CH331690A - Method of introducing hydroxyl groups into steroids - Google Patents

Description

  

      Process for the introduction of hydroxyl groups in steroids The present invention relates to a process for the introduction of hydroxyl groups in steroids by aerobic fermentation of a mycelium in the presence of the steroid, in which a critical adjustment of the ratio of the reaction participants and control of the reaction time is followed Purification of the oxidized steroid will be easier.

   This procedure is a further training in Switzerland. Patent No. 329191 and is characterized in that at least 0.5 g of My celium, calculated as the dry product, is used per gram of the steroid and the oxidized steroid obtained is isolated. According to the method according to the present invention it is possible to obtain hydroxyl groups in an effective, economical and technically satisfactory manner. B. to be introduced into the 11-position of 11-desoxvsteroids.



  In particular, mushrooms of the genus llucorales come into consideration. The method is mainly important for the conversion of steroids unsubstituted in the 11th position into 11-oxysteroids. In addition to the 11th oxy group, hydroxyl groups are sometimes introduced in other positions at the same time. This may not be undesirable as the introduction of hydroxyl groups in other positions of the steroid nucleus can also lead to valuable therapeutic products or intermediates, e.g.

   B. sol- just that have an oxy group in the 17-position. If such additional groups are not desired, they can easily be removed again. If there are oxy groups in the molecule of the steroid to be oxidized that can be oxidized to the keto group, they can, if appropriate (e.g.

   B. if very high yields of oxygen in the 11-position ent containing product are desired), protect against attack by the oxidizing fungi by converting them, for example, by esterification, etherification, halogenation or the like into a group that can be converted back into lets the Oxy group transform back.



  A wide variety of steroids can be used as starting materials, but especially those that are unsubstituted in the 11-position.



  The steroids can contain various substituents, e.g. B. in position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16 or 17, especially methyl groups in positions 10 and 13, keto, oxy or acyloxy groups in positions 3, 7 or 12; 17 side chains, of which the progesterone and corticosterone (ketol) side chains are particularly mentioned;

   a 17-keto group, a 17-oxy group; Furthermore, one or more double bonds in position 4, 5, 6th, 7, 8, 9 (11), 11 (12), 16 (17) and other positions or double bonds which are saturated by addition of halogen or hydrogen halide can also be used were to be present. Diene adducts, e.g. B. maleic acid ester adducts of steroids with a system of conjugated Dop pelbindungen, z. B. in the 5,7 position come into consideration.

   The presence and absence of a double bond in the 9 (11) or 11 (12) position of the nucleus is not a critical factor for the process, although steroids with an 11-methylene group, i.e. steroids with two hydrogen atoms on carbon atom 11, are used have, for reasons of economy and to avoid unnecessary conversions of saturated in unsaturated compound preferred; however, the fermentation itself can be carried out with equal ease with saturated or unsaturated compounds.



  Examples of steroids that come into consideration as starting materials are, for. E.g .: progesterone, 9 (11) - or 11 (12) -dehydroprogesterone, 7,9 (11) -bisdehydroprogesterone, 17-oxyprogesterone, 17a-progesterone, testosterone, pregnenolone, 3-oxy-5-pregnen-20-one , Pregnenolone, 3ss-oxy-5,16-pregnadien-20-one, acyloxypregnenolone, such as pregnenolone acetate, 3-oxy-5,6-oxydo-pregnan-20-one (a- or ss-oxydo),

            3-oxy-5-chloro-pregnan-20-one, 5,6-oxydo-pregnan-3,20-dione (a- or ss-oxydo), 4-bromo- and 4-chloro-pregnan-3,20 -dione, 5-chloro-pregnan-3,20-dione, 3-keto-pregnan-20-ol, 3-keto-allopregnan-20-ol, 3ss-oxy-16,17-oxydo-21-acetoxy-5 -pregnen- 20-one, 3ss-Oxy-16,17-oxydo-5-pregnen-20-one, 3ss-Oxy-5,6,21-tribromo-16,17-oxydo- pregnan-20-one, 3ss -Oxy-16-bromo-17-oxy-5-pregnen-20-one,

            3ss-Oxy-16-chlor-17-oxy-5-pregnen-20-one, 3ss-Oxy-5 (6), 16 (17) -dioxydo-pregnan-20-one, 3ss-Oxy-5 (6) , 16 (17) -dioxYdo-21-bromo- pregnan-20-one, 3ss-Oxy-5 (6), 16 (17) -dioxydo 2l-acetoxy-pregnan-20-one, 3ss-Oxy-5 (6 ), 16 (17) -dioxydo =? 1-oxy- pregnan-20-one, 11-deoxycorticosterone, d9 (11) - or 411 (1 ') - deoxyeortieosterone,

            11-deoxy-17-oxy-eorticosterone and acyloxy derivatives, such as the acetoxy derivative, 21-ox, 7-pregnenolone and 21-acyl derivatives, e.g. B. the acetate, 17,21-Dioxy-pregnenolone and 17,21-Diacy loxy derivatives thereof, for.

   B. the diacetoxy derivative, androstenedione, androstan-17-ol, 9 (11) - or 11 (12) -dehydroandrostenedione, 3-oxy-9 (11) - or -1 1. (12) -pregnen-20 -one, 3,21-Dioxy-9 (11) - or -11 (12) -pregnen- 20-one, 3,17,21-Trioxy-9 (11) - or -11 (12) -pregnen- 20 -one,

            4-Androsten-3-ol-17-one and its 3-acyl derivatives, e.g. B. the acetate, 5-androsten-3-ol-17-one and its 3-acyl derivatives, z. B. the acetate, ergosterol, stigmasterol, stigmastanol and the 3-acyl derivatives, z. B.

   Acetates, the same; Ergostenone, Stigmastenon, Stigmastanon, Cholestenone, Cholic Acid, Deoxy eholic acid, Lithocholic Acid, Cholanic Acid, Noreholanic Acid, Bisnorcholanic Acid, Cholenic Acid, Norcholenic Acid, Bisnorcholenic Acid, and 3-Oxy-, 3-Keto-, 3,7-Dioxy-,

          3,7-diketo-, 3,7,12-trioxy-, 3,7,12-triketo-derivatives, 9 (11) - or 11 (12) -unsaturated esters, thiolesters, as well as other derivatives of these acids.



  It is expedient to use steroids with up to 22 carbon atoms in the carbon skeleton of the steroid nucleus, in particular those with a side chain containing two carbon atoms in the 17-position and a methylene group in the 11-position. The 10-nor-methyl-, 13-nor-methyl and the 10,

  1.3-bisnor-methyl forms of all of the above-mentioned steroids are also suitable for the present fermentation process. If the 11-position is already oxidized or substituted, the predominant product can additionally be oxidized in another position.

   The 16-dehydro form of all the steroids mentioned can also be used. In all of these substances can be especially. by Mueorales fungi, in particular Rliizopus and Cunningliamella, especially Rhizopus nigrieans, Rhizopus arrhizus and Cunninghaniella blakesleeana, introduce hydroxyl groups.



  The strains Rhizopus arrhizus, ATCC (American Type Culture Collections) <B> 111 </B> 45, here often referred to as RH 176, and R.hizopus nigrieans, ATCC 6227b, give very good yields of the desired oxy steroids. But in certain cases, under special circumstances, other genera and species can also be used to advantage.



  Fungi suitable for the invention (including mutants) are available, for example, from Northern Regional Research Laboratories, Peoria, Illinois, or from the American Type Culture Collection (ATCC), Vashington, D.C. or from Centraalbureau voor Schimmeleultur Baarn, Holland. But they can also be obtained from natural sources using known microbiological methods.

    The culture media in which the mushrooms are grown should contain sources of assimilable carbon, nitrogen and minerals. Considerable growth and development can take place even under conditions that are worse than optimal.



  Sources of assimilable carbon can be carbohydrates such as starches, gelatinized starches, dextrin, sugar, molasses such as cane, beet and sorglium cereal, glucose, fructose, mannose, C-galactose, 3laltose, Saeeharose, lactose, pentoses, amino - acids, peptones or proteins and also carbon dioxide, glycerin, alcohols, acetic acid,

         Sodium acetate, citric acid, sodium citrate, lower and higher fatty acids or fats can be used.



