DEC0009818MA - - Google Patents

Description

FEDERAL REPUBLIC OF GERMANY

Registration date: August 16, 1954 Btekaiimt made on August 2, 1956

GERMAN PATENT OFFICE

The subject of the invention is a new process for the production of oxidation products of the steroid series, especially for breaking down the side chain of pregnane compounds and for producing un- -saturated compounds of the androstane series by biochemical means.

'. That ^; The process is characterized in that, on saturated or unsaturated androstanes or pregnanes which have a free or protected oxy- or oxogru'ppe in the 3- and 17- or in the <2O-position, cultures of Fusarium solani, Fusarium caucasicum, Rhizopus suinus, Venturia chlorospora or Venturia linicerae or the enzymes contained therein. . - ^; .-i.

The nutrient media known per se for this purpose are suitable for the cultivation of the fungi mentioned, eg. B. those with Sugars, such as glucose or lactose, with peptones, ■ corn steep liquor or soy products as well as with mineral salts, or synthetic nutrient solutions. One works particularly under aerobic conditions, for example in a shaking culture or in the case of submerged growth with stirring and a supply of air. The Eumycetes differ from other microorganisms; E.g. the bacteria, through good Growth under relatively simple growing conditions. The procedural reaction takes place in the described mushroom culture or with the contained / possibly enriched or depleted

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separate enzymes instead, in the simplest case in a slurry of the separated mushroom mycelium, the homogenized mushroom mycelium or in its filtrates or water-containing extracts. The starting materials used for the new process are saturated or unsaturated compounds of the pregnane and androstane series, e.g. B. progesterone, ii-dehydroprogesterone, ii-ketoprogesterone, ii-, 12- or 14-oxyprogesterone, 5- or 4 "pregnen-3-ol-2O-ones, 5-pregnen-3, 20-diols, ii-deoxycorticosterone , Corticosterone, 11-dehydrocorticosterone, 4- or 5-androstene-3, 17-dione, testosterone, 5-androsten-3-ol-i7 "On, adrenosterone, pregnan-3, 20-dione, pregnan-3-ol- 2O-one, androstan-3, 17-dione, allopregnan-3, 20-dione, 3 (3-acetoxyallopregnan-2O-one or corresponding compounds with protected oxy or oxo groups is an esterified or etherified oxy group with an aliphatic, aromatic or heterocyclic carboxylic acid, for example acetic acid, propionic acid, benzoic acid or furancarboxylic acid, for example the tetrahydropyranyloxy, benzyloxy or triphenylmethoxy group in particular of a dihydric alcohol, such as the ethylenedioxy group contain toffe double bonds, e.g. B. in 4-, 5 ". 6-, 7-, 8-, 9 (11) -, 11- or 14-position, or have additional substituents, such as free or protected oxy, oxo or carboxyl groups, and also epoxy groups or halogen atoms, for example in the 4-, 5- / 6-, 7-, 8-, 9-, 11-, 12-, 14-, 15- or 21-position. The abovementioned starting materials are of any steric configuration The process can also be carried out with mixtures which contain one or more of the above-mentioned starting materials - for example, starting from the neutral part formed during cholesterol oxidation, in particular after subsequent dehydration of the 3-position oxy group, inter alia the i, 4 "androstadiene-3, 17-dione.

The process products can be separated off by known methods. she can for example by extracting the reaction mixture with an organic solvent such as methylene chloride or ethyl acetate. For the further purification of the extract obtained in this way are suitable particularly chromatographic methods, e.g. adsorption on aluminum oxide or silica gel, application of distribution methods, e.g. the countercurrent process, or separation with the so-called "Girard reagents" such as trimethylammonium or pyridinium acetic acid hydrazide. Subsequently Finally, in place of the purification, recrystallization is preferably carried out from organic or aqueous-organic solvents.

