DEU0003001MA - - Google Patents

Description

FEDERAL REPUBLIC OF GERMANY

Registration date: September 30, 1954. Advertised on August 23, 1956

GERMAN PATENT OFFICE

The invention relates to a new process for the fermentative degradation of the 17-hour cell Side chain of 20-ketopregnans and 20-ketoallopregnans with formation of 17-ketotestanes (normal form), 17-ketoandrostanes and 20-oxypregnanes.

The breakdown of the side chain in position 17 of Steroid compounds with chemical means with the formation of 17-keto steroids, in particular of 17-KeitoandiOstanen and 17-Ketotestanen, is well known, but this way of working usually requires a number of measures, such as education a 17 (2o) double bond and oxidation of this double bond. At a Such oxidative degradation is the steroid nucleus often in other positions, especially double bonds attacked, which can lead to high losses. To avoid these losses, be such positions or groups are protected in a known manner, which in turn is at least two additional Action required. To describe z. B. Bergmann and Stevens (Journ. Org. Chem., Vol. 13, 1948, p. 10) the ozonolysis of the 22-enol acetate des 5, 7-bisnorchola, diene-3 /? - 0'l-22-al-3-acetate, which is protected by a 5, 8-position maleic anhydride adduct group, whereby the Maleic acid anhydride adduct of 5, 7-Androsta.dien-3 / J-ol - ^ - on-S-acetate is obtained.

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In accordance with the present invention, a '20 ketopregnane is made or 20-ketoallopregnan by the action of a fungus of the genus Penicillium in a 17-ketoandrostane or 17-ketotestane converted. The obtained 17-ketoandrostanes and 17-ketotestanes are valuable drugs or valuable Intermediate products for their manufacture.

The method according to the invention provides a number of valuable products, e.g. B. Progesterone, when exposed to Penicillium lilacinum, 4-androstene-3, 17-dione, known to have androgenic properties. 4-Androstene-3, 17-dione can turn into testosterone be transferred (Stavely, USA.-Patent No. 2 288 854) and the latter by hydrogenation into the therapeutically valuable dihydrotestosterone (Symposium on Steroids in Experimental and Clinical Practice, The Blakistan Company, New York, 1951, p. 375). The fermentation of ii-ketoprogesterone produces this in an analogous manner known 4-androstene-3, 11, 17-tricti (adrenosterone), which is a valuable starting material for the production of 11 / J-oxytestosterone.

The starting compounds for the present process are 20-ketopregnane and 20-ketoallopregnane. The cyclopentaopolyhydrophenanthrene core with a side chain in the 17-position can be used in other positions also carry keto or hydroxyl groups, especially in positions 3, 6, 7, 8, 11, 12, 14, 17 and 21, and can be in different Positions, especially in positions 4 and 5, contain double bonds.

Typical starting materials are the following: Pregnan - 3 α (or ß) - oil - 20 - one, 5 - Pregnen - 3a -

.35 (or /?) - ol-2O-one, Pregnan-3 α (or /?) - ol-ii, 20-dione, Pregnan-3a, 11 α (or 3 / ?, 11 /? Or 3 / ? ,, iicr or 3 a, 11 ß) -diol-20-one, progesterone, I7a-oxyprogesterone, ii-ketoprogesterone, 11 a-oxyprogesterone, 11 α, I7a-dioxyprogesterone, ii-keto- ^ a-oxyprogesterone, 11 a-acetoxyprogesterone, ii / 3-oxyprogesterone, ^ a-oxyprogesterone, 4-P rain-14 a, 17 a, 2i-triol-3, 20-dione, i4a-oxy-ii-deoxycorticosterone; 4-pregnen-i7a, 2i-diol-3,20-dione ("Reichstein's compound S"), corticosterone, cortisone, cortisone acetate, hydroeortisone ("Kendall's compound F") and 4 - pregnene - 11α, 17 a , 21 - triol 3, 20-dione (»ii-EpiverbverbindungenF«) Pregnan-3, 11-20-trione, Pregnan-i7a-ol-3, 11, 20-trione, Pregnan-3, 12, 20-trione, Pregnan-i7a-ol-3, 20-dione, AlIo ^ pregnan-3, 11-20-trione, Allopregnan-3, 20-dione, 5 "Pregnen-3 a-ol-20-one, Allopregnan-3 α ( or ß) -ol-20-011, Pregnan-3 α, 12 α, 2i-trioJ-20-one, Pregnan-3 a, 17 a-diol-20-00, 6 a- and. 6 ^ -Oxy- Progesterone, Pregnan-3, 6) 20-trione.

