DE1048579B - Process for the fermentative hydroxylation of the Ila position of steroids with up to 22C atoms and / or for the degradation of the 17-position side chain of these steroids - Google Patents

Description

\ f

FEDERAL REPUBLIC OF GERMANY

GERMAN

INTERNAT. KL.

C 07 C

PATENTA.MT

EXPLAINING PAPER 1048 579

U4735IVb / 12o

REGISTRATION DAY 16 AUGUST 1 95 7

NOTICE THE REGISTRATION AND ISSUE OF THE EDITORIAL: JANUARY 15, 19 5 9

The invention relates to a new method for Proactive hydroxylation of the 11 α-position of Steroids with up to 22 carbon atoms and / or to break down the 17-position side chain of these steroids.

The steroids to be treated according to the invention have the basic structure of 10,13-dimethylcyclopcntanopolyhydrophilicanthrene and must, if hydroxylation of the 11-formation is to be achieved, in this Position contain a methylene group.

The inventive method consists in that on a steroid of the type mentioned a fungus of known genus Sporotrichum can act, with steroids with an 11 methylene group in 11a position are hydroxylated. There can also be a breakdown of the 17-position side chain with the formation of a 17 / J-oxyandrostane can be achieved. Can't do any in the 11 position Oxygen function are introduced, then only occurs Degradation of the 17-position side chain.

For the degradation of the side chain, an aeration rate of at least 5 liters per minute per 100 liters of fermentation medium (5 ° / ₀ ) is required. If the aeration rate is reduced to about 1 ⁰ Z ₀ (11 air per minute per 100 liters of medium), then in the case of steroids with a 17-position side chain, selective hydroxylation of the 11-position occurs without the 17-position side chain being degraded by fermentation.

The breakdown of the permanent side chain of steroid compounds by the application of chemical measures is known. However, these are only minor. Yields obtained. In the present process, the yield is however high; the essential single procedural stage is simple, and it is advantageous that the 11 permanent Methylene group at the same time to form an 11 a -oxy steroid is oxidized. The inventive method is not only new and advantageous in the production of oxidized Steroids, the effectiveness of Sporotrichum is similar to that of other fungi in the introduction of Oxygen in the 11-hour storage of substrates and steroids, such as 1-dehydrosteroids and steroids of the androstane series think. Another advantage of steroid oxidation with Sporotrichum is that the formation of by-products compared to that of Microorganisms that also introduce hydroxyl groups is very much reduced.

In carrying out the method according to the invention, the selected steroid substrate becomes the most appropriate is dissolved in a solvent such as acetone or dimethylformamide, the action of a species of fungus Genus Sporotrichum or the oxidizing enzymes obtainable therefrom. To Clements and Shear, Genera of Fungi, Hafner Publishing Co., New York, 1954, belongs to the genus Sporotrichum to the family Moniliaceae of the order Monoliales of the class Deuteromycetes (Fungi imperfecti). To the procedure

for fermentative hydroxylation

the 11 α-position of steroids

with up to 22 carbon atoms and / or

to break down the 17-position side chain

these steroids

Applicant:

The Upjohn Company, Kalamazoo, Mich. (V. St. A.)

Representative;

Dr. W. Beil and A. Hoeppener, patent attorneys, Fraukfurt / M.-Höchst, Antoniterstr. 36

Claimed priority: V. St. v. America August 21, 1956

Peter Dietrich Meister, Kalamazoo, Mich.,

and Adolph Weintraub, Brooklyn, N.Y. (V. St. A.),

have been named as inventors

Fungi of the genus Sporotrichum examined which are suitable for the method according to the invention include the Species Sporotrichum sulfurences A.T.C.C. 7159 (catalog number of the American Type Culture Collection), Sporo-

iridium camis A.T.C.C. 11501, Sporotrichum carnis A.T.C.C. 15 507, Sporotrichum epigaeum A.T.C.C. 7145, Sporotrichum bombyeimim A.T.C.C. 7139, Sporotrichum folicola Oud., Sporotrichum pulviniforme Thorn, Sporotrichum olivaceum Fr., Sporotrichum globuliferum,

Sporotrichum maritimum, Sporotrichum anthophüum, Sporotrichum griseolum, Sporotrichum roscolum Oud. et Bcijerinck, Sporotrichum poae PK., Sporotrichum equi, Sporotrichum Beurmanni M. et Ram. and Sporotrichum Schencckii, A.T.C.C. 10 268 and the like

The fermentation technology, the media, the inoculation process as well

the additions of the substrate generally correspond to the

z. B. by Murray and Peterson in the USA.