With the new biochemical process, for example, progesterone and 5-pregnen-3 (5-ol-2o-one, n-deoxy-corticosterone or 4-androstene-3, 17-dione can be obtained using e.g. Fusarium solani in just one process step and with high yield into the 1,4-androstadiene-3, 17-dione. This chemical conversion is only possible in a multistage process with a significantly lower overall yield. 17-dione is an important starting material for the synthesis of oestrone, which can be obtained therefrom in a single process step: from the saturated oxy in the 4 (5) and 5 (6) positions and a free or protected oxy in the 3 position - or oxo group-containing pregnane compounds, depending on the duration of the incubation, the corresponding saturated 17-ketones or their derivatives dehydrated in ring A, for example the 1,4-androstadiene-3, 17-diones, are obtained analogously from the corresponding saturated androstane compounds ndings of their derivatives dehydrated in ring A. With prolonged incubation, a new compound, 1,2-dehydrotestolactone from F. = 219 to 220 ° and the specific rotation [α] ² _D ² = -49 ± 3 ° (c = 1.025 ^m CHCl ₃ ), which also has a strong band in the ultraviolet spectrum at 242 ταμ (ioge = 4.23) and contains 75.70% carbon and 8.05 % Hydrogen (calculated for C ₁₉ H ₂₄ O ₃ : C 75.97; H 8.05%). With other fungi, for example Rhizopus suinus, the well-known testolactone is also obtained in place of this compound.

The products of the process can serve as medicinal products or as intermediate products for their manufacture.

The invention is illustrated in more detail by the following examples.

example

4 liters of a nutrient solution, consisting of 30 cm ^{3 of} corn steep liquor, 50 g of peptone, 200 mg of raw glucose and tap water, are evenly distributed in 13 Erlenmeyer flasks with a capacity of 11 each and sterilized. The p _H of these solutions is 6.4. They are inoculated with a culture of the fungus Fusarium solani and with. Shaken mechanically at 25 °. After 48 hours the fungus has multiplied strongly. ^{A solution of ig progesterone in 45 cm 3 of} acetone is distributed over the 13 Erlenmeyer flasks under sterile conditions. Shake for a further 48 hours at the temperature mentioned above and then filter off the mycelium. The p _H of the culture filtrate is now 7.9.

The solution is extracted once with 1500 cm ³ , twice with 1000 cm ³ each and finally twice with 500 cm ³ each of methylene chloride. The extract is washed twice with 300 cm ³ 0.1 η-hydrochloric acid, twice with 300 cm ³ i ° / _o sodium bicarbonate solution and three times with 300 cm ³ of water, dried over sodium sulfate and evaporated in vacuo. The partially crystalline residue (1.14 g) is chromatographed on a column prepared with 30 g of aluminum oxide in a benzene-petroleum ether (6: 4) solution using the continuous flow method.

The benzene-petroleum ether and the benzene eluates are combined and recrystallized from acetone-petroleum ether. Colorless, rhombic plates which are uniform by paper chromatography and have a F. = 145 to 146 ⁰ ; [a] _D = + 110 ° ± 4 ° (CHCl ₃ ), +112 ⁰ ± 4 °

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(Alcohol). . This compound is the well-known i, 4-androstadien-3, 17-dione. Yield about 80%. If, instead of the progesterone solution, a solution of ι g of n-deoxycorticosterone or of 1 g of 4-androstene-3,17-dione in 45 cm ^{3 of} acetone is added to a corresponding culture of Fusarium solani, the culture is treated and worked up in the manner described above, in this way, the 1,4-androstadiene-3,17-dione is likewise obtained in high yield.
If, in the above example, the culture of Fusarium solani is replaced by a culture of Fusarium caucasicum produced on the same nutrient medium, progesterone is converted into 1,4-androstadiene-3,17-dione with the same high yield.

Example 2

^{A solution of 20 μg of 5-pregnen-3 / S-ol-2O-one in 37 cm 3 of} methanol is added under sterile conditions to 4 liters of a culture of Fusarium solani, which was prepared as described in Example 1. The culture is shaken for a further 48 hours at 26 ^0, filtered off the mycelium on it and extracted the culture filtrate as described in Example 1 with methylene chloride.

