US2649400A

US2649400A - Steroids

Info

Publication number: US2649400A
Application number: US306929A
Authority: US
Inventors: Herbert C Murray; Durey H Peterson
Original assignee: Upjohn Co
Current assignee: Pharmacia and Upjohn Co
Priority date: 1952-08-28
Filing date: 1952-08-28
Publication date: 1953-08-18
Anticipated expiration: 1970-08-18

Description

Patented Aug. 18, 1953 UNITED STATES PATENT OFFICE STEROIDS corporation of Michigan No Drawing. Application August 28, 1952,

Serial No. 306,929

30 Claims. 1

The present invention relates to a novel method for the introduction of oxygen into a steroid molecule. This application is a continuationin-part of our prior-filed applications Serial No. 297,242, filed July 5, 1952, Serial No. 7 ,9 filed. February 23, 1952 and issued as Patent 2,662,769, and Serial No. 180,496, filedAugust 19, 1950, and now abandoned, and relates more especially to an aerobic fermentation process wherein fermentation and oxygenation of Steroids may be accomplished by means of fungi 0f the genus Penicillium.

It is already known to introduce oxygen nto a steroid molecule, particularly into the eleven position, for the production of intermediates useful in the preparation of therapeutic compounds, or for the production of the drugs themselves, which intermediates or drugs have an 0X3- genated structure, particularly an eleven oxygenated structure, among which may, for example, be mentioned corticosterone, ll-dehydrocorticosterone (compound E, cortisone), and l7-hydroxycorticosterone (compound F). Such result has been accomplished, heretofore, only through highly involved organic synthesis necessitating a considerable number of steps. More recently, the introduction of oxygen into a steroid molecule by means of Mucorales fungi has been described by Murray and Peterson, United States Patent 2,602,769, issued July 8, 1952. ditionally been found that fungi 0f the genus Penicillium, e. g., Bem'cillium a'anthinellum, are useful in the oxygenation of steroids, including eleven desoxy steroids, although not necessarily producing the same result as the fungi of the Mucorales order,

It is an object of the present invention to provide a novel method for the introduction of oxygen into a steroid molecule. Another object of the i vention is the provision of such method whereby an eleven desoxy steroid (the term eleven desoxy steroid is employed throughout to indicate a steroid which contains no oxygen in the eleven position) is converted to an eleven oxygenated steroid by the action of a species of fungus of the genus Penicilliuni. Another object of the invention is the provision of a p ocess for the introduction of oxygen into at least the eleven position of an eleven desoxy steroid through the action of fungus of the genus Peni" cilliuni. Another object of the invention is t0 provide a process of oxygenating 3-keto steroids by means of Penicillia. Still another object of the invention is the provision of a process of modifying steroid structure by means of Peni It has adyields of eleven oxygenated cillia. Other and more particularly objects 01' the invention will become apparent hereinafter.

It has now been found that eleven desoxy steroids, which contain the cyclopentanopolyhydrophenanthrene nucleus, especially the 10,13 dimethylcyclopentanopolyhydrophenanthrones, can be readily converted in high yields to. corresponding oxygenated steroids by subjecting the steroid compound to the action of a species of fungus of the genus Penicillium. By the method of the present invention, an efiicient, economical, and commercially satisfactory method of introducing oxygen into the eleven position of an eleven desoxy steroid molecule is provided. Accordingly, a novel and simple approach to the production of eleven oxygenated steroid drugs is afforded, which is, as previously stated, of great importance to the chemical, pharmaceutical and medical professions, and of especial value in the treatment of physiological abnormalities known to be beneficially affected only by such eleven oxygenated drugs.

The method of the present invention, in its broader aspects, consists in fermenting a steroid or an eleven desoxy steroid by means of a species of fungus of the genus Penicillium. Another way of expressing one of the results of the process of the present invention is to say that the steroid is oxygenated since an oxygen atom is introduced thereinto. Other positions as well as the eleven position of the steroid molecule may undergo transformation due to the action of the fungus, but such transformations are not to be regarded as undesirable, since the introduction of oxygen into other portions of the steroid molecule may result in valuable therapeutic products or intermediates, for example those containing a hydroxy group at the 17 position. In case such additional groups are not considered desirable, methods are available for the removal of such groups with facility. An important advantage of the present invention is the oxygenation of eleven desoxy steroids in the eleven position. I-Iydroxy groups which are themselves capable of oxidation to keto groups, when present in the molecule of a steroid to be oxygenated, may, if considered necessary, as where exceedingly high hydroxysteroid product are sought to be produced, be protected from attack of various types, including attack by the oxidizing fungi, by conversion, as for example by esterification, etherification, halogenation, or the like, to a group which is reconvertible to a hydroxy group. However, such procedure is not a prerequisite to the introduct of oxygen, especially eleven oxygen, into a hydroxysteroid by the method of the present invention.

