BE549702A - - Google Patents

Description

       

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   It is known that steroids having a natural configuration can be oxygenated or dehydrogenated with the aid of microorganisms, ie introducing hydroxyls or oxo groups or double bonds, or transforming hydroxyls into oxo groups. However, the applicant has made - the surprising observation that d, # - steroids behave differently with respect to these microorganisms, practically

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 only the d form, that is to say the enantiomorphic form corresponding to natural steroids, being oxidized, while the formal remains unchanged.

   This finding has therefore led to a new process of racemate cleavage which makes it possible to obtain, alongside oxidized d-steroids, the # -steroids which, until now, had only been encountered in rare cases. .



   This new process is characterized by the fact that oxidizing enzymes produced by means of aerobic cultures of microorganisms are made to act on d, # - steroids, and that at least one of the enantiomorphic forms is isolated. Oxidizing enzymes are those which are capable of either introducing oxygen into the steroid molecule or removing hydrogen from it.



   Starting materials for the new process are generally d, # - saturated and unsaturated steroids, bearing any substituents, for example d, # - compounds of the cholestane, coprostane, cholane, spirostane, d series. furostan, bufanolide, cardanolide, estrane, especially those of the pregnan, androstane and testan series, as well as their higher and lower counterparts, for example A-nor, D-homo, and 19 -nor-corresponding compounds Double bonds are found, for example, in position 1 and / or 4, 5, 6, 7, 9, 11, 14, 15 and / or 16.

   Suitable substituents are in particular hydroxyl, oxo or

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 EMI3.1
 free or functional modified carboxylic groups, for example ester, ether, th ') ester, thioether, thiol- groups
 EMI3.2
 and thion-ester, acetal, mercaptal, ketal, hydrazone, semi-carbasone and enolic groups, for example in position
 EMI3.3
 2, 3, 6, 7, 11, 12, 16, 17, 18, 19, 20 and 21, as well as
 EMI3.4
 halogen atoms, in particular chlorine or fluorine, for example in position 9 and 17.

   It is particularly advantageous to implement this new process in the case of
 EMI3.5
 d1? -pregnanlqueso compounds Specific starting substances in this series are, among others, d, / -progesterone, d $ '? e-17a-progesterone, d, -l6a-hydrosy -progesterone, d, t : -17a- hydroxyprogesté3 * on, d, -e-11-oxo-progesterone, d, '- lla- as well as d, * 113-hydroxy-progeoterone, d, t-9,11- or - ll, 12-dehydi> o-prosesteroneJ, d..t-19-o, w-progeeterol'H3, d, '' 6'-ll-O3s: ō17a-h.yd3.'ioxîy¯pi 'ogesterone, d, -I-11a- as well as d..t: 11-hYdOXY-17a-hYdroXy-progeatone, d, if-9-chloro- or -9-fluoro-lip, i7a-dîhsrdrosy- progesteron, dj, f-11, 18-di-hydrorcy-progesterone, d, -11,17,18-trlhYdroxy-proggterone, d .. ';

  -11-hYàroXY-18-oxo-progesterone, d..9-chloro- or -9-fluoro-lip-hydroxy-l8-oxo-progeatone, Ci, - {- II, 18-dloxo-progesterone, d, e-19-nor-progeatone, d, -19-nor-lip- hydro: ty-l8-oxo-progesterone, d, C-cortexone, d, 118-hydroxy- and d, -l8 -oso-cort @ sone, d, t-oortîsone, d, if # hydrocortisone, d, t-17a; -hyctroxycortéxone, d, if-aldosterone, d, -pregnenolone, 1- dehydro-compounds corresponding

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 EMI4.1
 laying agents, for example d, -3-dhydro-progest: one, la d, - ;; ": l-dehydro-17-a-hydroxy-progesterone, la d, 1 - dehyd = o-1-oxo- - proge3teror.e, d, 1-dhydr o-11-a-hydro: y - p9ogestrone or d, 1-dehydro-ll-hydroxy-proge6terone, α, f-androstene-dia, d, f -l'7-methyl-androatène-diol, d, {- dehydro-epi- andro3teror.e, or their functional derivatives.

   Particularly important starting substances for the synthesis of
 EMI4.2
 aldostere are, for example, I8 - 11-.actone of d, '- p 3,2C dioo-.1 ° -hydroy-prgnérie-18-oique, d ^ leA4¯ 3,18,20 - cr3.oxo-1.-hydroay-prgn: ne or its 18,11-cyclo-semi-acetal and d, - & -3,20-dioxo-lll8-dihydroxy-prëgnene. The corresponding compounds hydroxylated at position 17a should also be indicated.



   The present process is advantageously carried out by causing the aerobic culture to act on the starting substance.
 EMI4.3
 of a single microorganism. Depending on the microorganism used, one obtains, for example, the d-steroid comprising a hydroxyl in position 6p, 7a, 7P, 8, IIa, lip, 14, 15a, 15P, i6a, 17a or 21, or comprising a double bond in position 1,2 and / or 4,5,
 EMI4.4
 or comprising an A 4¯3-ketonic and / or 17-keto group.



  However, it is also possible to proceed by causing several cultures to act on the starting substance in an operating phase. It has then proved advantageous to cause the various cultures to act successively over time.



   All aerobic cultures of microorganisms that

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 EMI5.1
 are capable of oxidizing steroldems, i.e. introducing hydroxyls or double bonds, or converting hydroxyls to oxo groups, suitable for the present process.



  Some categories that are envisaged for the process have been indicated for example below.
 EMI5.2
 Position 6 Trichothecium roseum Lenzites abietina Rhisopus arrhizus
 EMI5.3
 G110cladium catenulatum Gliocladium deliquescens Position 70 Curvularia lunata
Curvularia pallescens
 EMI5.4
 Curvularîa falcata Curvularia fallax
 EMI5.5
 .Psziza spec.