To carry out the process according to the invention, the selected 20-ketopregnane or -Allopregnan expediently in a solvent, like acetone, exposed to the culture of a fungus of the genus Peiiicilliunr. The classification and definition of Penicillium as used here corresponds to that of K. B. Raper and C. Thorn "Α Manual of the Penicillia", Williams and Wilkins Company, Baltimore,

1949. Of the species of the genus Penicillium which are suitable for the fermentation of steroids, be those of the subclasses Monoverticillate, Asymmetrica - divaricata, Asymmetrica - velutina, Asymmetrica - Janata, Asymmetrica - funiculoea, Asymmetrica-fasciculata, Biverticillata-symmetrica and Polyverticillata, e.g. B. Penicillium adametzi, brevi-compactum, camemberti, canescens, capsulatum, charlesii, citrinum, clavi forms, commune, cyclopium, decumbens, digitatum, duclauxi, etxpansum, frequentans, funiculosum, fascum, gladioli, granulatum, herquei, implicatum janthinellum, lavendulum, levitum, lilacinum, luteum, nigricans, notatum, novae-zeelandiae, ochraceum oixalicum, pallidum, purpurogenum, purpurogenum var. rubrisclero'tium, raistrickii, roqueforti, roseo-, purpureum, rugulosum, terrestre, thomii, urticae and; viridicatum called.

The fungi are cultivated for the purposes of the present invention in or ¹ on a nutrient medium which is favorable for their development. Solid culture media can be used, but preference is given to those that allow mass cultivation under aerobic conditions.

Moist, solid, small-sized nutrient media, such as bran, cereal grains, cereal semolina, wood chips, Shavings, sawdust, corn husks, fibers such as copra, chestnuts or lupine seeds can be used will. They can be extracted with alcohol, ether or other organic solvents, to avoid unwanted impurities and growth-inhibiting substances prior to fermentation to remove. The carriers can contain added growth factors and nutrients, if desired and can be in layers or trays with or without auxiliary ventilation, in towers, as in the case of the Making vinegar, or with movement, e.g. B. in a rotating drum can be used. Liquid culture media, like wort, are good for aerobic layers and even better for aerobic submerged fermentation conditions. Appropriately, the culture media should contain substances which provide carbon, nitrogen and minerals, although of course also under Considerable growth can occur under less than optimal conditions.

Available carbon can be obtained from carbohydrates, starches, gelatinized starches, dextrin, Sugars, molasses of cane, beet and sorghum sugar, glucose, fructose, mamoee, galactose, Maltose, suerose, lactose, pentoses, amino acids, peptones or proteins come from. Carbonic acid, glycerine, alcohols, acetic acid, sodium acetate, citric acid, sodium citrate, Lower fatty acids, higher fatty acids, or fats are examples of other materials that are assimilable Can provide carbon for the mushrooms' energy needs. Sometimes there are mixtures from various carbon sources beneficial. . ·.,

Assimilable nitrogen can be obtained from soluble or insoluble vegetable or. animal proteins, soy flour, lactalbumin, casein, egg albumin, Peptones, polypeptides or amino acids,

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Urea, ammonium salts, on base exchange resins or zeolite bound ammonia, ammonium chloride, sodium nitrate, potassium nitrate or morpholine originate, whey, soluble residues from the distilleries, corn steeping water or yeast extract have also proven to be very suitable.

The mineral components of the nutrient media can be naturally present or added Substances that serve aluminum, calcium,

ίο Chromium, cobalt, copper, gallium, iron, magnesium, Supply molybdenum, potassium, scandium, uranium and vanadium. Sulfur can be replaced by sulfates, Alkyl sulfonates, suilfocxyate, sulfamates, sulfinates, free sulfur, hyposulfite, persulfate, thiosulfate, methionine, cystine, cysteine, thiamine or biotin to be delivered. Phosphorus, preferably in pentavalent form, expediently in concentrations from about ο, οοι- to o, o7molar and preferably from about 0.015 to 0.02 molar, can be used as ortho-, meta- or pyrophosphate salts or esters, phytin, Phytic acid, phytates, glycerophosphates, sodium nucleinates and / or maize steeping water, casein or ovovitelline be present. It is desirable to have traces of boron, iodine and selenium present. The Bot is preferably added in the form of boric acid or sodium borate, especially after the Germination and during the first growth of the fungus.