The steroids operative in the method of the present invention are not limited as to type or number of substituents, and for operativeness in the process need only contain a nuclear unoxygenated or oxygenatable position, such as, for example, an unoxygenated eleven position; illustratively, a methylene group, as in an eleven desoxy steroid. Such compounds contain the nucleus:

which may in addition contain substituents or combinations of substituents about the nucleus, as in the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, l3, 14, 15, 16, and 17 positions, especially 10,13-dimethyl groups, 3,7, or 12 keto, hydroxy, or acyloxy groups; l7-side chains of which the progesterone and corticosterone (Ketol) side chains deserve special mention; a 17 keto group; a 17 hydroxy group, and the like; as well as double bonds in the 4, 5, 6, 7, 8, 9(11), 11(12), 16(1'7) and other positions, or combinations of positions, about the nucleus; or double bonds saturated by addition thereto of halogen or hydrogen halide; adducts of dienophiles such as maleic acid, maleic anhydride, or maleic acid esters with steroids having a conjugated double bond system, as at 5,7; and other substituents and combinations of substituents, double bonds and so forth too numerous for special mention, a great many of which are known in the steroid art. The presence or absence of unsaturation at the 9(11) or 11(12) positions of the nucleus is not a critical factor in the method of the present invention,

for while it is preferred to apply the process to a steroid having an eleven methylene group, i. e, a steroid having two hydrogen atoms at carbon atom eleven or no unsaturation in the 9(11) or 11(12) positions, for reasons of economy and to obviate unnecessary transformations of saturated to unsaturated compounds, the fermentation may be applied with equal facility to either the saturated or unsaturated compounds.

Representative steroids which may be fermented by the method of the invention include, for example, progesterone, 9(11) or 11(12) -dehydroprogesterone, 7,9(11) bisdehydroprogesterone, l7-hydroxyprogesterone, 17a-progesterone, testosterone, pregnenolones, 3-hydroxy-5- pregnene-ZO-one, pregnenolone, 3fi-hydroxy-5J6- pregnadiene-ZO-one, acyloxy-pregnenolones such as pregnenolone acetate, 3-hydroxy-5,6-oxidopregnane-ZO-one (aor fi-oxido), 3-hydroxy-5- chloropr gnane-2o-one, 5,6,-oxidopregnane-3,20- dione (aor B-oxido), 4-bromo and l-chloropregnane3,20-dione, 5-ch1oropregnane-3,20-di one, 8-ketopregnane-20-ol, 3-keto-allopregnane- 20-01, Se-hydroxy-ldl'Z-oxido 21 acetoxy-5- pregnene-ZG-one, 3fi-hydroxy 16,17 oxide-5- pregnene-20-one, 3B-hydroxy-5,6,2l-tribromo- 16,17-oxidopregnane-20-one, 3,3-hydroxy 16 bromo-17-hydroxy-5-pregnene-20-one, 3B hydroXy-16-chloro-17 -hydroxy-5pregnene-20- one, Bp-hydroxy-MS), 16(17) -dioxidopregnane-2Q- one, hydroxy-5(6),16 (17)-dioxido-21-brom0- mented by the method of this invention.

pregnane-ZO-one, 3shydroxy-5(6),16(17) dioxide-21-acetoxypregnane-20-one, 3(3-hydroxy- 5(6) ,16(l7) -dioxido 21 hydroxypregnane 20- one, ll-desoxycorticosterone, delta-90.1) or 11(12) -desoxycorticosterone, ll-desoxy-l'l hydroxycorticosterone and acyloxy derivatives, such as the acetoxy derivative, thereof, 21hydroxypregnenolone and Zl-acyl, e. g. acetyl, esters thereof, 17,21-dihydroxypregnenolone and 17,21-diacyloxy derivatives thereof, e. g. the diacetoxy derivative, androstenedione, androstan- 17-01, 9(11) or 11(12) dehydroandrostenedione, 3-hydroXy-9(1l) or 11(12) -pregnen20ones, 3,21-clohydroXy-9(11) or 11(12)-pregnen-2D- ones, 3,l7,2l-trihydroxy-9(ll) or 1l(12)-pregnen-20-ones, 4-androsten-3-ol-17-one and 3-acyl, e. g. acetyl, esters thereof, 5-androsten-3-ol-l7- one and 3-acyl, e. g. acetyl, esters thereof; ergosterol, stigmasterol, stigmastanol, and 3-acyl, e. g. acetyl, esters of the foregoing; ergostenone, stigmastenone, stigmastanone, cholestenone, cholic acid, desoxycholic acid, lithocholic acid, cholanic acid, norcholanic acid, bisnorcholanic acid, cholenic acid, norcholenic acid, blSIlOChO- lenic acid, and 3 hydroxy-, 3 keto-, 3,7 dihydroxy-, 3,7-diketo-, 3,7,l2-trihydroxy, 3,7,12- triketo-, 9(11) or 11(l2)-unsaturated, ester, thiolester, and further derivatives of the foregoing acids, and the like. Suitably a steroid having up to and including 22 carbon atoms in the carbon to carbon skeleton or a steroid having a two carbon atom side chain at the 17 position and an eleven methylene group may be used. The 10-ncr-methyl, the l3-nor-methyl, and the 10,13-bisnor-methyl forms of each of the above steroids, in which either one or both of the 18 and 19 position angular methyl groups are replaced by hydrogen, are included within the purview of those steroids which may be fer In the event that the ll-position is already oxygenated or substituted, the dominant product may be oxygenated additionally in another position. The lfi-dehydro form of each of the above steroids is likewise included. Within the purview of this invention is the fermentation of D-homosteroids, otherwise known as perhydrochrysenes, for example, D-homo-el-androstene-a,l7a-dione, D-homo-testosterone, D-homo-l7aa-methyltestosterone, D-homo-17aB-methy1testosterone, 17amethyl-17a-hydroxy-D-homo 4 androstene- 3,17-dione, and their 4,5-dihydro, ring A saturated analogs, D-homo-androstenones (3aor 35-hydroxyandrostane-17a-one), and. D-homoepidehydroandrosterone (3B-hydroxy-D-homo- 4-androstene-16a-one). All of these are amenable to fermentation with Penicillium fun i in accordance with the presented examples.

