2,721,828 United States Patent Office Patented Oct. 25, 1955

2 tained by subjecting starting steroids to the fermenta 2,721,828 tion process of this invention. For example, androstane 3,11,17-trione (male hormone activity) may be obtained PROCESS FOR PRODUCTION OF by fermentation of allopregnane-3,11,20-trione; etio 17-KETOSTEROOS cholane-3,11,20-trione (general anesthetic activity) from Herbert C. Murray, Hickory Corners, and Darey H. Peter pregnane-3,11,20-trione; 3c- or 3.3-hydroxyeticholane son, Kalamazoo Township, Kalamazoo County, Mich., 17-one (anesthetic activity) from the fermentation of assignors to The Upjohn Company, Kalamazoo, Mich., 3a- or 36-hydroxypregnane-20-one or from pregnane a corporation of Michigan 3,20-dione by the side chain fermentation of this in No Drawing. Application October 1, 1953, 10 vention and subsequent reduction with sodium boro Serial No. 383,701 hydride or lithium aluminum hydride; etiocholane-3,6,17 trione (which may be brominated to 4-bromoetiocholane 20 Claims. (C. 195-51) 3,6,17-trione and dehydrobrominated to give 4-andro stene-3,6,17-trione of estrogenic activity) from preg The present invention relates to a novel process for 5 nane-3,6,20-trione by side chain fermentation; adreno the fermentative degradation of the 17-side chain of sterone from the fermentation of 11-ketoprogesterone, 20-oxygenated steroids, especially 20-ketosteroids to cortisone or cortisone acetate; and other like active 17 yield 17-ketosteroids, especially 17-ketoandrostane and ketosteroids. 17-ketoetiocholane compounds. The starting steroid compounds of the present applica The process of the present invention comprises sub 20 tion are the 20-oxygenated steroids, and preferably the jecting a 20-oxygenated steroid, especially a 20-keto 20-hydroxy steroids and the 20-ketosteroids. The cyclo steroid to the action of a fungus of the genus Gliocladium, pentanopolyhydrophenanthrene radical with a 176-side to produce a 17-ketoandrostane compound. chain may possess keto groups or hydroxy groups in The process of degradation of the 17-side chain of other positions, especially positions 3, 6, 7, 8, 11, 12, steroid compounds to give 17-ketosteroids, especially 17 25 14, 17, and 21, and may have double bonds in various ketoandrostanes and 17-ketoetiocholanes by chemical positions especially in positions 4 and 5. Representa means is well known, but these procedures usually in tive starting materials include: 3ox- and 3,3-hydroxypreg volve a number of steps such as formation of a 17 (20) mane-20-one, 30- and 3,3-hydroxy-5-pregnene-20-one, 3oz double bond and oxidation of this double bond. In and 36-hydroxypregnane-1120-dione, 3.cx,110-, 3a, 116-, such oxidative degradation the steroid nucleus is often 30 3,3,11a- and 36,116-dihydroxypregnane-20-one, proges attacked in other positions, especially double bonds with terone, 11-ketoprogesterone, 110-hydroxyprogesterone, resulting high losses. In order to avoid such losses, 11o-acetoxyprogesterone, 11.f3-hydroxyprogesterone, 14a protection of such positions or groups, which involves hydroxyprogesterone, 140,170,21-trihydroxy-4-pregnene at least two additional steps, is practiced in the art. 3,20-dione, 14oz,17ox - dihydroxy - 21-acetoxy-4-pregnene For example, Bergmann and Stevens, J. Org. Chem. 13, 35 3,20-dione, 11a, 17a-dihydroxyprogesterone, 17a-hydroxy 10 (1948) show the ozonolysis of 22-enol acetate of 11-ketoprogesterone, 3,3,17a,20-allo-pregnanetriol (Reich 36-acetoxybisnor-5,7-choladien-22-all protected by a 5,8 stein's compound “O'”); 17 oz,21-dihydroxy-4-pregnene maleic anhydride adduct group to yield the maleic an 3,20-dione (Reichstein's compound “S”), corticosterone, hydride adduct of 36-acetoxy-5,7-androstadiene-17-one. cortisone, cortisone acetate, dihydrocortisone (Kendall's It is an object of the present invention to provide a 40 compound "F") and 11a, 17a,21-trihydroxy-4-pregnene direct method for the degradation of a 17-side chain 3,20-dione (11-epi "F' pregnane-3,11,20-trione, 17 oz of 20-oxygenated steroids, especially 20-ketosteroids by hydroxypregnane-3,11,20-trione, pregnane-3,12,20-trione, the action of a fungus of the genus Gliocladium. An pregnane-3,20-dione, 