US3013945A

US3013945A - 11alpha-hydroxylation of steroids by beauveria

Info

Publication number: US3013945A
Application number: US762926A
Authority: US
Inventors: Havsky Jan; Hershel L Herzog
Original assignee: Schering Corp
Current assignee: Merck Sharp and Dohme Corp
Priority date: 1958-09-24
Filing date: 1958-09-24
Publication date: 1961-12-19
Anticipated expiration: 1978-12-19

Description

United States Patent 3,013,945 Ila-HYDROXYLATION 0F STEROIDS BY BEAUVERIA Jan Ilavsky, Pompton Plains, and Hershel L. Herzog,

Mountain View, N.J., assignors to Schering Corporation, Bloomfield, N.J., a corporation of New Jersey No Drawing. Filed Sept. 24, 1958, Ser. No. 762,926

30 Claims. (Cl. 19551) The present invention relates to the microbiological treatment of steroids to effect a selective chemical modification thereof. More particularly, this invention relates to the eifective and substantial microbiological transformation of eleven desoxy steroids to form the corresponding ll-hydroxy derivatives thereof.

Heretof'ore-it has been known to introduce oxygen into the eleven position of a steroid nucleus. Among the processes suggested for accomplishing this oxidation, the biochemical have been of'particular interest, since it has made possible the direct introduction of oxygen at this point, avoiding the plurality of steps inherent in the highly involved onganic syntheses, otherwise necessary. Biooxidation is accomplished by subjecting a steroid containing an eleven methylene group to the action of oxygenating enzymes produced by various microorganisms. Such bio-oxidative techniques have hitherto presented certain difiiculties. of oxidation products may result which are only difliculty separated, and, in any event, necessitate the inclusion of an added step in the process. Further, the yield of prod uct, such as ll-epi-hydrocortisone, expressed as a proportion of the starting material such as Substance S, may be significantly small. Similarly, and most significantly, the concentration of steroid substrate and necessary period of time required to effect even a reduced yield of oxygenated product by certain of the known bio-oxidative procedures would be such as to render these techniques prohibitive. Also certain of 'the known hydroxylating microorganisms are so difiicultly developed and sustained as to seriously limit their commercial feasibility. Hence, the process of the present invention is believed to present a significant advance over methods previously proposed, since it presents a procedure whereby significantly great concentrations of an ll-desoxy steroid substrate undergoes a substantial and often complete conversion to its corresponding ll-hydroxylated derivatives.

Accordingly, the present invention provides a novel, efficient and expeditious procedure for the production of '1 l-hydroylated steroids, which is of material significance commercially, and which comprises subjecting eleven desoxy steroids to the oxidative action of an easily developed and l sustained hydroxylating fungus of the class Fungi lmperfecthiorder Moniliales, family Mon'iliaceae,

genus "Beauveria, and particularly. and indeed most desirably those isolates of the strain ATCC 13144. These fungi are unique in theirspecificity in effecting normally the substantial or complete conversion of steroid substrate containing an eleven methylene group'and producing the corresponding elevenialpha hydroxy compound thereof in percentages by weight based on the amount of 11- desoxy steroid substrate, of fifty percent and higher.

These fungal organisms including their mutants are stable and easily grown and can be obtained, as indicated, from known sources, such as .the American Type Culture Collection (ATCC), Washington, DC. Alternatively, they can be obtained from natural sources using techniques known to microbiologists. I I