In the process of the present invention, the operational conditions and reaction procedure and details may be those of parent application Serial No. 180,496, filed August 19, l 50, utilizing the action of a species of fungus of th genus Penicillium. The genus Penicillium belongs to the family Aspergillaceae of the order Aspergillales (Plectascales) The classification and definition of Penicillium as herein employed is that of Raper, K. B., and Thom, C., A Manual of the Penicillia, Williams and Wilkins Company, Baltimore, 1949. Among species of the genus Penicillium useful in the fermentation of steroids may be mentioned those of the group or subgenus: Mono-verticillata, Asymmetrica-divaricata, Asymmetrica-velutin Asymmetrica-lanata, Asymmetrica funiculosa, Asymmetrica fasciculata, Biverticullata-symmetrica, and Polyverticallata, for example: Penicillium adametzi, brevi-compactum, camemberti, canescens, capsulatum, charlesti, citrinum, c'laozforme, commune, cyclopium, decumbens, digitatum, duclaumi, erpansum, frequentans, funiculosum, fuscum, gladioli, granulatum, herquei, implicatum, italz'cum, y'anthinellum, Zavendulum, Zevitum, lilacinum, Zuteum, nigricans, notatum, novae-zee-Zandiae, ochraceum, oxalicum, pallzdum, purpurogenum, purpurogenum var. rubrisclerotium, raistrz'ckii, roqueforti, roseo-purpureum, ruglosum, terrestre, thomiz', urticae, and 'ui-rdicatum.

Culture of the fungi, for the purpose and practice of the present invention, is in or on a medium favorable to the develpment of the fungi. Solid media may be utilized, but the preferred media are those which permit quantity growth under aerobic conditions. Moist solid particulate media such as bran, cereal grains, cereal grits, wood chips, shavings, sawdust, cornhusks, fibrous material, such as copra, chestnuts, or lupine seeds may be used. These can be extracted with alcohol, ether or other organic solvents, to remove objectionable contaminants and growth inhibitors prior to fermentation. The cariers may optionally contain added growth factors and nutrients and may be used in layers or trays with or without auxiliary aeration, in towers as in the vinegar process or under conditions of agitation as for example by tumbling in a rotating drum. Liquid media, illustratively brewers wort, are well adapted to use under aerobic layer or more especially aerobic submerged fermentation conditions.

Suitably the media should contain sources of available carbon, nitrogen and minerals although of course there can be significant growth and development under less than optimum conditions.

Available carbon may be from carbohydrates, starches, gelatinized starches, dextrin, sugars, molasses as of cane, beet and sorghum, glucose, fructose, mannose, galactose, maltose, sucrose, lactose, pentoses, amino acids, peptones or proteins. Carbon dioxide, glycerol, alcohols, acetic acid, sodium acetate, citric acid, sodium citrate, lower fatty acids, higher fatty acids, or fats are illustrative of other materials which provide assimilable carbon for the energy requirements of the fungi. Mixtures of various carbon sources are sometimes advantageous.

Nitrogen in assimilable form may be provided by soluble or insoluble vegetable or animal proteins, soybean meal, lactalbumin, casein, egg albumin, peptones, polypeptides or amino acids, urea, ammonium salts, ammonia trapped on base exchange resins or on zeolites, ammonium chloride, sodium nitrate, potassium nitrate, morpholine. Whey, distillers solubles, corn steep liquor, or yeast extract have been useful.

As mineral constituents the media or menstruum may contain, naturally present or added, available aluminum, calcium, chromium, cobalt, copper, gallium, iron, magnesium, molybdenum, potassium, scandium, uranium, vanadium, and boron. Sulfur may be provided by sulfates, alkyl sulfonates, sulfoxylates, sulfamates, sulfinates, free sulfur, hyposulfite, persulfate, thiosulfate, methionine, cystine, cystein, thiamin or biotin. Phosphorus, preferably pentavalent, suitably in a concentration at or about 0.001 to 0.07 molar and particularly at or about 0.015 to 0.02, may be present as ortho-, meta-, or pyrophosphates, salts or esters, phytin, phytic acid, phytates, glycerophosphate, sodium nucleinate, casein or ovovitellin. Boron, iodine and selenium in traces may be advantageous. Desirably boron, in the form of boric acid or sodium borate, borax, may be present or added especially after germination and early growth of the fungus.

Other accessory growth factors, vitamins, auxins and growth stimulants may be provided as needed or desired.

While solid or liquid media may be utilized, liquid media is preferred as it favors mycelial growth.

Suspending or mycelial carriers such as filter earths, filter aids, finely divided cellulose, wood chips, bentonite, calcium carbonate, magnesium carbonate, charcoal, activated carbon or other s'uspendable solid matter, methyl cellulose, carboxymethyl cellulose or alginates may be added to facilitate fermentation, aeration and filtration.

The selected species of fungus is grown either in light or darkness on a medium containing available carbon, illustratively carbohydrates such as sugars or starches; assimilable nitrogen, illustratively'soluble or insoluble proteins, peptones or amino acids; and mineral constituents, illustratively phosphates and magnesium sulfate; and other art recognized, desirable or adventitious, additions. The medium may desirably have a pH before inoculation of between about 2.8 and 8.8 although a higher or lower pH may be used. A pH of about 4 to,6 is preferred for the growth of Penicillium. Low pH values inhibit bacterial contamination and facilitate sterilization. For example, at a pH of 2 to 3, effective sterilization of media may be accomplished by heating the media for thirty minutes at degrees centigrade, whereas, at a pH of 4 to 4.5, thermal sterilization may require superatmospheric pressure. Alternatively or concomitantly, bacterial contamination may be retarded by the presence of antiseptic or antibioticagents such as benzoates, sulfites, pencillin or circulin.