  A pulp made from Mexican yours roots can also be used as a medium for fermentation. . As a source of assimilable nitrogen. come soluble or insoluble vegetable or animal proteins, soy flour, Lactalbu-; min, casein, egg albumin, peptones, polypeptides or amino acids, urea, ammonium salts or base exchange resins, e.g. B. Zeolite, adsorbed ammonia into consideration.

    Whey, residues from alcohol distillation from maize mash, soaking liquid from starch production for maize or yeast extract are also suitable.



  The medium can naturally contain assimilable calcium, cobalt, copper, gallium, iron, magnesium, manganese, molybdenum, potassium, scandium and vanadium as minerals.

    Sulfur can be supplied by sulfates, alkyl sulfonates, sulfoxylates, sulfinates, sulfides, free sulfur, thiosulfate, methionine, cystine, cysteine, thiamine or biotin.

   Phosphorus can be in the form of ortho-, meta- or pyrophosphoric acid salts or esters, phytin, phytate, glycerophosphate, sodium nucleinate, casein or ovovitellin, advantageously in an approximately <B> 0.001 </B> to 0.07- , preferably 0.015 to 0.02 molar concentration be present. Traces of boron and iodine may be desirable.



  Other additional growth factors such as vitamins, auxins, and stimulants can also be added. For example, while R.hizopus nigricans supplies itself with vitamins, the organism Phycomyces blakesleeanus requires thiamine or a mixture of thiamine-pyrimidine and thiamine-thiazole or 4-methyl-5- (ss-oxy-ethyl) -thiazole as a growth factor.



  You can use suspending agents or mycelium carriers, such as filter earth, filter aids, finely divided cellulose, wood chips, bentonite, calcium carbonate, charcoal, activated charcoal or other suspendable solids, methyl cellulose, carboxymethyl cellulose or alginates, for fermentation, aeration and filtration to facilitate.



  During the aerobic growth of various Mucorales, in particular Rhizopus, Mucor and Mortierella, fat is synthesized. This fat synthesis can be controlled by reducing the amount of carbohydrates available to the mushrooms and using a medium in which the energy source consists primarily of protein or protein hydrolyzate.



  The pH of the solution is usually adjusted to about 4.5-5.9 before inoculating with the fungal organism. But you can also use higher or lower p11 values. If the autoclave is heated to 4.5 kg / cm2 of pressure for 30 minutes in order to create aseptic conditions, the pH drops from 6.5 to around 5.5-5.9, and this decrease should be taken into account if man the pu. before sterilization, so that one has optimal conditions for growth after the same.

   Antibiotics or antiseptics such as penicillin or sulphites can also be added to prevent or retard the growth of foreign organisms.



  The inoculation of the nutrient medium with the fungus, e.g. B. of the genus Mueorales, can be done in any suitable manner. Usually spores of the organism are used for this, but living mycelium can also be used. The inoculate can be mixed, homogenized or otherwise well dispersed before, during or after the dilution, for example in a Waring mixer. The fungus develops easily at around room temperature (20-28 C); but a relatively large temperature range of z.

   B. about 15-45 C applicable. However, growth is slower at the lower temperatures.



  The duration of cultivation before exposing the parent steroid to the action of the fungus is not critical. So you can use the steroid either before the Sterilisa tion, when inoculating the medium or some time, for. B. 24-48 hours, add later. In practice it has been shown that the steroid can be added even 5 days after the inoculation without the effect being impaired. After 24 hours of culture of the fungus, the p1.1 of the nutrient medium has dropped considerably, usually to around 3.5-5.2, depending on the buffer content of the same.

   A pH of about 3.0-6.8 has been found suitable for the conversion. Preferably, the steroid is added to the culture at this point. The preferred mode of operation is to add the steroid to the culture medium after an incubation time of 0 to 16, 24 or 48 hours, since at this stage the oxidizing effect or the oxidizing enzyme mechanism appears to be fully developed.



  As mentioned, the steroid is added in such amounts that per g of the same at least 0.5 g of mycelium, calculated as a dry product, are present in the medium, e.g. B. up to about 0.6 g / l or even 0.8 g / l. Higher concentrations inhibit the oxidative effect somewhat. The steroid can be added in purified or raw form or, for example, in the form of a mixture with fat or solvent.

   The manner in which the starting steroid is added is not critical and can be carried out in any suitable manner which ensures intimate contact of the steroid with the fermentation medium. So you can grow the microorganism in the presence of the finely ground steroid crystals, disperse the steroid in the fungal medium and the like.

   For this purpose, surface-active substances, dispersants or suspending agents such as the branded products Spans (hexitol anhydride esters of long-chain fatty acids, for example sorbitan monolaurate), Tweens (polyoxyalkylene esters of hexitol anhydride esters of long-chain fatty acids), aerosols (dioctyl-sulfanolsulfonium), alkylarysulfonium sodium sulfonium add.

   If the fermentation medium contains solid particles, these can be steroid-coated, impregnated or mixed. Preferably, however, the steroid is added to the fungus medium or the fermentation liquid in the form of a solution in a water-soluble organic solvent, such as ketones, acetone, alcohols, methanol, ethanol, isopropanol, benzyl alcohol or even a solution which is only slightly soluble in water is to,

    and mixes thoroughly so that a suspension or dispersion of fine steroid particles is formed and maximum contact of the medium with the steroid to be oxidized is ensured. The steroid or its solution can be homogenized with water, medium or other aqueous material to achieve a fine dispersion. The culture can be submerged or on the surface, but submerged culture is preferred.



  The temperature during the oxidation of the steroid does not have to be different from that before the steroid addition, it should only be kept in a range in which the oxidizing effect of the fungal organism is preserved.



  The effectiveness of the hydroxylation depends on the ventilation. It is best to use controlled ventilation by moving and / or bubbling air through the culture medium. The rate of oxidation of the steroid depends on the amount of ventilation and the amount of fungus growth at the moment of the steroid addition. It has been found that conversion is faster if the amount of ventilation is well controlled and the fungus is grown before the addition of the steroid Steroids 16-24 is precultured.

   Aeration can also be achieved by surface culture, with or without additional ventilation, e.g. B. by shaking. When the fermentation is carried out under sub-internal conditions, ventilation can be achieved by passing air bubbles in fine and / or coarse form through the medium. Aerobic conditions can be brought about not only by supplying air, but also by using other oxygen sources, such as mixtures of oxygen and nitrogen or hydrogen superoxide.

   When using hydrogen superoxide or when the ventilation is strong, one can at best add catalase in order to prevent destruction of the oxidative action of the fungus. The hydrogen peroxide can also be formed in situ by ultraviolet radiation.

   When using air for ventilation, the air supply advantageously takes place in an amount of 6 to 8 millimoles per hour per liter of sulfite, determined according to the method of Cooper, Fernstrom and Miller, Ind. Eng. Chem., 36, 501 (1944) .Optimal results are obtained when the oxygen uptake, determined by the method mentioned, is 7 millimoles per liter per hour. In certain circumstances it is desirable to have different aeration rates in the cultivation of the fungus and the oxidation of the steroids.

   The ventilation is expediently controlled by applying overpressure of, for example, 2.1 Ata or more or negative pressure of, for example 0.7 Ata. The addition of catalysts, such as ascorbic acid, glutamic acid, citric acid, lactic acid, tyrosine and tryptophan, makes it easier to absorb oxygen. Excessive or insufficient ventilation will reduce the yield of oxidized steroid.



  You can also add the foaming preventive agents such. B. glyceride oils and waxes, soybean oil, castor oil, bacon oil, olein, sulfonated oils, sulfonated castor oil, sulfonated olive oil, brominated bicinus oil, mineral oil, oleic acid, bacon oil plus 3% octadecanol or dioctylphenylphosphonide in quantities of 0.1-0, 5%.

   Using suitable devices, the foam and the air it contains can be returned to the mixture in order to facilitate ventilation.



  The fermentative hydroxylation of steroids can be carried out with irradiation with actinic, e.g. B. visible or ultraviolet, light in a non-lethal dose or under alternating exposure and darkness. In certain cases, such as Phyeomyces blakesleeanus, strong light has an inhibiting effect.



  The time required for the biological introduction of the hydroxyl group into the steroids changes, depending on the desired result, for example. from one to 70 hours. The oxidation begins practically immediately after addition to the culture of a fully cultivated fungus. A mushroom is usually fully cultivated after a period of 4-24 hours, depending on the degree of ventilation.