17a-hydroxypregnane-3,20 - dione, other object of the present invention is the provision allopregnane-3,11,20-trione, allopregnane-3,20-dione, 30 of a process for the production of 17-ketoandrostane and hydroxy-5-pregnene-20-one, 3c - and 33-hydroxyallo 17-ketoetiocholane compounds which are useful in 45 pregnane-20-one, 3c, 120,21-trihydroxypregnane-20-one, therapy or as intermediates for the synthesis of thera 3oz,17a-dihydroxypregnane-20-one, 6cy- and 6p3-hydroxy peutically useful products from 20-oxygenated steroids. progesterone, pregnane-3,6,20-trione, 21-methylproges Other objects and uses of this invention will be apparent terone, 21-ethylprogesterone, 36,20-dihydroxy-20-methyl to those skilled in the art to which this invention pertains. allopregnane, 38,20-dihydroxy-20-methyl-5-pregnane, The method of the present invention is able to fur 50 3,3,20,21-trihydroxy-20-methyl-5-pregnene, 36-hydroxy nish a variety of useful products. For example, proges 21-ethyl-5-pregnene-20-one, 36,17a-dihydroxy-17 3-(1,2,3- terone subjected to the action of Gliocladium catenu trihydroxypropyl)-androstane, and the like. latum gives 4-androstene-3,17-dione possessing known In carrying out the process of the present invention the androgenic properties, and also known 6,3-hydroxy-4- selected 20-ketosteroid, suitably in a solvent, for example, androstene-3,17-dione IBalant and Ehrenstein, J. Org. 55 is subjected to a growth of a species of fungus of the Chem. 17, 1587 (1952)). The resulting 4-androstene genus Gliocladium. The genus Gliocladium belongs to 3,17-dione can be converted to testosterone in accord the family Aspergillaceae of the order Plectascineae of ance with stavely, U. S. Patent 2,288,854, issued Octo the class Ascomycetes. The classification and definition ber 11, 1941, and the latter by hydrogenation to dihydro of Gliocladium as herein employed is that of Raper, testosterone of therapeutic use (Symposium on Steroids in 60 K. B., and Thom C., "A Manual of the Penicillia,” Experimental and Clinical Practice, The Blakiston Com Williams and Wilkins Company, Baltimore, 1949. Among pany, New York, 1951, p. 375). The other fermenta species of the genus Gliocladium useful in the fermenta tion product, 6B-hydroxy-4-androstene-3,17-dione, can tion of steroids may be mentioned the Gliocladium rose be oxidized by chromic acid to 4-androstene-3,6,17- tri un series, Gliocladium roseum, Gliocladium penicil one which possesses estrogenic activity IButenandt, Ber. 65 loides, Gliocladium vermoesceni (i. e., ATCC 10522); 69, 1163 (1936). Similarly other physiologically and Gliocladium catenulatum series, Gliocladium catenula pharmacologically active 17-ketosteroids or intermediates tum, Gliocladium fimbriatum; Gliocladium deliquescens for the synthesis of such 17-ketosteroids may be ob series, Gliocladium deliquescens (i. e., ATCC 10097), 2,721,828 3. 4. Gliocladium nigro-virescens (C. B. S. v. Beijima Hol wated carbon or other suspendable solid matter, methyl land); Gliocladium luteolitin, Gliocladium flaviiil, ceilulose, carboxymethyl cellulose or aginates may be Gliocladium ciboilii and Gliociadiini yiride. Of these aided to facilitate fermentaticin, aeration and filtration. species, especially preferred for the practice of the preSeit The selected species of fungus is grown on a medium invention are Gliociadiitii1 catentitlatun, American iype suitabiy containing assimilabie non-steroidal carbon, Culture Collection -uinber i0,523, Glioclaidiitiin rosettii, illustratively carbohydrates, silich as Sugar or starches, American Type Cliittire Collection fumber 10,521, assianiiable nitrogen, illustratively soil be or insoluble Gliocladium deliquescens (Centraalibureau voor Schim proteins, peptones or amino acids; and mineral constitu melcultur, Baarn, Holland) and Gliocladiitii iiite is a cris, is y phosphates and magnesium Sulfate, and (Centraal bureau voor Schim meicultur Saarn, zed, desirable additions. The medium the present invention, is in or on a mediurn favorable to the development of the fingi. Solid media may be uti lized, but the preferred media are those which permit quantity growth under aerobic conditic;is. ist scid the fungai growth-supporting