Depending on your needs or wishes, you can add other 'additional growth factors, vitamins, Provide auxins and growth stimulants.

Although one can use solid as well as liquid culture media, a liquid culture media is preferred, since it favors myceh growth.

To facilitate fermentation, aeration and filtration, one can use suspending agents or Mycelium carriers, such as filter earth, filter aids, finely divided cellulose, wood chips, bentonite, calcium carbonate, Magnesium carbonate, charcoal, activated charcoal or other suspendable solids, Add methyl cellulose, carboxymethyl cellulose or alginates. The selected genus of mushrooms is based on pulled a nutrient medium, the expediently assimilable carbon, z. B. Carbohydrates, such as sugars, starches, assimilable nitrogen, e.g. B. soluble or insoluble proteins, peptones or amino acids, and mineral components such as phosphates and magnesium sulfate, as well contains other known desirable additives.

The medium may advantageously have a p _H of about 4 to 8 or fewer or more hold before inoculation. For the culture of Penicillium, a P ₁₁ between about 4 and 6 is preferred.

The inoculation of the culture medium with the selected fungus of the genus Penicillium can be carried out in any suitable manner. Way to be carried out. Penicillium develops in a temperature range of about 20 to 38 ^° , temperatures of about 25 to 32 ^{° being} preferred.

The periods of development of the fungal growth before the addition of the steroid to be fermented does not seem to be critical. For example, you can use the steroid before sterilization can take place by the action of heat or in some other way, add to the nutrient medium or in the Moment when the culture medium is inoculated, or for some time, e.g. B. 24 or 48 hours later. The steroid to be fermented can be added in any suitable concentration, but is off for practical reasons a concentration of the steroid substrate of about or up to 0.6 g / l or even 0.8 g / l nutrient medium is satisfactory, and 2 g / l can be used, even higher concentrations Depending on the particular steroid being allowed if there is some hindrance from development wants to accept the mycelium. The addition of the to be fermented. Steroid substrate can be done in any arbitrary way, in particular in such a way that between steroid substrate and mushroom creates the largest possible contact area. This can be done using the steroid substrate either alone, with a dispersing agent or dissolved in an organic solvent in the fungus medium disperse or suspend. One can have both a surface and a submerse Use culture, but the latter is preferred. One can also use those formed during the cultivation of the mushroom Separate fermenting enzymes from the fungus or from the nutrient medium, with the steroid or mix a solution of the dispersion of the same and subject the mixture to aerobic conditions, to cause the steroid to ferment.

The temperature during fermentation of the steroid can be the same as it is for that Mushroom culture proved suitable. It just has to be kept in such a range that the Life, active growth or enzyme activity of the fungus is preserved.

While any form of aerobic incubation is appropriate for the growth of the selected fungus and fermentation of the steroid substrate, the effectiveness of steroid fermentation is dependent on aeration. Therefore, the aeration is usually controlled, be it by shaking and / or blowing air into the fermentation medium. Aeration can be done by surface culture or under submerged fermentation conditions. Aerobic conditions include not only the use of air to introduce oxygen, but also other sources or mixtures containing oxygen in, free or freely settable form. If air is used as the ventilation medium, a suitable degree of ventilation results in about 4 to 20 millimoles and preferably about 6 millimoles of oxygen per hour per liter, determined by the method of Cooper, Fernstrom and Miller (Ind. Eng. Chem., Vol. 36, 1944 , P. 504). The ventilation can be achieved by applying positive or negative pressure, e.g. B. 0.7 to 2.1 kg / cm ² abs., Be set. Oxygen uptake can be facilitated by the presence of various substances such as ascorbic acid, glutamic acid, citric acid, lactic acid, tyrosine or tryptophan.

