to mixture
Incubate in the temperature range of 45 ° C for 0 to 14 days. During the culture, the mixture is contacted with the molecular enzyme, preferably agitated. 1-
Dehydrogenation rates typically decrease over time. Bioconversion proceeds to completion rates of 98-100% in less than 24 hours using 10 g of substrate per.

Examples of various procedures that can be used include:

(a) A steroid and menadione are suspended in an aqueous solution having a weak buffering degree, and then dry cells are added. Surfactants such as Tween 80 are added at low concentrations, eg 0-5%, to facilitate suspension of the steroid.

(b) Suspend cells in an aqueous buffer system, followed by addition of electron carrier dissolved in ethanol, methanol, acetone (up to 5% of final volume). The steroid substrate can be added as a dry powder or can be dissolved (suspended) in a water-miscible organic solvent such as dimethylformamide, ethanol, methanol, acetone, dimethylsulfoxide or an immiscible organic solvent such as toluene.

(c) The dried cells are rehydrated in a small amount of buffer solution, and then a buffer solution and an organic solvent are further added. Addition of ethanol, acetone, xylene, butyl acetate, methylene chloride or toluene etc. gives a final organic solvent content of 0-95% (vol / vol). The amount of aromatic hydrocarbon used is about half the amount required to dissolve the most soluble of the starting steroid or product steroid, and the amount of the most sparingly soluble of the starting steroid or product steroid is dissolved. About 4 of the amount required for
The range is up to double. Steroids and electron carriers are added to initiate the reaction. It is preferred to add the aromatic hydrocarbon along with the steroid. However, this may be added later or at an earlier time.

(d) The wet cell cake is suspended in an aqueous solution having a weak buffering degree, or the fermentation liquor is diluted with the same aqueous solution, and then an exogenous electron carrier, steroid and aromatic hydrocarbon are added.

Useful compounds in practice of the substrate range present invention, 3-keto - [delta ⁴ - belong to pregnene steroid of - androstene and 3-keto - [delta ^4. It is recognized that the substrate for steroid-1-dehydrogenase has saturation between carbons C1 and C2 of the A ring and has a hydroxyl or keto group at the 3-position of the A ring. Members of the Androsten family include:

1) Androsta-4-ene-3,17-dione 2) Androsta-4,9 (11) -diene-3,17-dione and its 6α-fluoro, 6α-methyl or 16-methyl derivative 3) 11β -Hydroxyandrosta-4-ene-3,17-
Diones and Derivatives Thereof The 3-keto-Δ ⁴ -pregnene steroids that can be used are as follows.

1.17 α-Hydroxypregne-4-en-20-yne-
3-one and its 16-methyl derivative 2.11β, 21-dihydroxy-pregner 4,17 (20) -dien-3-one and its 6α-methyl derivative 3.20-chloro-pregna-4,9 (11) , 17 (20) -trien-21-ar-3-one 4. Several groups of 3,20-diketo-Δ ⁴ -pregnene, including:

a) 11,17,21-trihydroxy compounds such as hydrocortisone and its 6α-methyl derivative b) 9β, 11β-epoxy-17,21-dihydroxy compounds such as 9β, 11β-epoxy-17,21-dihydroxy-16β -Methyl-pregne-4-ene-3,20-dione c) 3,20-diketo-4,9 (11) -pregnesenes, such as 17α, 21-dihydroxy-pregna-4,9 (11) -diene- 3,20-dione and its 16α-methyl, 16β-methyl or 16α-hydroxy derivatives or 17α-acetate ester d) 3,20-diketo-4,9 (11), 4,9 (11), 16-pregnenetriene , Such as 21-hydroxy-pregna-4,9 (11), 16
-Triene-3,20-diones and their 6α-fluoro derivatives 21-ester derivatives of steroids containing 21-hydroxyl groups (# 2 and # 4) also serve as substrates. Preferred 21-esters consist of lower fatty acids such as acetic acid and lower alkyl or aryl groups such as monocyclic carboxylic acids such as benzoic acid.

The bioconverted product and unconverted substrate can be recovered from the mixture by conventional means. Steroids can typically be recovered by filtration followed by extraction of the filter cake with an organic solvent such as acetone or methylene chloride. Alternatively, the whole bioconversion mixture can be extracted by mixing with a water immiscible solvent such as butyl acetate or methylene chloride. The product is then isolated from the organic solvent.