   If the steroid is added to the fermentation medium at the same time as inoculation, longer times, including the growth period, are required for a high degree of conversion, which is probably due to the fact that the oxidizing effect of the fungal enzyme system is only fully developed after a certain culture time . Accordingly, when the steroid is added, times of 8-72 hours can be expected at the same time as the vaccination.

   On the other hand, if the steroid of the fermentation liquid is used after a previous aerobic culture, e.g. B. after 16 to 24 hours, too, the conversion of the deoxysteroid into the oxysteroid begins immediately and within 1-72, preferably 24 hours or even less, high yields are obtained.

   It should be noted that if the deoxysteroid is exposed to the oxidizing effect of the fungus or its enzymes for a long period of time, a somewhat greater conversion into polyoxy derivatives takes place; however, in each case a hydroxyl group is usually introduced in the 11-position.



  The mycelium is quantitatively determined as dry matter by drying an aliquot to constant weight at 60 ° C. and 736 mm Hg. The same results are obtained when drying at 60 ° C. without vacuum. The determined dry weight varies somewhat depending on the way in which the mycelium was isolated. If the medium is filtered through filter paper, insoluble material is retained along with the mycelium, while filtration through a fine wire mesh often does not leave all of the mycelium behind.

   Differences in the filter material can affect the found mycelium weight by up to 1 g / 1. Varying amounts of the inoculation material gave, calculated on a dry basis, 0.8-6.0 g of mycelium per liter in 24 hours, while after 48 hours the mycelium weight was 1-11 g / l. With the same inoculation, medium, time, temperature and ventilation, the results are very reproducible.

   Under optimal culture conditions, addition of the steroid after 24 hours of mushroom culture and a conversion time of 48 hours, about 7 g of dry mycelium are obtained per liter of medium.



  An addition of 0.5 g steroid, e.g. B. progesterone, per liter of a 24-hour culture of the fungus in the fermentation medium, followed by a further 24-hour conversion period, resulted in less than 2 o / a conversion of progesterone to oxyprogesterone with simultaneous formation of 1 gl @ lycelitim per liter, calculated on a dry basis.

   Increasing the concentration of my celium to 3.7 g / l, 401 / o of the steroid was converted, and an even higher inoculation, which gave a concentration of about 6 g / l my celiuni after 30 hours of conversion, resulted in about one 86% conversion of the steroid into oxysteroid.



  With an amount of at least 0.5 g / 1 llycel on a dry basis, with a fermentation of at least 3 hours, you get satisfactory oxysteroids which can be easily purified by crystallization. Better results are obtained with a fermentation time of at least 5 hours. With a concentration of the steroid substrate of up to 2 g / 1 of the medium and 1-20 g / 1 l @ lycelium (on a dry basis) a conversion period of 5 to 72 hours is desired.



  After the end of the oxidative fermentation, the hydroxylated steroids, eg. B. 11-oxy-steroids are isolated in any appro priate manner from the fermentation broth. In most cases, the converted steroid has the same number of carbon atoms in the carbon skeleton as the starting material.

   A particularly preferred way of obtaining the oxysteroids consists in extracting the reaction mixture (fermentation liquid -I- lly cel) with a water-immiscible solvent, such as a halogenated hydrocarbon, e.g. B. t4lethylene chloride, ethylene chloride, chloroform, ether, ester, amyl acetate or the like.

    Another suitable extraction method is to separate the cells from the fermentation liquid and separate them with a water-miscible or immiscible liquid, e.g. B. acetone, to extract while extracting the fermentation liquid freed from the mycelium with solvents which are not soluble in water. The cells can be disrupted before or during the extraction, for example by exposure to heat or chemicals. If you use heat to disrupt the cells, you can use the solvent, e.g.

   B. methylene chloride, introduce as vapor or liquid into the mixture, whereby the extraction ver runs with high efficiency. The Oxy steroids can also vate Deri, such as. B. esters or urea adducts are isolated. The extracts can, conveniently after they have been washed successively with an alkaline or sodium bicarbonate solution and water, combined, after which the solvent is expelled with a stream of air, oxygen or in a vacuum or the like and the residue is taken up in fresh organic solvent, to recrystallize or purify it.

   Fats and steroids can be separated from each other via urea accumulation products, which arise when the raw solid products are brought together with urea solutions. A further crystallization can, if desired, from organic solvents, e.g. B. alcohol. The crystalline product can be identified by known methods, e.g. B. by elemental analysis, melting point, infrared spectrum, X-ray diagram, ultraviolet spectrum, micro-combustion u. at the. Furthermore, the crystalline material or extracts of non-crystalline material can be identified, characterized and isolated using the methods of paper chromatography.

    In addition to the oxy steroids, other fermentation products such as citric acid, fumaric acid, glueonic acid, ttaconic acid, lactic acid, enzymes and various antibiotics can advantageously be obtained as by-products from the fermentation solution.

   The addition of steroid in the above concentration and times to the media and under conditions such as those for the production of fumaric acid in US Pat. No. 2326986 by Waksman and No. 2327191 by Kane et al. or for the production of lactic acid, USA patent No. 2132712, allows the production of oxysteroids and valuable acids as by-products.

   The oxysteroids formed in the heterofermentation processes can be separated from the by-products from the fermentation mixture before or after the end of the fermentation. After removing the oxysteroid, e.g. With methylene chloride or triglyceride oil, for example, fermentation can be allowed to continue until the end.

      <I> Example 1: </I> Dioxyprogesterone To 4 liters of a 32-48 hour old culture of RH 176 (Rhizopus arrhizus strain), 1 g of progesterone in 50 cubic meters of acetone is added, so that a suspension of the steroid is formed. The culture is then incubated at room temperature for 48 hours. After this time the pl, of the medium is 3.5.

   The fermentation liquid and the mycelium are extracted one after the other with three liters each, then with two liters and again with one liter of methylene chloride. The methylene chloride extracts are combined, washed twice with 400 em3 2 / o sodium bicarbonate solution each and three times with 500 cm3 distilled water each time.

   The solvent is evaporated to dryness in a vacuum and the solids are taken up in 50 cm3 of methylene chloride. The solution is placed in a 100 cm3 beaker and evaporated using a stream of air. The residue (1.585 g) is dissolved in 5 cubic meters of hot methanol and allowed to cool slowly to room temperature. 75 mg of crystals with a melting point of 245 to 249 ° C. are obtained. These crystals are referred to as U-I in the following.

   It is dissolved in 5 cubic meters of methanol and filtered into a centrifuge tube. After cooling, 23 mg of the U-I crystals are obtained. The paper chromatography of these crystals according to Zaffaroni using diethylene glycol monoethyl ether as the stationary phase and methyl cyelohexane as the mobile phase reveals a single spot that is more polar than progesterone.

    The micro-combustion indicates that two hydroxyl groups have been attached to the progesterone molecule.



  Analysis Ber. for progesterone: C 80.23; H 9.55 dioxyprogesterone: C 72.8; H 8.7 U-I found: C 72.8; H 8.6. The compound U-I shows more than one oxy group in the infrared analysis, but, as the infrared spectrum also shows, retains the original 3-keto-d4 and 20-keto structure of progesterone.



  The acetylation of the compound UI with 0.3 cm3 of pyridine and 0.3 cm3 of acetic anhydride for 9.0 mg of the compound, whereby the mixture is left to stand for 16 hours at room temperature, 12.5 cubic meters of water are added and, after standing for one hour, it is cooled at room temperature, to cause crystallization, 6.7 mg of crystalline diacetate of melting point 145-148 ° C. and 154-155 ° C., which is designated as U-II, 6,11-diacetoxy-progesterone, are obtained.

    The infrared underlining confirms the absence of the hydroxyl group and the presence of new acetoxy groups.



  The combined mother liquors, which still contain 1.5 g of solid fractions of the first crystalline compound U-I, are freed from the solvent by ventilation, dissolved in 50 cm3 of benzene and chromatographed over alumina (A1203) as shown in Table I. To do this, 50 g of alumina washed with acid and dried at 45 C are used as the adsorbent and the column is developed with 100 cm3 portions of solvent.

   The specified solvent composition relates to the total volume of the mixed solvent in each portion or fraction. In the paper chromatogram fraction A (19 and 20 in Table I) shows only one point which is more polar than progesterone, but less polar than compound U-1, which occurs in fractions 21-33.