2 721 828 and 2 602 769.

In contrast, the ventilation speed is critical;

low oxygen delivery rates, e.g. B. for Sporotrichum sulfurescens from about 0.5 to 21 air each Minute per 1001 fermentation medium only cause the introduction of oxygen into the 11 α-position, but not the simultaneous one fermentative degradation of the 17 permanent side

809 729/282

chain of pregnant women For the ferro en tative Side chain degradation involves greater oxygen delivery rates and / or higher temperatures and sometimes longer reaction times are necessary. At a Ventilation speed of 51 or more per minute each 1001 medium is a degradation of the 17-position side chain reached by pregnancies with Sporotrichum sulfurescens at 20 to 28 ° C. For the introduction of oxygen in the 11 α-position of steroids without 17 positions Side chain the ventilation speed is not critical, and it can be 0.5 to 10 or 201 air per minute 1001 medium can be used.

With regard to the hydroxylation of the 11α-position, it has been found that optimal conditions are achieved if, after inoculating the reaction vessel with the selected sporotrichum species, it is allowed to grow for 24 to 72 hours before the addition of the steroid. While shorter growing cycles can also be used, the yield will be significantly reduced. The optimum temperature during growth is 20 to 28 ^° C., but a range from 15 to 32 ^° C. can also be used. It was found to be advantageous to lower the temperature to around 20 ° C before adding the steroid. If a breakdown of the side chain is to be avoided, a reaction time of about 24 hours and a ventilation rate of 0.5 to 2 liters per minute per 100 liters of medium are most suitable for the various Sporotrichum species. If, however, a breakdown of the side chain is to be achieved in addition to the 11'-hydroxylation, or if there is no side chain at all, higher oxygen supply rates, namely 5 to 201 per minute per 1001 medium, and longer reaction times, ie 24 to 96 hours, are more advantageous.

The following examples explain the process according to the invention:

example 1

17a-methyl-1,4-androstadien-ll a, 17 £ -diol-3-one
(lla-oxy-1-dehydromethyltestosterone)

1001 of a medium containing 1% dextrose, known under the trade name Cerelose, 2% corn steep liquor (60% solids) and tap water were adjusted to a pn value of 5 with a 25% sodium hydroxide solution. Then 400 ecm of lard oil octadecanol, which contains about 1% octadecanol, was added as an antifoam agent. This medium was sterilized for 45 minutes at a bracket of 1.4 kg / cm ^a and inoculated with 61 of a 24 hour old culture of Sporotrichum sulfurescens from Beyman; this strain came from the Centraalbujeau voor Schimmelcultures, Baarn, Holland. The medium was stirred with a stirrer at a speed of 200 revolutions per minute and sterile air was supplied at a speed of 2 per minute. After 24 hours, 16 g of 1-dehydromethyltestosterone, which contained 9% methyltestosterone, dissolved in 200 ecm hot absolute ethanol, were added. The _pH value was now 4.8, the fermentation was interrupted after 24 hours; At this point in time, the pn value was 5.1. The fermentation solution was then filtered through a Knight Ware vacuum filter (50.8 cm). The tank and filter were washed with water. The mycelium on the filter was dried by sucking 121 acetone through twice and extracted twice with 121 methylene chloride each time. The filtered fermentation solution was extracted once with 241 methylene chloride, a second time with the 481 extract solution from the extraction of the mycelium cake and twice more with 241 methylene chloride each time. The combined fermentation solution extracts were washed with 121 2% sodium carbonate and then with 24 liters of water and then dried with anhydrous sodium sulfate. The dry extract was concentrated to about 11 by flat evaporation and then evaporated to dryness under a suction cup. the dry residue weighed 330 g It was dissolved in 780 cc of benzene and passed through an alumina column. (the alumina - 1000 g - was washed with acid and dried at 120 ⁰ C) were the eluate fractions listed in the table of per 780. ecm received.