The time required for the steroid to ferment varies depending on the mode of operation

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something. If the steroid substrate is present at the time of inoculation of the medium, it can range from 8 to Take 72 hours. However, one adds the steroid to the fungus after it has already grown significantly is e.g. B. after 16 to 24 hours, at the most favorable temperature, the conversion begins of the steroid substrate immediately and high yields are obtained over the course of 1 to 72 hours. In the Usually the result of 24 hours is satisfactory.

After the steroid fermentation has ended, the fermented steroid obtained is removed from the reaction mixture isolated. A particularly cheap insulation s method consists in extracting the fermentation mixture including the mycelium with a water-immiscible organic solvent for the steroid, such as methylene chloride, Chloroform, carbon tetrachloride, ethylene chloride, trichlorethylene, ether, amyl acetate or Benzene and the like. The fermentation liquor can be separated from the mycelium and mixed individually with suitable ones Extract solvents. The mycelia can be mixed with either water or water-immiscible solvents can be extracted, with acetone shown to be effective has. The fermentation liquid freed from the mycelium can be immiscible with water Solvent to be extracted. One can use the extracts either before or after washing with alkaline solutions, for example sodium bicarb on at, combine, in a suitable manner, for. B. Dry over anhydrous sodium sulfate and recrystallize from organic solvents or chromatography the purified 17-ketosteroid obtained from the other fermentation products separate.

The following examples are intended to explain the process according to the invention in more detail.

example 1

Fermentation of progesterone and isolation of 4-androstene! -3, 17-dione and 4-pregne.n-2o /? - ol-3-one. A culture medium is prepared which contains 20 g of enzymatically digested lactalbumin (edamine), 3 g contains corn steep liquor and 50 g technical dextrose with tap water to 1 1 diluted, and provides the p _H to 5.85. 1.2 1 of this sterilized medium are inoculated with Penicillium lilacinum thorn (American Type Culture Collection No. 10 114) and ^{incubated for 48 hours at 26 0} , venting and stirring in such a way that the oxygen uptake is 6.3 to 7 millimoles per hour and liter Na ₂ SO ₃ , determined by the method of C 00per, Fernstrom and Miller (Ind. Eng. Cheim., Vol. 36, 1944, p. 504) is. ^{6 g of progesterone, dissolved in 50 cm 3 of} acetone, are suspended in this nutrient medium, which contains Penicillium lilacinum thorn grown for 4 hours. After a further 24 hours of incubation under the same: temperature and ventilation conditions, the liquid and mycelium are separated. The mycelium is filtered off, twice with about its volume each

; Washed aoeton and then extracted twice with its volume of methylene chloride. The solvents containing aoetone and methylene chloride extracts are added to the filtrate. The solution is then extracted twice with half its volume of methylene chloride each time and then twice with a quarter of its volume of methylene chloride each time. The combined methylene chloride extracts are washed twice with one tenth of their volume each of 2% strength aqueous sodium bicarbonate solution and then twice with one tenth of their volume of water each time. After drying the methylene chloride extract over about 3 to 5 g of anhydrous sodium sulfate per liter of solvent and filtering, the solvent is distilled off. The residue obtained in this way weighs 5.88 g and is redissolved and chromatographed over 150 g of alumina, using 600 cm ^{3 of} solvent each time, as indicated in Table I.

Table I.

fraction solvent benzene ^l 9 I. Fixed E luat
in mg I. benzene ^X 9 I. 98.2 2 Benzene-Ether 19: 1 9 I. 47.6 0 Benzene-Ether 19: 1 9 I. 18.8 4th Benzene-Ether 9: 1 I. I. 132.8 5 Benzene-Ether 9: 1 I. I. 154.2 6th Benzene-ether 1: 1 I. I. 27.5 7th Benzene-ether 1: 1 I. I. 302.7 8th ether 1267.0 9 ether ¹ p : ι 1160.6 IO Ether — chloroform 402.2 II Ether — chloroform 97> 9 12th Ether - chloroform, 69.4 13th Ether — chloroform 180.7 14th Ä t h er --- ChI orof orm .356.1 15th . Ether — chloroform 101.1 16 Ether — chloroform 95.3 17th Ether — chloroform 32.6 18th chloroform 12.9 19 to 22 Chloroform-acetone 34.9 23 acetone 5.8 24 Methanol 24.6 25th Methanol 210.3 26th 7, ²