  Position 7:, Rhisopus arrhisus Proactinomyces rosées Position 8 Curvularia palieseens Pleospora Gaeumanni Helioostylum piriforme
 EMI5.6
 Position 11a; Rhisopus iiigrîcans Rhiopus arrhizus Aspergillus niger Aspergillus, ochraceus Penicillium notatum

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 Penicillium adametzi Penicillium janthinellum Mucor mucedo
 EMI6.1
 Lensites sepiarla Tilletia tritici Neurospora sitophila Neurospora crassa
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 Position 119 Curvularia lunata Curvularia pallescens Curvularia fallax Curvularia brachyspora
 EMI6.3
 Cunnlnghamel1a Blakesleena
Streptomyces fradiae Stigmlna platani Position II}:

   Sclerophoma entoxylina Parasitella simplex Helicostylum piriforme
Mucor griseocyanus
 EMI6.4
 Mucor parasiticus Position 15 [alpha] Gibberella baccata Nectria cinnabarina
 EMI6.5
 HorModendrium viride Position 15ss: Lenzites abietina Spicaria Position 16 [alpha]: Didymella Vodakii

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 EMI7.1
 Actinomycetes A 6246, A 77 * 6 * A 7747. A 7451,
 EMI7.2
 A 7486 kept at the Swiss Federal Institute of Technology in Zurich
 EMI7.3
 Position 17a:

   Trichothecium roseum Cephalothec1um roseum Oucurbitaria laburni Lspthosphaeria maculans Trichoderma lignorum Trichoderma glacum Acrospeira lavis
 EMI7.4
 Lophotrichua hartînîl Melanospora parasitica Thielavia terricola Dactylium dendroldes
 EMI7.5
 Position 21 Ophiobolu3 herpotrichus Sclerotinia fruoticola
 EMI7.6
 Wojnowlela graminia
Hendersonia r'ubi
Hendersonia abietus
Dilophosphora spec.



   Phoma hibernica
Septoria aesculi
Aspergillus niger
 EMI7.7
 , Eshydrogenation in ll '2: Fusr1um solani Fsar1m cauca.a1cum Calonectria decorates Alternaria passiflorae

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 Ophiobolus heterostrophus Ophiobolus Miyabeanus Didymella lycopersici Corynebacterium simplex
 EMI8.1
 Bacillus sphaer1cus Baeillus subtilis
 EMI8.2
 % cobacteria Position 3 and / or 17 Proactinomyces erythropolis
Proactinomyces aquosus
Proactinomyces restrictus
Proactinomyces roseus
Azotobacter
 EMI8.3
 94yeobacteria Mlcrococcua dehydrogenans Corynebacterium mediols.num Alca11gerHa faecalis Yeast and oxidizing bacteria Pseudomonas
 EMI8.4
 Flavobac terium

  androstenedionleum Plavobacterlum carbonilicum Actinomyces albus Aspergillus niger
 EMI8.5
 Phycomyces blakesleeanus Penicillium chrysogenum.

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     @As it emerges from the above list, the microorganisms indicated are fungi or bacteria and belong, for example, to the classes of phycomycetes, ascomycetes, basidiomycetes, Fungi imperfecti or actinomycetes.



   For carrying out the process, the starting substances can be incubated under aerobic conditions known per se with cultures of the microorganisms indicated.



  Growth takes place in a surface culture or, which is technically advantageous, in a submerged culture, in which case one shakes or shakes. The cultures contain assimilable carbon, in particular carbohydrates, as well as, where appropriate, growth substances, for example corn swelling water or beer wort, and inorganic salts.

   It is therefore possible to use natural, synthetic or semi-synthetic nutrient solutions. A practically very simple process will be described in what follows without the invention being limited for all that to the indications provided. The organisms are cultivated in similar apparatus and under conditions. those which are known in the manufacture of antibiotics as the submerged culture process.



  After the cultures have grown, the indicated starting substances are added in the form of a fine dispersion or solution, for example in methanol, acetone or ethylene glycol, and the incubation continues. Finally, we separate the mycelium,

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 extracts the filtrate and / or the mass of mycelium and isolates from the extract in a manner known per se, the d-steroids and / or the # -steroids, for example by partitioning between solvents or mixtures of immiscible solvents with each other, by adsorption, chromatography, crystallization, transformation into functional derivatives, such as Girard compounds and the like.

   These same reactions can also be carried out by first separating the active enzymes from suitable aerobic cultures of the organisms indicated and using them to the exclusion of the growing cultures. It is thus possible, for example, to separate the mycelium formed from suitable aerobic cultures of the organisms indicated, to suspend it in water or in buffer solutions, to add to these suspensions the starting substances indicated. , then incubate.



   If several microorganisms are to act in one operating phase, the following procedure can be carried out, for example, After development of the cultures of the first organism, the starting substances indicated are added, in the form of a fine dispersion or of solution, for example in methanol , acetone or ethylene glycol, and continue incubation until maximum reaction is reached. A grown culture of the second organism and, if necessary, nutrients are then added to the reaction mixture, without filtering or isolating the reaction product.

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 and appropriate growth slugs, then continue incubation.

   If necessary, this is repeated with a third microorganism. The progress of the various oxidations can be followed by chromatography on paper.



     The products of the process can be used as medicaments or as intermediates for the preparation of other valuable products. The new process has proved to be particularly interesting for the synthesis of! @ Aldosterone.



  Thus, for example, we obtain in a single phase, with a good yield, d-aldosterone, by causing an aerobic culture of Ophiobolus herpotrichua to act on d, # - # 4-3,18, 20-trioxo-11ss-hydroxy-pregnene or on its 18,11-cyclo-semi-acetal.



  When operating by means of a purely chemical process, approximately 7 stages are necessary for this synthesis, starting from the same initial product.



   In general, the resolution of racemates according to this new process is particularly straightforward because the hydroxylated or dehydrogenated derivatives of one of the antipodes can easily be separated from the other antipode Unchanged, owing to their high polarity.



   Already since the classic researches of Pasteur, it is true that a microbiological method was occasionally used to obtain one of the antipodes from racemates. Usually ordinary fungi or bacteria were used which assimilated the substances of

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 starting point and which, in particular, assimilated the natural antipode (d) faster than the non-natural antipodes (±}. According to this process, in order to obtain an optically pure preparation, it was therefore necessary to treat microbiologically up to 'that at least one of the forms is completely destroyed, especially the one which, most of the time, was the most interesting from a biological point of view.

   On the contrary, the process of the present invention provides, even in the case of a reaction which is only partial, the modified antipode, which, most of the time, represents that which is the most interesting from the point of view. biological, in an optically pure form, and this in the form of a derivative which is practically valuable in itself or for other synthetic purposes, and saves chemical transformations. If one of the antipodes is completely transformed, following this process, the other is also present in an optically pure form.



   The present invention relates to a process for splitting racemates into their constituents.



   The invention is described in more detail in the non-limiting examples which follow in which the temperatures are indicated in degrees centigrade.

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 EMI13.1
 



  E: r. Example 1.



   In five conical flasks of 500 cm 'capacity, 120 cm of an aqueous solution are sterilized in each.
 EMI13.2
 70% beer mate and inoculated with Ophiobolus herpotrilus. Or shake the flasks at 270. After 4 days, and under sterile conditions, a 30 mg solution is added to each of the well-developed cultures. of dj, progesterone in 1.5 ctt (acetone and continue to stir at the same temperature.