The following are the results of different bioconversions showing the superiority of the method of the invention over prior art methods. Bioconversion was performed using the conditions detailed above.

Example 1 Preparation of dry cells Bacterium cyclooxidans (ATCC 12673) was placed in a shake flask containing a medium of pH 7.0 consisting of cererose, peptone and corn steep liquor (6 g / each).
Was inoculated. The culture medium was cultivated on a rotary shaker at 28 ° C. until the disappearance of glucose occurred. At this point cortisone acetate (0.5 g /) was added and the flask was incubated for a further 16 hours. The cells were harvested by centrifugation, washed twice with water and then placed in a low vacuum oven at 45 ° C until dry.

Bioconversion of 6α-methylhydrocortisone 0.5 g of dried bacterial cells prepared as described above was added to 50 mM of pH 7.5.
It was rehydrated while stirring in 50 ml of phosphate buffer. 0.02 as menadione as ethanol solution (8.6mg / ml ethanol)
It was added to the bacterial suspension at a level of 5 ml / 50 ml. The substrate was added as a dimethylformamide (DMF) solution (100 mg of 6α-methylhydrocortisone per ml of DMF) to a final bioconversion concentration of 0.5 g /. The mixture was incubated at 28 ° C with agitation. After culturing for 4 hours, 91% of the steroid was converted. 6
α-Methylprednisolone was recovered by conventional means.

Example 2 Androsta-1,4,9 (11) -triene-3,17-dione production a) Preparation of biocatalyst Containing 6 g of glucose, 6 g of corn steep liquor and 6 g of spray-dried lard water. In a culture medium in a shake flask, the Altrobacter simplex (ATCC6
946) was grown. The culture medium was cultivated on a rotary shaker at 28 ° C. until the disappearance of glucose occurred. At this point, cortisone acetate (0.5 g /) was added to induce steroid-1-dehydrogenase synthesis. After culturing overnight, cells were introduced by low speed centrifugation. The bacterial pellet was placed in a low vacuum oven at 55 ° C for 24 hours for drying. After 24 hours, the dried material was transferred to an airtight container and stored at 5 ° C until needed for bioconversion.

b) Bioconversion of androsta-4,9 (11) -diene-3,17-dione 10 g / dry cell was hydrated by stirring for 30 minutes in 50 mM phosphate buffer of pH 7.5. Then 86 mg of menadione as a dry powder
The final concentration of / was added to the bacterial suspension. Finely ground antrosta-4,9 (11) -diene-3,17-dione was slurried in dimethylformamide to give a steroid 2.
5 g and 2% (v / v) DMF were added to the bioconversion mixture. Stir the mixture at 31 ° C in the presence of air for 24
Incubated for hours. After cultivation, Androsta-1,4,9 (11)
-Triene-3,17-dione was recovered by conventional means.

Example 3 Following the conditions described in the previous example, the steroid compounds below were exposed to dry cells of A. simplex to give the corresponding 1,2
A dehydro derivative was obtained.