Inoculation of the fungal growth-supporting medium with the selected fungus of the genus Penicillium may be accomplished in any suitable manner. Penicillia grow over a wide range of temperatures from about eight degrees centigrade to about 35 degrees centigrade and preferably at or about room temperature or between about fifteen degrees centigrade and 30 degrees centigrade,

The developmental period of fungal growth required before the steroid to be fermented is exposed to the fungus does not appear to be critical. For example, the steroid may be added either before thermal or other sterilization of the medium, at the time of inoculating the medium with the selected Pencillium species, or at some time, as 24 to 48 hours, later. The steroid to be fermented may be added at any suitable concentration although for practical reasons steroid substrate at a concentration of about or up to about 0.6 gram per liter or even 0.8 gram per liter of medium is satisfactory and two grams per liter is operative although higher concentrations, depending upon the particular steroid, may be used with some inhibition of mycelial development. Either a purified steroid, a crude material containing steroid, or a steroid material comprised of or consisting predominantly or essentially of steroid, for example, a mixture of steroid and fat or solvent, may be used as substrate. The addition of steroid substrate to be fermented may be accomplished in any suitable manner especially so as to promote a large surface of contact of the steroid substrate with theoxygenating activity of the fungus, such as by dispersing the steroid substrate, either alone, with a dispersing agent, or in solutionin a water-miscible organic solvent, by mixing or homogenizing the steroid substrate with the fungal medium to form a suspension or dispersion of steroid. Either submerged or surface culture procedures may be used with facility, although submergedculture is preferred. Alternatively, steroid fermenting enzymes of a growth of the fungus may be separated from the fungus or medium, admixed with the steroid or a solution or dispersion thereof, and the mixture subjected to aerobic conditions to accomplish fermentation of the steroid.

The temperature during the period of fermentation of the steroid may be the same as that found suitable for fungal growth. It need be maintained only within such range as supports life,;active growth, or the enzyme activity of the fungus.

While any form of aerobic incubation is satisfactory for the growth of the selected fungus or fermentation of the steroid substrate, the efficiency of steroid fermentation is related to aeration. Therefore, aeration is usually controlled, as by agitation and/or blowing air through the fermentation medium. Aeration may be effected by surface culture or under submerged fermentation conditions. Aerobic conditions include not only the use of air to introduce oxygen, but also other sources or mixtures containing oxygen in free or liberatable form. In using air as the aerating medium, a desirable rate of aeration is about four to twenty millimoles of oxygen per hour per liter as determined by the method of Cooper, Fernstrom and Miller, Ind. Eng. Chem., 36, 504 (19%). In the accompanying working examples, aeration concomitant with agitation and stirring corresponds to four, eight or twenty millimoles of oxygen per hour per liter at stirring speeds of 1'76, 250, or 360 revolutions per minute, respectively. Under some conditions it is desirable to utilize different rates of aeration during the fungus growing or developing stage as contrasted with the steroid fermentation stage. Aeration is suitably modified by using superatmospheric or subatmospheric pressures, for example thirty pounds per square inch or ten pounds per square inch absolute. Oxygen uptake may be facilitated by the presence of various catalysts such as ascorbic acid, glutamic acid,

citric acid, lactic acid, tyrosine, or tryptophane.

The time required for the fermentation of steroids varies somewhat with the procedure. When the steroid substrate is present at the time of inoculation of the medium, periods of from eight to 72 hours may be used. However, when the steroid is added to the fungus, after substantial aerobic growth of the fungal organism, for example after 16 to 24 hours at optimum temperature, the conversion of steroid substrate begins immediately and high yields are obtained in from one to 72 hours, 24 hours being generally satisfactory. The steroids may be fermented in a simultaneous or sequential heterofermentative procedure resulting in other useful products, which are recoverable according to procedures known in the art, including enzymes and acids, for example amylase, invertase, lipase, maltase, protease, proteolytic enzymes, rennet, urease, citric acid, fumaric acid, gluconic acid, itaconic acid, kojic acid and oxalic acid. These fermentation products may be separated from the fermentation beer at the same time, before or after the fermentation is complete with respect to the steroid fermentation products. The Penicillium fermentation processes as illustratively disclosed in United States Patents 2,458,495 and 2,538,721 or aerobic processes using other penicillia such as P. cyclom'um, P. nigricans, P. motatum and P. roqaeforti, with the addition of steroids as above described, are useful in the simultaneous oxygenation of steroids and the production of Penicillium fermentation products.

After completion of the steroid fermentation, the resulting fermented steroid is recovered from the fermentation reaction mixture. An especially advantageous manner of recovering the fermented steroid involves extracting the fermentation reaction mixture, including the fermentation liquor and mycelia with a water-immiscible organic solvent for steroids, for example, methylene chloride, ethylene chloride, trichloroethylene, ether, amyl acetate, and the like. The fermentation liquor and mycelia may be separated and then separately extracted with suitable solvents. The mycelia may be extracted with either watermiscible 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, as for example over anhydrous sodium sulfate, and the purified fermented steroid obtained by recrystallization from organic solvents or by chromatography.

The following examples are illustrative of the process of the present invention and are not to be construed as limiting.

