JPH0824596B2 - Steroid conversion process - Google Patents

Description

Detailed Description of the Invention

[0001]

INDUSTRIAL APPLICABILITY The first case in which coticosteroids were used for therapy was in the 1950s, when cotisone acetate treatment was introduced for rheumatoid arthritis.

In subsequent studies, insertion of unsaturation at the 1,2-position of hydroco-thizone and co-thizone increased the effectiveness of the resulting steroids, prednisolone and prednisone, and reduced drug-induced salt retention. It was proved to be. Most other steroids used for the treatment of diseases which respond to coticosteroids have since been synthesized with double bonds at the 1,2-position of the steroid molecule. In 1977, two US patents were issued showing a new method for the synthesis of co-thicosteroids from sterol precursors. US Patent No. 4,035,23
No. 6 describes a method of making 9α-hydroxyandrostenedione through the fermentation of sitosterol, stigmesterol or cholesterol. US Patent No. 4,
041,055 reveals a general method for synthesizing medically useful coticosteroids from this androstene. The intermediates treated in this chemistry are 3-keto-Δ
^Can have ^{4,9 (11)} configuration.

[0003]

The prior art and the problems to be solved by the invention are as follows.
A prior art method which revealed the biological conversion of 1,2-saturated steroids to the corresponding 1,2-dehydrosteroids.

US Pat. No. 2,837,464 “Method for producing dienes by Corynebacterium” Description of 1-dehydrogenation of steroids in fermentation broth by Arthrobacter (Corynebacterium) simplex.

US Pat. No. 3,360,439 entitled "Process for Making 1-Dehydrosteroids". A. pre-treated with a lower alkanol or a lower alkanone such as acetone prior to mixing with the substrate and hydrogen carrier. Description of 1-dehydrogenation of steroids by using Simplex cells.

Charney. W. and Herzog. H., 1967, microbial conversion of steroids. Academic Press, New York
Ku, pp. 4-9, 236-261. Historical background on 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 Bi
oengineering Volume 21 1979. The authors found Nocardia rhodocrous in a mixture of 50% benzene-heptane.
The study of 1-dehydrogenation of 4-androstene-3,17-dione by Dr. 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 Arthrobacter simplex. The enzyme in N. rhodochras must be different from that produced by 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. Oxygen is an essential reagent for the 1-dehydrogenation reaction. The solvent (50% benzene-heptane) is flammable in the presence of oxygen. 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, 1119-1126, 198
0 years. The same laboratory studied the 1-dehydrogenation of hydroco-thizone 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.
10% methanol-water or 10% propylene glycol-
A water-miscible solvent such as water was chosen as the preferred solvent. The prior art did not disclose or suggest this improved method.

[0009]

In the invention of the parent application of the divisional application of the present application, air-dried or heat-dried microbial cells capable of catalyzing 1-dehydrogenation of steroids are used, and in an aqueous environment, particularly exogenous electrons are used. In the presence of the receptor, more efficient conversion of 1,2-saturated steroids to the corresponding 1,2-dehydro derivatives is obtained than is obtained by the best known prior art methods.

The present invention discloses a method involving adding an aromatic hydrocarbon as a water-immiscible solvent to an aqueous slurry comprising A. simplex cells, an exogenous electron acceptor and a steroid to be dehydrogenated. And claimed. Stir the slurry and supply oxygen to the extent that the oxygen content of the gas cavity is lower than the minimum combustion amount of oxygen,
Alternatively, the reaction is performed at a temperature lower than the flash point of the organic solvent.

These procedures are superior to the 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, A. The amount of 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. Furthermore, the solvent procedure is superior to the prior art for dehydrogenating steroids with other microbial activity such as Nocardia rhodochras. This is because the reaction catalyzed by A. simplex can be carried out with a stable and inexpensive electron acceptor like menadione.

Microorganisms The microorganisms that can be used in the present method are any of the many known microorganisms known as 1-dehydrogenating bacteria. Such microorganisms are described in Charney. W. and Herzog. H., 1967, "Microbial conversion of steroids" (Academic Press, New York). Yo
Ku), is named.