No. Name 1. Androst-4-ene-3,17-dione 2. 6α-Fluoro-androsta-4,9 (11) -diene-3,17-dione 3.6α-Methyl-androsta-4, 9 (11) -diene-
3,17-dione 4.16β-methyl-androsta-4,9 (11) -diene-
3,17-dione 5.17α-hydroxypregne-4-en-20-yne-
3-one 6.17 α-hydroxypregna-4,9 (11) -diene-20
-In-3-one 7.17α-hydroxy-16β-methyl-pregna-4,9
(11) -Dien-20-yn-3-one 8.11β, 21-dihydroxy-pregna-4,17 (20) -dien-3-one 9.21-acetoxy-11β-hydroxy-pregna-4,
17 (20) -Dien-3-one 10.6α-methyl-11β, 21-dihydroxy-pregna-4,17 (20) -dien-3-one 11.20-chloro-pregna-4,9 (11) , 17 (20) -trien-21-ar-3-one 12.hydrocortisone 13.6α-methylhydrocortisone 14.21-acetoxy-11β, 17-dihydroxy-16β
-Methyl-pregna-4-ene-3,20-dione 15.21-acetoxy-9α-fluoro-11β, 17-dihydroxy-16β-methyl-pregna-4-ene-3,20
-Dione 16.21-acetoxy-9β, 11β-epoxy-17-hydroxy-16β-methyl-pregne-4-ene-3,20-
Dione 17.21-acetoxy-17-hydroxy-pregna-4,9
(11) -Diene-3,20-dione 18.21-acetoxy-16α. 17-Dihydroxy-pregna-4,9 (11) -diene-3,20-dione 19.21-acetoxy-17-hydroxy-16α-methyl-pregna-4,9 (11) -diene-3,20-dione 20.21-Benzyloxy-17-hydroxy-16β-methyl-pregna-4,9 (11) -diene-3,20-dione 21.21-acetoxy-17-hydroxy-16β-methyl-pregna-4,9 ( 11) -Diene-3,20-dione 22.21-acetoxy-pregna-4,9 (11), 16 (17) -triene-3,20-dione 23.21-acetoxy-6α-fluoro-pregna-4 , 9
The corresponding products obtained from the (11), 16-triene-3,20-dione conversion are:

No. Product 1a Androsta-1,4-diene-3,17-dione 2a 6α-fluoro-androsta-1,4,9 (11) -triene-3,17-dione 3a 6α-methyl-androsta- 1,4,9 (11) -triene-3,17-dione 4a 16β-methyl-androsta-1,4,9 (11) -triene-3,17-dione 5a 17α-hydroxy-pregna-1,4 -Diene-20
-In-3-one 6a 17α-hydroxy-pregna-1,4,9 (11) -trien-20-in-3-one 7a 17α-hydroxy-16β-methyl-pregna-1,
4,9 (11) -trien-20-in-3-one 8a11β, 21-dihydroxy-pregna-1,4,17 (20)
-Trien-3-one 9a21-acetoxy-11β-hydroxy-pregna-
1,4,17 (20) -trien-3-one and 11β, 21-dihydroxy-pregna-1,4,17 (20) -trien-3-one 10a 6α-methyl-11β, 21-dihydroxy-pregna 1,4,17 (20) -trien-3-one 11a 20-chloro-pregna-1,4,9 (11), 17 (20) -tetraen-21-ar-3-one 12a prednisolone 13a 6α-methyl -Prednisolone 14a 21-acetoxy-11β, 17-dihydroxy-16β
-Methyl-pregna-1,4-diene-3,20-dione, and 11β, 17,21-trihydroxy-16β-methyl-pregna-1,4-diene-3,20-dione 15a21-acetoxy-9α -Fluoro-11β, 17-dihydroxy-16β-methyl-pregna-1,4-diene-
3,20-dione and 9α-fluoro-11β, 17,21-trihydroxy-16β-methyl-pregna-1,4-diene-
3,20-dione 16a 21-acetoxy-9β, 11β-epoxy-17-hydroxy-16β-methyl-pregna-1,4-diene-3,2
0-dione and 9β, 11β-epoxy-17,21-dihydroxy-16β-methyl-pregna-1,4-diene-3,20-
Dione 17a 21-acetoxy-17-hydroxy-pregna-1,
4,9 (11) -triene-3,20-dione and 17,21-dihydroxy-pregna-1,4,9 (11) -triene-3,20-dione 18a 21-acetoxy-16α, 17-dihydroxy- Pregna-1,4,9 (11) -triene-3,20-dione and 16α, 1
7,21-Trihydroxy-pregna-1,4,9 (11) -triene-3,20-dione 19a 21-acetoxy-17-hydroxy-16α-methyl-pregna-1,4,9 (11) -triene -3,20-dione and 1
7,21-Dihydroxy-16α-methyl-pregna-1,4,9
(11) -Triene-3,20-dione 20a 21-benzyloxy-17-hydroxy-16β-methyl-pregna-1,4,9 (11) -triene-3,20-dione 21a 21-acetoxy-17-hydroxy -16β-Methyl-pregna-1,4,9 (11) -triene-3,20-dione and 1
7,21-Dihydroxy-16β-methyl-pregna-1,4,9
(11) -Triene-3,20-dione 22a 21-acetoxy-pregna-1,4,9 (11), 16-tetraene-3,20-dione and 21-hydroxy-pregna
1,4,9 (11), 16-tetraene-3,20-dione 23a21-acetoxy-6α-fluoro-pregna-1,
4,9 (11), 16-Tetraene-3,20-dione and 6α-Fluoro-21-hydroxy-pregna-1,4,9 (11), 16-tetraene-3,20-dione Example 4 Two Different steroids were bioconverted by A. simplex cells in the fermentation broth and dried A. simplex cells. After each bioconversion was judged to be complete (indicated by no further reduction of residual substrate level), the steroids of the mixture were extracted and isolated, yield data for each condition was obtained. The table below summarizes these results.