Fractions 9 to 11 are combined and evaporated. 1.66 g of solids are obtained, which ^{are washed twice with 5 cm 3 of} hexane each time (known under the trade name “Skellysolve B”). The remaining crystals, 1.58 g, m.p. 172 to 175 ⁰ , are dissolved in 10 cm ^{3 of} hot acetone, the solution is filtered and concentrated to 5 cm ³ . After standing overnight in the refrigerator, 1.2 g of crystals with a melting point of 175 to 177 ^{° are obtained} . These crystals are recrystallized from 4 to 5 cm ^{3 of} acetone and 3.5 cm ^{3 of} “Skellysolve B”, whereby 899 mg of crystalline 4-androstene-3, 17-dione with a mp = 175 to 176.5 °; [α] " _D + 194 (c = 0.981 in chloroform).

Analysis for C ₁₉ H ₂₆ O ₃

Calculated C, 79.69, H, 9.15;

found C, 79.53, H, 8.94.

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The parliamentary groups; 13 and 14, which contain 537 mg of solid substance, are combined, dissolved in 50 cm ^{3 of} benzene and chromatographed again over 25 g of clay. Fractions of 50 cm ^{3 are} collected as shown in Table II.

Table II

fraction solvent ^l 9 I. Solid eluate
in mg I. Benzene-ether 1: 1 ^l 9 I. 7.0 2 Benzene-ether 1: 1 9 I. 5.2 3 ether 9 1 2.1 4th ether I. ι · 24.8 5 Ether — chloroform I. I. 48.1 6th Ether — chloroform I. I. 8O, 3 7th Ether — chloroform 87.6 comb. 8th Ether — chloroform 7O, 4 9 Ether — chloroform 51.0 IO Ether — chloroform 19: 1 37.5 II Ether — chloroform ! 9.7 12th Chloroform. 9> ^T 13th chloroform 24.8 14th chloroform 5, o 15th Chloroform-acetone 4.8 16 acetone 4.0 17 and 18 Methanol 13.3

Fractions 4 to 11 are combined and 3 cm ³ of methylene chloride and 1 cm ³ »SkellysolveB" recrystallized to give 114mg crystals, mp = 151-152 ^{0 and} by infrared and microanalysis as 4-pregnene-2o twice a -ol-3-one to be identified.

B e i s ρ i e 1 2

Fermentation of ii-deoxycorticosterone acetate

Made from 0.5% peptone, 2% dextrose, 0.5% soy flour, 0.5% monobasic potassium phosphate, 0.5%

Sodium chloride and 0.3% yeast extract is prepared by diluting with tap water to 1 1 a breeding ground forth, the p _H is set to 5.9 post-sterilization. 3 1 of this culture medium are loaded with Penicillium canescens (ATCC No. 10419)

inoculated and incubated for 24 hours at 28 ⁰ , with a rate of 7 millimoles per liter of Na ₂ SO ₃ and hour, determined by the method of Fernstrom and Miller (Ind. Eng. Chem., Vol. 36, 1944, p. 504), ventilated. In this nutrient medium, which contains a 24-hour old culture of Penicillium canescens, 1 g of II-deoxycorticosterone acetate, dissolved in 20 cm ^{3 of} absolute ethanol, is suspended. After a further 24 hours of incubation under the same temperature and ventilation conditions, the solution and the mycelium are extracted and chromatographed as in Example 1, using 4-androstene-3, 17-dione and 4-pregnen-20α, 2i-diol-3 -on receives.

Example 3

Fermentation of I4ct-Oxyprogesterone Make a culture medium by adding 20 g of corn steep liquor, 20 g of dextrose, 1 g of a