  Fraction A is dissolved in 2 cm hot methanol and filtered. After cooling overnight, 171 mg of crystals of a compound designated U-III, which melts at 166 to 167 ° C., are obtained.

   After recrystallization from 98.7 mg of U-III, 11a-oxy-progesterone, melting point I.66-167 C; [a] D = + 175.9 (c = 1.0127 in chloroform). Microburns confirm the deposition of one atom of oxygen on progesterone.



  Analysis Ber. for C2113.03: C = 76.40; H = 9.10 U-III found: C = 76.66; H = 8.92. The infrared analysis shows the presence of a hydroxyl group and the original progesterone molecule.



  The position of the oxy group in U-III is determined as the 11-position by oxidizing 30 mor U-III in 0.5 cm3 glacial acetic acid using 1 em3 glacial acetic acid containing 5 mg Cr03 and 0.005 cm3 water by allowing it to stand for one hour.

   A few drops of methanol are then added and, after 10 minutes, the solution is diluted to 45 cm3 with water, the solution is extracted three times with 15 cm3 of methylene chloride each time and the methylene chloride is then evaporated. The residue is recrystallized from 0.5 cm3 of methanol, 19 mg of a triketone, U-IV, having a melting point of 172-175 C;

    [a] D = -I-158 in chloroform and -f-143 in acetone. The triketone structure is confirmed by the Inlra.rotanalyse. Further investigations in the infrared spectrum show that the new keto group can apparently only be present in positions 1, 7, 11 or 12. But there is no known natural or synthetic 1-keto steroid.

   The physical constants of oxyprogesterone U-III do not agree with those of 12a-oxyprogesterone. match (m.p. 200-203 C, [a1'7 = + 205 (c = 1.0 in acetone); Wett:

    stein, Helv. Chim. Acta., 31, 1963 [1945]), not even with those of 12p) '- oxy-progesterone (melting point 164--167 C, melts again after cooling at 195-198 C; [a] D = + 205 [c = 0.502 in acetone]; Reiehstein CA 36, 2261 [1942]). So I still have the 6 and 11 positions for the new keto group of the compound U-VI.



  A comparison of the physical constants shows that the triketone (compound U-VI) is 11-keto-progesterone (melting point 172 to 175 ° C; [a] D = -f- 258 8 [c = 0.9 in acetone] ; Reichstein, Helv. Chim. Acta, 23, 684 [1940]; ibid. 26, 721 [1942]).

   A comparison of the product with real 11-ketoprogesterone by means of infrared spectra, X-ray diffraction, paper chromatography and melting point analysis shows the identity of the compounds.



  The physical constants and infrared spectra of the compound U-III agree with those of 11fl-oxy-progesterone (melting point 187-188; [a] D = -! - 222-225 [c = 1.75 in acetone]; Shoppee and Reichstein, Helv. Chim. Acta, 24, 351-60 [1941]) do not agree.

   Since U-VI shows that the oxygen introduced is in the 11-position, U-III must be 11a-oxy-progesterone.



  20 mg of the compound U-III are acetylated using 0.6 cm 3 of pyridine and 0.6 cm 3 of acetic anhydride. After 16 hours of standing at room temperature, 25 cms of water are added. After one hour, cooling is carried out to induce crystallization, the crystals formed are washed with water and dried. 16.1 mg of a monoacetate, 11a-acetoxy-progesterone, U-V, of melting point 175-177 C. are obtained.

   Infrared analysis indicates the absence of the hydroxyl group and the presence of a new acetoxy group.



  Analysis Ber. for C23H3204: C, 74.00; H 8.85. Found: C 74.33; H 8.78.
EMI0009.0056
  
    <I> Table <SEP> I </I>
<tb> A120 <SEP> Chromatography <SEP> of <SEP> biologically <SEP> converted <SEP> progesterone
<tb> (under <SEP> use <SEP> of the <SEP> organism <SEP> RH <SEP> 176)
<tb> Eluted
<tb> fraction <SEP> solvents <SEP> solids <SEP> paper chromatography
<tb> mg
<tb> 1 <SEP> Benzene <SEP> 0
<tb> 2 <SEP> Benzene <SEP> 182.2 <SEP> No <SEP> stains
<tb> 3 <SEP> benzene <SEP> plus <SEP> 5 <SEP>% <SEP> ether <SEP> 123.0
<tb> 4 <SEP> benzene <SEP> plus <SEP> 5% <SEP> ether <SEP> 246.6
<tb> 5 <SEP> Benzene <SEP> plus <SEP> 10% <SEP> Ether <SEP> 162.9 <SEP> No <SEP> stains
<tb> 6 <SEP> benzene <SEP> plus <SEP> 10% <SEP> ether <SEP> 18,

  0
<tb> 7 <SEP> Benzene <SEP> plus <SEP> <B> 10% </B> <SEP> ether <SEP> 11.2
<tb> 8 <SEP> Benzene <SEP> plus <SEP> <B> 500/9 </B> <SEP> Ether <SEP> 128.2 <SEP> No <SEP> stains
<tb> 9 <SEP> benzene <SEP> plus <SEP> 50% <SEP> ether <SEP> 33.6
<tb> 10 <SEP> ether <SEP> 16.1
<tb> 11 <SEP> ether <SEP> 15.4
<tb> 12 <SEP> ether <SEP> plus <SEP> 5% <SEP> CHCIs <SEP> 3.8
<tb> 13 <SEP> ether <SEP> plus <SEP> 5% <SEP> CHC13 <SEP> 2.0
<tb> 14 <SEP> ether <SEP> plus <SEP> 10% <SEP> CHC13 <SEP> 3,4
<tb> 15 <SEP> ether <SEP> plus <SEP> 10% <SEP> CHC13 <SEP> 3,6
<tb> 16 <SEP> ether <SEP> plus <SEP> 50% <SEP> CHC13 <SEP> 3.5
<tb> 17 <SEP> ether <SEP> plus <SEP> 50% <SEP> CHC13 <SEP> 2.1
<tb> 18 <SEP> CHCl3 <SEP> 2.5
EMI0010.0001
  
    <I> Table <SEP> I </I> <SEP> (continued)

  
<tb> Eluted
<tb> fraction <SEP> solvents <SEP> solids <SEP> paper chromatography
<tb> mg
<tb> Group <SEP> A <SEP> (19 <SEP> -f- <SEP> 20)
<tb> 19 <SEP> CHC13 <SEP> 222,6 <SEP> Only <SEP> a <SEP> spot, <SEP> the <SEP> more polar <SEP> is <SEP> than
<tb> 20 <SEP> CHC13 <SEP> plus <SEP> 50 / a <SEP> acetone <SEP> 167.9 <SEP> progesterone.

   <SEP> Mixture <SEP> of <SEP> 2 <SEP> flek lien, <SEP> 1 <SEP> equals <SEP> like <SEP> fraction <SEP> 20, <SEP> der
<tb> other <SEP> more polar <SEP> than <SEP> fraction <SEP> 20
<tb> 21 <SEP> CHC13 <SEP> plus <SEP> 5 <SEP>% <SEP> acetone <SEP> 23.5
<tb> 22 <SEP> CHC13 <SEP> plus <SEP> 10% <SEP> acetone <SEP> 17.3
<tb> 23 <SEP> CHC13 <SEP> plus <SEP> 10% <SEP> acetone <SEP> 8.7 <SEP> Only <SEP> slow <SEP> moving <SEP> spot
<tb> 24 <SEP> CHC13 <SEP> plus <SEP> 50% <SEP> acetone <SEP> 30.9
<tb> 25 <SEP> CHC13 <SEP> plus <SEP> 50% <SEP> acetone <SEP> 6.1
<tb> 26 <SEP> acetone <SEP> 13;

  0 <SEP> Only <SEP> slowest <SEP> moving <SEP> spot
<tb> 27 <SEP> acetone <SEP> 8.4
<tb> 28 <SEP> acetone <SEP> plus <SEP> 5% <SEP> methanol <SEP> 25.0
<tb> 29 <SEP> acetone <SEP> plus <SEP> 5% <SEP> methanol <SEP> 4,6
<tb> 30 <SEP> acetone <SEP> plus <SEP> 10% <SEP> methanol <SEP> 7.4
<tb> 31 <SEP> acetone <SEP> plus <SEP> 10% <SEP> methanol <SEP> 1.4
<tb> 32 <SEP> acetone <SEP> plus <SEP> 50% <SEP> methanol <SEP> 8.1
<tb> 33 <SEP> acetone <SEP> plus <SEP> 50% <SEP> methanol <SEP> 5,3 <I> Example 2: </I> Oxy-progesterone For a 24-hour culture of the organism RH 176 ( Rhizopus arrhizus strain) in 4.0 1 medium is added 1 g of progesterone in 50 cm3 of acetone.