   After 4 days, the mveelium is separated and washed with water and ethyl acetate. After combining them, the filtrates are extracted four times with 200 cm3 of ethyl acetate each time. The combined extracts are washed with water, dried and evaporated. From an estimate
 EMI13.3
 carried out by chromatography on paper, the residue (160 mg) contains approximately equal amounts of two compounds which behave like cortexone IIIm (Mao;): y-corticoaterone resp. progesterone). These two compounds are separated by a preparation chrom4qtograpille: /, PD7> paper, with the Bush B 3 system.

   The areas corresponding to the cortexone are cut and eluted with 400 in.3 of 50% methanol. The methanol is then removed in vacuo. The aqueous solution which remains is extracted four times with 50 cm 3 of chloroform each time, after having combined them, the extracts are washed with water, dried and evaporated in vacuo. The residue is crystallized
 EMI13.4
 in a mixture of acetone and ether. and we oht1.b toua

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   pu.-ce, d-cortexone with a melting point of 140 - 143.



    [[alpha]] D = + 180 / in ethanol). The zones of the preparation chromatogram on paper which correspond to progesterone are treated in the same way. 92 mg of a crude extract are obtained, from which there is obtained, by repeated crystallization from a mixture of acetone and petroleum ether, pure # -progesterone, melting point 122 - 125; [[alpha]] D = - 190 (in acetone).



    ¯Example 2.



   In a stirring vessel, inoculated with Cunninghamella Blakesleena 500 cm3 of sterile beer wort and stirred 3 days to 27. Under sterile conditions, a solution of 150 mg of d, # - cortexone in 8 cm3 of acetone was then added to the well-grown culture. At the same time, in a second container 500 cm3 of sterile beer wort are inoculated with Trichothecium roseum. The two cultures are agitated. for 3 days at 27 and then combine them under sterile conditions, after which stirring is continued at the same temperature.



  After 3 days, the mycelium is separated and washed with water and ethyl acetate. After having combined the filtrates, they were entered as described in Example 1. According to an evaluation carried out on paper, the residue obtained (165 mg) is essentially formed of 3 compounds which correspond respectively to cortexone, to hydrocortisone, and cortisone. This residue is chromatographed according to the

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 method by passing through 8 g of silica gel, eluting first with chloroform, then with a mixture of chloroform
 EMI15.1
 rt of acetone (99 1 L and finally with a mixture of chloroform and acetone (1: 1,;.

   The various fractions (20 cm 3 each) are subjected to chroniatographic examination on paper.



  The fractions eluded with chloroform contain impurities, while. in the fractions eluted with the mixture of chloroform and acetone (99: 1) there is a compound corresponding to cortexone. These fractions are combined and evaporated.



  By crystallization from mixtures of acetone and ether,
 EMI15.2
 we get the - & cort3òne with a melting point of 138 - 112 ().



  [a] p - 1150 (in ethanol). The other fractions studied with the mixture of chlo20form <s and. acetone (1x1) are basically made up of d-9 d-hydrocortisone and d-cortleone. They are combined, evaporated and the constituents separated using a preparation process on paper (p'popylenegly-col-t "luene system. The treatment of the areas of paper respectively containing the d-hydrocortisone and α-cortisone a 11reÙ "as described in Example 1. By crystallizing the two extraction residues from mixtures of acetone and iaopropyl ether, d-hydrocortisone of a melting point of
 EMI15.3
 205 - 210 ai D 165 (in ethanol) and d-cortisone with a melting point of 210 - 216, [a] D "- <- 195 (in d 1oxa.nne).

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   Example 3.



   In a shaking container, dissolved in 500 cm3 of tap water 1 g of sodium nitrate, 0.5 g of primary potassium orthophosphate, 0.25 g of magnesium sulfate-heptahydrate, 0.25 g of potassium chloride, 25 g of raw glucose and 0.5 g of Difco yeast extract, brought together to a pH of 5 and sterilized. This nutrient solution is inoculated with Didymella lycopersici and stirred for 3 days at 27. A solution of 120 mg of d, # - cortisone in 5 cm3 of methanol is then added to the whole, under sterile conditions. Stirring is continued for 4 days at 27, then the mycelium is filtered off and washed with water and ethyl acetate.



    After combining the filtrates, they are extracted as described in Example 1. As can be seen from chromatographic examination on paper, the extraction residue contains a product which behaves like the starting material, and l-dehydrocortisone. Using preparative chromatography on paper / propylene glycol-toluene system), the constituents of this mixture are separated.

   By recrystallization from acetone, d-1dehydro-cortisone with a melting point of 231 - 234, [[alpha]] D = + 1700 (in dioxane) is obtained By repeated crystallization from a mixture of acetone and isopropyl ether, pure "-cortisone with a melting point of 211-217, [[alpha]] D = - 1900 (in dioxane) is obtained.

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   Example 4.



     A nutrient solution is prepared containing, for one liter of tap water, 20 g of peptone, 5 cm3 of
 EMI17.1
 corn swelling (Cornsteep-l1quor) and 10 g of raw glucose and the pH of which is adjusted to 6.3. We py @ nd! 4 conical flasks with a capacity of 500 cm3 and sterilize in each of them 120 cm3 of the above nutrient solution and inoculate them
 EMI17.2
 with Wojnow1cia gram1nis. These cultures were stirred for 4 days at 27, after which was added, under sterile conditions, each time a solution containing 30 mg of
 EMI17.3
 (18- # p-llgj-lactone du U 4.x ".% 1 3, nGlJr'tlJ.o.ad .L.1. Iâ '., Doà pgli" w' 18-oic in 1, 5 char of methanol.

   Stirring is then continued <3 "at the same temperature. After 8 days, the
 EMI17.4
 mycelium washes it with water and ethyl acetate, combines the filtrates and extracts them as described in Example 1. The extraction residue (140 mg) contains, according to an evaluation
 EMI17.5
 chyomstogra.phique on paper, a product analogous to the starting material, as well as .8 -x.llwlacton of Â! -3,20- dioco - .. (, 1-dihydror-prgnéne 18-ou. The constituents of this residue are separated using preparative chromatography on paper (forraamide -benzene -chloroform system) # As described in Example 1, the two components and the extract are eluted on paper.

   By crystallization in a mixture
 EMI17.6
 acetone and ether, tl8yll.p .acton of d-A -'320-dioxo-llp21-dihydroxy-prëgnene-l8-oic of a

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 mp 205-222, 1a] - '= 1170 (in acetone).



   Example 5.