Bacteria that perform 1-dehydrogenation of steroids are
It is divided into two groups as defined in the 8th edition of "Burger's Manual of Data Mining Biology". The procedure described herein is "Part 17 Actinomycetes and Related Organisms".
Have been successfully demonstrated for species from the genera of Arthrobacter and Corynebacterium contained in. 1-Dehydrogenated species of some other genera of Part 17 such as Nocardia, Mycobacterium, Streptomyces and Bacteria 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. US Patent No. 3,065,146
The bacteria cyclooxidans, included in 1-Dehydrogenation Bacteria-ATCC No. 12673, was tested as a useful microorganism. Bacteria used extensively for 1-dehydrogenation of steroids are Arthrobacter simplex, ATCC 6
946, which is disclosed in US Pat. No. 2,837,464. As such, many of the following use this microorganism to exemplify the method. However, it should be understood that the method involves the use of any 1-dehydrogenating microorganism.

Growth of Microorganisms 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 starch, etc.). b) carbon and 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, phosphates, sulphates, manganese, copper supplied by less purified medium components (such as Corn-preca). Cobalt, molybdenum, etc. Living organisms require oxygen present in the atmosphere for growth.
The growth temperature range is 10 to 45 ° C, and 28 to 37 ° C 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 cothizone acetate was added to 0.005% (% w / v) or higher level induces the steroid-1-dehydrogenase activity of the 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 medium grown on nutrients such as lard oil rapidly initiates steroid-1-dehydrogenase synthesis, whereas glucose-grown culture medium requires substrate depletion prior to enzymatic synthesis. is there. 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 agglutination, filtration, and ultrafiltration. The isolated cells have a water content in the range of about 1 to about 10%,
It is dried under reduced pressure at 1-85 ° C (preferably 55-75 °), or by air drying while 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 transferred into a polyacrylamide gel and collagen as described in "Methods of Enzymology", Volume XLIV, 1976 (Academic Press, New York), pages 11-317. It is also prepared by immobilizing dry cells by entrapping the cells or covalently binding the cells to a polyelectrolyte 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 bacterial amount is in the range of 0.1 to 50 g per liter, and the steroid is added at a weight ratio of 0.05 to 5.0 (steroid: bacterial cell). When bioconversion is carried out in an aqueous environment, a microbial load of 8-10 g / l for 5-10 g steroid / l is preferred. To stimulate the reaction, a catalytic amount (eg 5 × 10 ^-14 M menadione) of exogenous electron carrier is added. Useful compounds are menadione (2-methyl-1,4-naphthoquinone), phenazine methosulfate, dichlorophenol-indophenol, 1,4-naphthoquinone, menadione bisulfite, ubiquinones (coenzyme Q ) And vitamin K type compounds. The mixture is incubated in the temperature range of 5-45 ° C for 0-14 days. During the culture, the mixture is contacted with molecular oxygen, preferably agitated. The 1-dehydrogenation rate typically decreases with time. Bioconversion proceeds to completion rates of 98-100% in less than 24 hours using 10 g of substrate per liter.

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. Tween 80
A surface active agent such as is added at a low concentration, for example 0 to 5%, to facilitate suspension of the steroid.

(B) Suspending bacterial cells in an aqueous buffer system,
Subsequently, ethanol, methanol, an electron carrier dissolved in acetone are added (up to 5% of the final volume).
The steroid substrate is added as a dry powder or dissolved (suspended) in a water-miscible organic solvent such as dimethylformamide, ethanol, methanol, acetone, dimethylsulfoxide or an immiscible organic solvent such as toluene. )it can.

(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. Ethano
Additions such as toluene, acetone, xylene, butyl acetate, methylene chloride or toluene give 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. It is in the range of up to about 4 times the amount required. 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 moist bacterial cake is suspended in an aqueous solution having a weak buffering degree, or the fermentation broth is diluted with the same aqueous solution, and then exogenous electron carrier, steroid and aromatic hydrocarbon are added. To do.

