Unitedto up O States - Patenta 1- Office Patented July2,844,513 22, 1958 2 one, d, 1-1-dehydro-17 oz-hydroxy-progesterone, d,1-1-dehy 2,844,513 dro-11-oxo, 11c-hydroxy- or 116-hydroxy-progesterone, dl-androstene-diol, d.l-17-methyl-androstenediol, d.l-de PROCESS FOR SPLTTING RACEMATES hydro-epi-androsterone or their functional derivatives. Albert wettstein and Ernst Vischer, Basel, and Charles 5 Especially important starting materials for the aldoster Meystre, Arlesheim, Switzerland, assignors to Ciba one synthesis are, for example, d.l-A-3:20-dioxol 18-hy Pharmaceutical Products Inc., Summit, N.J. droxy-pregnene-18-acid-lactone-(18->11), d.l-A-3:18:20 M trioxo-11p-hydroxypregnene or its 18:11-cyclo-semiacetal No Drawing. Application July 18, 1956 and d.1 - A - 3:20 - dioxo -116:18-dihydroxy-pregnene. Serial No. 598,542 10 There may also be mentioned the corresponding com Claims priority, application Switzerland July 20, 1955 poundsThe processhydroxylated is advantageously in the 17a-position. carried out by subject 12 Claims. (C. 195-3) ing the starting material to the action of an aerobic cul ture of a single micro-organism. It is known to oxygenate or dehydrogenate with the 15 Depending on the micro-organism used, there is ob aid of micro-organisms steroid compounds having a nat- tained, for example, the d-steroid containing a hydroxyl ural configuration, that is to say, to introduce hydroxyl group in the 66-, 7c, 76, 8-, 11a, 116-, 14-, 1.5oz-, 156-, or oxo groups or double bonds into these compounds, 16a, 17a- or 21-position, or containing a double bond or convert hydroxyl groups into oxo groups. in the 1:2- and/or 4:5-positions or with a A-3-keto The present, invention is based on the unexpected ob-20 and/or 17-keto-grouping. Alternatively, the starting servation that di-steroid compounds behave differently material may be subjected in one operation to the action with respect to these micro-organisms in that substan- of several cultures, it being of advantage to subject the tially only the d-form, that is to say, the enantiomorphic starting material to the action of the individual cultures form corresponding to the natural steroids, is oxidized, in succession. and the 1-form remains unchanged. This observation has 25 There are suitable for the present process all aerobic led to a new process for splitting racemates, in which cultures of micro-organisms which are capable of oxid there are obtained, in addition to the oxidized d-steroids, izing steroid compounds, that is to say, introducing hy 1-steroids of which only a few instances are known. droxyl groups or double bonds or of converting hydroxyl Accordingly, this invention provides a process for groups into oxo groups. A few types of micro-organisms splitting d.l-steroids into their enantiomorphic forms, 80 for use in the present process are given by way of ex wherein d.l-steroids are subjected to the action of oxid- ample below: izing enzymes produced by the aerobic culture of micro organisms, and at least one of the enantiomorphic forms 69-PE, ecium roseum is isolated. Oxidizing enzymes are such as are capable. 35 Lenzites abietina either of introducing oxygen into the steroid or of re Rhizopus arrhizus moving hydrogen therefrom. Gliocladium catenulatum Suitable starting materials for the new process: are in Gliocladium deliquescens general saturated or unsaturated d.l-steroids substituted 7a-position: in any desired manner, for example, d.l-compounds of 40 Curvularia lunata the cholestane, coprostane, cholane, spirostane, furostane, Curvularia palescens butanolide or cardanolide series, and especially those of Curvularia falcata the pregnane, androstane or testane series, and also their Curvularia fallax higher and lower homologues, for example correspond Peziza spec. ing A-nor-, D-homo- or 19-nor-compounds. Double 76-position: bonds may be present, for example, in the 1- and/or 4-, 45 Rhizopus arrhizus 5-, 6-, 7-, 9-, 11-, 14-, 15, and/or 16-positions. As Proactinomyces roseus substituents there come into consideration free or func tionally converted hydroxyl, oxo or carboxyl groups, PE allescen such as ester, ether, thioester, thioether, thiol-ester and 50 Pleospora gaeumannip s thion-ester, acetal, mercaptal, ketal, hydrazone, semi Helicostylum piriforme carbazone and enol groups, for example, in the 2-, 3-, 11oz-position: 6, 7, 11-, 12-, 16-, 17-, 18-, 19, 20- and 21-positions, Rhizopus nigricans and also halogen atoms, especially chlorine or fluorine, Rhizopus arrhizus for example, in the 9- and 17-positions. The new proc 55 Aspergillus niger ess is of special interest as applied to d,l-pregnane com Aspergillus ochraceus pounds. Examples of starting materials of this series . Penicillium notatum are, inter alia, d.l-progesterone, d.l-17o-progesterone, Penicillium adametzi d-16oz- hydroxy-progesterone, d.l-17 oz-hydroxyprogester Penicillium janthinellum one, d.l-11-oxoprogesterone, d.l-11a- and -116-hydroxy 60 Mucor nucedo progesterone, d.l-9:11- or 11:12-dehydroprogesterone, d.l- Lenzites sepiaria 