DE1493095C - Process for the production of 3 keto delta high 1.4 steroids of the androstane or pregnane series - Google Patents

Description

The present invention relates to a process for the preparation of 3-keto-! '^ - steroids of the androstane or pregnane series. In particular, the subject of the present invention is a microbiological process by means of which using the new microorganism Nocardia lucida n. Sp. a double bond in position 1 of a 3-ketol ⁴ steroid of the androstane and pregnane series can be introduced. Nocardia lucida n. Sp. was deposited with the Institute of Microbiology of the Rutgers University (USA) with the index number IM 3890 and with the Commonwealth Mycological Institute, Ferry Lane, Kew (Surrey, England) with the index number IMI 113.416.
. It is known that numerous microorganisms of the genus Nocardia are able to introduce a double bond in position 1 into the steroid molecule (British patent 874 188, German patent 1 093 792, French patent 1 270 506, Indian patent 62 483). However, one cannot conclude from this that all microorganisms of the above-mentioned genus cause said dehydration, since it has been proven that the strains of Nocardia rugosa, N. Brasiliensis, N. caprae and N. frabozae are unable to introduce the double bond in position 1.

The new microorganism used in the process according to the invention, which is also called Nocardia lucida n. Sp. belongs to the genus Nocardia Trevisan and produces enzymes which cause dehydration in position 1 of the 3-keto / l ⁴ -steroids of the androstane or pregnane series, the corresponding "!" - ^ - steroids with high conversion yields can be obtained.

The new microorganism also has the surprising property that it acts as a carbon source in addition to the usual carbohydrates, mixtures of hydrocarbons with preferably Can utilize 12 to 18 carbon atoms.

Microscopic appearance

The new microorganism N. lucida forms on

ίο Potato Glucose Agar initially a mycelium with very long (25 to 30 μ x 1.0 to 1.5 μ), often crooked, poorly branched, unseptate hyphae. After about For 24 hours the hyphae become shorter and septate, and the first fragments appear as bacteroid-like Elements about 3 x 0.8 μ in diameter.

A complete fragmentation into coccoid-like elements of about 1 μ in diameter is only possible around the 40th hour proven. Such coconut-like elements often appear to be characteristically arranged.

The formation of aerial mycelium could not be detected in any culture medium. The mycelium is gram-positive and not resistant to acid. The microorganism grows well at a temperature of 28 to 30 ^° C. and does not produce any soluble pigments on the usual nutrient media.

Macroscopic appearance

In the table, the observed at the indicated culture soil properties are given, the 'observations were made after a 7-day incubation at 28 to 30 ⁰ C.

Culture medium growth sparse color Remarks Czapek agar ¹ ) plentiful, smooth, from whitish to light pink Bennet agar ¹ ) shiny patina with Salmon pink clean edge no growth . < Glycine-Glycerin-Agar ¹ ) very sparse Glycerine peptone Pink, very bright Potato agar ¹ ) plentiful, smooth, Emerson agar shiny patina Salmon pink no growth Starch agar and Pridham Salts ² ) plentiful, smooth, ". Glucose agar shiny patina Salmon pink Yeast extract ¹ ) plentiful, smooth, Glucose Potato Agar ³ ) shiny patina Salmon pink sparse Nutrient agar ¹ ) moderate Pale pink Meat extract starch
Pentnn-Aßfar ¹ ) moderate very light pink no starch hydrolysis X \ * LJ LKJ km xlgUl I
Gelatin agar ¹ ) well, dark broth, pink very light pink no gelatin liquefaction Yeast broth ¹ ) Precipitation on the Light pink floor sparse milk colorless the milk becomes alkaline, no coagulation

') Wa k s m a n, p. Α., "The Actinomyces", Vol. II, pp. 328 to .134.

^z ) P ridham, T. C, "Antibiotics Annual", 1956/1957, pp. 947 to 953.