medium particulate media, Such as bi'an, Cereal grains, cereal grits, Seiected giS of the genus Gliocladium may be wood chips, shavings, sawdust, cornhusk accoranipiished in any suitable anner. Gliocladium grow rial, such as copra, chestnuts, or upine seeds inay be over a range froii) about twenty to about 38 degrees used. These can be extracted with alcohol, ether or centigrade, with a temperature between about 25 to about other organic solvents, to remove objectionable contami 20 35 degrees pieferred. nants and growth inhibitors prior to fermentation. The The developmental period of fungal growth required carriers may optionaily contain added growth factors and before the Steroid to be ferinented is exposed to the fungus nutrients and may be used in layers cr trays with or with does not appear to be critica. For example, the steroid out auxiliary aeration, in towers as in the vinegar process i.ay be added either before thermal or other steriliza or under conditions of agitation as for example by tum 25 tion of the medium, at the time of inoculating the me bling in a rotating drum. ; liquid inedia, illustratively diuri with the selected Giocladiurn species, or at Some brewer's wori, are well adapted to use under aerobic layer time, for exampie, 24 or 40 hours, later. The steroid or more especially aerobic submerged feinentation con to e ferniented may be added at any suitabie concen ditions. Suitably the finedia should contain Sources of tration although for practical reasons steroid substrate available carbon, nitrogen and lininerais although of 30 at a concentration of about or up to about 0.6 grain course there can be significant growth and development per liter or ever, 0.8 gram per iter of medium is satis under less than optimun conditions. factory and two granis per liter is operative although Available carbon may be from carbohydrates, starches, higher concentration, depending on the particular steroid, may be used with some inhibition of mycelial develop gelatinized starches, dextrin, Sugars, molasses as Gf cane, beet and Sorghun, glucose, fructose, annose, galactose, test. The addition of Steroid substrate to be fermented maltose, Sucrose, actose, pentOSes, aimino acids, peptories may be accomplished in any suitable manner especially or proteins. Carbon dioxide, glycero, alcohols, acetic so as to promote a large surface of contact of the steroid acids, sodium acetate, citric acid, sodium citrate, lower substrate with the fungus, such as by dispersing the fatty acids, higher fatty acids, or fats are illustrative of steroid Substrate, either alone, with a dispersing agent, other materials which provide assimilable carbon for the 40 r in solution in an organic solvent by mixing or homog energy requirements of the fungi. ?ixtures of various eizing a steroid substrate with a fungal medium to form carbon sources are sometimes advantageous. a Suspension or dispersion of steroid. Either Subrinerged Nitrogen in assimilable form may be provided by or surface culture procedures may be used with facility, soluble or insolubie vegetable or animal proteins, soy although submerged culture is preferred. Alternatively, bean meal, lactalbumin, casein, egg alburnin, peptones, steroid fermenting enzymes of a growth of the fungus polypeptides or amino acidis, urea, aiminonium saits, aim may be separated from the fungis or medium, admixed s monia trapped on base exchangeSY resins or on Zeclies, ann with the steroid or a solution or dispersion thereof, and monium chloride, sodiuin nitrate,3. potassius nitrate, or the mixture Sibjected to aerobic conditions to accomplish morpholine. Whey, distiliers solubies, corn steep liquer, fermentation of the steroid. or yeast extract have been useful. The temperature during the period of fermentation of As mineral constituents the media or meinstrum may 50 the steroid may be the same as that found suitable for contain, naturally present or added, available aiminim, fungal growth. it need be maintained only within Such calcium, chronium, Cobai, copper, gallium, iron, agne range as supports iife, active growth, or the enzyme ac sium, molybdenurn, potassiuim, Scandium, iranium and tivity of the fungus. vanadium. Sulfur