[0024] Useful compounds in practice of the substrate range present invention, 3-keto - △ ⁴ - belongs to △ 4-pregnene steroid of - androstene and 3-keto. It is recognized that the substrate for steroid-1-dehydrogenase has saturation between the carbons C ₁ and C ₂ 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-dione and its derivatives

The 3-keto-Δ ⁴ -pregnene steroids that can be used include the following: 1. 17α-Hydroxypregn-4-en-20-yn-3-one and its 16-methyl derivative 2.11β, 21-dihydroxy-pregna-4,17 (20) -diene-
3-one and its 6α-methyl derivative 3. 20-chloro-pregna-4,9 (11), 17 (20) -triene-21-
All-3-on 4. Several groups of 3,20-diketo- △ ⁴ -pregnene, including:

A) 11,17,21-trihydroxy compound,
For example, hydroco-thizone and its 6α-methyl derivative b) 9β, 11β-epoxy-17,21-dihydroxy compound,
For example, 9β, 11β-epoxy-17,21-dihydroxy-16β-methyl-pregne-4-ene-3,20-dione c) 3,20-diketo-4,9 (11) -pregnesene, for example
17α, 21-Dihydroxy-pregna-4,9 (11) -diene-3,20-
Diones and their 16α-methyl, 16β-methyl or 16α-hydroxy derivatives or 17α-acetate esters d) 3,20-diketo-4,9 (11), 4,9 (11), 16-pregnetrienes, for example 21-Hydroxy-pregna-4,9 (11), 16-triene-3,20-dione and its 6α-fluoro derivative

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 the prior art methods. Bioconversion was performed using the conditions detailed above.

[0031]

EXAMPLES Reference Example 1 Preparation of dry cells In a shake flask containing a medium of pH 7.0 containing cererose, peptone and Konstanch plicar (6 g / l each), bacteria cyclodextrin ( ATCC 12673) was inoculated. The culture medium was cultivated on a rotary shaker at 28 ° C until disappearance of glucose occurred. At this point, Cotisone acetate (0.5 g / l) 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-methylhydroco-thizone 0.5 g of dried bacterial cells prepared as described above was added to 50 m of pH 7.5.
It was rehydrated with stirring in 50 ml of M phosphate buffer. Menadione as an ethanol solution (8.6mg / ml ethanol)
It was added to the bacterial suspension at a level of 0.025 ml / ml. The substrate was added as a dimethylformamide (DMF) solution (100 mg of 6α-methylhydroco-thizone per ml of DMF) to a final product conversion concentration of 0.5 g / l. The mixture was incubated at 28 ° C. with stirring. After 4 hours of culture, 91% of the steroid was converted. 6α-Methylprednisolone was recovered by conventional means.

Reference Example 2 Androsta-1,4,9 (11) -triene-3,17-dione production a) Preparation of biocatalyst Glucose 6 g / l, Konstanh plicar 6 g / l and spray Altrobacta simplex (ATCC 694) was added to the medium in a shake flask containing 6 g / l of dried lard water.
6) was grown. Glucose the culture medium on a rotary shaker.
Incubation was carried out at 28 ° C. until the disappearance of smut occurred. At this point, co-inducing to induce steroid-1-dehydrogenase synthesis
Thizone acetate (0.5 g / l) was added. After culturing overnight, cells were introduced by low speed centrifugation. The fungal pellets were 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) Androsta-4,9 (11) -diene-3,17
-Bioconversion of dione. Dry cells (10 g / l) were added in 50 mM phosphate buffer (pH 7.5) 3
Stir for 0 minutes to hydrate. Menadione was then added as a dry powder to the bacterial suspension to a final concentration of 86 mg / l. Finely ground androsta-4,9 (11) -diene-3,17-dione was slurried in dimethylformamide to give steroids per liter
2.5g and 2% (v / v) DMF levels were added to the bioconversion mixture. Stir the mixture at 31 ° C. in the presence of air, 2
Cultured for 4 hours. After culturing, Androsta-1,4,9 (11)
-Triene-3,17 dione was recovered by conventional means.