EXAMPLE 1 Raulins medium having a composition of fifty grams of dextrose, three grams of tartaric acid, three grams of ammonium nitrate, 0.4 gram of dibasic ammonium phosphate, 0.5 gram of potassium carbonate, 0.2 gram of ammonium sulfate, 0.05 gram of zinc sulfate, 0.05 gram of ferrous sulfate, one gram of sodium acetate, and 0.3 gram of magnesium carbonate diluted to one liter with distilled water was adjusted to a pH of 7.0 with hydrochloric acid and sodium hydroxide. Twelve liters of this medium was sterilized by heat. The pH after sterilization was 6.35. This sterile medium was maintained at room temperature and inoculated with spores of Penicillium adametzi, ATCC 10407; the aeration was adjusted to one liter per minute and the agitation to 200 revolutions per minute. After 66 hours of growth, the pH was 3.75 and three grams of progesterone dissolved in milliliters of acetone was added. After 47 hours of bio-conversion time the fermentation was stopped. The final pH was 3.4.

The mycelium was separated from the whole beer by squeezin through gauze. The separated mycelium was extracted twice, each time with a volume of acetone approximately equal to the volume of the mycelium and again extracted twice, each time with a volume of methylene chloride approximately equal to the volume of the mycelium. The acetone and methylene chlo- ,ride extracts including solvent were added to the beer filtrate. The mixed extracts and beer filtrate were then extracted four times, each time with three liters of methylene chloride. The combined methylene chloride extract was washed twice, each time with one-tenth by volume portions of a two percent aqueous solution of sodium bicarbonate and then twice with one-tenth by 9 volume portions of water. The methylene chloride extract was dried with anhydrous sodium sulfate and then concentrated to a small volume on a steam bath.

The concentrated extract, freed of solvent, weighed 1.6388 grams. This residue was dissolved in 100 milliliters of benzene and chromatographed over 50 grams of alumina (hydrochloric acid washed, water washed, and dried at 120 degrees centigrade) using IOU-milliliter portions of developing solvent as indicated in Table I. Paper chromatography analysis using a toluene-propylene glycol system demonstrated production of a steroid component having a mobility similar to but different from 14a-hydroxyprogesterone. Fractions 13 through 16 were combined, dissolved in methylene chloride, filtered, and evaporated to a syrup of three milliliters. To this syrup, thirty milliliters of ether was added with stirring to effect immediate precipitation of small white crystals Weighing 0.425 gram, melting at 200 to 208 degrees centigrade, and having a melting point upon recrystallization of 208 to 209 degrees centigrade, an [(11 of plus 43 degrees 1.00 in chloroform), and an analysis of 0251-13404. Infrared spectrum showed this compound to contain a 3-keto-A4 grouping, 20-keto, an additional carbonyl group and probably an ether group. This compound has estrogenic activity.

Chromatographic analysis Table I Fraction Solvent fififf gfig benzene-ether 10:1

benzene-ether 1:1.

. do ethcgchloroform 10:1

o ether-chloroform 1- .d

EXAMPLE 2 Otherwise in the same manner as Example 1, the inoculant was Penicillz'um raz'strz'clciz, ATCC 10490.

EXAMPLE 3 Otherwise in the same manner as Example 1, the medium was adjusted to pH 4.2 before sterilization and the inoculant was Penicilli'am canescens, ATCC 10419.

EXAMPLE 4 Otherwise in the same manner as Example 1, the medium was adjusted to pH 7 before sterilization, the inoculant was Penicillium urticae, ATCC 10120, and the agitation was 160 revolutions per minute. After 66 hours of growth, three grams of progesterone dissolved in 160 milliliters of acetone was added and fermentation was continued for 47 hours at which time the pH was 4.2.

Extraction as in Example 1 produced 4.196

grams of crude extract solids. This residue was dissolved in 300 milliliters of benzene and chromatographed over grams of alumina (hydrochloric acid washed, water washed, and dried at 120 degrees centigrade for four hours) using 300-milliliter portions of developing solvent as indicated in Table II.

Fractions 20 and 21 were combined and crys tallized from three milliliters of methylene chloride by the addition of forty milliliters of ether to produce fine white crystals which were filtered, and washed with ether to give 745.7 milligrams of crystals melting at 205 to 218 degrees centigrade. Recrystallization from 25 milliliters of ethyl acetate gave 632 milligrams of crystals of melting point 210 to 228 degrees centigrade. Recrystallization twice more from methylene chloride-ether (1:10) gave 517.5 milligrams of crystalline product having a melting point of 234 to 235 degrees 'centigrade, and progestational activity. According to infrared spectra, this is a new monohydroxyp-rogesterone.

Fractions 5 through 10 were combined and crystallized from five milliliters of methylene chloride by the addition of fifty milliliters of ether. After refrigerating for two hours, the crystals were filtered and washed with ether. Recrystallization from fifteen milliliters of ethyl acetate followed by recrystallization from seven milliliters of ethyl acetat gave 0;259 milligram of allopregnanedion having a melting point of to 198 degrees centigrade.

Chromatographic analysis Table II Eluate Solids,

benzene o benzene-ether 10:1.

EXAMPLE 5 Otherwise in the same manner as Example 1, the medium was adjusted to pH 4.5 before sterilization, the inoculant was Penicilli um Zateam, ATCC 10465, and the agitation was 250 revolutions per minute. After a culture development time of 96 hours, progesterone was added for a bioconversion time of 92 hours and the resulting fermented steroid was recovered.