basic potassium phosphate, 2 g sodium nitrate, 0.5 g magnesium sulfate, 0.2 g potassium chloride, 0.01 g ferrous sulfate and 2 g sodium acetate diluted to ι 1 with tap water. After sterilization, 2 liters of this nutrient medium ^{are distributed in proportions of 100 cm 3} each in 20 shake flasks and inoculated with Penicillium lilacinum. To a 24-hour culture of the fungus at 26 ⁰ are added to each bottle 10 mg I4ct-Oxyprogesteron, prepared by the method of the patent application 2024 U IVb / 120th After 48 hours of incubation with shaking with the surrounding air at 26 ⁰ , the contents of the bottles are combined and extracted with methylene dichloride; the extracts are evaporated to give 785 mg of solid residue. This is dissolved in 35 cm ^{3 of} benzene and chromatographed over 40 g of clay. Fraction 24 (chloroform) weighs 103 mg and contains 22% 4-androsten-i4a-ol-3, 17-dione. Fraction 25 (acetone) weighs 116.5 mg and contains 16% 4-androsten-i4a-ol-3, 17-dione. This fraction is dissolved in ι cm ^{3 of} ethyl acetate and allowed to evaporate at room temperature. After repeating this process several times, crystallization occurs. The fraction is then with 2 cm ^{3 of} ether and less - ·. gen drops of acetone triturated to give 10 mg of 4-androstene-I4A ¹ ol-3, 17-dione, mp = 252-258 ⁰ is obtained.

In the same way as in Example 3, 14a-oxy-n-deoxycorticosterone is obtained by fermentation or 4- Pregnen- 14 a, 17 a, 21-triol-3, 20-diene with Penici'llium lilacinum 4-AndiOsten-140-01-3, 17-dione (gonadal active).

B e i s ρ i e 1 4 Fermentation of n-deoxycorticosterone acetate

with Penicillium nigricans

In the same way as in Example 1, when using Penicillium nigricans (ATCC No. 10115) and ii-deoxycorticosterone acetate as Starting steroid 4-androstane-3, 17-dione and 4-pregnen-20α, 2i-diol-3-one.

Example 5 Fermentation of ii-deoxycorticosterone acetate

with Penicillium charlesii

In the same way as in Example 1, when using Penicillium charlesii (ATCC No. 8730) and ii-deoxycorticosterone acetate 4-androstene-3,17-dione and 4-pregnen-20a, 2i-diol-3-one. - -; In the same way give the 21-acyloxy esters des ii-deoxycorticosterone, e.g. B. the propionate, Butyrate, isobutyrate, valerate, hexanoate, benzoate, phenyl acetate and other esters of ii-deoxycorticosterone when treated with a fungus of the genus Penicillium 4-androstene-3, 17-dione and the corresponding 4 ~ pregnen-20 et, 2i-diol-3-one.

Example

Fermentation of progesterone with Penicillium brevi compactum "In the same way" as in the examples is obtained using Penicillium brevi compactum

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(ATCC No. 9056) from progesterone 4-androstene-3, 17-dione and 4 "pregnen-2Oa, 21-diol-3-o.11.

Example 7

Fermentation of 11-deoxycorticosterone acetate with Penicillium lividum

In the same way as in Example 1, when using Penicillium lividum (ATCC No. 10102) and 11-deoxycorticosterone acetate, 4-androstene-3,17-dione and 4-pregnen-2Oa, 21-diol-3-one are obtained. In an analogous manner, other 21-esters of ii-deoxycorticosterone, such as propionate, butyrate, isobutyrate, valerate, hexanoate, benzoate or phenyl acetate, on treatment with a fungus of the genus Penicillium give 4 ~ androstene-3, 17-dione and the corresponding 4- Pregnen-2o a, 21-diol-3-one-21-acylate.

Examples

Fermentation of ii-ketoprogesterone
with Penicillium expansum

Using the same procedure as in Example 1, Penicillium expansum is obtained (ATCC No. 7861) and n-ketoprogesterone the 4-androstene-3, 11, 17-trione (adrenosterone).

Example 9

Fermentation of progesterone

The procedure is the same as in Example 2 using Penicillium frequentans (ATCC No. 10444) and progesterone and receives 4-androstene-3, 17-dione.

Example 10
Fermentation of i7a-oxyprogesterone

The procedure is the same as in Example 1 using of Penicillium lilacinum (ATCC No. 10114) and I7a-oxyprogesterone and receives 4-androstene-3, 17-dione.

Example 11

Fermentation of 11 α, I7 <x -dioxyprogesterone

It works the same as in Example 2 by comparison - (. ATCC No. 10506) ₀ turn of Penicillium thomii and na, I7a-Dioxyprogesteron and receives 4-androstene-ii 01-0I-3, 17-dione.