Representative of the 'II-desoxy steroids for use in the practice of ,our invention are: 4-pregnene-17u,21-diol-3, ZO-dione (.11-desoxy:17a-hydroxy-cortisone, Reichsteins Substance C), 1,4-pregnadiene-17a,21-diol-3,20-dione (1- Thus, by way. of illustration, a number i dehyd'ro-l1-desoxy-17a hydroxycortisone), 16amethyl- Patented Dec. 19, 1961 allopregnane-l7a,2l-diol 3,20 dione, 16 8 methylallopregnane-17a,21-diol-3 ,20-3,20di0ne, 16a t butylallopregnane-17a,21-diol-3,20-dione, 16fl-ethylallopregnane- 17a,21-diol-3,20- dione, 16B-methyl-4-pregnene 17,2ldiol-3,20-dione, 16a-methyl-1,4-pregnadiene-17a,21-diol- 3,20-dione, l6,8-methyl-l,4 pregn adiene 170;,21 diol- 3,20-dione, 16a-t-butyl-1,4-pregnadiene 17,21 diol 3, 20-dione, 16,8:ethyl-1,4-pregnadiene-17a,2l diol 3,20- dione, pregnane-l7a,2l-diol 3,20-dione, allopregnane-l7a, 2ldiol-3,2 0-dione, pregnane-17a,2l-diol-3,20-dione, 21- acetate, 16umethyl-4-pregnene-17a,21-diol-3,20-dione, 1, 4,6-pregnat1iene-17a,21-diol-3,20-dione, as well as the 21- acyl (e.g. acetate, propionate) derivatives and particu-- larly the acyl derivatives of the lower alkanoic acids thereof. It will be apparent that the 1.1-desoxy steroids which can be hydroxylated at the ll-carbon atom of the steroid nucleus by the process herein described are not narrowly delimited, but encompass those ll-desoxy compounds having a cyclopentanoperhydrophenanthrene nucleus e.g. ll-desoxy sterols, bile acids, cardiac aglycones, saponins, sex hormones, D-vitamins, androstanes and preferably the ll-desoxy androstenes, such as the 4- androstenes, 1,4-androstadienes e.g. 4-androstene-3,17- dione, 1,4-androstadiene-3,17-dione, testosterone and the .like and particularly those wherein the A-ring contains preferably a 3-keto configuration and a cortical side chain containing Z-carbon atorns attached to the D-ringat C-l7. -In addition, it is often desirable that at least one double bond be present (e.g. at A in the A-ring). In general, however, the more preferred compounds for use in the practise of our invention are the ll-desoxypregnanes, allopregnanes, and unsaturated analogues thereof (including within the term pregnanes and allopregnanes as employed herein, and unless otherwise explicitly indicated, the substituted derivatives thereof as well), and particularly those compounds'represented by the structure:

CHZOR wherein R is hydrogen or an acyl radical derived, preferably, from a lower alkanoic acid; andR' is hydrogen or a lower alkyl; R is hydrogen or allo-hydrogen, and R" is preferably allo-hydrogen when R is alkyl, and the l-pregnene, 4-pregnene, 1,4-pregnadiene and 1,4,6-preg- I natriene analogues thereof- The'product in each instance is the corresponding 11o;- hydroxy compound, which is formed in high and in some instances quantitative yield, In particular, Reichstein's Substance S, and its respective l6a-methyl and 16B methyl :derivatives are converted to 4-pregnene-11a,17a,21-t1iol- 3,20-dione and 16a-methyl-4-pregnene-11u,17u,2 1-triol- 3,20-dione and 16{3-methyl-4-pregnene-11a,l7a,21-trio1- 3,20-dione respectively in yields in excess of It should be noted that where the steroid substrate contains a C-21 acetate radical, this group is generally hydrolyzed,

20-one, the preparation of which is described hereinafter,

as well as the corresponding l6-ethyl, l6-tert. butyl and a like homologues thereof.

The ll-desoxy and 11oxygenated steroids disclosed herein containing alkyl substituents in the -16 position together with processes for their production are the invention of Eugene P. Oliveto and Richard Rausser and are not claimed in the present application apart from our invention which is limited to a novel process wherein ll-desoxy steroids are transformed by a species of fungus of the genus Beauveria to the corresponding lla-hydroxylated derivatives.