may be provided by Sulfates, aiky While any form of aerobic incubation is satisfactory sulfonates, sulfoxylates, sulfamates, Sufinates, free sui 55 for the growth of the selected fungus and fermentation fur, hyposulfite, persulfate, thiosulfate, methionine, cys of the steroid substrate, the efficiency of steroid fermenta tine, cystein, thiamin or bictin. Phosphorus, preferably tion is related to aeratio. Therefore, aeration is usual pentavalent, suitably in a concentration at Cr about 0.001 Iy controi!ed, as by agitation and/or blowing air through to 0.07 molar and particizlarly at or about 0.015 to 0.02, the ferinentation medium. Aeration may be effected by may be present, Suitably as ortho-, reta-, or pyrophos 60 surface culture or under submerged ferrentation condi phates, salts or esters, phytin, phytic acid, phytates, gly tions. Aerobic conditions include not only the use of cerophosphate, Sodium nucleinate, and/or corn steep air to introduce oxygen, but also other sources or mix iiquor, casein or ovovitellin. Boron, iodine and Selenium tures containing oxygen in free or liberatable form. In in traces may be advantageous. Besirably boron, in the using air as the aerating medium, a desirable rate of aera form of boric acid or sodium borate may be present or 65 tion is about four to twenty inilinoises and particularly added especially after germination and early girowth of about six millimoies of oxygen per hour per liter as de the fungus. termined by the method of Cooper, Fernston and Miller, Other accessory growth factors, vitamins, auxins and ind. Eng. Chem. 36, 504 (1944). Aeration is suitably growth stimulants may be provided as needed or desired. modified by tising Superatnospheric or Slibatinospheric While seid of liquid media finay be utilized, a liquidi pressures, for exampie, thirty pounds per square inch or medium is preferred as it favors inycelia growth. ten pounds per square inch absolute. Oxygen uptake Suspending or mycelial carriers such as fitter earths, ray be facilitated by the presence of various agents such filter aids, finely divided cellulose, wood cihips, bentonite, as ascorbic acid, glutamic acid, citric acid, lactic acid, calcium carbonate, magnesium carbonate, charcoal, acti 75 tyresine, or tryptophane. 2,721,828 5 6 The time required for the fermentation of steroid graphed over 150 grams of alumina using 300 milliliter varies somewhat with the procedure. When the steroid portions of solvent as indicated in Table I. substrate is present at the time of inoculation of the medium, periods of from eight to 72 hours may be used. TABLE I However, when the steroid is added to the fungus, after substantial aerobic growth of the fungal organism, for Fraction Solvent Eluatemilligrams solids, example, after 16 to 24 hours at optimum temperature, the conversion of steroid substrate begins immediately 257 and high yields are obtained in from one to 72 hours, 24 48 144 hours being generally satisfactory. 10 67 benzene-ether O After completion of the steroid fermentation, the re - - - - -do------53 sulting fermented steroid is recovered from the fermenta benzene-ether 1:1 175 tion reaction mixture. An especially advantageous man 993 ner of recovering the fermented steroid involves extract ing the fermentation reaction mixture, including the fer mentation liquor and mycelia with a water-immiscible organic solvent for steroids, for example, methylene chlo ride, chloroform, carbon tetrachloride, ethylene chloride, trichloroethylene, ether, amyl acetate, benzene, and the ---do-- like. The fermentation liquor and mycelia may be sep chloroform arated and then separately extracted with suitable solvents. The mycelia may be extracted with either water miscible or water-immiscible solvents, acetone being effective. The fermentation liquor, freed of mycelia, may be extracted with water-immiscible solvents. The extracts can be combined, either before or after washing with an alkaline solution, illustratively sodium bicarbonate; suitably dried, Fraction 7 was evaporated and the thus obtained crys as for example over anhydrous sodium sulfate; and the tals were twice recrystallized from one milliliter of meth purified fermented steroid obtained by recrystallization anol to yield 102 milligrams of 4-androstene-3,17-dione, from organic solvents or by chromatography to isolate 30 melting point 169 to 171 degrees centigrade. the 17-ketosteroids from the other fermentation products. Analysis: Calculated for C19H26O2: C, 79.68; H, 9.15. The following examples are illustrative of the process Found: C, 79.53; H, 8.84. of the present invention and are not to be construed as . Fractions 8 and 9 (254.9 milligrams) were rechromato limiting. graphed over 12 grams of alumina using 25-milliliter por tions of solvent as indicated in Table II. Example I.