(a) The definition of a useful steroid is either a Δ ¹ compound that can be used in subsequent syntheses or a 1,2-dihydro substrate that can be recycled to another biotransformation to make the product.

(b) Significant amounts of other undesired steroid molecules were also recovered.

Example 5 Altrobacter simplex was grown in a 5 "microfarm" fermentor to induce the synthesis of steroid-1-dehydrogenase and harvested by centrifugation. A portion of the recovered fungal paste was subjected to two different separation methods. The first is the procedure described herein, which is to dry the cells under reduced pressure at 55 ° C. The second method is US Pat. No. 3,360,43 for the preparation of acetone dried cells.
It was the procedure recommended in No. 9. The fungus was mixed with acetone, harvested and dried under reduced pressure at 5 ° C. The dry fungus preparation was then used to bioconvert the Δ ^{9,11 -androstenedione} in a shake flask with 10 g of fungus per 10 g of steroid.

Example 6 Instead of the 6α-methylhydrocortisone of Example 1 or the androsta-4,9 (11) -diene-3,17-dione of Example 2, the substrates listed below were used, with corresponding enumeration The product obtained is obtained.

Substrate 1.2 1-acetoxy-9α-fluoro-11β, 16α, 17
-Trihydroxy-pregna-4-ene-3,20-dione 2.21-acetoxy-6α, 9α-difluoro-11β,
16α, 17-trihydroxy-pregna-4-ene-3,20
-Dione-16,17-acetonide 3.21-acetoxy-6α-fluoro-11β-hydroxy-16α-methyl-pregna-4-ene-3,20-dione 4.21-acetoxy-6α-fluoro-11β, 17 -Hydroxy-pregna-4-ene-3,20-dione 5.21-acetoxy-6α, 9α-difluoro-11β,
17-dihydroxy-16α-methyl-pregna-4-ene-3,20-dione 6.21-acetoxy-9α-fluoro-11β, 16α, 17
-Trihydroxy-pregna-4-ene-3,20-dione-16,17-acetonide 7.2 1-acetoxy-9β, 11β-epoxy-6α-fluoro-16α, 17-dihydroxy-pregna-4-ene-3 , 20-dione-16,17-acetonide 8.21-acetoxy-9β, 11β-epoxy-16α-
Hydroxy-pregna-4-ene-3,20-dione 9.21-acetoxy-9β, 11β-epoxy-16α, 17
-Dihydroxy-pregna-4-ene-3,20-dione-
16,17-Acetonide Product 1.21-acetoxy-9α-fluoro-11β, 16α, 17
-Trihydroxy-pregna-1,4-diene-3,20-dione and 9α-fluoro-11β, 16α, 17,21-tetrahydroxy-pregna-1,4-diene-3,20-dione 2.21- Acetoxy-6α, 9α-difluoro-11β,
16α, 17-trihydroxy-pregna-1,4-diene-
3,20-dione-16,17-acetonide and 6α, 9α-fluoro-11β, 16α, 17,21-tetrahydroxy-pregna-1,4-diene-3,20-dione 16,17-acetonide 3.21 -Acetoxy-6α-fluoro-11β-hydroxy-16α-methyl-pregna-1,4-diene-3,20-dione and 6α-fluoro-11β, 21-dihydroxy-16
α-Methyl-pregna-1,4-diene-3,20-dione 4.21-acetoxy-6α-fluoro-11β, 17-hydroxy-pregna-1,4-diene-3,20-dione and 6
α-fluoro-11β, 17,21-trihydroxy-1,4-diene-3,20-dione 5.21-acetoxy-6α, 9α-difluoro-11β,
17-dihydroxy-16α-methyl-pregna-1,4-diene-3,20-dione and 6α, 9α-difluoro-11
β, 17,21-trihydroxy-1,4-diene-3,20-dione 6.21-acetoxy-9α-fluoro-11β, 16α, 17
-Trihydroxy-pregna-1,4-diene-3,20-dione-16,17-acetonide 7.21-acetoxy-9β, 11β-epoxy-6α-fluoro-16α, 17-dihydroxy-pregna-1,4 -Diene-3,20-dione-16,17-acetonide 8.21-acetoxy-9β, 11β-epoxy-16-hydroxy-pregna-1,4-diene-3,20-dione and 9
β, 11β-epoxy-16α, 21-dihydroxy-pregna-1,4-diene-3,20-dione 9.21-acetoxy-9β, 11β-epoxy-16α, 17
-Dihydroxy-pregna-1,4-diene-3,20-dione-16,17-acetonide Example 7 Biotransformation of 11β-hydroxy-androsta-4-ene-3,17-dione described in Example 2. 1 g of the dried A. simplex cells thus prepared was resuspended in 100 ml of 50 mM phosphate buffer having a pH of 7.5. Menadione dissolved in 3A ethanol was added to a final concentration of 86 mg /. 0.5 g of 11β-hydroxy-androstenedione was added to the flask. The mixture was cultivated on a rotary shaker at 31 ° C. Two products were obtained by thin layer chromatography of the methylene chloride extract when harvested after 1 day of culture. About 95% of the steroids were present as 11β-hydroxy-androsta-1,4-diene-3,17-dione. The rest was unconverted substrate.