Reference Example 3 According to the conditions described in the previous Reference Example, the following steroid compounds were treated with A. Exposed to the dry cells of the simplex and corresponding
A 2-dehydro derivative was obtained. Number name 1. Androsta-4-en-3,17-dione 2. 6α-Fluoro-androsta-4,9 (11) -diene-3,1
7-Zeon 3. 6α-methyl-androsta-4,9 (11) -diene-3,17-
Zeon 4. 16β-methyl-androsta-4,9 (11) -diene-3,17
-Zeon 5. 17α-Hydroxypregne-4-en-20-in-3-one 6. 17α-Hydroxypregna-4,9 (11) -dien-20-in-3-one 7. 17α-Hydroxy-16β-methyl-pregna-4,9 (11)
-Diene-20-in-3-on 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-4,1
7 (20) -dien-3-one 11. 20-Chloro-pregna-4,9 (11), 17 (20) -triene-2
1-al-3-one 12. Hydroco-Thizone 13. 6α-Methylhydroco-thizone 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 (11),
16-triene-3,20-dione

The corresponding products obtained from the conversion are: Numbered name before 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-dien-20-in-3-one 6a. 17α-Hydroxy-pregna-1,4,9 (11) -trien-20-yn-3-one 7a. 17α-Hydroxy-16β-methyl-pregna-1,4,9
(11) -Triene-20-in-3-one 8a. 11β, 21-dihydroxy-pregna-1,4,17 (20)-
Trien-3-one 9a. 21-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) -tetraene-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 15a. 21-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-3,20-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, 17,21-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 17,21-dihydroxy-16α-methyl-pregna-1,4,9 (11 ) -Trien
-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 17,21-dihydroxy-16β-methyl-pregna-1,4,9 (11 ) -Trien
-3,20-dione 22a. 21-acetoxy-pregna-1,4,9 (11), 16-tetraene-3,20-dione and 21-hydroxy-pregna-1,4,9 (1
1), 16-Tetraene-3,20-dione 23a. 21-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-
Zeon

Reference Example 4 Two different steroids were used in A. Simplex cells and dried A. Biotransformed by Simplex cells. After each bioconversion was determined to be complete (indicated by no further reduction in residual substrate level), the steroids in the mixture were extracted and isolated to give yield data for each condition. The table below summarizes these results.

[0038]

[Table 1] 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 bioconversion to produce the product. b) Significant amounts of other undesired steroid molecules were also recovered.

Reference Example 5 Altrobacter simplex was grown in a 5 l "micropharm" fermentor to induce steroid-1-dehydrogenase synthesis and harvested by centrifugation. A portion of the recovered bacterial paste was subjected to two different separation methods. The first is the procedure described in the present specification, in which the cells are dried 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. Then, using the dry fungus preparation, add 10 g / l of fungus and 1 / l in a shake flask.
The biotransformation of Δ ^{9,11 -androstenedione} was carried out with 10 g of steroid per ^gram .

A. dried by different methods Comparison of bioconversion ability of simplex cells Bioconversion activity ⁺ At the time of sampling Residual substrate (produced product g) Bacterial morphology (hours) (%) Time / bacteria g Acetone dry 1 98.7. 013 4 95.5. 011 24 84.1 ^* . 007 Heat drying 1 85.1. 149 4 23.2. 192 24 7.2 ^* . The 039 (+) bioconversion activity was calculated as follows. Resulting product g / incubation time / added cells g = activity (*) The residual level did not decrease even after further culturing.

Reference Example 6 6α-Methylhydroco-thizone of Reference Example 1 or Reference Example 2
Substituting Androsta-4,9 (11) -diene-3,17-dione with the substrates listed below gives the corresponding listed products. Substrate 1. 21-acetoxy-9α-fluoro-11β, 16α, 17-trihydroxy-pregna-4-ene-3,20-dione 2. 21-acetoxy-6α, 9α-difluoro-11β, 16α, 1
7-Trihydroxy-pregna-4-ene-3,20-dione-16,17
-Acetonide 3. 21-acetoxy-6α-fluoro-11β-hydroxy-1
6α-Methyl-pregn-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. 21-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, 1
7-trihydroxy-pregna-1,4-diene-3,20-dione-1
6,17-acetonide and 6α, 9α-fluoro-11β, 16α, 17,2
1-Tetrahydroxy-pregna-1,4-diene-3,20-dione
-16,17-acetonide 3. 21-acetoxy-6α-fluoro-11β-hydroxy-1
6α-methyl-pregna-1,4-diene-3,20-dione and 6α-
Fluoro-11β, 21-dihydroxy-16α-methyl-pregna
-1,4-diene-3,20-dione 4. 21-acetoxy-6α-fluoro-11β, 17-hydroxy-pregna-1,4-diene-3,20-dione and 6α-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-11β, 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 9β, 11β-epoxy-16α, 21-dihydroxy-pregna-1,4-diene-3,
20-dione 9.2 1-acetoxy-9β, 11β-epoxy-16α, 17-dihydroxy-pregna-1,4-diene-3,20-dione-16,17-acetonide