   After incubating for 24 hours and shaking with air, the fermentation solution is extracted as in Example 1. The methylene chloride extracts are washed twice with 300 cubic meters of 2% strength sodium bicarbonate solution and twice with 500 cubic meters of distilled water, and the combined extracts are dried over as little anhydrous sodium sulfate as possible.

   6 cm3 of this solution, corresponding to 2 mg of the original progesterone, are investigated using the same solvents as in Example 1 on paper chromatography. It shows that strong conversion into 11a-oxy-progesterone. has taken place. The remaining methylene chloride extract is evaporated to dryness in a stream of air.

   The solid residue (1.584 g) is dissolved in 100 cm3 of benzene and an insoluble fraction of 145 mg is filtered off. The melting point of this compound is 190 to 230 ° C. The insoluble fraction gives a total of 90 mg of crystalline compound U-1 with a melting point of 230-240 ° C. by dissolving 145 mg of the same in 20 cm3 of acetone, narrowing the solution to 10 cm3 and cooling.

    In paper chromatography according to Zaffaroni and the determination of the melting point, the crystals turn out to be the dioxy compound, 6,11-dioxy-progesterone, compound U-I. The filtrate, which contains 1.439 g of solid material, is chromatographed over a clay column (A1203) using the technique described in Example 1 (Table II)

  . 100 cm3 portions of the solvent are used to develop the column. Fraction B of the chromatographic separation (Table II) contains 571 mg of 11a-oxy-progesterone (compound U-III) and is dissolved in 20 em3 of methanol, then treated with 20 cm3 of water, it is warmed and cooled in the refrigerator. Another 20 cubic meters of water are added and cooling is continued.

   Crystallization occurs and 300 mg of dry crystals of 11a-oxyrogesterone (compound U-III) with a melting point of 165-166 ° C. are obtained.



  A compilation of a number of biological oxidations is given in Table III.
EMI0011.0007
  
    <I> Table <SEP> II </I>
<tb> A1203 <SEP> Chromatogram <SEP> of <SEP> biologically <SEP> converted <SEP> progesterone
<tb> under <SEP> use <SEP> of the <SEP> organism <SEP> RH <SEP> 176
<tb> Eluted
<tb> fraction <SEP> solvents <SEP> solids <SEP> paper chromatogram
<tb> mg
<tb> 1 <SEP> Benzene <SEP> 208
<tb> 2 <SEP> Benzene <SEP> 12
<tb> 3 <SEP> benzene <SEP> plus <SEP> 5% <SEP> ether <SEP> 4
<tb> 4 <SEP> benzene <SEP> plus <SEP> 5% <SEP> ether <SEP> 14
<tb> 5 <SEP> benzene <SEP> plus <SEP> 10% <SEP> ether <SEP> 37
<tb> 6 <SEP> benzene <SEP> plus <SEP> 10% <SEP> ether <SEP> 19
<tb> 7 <SEP> benzene <SEP> plus <SEP> 50% <SEP> ether <SEP> 7
<tb> 8 <SEP> benzene <SEP> plus <SEP> 50% <SEP> ether <SEP>

  19th
<tb> 9 <SEP> ether <SEP> 4
<tb> 10 <SEP> ether <SEP> 16
<tb> 17. <SEP> ether <SEP> plus <SEP> 5% <SEP> CHC13 <SEP> 8
<tb> 1.2 <SEP> ether <SEP> plus <SEP> 5 <SEP> 0 / a <SEP> <B> CHCI3 </B> <SEP> 4
<tb> 13 <SEP> ether <SEP> plus <SEP> 10% <SEP> <B> CHCI3 </B> <SEP> 0
<tb> 14 <SEP> ether <SEP> plus <SEP> 10% <SEP> <B> CHCI3 </B> <SEP> 0
<tb> 15 <SEP> ether <SEP> plus <SEP> 50% <SEP> CHC13 <SEP> 4
<tb> 16 <SEP> ether <SEP> plus <SEP> 50% <SEP> <B> CHCI3 </B> <SEP> 2
<tb> 17 <SEP> CHCl3 <SEP> 11 <SEP> fraction <SEP> B <SEP> (19 <SEP> + <SEP> 20)
<tb> 18 <SEP> <B> CHCI3 </B> <SEP> 265 <SEP> Only <SEP> stain <SEP> from <SEP> 11a-Oxy-proge 19 <SEP> CHCl3 <SEP> plus <SEP > <B> 5 ()

  / o </B> <SEP> acetone <SEP> 235 <SEP> steron <SEP> plus <SEP> traces <SEP> of <SEP> three other <SEP>
<tb> 20 <SEP> CHC13 <SEP> plus <SEP> <B> 51 / o </B> <SEP> acetone <SEP> 60 <SEP> <SEP> slower <SEP> moving <SEP> Ver 21 <SEP> CHC13 <SEP> plus <SEP> 10% <SEP> acetone <SEP> 16 <SEP> bonds
<tb> Mixture <SEP> of <SEP> bioxy-progesterone
<tb> 22 <SEP> <B> CHCI3 </B> <SEP> plus <SEP> 10% <SEP> acetone <SEP> 22 <SEP> and <SEP> two <SEP> more <SEP> polar <SEP > Connections
<tb> 23 <SEP> <B> CHCI3 </B> <SEP> plus <SEP> 50% <SEP> Aeeton <SEP> 27 <SEP> I <SEP> Same as <SEP> as <SEP> fraction <SEP > 22,

   <SEP> but <SEP> less 24 <SEP> CHCl3 <SEP> plus <SEP> 50% <SEP> acetone <SEP> 37 <SEP> less <SEP> the <SEP> 2 <SEP> spots <SEP> of <SEP> 22 <SEP> and <SEP> more
<tb> of a <SEP> third <SEP> and <SEP> fourth
<tb> 25 <SEP> acetone <SEP> 29 <SEP> compound <SEP> less <SEP> polar <SEP> than <SEP> die
<tb> 26 <SEP> Aeeton <SEP> 43 <SEP> 2 <SEP> spots <SEP> in <SEP> 22, <SEP> but <SEP> more polar <SEP> than
<tb> 11a-oxy-progesterone
<tb> 27 <SEP> acetone <SEP> plus <SEP> 5% <SEP> methanol <SEP> 16
<tb> 28 <SEP> acetone <SEP> plus <SEP> 5% <SEP> methanol <SEP> 24
EMI0012.0001
  
    <I> Table <SEP> II </I> <SEP> (continued)

  
<tb> Eluted
<tb> fraction <SEP> solvents <SEP> solids <SEP> paper chromatogram
<tb> mg
<tb> 29 <SEP> acetone <SEP> plus <SEP> 10% <SEP> methanol <SEP> 4
<tb> 30 <SEP> acetone <SEP> plus <SEP> 10% <SEP> methanol <SEP> 0
<tb> 31 <SEP> acetone <SEP> plus <SEP> 501 / o <SEP> methanol <SEP> 1
<tb> 32 <SEP> acetone <SEP> plus <SEP> 501 / o <SEP> methanol <SEP> 23
<tb> 33 <SEP> methanol <SEP> 9
<tb> <I> Table <SEP> III </I>
<tb> Fermentation <SEP> <SEP> fractions obtained from <SEP> of the <SEP> A1203 column <SEP>
<tb> Inserted <SEP> volume <SEP> nset <SEP> of the <SEP> incubation- <SEP> progesterone <SEP> lla-oxy- <SEP> dioxy- <SEP> other
<tb> Transmutation progesterone <SEP> time <SEP> progesterone <SEP> progesterone
<tb> mg <SEP> mixture <SEP> h <SEP> mg <SEP> mg <SEP> mg <SEP> products
<tb> 1 <SEP> mg
<tb> 1000 <SEP> 4 <SEP> 48 <SEP> 0 <SEP> 391 <SEP> 142

  <SEP> 18
<tb> 1200 <SEP> 4 <SEP> 67 <SEP> 0 <SEP> 507 <SEP> 222 <SEP> 95
<tb> 1000 <SEP> 4 <SEP> 24 <SEP> 0 <SEP> 571 <SEP> 225 <SEP> 94
<tb> 1000 <SEP> 4 <SEP> 30 <SEP> 0 <SEP> 471 <SEP> 265 <SEP> 120
<tb> 3000 <SEP> 10 <SEP> 24 <SEP> 1520 <SEP> 1260 <SEP> 76 <I> Example 3: </I> 11-oxy-steroids In the same way as stated above, the in Table IV oxidized steroids in the 11-position. The details can be found in Table IV.