³ J glucose potato agar: 200 g peeled potatoes, 20 g glucose, 20 g agar, distilled water, 1000 ml at pH 7.2.

Biochemical properties, ^.

Starch: no hydrolysis; a)

Gelatin: no liquefaction; b) nitrates: reduction to nitrites; good recovery

the nitrates for growth; 5 c)

Milk: the milk becomes alkaline; no peptoni- d)

ization, no coagulation; . 7. N.

Melanin: hardly detectable formation; a) indole: no formation;

Tyrosine: tyrosine is broken down; io b)

Generation of H ₂ S: positive lead acetate reaction; c)

Acid production from carbohydrates: positive from d)

Glucose, fructose, sorbitol, glycerin and mannitol, 8. N.

negative from xylose, maltose, lactose, sucrose, a)

Arabinose and inositol. 15 b)

The above hydrocarbons can c)

can be used well for breeding. Phenol becomes 9th N.

not recovered. a)

Identification of the tribe,.

The description of the examined microorganism- c)

mus corresponds to the genus Nocardia Trevisan d)

(Bergey's Manual of Determinative Bacteriology— 10. N.

Seventh Edition 1957, pp. 713 to 715). a)

From the analytical key of the Nocardia- 25 b) genus by Waksman and H e η r i c i (Wa k s-

man, S.A., "The Actinomycetes", Vol. II, 1961). c)

results for the examined microorganism d)

a systematic 11th N.

Position. Nevertheless, Nocardia lucida 3 ° a) differs from the strain Nocardia rubra in several ways

Properties. In the following, the at b)

Comparison of the properties of Nocardia lucida c) n. Sp. obtained with those of the known species

Information given. 35

The new strain Nocardia lucida distinguishes d)

from: e)

1. Nocardia rubra:

a) because he does not have a mycelium with a dull, wrinkled patina 12. N. a vivid red color, 4 ° a)

b) because it forms hyphae longer than 10 to 15 μ,

c) because it forms acids from sorbitol. b)

2. N. opaca: c)

a) because the mycelium on glycerine potato agar d) is not dry, rough, folded and leather-colored, 45 e)

b) because there are no clumps of on potato agar
Forms aerial hyphae, 13th N.

c) because it does not utilize phenol, a)

d) because he has much longer hyphae (up to 25 to 30 μ). b) forms. 50 c)

3. N. polychromogenes: d)

a) because it does not form a whitish aerial mycelium,

b) because it is not 70 to 100 μ long, very ver. 14. N. forms branched threads, a)

c) because it does not hydrolyze the starch. 55 b)

4. N. globerula:

a) because it does not form threads 1 · 2 to 9 μ, c)

b) because the mycelium is not colored orange or brown-orange d), 15. N.

c) because it reduces nitrates. 6 ° a)

5. N. coeliaca: b)

a) because he does not have short rods of 0.8 · 5 μ

forms, c)

b) because it does not form dry 1 6. N. kenes folded mycelium with orange ^{color on glycerine potato agar, 6} 5 a)

c) because it reduces nitrates,

d) because he has acids from glucose and glycerin

forms. b)

blackwellii:

because it does not form a white aerial mycelium,
because it does not form colorless colonies on potato agar,

because it forms acids from mannitol and sorbitol, because it reduces nitrates,
asteroides:

because it does not form mycelium of yellow or orange color,

because it does not form a white aerial mycelium,
because it forms acids from mannitol,
because it breaks down the crystals of tyrosine,
leishmanii:

because it does not form aerial mycelium,
because he does not coagulate the milk,
because it is acid-proof.
formica:

because the hyphae of the mycelium fragment into cocoid-like shapes,
because it does not form aerial mycelium,
because it does not form a brown soluble pigment, because it uses paraffin,
gardneri:

because it does not liquefy gelatine,
because it forms acids from glucose or mannitol,

because it has no cream-colored mycelium,
because it does not form aerial mycelium,
crythropolis:

because he hyphae longer than 11 μ (up to 25 to 30 μ) having,

because it forms cokcoid-like elements,
because it does not form dry, wrinkled, orange-colored mycelium on glycerine potato agar,

because it reduces nitrates to nitrites,
because it forms acids from glucose and glycerine,
rugosa:

because it has no mycelium with a thick, folded, cream-colored layer,
because he does not coagulate milk,
because it does not liquefy gelatine,
because it reduces nitrates,
because it does not form acids from arabinose and produces acids from sorbitol,
brasiliensis:

because it does not form a leather-colored aerial mycelium, because it does not liquefy gelatine,
because it does not form acids from inositol,
because it does not form folded, yellow-colored mycelium on glucose-yeast extract agar,
caprae:

because it does not form aerial mycelium,
because it does not form brown soluble substances,

because he does not coagulate milk,
because he uses paraffin,
petroleophila:

because it does not form a white aerial mycelium,
because it does not form yellow-white colonies on peptone meat extract agar,
because it grows on potatoes, corallina:

because there is no raised, dry mycelium with yellowish brown on glycerine potato agar or coral wrinkles, because it forms hyphae longer than 10.μ (up to 25 to 30 μ),

c) because he does not use phenol,

d) because there is no pink layer on the milk surface forms,

e) because it forms acids from glucose.
17. N. salmonicolor:

a) because on peptone meat extract agar there is no folded, yellow, orange-red Mycelium has faded color,

b) because it forms acids from glucose and glycerine,

c) because there is no raised, warty, brittle, shiny, leather-colored mycelium on potatoes, the color of which then changes to orange and blood-red color.

From this it can be concluded that the microorganism examined is different from the previously known Species is different, and therefore the name Nocardia lucida n. Sp. given.

The microorganism N. lucida can be preserved by freeze drying, using milk or Milk serum can be used as a suspending agent. He can also by successive Cultivation can be kept on glucose-potato agar.

The present invention is a process for the preparation of 3-keto-steroids of the androstane I ¹ ^ pregnane or by incubation of a corresponding 3-keto-l ⁴ -steroid with a culture of the genus Nocardia and under submerged aerobic conditions in a carbon -, nutrient medium containing nitrogen source and mineral salts, which is characterized in that the incubation with a culture of Nocardia lucida n. Sp. is carried out. It is the 3-keto-I extracted resulting ¹ "^ steroid in a known manner.

As a carbon source, starch, dextrin, molasses can be used. Sucrose, glucose, maltose, glycerin, Vegetable oils, cereal and legume flour, cereal softening and other substances customary therefor be used. In addition to some of the nitrogen-containing substances mentioned above, the nitrogen source is used also casein, ammonium salts, peptones, meat and fish meal or others for this Purpose commonly used substances in question. The inorganic salts can be sulfates, phosphates, . Chlorides or nitrates of sodium, potassium, magnesium, iron or cobalt as well as calcium carbonate. The use of surface-active substances such as "Tween 80" (registered trademark for polyoxyethylene sorbitan monooleate), is also useful.

It has also surprisingly been found that the above-mentioned carbon sources completely can be replaced by crude gas oil-paraffin mixtures. The microorganism preferably uses Mixtures of hydrocarbons containing 12 to 18 carbon atoms.

The microorganism is cultivated under submerged and aerobic conditions in rotary flasks or in fermenters at a temperature between 24 and 34 ° C., preferably at 30 ^° C., for a period of 30 to 50 hours. The pH of the nutrient medium must be between 7 and 8, preferably between 7.5 and 7.6. The pH value remains practically constant during fermentation.

The starting steroid can be the microorganism culture either in solid form or in a suitable one organic solvents such as methyl alcohol, ethyl alcohol, acetone, dimethylformamide or their analogues, added in solution. The fermentation takes place under the same conditions carried out for 20 to 50 hours.