   In 4 conical flasks, 12C cm3 of the nutrient solution described in Example 4 are introduced each time, then
 EMI18.1
 sterilized. Seed with Ophiobolus herpotricU6 and shake the cultures for 5 to 27 days. Then added to each of the cultures, under sterile conditions,
 EMI18.2
 a solution of 30 mg of d9 - ,. 8,20-trioo-â.-Idroy¯ pregnene or its 18,11-cyclo-semi-acetal in 1.5 car of methanol and continue to stir at the same temperature.



  After 10 days, the mycelium is separated, washed with water and ethyl acetate, the filtrates are combined and extracted as described in example 1. Along with a product analogous to the material. initially, the extraction residue contains a compound which behaves like aldosterone. This residue is purified using preparative chromatography on paper (Bush C system), the elution and extraction being carried out as described in Example 1. In a mixture of acetone
 EMI18.3
 and ether gives crystalline d-aldosterone which melts in 162 - 168, .ct] =. 142 (in acetone "

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   Example 6.



   In a shaking container, 300 cm3 of the nutrient solution described in Example 4 are sterilized and inoculated with Wojnowicia graminis. After 4 days of stirring, the culture has developed well and a solution of 50 mg of d, # - # 4-3,18,20-trioxo-11ss-hydroxy-prégnene is added to it under sterile conditions. or its 18,11-cyclo-semi-acetaldans 3 cm3 of methanol. At the same time, in a second / stirring vessel, 300 cm3 of beer wort sterilized with Trichothecium roseum are inoculated.

   The two cultures were stirred for 4 days at 27, then combined under sterile conditions, and continued to stir at the same temperature. After 4 days, the mycelium is separated and washed with water and ethyl acetate. After having combined the filtratson extracts as described in Example 1.



   Besides a compound which behaves like the starting material, the extraction residue (55 mg) contains α-17 [alpha] -hydroxy-aldosterone. The latter is obtained in the unitary state after preparative chromatography on paper (propylene glycol-toluene system), the elution and extraction of the papers being carried out according to the indications given in Example 1.

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     Example 7
In a shaking container, 300 cm of beer wort are sterilized and inoculated with Trichotecium roaeum. The culture is stirred for 4 to 27 days, after which a solution of 60 mg of d, # - aldosterone in 3 cm3 of methanol is added under sterile conditions. Stirring is continued for 4 days at 27, then the mycelium is separated and washed with water and ethyl acetate. After combining them, the filtrates are extracted as described in Example 1. The extraction residue which, next to the starting material contains d-17 [alpha] -hydroxy-aldosterone, is separated with using preparatory paper chromatography (Bush C system).

   By elution from the corresponding areas of the papers according to the indications given in Example 1, d-17 [alpha] -hydroxy-aldosterone is obtained in unit form. It crystallizes in a mixture of acetone and ether. From the zones corresponding to the starting material *, by crystallization in a mixture of ether and acetone, la-aldosterone with a melting point of 162-168 and a rotatory power is obtained. specific [[alpha]] D = -1420 (in acetone).



   Example 8
In a shaking container, inoculated with Ophiobolus herpotrichus 300 cm3 of sterile beer wort and stirred at 27. After 5 days, add to the whole, in

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    under sterile conditions, a solution of 60 mg of d, - # 4-
 EMI21.1
 5,20-dioxo-ilpj, 18-dihydrosy-pregnens in 4 car of methanol and continue to stir at the same temperature. After 6 days, the mycelium is separated and washed with water and acetate
 EMI21.2
 d t ethyl.

   After combining them, the cossus filtrates indicated in Example 1 were extracted. Diaprés evaluated by paper chromatography., The residue consists of a compound analogous to the starting material, thus
 EMI21.3
 only by d-A ¯5J, 20 dloxo-lipl8,21¯til) ihydro> î3f¯prëgaàae which is separated using a preparative chromatogaph1G on speaking (propylee-glyool-toluene system). d, oL / -3,, 20 ... dioxo. "llp., 18j, 21-.trihydroxy-pygnèns crystallizes from a mixture of acetone and ether.



    Example
In a shaker container, sterilize 200 cm
 EMI21.4
 of a (.: olutl!: m nutrient which contains, for one liter of tap water, 10 g of raw glucose., 5 g of peptone, 3 g of extract
 EMI21.5
 of meat (O.Ko Lab .temco L 5 g of sodium chloride and 10 g of calcium carbonate, and which is adjusted to a pH of 7.5, then inoculated with a culture of a strain of Streptomyoes albus.

   We stir 2 days at 27 and then add to the whole, in
 EMI21.6
 under sterile conditions, a solution of 50 mg of d / L-a-estradiol in 2 cm3 of dioxane. Stirring is continued for 3 days at 27, after which the mycelium is separated and the

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 Cultured filtrate with methylene chloride. According to chromatographic examination on paper, the extraction residue
 EMI22.1
 is constituted by- compounds, corresponding to l1a-ostradlo1 and 1 ort a! e The constituents of the mixture are separated using preparative chromatography on paper and one obtains, by crystallization from aqueous methanol,

     of a
 EMI22.2
 part .e ae t = Wdio. with a melting point of 175-178, - ta] - -81 and, on the other hand, doestrous with a melting point of 25 '- 5g - + 101 & Example 10 A 200 cm5 of a c Ë ;:. After well-developed stirring of Pseudomonas, a solution of 50 mg of in 2 ml of dloxane is added under sterile conditions. We shake z8 tt: id's: 'r3: S 2'Yo and. then removing the culture with methylene chloride. According to a chromatographic examination on paper, the exaction residue behaves as
 EMI22.3
 androsterone and r.:.; "51.1. ' ? W .... ir 'CW vLE'. '.

   The constituents of this mixture are separated using a preparation chr-omatographiê on paper and 1 ion is obtained by crystallization
 EMI22.4
 in aqueous methanol, la) ja; 1droatéx "one with a melting point of 180-1820; [a] D m g2a is ë-3.17ioetondrostane with a melting point of 130-132". [a] p - - 109.

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  Example 11
 EMI23.1
 - .. ¯¯¯¯ - ¯¯¯ ..



  At 200 because of a well-developed culture of agitation
 EMI23.2
 According to a strain of Streptomyces fradia, a solution of 50 mg of d, -prgillnolone in 2 cm of acetone is added under sterile conditions. Stirred for 24 hours at 27 and then extracted the culture with methylene chloride.



  Based on chromatographic examination on paper, the residue
 EMI23.3
 extraction behaves like pregnenolone and progesterone. The constituents of the mixture are separated using preparative chromatography on paper and obtained, by crystallization from mixtures of acetone and ether
 EMI23.4
 petroleum, 7.a-prgrnolane with a melting point of 187-189, la] D = -250 and d-progestroae with a melting point of 125-127, [[alpha]] D = + 1900 - @
Example 12
 EMI23.5
 ..¯..m..¯, ... ¯¯¯.