EXAMPLE 6 Otherwise in the same manner as Example 1, the medium was adjusted to pH 4.0 before sterilization, and inoculated with Penicillzum expansum, ATCC 7861. Progesterone was added with no previous culture time. After a bioconversion time of 48 hours, the resulting fermented steroid was extracted and recovered.

11; EXAMPLE 7 Otherwise in the same manner as Example 1, the medium was adjusted to pH 6.65 before sterilization, inoculated with Penicillium notatum, ATCC 9479, and agitated at 250- revolutions per minute. Progesterone was added with no previous culture time. After a bioconversion time of 48 hours, the resulting fermented steroid was extracted and recovered.

EXAMPLE 8 In the same manner as Example 1, the starting steriod was l'la-hydroxyprogesterone and the resulting fermented steroid was recovered.

EXAMPLE 9 In the same manner as Example 1, the starting steroid was 1711,21-dihydroxyprogesterone and the resulting fermented steroid was recovered.

EXAMPLE 10 In the same manner as Example 1, the starting steroid was pregnane-3,20-dione and the resulting fermented steroid was recovered.

EXAMPLE 11 In the same manner as Example 1, the starting steroid was 21-hydroxy-4-pregnene-3,20-dione and the resulting fermented steroid was recovered.

EXAMPLE 12 In the same manner as. Example 1, the starting steroid was lfi-dehydroprogesterone and the resulting fermented steroid was recovered.

EXAMPLE 13 To twelve liters, of a medium having a composition of fifty grams of Cerelose. commercial dextrose, thirty grams of sucrose, twov grams of ammonium nitrate, one gram of monobasic potassium phosphate, 0.5 gram of magnesium sulfate heptahydrate, 0.01 gram of ferrous sulfate, 0.2 gram of zinc sulfate, 0.1 gram of manganese sulfate, and two grams of yeast extract diluted to one liter with tap water, adjusted to a pH of 6.65, sterilized, inoculated with Penicillium urtic-ae, ATCC 10120, and cultured for 48 hours. with agitation of 1'76 revolutions per minute, three grams of progesterone. dissolved in a minimum of acetone was added. Following a bioconversion time of 48 hours, the resulting fermented steroids were extracted and recovered as in Example 1.

EXAMPLE 14 Otherwise in the same manner as Example 1.3:, the medium was adjusted to aniniti-al pI-I of 3.5, the inoculant was Penicillium lilacinum, A'I'CC 10114, the agitation was 250 revolutions per minute, and the resulting fermented steroids wererecovered.

EXAMPLE 15 Otherwise in the same manner as Example 13-, the. medium was adjusted before sterilization to a. pH of 3.5, was inoculated with Penicillium brevi-compactum,.ATCC 9056, and agitated. at. 17.6 revolutions per minute. With no previous culture time, progesterone Was added. After a bioconversion time of 48 hours, the resulting fermented steroids were recovered.

EXAMPLE. I6

Otherwise in the same manner as Example 15-, the inoculant was. Penicillium citrinwm, ATCC 10105.

EXAMPLE 17 To twelve liters of medium having a comp'osi tion of five grams of soybean meal, twenty grams of Cerelose commercial dextrose, five grams of Pabst debittered brewers yeast extract, five grams of sodium chloride, and five grams of monoba'sic potassium phosphate diluted to one liter with tap water and adjusted to a pH of 4.0, sterilized, inoculated with Peniciliium thomii, A'ICC 10506, and cultured for 48 hours, three gramsof progesterone dissolved in a minimum ofacetone was added. Following a bioconversion time of 48 hours with agitation at 176 revolutions per minute, the resulting fermented steroids were extraoted and recovered as in Example 1, with aliquots being subjected to paper chromatography.

EXAMPLE18 In the same manner as'Example 1'7, the medium adjusted to an initial pH of 6.55, inoculated with Remcillium bvevi-compactum, ATCC 4056; and agitated at 250 revolutions per minute. produced fermented steroids which were recovered.

EEQAMPLE 19 Otherwise in the same manner as Example 1'7, the medium was inoculated with Penicillium lilacz'num, ATCC 10114, and agitated at: 17:6 revolutions per minute. With no previous culture time, progesterone was added. After a bioconversion time of 48 hours, the resulting fermented steroids were recovered.

EXAMPLE 20 In the same manner as. Example 17,. the medi-' um was adjustedto an initial pH of 4.1, inocu lated with Penicillium name zeelcmdz'ae, ATCC 10473, and agitatedat 17:6 revolutions per minute. With no previous culture time, progesterone was added. After a bioconversion time of 48'hours', the resulting fermented steroids were recovered:

EXAMPLE 21 In the same manner as Example 17, the medium was inoculated. with Pemicillium urticae', A'ICC 10120, and agitated at 250 revolutionsper minute. Progesterone was. added with no. previ-' ous culture time. After a bioconversion time of 48; hours, the resulting fermented steroids were recovered.

EXAMPLE'22- To. twelve liters of medium having a composition of twenty grams of; com steep liquor, twenty grams of dextrin, one gram of monobasic potassium phosphate, two. grams. of, sodium nitrate, 0.5 gram of magnesium sulfate, 0.2 gram of. potasssium chloride, 0.01 gram of ferrous sulfate, and two grams of sodium acetate diluted to one liter with tap water, adjusted. to a pH: of- 4.6, sterilized, and cultured for hours: Penicilnigricans, A'ICC 10211 5,. there: was added three grams of progesterone dissolved inl a minimum of acetone. Followinga bioconversion time of 48 hours with agitation at 250 revolutions per minute, the resulting fermented. steroidswere; extracted and recovered as. in: Example 1.v

Otherwise in thesa'me manner. as: Example 22; themedium Was adjusted before sterilization to a. pH. of 4.35, inoculated. with Penicillz'um. Gwali- 5136610476,- and agitated at: llfi'r'evolu tions permin-ute; Progesterone wasaaiddectwith 13 no previous culture time. After a bioconversion time of 48 hours, the resulting fermented steroids were extracted and recovered as in Example 1.