The conversion of the 3-keto. l ⁴ -steroids into the corresponding 3-keto-. I ¹ - ⁴ steroid is determined by thin layer chromatography (cf. Egon Stahl, Pharmac. Weekblad, 92, 1957, p. 829; Chemiker Zeitung, 82, 1958, p. 323; Van Dam et al., J. Chromatography, 4 , 1960, p. 26). The extraction

ίο can be carried out according to known methods, the fermentation broth with a suitable solvent, such as methyl isobutyl ketone, chloroform, Methylene chloride, trichlorethylene and their analogs are extracted.

The following examples explain the process according to the invention.

example 1
l ^lj * -Pregnadien-17 «, 21-diol-3, l 1,20-trione

A 3 day old culture of N. lucida on potato glucose agar bred is used to make two 300 ml Erlenmeyer flasks, each containing 50 ml of the following nutrient medium to be inoculated:

KCl Ig

NaNO ₃ 6 g

Anhydrous MgSO ₄ 0.3 g

Ca (H ₂ PO ₄ J ₂ · H ₂ O 4.0g

FeSO ₄ · 7 H ₂ O 20 mg

ZnSO ₄ -7 H ₂ O 5 mg

CoCl ₂ • 6 H ₂ O 5 mg

Mixture 1: 1 of gas oil (specific
Weight at 15 ° C / 4 ° C = 0.8203);
Flash point 72 ° C and of raw
Paraffin (density at 15 ⁰ C = 0.891;
Density at 8O ⁰ C = 0.772); Freezing point of the resulting mixture: 46 ° C 15 ml

Tap water 1000 ml

The pH value is adjusted to 7.5 to 7.6 with dilute sodium hydroxide solution, sterilization for 30 minutes at 120 C.
The gas oil-paraffin mixture is sterilized separately and added to the culture medium under sterile conditions.

Fermenting the culture at 3O ⁰ C on a shaker at 220 rev / min and an eccentricity of cm. 6

After 40 hours, add 25 mg to each flask

r ^t -Pregnen-17u, 21-diol-3, ll, 20-trione, in 1 ml of ethanol

dissolved, introduced, and the mixture is further fermented under the same conditions for 24 hours.

The chromatographic test (thin-layer plates made of silica gel, solvent benzene to ethyl acetate 70:30) shows almost complete disappearance of the starting product and the formation of a substance, the Rf value of which corresponds to that of l'- ⁴ -pregnadiene-17 «, 21-diol-3, 1 1-20-trions is equal.

Extraction, crystallization and identification are carried out in the following way: The content the two flasks are combined, vigorously shaken out with 50 ml of petroleum ether in a separating funnel, whereupon the petroleum ether layer is discarded. the

f> 5 broth is then again with methyl isobutyl ketone extracted; the organic extract is dried over anhydrous sodium sulfate, filtered and used Evaporated dry.

The dry residue is chromatographed on 1 g of silica gel. Eluting with 5% ethyl ether-ethyl acetate gives a crystalline product which ^{melts at 230 to 234 "J} C; [α]? = + 170 ° (1% in dioxane), ). _Max = 238 πΐμ, EJ" ° _m = 435.

The product obtained was found to be the same by chromatography and on IR examination of an authentic sample of l ¹⁴ -pregnadiene-17i /, 21-diol-3, l I-20-trione. Chromatographic examination shows that the conversion is complete and the yield is high. Yield: 75%

B is ρ ie 1 2
l'- ^4- Pregnadiene-17 ", 21-diol-3, ll -20-trione

The fermentation is carried out as described in Example 1 with the difference that the nutrient medium contains glucose (60 g / l) instead of the crude gas oil-paraffin mixture. The chromatographic analysis confirms the almost complete disappearance of the starting product with the simultaneous formation of a substance with an Rf value which is equal to that of l'- ⁴ -pregnadiene-17 «, 21-diol-3, ll-20-trione. Yield: 85%

•

Example 3
I ^1-4 -Pregnadien-l 7 ", 21-diol-3,11,20-trione

The fermentation is carried out as described in Example 1 with the difference that the nutrient medium has the following composition:

Glucose 20 g

Peptone *) 5 g

Tripton *): 5 g

Yeast extract 5 g

CaCO ₃ 2.5 g

Distilled water 1000 ml

*) Trade names for enzymatically hydrolyzed meat extracts.