  <I> Example 4 </I> 300 cm3 of a nutrient medium which contains 21 / o edamin, 5% dextrose and maceration liquid from maize and which has a pH of 6.5 are inoculated with spores of the organism RH 176 for 24 hours moved under aerobic conditions.

   From this medium, samples of 20 cm3 each are placed in sterile 100 em3 flasks and about 6 mg of the following steroid substrates dissolved in a maximum of 0.3 em3 acetone, so that a fine suspension of the steroid is formed, is added to each sample. After 24 hours, the contents of the flask are extracted in order to determine the change using a paper chromatogram. Table V lists the steroids treated as well as the prr just prior to the start of the extraction.

      
EMI0013.0001
    
EMI0014.0001
    
EMI0015.0001
    
EMI0016.0001
    
EMI0017.0001
  
    <I> Table <SEP> V </I>
<tb> steroid <SEP> end-p.
<tb> 1. <SEP> 4-androstene-3,17-dione <SEP> 3.75
<tb> 2. <SEP> Cholestenone <SEP> 4.12
<tb> 3. <SEP> Ergosterone <SEP> 3.9
<tb> 4. <SEP> Ergosterol <SEP> 3.6
<tb> 5. <SEP> 3f, 21-diaeetoxy-5-pregnen-20-on <SEP> 3.75
<tb> 6. <SEP> 3ss-acetoxy-5-pregnen-20-one <SEP> 3.9
<tb> 7. <SEP> Stigmasta-4,22-dien-3-on <SEP> 3,4
<tb> B. <SEP> Dehydroergosteryl-dioxido-maleic acid anhydride adduct <SEP> 3.85
<tb> 9. <SEP> 3-Acetoxy-5,7,9 (11) <SEP> -pregnatrien 20-one-maleic anhydride adduct <SEP> 3,8 <I> Example 5:

  </I> 11-Oxy-steroids The microorganisms are grown in a medium containing 20 g of edamin, 5 cm3 of maize fermentation liquid, 50 g of dextrose, diluted to 1 liter with tap water, and the pli of which was adjusted to 6.5 before it was sterilized at 120 C for about 20 minutes.



  The nutrient media are inoculated with cultures of the organisms grown from spores on a medium containing 10 1 / o dried malt extract and 2% agar. Each flask contained 30 cm3 of the medium.



  The flasks are shaken under aerobic conditions at room temperature for 24 hours, and then a certain amount of steroid substrate in the form of a solution containing 2 percent by weight per volume of acetone is added and shaken at room temperature for a further 24 hours. Use 6 mg of steroid for every 30 em3 of medium.



  The mixture is then extracted three times with methylene chloride, each time using an equal volume of methylene chloride as that of the aqueous phase. The extract is separated off, washed with 2% sodium bicarbonate solution and distilled water. The solvent layer is evaporated and the residue is checked by the paper strip chromatography method. The steroids used were all oxidized by the organism used. Table VI shows the fungus tested and the steroid substrate used.

    
EMI0017.0020
  
    <I> Table <SEP> VI </I>
<tb> Steroid: <SEP> Progesterone
<tb> American.
<tb> organism <SEP> culture type collection
<tb> No.
<tb> 1. <SEP> Mucor <SEP> rouxii <SEP> 4857
<tb> 2. <SEP> Mucor <SEP> griseo-cyanus <SEP> (-h) <SEP> 1207a
<tb> 3. <SEP> Mucor <SEP> griseo-cyanus <SEP> (-) <SEP> 1207b
<tb> 4. <SEP> Mucor <SEP> hiemalis <SEP> var. <SEP> albus <SEP> 6800
<tb> 5. <SEP> Mucor <SEP> hiemalis <SEP> 8690
<tb> 6. <SEP> Mucor <SEP> mucedo <SEP> 9836
<tb> 7. <SEP> Mucor <SEP> mucedo <SEP> 9635
<tb> B. <SEP> Mucor <SEP> mucedo <SEP> 7941
<tb> 9. <SEP> Rhizopus <SEP> nigricans <SEP> 10404
<tb> 10. <SEP> Rhizopus <SEP> nigricans <SEP> 7577
<tb> 11. <SEP> Rhizopus <SEP> nigricans <SEP> (-I-) <SEP> 6227a
<tb> 12. <SEP> Rhizopus <SEP> nigricans <SEP> (-) <SEP> 6227b
<tb> 13. <SEP> Rhizopus <SEP> chinensis <SEP> (-) <SEP> 1227b
<tb> 14.

   <SEP> - <SEP> Rhizopus <SEP> japonicus <SEP> 8446
<tb> 15. <SEP> Rhizopus <SEP> oryzae <SEP> 10260
<tb> 16. <SEP> Rhizopus <SEP> oryzae <SEP> 9363
<tb> Steroid: <SEP> 11-deoxycorticosterone
<tb> 17. <SEP> Mucor <SEP> rouxii <SEP> 4857
<tb> 18. <SEP> Mucor <SEP> griseo-cyanus <SEP> (-I-) <SEP> 1207a
<tb> 19. <SEP> Mucor <SEP> griseo-cyanus <SEP> (-) <SEP> 1207b The test of the aforementioned organisms with 11-deoxycorticosterone, 17-oxyprogesterone and 11-deoxy-17-oxycorticosterone gives same results, i.e. introduction of a hydroxyl group into the steroid nucleus.



  The same result is obtained when treating progesterone, 11-deoxy-corticosterone, 11-deoxy-17-oxycorticosterone and 17-oxyprogesterone with the following fungi:

       Mucor genevensis, plumbeus, parasiticus, dubius, javanicus, adventitius, microsporus, rouxianus, racemosus;

          Rhizopus reflexus, shanghaiensis, tritici, cohenii, delemar, tonkinensis, kazanensis; Phycomyces blakesleeanus, theobromatus; Absidia glauca;

          Zyghrynchus moelleri, heterogamus and Syncephalus nodosa. <I> Example 6:

  </I> 11a-Oxy-progesteron A medium is made up of 20 g edamin (enzymatically degraded lactalbumin), 3 g maceration liquid from corn and 50 g dextrose, diluted to 1 liter with tap water and diluted to pH 4.3-4, 5 set, manufactured. 12 liters of this sterilized medium are mixed with Rhizopus nigricans (-);

       ATCC No. 6227b and cultivated for 24 hours at 28 C by venting and stirring so that the oxygen uptake is 6.3-7 millimoles per liter of Na 2 SO 3 per hour, according to the method of Cooper, Fernstrom and Miller, Ind. Eng. Chem., 36, 504 (1944). 6 g of progesterone in 150 cm3 of acetone are then added to this medium, so that the steroid is suspended in the medium. After culturing for a further 24 hours under the same temperature and ventilation conditions, the liquid and mycelium are extracted.

   The mycelium is filtered off, washed twice with about its volume of acetone and then extracted twice with about its volume of methylene chloride. The acetone and methylene chloride extracts are added to the liquid and the mixture is extracted successively twice with half its volume of methylene chloride and then twice with a quarter volume of methylene chloride each time.

   The combined methylene chloride extracts are washed with one tenth of their volume of 2% sodium bicarbonate solution and then twice with one tenth volume of water each time. After drying the methylene chloride solutions with about 3-5 g of anhydrous sodium sulfate per liter and filtering, the solvent is distilled off. The residue is taken up in as little methylene chloride as possible, filtered and the solvent evaporated.

   The raw crystals obtained are dried and then washed five times with 5 cm3 of ether per gram of raw crystals; Yield 5.072 g of crystals with a melting point of 165-168 C. After recrystallization from methanol, 311 mg of 11a-oxy-progesterone with a melting point of 166-168 C, [a] D = + 180 (c = 1.0127 in chloroform) are obtained ; k242 46.72.