-Fermentation of progesterone and isolation 35 of 4-androstene-3,17-dione and 66-hydroxy-4-andro TABLE II stene-3,17-dione Fraction Solvent risis, A medium was prepared of twenty grams of Edamine enzymatic digest of lactalbumin, three grams of corn ------benzene-ether i:1. 0. steep liquor, and fifty grams of technical dextrose diluted 50.8 24.7 to one liter with tap water and adjusted to a pH of 5.85. 7.6 Twelve liters of this sterilized medium was inoculated 3.2 0. with Gliociadium cantenulatum, American Type Culture 78------ether-chloroform mixtures; 0.5 Collection No. 10523, and incubated for 48 hours at a chloroform; acetone. temperature of 26 degrees centigrade using a rate of aera 19------methanol------3.4 tion and stirring such that the oxygen uptake was 6.3 to 7 millimoles per hour per liter of Na2SO3, according The fractions 2, 3, and 4 were evaporated to yield to the method of Cooper, Fernstrom and Miller, Ind. 193.1 milligrams of crystals which were shown by infra Eng. Chem. 36, 504 (1944). To this medium contain 50 red spectrum and papergram studies to contain 66-hy ing a 48 hour growth of Gliocladium catenulatum was droxy-4-androstene-3,17-dione. To isolate the 66-hy added three grams of progesterone in fifty milliliters of droxy-4-androstene-3,17-dione the combined fractions 2 acetone to provide a suspension of the steroid in the cul to 4 were dissolved in five milliliters of acetone, filtered ture. After an additional 24 hour period of incubation and evaporated to two milliliters. Upon the addition of under the same conditions of temperature and aeration, 55 3 milliliters of Skellysolve B (a mixture of hexanes), 38 the beer and mycelium were separated. The mycelium milligrams of crystals A of melting point 184-290 de was filtered, washed twice, each time with a volume of grees centigrade were recovered. The second crop of acetone approximately equal to the volume of the my crystals B 50 milligrams and a third crop of crystals C celium, and extracted twice, each time with a volume were obtained from the mother liquors by refrigeration of methylene chloride approximately equal to the volume 60 over night. Crystals B and C were combined, dissolved of the mycelium. The acetone and methylene chloride in four milliliters of ethyl acetate, filtered, and evap extracts including solvent were added to the beer filtrate. orated to one milliliter. Upon the addition of 2.5 milli The mixed extract and beer filtrate were extracted suc liters Skellysolve B and refrigeration over night, 42.8 cessively with two one-half volume portions of methylene milligrams of crystals D of melting point 176 to 185 de chloride and then with two one-fourth volume portions 65 grees centigrade were recovered by filtration. This prod uct D was recrystallized from ethyl acetate and Skelly of methylene chloride. The combined methylene chlo solve B as above to yield 30.4 milligrams of crystals E, ride extracts were washed with two one-tenth by volume of melting point 186 to 188 degrees centigrade. Crystals portions of a two percent aqueous solution of sodium A, 38 milligrams and crystals E, 30.4 milligrams, were bicarbonate and then with two one-tenth by volume por 70 dissolved in four milliliters of ethyl acetate, combined tions of water. After drying the methylene chloride ex and evaporated to one milliliter. Upon the addition of tracts with about three to five grams of anhydrous sodium five drops of Skellysolve B, crystals were formed. After sulfate per liter of solvent and filtering, the solvent was 45 minutes at room temperature, followed by filtration, distilled from the filtrate. The residue thus obtained 43.6 milligrams of 66-hydroxy-4-androstene-3,17-dione weighed eight grams and was redissolved and chromato 75 were obtained. - 2,721,828 7 - 8 Analysis: Calculated for CH2O: C, 75.46; H, 8.67. Gliocladium catenulatum, ATCC 10523, and pregnane Found: C, 75.39;H, 8.47. 