fraction
No. solvent benzene volume Solids
weight Benzene - 10% ether in ecm hi g 15th 1–2 Benzene —30 7 “Ether 1560 250.49 3-4 Benzene - 50% ether 1560 6.07 5-6 Benzene - 50% ether 1560 5.61 7th ether 780 2.15 20th 8th ether 780 1.81 9 Ether - 5% chloroform 780 6.13 10 Ether - 5% chloroform 780 14.99 11 Ether -10% chloroform 780 4.08 12th Ether -10% chloroform 780 1.35 25th 13th Ether - 30% chloroform 780 0.38 14th Ether - 30% chloroform 780 0.18 15th Ether - 50% chloroform 780 0.18 16 Ether - 50% chloroform 780 0.25 17th chloroform 780 0.42 30th 18th chloroform 780 1.14 19th Chloroform - 5% acetone 780 0.74 20th acetone 780 0.25 21 Methanol 780 7.50 22nd 780 8.05 35 23 780 2.20

Fraction 22 was evaporated to give 8.05 g of solids. These were in a mixture of 100 ecm Ethyl acetate and 50 ecm of methanol dissolved. The solution obtained was nitrated and the filtrate was concentrated to 35 ecm, 5.52 g of crystals having a melting point of 249.5 to 252.5 ° C. were obtained. These were once again made 70 ecm methanol - ethyl acetate (6: 1) recrystallized. 4.03 g of crystalline 17a-methyl-1..4-androstar were thereby obtained

dien-llo, 17 _i S-diol-3-onni with a melting point of 250 to 253 ⁰ C and an optical rotation of [a] £ = -33 ° (in chloroform); A ^ = 249 and 264 ΐπμ; E 17100 and 11000; Infrared: OH, 3640 cm- ¹ , 3410 cm- ¹ ; conjugated ketone: 1660 cm ' ¹ ^ ¹ - ⁴ C = C; 1617, 1600 cm- ¹ ; the. The substance obtained is superior to that of methyltestosterone in its anabolic effectiveness.

Analysis for C ₂₀ H ₂₈ O ₃ :

Calculated .... C 75.91, H 8.92; found C 76.17, H 9.15.

Identical results are obtained using Sporotrichum sulfurescens, American Type Culture Collection No. 7159 obtained.

Example 2

(1 la-oxy-1-dehydrotestosterone) The fermentation of a dispersion of 3 g of 1,4 androstadien-17jl? -Ol-3-one (1-dehydrotestosterone) with Sporotrichum sulfurescens A / IX.C. No. 7159 in 121 medium of Example 1 and subsequent extraction in the manner indicated in Example 1 using equiva-

Lent amounts of solvent yielded 9.2 g of extraction

5 6

Substances dissolved in 220 ecm of benzene and over 350 g of alumina Analysis for C ₁₉ H ₂₁ O ₃ :

were chromatographed: The column was calculated twice with C 75.95, H 8.05;

310 ecm each of the following solvents and solutions found C 75.7S, H 8.33.