The chromatographic test shows the almost complete disappearance of the starting product and the formation of a product with an Rf value equal to that of l'- ⁴ -pregnadiene-17ii, 21-diol-3, l 1,20-trione. Yield: 80%.

Example 4
L ^'4 -Pregnadien-ll / i, 17 ", 21-triol-3,20 dione-

500 ml of nutrient medium of the same composition as in Example 1 are placed in 2000 ml flasks sterilized, which are provided with three breakwaters. You will be inoculated with a mycelium suspension, cultured by washing for half a day, 3 days in a Roux flask on potato-glucose agar Culture was won.

The flasks are placed on a shaker at 120 rpm and eccentricity at 28 ° C. for 36 hours incubated from 70 mm. 300 ml of that culture are used to inoculate 3 liters of one sterilized in 5-liter laboratory fermenters Nutrient medium of the same composition.

The fermentation is carried out at a stirring speed of 300 rpm with an air supply of 11 l / min at a temperature of 30 ° C.

After 48 hours of fermentation, 1.5 g of I ⁴ - products - I l /> \ 17 <i, 21 - triol - 3.20 - dione. dissolved in 60 ml of methyl alcohol, added.

Fermentation is then carried out for a further 40 hours under the same conditions. The chromatographic analysis on a thin layer of silica gel, being used as a chromatographic System used chloroform to methanol to water 92.5: 7.5: 0.5 confirms this Disappearance of the starting compound and the formation of a product whose Rf value corresponds to that of the Prednisolone is the same.

The broth is extracted with methyl isobutyl ketone, the organic extract over sodium sulfate dried and then used for. Evaporated dry. The dry residue is between 500 ml of petroleum ether distributed with a boiling range of 60 to 80 ° C and 600 ml of 80% aqueous methanol.

The exact methanol is separated off and evaporated to a small volume in vacuo.

The concentrate is extracted with 600 ml of methyl isobutyl ketone, then washed with a little water, whereupon the organic extract is evaporated to a volume of about 5 ml; then 20 ml Ethyl ether is added and the mixture is placed in a refrigerator to crystallize.

1.3 g (87%) / l'- ⁴ -pregnadiene-l Ιβ, Πα, 21-triol-3,20-dione, which melts at 240 to 241 ° C; [«]? = + 100 ° (1% dioxane), / _mflX = 242 m, x, E \ t = 415; With an authentic sample of I ^{1 <4} -Pregna ·: dien-II / U7 «, 21-triol-3,20-dione, it turns out to be identical and different from ^ -n in the chromatographic test

Example 5 9 "-Fluor-1 ^1A -pregnadiene-1 l / U7", 21-triol-3,20-dione

The procedure is as in Example 1 with the difference that when the culture has reached the age of 48 hours, 25 mg of 9 '-fluoro-, f-pregnene-11 beta, 1 7u, 21-triol · - 3,20 dione are added, which are dissolved in 1 ml of methanol in the presence of 0.1 ml of a 20% aqueous solution of polyoxyethylene sorbitan monooleate ("Tween 80"). After 40 hours of incubation, the chromatographic analysis confirms the complete disappearance of the starting product and the formation of a product whose Rf value corresponds to that of the 9fi-fluorine-. I ¹⁴ -pregnadien-l l / i, 17 ", 21-triol-3,20-dione is the same.

It is then extracted with methyl isobutyl ketone and then partitioned between 80% aqueous methanol and petroleum ether, whereupon the methanol is evaporated off and the residue is extracted again with methyl isobutyl ketone, as in Example 4. ^{The 9u-fluorol'-4-} pregnadiene-llfi, 17 ", 21-triol-3,20-dione, which melts at 274 to 275 ° C., is obtained in a 71% yield; [«] '?' = +94 (dioxane): /, = 238 ΐημ. El ¹ ;: = 404.