  If in the example above, a culture of Aspergillus niger was used to transform the dl-pre @ 1enolonGI instead of the culture of the strain of Streptomyces fradiae and which was incubated and processed as indicated, we ,
 EMI23.6
 can then also obtain, -pregnenolone with a melting point of 185-1880, [a3p "-23 and d-progesterone with a melting point of 123-126, [aj D = + 191.

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 EMI24.1
 EK & sple 13
 EMI24.2
 A nutrient solution is prepared which contains,
 EMI24.3
 for a liter of tap water, 2.6 g of tartaric resolves, 2 6 g of 8? ''? '3rGf of ammonium, 0117 g of C31.'a.' at'. áfn.ilt3'iâ'U:

  '? s 0.4 g of secondary ammonium phosphate, 0, k- g of potassium carbonate, 0.27 g of magnesium carbonate, 50 g of glucose and 1 g of yeast extract (Difoo) and adjust the pH to, C. In a conical flask 120 μl of this solution are sterilized, inoculated with Rhizopus nigrieans and stirred at bzz. Have?; After 2 days, a solution of 30 mg of dan -progesterone in 1.5 cm of salt is added to the whole, in sterile O 2, 'onN, and the stirring is continued for 2 days at'. The mycelium is then separated and washed at 1 c: with and with methylene chloride. After having combined it, the extract is extracted:!. 1t:;. "" '. 7.a..S'v..T - .; 's "n. to 5 1? 0} .1!' isos with methylene chloride, 18.l them:; ::. tt: '2.it,; j to 1 at \ $ read dries and evaporates them.

   An eissaien chcatogph1qu ur paper of the reú: 1 ..: '! Ü of extraction against cua this last sst constituted with party-as zip? to nearly equal by compounds which correspond respectively to progesterone st è la 11a-nYúro ;;: y-proges-; 6 ... i0i3.L'o 0: repairs the constituents of the mixture at â3t.k2 of a preparative chromatography on paper is obtained by crystallization from dan mixtures of dtacetcne and L 4t G 4ne.i, '. dIU part, la / progea0rOnê <3 * a melting point of 118-120, C cI -187 1 (in dioxane) e, tauure part, the â-l1.: - hydroxy-p: rog6stél "' one with a melting point of 69-1 '. [a] 1) = + 176 (in chloroform).

 <Desc / Clms Page number 25>

 



   Example 14 A nutrient solution is prepared containing, for one liter of tap water, 10 g of cane sweat; 10 g of
 EMI25.1
 Trypton-Difoo, 2 g of sodium nitrate; 1 g of secondary potassium phosphate, 0.5 g of magnesium sulfate, 0.5 g of potassium chloride, and 10 mg of ferrous heptal sulfate.
 EMI25.2
 hydrata, adjusts the pH to 7PO and then added 2.5 g of calcium carbonate. In a conical flask one. sterilize 120 eu 3 of this solution, inoculate with Curvularia lunata and stir at 27. After 2 days the mycelium is separated, washed with little water and suspended in 120 cm3 of distilled water.

   To this suspension was added a solution of 30 mg
 EMI25.3
 of in 1.5 com of adtone and shakes for 30 hours at 27. Extracted el1.suite le, mdiangs 4 times with ethyl acetate. The extracts are washed with water, dried and evaporated. From an ehromato- evaluation
 EMI25.4
 graphically, the extraction residue is essentially made up of two substances which correspond respectively to cortexone and corticosterone.

   Using preparative paper chromatography and subsequent crystallization in mixtures of acetone and ether
 EMI25.5
 from petroleum, we obtain / C-eortexone with a melting point of 138-141 la] D - -175 (in absolute alcohol) and d-oort1coBtone with a melting point of 178-181, [ a + 220 (in absolute alcohol).

 <Desc / Clms Page number 26>

 
 EMI26.1
 



  ..ti> C: .a). 5.



  ¯¯Wl.). "" # ;: Yoo & II., P. "" R ... a "" "mt Adding ,, to one r1 -" '- -' ,: "i" "" Sa \ ' ; "'- Ó',. Y" t -: - 1 of Curvularia lunata in 120 m3 of distilled water,} suspension which was obtained corsas 5s'i}, 1! 3. f "::; J" mpJ, D tt.! A itaic '. :: a solution \ .1 =! 1O} "i ing ds ë'-T.7chyës'cxy-oo? T: o êôv-: j" j..5 '- ".......... t ... 1 ... . ':' i Gte1: 1 operating CC:. 'LU \ 3 tr.-''.-. vï'i, Fl.;: 7 "''. ¯ avoii3 sg: 1.; 0 posing z days 37 1 ': \ t't') .- f "JjJh'i .1." ... hYJ "': t.1 ... \,.:; ¯f: .. = r, -: ¯. ...;:; c'L "$ rlÇ1't .... \ ';); ::." I bzz evaluation sootuik'; jï3 ehoEË'bc; 3iH Dtii? i efôstîrfcl! # raise waters ubaui / ov . which 6û \ '. jHcdnt at 17o> -h2yâïfos; jr- ccr'csxon e;) t at 17 (z-11tlo1:.: - i .eJG ::' i; .. c ::; j ' 1Í / (.) L ;? 0 - ,.

   S>. : t mix he has it ... 1t!> "1h'-¯) $ .: L..c. ':' r :.:.: .. ¯: - r '..s6 ::. v >, 5? .. -. ';' ,. e Y .. ¯ :: 4n 5: iv3 on paper and l5on Oi J \ a "1 ¯ ....... f ':: ¯": = 3 ': I <......... 1. Ï>.' .. '' .. SF; '' L.:,...;'<- ':: ":::: ;; t3, a. $ i. "t '."'. k â; n
 EMI26.2
 from a manure point '& -. SILO-- -l :: as [au- -: i! SO "'1 absolute alcohol]' and other; '. 1.'iy..m: ¯' .f '-.;,': 7 , ..x- "-,.: é: <. '. rt ..!. Pi-: f' k.ic i.ji2; 'i':; ryC7: r'i3aa0â ';> from here; ; r: 3:; ..-- :; <.- ..: - v: a.i3 'i :: 09-23.2, [aj # 171e
 EMI26.3
 (in "," ,, .. 1 absolute).