3,6,20-trione produced etiocholane-3,6,17-trione. Example 2.-Ferinentation of ill-desoxycorticosterone Fxample II.-Fermentation of 66-hydroxyprogesterone In the saine manner as given in Example 1, using 5 in the sailine manner as given in Example 1, using Gliocladium catenuiaitina, American Type Culture Col (Gliocladium catenulatum, ATCC 10523, and 6,3-hydroxy lection No. 10523, and 1-desoxycorticosterone as a start progesterone produced 6,3-hydroxy-4-androstene-3,17-di ing steroid produced 4-androstene-3,17-dione and 66-hy Oc. droxy-4-androstene-3,17-dione. Example 2.-Fermentation of pregnane-3,20-dione Example 3.-Fermentation of 11-desoxycorticosierone . 0 in the same manner as given in Example 1, using (ICeiate Gliocladium catenulatun, ATCC 10523, and pregnane in the same manner as given in Example 1, using Glio 3.20-dione produced etiocholane-3,17-dione. cladium roseum, American Type Cuiture Collection No. Example 13.-Fermentation of allopregnane-3,11,20 10521, and 11-desoxycorticosteroine acetate produced 15 tione 4 - androstene - 3,17-dione and 66 - hydroxy - 4 - an -- drostene - 3, 17 - dione. Similarly, cither esters of 11 . the same nanner as given in Example 1, using desoxycorticosterone illustratively the propioinate, bu Cliocladium catenulaturin, ATCC 10523, and allopreg tyrate, isobutyrate, Valerate, hexanoate, benzoate, and Eane-3,11,20-trione produced androstane-3,11,17, trione. phenylacetate yield 4-androstene-3,17-dione and 6,3-hy 20 Example 14.-Fermentation of Reichstein's compound S droxy-4-androstene-3,17-dione upon treatment with a (17c,21-dihydroxy-4-pregnene-3,20-dione) fungus of the genus Giocladium. in the same manner as given in Example 1, using Example 4.-Fermentation of ll-desoxycorticosterone 6iocladium catenulatum, ATCC 10523, and Reichstein's aCetate 25 compound S (17a,21-dihydroxy-4-pregnene-3,20-dione) In the same manner as given in Example 1, using produced 4-androstene-3,17-dione and 6,3-hydroxy-4- Gliocladium deliquescens, Centraalbureau voor Schim androstene-3,20-dione. meicultur, Baarn, Holland, and 11-desoxycorticosterone Example 15. Fermentation of cortisone acetate produced 4-androstene-3,17-dione and 6,3-hy droxy-4-androstene-3,17-dione. 30 in the same manner as given in Example 1, using Similarly, other esters of 11-desoxycorticosterone, i Giocladium catentilatum, ATCC 10523, and cortisone lustratively the propionate, butyrate, isobutyrate, Valerate, produced 4-androstene-3,11,17-trione (and renosterone). hexanoate, benzoate, and phenylacetate yield 4-andro Example 16-Fermentation of cortisone acetate stene-3,17-dione and 63-hydroxy-4-androstene-3,17-dione In the Saine manner as given in Example 1, using upon treatment with Gliocladim deliquescens. 35 Gliocladiuin catentilattirin, ATCC 10523, and cortisone Example 5-Ferineintation of 11-desoxycorticosterone acetate produced 4-androstene-3,11,17-trione. (ICetate Example 17-Ferinentation of 11a, 17a,21-trihydroxy-4- In the same manner as given in Example 1, using pregnene-3,20-dione Gliocladiitin luteolitia, Centraalbureau voor Schimmel 40 in the same manner as given in Example 1, using cultur, Baarn, Holland and 11-desoxycorticosterone pro Giocladium cateinulatun, ATCC 10523 and 11a, 17a,21 pionate produced 4-androstene-3,17-dione and 6,3-hy trihydroxy-4-pregnene-3,20-dione produced 11 a-hydroxy droxy-4-androstene-3,17-dione. Similarly, other esters of 11-desoxycorticosterone, illustratively the propionate, 4-androstene-3,17-dione. butyrate, isobutyrate, valerate, hexanoate, benzoate, and 45 Evailple 18-Fermentation of 3,3-hydroxy-21-ethyl-5- phenylacetate yield 4-androstene-3,17-dione and 6,3-hy pregnene-20-one droxy-4-androstene-3,17-dione upon treatment with Glio in the same manner as given in Example 1, using cladium luteolun. Gliocladium catentilatun, ATCC 10523, and 3.3-hydroxy Example 6.