Example 6

9a-fluoro-1'- ⁴ -pregnadiene-1 l / i, 16u, 17 ", 21-tetrol-3,20-dione

The procedure is as in Example 1 with the only difference that if the culture is the age of 48 hours has reached 25 mg of 9 "-Fluor- f ^ -pregnen-11 ^, 16", 17 ", 21 -tetrol-3,20-trione be added in 1 ml of methanol in the presence of 0.1 ml of a 20% aqueous polyoxyethylene sorbitan monooleate solution are resolved. After 40 days of incubation, chromatographic analysis confirms the disappearance dos starting product and the formation

109 608 93

of a product whose Rf value corresponds to that of the 9 «- fluorine -. I ¹ - ⁴ - pregnadien - 11 / U6 «, 17u, 21 - tetrol-3,20-dione is the same. The extraction carried out by the same method as described in Example 4 leads to the separation of the 9a-fluorine. I ¹⁴ -pregnadienll / U6u, 17a, 21-tetrol-3,20-dione in 60% yield, which ^{melts at 260 to 266 0} C; [α]? = + 67 ° '(l% dioxane), l _max = 239 ηΐμ, EJ ^ = 385. Three thin-layer chromatograms on silica gel show that the product is identical to triamcinolone.

B e i s ρ i e 1 7

9u-fluorine-, l ^K4 -pregnadiene-l l / f, 16u, 17a, 21-tetrol-3,20-dione

The procedure is as in Example 1, with the difference that when the culture has reached the age of 30 hours, it is diluted in a ratio of 1 part of culture broth to 2 parts of sterile tap water. After dilution, 25 mg of 9 fluorine, l ⁴ -pregnene-ll £, 16a, 17u, 21-tetrol-3,20-dione, dissolved in 0.125 ml of dimethylformamide in the presence of a drop of 20% strength, are added to each flask aqueous polyoxyethylene sorbitan monooleate solution added. It is then fermented for another 40 hours. The chromatographic analysis confirms the disappearance of the starting product and the formation of a product whose Rf value corresponds to that of 9'-fluoro-, l'- ⁴ -pregnadiene-llß, 16a, 17a, 21-tetrol-3,20-dione. The 20 // hydroxy derivative is practically absent.

Extraction by the method of Example 4 leads to the separation of the 9a-fluorine, l ^1A -pregnadienll / i, 16 ', 17a, 21-tetrol-3,20-dione. Yield: 68%

35 examples

- l'- ^4- Pregnadiene-17a, 21-diol-3, ll, 20-trione

The procedure is as in Example 1 with the only difference that in the composition of the nutrient medium the NaNO ₃ and the Ca (H ₂ PO _{4 I 2 are} replaced by (NH _{4 I 2} HPO ₄ in a concentration of 4.7 g / l The chromatographic analysis confirms the complete disappearance of the starting product and the formation of a product whose Rf value ^{corresponds to that of / 1A} -Pregnadiene-17u, 21-diol-3,11,20-trione Extraction leads to the separation of the prednisone in a yield of 65% -

50 Example 9

17 "-Methyl- / l ¹ ' ⁴ -androstadien-3,17 / i-diol-3-one

The procedure is as in Example 1 with the difference that when the culture is the age of 48 hours has reached 25 mg per flask 17a-MethyI- / i ^ -androsten-Sj ^ / f-dioW-on, dissolved in 0.125 ml of dimethylformamide in the presence of a drop of a 20% aqueous polyoxyethylene sorbitan monooleate solution, be added. It is then fermented for a further 24 hours. The chromatographic Analysis confirms the formation of a product whose Rf value corresponds to that of 17 «-methyl-, l '* -androstadien-3,17 / i-diol-3-one is equivalent to.