  E; '- iin}) 1> oe 16 In a r6c: 1.i'Ü1t ià shakes;?;, On'? L :: d3'i, ï'iLéw .in 500 emï, u of the tap., 1 g of sodium nitrate "O / J 5 g of primary potassium orthophosplate ,, 0, 2 g of magnesium sulfate-heptahydra te, 0.25 g of potassium chloride, 25 g of raw glucose and 0, 5 g of yeast extract 17i'eap brings the whole to a pH of 5 and sterilizes. This nutrient solution is inoculated with Didymella lycoperslci and stirred for 3 days at 27
 EMI26.4
 Then added, under sterile conditions, a solution of

 <Desc / Clms Page number 27>

 
 EMI27.1
 120 mg of d. Jr¯l7aAvjûvoiz $ ** QQX <tQQB t4von & in QtnJ remove metliancl. We shake 4 LG again ''! '4S 2E does not separate the mycelium? wringing and washing with water and ethyl acetates, After having combined the filtrates, they entered plush described in 1! simple 1.

   The extraction residue is constituted., As shown by an examination 1: d ".t: ï't7r2i: ïC'S ..; '. T.:w3î,' uw on paper by a product which s's eoraports will cost! MEt: igr '. & From depart and by "f¯uchyF.) T7'w''t 4i" 1, V1-hyd.PwaiJ.L YJ S9V: .7'Lfiw.Jej.YS.i We separate the C: G s "of: LrGL! .s': zê: 3 this mixture l5aide of a Cl preparation on paper chromatography (propyl & neglyeol¯to3.uèsi system).



  By recris tallisatioa in E3. '' "". È.'t ', C7g OR obtains on the one hand the 3.wy¯l-dëhyd ::' esa.bÏo :: ma 'aca ;; E: = tun: of a melting point it:. i ... ', 3''j' '[a 4- 98 (<3ar4s the dioxsume) and, other pa2> tj, lal7c; - hydrox? -corticoatdrone with a melting point of 2-. .:. arl [o3D -i68o in absolute alcohol).
 EMI27.2
 Example 17
 EMI27.3
 Retort described in the previous example, we J; ':; xJ.lâ' ut .. 'culture of Didymella a:;' Jt'àiif? Ï: î.é. 5''i 7 then added a solution of 120 mg of 21-trimthylac4tat d, 17a-hydoxycoytiûogteron® in 5 mg of acetone.

   After stirring for 4 days at 27, the reaction product G'c; 'ff121' '3: E:' c ', -11t Clv is isolated. ..II111I '..' T '? ! The extraction residue is essentially constituted, as shown by an evaluation carried out by chromatography on paper, by '' 1: 3uë products which occur.

 <Desc / Clms Page number 28>

 
 EMI28.1
 respectively behave as the starting material. and as the tritnethylaoêtE1.te of l-dcµhydrQ¯17āhyd: i? o.xy * corti-¯ c03t6rcn: e :.

   The constituents of this mixture are separated with the aid of a preparation chpomaogpaphie on paper and one obtains, by crystallization from ethyl acetate, on the one hand: 2;:. Zrxhy .aca, of the -17a-hycro.ycr .- ,: o 4t = 'on
 EMI28.2
 
 EMI28.3
 of a painting of i us 5 or of 231-232, fa3p = '-ll5 (in the dîo, xa.rr,: e) and on the other hand, the G "lW.d.!'. 3 '4 ± 1,. & GVGS 'of d-.xvtdr> a.'Ex.ydrsytc; y.coticosteo: ae of a fusion paint of 233236 p [al 4-1030 (in chloroform).
 EMI28.4
 Example 18
 EMI28.5
 #J> J> MM t * K M T ta * B # * a We inoculate with Didymslla .yop:.:. G 120 because of the nutrient solution described in Example 4 and stir for 2 days at 27. Then added, under conditions 1 ':,'; é '. ": Lles, a solution of 30 mg of ± -aldo8teron # in 1.5 cm' c'Pacétont'3 and continued stirring with mime h ': ml}:) (5- erasure.

   After 3 days, the mycelium is separated and washed with water and i. 1 taaé.: - ate of ethyl. After having combined the filtrates, it is extracted as described in Example 1. As shown in exaI: 1.an chromatogj =, aphiquê on paper the residue ± the extraction consists of a product which behaves oc- maes:.: 3.0'I'Oiscf and by.-t'dt'3ytiG-, iLO'Fx'OL, We separate this-si two products with the help of a preparation chromatography t:

  u? paper (Bush C system) and crystallizes them

 <Desc / Clms Page number 29>

 then in a mixture of acetone and ether. We obtain
 EMI29.1
 lat-aldosterone (melting at 110 '/ 158-165', [UID = -i4l 0 [in acetone]) and d-1-dêb-ydro-aldosterone (bands in the infrared, in chloroform , among others at 2t82gp 2j95gp 5p87g, 6,01n. 6.17. 6p24J.J '6e9lg, 7el5ge' (, 3llt ..



  7p77g 9, ten, 10, 68, and 11, 28 | i). d-1-dehydroaldosterone is acetylated in the usual manner using a mixture of pyridine and acetic anhydride. We obtain a mixture of two, products which, at the
 EMI29.2
 chromatography on paper with the pr.opylàn0g1ycol-toluÈ / i.'H '; system, gives "respectively, R values of Oe7O Ot O, 15v these two products are respectively lô 18,21-diao & tat and do la dl dehydro The latter is obtained in a pure crystalline form by treating the crude mixture of acetates with absolute ether.

   Fusion point
 EMI29.3
 182-18 ::,, bands in 1 t infra '.: \. "Oug in Io methyltnô chloride, among others at 2p81., P 5.74j} ,,; 6, Olv ,, 6 16pe bzz 7p31 $ 8 , \\ 17t 8iJ93J., Y 8.98211.J! 9.1'72.I 'lO, 03 !, lO, 221.J! LOp62t.ll 11.32, and 12.29.



    Example 19
 EMI29.4
 One prepares, cornai described dangllexeiaple 6th t 120 cm '' of united -culture of Didymella lycopêreio1. To this is added, under sterile conditions, a solution of 25 mg of d, E -EOl't1i3oi1e in 1.5 µm3 of methanol. We shake for

 <Desc / Clms Page number 30>

 
 EMI30.1
 3 plays at 27 and then separated the Mycelium. On Go checkered water and ethyl acetate. After stripping the filtrates, the extract described in the example is obtained. According to a chroma graphic examination on p-apîjr, the distraction residue is formed from l-dehyâro-cortiBone ot a product which ss CO! I1po ;:. t like 0 !! separates ava dziuz on steps by chromatography of the preparation on paper.

   We obtain d um; on the one hand d-1-dehydro-cor-tisone [melting point 2j51 "2j54, [a! D - + 1690 (in âioxanna) j and, on the other hand, E -oortisone [melting point = 215, [ 0.1D - "203- (in dioxane)].



   Example 20
With a culture of Trichothecium roseum, we seed 120 cm3 of a sterilized nutrient solution of Czapek-Dox and stir for 5 days at 27. Then added to the whole, under sterile conditions, a solution of
 EMI30.2
 30 mg of d, Z-aldootone in le5 am3 of acetone and continues to stir at the same temperature. After 4 days, the mycelium is separated and washed with water and ethyl acetate.