A variety of procedures can be employed to prepare the l6-alkyl steroids. Illustratively, 16w or 16,8-alkyl-11- desoxyallopregnanes can be prepared from l6a-alkyl or 16fl-alkyl pregnenolones, such as for example, 16a-methyl pregnenolone, 16,8-tert.-butylallopregnenolone and the like by initially hydrogenating the 16-alkyl-pregnenolone over such conventional catalytic agents as palladium on charcoal to cause the formation of a solid precipitate, the 16ocor 16 3-alkylallopregnane-3fi-ol-20-one, e.g. 16:1- or l6,8-methylallopregnane-3B-ol-20-one. In order to introduce an hydroxyl group at 0-17, the C-20 keto group of this latter compound is converted to the enol-acetate by refluxing said 16-alkylallopregnane-3fl-ol-ZO-one with acetic anhydride and a strong acid, such as ptoluenesulfonic acid or perchloric acid, for example. It is preferred not to isolate the enol-acetate but rather to react said substance in situ with a peroxy-acid, such as for example, peracetic acid, perbenzoic acid, monoperphthalic acid, or pertrifluoroacetic acid, whereby a second intermediate, a 17,20-epoxide, preferably not isolated, is obtained. Treatment of the reaction mixture with alkali hydrolyzes the epoxide in situ to cause the production of the crude, solid, 17a-hydroxylated product which can be separated out suitably by filtration and subsequent crystallization from a medium such as methanol-water. The crystalline product is lo-alkylallopregnane-iifi,l7a-diol 20-one. Acyloxylation, e.g. acetoxylation, of the C-21 methyl group is effected in the conventional manner as noted hereinabove, such as by bromination of the 0-21 methyl group, followed by reaction of the bromo-derivative with, for example, sodium or potassium acetate, whereby IGoc-aikYlallopregnane-Sfl,l7a,2l-triol-ZO-one 21-acetate is formed.

The product so obtained is oxidized sequentially at the 3C position by known standard procedures such as for example with N-bromoacetamide, N-bromosuccinimide or chromic acid to yield upon subsequent crystallization from acetone-hexane, 16-alkylallopregnane 17a,21-di0l-3,20- dione ZI-acetate. This latter compound can be saponified so as to produce the corresponding C-21 alcohol, 16- alkylallopregnane-l7a,21-diol-3,20-dione, by reaction with hydrolytic agents such as aqueous methanolic potassium bicarbonate or with such reagents as sodium carbonate, sodium hydroxide, sodium alkoxides (e.g. sodium methoxide, sodium ethoxide) and acids such as p-toluenesulfonic acid.

Microorganisms such as for example Flavobacterium dehydrogenans var. hydrolyticum can also be employed for this purpose using standard procedures such as that disclosed in Union of South Africa Patent 3462/55 and described in detail hereinafter. The crude product thus formed is desirably separated out by filtration and crystallized from acetone-hexane. The one hydroxyl group is then, if desired, introduced into the Ila-position of said 16-alkylallopregnane l7oc,2l-diol-3,20-dione by the process of our invention employing a microorganism of the genus Beauveria.

The resultant products are valuable intermediates in the formation of the corresponding llfi-hydroxy and 11- keto-1,4-dienes which are valuable therapeutically active compounds useful in the treatment of inflammatory diseases such as arthritis. To effect the production of the Ila-hydroxy 1,4-dienes the 16a-alkylallopregnane-lla, l7a,21-triol-3,20-dione is initially reacted with chromium trioxide in pyridine to cause the oxidation of the 1105 hydroxy to the ll-keto derivative, l6a-alkylallopregnane- 17a,21-diol-3,11,20-trione. This compound is then converted to the corresponding 1,4-diene by dihalogenation, preferably dibromination which occurs at the 2-carbon and 4carbon linkage of the A-ring. Didehydrobromination is then effected with a basic agent, preferably dimethylformamide, to produce the desired 1,4-diene, 16aalkyl-1,4-pregnadiene-l7,21-diol-3 ,l1,20-trione.

Alternatively, the 16-alkylallopregnane-17a,21-dio1-3, 20-dione 21-acylate can be dehydrogenated initially in the A-ring in order to effect the preparation of the corresponding ll-desoxy 1,4-diene by the procedure described immediately above whereby the l6-alkylallopregnane-l7a, 21-dio1-3,20-dione 2l-acetate is dihalogenated preferably with bromine, to form the intermediate 2,4 dibromo-l6- alkylallopregnane-l7m,2l-diol-3,20-dione 2l-acetate. Didehydrobromination with basic agents, preferably dimethylformamide, produces the 21-acetate of l6-alkyl- 1,4-pregnadiene-l7a,21-diol-3,20-dione, which can then be saponified so as to produce the corresponding C-21 alcohol, 16-alkyl-l,4-pregnadiene-l7a,2l-diol-3,20-dione, by employment of standard hydrolytic agents such as those described above. The hydroxyl group is then introduced into the Ila-position thereof by the process of my invention as described in detail hereinafter employing a microorganism of the genus Beauveria. The resultant 16- alkyl-l,4-pregnadiene 11u,17a,21-t1'iOl-3,20-diOne can, of course, be again esterified at the 0-21 position if desired by standard acyloxylation, e.g. alkanoyloxylation, procedures such as that noted hereinabove, e.g. reaction with acetyl chloride and pyridine and sequentially oxidized with chromic acid to the corresponding ll-keto derivative, l6-alkyl-l,4-pregnadiene-17a,21 triol-3,1l,20-trione which is a valuable therapeutic agent in the treatment of anti-inflammatory disease, such as arthritis.