      Analysis: Ber. for C21H3003: C 76.40; B 1-1 9.10 Found: C 76.77; H 8.92 <I> Example i </I> 11a, 17a, 21-trioxy-4-pregnen-3,20-dione (compound epi-F) from 17a, 21-dioxy-4-pregnen-3, 20-dione (compound S) Following the procedure of Example 6, after 96 hours of treatment of 2 g of 17a, 21-dioxy-4-pregnen-3,> 0-dione with Rhizopus nigricans ATCC 6227b and extraction, 4.988 g of a semi-crystalline extract are obtained.

   This dried extract is rubbed four times with 6 cm3 of ice-cold methylene chloride each time. 630 mg of insoluble crystals with a melting point of 194-199 C. remain. After un-azrystallization from 3 cm3 of methanol and 10 cm3 of ether, 404 mg of crystals with a melting point of 205 to 210 ° C. are obtained.

   Two further recrystallizations from the same solvents give 132 mg of 11a, 17a, 21-trioxy-4-pregnen-3,20-dione with the constant m.p. 209-212 C and 217 to 219 C depending on the crystal form [a] D = -f- 113 (e = 1.568 in methanol). Analysis Ber. for C21113005: C, 69.58; H 8.35 Found: C 69.26; H 8.34 17a-Oxy-21-acetoxy-4-pregnen-3,20-dione can also be used as the starting material with equally good results.



  The oxidation of 1.1a, 17a, 21-trioxy-4-pregnen-3,20-dione with Cr03 in glacial acetic acid with. subsequent extraction with ether and washing with. Water gives androsterone of m.p. 217-220 C.



  Analysis Ber. for C10112403: C 75.96; H 8.05 Found: C 75.61; H 8.10 The 11,21-diacetate of compound epi-F melts at 198-202; [a] D = + 115 (c = 1.145 in chloroform), k240 33.33. The infrared spectrum confirms the structure.

    Analysis Ber. for C25112407: C 67.24; B 1V7.67 Found: C 67.43; H 7.94 Example 8: 14a-Oxy-progesterone Under the same fermentation and extraction conditions as in Example 6, 12 l of the medium that contains a 20-hour culture of Mucor parasitious, ATCC 6176, are added , contains 3 g of progesterone in as little alcohol as possible.

   After a further 48 hours of culture, the extraction of the liquid and the myelium yields 1.667 g of an oily residue which is dissolved in 150 cm3 of benzene and chromatographed over 220 g of clay (washed with acid and dried at 120 ° C.), 200 cm3 portions being made the solvents shown in Table VII were used.

    
EMI0019.0015
  
    <I> Table <SEP> VII </I>
<tb> eluate fraction <SEP> solvent <SEP> solids
<tb> in <SEP> mg
<tb> 1 <SEP> Benzene <SEP> 73.0
<tb> 2 <SEP> Benzene <SEP> 26.5
<tb> 3 <SEP> benzene <SEP> plus <SEP> 5% <SEP> ether <SEP> 28.0
<tb> 4 <SEP> Benzene <SEP> plus <SEP> <B> 51/9 </B> <SEP> ether <SEP> 21.5
<tb> 5 <SEP> benzene <SEP> plus <SEP> 20% <SEP> ether <SEP> 35.0
<tb> 6 <SEP> benzene <SEP> plus <SEP> 201 / o <SEP> ether <SEP> 27.5
<tb> 7 <SEP> benzene <SEP> plus <SEP> 50% <SEP> ether <SEP> <B> 17.0 </B>
<tb> 8 <SEP> benzene <SEP> plus <SEP> 50% <SEP> ether <SEP> 6.5
<tb> 9 <SEP> ether <SEP> 4,5
<tb> 10 <SEP> ether <SEP> 5.0
<tb> 1.1 <SEP> ether <SEP> plus <SEP> 5% <SEP> CHC13 <SEP> 7.5
<tb> 12 <SEP> ether <SEP> plus <SEP> 50 / a <SEP> <B> CHCI3 </B> <SEP> 9.5
<tb> 13 <SEP> ether <SEP> plus <SEP> 101 / o <SEP> <B> CHCI3 </B> <SEP> 1839,

  0
<tb> 7.4 <SEP> ether <SEP> plus <SEP> 10% <SEP> <B> CHCI3 </B> <SEP> 605.5
<tb> 15 <SEP> ether <SEP> plus <SEP> 20% <SEP> <B> CHCI3 </B> <SEP> 356.0
<tb> 16 <SEP> ether <SEP> plus <SEP> 20% <SEP> CHC13 <SEP> 336.5
<tb> 17 <SEP> ether <SEP> plus <SEP> 50% <SEP> <B> CHCI3 </B> <SEP> 331.0
<tb> 18 <SEP> ether <SEP> plus <SEP> 50% <SEP> CHC13 <SEP> 269.5
<tb> 19 <SEP> ether <SEP> plus <SEP> 50% <SEP> <B> C1-1C13 </B> <SEP> 94.0
<tb> 20 <SEP> <B> CHCI3 </B> <SEP> 68.0
<tb> 21 <SEP> <B> CHCI3 </B> <SEP> 22.0
<tb> 22 <SEP> CHCl3 <SEP> 15.0
<tb> 23 <SEP> CHCl3 <SEP> plus <SEP> 5 <SEP>% <SEP> acetone <SEP> 18.0
<tb> 24 <SEP> <B> CHCI3 </B> <SEP> plus <SEP> 5 <SEP>% <SEP> acetone <SEP> 8.0
<tb> 25 <SEP> <B> CHCI3 </B> <SEP> plus <SEP> <B> 10 </B> <SEP>% <SEP> acetone <SEP> 12,

  5
EMI0019.0016
  
    Eluate fraction <SEP> solvent <SEP> solids
<tb> in <SEP> mg
<tb> 26 <SEP> <B> CHCI3 </B> <SEP> plus <SEP> 10% <SEP> acetone <SEP> 9.0
<tb> 27 <SEP> <B> CHCI3 </B> <SEP> plus <SEP> 50% <SEP> acetone <SEP> 26.5
<tb> 28 <SEP> CHCl3 <SEP> plus <SEP> 50% <SEP> acetone <SEP> 29.5
<tb> 29 <SEP> acetone <SEP> 62.0
<tb> 30 <SEP> methanol <SEP> 77.0 Fraction 13 is dissolved in 20 cm3 of acetone and filtered to separate insoluble suspended material. The clear filtrate is concentrated and ether is added dropwise until crystallization begins. On cooling, 585.5 mg of crystals with a melting point of 182-194 ° C separate out.



  A portion of 85 mg of these crystals is sublimed for 2 days at 165 ° C. in a vacuum of 0.05 mm Hg, 5 mg of an oily residue remaining. The sublimate is recrystallized from 3 cm 3 of acetone by adding hexane and 14a-oxy-progesterone with a melting point of 195 to 200.5 ° C. [a123 = + 188 (c = 1.036 in chloro form) is obtained.



  Analysis Ber. for CziH3004: C, 76.40; H 9.15 Found: C 77.02; H 9.55 <I> Example 9: </I> 14-Oxy-progesterone Under otherwise the same conditions as in Example 6, 5 l of a medium containing a culture grown at 25 ° C. for 24 hours are added. Mucorgrieseo cyanus, ATCC 1207a, contains 5 g of progesterone dissolved in as little ethanol as possible. After another 24 hours of culture, the extraction yields 4.815 g of solid products. These are rubbed with Skellysolve B, after which 3.322 g remain, which are freed from fat with ether.

   The residue, which is insoluble in ether, is chromatographed over 85 g of activated alumina (washed with hydrochloric acid, rinsed with water and dried for 4 hours at 120 ° C.) using 170 cm 3 of solvent per table VIII.