-Fermentation of 11-ketoprogesterone 21-ethyl-5-pregnene-20-one produced 3 (3-hydroxy-5-andro 50 Stene-17-one. In the same manner as given in Example 1, using it is to be understood that the invention is not to be Gliocladium catenulattin, ATCC 0523, and 11-keto limited to the exact details of the procedure shown and progesterone produced 4-androstene-3,11,17-trione (ad described as obvious inodifications and equivalents will renosterone). be apparent to one skilled in the art, and the invention is Example 7-Fermentation of 21-methylprogesterone therefore to be limited only by the scope of the appended clains. In the same manner as given in Example 1 using We claim: Gliocladium catenulaiun, ATCC 10523, and 21-methyi 1. A process for the production of a 17-ketosteroid, progesterone produced 4-androstene-3,17-dione. which comprises: growing a fungus of the genus Glio 60 cladium under aerobic conditions, in the presence of a Example 8.-Fermentation of Reichstein's Compound 'O' nutrient medium and a 20-oxygenated steroid selected ... (3,3,17a,20-allopregnanetriol) from the group consisting of 20-ketosteroids and 20-hy in the same manner as given in Example 1, using droxysteroids. Giocladiuin roseum, ATCC 10521, and 36,17a,20-allo 2. A process for the production of a 17-ketosteroid, pregnanetriol, produced 3-hydroxyandrostane-17-one. 65 which comprises: growing a fungus of the genus Glio cladiuin irider aerobic conditions, in the presence of a Example 9.-Fertinentation of 36,20-dihydroxy-20-methyl nutrient medi: in coil airing assimilable non-steroidal car 5-piegnene bon and a 20-oxygenated Steroid selected from the group In the same manner as given in Example 1, using consisting of 20-keicSteroids and 20-hydroxysteroids and Gliocladian catenulatun, ACC 10523, and 36,20-di 70 recovering the resulting 17-ketosteroid. hydroxy-20-methyl-5-pregnene produced 3,3-hydroxy-5- 3. A piccess for the production of a 17-ketosteroid, which comprises: growing a fungus of the genus Glio androstene-17-one. cladiuin under aerobic conditions with agitation in the Exampie 10.-Ferinentation of pregnane-3,6,20-trione presence of a nutrient medium containing assimilabie non In the same manner as given in Example 1, using Steroidal carbon, nitrogen and phosphorus and a A4-20 2,721,828 9 10 oxygenated steroid selected from the group consisting of 13. A process for the production of a 17-ketoandrostane 20-ketosteroids and 20-hydroxysteroids and recovering the which comprises: growing a fungus of the genus Glio resulting 17-ketosteroid. cladium under aerobic conditions in the presence of a 4. A process for the production of a 3,17-diketosteroid, nutrient medium containing assimilable non-steroidal car which comprises: growing a fungus of the genus Glio bon and 20-oxygenated allo steroid selected from the cladium under aerobic conditions and with agitation in group consisting of 20-ketosteroids and 20-hydroxysteroids the presence of a nutrient medium containing assimilable and recovering a thus-produced 17-ketoandrostane. carbohydrate, nitrogen and phosphorus and a 3,20-diketo 14. A process for the production of a 17-ketoandrostane Steroid and separating the thus-produced 3,17-diketo which comprises: growing Gliocladium catenulatum un steroid. O der aerobic, Submerged conditions and with agitation in 5. A process for the production of a 17-ketosteroid the presence of a nutrient medium containing assimilable which comprises: growing a fungus of the genus Glio carbohydrate, nitrogen and phosphorus and a 20-ketoallo cladium under aerobic, submerged conditions in the pres pregnane and recovering a thus-produced 17-ketoandro ence of a nutrient medium containing assimilable carbo Stane. hydrate, nitrogen and phosphorus and a 20-ketosteroid 5 15. A process for the production of androstane-3,11,17 and recovering the thus-produced 17-ketosteroid. trione which comprises: growing Gliocladium catenulatun - 6. A process for the production of a 17-ketosteroid under aerobic conditions and with agitation in the pres which comprises: growing a fungus of the genus Glio ence of a nutrient medium containing assimilable carbo cladium under aerobic conditions, with agitation, in the hydrate, nitrogen and phosphorus and allopregnane presence of a nutrient medium containing assimilable non 20 3,11,17-trione, and recovering a thus-produced 17-keto Steroidal carbon, nitrogen and phosphorus and a 20-keto androstane. Steroid and isolating the resulting 17-ketosteroid. 