Further, l6fl-alkyl-l7a-hydroxy 4 pregnenes for use in the process of our invention can be prepared in the following manner wherein a 16-a1kyl-5,1G-pregnadiene- 3fl-ol-20-one is employed as the starting material. This compound is initially epoxidized at the 16(17)-linkage of the D-ring, with hydrogen peroxide. The 3-C hydroxyl group of this epoxy compound is then acetylated with acetic anhydride in a basic reaction medium, e.g. pyridine, to yield 3 fl-acetoxy-16fi-methyl-l6a,17a-epoxy-5- pregnene-ZO-one. Although acetylation is shown and is indeed the preferred procedure, other acylations such as for example with propionic anhydride to cause the formation of the corresponding 3fi-propionoxy derivative can also be performed.

The 3p-alkanoyloxy compound thus produced is then reacted with glacial acetic acid and a hydrogen halide, e.g. hydrogen chloride, hydrogen bromide, to effect the preparation of the alkylene derivative, for example, the 16-methylene compound, 35 acetoxy 16 methylene-$- pregnene-lh-ol-ZOone, which is hydrogenated by standard procedure using, for example, palladium on charcoal, to form the corresponding l6-alkyl derivatives, e.g. 3;?- acetoxy-l-fi-methyl-S-pregnene-l7a-ol 20 one. This compound can then be acyloxylated in the 0-21 position of the D-ring by halogenation, preferably bromination, of the C-21 methyl group, followed by reaction of the bromo-derivative, thus produced with sodium or potassium acetate, butyrate or the like, in the usual manner, to give the 313,21-dialkanoyloxy-1Gp-alkyI-S-pregnene-17a-ol- ZO-one. This compound when treated with a hydrolytic agent, e.g. sodium bicarbonate, sodium hydroxide, toluenesulfonic acid, followed by oxidation of the 3-hydroxyl group, as by chromic acid in acetone, and rearrangement of the 5,6-double bond to 4,5 by the action of acid or when treated with amicroorganism such as Flavqbacterium dehydrogenans var. hydrolyticz zm 'will yield 165- alkyl-17a,21-dihydroxy-4-pregnene 3,20 dione. Subsequent exposure to the enzymatic activityof a strain of Beauveria in accordance with the practice of our inven- 'l7a,2l-dihydroxy-4-pregnene 3,20-dione'of the 11a hydroxyl group. These resultant compounds, the 16fl-alkyll1 ,17a,2l-trihydroxy-4-pregnene 3,20 diones are valuable intermediates in'the formation of the corresponding lIB-hydroxy-4-monoenes and the ll-keto 4-monoenes which compounds are therapeutically useful in the treatment of inflammatory diseases,'such asfor example, ar-

thritis, or can be convertedto the corresponding 1,4-dienes, 16p-alkyl-11a,17a,2l-trihydroxy 1,4 pregnadiene- 3,20-diones by reaction with Corynebacterium simplex according to procedures described in United States Patent 2,837,464; these compounds can then be readily converted to thecorresponding ILB-hydroxyl or 1l-keto-l,4-dienes which are therapeutically active anti-inflammatory compounds useful in the treatment of arthritis and like diseases.