Example 8 According to the conditions of Example 7, the following androstenedione derivative with modified C-11 position can be 1-dehydrogenated to the following product.

Substrate → Product (1) 11β-hydroxy-16β-methyl-androsta-
4-ene-3,17-dione → 11β-hydroxy-16β-methyl-androsta-1,
4-diene-3,17-dione (2) 11β-hydroxy-16α-methyl-androsta-
4-ene-3,17-dione → 11β-hydroxy-16α-methyl-androsta-1,
4-diene-3,17-dione (3) 6α-fluoro-11β-hydroxy-androsta-4-ene-3,17-dione → 6α-fluoro-11β-hydroxy-androsta-
1,4-diene-3,17-dione (4) 6α-methyl-11β-hydroxy-androsta-
4-ene-3,17-dione → 6α-methyl-11β-hydroxy-androsta-1,
4-diene-3,17-dione (5) 11α-hydroxy-androst-4-ene-3,17
-Dione → 11α-hydroxy-androsta-1,4-diene-3,1
7-dione (6) Androsta-4-ene-3,11,17-trione → Androsta-1,4-diene-3,11,17-trione Example 9 1) In reaction vessel (1) Then put the following together.

a) 50 mM KPO ₄ buffer (pH 7.5) 0.9 b) A. simplex dried cells 3.66 g c) menadione 0.08 g d) 21-acetoxy-pregna-4,9 (11), 16-triene-3,20- Dione 8g e) Toluene 100ml 2) Stir at 15cal / min.

3) Air is sent as needed to keep oxygen at about 3-6% in the gas space of the reaction vessel.

5) After 25 hours, collect the toluene phase.

In this example, the steroids in the toluene phase were as follows.

94.3% 21-acetoxy-pregna-1,4,9 (11), 16-tetraene-3,20-dione 4.2% 21-hydroxy-pregna-1,4,9 (11), 16-tetraene-3,20 -Dione 1.5% 21-acetoxy-pregna-4,9 (11), 16-triene-3,20-dione The following range of variables can be used for the above steps.

Step 1) a) The buffer pH can be in the range of 6-10.

b) Reduce the amount of A. simplex to a level sufficient to lead to the end of the reaction. Typical levels are 0.05 to 1.0 g / g steroid. The microbial source of the enzyme is Arthrobacter (Coronebacterium) simplex (ATCC6946) or bacterium cyclooxidans (A
TCC12673).