      
EMI0020.0001
  
    <I> Table <SEP> VIII </I>
<tb> eluate fraction <SEP> solvent <SEP> solids
<tb> in <SEP> mg
<tb> 1 <SEP> Benzene <SEP> 1.1
<tb> 2 <SEP> Benzene <SEP> 0.4
<tb> 3 <SEP> benzene <SEP> plus <SEP> 5 <SEP>% <SEP> ether <SEP> <B> 3,9- </B>
<tb> 4 <SEP> benzene <SEP> plus <SEP> 5 <SEP>% <SEP> ether <SEP> 1,3
<tb> 5 <SEP> benzene <SEP> plus <SEP> 10 <SEP>% <SEP> ether <SEP> 6 <SEP> benzene <SEP> plus <SEP> 10 <SEP>% <SEP> ether <SEP > 96.0
<tb> 7 <SEP> benzene <SEP> plus <SEP> 50 <SEP>% <SEP> ether <SEP> 259.6
<tb> 8 <SEP> benzene <SEP> plus <SEP> 50 <SEP>% <SEP> ether <SEP> 21.5,8
<tb> 9 <SEP> ether <SEP> 143.5
<tb> 10 <SEP> ether <SEP> 84.3
<tb> 11 <SEP> ether <SEP> plus <SEP> 5% <SEP> CHCI3 <SEP> 104.4
<tb> 12 <SEP> ether <SEP> plus <SEP> 5% <SEP> CHCI3 <SEP> 66.9
<tb> 13 <SEP> ether <SEP> plus <SEP> 10% <SEP> CHCI3 <SEP> 18,

  5
<tb> 14 <SEP> ether <SEP> plus <SEP> <B> 100 / a </B> <SEP> CHCI3 <SEP> 8.1
<tb> 15 <SEP> ether <SEP> plus <SEP> <B> 500/9 </B> <SEP> CHCI3 <SEP> 2.4
<tb> 16 <SEP> ether <SEP> plus <SEP> 501 / o <SEP> CHCI3 <SEP> 11.6
<tb> 17 <SEP> ether <SEP> plus <SEP> 501 / o <SEP> CHCI3 <SEP> 13.2
<tb> 18 <SEP> ether <SEP> plus <SEP> <B> 50% </B> <SEP> CHCI3 <SEP> 9.4
<tb> 19 <SEP> CHC13 <SEP> 39.0
<tb> 20 <SEP> CHCl3 <SEP> 474.4
<tb> 21 <SEP> CHC13 <SEP> 81.6
<tb> 22 <SEP> CHCI3 <SEP> 40.5
<tb> 23 <SEP> CHC13 <SEP> plus <SEP> 5 <SEP>% <SEP> acetone <SEP> 2-1,1
<tb> 24 <SEP> acetone <SEP> 22.7
<tb> 25 <SEP> methanol <SEP> 65.2 The eluted solids of fraction 22 give 20 mg of crystals with a melting point of 230-238 C.



  The eluted solids from fractions 19 and 20 are combined and recrystallized from acetone, 382.5 mg of crystals having a melting point of 179-188 ° C. being obtained. These are recrystallized from 2 cubic meters of methanol and 14-oxy-progesterone with a melting point of 180 to 17 ° C, [a] D = + 200 (c = 0.4857 in chloro form) is obtained.



  Analysis Ber. for C21113003: C, 76.40; H 9.15 Found 0 76.50; 11 9.31. <I> Example 10 </I> 14-Oxy-deoxycorticosterone acetate Under otherwise identical conditions as in Example 6, 1.13 g of deoxycorticosterone acetate are added to 4 liters of a medium which contains a 24-hour culture of Mucor griseocyanus ATCC 1207a 100 em3 acetone. After a conversion time of 24 hours, the extraction gives 2.5 g of oily crystals.

   These are extracted exhaustively with Skelly solo e B and then with ether, after which 0.64 g of brown crystals remain. These are dissolved in 12 cm3 of hot methanol, filtered and cooled, giving <B> 0.2989 </B> g of white crystals of 14a-oxy-deoxycorticosterone that soften at 173 ° C. and melt at 175-176 ° C. have characteristic infrared spectra in mineral oil and chloroform.

    
EMI0020.0029
  
    analysis
<tb> Ber. <SEP> for <SEP> C21H3004: <SEP> C <SEP> 72.83; <SEP> 1I <SEP> 8.73
<tb> (with <SEP> 50 <SEP> C <SEP> dried) <SEP> found. <SEP>: <SEP> C <SEP> 70.37; <SEP> H <SEP> 8.70
<tb> C <SEP> 70.64; <SEP> H <SEP> <B> 8.57 </B>
<tb> (with <SEP> l_00 <SEP> C <SEP> dried) <SEP> found: <SEP> C <SEP> 71,14; <SEP> H <SEP> 8.62 The crude extract leached with Skelly solve B and ether can also be crystallized from acetone to convert 14a-oxy-deoxycortieosterone with a melting point of 167 to 170.5 C [a] " = + 190 (c = 0.3341 in chloroform).



  Analysis: Ber. for C..1113004: C 72.83; H 8.73 Found: C 72.83; H 8.48. The 21-acetate of the 14-oxy-desoxyeortico- sterons melts at 160-162 C. Its structure is confirmed by the infrared spectrum. <I> Example 11 </I> Introduction of an oxy group in 10-normethylprogesterone under the same culture and extraction conditions as in.

   Example 6 is added to 31 of a 24-hour culture of Rhizopus nigricans, ATCC 6227b, 1 g of 10-normethylprogesterone (19-normethylprogesterone) dissolved in as little ethanol as possible. After 24 hours of conversion, the extraction gives 1.83 g of a crude oily residue. The same is cleaned by washing four times with 5 ems of diethyl ether each time, with crystalline hydroxylated 10-normethylprogestone remaining.

   The crystals can also be dissolved in 3.5 ems of hot methanol, filtered and the product allowed to crystallize on cooling.



       Hydroxylated 10-normethylprogesterone can also be obtained with Rhizopus arrhizus (ATCC 11145), Mucor griseo-eyanus, Mitcor parasitieus or Helicostylum piriforme. Place of Rhizopus nigrieans.



  Other microorganisms that can be used successfully according to the examples to introduce oxy groups in steroids, especially 71-deoxysteroids, but which do not necessarily give the same results as those of the genus Mucorales, are various strains of Penieillium, z. B. P-100, Aspergilli, e.g.

   B. Aspergillus niger, Neurospora sitophila, Neurospora erassa, Oospora lactis, Polyoporus abietinus, Etomophthorales (Ento- inoplitlioi-a eoronata). The fermentation process with Penicillium as described in US Pat. No. 2458495, e.g.

   B. using the Penieillium Ehrysogenum Thom or other Penieillium species, such as P. cy clopium, P. nigricans, P. notatum and P. roque- forti, see suitable for the simultaneous oxidation of steroids and the production of fermentation products of the Penicilliums.

 

Claims (1)

Claim process for the introduction of hydroxyl groups into steroids by aerobic cultivation of fungi in the presence of the steroids, characterized in that at least 0.5 g of mycelium, calculated as the dry product, is used per gram of the steroid and the oxy-steroid obtained is isolated. SUBClaims 1 .. Method according to claim, characterized in that the starting steroid has a 1-1-1 #. Ethylene group and up to 22 carbon atoms in the carbon skeleton. '?. Method according to patent claim and sub-claim 1, characterized in that the fermentation takes at least 3 hours. 3. The method according to claim and dependent claims 1 and 2, characterized in that the fermentation is carried out with a fungus of the Mucoraceae family. 4th Method according to claim and dependent claims 1 to 3, characterized in that the fermentation is carried out with a mushroom of the genus Mucor. 5. The method according to claim and dependent claims 1 to 3, characterized in that the fermentation is carried out with a fungus of the genus Phycomyees. 6. The method according to claim and dependent claims 1 to 3, characterized in that the fermentation is carried out with a fungus of the genus Rhizopus. 7. The method according to claim and dependent claims 1 to 3, characterized in that progesterone is used as the starting steroid. B. The method according to claim and dependent claims 1 to 3, characterized in that 17a-oxy-progesterone is used as the starting steroid. 9. The method according to claim and dependent claims 1 to 3, characterized in that the starting steroid used is 17a, 21-dioxy-progesterone. 10. The method according to claim and dependent claims 1 to 3, characterized in that 16-dehydroprogesterone is used as the starting steroid. 11. Method according to claim and dependent claims 1 to 3, characterized in that 21-oxy-4-pregnen-3,20-dione is used as the starting steroid. 12. The method according to claim and dependent claims 1 to 3, characterized in that the starting steroid used is 3ss-oxy-5,16-pregnadien-20-one.