EXAMPLE 24 A medium was prepared from five milliliters of corn steep liquor, twenty grams of Edamine commercial lactal'bumin digest and fifty milligrams of Cerelose commercial dextrose per liter of tap water and adjusted to a pH of between about 5.5 and 5.9. To four liters of this medium containing a 24-hour growth, at room temperature with aeration, of Penicillium y'anthinellum, ATCC 10455, was added one gram of progesterone in fifty milliliters of acetone. After a biocon-' version time of 48 hours at room temperature with aeration, the fermented steroid was extracted and recovered as in Example 1.

EXAMPLE 25 To twelve liters of medium having a composition of twenty grams of corn steep liquor, twenty grams of dextrose, one gram of monobasic potassium phosphate, two grams of sodium nitrate, 0.5 gram of magnesium sulfate, 0.2 gram of potassium chloride, 0.01 gram of ferrous sulfate, and two grams of sodium acetate diluted to one liter with tap water, adjusted to a pH of 4.7, sterilized, and cultured with Penicillium 10mth-mellum, ATCC 10455, with agitation at 176 revolutions per minute, there was added three grams of progesterone dissolved in a minimum of acetone. Following a bioconversion time of 48 hours, the resulting fermented steroids were extracted and recovered as in Example 1.

It is to be understood that the invention is not to be limited to the exact details of operation or exact organisms and compounds shown and described, aS obvious modifications and equivalents will be apparent to one skilled in the art, and the invention is therefore to be limited only by the scope of the appended claims.

We claim:

1. A process for the introduction of oxygen into a steroid which comprises: growing a Penicillium under aerobic conditions in the presence of a fermentation medium containing asssimilable nonsteroidal carbon and a steroid having an eleven methylene group and recovering the resulting oxygenated steroid.

2. A process of fermenting a steroid which comprises: growing a Penicillium under submerged aerobic conditions in a fermentation medium containing assimilable non-steroidal carbon and a steroid having an eleven methylene group, and recovering the resulting oxygenated steroid.

3. A process of fermenting a steroid which comprises: growing a Penicillium under submerged aerobic conditions in a fermentation medium containing carbohydrate and a steroid having an eleven methylene group, and recovering the resulting oxygenated steroid.

4. A process of oxygenating a steroid which comprises: aerobically subjecting a steroid containing an eleven methylene group to the oxygenating activity of a growth of Penicillium and isolating the resulting oxygenated steroid.

5. A process of fermenting a steroid which comprises: growing a Penicillium under aerobic submerged agitated conditions in a fermentation medium containing assimilable non-steroidal carbon and a steroid substrate, consisting essentially of steroid having an eleven methylene 14 group, and recovering the resulting fermented steroid.

6. A process of oxygenating a steroid which comprises: dispersing a steroid substrate, consisting essentially of a 3-keto steroid, in an aqueous nutrient medium, and growing a Penicillium in said medium under aerobic conditions.

7. A process of fermenting a steroid which comprises: dispersing a steroid substrate, consisting essentially of a 3-keto steroid, in an aqueous nutrient medium, growing a Penicillium in said medium under aerobic conditions, and isolating the resulting fermented steroid.

8. A process of fermenting a steroid which comprises: aerobically contacting a growing Penicillium with a steroid substrate, consisting essentially of a steroid having up to and including 22 carbon atoms in the carbon to carbon skeleton, and recovering the resulting fermented steroid.

9. A process of o'xygenating a steroid which comprises: growing a Penicillium under aerobic agitated conditions in a nutrient fermentation medium containing a steroid having an eleven methylene group, and isolating the resulting oxygenated steroid.

10. A process of oxygenating a steroid which comprises: growing a Penicillium under aerobic agitated conditions in a nutrient fermentation medium containing carbohydrate and a steroid and extracting the resulting oxygenated steroid.

11. A process which comprises: growing Penicillium chrysogenum under aerobic agitated conditions in a nutrient fermentation medium containing carbohydrate and steroid and extracting the resulting fermented steroid.

12. A process which comprises: dispersing a steroid substrate, consisting essentially of a steroid, having an eleven methylene group and up to and including 22 carbon atoms in the carbon to carbon skeleton, in a fermentation medium and subjecting such dispersed steroid to the actionof viable Penicillium under aerobic agitated conditions andseparating the resulting fermented steroid.

13. A process comprising growing a species of fungus of the genus Penicillium under aerobic agitated conditions in a nutrient fermentation medium containing a steroid substrate, consisting essentially of a steroid having an eleven methylene group and up to and including 22 carbon atoms in the carbon to carbon skeleton, and recovering the resulting oxygenated steroid.

14. A process comprising growing a species of fungus of the genus Penicillium under aerobic conditions in a nutrient fermentation medium containing progesterone.

' 15. A process comprising dispersing progesterone in a nutrient fermentation medium, and growing Penicillium chrysogenum in said medium under aerobic conditions.