Similarly, 1oa-alkyb17a-hydroxy-4-pregnenes can be prepared from 16a-alkyl-pregnenolone-3 -acetate (e.g. l6u-methyI-S-pregnene-3;8-ol-20-one 3-acetate as well as the corresponding propionate, butyrate or the like), by

sequentially halogenatinge.g. chlorinating this latter compound in the 5-C and 6-C positions, in a-basic medium such as pyridine, lutidine or the like; introducing the hydroxy group into the 17-C position by the method described above, that is by enolacetylation of the 20-C keto group with. acetic anhydride and p-toluenesulfonic acid or the like and sequential reaction of this 20-enol-acetate, preferably in situ, with an acid such as peracetic acid or perchloric acid to form the corresponding 17,20-epoxide. This compound too is treated in situ with alkali, e.g.

potassium bicarbonate, and hydrolyzed thereby to yield the 16a-alkyl-S,6-dichloropregnane-3,l7a-diol 20 one 3- acetate. Acyloxylation of the 0-21 position is then effected in the standard manner by bromination of the (3-21 methyl group and subsequent reaction thereof with sodium or potassium acetate or the like. The resulting compound, 16a-alkyl-5,6-dichl0ro pregnene 3 ,9,17a,21-

triol-ZO-one Zl-acetate, is then oxidized at the 0-3 position' of the A-ring and hydrolyzed at C-21 by means of a microorganism, e.g. Flavobacterium dehydrogenans var. hydrolyticum, as noted above, to yield 16'a-alkyl-5,6-dichloro-pregnane-17,21-diol-3,ZO-dione. This latter compound is readily dehalogenated employing well-known procedures e.g. zinc dust in an acid medium such as acetic acid, whichresults in the formation of a double bond at the 4(5)-carbon linkage, the product being 16a-alkyl-4- produce the desired 16a-alkyl-4-pregnene-l7u,2l-diol-3,

20-dione.

This compound however prepared, when subjected to the enzymatic activity of a species of the microorganism of the genus Beauveria according to the process of our invention as described herein, results in the lla hydroxylation thereof to produce the valuable intermediate 1'6a-alkyl4-pregnene-l1a,l7a,21-triol-3,20 dione which can be converted by known procedures to the corresponding 11-keto-4-monoenes (eg. by oxidation with chromic acid), their corresponding 11-keto-1;4-dienes, or if desired, the 11fi-hydroxy-4-monoenes or llfl-hydroxy- -1,4-dienes, all of which are'valuable agents in the'treatment of inflammatory diseases such as arthritis, as noted above.

The hydroxylation process of our invention is conveniently accomplished by cultivating the microorganism; for example the preferred -'Beauveria=( ATGC 1 3144), under .76 aerobic conditions, on a suitable mediumsuchas described hereinafter,-in-intimate'admixture with-gthe 11- desoxy steroid, such as Substance S, the cultivation or growth of the fungus being continued until the'desired enzymatic hydroxylation'is effected. Alternatively, and

indeed preferably, the process -ofthe present invention is effected by growing amicroorganism in-a suit-able'fermentation medium under aerobic conditions, and then separating the cells of the microorganism 'sotcultivatedtherefrom. The ll-desoxy steroid, such as for example'the Substance S referred to hereinabove, which is to be hydroxylated, is-then added to these cells under'acrobic conditions, for a period of-time suflicient to elTect-the desired oxygenation. Thislatter procedure results in a marked simplification of the recovery step, but while-the latter method is deemed the most desirable the order of addition is not critical. Thusthe ll-desoxysteroid can for example also be added-to the growth medium-which is then inoculated with the fungus.-

The eleven desoxysteroid starting material-is introduced into the nutrient, medium by' known standard procedures, as for example, by forming -a-suspension thereof inwater, or bypreparinga solution or'suspension thereof, in a'solvent such as methanol, ethanol, acetone, propylene glycol, dimethylformamide, or *dimethylacetamide, or

other 1 water-miscibleorganicsolventwhich is nontoxic to the microorganism.

It may, in addition, he I added in a finely divided form such as a solid micronizedpowder.

It is ordinarilypreferredthat the steroid be present in very finely divided'form in order'to expose the greatest'surface 'area therefor and thus permit maximumcontact with the oxygenating .enzyme produced' by the microorganism and :the most efiicient conversion of eleven vdesoxy steroid to itsleleven-alpha hydroxylated derivative.

duced in a continuous. or intermittcnhmanner over ape- .riod of'time.