Medium mixtures developed: benzene, benzene - SO ⁰ I ₀ ether,

Ether, ether - IQ ⁰ Z ₀ chloroform, chloroform - 10% 5 One mixed 100 mg 1,4-androstadien-lla-ol-3,17-dione acetone, chloroform - 20% acetone, chloroform - 50% with 0, 5 ecm acetic acid anhydride and 2 ecm pyridine, allowed AcCtOn ₁ ACeIOn ₁ AcCtOn -10% methanol, acetone - 20% the mixture to stand for 18 hours, diluted with 50 ecm methanol and methanol. The acetone i0 _^0/0 -Methanol- water, extracted five times with 25 cc of ether, gave verFraktionen as Eluatrückstaiid 2.64 g of crystalline united the ether extracts and washed them twice with material from dom after two UmkristalKsation from i '10 each ecm 2% hydrochloric acid, once with 10 ecm water, methanol-ether (1: 1) 1.844 g 1,4-Androstadieti-11 <T, once with 10 ecm 2% sodium bicarbonate solution and 17 / I-diol-3-one obtained became. A portion of this material was rinsed once more with lOccira water and then dried over twice more from chloroform-ether (1: i) to anhydrous sodium sulfate. It was then filtered and crystallized and gave analytically pure Ila-Oxy-1-de- and the solvent was evaporated. 105.5 mg of hydrotestosferon with a melting point of 174 to 15 l, 4-And: Tostadien-IIIa- »I-3,17-dione-II-acetate with a 175.5 °; [a] _D = -7 "(concentration = 1.0 in chloroform); melting point from 195 to 197 ° C; \ o] d = + 112 °.
[M] /, = -21 °; A ^ = 249 ηιμ; Ε = 17 100.

The infrared spectrum confirmed the assumed analysis for C ₂₁ H ₁₆ O ₄ :

Structure. Calculated ... C 73.66, H 7.66;

\ λ i- η τι r \ a ° found C 73.35, H 7.81.

Analysis for C ₁₉ H ₂₆ O ₃ : ^&

Calculated ... X 75.46, H 7.82;

found C75.14, H8.91. Example 4

A mixture of 200 mg lla-oxy-1-dehydrotesto- Ha-oxytestosterone
sterone, 20 ecm dry pyridine and 0.6 ecm acetic acid as 12 l of a medium were prepared, the per liter of anhydride was heated to room temperature for 18 hours. Solution 2 g (NH ₄ ) _a SO ₄ , 1.0 g Κ _? 0 ₄ , 30 g. "Cereal solution, then diluted in 200 ecm of water and dextrose five times, 0.01 g of FeSO ₄ , 0.5 g of MgSO ₁ , 0.3 g of ZnSO ₁ , extracted with 100 ecm of diethyl ether each time. 5 g K Cl and tap water contained; the medium was ether extracts were sterilized with 2% hydrochloric acid in by heating and cooled and with an amount of 50 ecm, once with 50 ecm water, once with 30 24-hour-old culture of Sporc-trichum epigaeum with 50 ecm 2% sodium bicarbonate solution and also inoculated ATCC 7145. After a further 24 hours, washed once with 50 ecm water, dried with anhydrous growth at an aeration rate of 11 sodium sulfate, filtered and the solvent air per minute (12 1 medium each time) was 3 g progesterone , evaporated at room temperature. One obtained crude solution in 50 ecm acetone, added. The incubation and crystals, which were recrystallized twice from ethyl acetate 35 aeration was continued at room temperature for 48 hours and were 206.7 mg l ^ -Androstadien-lla. ^ / S-diol- continued.

3-on-II «, 17 /? - diacetate with a melting point of 227.5 to Die as in Example 1 using an equivalent 228 ^° C. X »t = 245 ΐημ; E = 17,800; Extraction carried out [a] v amounts of solvent gave = + 47 ° (concentration = 1.0 in chloroform). The infra-5.75 g of extraction substances, the three times with 20 ecm ether red spectrum each confirmed the assumed structure. 40 hexane (5: 1) were triturated and 1.45 g of crystalline Ai-iaiiTcr. for r H η · Ιία-oxytestosterone from melting point Ϊ78 to 182 ⁰ C;

Btechn "". C 7i.48, H 7.82; M »= + ^ (chloroform).

found C 7180 H 8 22 Identical results are obtained if one

Progesterone of the fermentative action other Sporo-

Example 3 * 5 trichum species, such as Sporotrichnm sulfurescens, Sp. Carnis,

..., ...,. , O4TJ- Sp. Bombycinum or the like.