Reference Example 7 Biotransformation of 11β-Hydroxy-androsta-4-ene-3,17-dione A. Produced as described in Reference Example 2 1 g of dried simplex cells 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 / l. 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.

Reference Example 8 According to the conditions of Reference Example 7, the following androstenedione derivative having a 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-androsta-4-ene-3,17-dione → 11α-hydroxy-androsta-1,4-diene-3,17
-Dione (6) Androsta-4-ene-3,11,17-trione → Androsta-1,4-diene-3,11,17-trione

Example 1 1) In a reaction vessel (1 l) combine the following: a) 50 mM KPO ₄ buffer (pH 7.5) 0.9 l 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 / l min. 3) Air is sent as needed to keep oxygen at about 3-6% in the gas space of the reaction vessel. 4) Hold the temperature at 28 ° C ± 1 ° C. 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-Zeon

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) A. The amount of simplex is reduced to a level sufficient to guide the reaction to completion. Typical levels are 0.05 to 1.0 g / g steroid. The microbial source of oxygen is Arthrobacter (Corynebacterium) simplex (ATCC 6946) or Bacterium cyclooxidans (ATCC 12673).

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

C) The electron acceptor and the following can be selected. Menadione (2-methyl-1,4-naphthoquinone), menadione bisulfite, 1,4-naphthoquinone and other vitamin K type compounds. The level of electron acceptor is determined by consideration of price and reaction rate. The reaction rate is linearly proportional to the concentration of menadione. Typical levels are about 5 to about 40 g / kg steroid.

D) The steroid concentration should be as high as possible so long as there is efficient conversion. Steroids
3-keto - [delta ⁴ - androsten or 3-keto - [delta ⁴ - belongs to pregnen system, preferably with a 5 g / l or more solubility in organic solvents.

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. Aromatic hydrocarbons are needed to dissolve the most sparingly soluble starting or product steroids from about half the amount needed to dissolve the most readily soluble starting or product steroids. It is in the range of up to about 4 times the amount. 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%
Is. In the case of xylenes, it is possible to carry out the reaction at a temperature lower than the flash point. m-xylene, o-xylene and p-
The flash points of xylene are 29, 32 and 39 ° C, respectively.

Step 4) The reaction temperature can be from about 0 to about 45 ° C. Step 5) The reaction is typically run for 2 days. It can react for hours to weeks.

Example 2 Comparison of Toluene Procedure and Aqueous Procedure (A) Procedure The following are combined. 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. Two
Take two 5 ml fractions and place in a 125 ml flask. Add the following to each flask: 5 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) Stir for 4 days on a shaker. After 4 days, extract with 25 ml of toluene. Analyze extracts.

[0055] (B) Results Procedure based electrolyte aqueous toluene androsta-4,9 (11) - diene-3,17-dione 17.7% 0.00% androsta -1,4,9 (11) - triene -3, 17-dione 82.3% 100.00%

Example 3 According to the conditions described in Example 1, A. Expose the following steroid compounds to dry cells of Simplex
A 2-dehydro derivative was obtained. Number name 1. Androsta-4-en-3,17-dione 2. 6α-Fluoro-androsta-4,9 (11) -diene-3,1
7-Zeon 3. 6α-methyl-androsta-4,9 (11) -diene-3,17-
Zeon 4. 16β-methyl-androsta-4,9 (11) -diene-3,17
-Zeon 5. 17α-Hydroxypregne-4-en-20-in-3-one 6. 17α-Hydroxypregne-4,9 (11) -dien-20-in-3-one 7. 17α-Hydroxy-16β-methyl-pregna-4,9 (11)
-Diene-20-in-3-on 8. 21-acetoxy-11β-hydroxy-pregna-4,17
(20) -Dien-3-one 9. 20-chloro-pregna-4,9 (11), 17 (20) -triene-
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-Benzyloxy-17-hydroxy-16β-methyl-
Pregna-4,9 (11) -diene-3,20-dione 13. 21-acetoxy-17-hydroxy-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 (11),
16 (17) -triene-3,20-dione