  After combining them, the filtrates are extracted as described.
 EMI30.3
 in Example 1. According to a chrometographic examination on paper, the extraction residue is essentially constituted by a product which behaves like aldosterone, and by
 EMI30.4
 17o-hydroxy-aldosterone. We separate these two substances

 <Desc / Clms Page number 31>

 
 EMI31.1
 1.c :: of a preparative chromatography on paper (Bush system 0) We obtain oFn AJ "'G6dL4 IICroata7 and d¯17a-hydroxy-aldostërone.



  Example 21 With a culture of Ophiobolus horpo4cirîchuo, we
 EMI31.2
 
 EMI31.3
 inoculate 120 cm "6th sterile beer must and stir for
 EMI31.4
 3 days to 27. We then add to the whole, under conditions
 EMI31.5
 sterile, a solution of 40 mg of (1 $ -----, .r..8j..g'otx du d, L¯A l ..3ndq o0--3.p-hydrQyxgna.3.l8- oic in 1.5% Acetono and continue to stir at 27. After 6 days the mycelium is separated and washed with water and ethyl acetate. After connecting the filtrates, they are washed. traits '#olLttl ":' described in 1 Example 1.

   According to an examination ahr omztogra3:.: Q, i on paper 3 the residue <38xtraotion is constituted by a product, which behaves as the starting material, and by the (l8 3..j ...; o :, s du 3at-> dco-- llpj We separate l @ s two substances using uno ahx 7o: g'oraph of preparation on t Iton paper obtains the (l8 ## - llp) -lacton @ au , '3 p acâ-. D.cCto-llp-hydroxy-prgs: t.8.o: cu' [a] D -IS1! -0) and la (18 # -ll) -. Lsctone of c- ba 3 0-d. r-.3., 2.-c.kZ; crox .. rgri: ea.8 ... o; qu, Lcc -? '7 j.
 EMI31.6
 



  Example 22
 EMI31.7
 With a dtOph3.oboiu herpotrîchue culture, we

 <Desc / Clms Page number 32>

 inoculate 120 cm3 of sterile beer wort and stir for 3 days at 27. Then added to the whole, under sterile conditions, a solution of 40 mg of d, # - # 4-3,18,20-trioxo-11ss-hydroxy-pregnene or bran in 1.5 cm3 of acetone and continue shake 27. After 6 days, the mycelium is separated and washed with water and ethyl acetate. After combining the filtrates, they are treated as described in Example 1.

   According to a chromatographic examination on paper, the extraction residue consists of a product which behaves like the starting material, and of aldosterone. These two substances were separated using preparative paper chromatography to obtain ± -44 or its 18,11-cyclo-semi-acetal, as well as d-aidosterone ([[alpha] ] D = + 142).



   The starting material denied in use can be prepared for example according to the procedure described in Belgian patent No. 547,904 of May 17, 1956 in the name of Mr. Tadéus REICHSTEIN and having the title: "Process for the preparation of oxygenated steroids and new compounds thus obtained .



   Example 23
Using a culture of Ophiobolue herpotrichus, 120 cm3 of sterile beer wort are inoculated and stirred for 3 days at 27. Then added to the whole, under conditions

 <Desc / Clms Page number 33>

 
 EMI33.1
 sterile, a solution of 40 mg of dl.19-nor-prOgestéron in 1.5 cm of acetone and continued to stir at 27. After 3 days the mycelium is separated and washed with water and
 EMI33.2
 ethyl aoét8t. After combining the filtrates, they are extracted as described in Example 1.

   According to a chrornatographic examination on paper, the entry residue is oon: 3tituated by a product which behaves as the starting material and by dg the 19-nor-oort0xoneu These two substances are separated with the aid of preparation on paper ohromatogr-aph10 and dQ19-nor-cortexon (tal M - <. l43) and dQ19-nor-cortexon were obtained. This cleraîèrg is acetylated in the usual manner using a mixture of pyridine ot c1 tan .. acetic hydride. 21-acetate present a rotatory power
 EMI33.3
 spoifique [a] D + 145.



  Example 24 With the aid of an ô sophiobolua heI'potl "1chua culture, 120 cm 3 of sterile beer wort are inoculated and stirred for 3 days at 27. The whole is then added in containers.
 EMI33.4
 sterile ditions, a solution of 40 mg of d3 / ± ¯9o> ± luoro-lip-hydroxy -progesterone in 1.5 cm 3 of acetone and continue to stir at 27. After 4 days, the mycelium is separated and washed with water and ethyl acetate. After combining the filtrates, they are extracted as described in Example 1.



  According to an chromatographic examination on paper, the residue

 <Desc / Clms Page number 34>

 extraction consists of a product which behaves like the starting material, and of 9 [alpha] -fluoro-corticosterone. These two substances are separated using preparative chromatography on paper and
 EMI34.1
 1a ± -9a-tluoro-ll-hydroxy-progesterone (la] D - -190), and 9a-fluoro-eortieosterone, The latter is acetylated in the usual manner using a mixture of pyridine and acetic anhydride. 21-monoacetate has a specific rotatory power [[alpha]] D = + 187.



   Example 25
Using a culture of a species of the genus Rhizopus, 120 cm3 of sterile beer wort are inoculated and stirred for 3 days at 27. Then added to the whole, under sterile conditions, a solution of 30 mg of d, # - cor-
 EMI34.2
 texone (11-deoxy-cortlooBtarone) in 1.5 cam3 of acetone and continues to stir at 27. After 4 days, the mycelium is separated and the culture filtrate is extracted as described in the previous examples. chromatography on paper,

   the extraction residue consists essentially of a product which behaves as the starting material and of 6p-hydroxy-cortexone. These two substances are separated using preparative chromatography
 EMI34.3
 on paper. We obtain ± -aortexon0 (la] D = -175) and d-6p-hydroxy-cortexone ([[alpha]] D = + 100).

 <Desc / Clms Page number 35>

 



   Example 26
Using a culture of a species of the genus Peziza bearing the reference number M 26 at the Swiss Federal Institute of Technology in Zurich, 120 cm3 of sterile beer wort are inoculated and stirred for 3 days at 27. Then added to the whole, under its sterile conditions, a solution of 30 mg of
 EMI35.1
 d, ±> cor.txo: 1.e (11-deJ13oxy-certicogtêl "on0) in 1.5 cm 3 of acetone and continue to stir at 27. After 4 days, the mycelium is separated and the culture filtrate as described in
 EMI35.2
 1 (1i.fmpl p:; ', acé: dent.