l ^ Androstacben-ila-ol-Sn-dKm ^ _{glekher Wefae wk} | ^ _{Application of the preceding}

To 121 medium, which per 100 ecm of water 1.2 g of maize process with other substrates, such as deoxycortico

swelling solids and 1 g of commercial dextrose, steron, 4-pregnen-17a, 21-diol-3,20-dione, 17a-oxyproge

its pH value with sodium hydroxide solution to 4.5 sterone 50, lla-oxyprogesterone, 4-pregnen-lla, 21-diol-3.20-

had been stclled and in which there was a 24 hour old dione or testosterone, the same result, namely

Culture of Sporotiichum bombycinum ATCC 7139 lla-oxytestosterone obtained,
found, 3 gl / i-androstadiene-S. ^ - dione, dissolved in

100 ecm acetone, added. After 24 hours of incubation, Example 5
with a ventilation rate of 1 liter of air per 55 lla-OxvDroe-esteron
Minute the fermentation solution and the mycelium with Me- uxyprogesreron
ethylene dichloride extracted, whereby 6.18 g of brown oily solids were obtained in the same manner as in Example 4. The solids were dissolved in the same medium with a 24 hour old culture of 200 ecm benzene, inoculated over 180 g of alumina chromato-Sporotrichum epigaeum ATCC 7145. After graphite and twice with 200 ecm chloroform each, two to a 24-hour growth period, 3 g promal with 200 ecm chloroform + 10% acetone and one yesterone, dissolved in 50 ecm acetone, were added. The painting was developed with 200 ecm chloroform - \ - 30% acetone. The ventilation rate was kept to about one tenth of the solids obtained from the combined eluates, the ventilation rate in Example 4, namely 1.53 g, were recrystallized twice from chloroform, at about 100 to 110 ecm of air per minute. After an incubation time of 48 hours whereby the crystallization was brought about by dilution with an incubation time of 48 hours, the ether was extracted. 1.153 g of 1,4-androstadiene were obtained using equivalent proportions of solvent lla-ol-3,17-dione with a melting point of 212 to 214 ^° C .; carried out as in Example 1. That in this way [ΔJD = + 86.5 ° (concentration = 1.01 in chloroform); The raw material obtained, about 6g, was processed four times with Ai, ^ = 2.48 ΐημ; E = 17,500. The infrared spectrum was triturated with 20 ecm of ether-hexane (5: 1), which confirmed the assumed structure. 70 stalline lla-oxyprogesterone from melting point 166 to

7 8

168 ⁰ C and an optical rotation of [α] "= 180" (in example 8

Chloroform). _{Λ1 t} - _oni ~.

'11 ", 17 / 9,21 -Tnoxy

Example 6

.... ,. ..., _ ,,. , "1001 of a medium containing 1.0 _{% / 0} Ccrelosedcxtrose, 2.0%

M-Androstadien-lla. ^ - diol-S-on _{5 Corn coke water (60} o _{/ o} solids) and tap water

121 medium of the type described in Example 4 were adjusted with 5% sodium hydroxide solution by the method of Example 1 with a pn value of 4.9. 400 ecm of speck oil octadicanol were inoculated as antifoam ATCC 7145 to this mixture of 24 hour old culture of Sporotrichum epigaeum, and then 3 g of 1,4-pregnadiene agent were added. This medium was added at 21-ol-3,20-dionine / 100 ecm acetone for 45 minutes. Then i ° a pressure of 1.4 kg / cm ^ä sterilized with 6 1 a was incubated and aerated (600 cc per minute) and 48 hours old growth of Sporotrichum sulfudann extracted four times with 31 methylene dichloride; rescens vaccinated by Beyman. This Sporotrichum thereby obtained 4.81 g of extract materials. These were strain, was obtained from the Centraalbureau voor Schimmeld three times with 20 ecm of ether-acetone (5: 1) triturated cultures, Baarn (Holland). The medium was stirred and then gave 1.22 g of crystalline 1,4-androstadiene-iS at a speed of 200 rpm and 11a, 17 /? - diol-3-one with a melting point of 170 to 174 ° C. sterile air through a sprinkler at a speed