19-oxoprogesterone, d.l-11-oxo-17a-hydroxy-progester Tilletia tritici one, d.l-11a- and 11B-hydroxy-17a-hydroxy-progesterone, Neurospora sitophila dl-9-chloro- or -9-fluoro-11p:17a-dihydroxy-progester one, d.1-116:18-dihydroxy-progesterone, d.l-11f8:17:18 Neurospora crassa trihydroxy-progesterone, d.1-116-hyroxy-18-oxo-proges 65 115-position: terone, d.l-9-chloro- or -9-fluoro-115-hydroxy-18-oxo-pro Curvularia lunata gesterone, d.l-11:18-dioxo-progesterone, d.l-19-nor-pro Curvularia pallescens gesterone, d,i-19-nor-11 (3-hydroxy-18-oxo-progesterone, Curvularia fallax d,l-cortexone, d.l-18-hydroxy- and 18-oxo-cortexone, d.l- Curvularia brachyspora cortisone, d.l-hydrocortisone, d.1-17a-hydroxy-cortexone, 70 Cunninghamella blakesleeana d.1-aldosterone, d.l-pregnenolene, the corresponding 1-de- . Streptomyces fradiae : hydro-compounds, for example, d.l-1-dehydroprogester- . Stigmina platani 2,844,513 3. 4r 14-position: be incubated in known manner under aerobic conditions Sclerophoma entoxylina with the cultures of the aforesaid micro-organisms. The Parasitella simplex growth may be carried out as a surface culture or, which Helicostylum piriforme is technically preferable, as a submerged culture, with Mucor griseocyanus agitation or stirring. The culture media contain as Mucor parasiticus similable carbon, especially carbohydrates, and if desired, 15o-position: growth promoting substances, for example, corn steep Gibberella baccata liquor or beer wort, and inorganic salts. Thus there Nectria cinnabarina may be used natural, synthetic or semi-synthetic nutrient Hornodendrum viride O solutions. The simplest way of carrying out the proc 156-position: ess may be illustrated as follows without limiting the in Lenzites abietina vention thereof: The organisms are cultivated in ap Spicaria paratus and under conditions known in the production 160-position: of antibiotics as the so-called deep tank process. After Didymella vodaki 15 the culture has developed, a starting material of the kind Actinomycetes ETH A 6246, A 7746, A 7747. A defined above is added in the form of a fine dispersion 7451, A7486 or solution, for example, in methanol, acetone or ethyl 17 cy-position: ene glycol, and incubation is continued. Finally the Trichothecium roseum filtrate and/or the mycelium mass is extracted, and the Cephalothecium roseum 20 d-steroid and/or the l-steroid is isolated in known man Cucurbitaria laburni ner from the extract, for example, by a demixing meth Leptosphaeria maculans od, adsorption, chromatography, crystallization, conver Trichoderina lignorum sion into functional derivatives such as Girard com Trichoderma glaucum pounds, or the like. The same reactions can also be Acrospeira levis 25 carried out by first separating the active enzymes from Lophotrichus hartini the aerobic cultures of the appropriate organisms and Melanospora parasitica using them apart from the growing cultures. Thus, for Thielavia terricola example, the mycelia formed by the aerobic cultivaticn Dactylium dendroides of the appropriate organisms are separated, Suspended 21-position: 30 in water or buffer solutions, the appropriate starting Ophiobolus herpotrichus materials are added to the suspensions and the latter Sclerotinia fructicola are incubated. Wojnowicia graminis When several micro-organisms are to be used in a Hendersonia rubi single operation, the process may be carried out as fol Hendersonia abietus 35 lows: After developing the culture of the first organism, Dilophosphora spec. the starting material is added in fine Suspension or in Phona hibernica solution, for example, in methanol, acetone, or ethylene Septoria aesculi glycol, and incubation is continued until the maximum Aspergillus niger reaction has taken place. Then, without previous filtra 1:2-dehydration: 40 tion or isolation of the oxidation product, a fully de Fusarium solani veloped culture of the second organism and if necessary, Fusarium caucasicum the appropriate nutrient substances and growth promot Calonectria decora ing substances are added to the reaction mixture, and Alternaria passiflorae the incubation is continued. If desired, this operation Ophiobolus heterostrophus 45 may be repeated with a third micro-organism. The Ophiobolus miyabeanus progress of the individual oxidations can be observed by Didymella lycopersici paper chromatography. Corynebacterium simplex The products of the process can be used as medica Bacillus sphaericus ments or as intermediate products for making medica Bacillus subtilis 50 ments. The new process is especially valuable in the Mycobacteria aldosterone synthesis. Thus, for example, by the action 3 and/or 17-position: of an aerobic culture of Ophiobolus herpotrichius on Proactinomyces erythorpolis d: 1-A-3:18:20-trioxo-1 16-hydroxy-pregnene or its 18:11 Proactinomyces aquiosus cyclo-semiacetal in a single stage d-aldosterone is ob Proactinomyces restrictus 55 tained in good. yield. When using a purely chemical Proactinomyces roseus process about 7 steps are required for this synthesis, using Azotobacter the same starting material.