Example 12
Fermentation of Pregnan-3, 6, 20-trion

The procedure is as in Example 2 using Penicillium novae zeelandiae (ATCC No. 10473) and Pregnan-3, 6, 20-trione and contains Testan-3,6,17-trione. Testan-3,6,17-trione is included Brominating 4-bromotestane-3, 6, 17-trione, which by splitting off hydrogen bromide into 4-androstene-3, 6, 17-trione passes over with estrogenic effect (Butenandt, Ber. D. German former Ges., Vol. 69, 1936, p. 1163).

Example 13 Fermentation of 6 / J-Oxyprogesterone

In the same way as in Example 1, when using Penicillium citrinum (ATCC No. 10105) ^and 6 / J-oxyprogesterone, 4-androstene-6 /? -Ol-3, 17-dione is obtained.

B e i s ρ i e 1 14

Fermentation of Pregnan-3, 20-dione

The procedure is the same as in Example 2 using Penicillium canescens (ATCC No. 10 419) and pregnan-3,20-dione and testan-3, 17-dione is obtained, which by reduction with sodium borohydride in testan-3 α ( or ß) oil passes over, which has an anesthetic effect.

Example 15 Example 16

Example 17 Fermentation of cortisone

Fermentation of allopregnan-3, 11, 20-trion

The procedure is as in Example 1 using Penicillium lilacinum thorn and AlIopregnan-3, ^τι > 20-trione and androstan-3, 11, 17-trione (male hormone activity) is obtained.

Fermentation of Reichstein's substance (4-Pregnen-i7a, 2i-diol-4-3, 20-diotii)

The procedure is as in Example 1 using Penicillium lilacinum (ATCC No. 10114) and Reichstein's substance S and receives 4-androstene-3, 17-dione and 4-pregnen-i7 <x, 20a, 21-triol-3-one.

In the same way as in Example 1, the action of Penicillium lilacinum (ATCC No. 10114) on cortisone 4-androstene-3, 11, 17-trion (adrenosterone).

Example 18 Fermentation of Cortisone Acetate

One works in the same way as in Example 1 and obtains by the action of Penicillium lilacinum (ATCC No. 10114) on cortisone acetate 4-androstene ~ 3, 11, 17-trion.

B e i s ρ i e 1 19

Fermentation of 4-pregnen-iia, 17a, 2i-triol-3, 20-dione

You work as in Example 1 and get through Exposure to Penicillium lilacinum (ATCC

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No. ίο 114) ^{urL <} i 4-Pregmem.-11 a, 17a, 21-triol-3,20-dione das 4-Androstan-11 a-ol-3, 17-dione.

Example 20
Fermentation of Pregnan-3 ". ^{1 x} «> 17 a-triol-20-one

You work as in example i and get by the action of Penicillium lilacinum (ATCC No. 14) on Pregnan-3 α, 11 α, 17 a-triol-20-one the Testan-3a, 11 a-diol-17-one.

Example 21

Fermentation of Pregnan-3, ^I2 > 20-trione

The procedure is as in Example 1 and, through the action of Penicillium lilacinum (ATCC No. 10 114) on Pregnan-3, 12, 20-trion dasTestan-3, 12, 17-trion.

Claims (4)

Patent claims: i. Process for the preparation of 17-keto testanes, 17-ketoandrostanes and 20-oxypregnanes by incubating 20 ketopregnans or allopregnans with the culture of a lower fungus in a nutrient solution under aerobic conditions with separation of the Reaction product, in particular by extraction, characterized in that the incubation with the culture of a fungus of the genus Penicillium. 2. The method according to claim 1, characterized in that that the culture solution used for the incubation is assimilable, not the starting steroid originating carbon, especially in the form of carbohydrates, as well as assimilable nitrogen and phosphorus contains and the culture is moved. 3. The method according to claim 1 and 2, characterized in that one is unsaturated in the 4-position 20-ketopregnane or 3, 20-diketopregnane, especially progesterone, ii-deoxycorticosterone or its 21-acylates, as well as I4a-oxyprogesterone, an allopregnan-3, 11, 20-trione or a 20-ketopregnane, in particular Pregnan-3, 20-dione or Pregnan-3, 6, 17-trione, used as starting compounds. 4. The method according to claim 1 to 3, characterized in that the incubation with a culture of Penicillium lilacinum, Penicillium canescens or Penicillium novae zeelandiae performs.