The extraction residue is chromatographed on a column prepared with 30 g of aluminum oxide in a benzene-petroleum ether (8: 2) solution using the continuous flow method. The first benzene-petroleum ether fractions are combined (about 140 mg) and recrystallized from acetone-petroleum ether. The received rhombic plates from F. = 145 to 146 ° represent the 1,4-androstadien-3,17-dione Fractions of the chromatogram contain a small amount of 5-androstene ~ 3ß-ol-i7-one.

Example 3

A solution of 20 g of the debrominated, hydrolyzed and then dehydrated neutral part formed in the chromic acid oxidation of cholesteryl acetate dibromide in 700 cm ^{3 of} acetone by the Oppenauer method is added to 40 1 of a culture of Fusarium solani, shown according to Example 1, in a conventional culture batch with stirring and aeration. After the incubation and work-up described in Example 1, 5.2 g of 1,4-androstadiene-3,17-dione are obtained.

Example 4

The solution of 1 g of I, 4 "androstadiene-3, 17-dione in 35 cm ^{3 of} acetone is added to 4 liters of a culture of Fusarium caucasicum prepared according to Example 1. After incubation for 7 days, the mixture is worked up as described in Example 1 and the 'extract obtained was purified according to the Entmischungsmethode. recrystallization from acetone, finally about 6o ° / ₀ of the novel compound 1, 2-Dehydrotestolacton mp = 219 to 220 ° and the specific rotation [α] = -49% ± ₅ ° (c = 1.025 in CHCl ₃ ); ultraviolet spectrum: X _max = 242m / *; log.Z '= 4.23. Microanalysis: C = 75.70, H = 8.05%.

The same compound is prepared under the conditions of Example 1 from progesterone, 5-pregnen-3, 20-dione, 5-pregnen-3/3-ol-2O-one, ii-deoxy-corticosterone or 4-androstene-3, 17-dione obtained by incubating for 6 to 10 days instead of 2 days.

Example 5

Three Erlenmeyer flasks of 200 cm ^{3 of} capacity, each containing 50 cm ³ of the sterile nutrient solution described in Example 1 are inoculated with Fusarium solani and shaken mechanically for 48 hours at 26 ^0th Are then added to each culture, a solution of 10mg per Allopregnan-3,20-dione in 0.5 cm ³ of acetone and shaking continued at 26 ^0th After 24 and 48 hours and after 7 days, one culture is filtered and extracted as described in Example 1. The three extraction residues 1 to 3 are examined by paper chromatography. Extracts 1 and 2 contain androstane-3,17-dione, while extract 3 contains 1,4 ~ androstadiene-3,17-dione.

If in the above example the allopregnan-3, 20-dione is replaced by a corresponding 3/3-acetoxy-allopregnan-20-one solution, this is also found in the extracts after incubation for 24 or 48 hours the androstane-3, 17-dione and after 7 days of incubation the 1, 4-androstadien-3, 17-dione. Replaces in the example above, allopregnane-3, 20-dione is found through androstane-3, 17-dione only unchanged starting material is used in the extracts after incubation for 24 or 48 hours after 7 days of incubation the 1,4-androstadiene-3,17-dione.

Example 6

4 1 of a so-called Czapek-Dox nutrient solution (composition: 2 g NaNO ₃ , ι g K ₂ HB ₄ , 0.5 g KCl, 0.5 g MgSO ₄ , 0.02 g FeSO ₄ , 50 g glucose in 1 1 water ) are evenly distributed in two shaking flasks, sterilized and inoculated with a culture of the fungus Rhizopus suinus. After 2 days of shaking at 26 ⁰ , the fungus has developed well, and a solution of 500 mg of 11-deoxycorticosterone in 15 cm ^{3 of} acetone is added to each vessel under sterile conditions. Shake for a further 4 days at the temperature described above and then filter off the mycelium. The culture filtrate is shaken out as described and the extract is washed, dried and evaporated. The residue (1 g) is separated in a countercurrent process. To do this, it is dissolved in 50 cm ^{3 of} absolute ethanol and 150 cm ^{3 of} water and extracted with 200 cm ^{3 of} benzene. The lower layer is separated and passes through four more separatory funnel, with 200 cm ³ at 25 ° / ₀ sodium ethanol saturated benzene are charged. This procedure is repeated four times with 200 cm ³ of saturated benzene with 25 ° / ₀ sodium ethanol, so that, finally, five and five benzene aqueous alcohol solutions are available. They are evaporated in a vacuum. The paper chromatographic investigation shows that the aqueous-alcoholic solutions contain only a few highly polar by-products, while the first three benzene solutions. Contain 650 mg of a compound which is slightly more polar than the starting material and has no reducing power. By recrystallization