   According to a chromatographic examination on paper .. the extraction residue is essentially constituted by a product which behaves like the starting material
 EMI35.3
 and by d's 7a-hydro3Ey-coî> t & one, These two oubstanocs are separated using dJ'.1: preparative chromatography on paper.



  We obtain from t-cor1:; (i!: Xona ([a) D = -173) and d-7a-hydroxy; -cortaxone (ïct] d3 + 155).



   Example 27
Using a culture of Lenzites abietina, 120 ounces of sterile beer wort are inoculated and stirred 3 days to 27. One then adds under sterile conditions, a
 EMI35.4
 solution of 30 mg of d,% -cortexone (11-deoxy-corticoctone) in 1.5 cm of acetone and continue to stir at 27. After 4 daysa, the mycelium is separated and the filtrate is extracted from

 <Desc / Clms Page number 36>

 
 EMI36.1
 Otlt \ .1I 'a3Tlli described in the previous examples. According to chromatographic paper on paper, the extraction residue is essentially constituted by a product which behaves as the starting material and by 15p-hydroxy-eortexone.



    These two substances are separated using chromatography
 EMI36.2
 preparation on paper. We obtain /, Vl-oortexone ([aJD r -170) and d-i5¯h'droxy eortexarie ([a.] D = + l4l).



  Example 28
 EMI36.3
 .aINWWibli aa
Using a culture of Glbberella baccata, 120 cm 3 of a nutrient solution of Czapek-Dox and are inoculated. shakes 3 days at 27. We then add to the whole, in
 EMI36.4
 conditions exited, a solution of 30 mg of d, l ... cortexone 3.1.t oxy a> rtiaoatrctke in 1.5 cm of acetone and continue to stir at 27. After 4 days, the mycelium is separated and the culture filtrate is extracted as indicated in the previous examples. Based on chromatographic examination on paper, the extraction residue is essentially constituted by a product which behaves like the material of departure, and by
 EMI36.5
 of: 5α-hydroxy-cortezone. These two substances are separated using preparative paper chromatography.

   L-cortexone ([a] D: = 1 -169) and d-15a-hydroxy-cortexone (cx1 + 195) are obtained.

 <Desc / Clms Page number 37>

 



   Example 29
Using a culture of Pleospora Gaeummani, 120 carbons of sterile beer wort are inoculated and stirred for 3 to 27 days. Then added to the whole, under sterile conditions,
 EMI37.1
 a solution of 30 mg of djb -cortexone (11-deoxy-corticosterone) (Jans 1.5 cm 3 of acetone and continues to stir at 27 0. After 4 days, the mycelium is separated and the culture filtrate as described in the previous examples.After a graphic chromate examination on paper, the extraction residue is essentially constituted by a product which behaves
 EMI37.2
 as the starting material and by 14a.M.hydroxy ... oortexone.



  These two substances are separated using chromatography
 EMI37.3
 d.3 preparation on paper. We obtain t -cortêXOD.e {[aïn -1-711.0 and d @ la d <-l4Q-hy <3roxy <-cortexone ([o.] + 179) -
Example 30 120 cm3 of a nutrient solution are sterilized
 EMI37.4
 which r.B'ferms for 1 liter of tap water, 10 g of glucose b? u '.:.) -5 g of peptone, 5 g of NaClj, 3 g of pxo Lab Lemco (ax of meat from trade) and 10 g of CaCO3.9 and which was 9m3ee. a pH of 75 using dilute sodium hydroxide solution, then inoculate with a culture of the Houshe d's Streptomyces? 7747 (Special botany institute of the Federal Polytechnic of Zurich).

   We

 <Desc / Clms Page number 38>

 stir for 2 days at 27 and then add, under sterile conditions, a solution of 30 mg of d, # - cortexone in 5 cm of acetone. Stirring is continued at the same temperature and after 2 days the mycelium is separated. The culture filtrate is extracted as described in Example 1.



  According to chromatographic examination on paper, the extraction residue consists of a product which behaves as the starting material and of 16α-hydroxy-cortexone.



  These two substances are separated using chromatography
 EMI38.1
 preparation on paper. Cortexone (Ea3D -171) and d-16a are obtained.Eydroxy-cortexo-ae cCaD + 11
The starting materials to be used according to the process can be prepared according to processes known per se, starting from tetracyclic intermediates which are accessible by total synthesis, for example from the cholestane, pregnan or testane series,


    

Claims (1)

Claims.¯ I. A process for splitting racematics EMI39.1 â, M-Btti2 * oïcifâ3 in their components t characterized by the fact that in fact act oxidizing enzymes produced using EMI39.2 aerobic cultures of microorganisms on <3, / '-' steroids and isolate at least one of the enantiomorphic forms. The present process can be further characterized by the following points EMI39.3 1) Hydroxylating enzymes are used 2) Dehydrating enzymes are used # a .. 3) Aerobic cultures of microorganisms are used. allow the introduction of a hydroxyl in position 6, EMI39.4 7. 8,: 1, li.9 15, 16, 1'7}, or \ 21. 4) Aerobic cultures of microorganisms are used which shovel to introduce oxo groups into EMI39.5 position 6 7j, JI: 1.1, 15 or 16. 5) ¯ # Aerobic cultures of microorganisms were used which allow the introduction of a double bond in position 1, 2 t r in position 4,5. 6) Aerobic cultures of microorganisms were used (3s qu: L make it possible to dehydrogenate the hydroxyls located in position 3 and / or 17 to oxo groups. 7) Several microorganisms are made to act in one <Desc / Clms Page number 40> operative phase. 8) As starting materials, d, # -pregnan compounds are used. 9) As starting substance d, # - progesterone is used. 10) As starting material d, # - cortexone is used. 11) As starting material d, # - cortisone is used. 12) The starting substance is used EMI40.1 (18 ## 11) -lactone of d, -A -5J, 20-dioxo lip-hydrox5 '-. Pregnene-18oic. 13) We use d, # - as starting substance EMI40.2 ti, 18p ü.M.o, o -..-. hydpos y-.p.gan or its 18,, 11-cyolo- seml-acetal. 14) As starting material d, # - EMI40.3 D -, 2C - dioto - li, .8d3.hydrot - prgt7.én. 15) As starting substances, compounds EMI40.4 posed hydroxyls in position 17as, and corresponding to those indicated under 12 to 14. 16) As starting substance d, # - EMI40.5 androstene-d: Lol, d, ± -17-methYl-androstene-d101, d, / v ddhydrc-épiaiid rootone or â, -pregnenolone. II. As new industrial products, the <Desc / Clms Page number 41> compounds obtained by carrying out the process defined above. Note: Added ten words.