With other substrates, namely 1,4-pregnadiency of 21 per minute supplied. After 24 hours of 3,20-dione, M-pregnadiene- ^ a ^ l-diol-S ^ -dione, 1,4-an growth, 25 gl7a, 21-dioxy-4-pregncn-3,20-dione drostadien- 17 /? - ol-3-one and l ^ -Pregnadien-lla-ol-S ^ O-di- (Reichstein's substance S), dissolved in 950 ecm hot absoon, the same result was obtained. 20% ethanol was added. Thereafter, the p _H value was

A reduction in the ventilation speed to 4.9. The fermentation stopped after about 24 hours UO up to 120 ecm per 12 1 medium resulted in example 6 broke. The ρπ value at this point in time was 4.6. with the same substrate, namely l / l-Prcgnadicn- ^ l-ol- The fermentation medium was replaced by a 50 cm Knight-Warc-3,20-dione, if all other conditions the same vacuum filter filtered. The container and the filter were were, l, 4-pr € ^ iadicn-lla, 21-diol-3,20-dic> r]. 25 washed with 201 water. The mycelium on the filter

was dried twice with 121 acetone and twice

Example 7 _{extracted with e 12} ] methyl chloride. The filtrate was

lla, 17 / ^ Dioxy-17 «-metlryl-4-androsten-3-one _with 24 liters of methylene chloride, then with the 481 extract

(llct-oxymethyltestosterone) the extraction of the mycelium and finally two more times

101 of a medium that extracts 1.0% cerelose dextrose, 30 with 24 liters of methylene chloride each time. The United

2.0 ° / o MaisqueUwasser (60 ° / ₀ solids) and line Gärmediumextrakte were washed with 241 water.

contained water, was mixed with 25 ⁰ Z ₀ Igor Natriumhydroxyd- The extract was in a flash evaporator to approximately solution adjusted to a _pH value of about. 5 This 11 concentrated and then under a hood to the mixture, 10 ecm of speck oil-octadecanol was evaporated as anti-dryness. The dry residue was

foaming agent added. This was followed by 45 minutes at 35 three times with 100 ecm each time of a 9: 1 ether-acetone solution

sterilized at a pressure of 1.4 kg / cm ² , rubbed off ^{to 28 ° C.} The remaining solids, consisting of let cool and weighed with 0.5 l of a 72 hour old wax from lla. ^ A ^ l-Trioxy ^ -pregnen-S ^ O-dione crystals, turns from Sporotrichum sulfurescens from Beyman 16 .08 g and had a melting point of 205 inoculated. This Sporotrichum strain was from Centraal to 21O ⁰ C.

bureau voor Schimmelcultures, Baarn (Holland). 40 Example 9

The fermentation medium was at a rate of .. , -, o- ^ · _Λ j j. r>

Stirred at 200 rpm and sterile air through a sprinkler lla, 17f-dioxy-4.androsten-3-one

fed at a rate of 0.2 liters per minute. (U a-Uxy testosterone)

After 24 hours of growth at 28 ° C, the Gc- The fermentation of a dispersion of 3 g of 17 / ?, 21-Di-

mixture was cooled to 2O ⁰ C, then 10 g Mcthyltestostcron, +5 oxy-4-pregnene-3,20-dione in 121 of the same medium as dissolved in 30 cc of dimethylformamide was added. in Example 8 with Sporotrichum sulfuresceiis ATCC 7159 The fermentation was interrupted after 48 hours. using a ventilation rate of The fermentation medium was nitrated through a 50 cm Knight Ware 21 air per minute (about 16 ° / _c ) and subsequent ex-vacuum filter and four times with 2.5 1 methylene traction in the same ratio as extracted in example 1 chloride. The combined extracts were added and yielded about 1.4 g of IIIa-oxytestosterone.
next with 1 1 2 ⁰ Z ₀ IgCm sodium bicarbonate, then with 2 1 MO