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Acetone petroleum ether is used to obtain the testolactone in needles with a F. = 203 to 206 °; [a] a '= + 40 ° (c = 1065 in chloroform) absorption bands in the infrared at 5.81, 5.98 and 7.17 μ (Nujol = specially purified Päraff oil).
Microanalysis for C ₁ ^ H ₂₆ O ₃ :

calculated ............. 'V C 75.46%, H 8.67%;

found C 75.26%, H 8.80%.

Example 7

Four Erlenmeyer flask of 200 cm ³ capacity, which each contain 50 cm ³ of the sterile nutrient solution described in Example 1, are inoculated with Fusarium solani and shaken mechanically for 48 hours at 26 ^0th The mycelium of the four cultures is then separated off individually and washed with water. The mycelia from two cultures are suspended directly in two Erlenmeyer flasks in 50 cm ^{3 of} distilled water each, while the other two mycelia are first ^{comminuted individually in a homogenizer with 50 cm 3 of} distilled water and only then transferred to an Erlenmeyer flask. To the two Myzelaufschlämmungen and the two Mycelhomogenaten is added per a solution of 10 mg of progesterone in 0.5 cm ³ of acetone and makes the sealed with cotton wool Erlenmeyer flask at - 26 ⁰ shake mechanically. After 36 hours, a mycelium slurry and a mycelium homogenate are filtered and extracted as described in Example 1. The paper-chromatographic investigation shows that the extraction residues mainly contain 1,4-androstadiene-3, 17-dione in addition to a little progesterone. After 7 days, the second mycelium suspension and the second mycelium homogenate are processed and examined in the same way. Only 1,2-dehydrotestolactone is present in the extracts obtained in this way.

Example 8

If, in example 6, instead of the culture of Rhizopus suinus, a culture of Venturia chlorospora or Venturia liniceräe and is used in the remaining 1 i-Desoxycorticosteroid incubated in the same way, testolactone with a melting point of 203 ° to 206 ° is obtained after a similar work-up.

Claims (3)

^{PATENTANSPKCCIIli:} ι. Process for the production of oxidized Steroids by incubating the steroids to be oxidized with a culture of a lower fungus . or the enzymes obtainable therefrom and separation the oxidation products, characterized in that androstanes or pregnanes, which contain a free or protected oxy or oxo group in the 3- and 17- or in the 20-position, with cultures of Fusarium solani, Fusarium caucasicum, Rhizopus suinus, Venturia chlorospora or Venturia liniceräe biologically oxidized. 2. The method according to claim 1, characterized in that that there are compounds of the Pregnan or Androstan series with a 4 (5) or 5 (6) position Double bond, especially progesterone, 5-pregnen-3/3-ol-2O-one, 11-deoxycorticosterone, 4'-Androsten-3, 17-dione, 5-Androsten-3 ^ -ol-17-one, also those in chromic acid oxidation of the cholesteryl acetate dibromide formed, entbrominated, hydrolyzed and then after the Oppenauer method. dehydrated neutral part as well as allopregnan-3, 2.0-dione or 3 /? - Acetoxy-allopregnan-2O-oii as starting materials used. 3. The method according to claim 1 and 2, characterized in that the culture of the fungus or the enzymes contained therein act on the starting steroid for 1 to 2 days or 6 to 15 days leaves. ■ Considered publications:
U.S. Patent No. 2602769;
Experientia, Vol. 8, 1952, p. 422;
Research (London), Vol. 6, 1953, p. 309. © 609 577/488 7. 56