16. A process for the production of a 17-ketoetiocho 7. A process for the production of a 17-ketosteroid lane which comprises: growing a fungus of the genus which comprises: growing a fungus of the genus Glio Gliocladium under aerobic conditions in the presence of cladium under aerobic, submerged conditions in the pres 25 a nutrient medium containing assimilable non-steroidal ence of a nutrient medium containing assimilable non carbon and a 20-oxygenated steroid selected from the Steroidal carbon, nitrogen and phosphorus and a 20-keto group consisting of 20-ketosteroids and 20-hydroxysteroids Steroid and extracting the resulting 17-ketosteroid. of the normal series and recovering a thus-produced 17 8. A process for the production of a 17-ketosteroid ketoetiocholane. which comprises: growing a fungus of the genus Gio 30 17. A process for the production of a 17-ketoetiocho cladium under aerobic, Submerged conditions with agita lane which comprises: growing Gliocladium catenulatum tion, in the presence of a nutrient medium containing as under aerobic conditions and with agitation in the pres Similable non-steroidal carbon, nitrogen and phosphorus ence of a nutrient medium containing assimilable carbo and a 20-ketosteroid and recovering the resulting 17-keto hydrate, nitrogen and phosphorus and a 20-ketopregnane steroid. 35 and recovering a thus-produced 17-ketoetiocholane. 9. A process for the production of a 17-keto-4-andro 18. A process for the production of an etiocholane-3,17 Stene which comprises: growing a fungus of the genus dione which comprises: growing a fungus of the species Gliocladium under aerobic conditions with agitation in Gliocladium catenulatum under aerobic conditions and the presence of a nutrient medium containing assimilable with agitation in the presence of a nutrient medium con non-Steroidal carbon, nitrogen and phosphorus and a A4 40 taining assimilable carbohydrate, nitrogen and a preg 20-OXygenated Steroid selected from the group consisting nane-3,20-dione and recovering a thus-produced etiocho of 20-ketosteroids and 20-hydroxysteroids and recovering lane-3,17-dione. a resulting 17-keto-A-androstene. 19. A process for the production of an etiocholane 10. A process for the production of a 4-androstene-3,17 3,6,17-trione which comprises: growing a fungus of the dione which comprises: growing a fungus of the genus 4.5 Species Gliocladium catenulatum under aerobic conditions Gliocladium under aerobic conditions with agitation in in the presence of a nutrient medium containing assimil the presence of a nutrient medium containing assimilable able non-steroidal carbon and a pregnane-3,6,20-trione non-Steroidal carbon, nitrogen and phosphorus, and a 3 and recovering a thus-produced etiocholane-3,6,17-trione. keto-A-20-oxygenated steroid selected from the group 20. A process for the production of etiocholane-3,6,17 consisting of 20-ketosteroids and 20-hydroxysteroids and : trione which comprises: growing a fungus of the species recovering a thus-produced 4-androstene-3,17-dione. Gliocladium catenulatum under aerobic conditions with 11. A process for the production of a 4-androstene-3,17 agitation in the presence of a nutrient medium containing dione which comprises: growing a fungus of the species assimilable carbohydrate, nitrogen and phosphorus and Gliocladium catenulatum under aerobic, submerged con pregnane-3,6,20-trione and recovering the thus-produced ditions with agitation in the presence of a fermentation etiocholane-3,6,17-trione. medium containing assimilable carbohydrate, nitrogen and phosphorus and a 3,20-diketo-A-steroid and recover References Cited in the file of this patent ing a thus-produced 4-androstene-3,17-dione. UNITED STATES PATENTS 12. The process of claim 11 wherein the 3,20-diketo A-steroid is selected from the group consisting of pro 60 2,649,400 Murray et al. ------Aug. 18, 1953 gesterone, 11-desoxycorticosterone and 21-carboxylic acid esters of 11-desoxycorticosterone.