16. A process comprising growing a species of fungus of the genus Penicillium under aerobic conditions in a nutrient fermentation medium containing l7a-hydroxyprogesterone.

17. A process comprising growing a species of fungus of the genus Penicillium under aerobic conditions in a nutrient fermentation medium containing 1%,2l-dihydroxyprogesterone.

18. A process comprising growing a species of fungus of the genus Penicillium under aerobicconditions in a nutrient fermentation medium containing pregnane-3,20-dione.

19. A process comprising growing a species of fungus of the genus Penicillium under aerobic 15 conditions in a nutrient fermentation medium containing a steroid selected from the group consisting of 21-hydroxy-4-pregnene-8,2.0-dione and ld-dehydroprogestercne,

20. A process which comprises: dispersing a steroid substrate, consisting essentially ofa steroid having an eleven methylene group and up to and including 22 carbon atoms in the carbon to carbon skeleton, in an aqueous fermentation medium and aerating and agitating said dispersion in the presence of a species of fungus selected from the group consisting of Penicil- Zium adametzi, Penicillium breoi-compactum, Penici'llium citrinum, Pencillium nigrzcans, Penicillium oaraZicum, and Penicillium rais tricki.

21. A process of oxygenating a steroid which comprises: growing a Penicillium under aerobic submerged agitated conditions in a fermentation medium containing assimilable nitrogen, phosphate, carbohydrate, and a steroid substrate, consisting essentially of a steroid havin an eleven methylene group, and recovering the resulting oxygenated steroid.

22. A process which comprises: dispersing a 3-keto steroid having an eleven methylene group in an aqueous fermentation medium containing assimilable nitrogen, phosphate and .carbohydrate, and therein growing under aerobic agitated conditions a species of fungus of the genus Penicillium.

23,. A process which comprises: growing a species of fungus selected from the group consisting of Penicillium adametzi, Pem'cz'lli'um brevicompactum, Penicillium citrinum, Penicillium m'gricans, Penicillium oxalz'cum, and Penicil- Zium y'anthinellum under aerobic .agitated conditions in a fermentation medium containin assimila-ble nitrogen, phosphate, carbohydrate, and a steroid substrate, consisting essentially of a steroid having an eleven methylene group and up to and including 22 carbon atoms in the carbon to carbon skeleton.

24. A process which comprises: growing a species of fungus selected from the group consisting of Penicillium adametzi, ,Penicillium. brevi-compactum, Penicillium citrinum, Penicillz'um nigriccms, Penicillzum oralicum, and Penicillium y'anthinellum under aerobic agitated conditions in a fermentation medium containing assimilable nitrogen, phosphate, carbohydrate, and progesterone.

25. A process which comprises; growing aspecies of fungus selected from the group consisting of Penicillz'um adametei, Penicillium brevicompactum, Penicillz'um citrinum, Penicillium nigricrms, .Penicillz'um oralicum, and Penicillium janthinellum under aerobic agitated "conditions in a fermentation medium containing assimilable 1'6 nitrogen, phosphate, carbohydrate, and I'Za-hY- droi /progesterone.

26. A process which comprises: growing a species of fungus selected from the group consisting of Penicillzum adametzi, Penicillium brevicommctcm, Pemcilzium citrz'num, Pen nigrisens, Pecic l icm cwali um, a d c ll um family/ indium pride; aerobic agitated conditions n a fermentation med um c n i assimilable nitro en r asphate, ca bo ydra e an 9 2 dilwdroxyrroecstercne A p ocess w ich com ri es: gr w a su cies at f ngus selected it m the grou co i ing pi Penici-Zlium ada'metgi, Penici'llium brevie m-pgietum, Penicillin cit-rinu-m, Penicillium uigric s, Penicillium oralilcum, and Penicillz'um mat Helium unde ear-ob agi ate on it ns a fermentation medium containing assimilable nitrogen, phosphate, carbohydrate, and pregnane-3,20-dione.

2.8.. A process which compri wing a sp cies of fungus sel c ed f om the r up con t: ing of Renici'liz'um wdametgj, Penici'llium brevicompactum, Peziz'cillium citrinum, Penicillium nigrtcans, Peniciliium oxali cum, and Penicillium y'amihinel'lum under aerobic agitated conditions in a fermentation medium containing assimilable nitrogen, phosphate, carbohydrate and a steroid selected from the group consisting of zl-hydroxy- 4-pregnene 3,20-dione and IS-dehydroprogesterone.

29. A process for the production of .an oxygenated steriod comprising the steps of aerobically growing a culture of an oxygenating strain of an organism of the Penicillium genus, and exposing a steroid to the oxygenating activity .of enzymes produced by the said culture.

'30. A process for th production of an eleven oxygenated steroid comprising the aerobic termentation of a nutrient-containing substrate, containing a steroid having an eleven methylene group, by means of a submerged growth of an organism of the genus *Penicillium.

HERBERT C. MURRAY. 'DUREY I-I PETERSON.

References Cited in the -fi1e of this patent UNI ED TA S PA T Name Date Murray et a1 July 8, 1952 OTHER REFERENCES Number (CODY

Claims

29. A PROCESS FOR THE PRODUCTION OF AN OXYGENATED STERIOD COMPRISING THE STEPS OF AEROBICALLY GROWING A CULTURE OF AN OXYGENATING STRAIN OF AN ORGANISM OF THE PENICILLIUM GENUS, AND EXPOSING A STEROID TO THE OXYGENATING ACTIVITY OF ENZYMES PRODUCED BY THE SAID CULTURE.