The corresponding products obtained from the conversion are as follows: Number name 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-yne
-3-ON 6a. 17α-Hydroxypregna-1,4,9 (11) -triene
-20-in-3-on 7a. 17α-Hydroxy-16β-methyl-pregna-1,4,9
(11) -Triene-20-in-3-one 8a. 21-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-ar-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 17,21-dihydroxy-16α-methyl-pregna-1,4,9 (11 ) -Trien
-3,20-dione 12a. 21-Benzyloxy-17-hydroxy-16β-methyl-pregna-1,4,9 (11) -triene-3,20-dione 13a. 21-acetoxy-17-hydroxy-16β-methyl-pregna-1,4,9 (11) -triene-3,20-dione and 17,21-dihydroxy-16β-methyl-pregna-1,4,9 (11 ) -Trien
-3,20-dione 14a. 21-acetoxy-pregna-1,4,9 (11), 16-tetraene-3,20-dione and 21-hydroxy-pregna-1,4,9 (1
1), 16-Tetraene-3,20-dione 15a. 21-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-
Zeon

The usefulness of 1,2-dehydrosteroids is well known. See, for example, U.S. Pat. No. 3,284,447. This patent demonstrates the utility of Δ 1,4,9 ⁽¹¹⁾ pregnenetrienes in the synthesis of carbon 16 substituted diuretic co-ticosteroids. US Patent 4,041,
No. 055 is a compound derived from a Δ ^1,4 -androstenedione derivative.
The synthetic method of thicosteroids is clarified, and the usefulness of 1,2-dehydroandrostenses is shown as an important intermediate in the production of medically useful steroids.

 ─────────────────────────────────────────────────── ———————————————————————————————————————— Inventor Timothy Wender Evans Three Rivers West Michigan, Michigan, USA 16710 (56) References CHEM. PHARM. BULL. 23 (1975) p. 2164-2166 BIOTECHNOL. BIOEN G. , 21 (1979) p. 2133-2145

Claims (6)

[Claims] 1. A water immiscible solvent consisting of an aromatic hydrocarbon selected from toluene, xylene, and benzene, and mixtures thereof, and menadione, 1,4-naphthoquinone, menacion bisulfite and other vitamins. K
1,2-saturated steroids consisting of exposing 1,2-saturated steroids to Arthrobacter simplex or bacterium cyclooxidans in the presence of exogenous electron carriers selected from
Improved method for conversion to 2-dehydrosteroids. 2. A process according to claim 1 wherein the aromatic hydrocarbon is toluene or xylene. 3. A water-immiscible solvent consisting of aromatic hydrocarbons, which is about half the amount required to dissolve the most soluble steroid substrate or product steroid, or the steroid substrate or product steroid. A process according to claim 1 present in about 4 times the amount required to dissolve the most sparingly soluble. 4. Exposure of a 1,2-saturated steroid to an Altrobacter simplex or bacterium cyclooxidans in the presence of a water-immiscible solvent of aromatic hydrocarbons and an exogenous electron carrier, in which case combustion 1,2-saturated steroids consisting of adding oxygen to keep the oxygen concentration below the minimum oxygen requirement for
A process according to claim 1 which is an improved process for conversion to 2-dehydrosteroids. 5. Exposure of a 1,2-saturated steroid to an Altrobacter simplex or a bacterial cyclooxidans in the presence of a water-immiscible solvent of aromatic hydrocarbons and an exogenous electron carrier, in which case Consists of performing the reaction at a temperature lower than the flash point of the organic solvent,
The process of claim 1 which is an improved process for converting 1,2-saturated steroids to 1,2-dehydrosteroids. 6. The method according to claim 1, 4 or 5, wherein the water-immiscible solvent is diluted with another water-immiscible solvent.