Water and washed over anhydrous sodium sulfate ^el "

dried. The extract then turned into Ua-Oxypregnan-3,20-dione in vacuo

Evaporated to dryness. The dry residue was in the fermentation of a dispersion of 5 g of Prcgnan-

100 ecm EthyJacctat dissolved and the solution 2 g activated charcoal 55 3,20-dione in 24 l of the same medium as in the example! admitted. The mixture obtained was filtered through di- with Sporotrichum epigaeum ATCC 7159 at a Beatomeenerde (known under the trade name Celite) ventilation rate of 250 ecm per minute resulted. The diatomaceous earth was washed out with 100 ecm hot Ila-Oxypregnan-3,20-dione from melting point 102 to ethanol and that with the washing solution 105 ⁰ C; [a] p = -1-83 ° (chloroform),
The combined filtrate was evaporated until crystallization began. The fermentation in 241 of the above medium continued at one point. The mixture was then returned to room temperature per minute aeration rate of 31 and then in a refrigerator to between 0 and 5 ⁰ C lla.iy / J-Dioxyätiocholan-S-one.

chilled. The crystals obtained in this way were The fermentation of the following methods

collected on a filter and dried. There were sulfuresceiis compounds of the pregnane series with Sporotrichum total of 5.1063 g of lla-Oxymethyltestosteron from 65 or other types Sporotrichum at Belüftungsge melting point receive 154 to 156 ⁰ C. speeds from 0.5 to 21 per 1001 medium according to the

In other experiments in which the Myccl and the method of Example 10 were extracted from 17a-oxypregnane fermentation medium, the compound 11a, 17a-dioxypregnane-3,20-dione testosterone became a 70 from the methyl-3,20-dione ° / ₀ yield of lla-oxymethyl a melting point of 191.5 ° C to J92,5, aus3a, 17a-Dioxytestosteron obtained. 70 pregnan-20-one the compound 3a, lla, 17a-trioxypregnan-

20-one from melting point 184 to 186 ⁰ C; [a] j> = + 52 ° (acetone) and from allopregnan-3,20-dione the compound llrt-oxyallopregnan-3,20-dione of melting point 197 to 200 ⁰ C; [a] _D = + 82 ° (chloroform).

Example 11
S-ketobisnor- ^ cholen-l la, 22-diol

Fermentation of a dispersion of 3 g of ketobisnor-4-cholen-22-ol in 121 medium with Sporotrichum sulfurescens A.'fCC 7159 by the method of Example 1 with subsequent extraction in the appropriate ratio gave 3-K.etobisnor-4-cb. olenlla, 22-diol, melting point 130 to 133 ° C; [ά] _ο = + 78 ° (chloroform).

Claims (5)

Patent claims: 1. Process for the fermentative hydroxylation of the Ila position of steroids with up to 22 carbon atoms ao and / or to break down the permanent side chain of these steroids by incubation with an under Microorganism growing under aerobic conditions, characterized in that the incubation is carried out with a microorganism of the genus Sporotrichum. 2. The method according to claim 1, characterized in that that for hydroxylation of the Ila position of pregnane derivatives during the incubation one Ventilation speed of up to 21 air per 100 1 Maintains fermentation medium per minute. 3. The method according to claim 1, characterized in that to achieve the degradation of the 17-position side chain of pregnan derivatives with the formation of a 17 /? - position hydroxyl group and, if there is a methylene group in the 11-position, for the simultaneous hydroxylation of the Ila-position maintains a ventilation rate of up to 5 to 15 liters of air per 100 liters of fermentation medium per minute. 4. The method according to claim 1 to 3, characterized in that the Sporotrichum for incubation Species Sporotrichum sulfurescens, Sp. Carnis, Sp. Epigaeum and Sp. Bombycinum used. 5. The method according to claim 1 to 4, characterized in that that the starting materials are progesterone, 1 - dehydrotestosterone, 1,4- androstadiene - 3,17- dione, l, 4-pregnadien-21-ol-3,20-dione, l-dehydro-17 <z-methyltestosterone or used methyltestosterone. Q 809729/282 1.59