Microbial Preparation: A. simplex cells are used in fermentation broth as is well known in the art, or cells are harvested, dried and modified by acetone treatment or fixation.

c) The electron acceptor can be selected from: Menadione (2-methyl-1,4-naphthoquinone), menadione bisulfite, 1,4-naphthoquinone and other vitamin K-type compounds The electron acceptor level is determined by price and kinetics considerations. The reaction rate is linearly proportional to the concentration of menadione. Typical levels are about 5 to about Kg steroids
It is 40 g.

d) The steroid concentration should be as high as possible, as long as there is efficient conversion.

Steroids are 3-keto-Δ ⁴ -androstene or 3-
It belongs to the keto-Δ ⁴ -pregnene system and preferably has a solubility of 5 g / or more in an organic solvent.

e) The aromatic hydrocarbon is selected from toluene, benzene or xylene. These solvents may be used together or may be diluted with another water immiscible solvent such as heptane or methylene chloride.

The amount of aromatic hydrocarbons is required to dissolve the most sparingly soluble starting or product steroid from about half the amount needed to dissolve the most readily soluble starting or product steroid. It is in the range of up to about 4 times.

In this example, toluene was added at a level sufficient to dissolve the steroid at the end of the reaction.

It is preferred to add the aromatic hydrocarbon together with the steroid. However, the aromatic hydrocarbon can be added later or at an earlier time.

Step 2) Stir with as high a stirring force as possible. The reaction rate is a strong function of stirring force.

Step 3) The reaction requires oxygen as the final hydrogen (electron and proton) acceptor. However, due to the presence of aromatic hydrocarbons,
An explosive environment exists in the gas space of the reactor. To overcome this disaster, it is advantageous to keep the oxygen concentration in the gas space well below the minimum oxygen content for combustion. In the case of benzene, the minimum oxygen content is 11.2%. In the case of xylenes, it is possible to carry out the reaction at a temperature lower than the flash point.
The flash points of m-xylene, o-xylene and p-xylene are 29, 32 and 39 ° C, respectively.

Step 4) The reaction temperature may be about 0 to about 45 ° C.

Step 5) The reaction is typically run for 2 days. It can react for hours to weeks.

Reference Example 10 Comparison of Toluene Procedure and Aqueous Procedure (A) Procedure Combine the following:

A. Simplex cells 0.4 g 50 mM KPO ₄ buffer (pH 7.5) 50 ml Stir for 2 hours or until a homogeneous slurry is obtained.

Take two 25 ml fractions and place in a 125 ml flask.

Add the following to each flask:

50 mM menadione in 3A alcohol 0.25 ml Androsta-4,9 (11) -diene-3,17-dione 0.2 g To one of the stoppered flasks is added 2 ml of toluene.

Wrist movement (list action) Shake for 4 days.

After 4 days, extract with 25 ml of toluene.

Analyze extracts.

(B) Result Example 11 According to the conditions described in Example 9, the following steroid compounds were exposed to the dried cells of A. simplex, and the corresponding 1,2
A dehydro derivative was obtained.

No. Name 1. Androst-4-ene-3,17-dione 2. 6α-Fluoro-androsta-4,9 (11) -diene-3,17-dione 3.6α-Methyl-androsta-4, 9 (11) -diene-
3,17-dione 4.16β-methyl-androsta-4,9 (11) -diene-
3,17-dione 5.17α-hydroxypregne-4-en-20-yne-
3-one 6.17 α-hydroxypregne-4,9 (11) -diene-20
-In-3-one 7.17α-hydroxy-16β-methyl-pregna-4,9
(11) -Dien-20-yn-3-one 8.21-acetoxy-11β-hydroxy-pregna-4,
17 (20) -dien-3-one 9.20-chloro-pregna-4,9 (11), 17 (20) -trien-21-al-3-one 10.21-acetoxy-17-hydroxy-pregna −4,9
(11) -Diene-3,20-dione 11.21-acetoxy-17-hydroxy-16α-methyl-pregna-4,9 (11) -diene-3,20-dione 12.21-benzoyloxy-17-hydroxy -16β-Methyl-pregna-4,9 (11) -diene-3,20-dione 13.21-acetoxy-17-dihydroxy-16β-methyl-pregna-4,9 (11) -diene-3,20- Dione 14.21-acetoxy-pregna-4,9 (11), 16-triene-3,20-dione 15.21-acetoxy-6α-fluoro-pregna-4,9
The corresponding products obtained from the (11), 16 (17) -triene-3,20-dione conversion are:

No. Product 1a Androsta-1,4-diene-3,17-dione 2a 6α-fluoro-androsta-1,4,9 (11) -triene-3,17-dione 3a 6α-methyl-androsta- 1,4,9 (11) -triene-3,17-dione 4a 16β-methyl-androsta-1,4,9 (11) -triene-3,17-dione 5a 17α-hydroxypregna-1,4 -Diene-20-
In-3-one 6a 17α-hydroxypregna-1,4,9 (11) -trien-20-in-3-one 7a 17α-hydroxy-16β-methyl-pregna-1,
4,9 (11) -trien-20-yn-3-one 8a21-acetoxy 11β-hydroxy-pregna-1,
4,17 (20) -trien-3-one and 11β, 21-dihydroxy-pregna-1,4,17 (20) -trien-3-one 9a 20-chloro-pregna-1,4,9 (11) , 17 (20) -Tetraen-21-al-3-one 10a 21-acetoxy-17-hydroxy-pregna-1,
4,9 (11) -triene-3,20-dione and 17,21-dihydroxy-pregna-1,4,9 (11) -triene-3,20-dione 11a 21-acetoxy-17-hydroxy-16α- Methyl-pregna-1,4,9 (11) -triene-3,20-dione and 1
7,21-Dihydroxy-16α-methyl-pregna-1,4,9
(11) -Triene-3,20-dione 12a21-Benzyloxy-17-hydroxy-16β-methyl-pregna-1,4,9 (11) -triene-3,20-dione 13a21-acetoxy-17-hydroxy -16β-Methyl-pregna-1,4,9 (11) -triene-3,20-dione and 1
7,21-Dihydroxy-16β-methyl-pregna-1,4,9
(11) -Triene-3,20-dione 14a 21-acetoxy-pregna-1,4,9 (11), 16-tetraene-3,20-dione and 21-hydroxy-pregna
1,4,9 (11), 16-tetraene-3,20-dione 15a21-acetoxy-6α-fluoro-pregna-1,
4,9 (11), 16-tetraene-3,20-dione and 6α-fluoro-21-hydroxy-pregna-1,4,9 (11), 16-tetraene-3,20-dione 1,2-dehydro The usefulness of steroids is well known. See, for example, U.S. Pat. No. 3,284,447. This patent reveals the utility of Δ 1,4,9 ⁽¹¹⁾ pregnenetrienes in the synthesis of carbon 16 substituted diuretic corticosteroids. U.S. Pat.No. 4,041,055 has Δ ^1,4 −
The method for the synthesis of corticosteroids from androstenedione derivatives is clarified, and the usefulness of 1,2-dehydroandrostenses is shown as an important intermediate in the production of medically useful steroids.

Claims (7)

[Claims] 1. A 1,2-saturated 3-ketosteroid, which comprises exposing a 1,2-saturated steroid to air-dried or heat-dried cells of a microorganism having a 1-dehydrogenation action. 2
-A method of converting to dehydrosteroids. 2. The method according to claim 1, wherein the air-dried or heat-dried microbial cells have a water content of about 1 to about 10%. 3. A microorganism having a 1-dehydrogenating action is Arthrobacter simplex or Bacterium cyclooxyd.
ans), the method according to claim 1. 4. In the presence of an exogenous electron carrier, 1-
Conversion of 1,2-saturated steroids to 1,2-dehydrosteroids consisting of exposing 1,2-saturated steroids to air-dried or heat-dried bacterial cells of dehydrogenating microorganisms The method according to claim 1. 5. An exogenous electron carrier is menadione, phenazine methosulfate, dichlorophenol indophenol, 1,4-naphthoquinone, menadione bisulfite, ubiquinones (coenzyme Q) or vitamin K type compounds. Method according to section 4 of 6. The air-dried or heat-dried microbial cell has a water content of about 1 to about 10%.
Method by paragraph. 7. The method according to claim 4, wherein the microorganism having a 1-dehydrating action is Arthrobacter simplex or bacterium cyclooxidans.