The contents of 20 flasks are extracted with 200 ml of methylene chloride, whereupon the solvent is evaporated and the residue is recrystallized from acetone, petroleum ether. Crystals of 17 "-Methyll'- ⁴ -androstadien-3,17 / ^ - diol-3-one are obtained, which melt at 160 to 162 ° C; [u] ?, ⁰ = +85 (CHCl ₃ ); /. """= 244 πΐμ, Ej °° _m = 260; 305 ηΐμ, Ej ^ = 175. Yield: 70%.

Example 10
17 (i-methyl- l'- ^4- androstadien-17 / ^ - ol-3-one

The procedure is as in Example 1 with the only difference that when the culture has reached the age of 48 hours, 25 mg per flask of ^{17'-methyl-I 4} -androsten-17β-ol-3-one, dissolved in 0.125 ml Dimethylformamide, are added. This is followed by fermentation for a further 24 hours. The chromatographic analysis confirms the formation of a product whose Rf value ^{corresponds to that of 17a-Methyk I 14} -androstadien-17 /? - ol-3-one. The extraction gives crystals of 17a-methyl-. I ¹⁴ -androstadien-17 / i-ol-3-ons, which melt at 163 to 165 ° C; Ia] V = 0 ° (CHCl ₃ ); l _max = 245 ΐημ, E ^ = 510. Yield: 72%.

Example 11

6a, 9u-difluoro-. I ¹⁴ -pregnadien-11 / J, 17 ", 21-triol-3,20-dione

The procedure is as in Example 1 with the difference that when the culture has reached the age of 40 hours, 25 mg per flask 6a, 9a-Difluor- ^ -pregnen-li / U7u, 21-triol-3,20-dione , dissolved in 0.125 ml of dimethylformamide in the presence of a drop of a 20% aqueous polyoxyethylene sorbitan monooleate solution, are added. It is then fermented for a further 48 hours. Chromatographic analysis confirms the formation of a product with an Rf value equal to that of 6a, 9f / -difluor1'- ⁴ -pregnadiene-II / U7u, 21-triol-3,20-dione. The contents of the 20 flasks are extracted with 500 ml of methyl isobutyl ketone, the extract is washed and evaporated to almost dryness. Towards the end of the concentration, the 6u, 9u-Dinuor- I ¹⁴ -pregna- ■ diene-1 Iftl7 «, 21-triol-3,20-dione, which melts at 220 to 223 ° C; Ia] ² S = + 88 ° (1% dioxane), ?. _mux = 237 ΐημ, Ej ^ _n , = 380. Yield: 86%.

Example 12
l ^l4 -pregnadiene-3,20-dione

The procedure is as in Example 1 with the difference that when the culture has reached the age of 40 hours, 25 mg per flask progesterone dissolved in 0.2 ml of dimethylformamide in the presence of a drop of 20 "/ _o aqueous solution of polyoxyethylene The culture is then fermented for 48 hours and then worked up in the usual way, giving l'- ^4- pregnadiene-3,20-dione.

Claims (4)

Patent claims: 1. Process for the production of 3-keto-I ¹⁴ -steroids of the androstane or pregnane series by incubating a corresponding 3-ketol ⁴ -steroid with a culture of the genus Nocardia under submerged and aerobic conditions in a nutrient medium containing carbon, nitrogen and mineral salts , thereby indicates that the incubation with a culture of Nocardia lucida n. sp. accomplished will. 2. The method according to claim 1, characterized in that a mixture is used as the carbon source of aliphatic hydrocarbons of the gas oil-paraffin type, which preferably have 12 to 18 carbon atoms contains is used. 3. The method according to claim 1 or 2, characterized in that the fermentation at a Temperature between 24 and 34 ° C, preferably at 30 C, is stirred. 4. The method according to claim 1 to 3, characterized in that the fermentation at a pH between 7 and 8, preferably 7.5 to 7.6, is carried out.