Optionally, thesteroid can be added atone time or:intro- Suitable nutrient mediator the cultivation of Beauveria include assimilable-carbomorganic and inorganic sources of nitrogen'together :with :minor amountsof inorganic salts and trace elements. The concentration of 'thesecon- .stituents can be varied within avide limits. sources of carbon appropriate for use in thesez-growth media are carbohydrates :such as dextrose, l glucose, istarch, inverted molasses and the like. Organic nitrogen sources Standard normally employed :.are .such substantially .proteinaceous materials as;corn steep liquor, :lactalbumiddigest, "yeast extract, or soybean .meal containing:--from approximately ten percent to fifty-percent protein. Sources of inorganic .bound nitrogen -.are represented by ammonium .nitrate, ,dibasicjammonium phosphate andthe'like. "Trace elements are supplied:by:the;-inclusion in the: culture medium of tap-water. .=Inorganic.salts :(e.g. suitable water-soluible salts of magnesium, zinc, potassium, sodium, ;.-phos- -phorous,-iron,:.and the:like) and;other:-suchtmaterials,;;for .example, nicotinamide, r. are normally present -in':the asources :of ;-assimilable zcarbonzand :organic nitrogen in ,amountszsufiicienttoassure. optimum growth ,ofthe; microorganism in the culture broth but can'be separately-added to the.growthamediaifsodesired.

The :iollowing vare examples of suitable aqueous pnurtrient media forausecinithe: presentdnvention:

:Mediurn Number ,1

Dextrose (Cerelose) ..djo r.. 11 Jan w t r .1 V ;.do .98 'P .55. Medium Number 2: l V

sne a 1011 Glucose, v 25 .KH PQ; gm 0. 5

;Tap watentooneliter.

See footnote at end or table.

7 Medium Number 3:

Sodium nitrate "gm-.. 2 K2HPO4 gm 1 MgSOflH O -gm 0.5 Yeast extract gm 1 KCl grn 0.5 Glucose gm 50.0

Tap water to one liter.

Percentages as referred to throughout this specification and unless otherwise explicitly indicated, are percentages by weight.

The pH of the culture medium is not critical, although it is known that fungi fare better in a slightly acid medium as opposed to an alkaline medium. Thus, a pH of 4 to 8 is thoroughly operative for proper growth of Beauveria, with a preferred pH being in the range of 5.3 to 7.5. The pH is adjusted to these ranges by addition of a suitable acid or alkaline material, such as, for example, sodium hydroxide or potassium hydroxide or hydrochloric acid. The addition of small quantities of anti-foaming agents, although not essential, is desirable, particularly in commercial scale operation.

To promote to growth of Beauveria and the biochemical transformation of the steroid substrate, Substance S, the culture is preferably shaken and/or aerated and stirred.

Thus the eleven desoxy steroids employed in the present invention can be suitably oxygenated in the following manner:

The spores as received from the culture collection, or vegetative growth of an oxygenating strain of Beauveria (ATCC 13144) suspended in 100 ml. sterile distilled water, are grown on an agar medium (e.g. 0.3% yeast extract, 1.0% cerelose, 0.1% corn steep liquor) at a preferred temperature of 22 C. to 28 C., although temperatures as low as C. or as high as 35 C. are not detrimental. After a period of time, about three to 10 days, and preferably about 7 days, sporulation occurs. The growth medium and spores are then diluted and washed with sterile water and after several washings, there remains a heavy spore suspension which is used for inoculation of the liquid fermentation medium as described above. The conversion from the spore stage to the vegetative mycelium stage generally occurs in 12 hours to 48 hours and can actually occur in as little as 3 hours. At the end of this growth period, which is measured by the appearance of a thick vegetative mycelium, an inoculum consisting of about 1% vegetative mycelium'is added to shake flasks containing the fermentation medium, and the flasks are shaken on a rotary shaker until substantial growth is observed (generally 12 hours to 24 hours). At this point, the eleven desoxy steroid substrate, finely divided or dissolved 'as described hereinabove is added to the culture medium. The steroid concentration in an alcohol medium, for example, is normally from 2 to 3 mg. of steroid per gram of solvent. Increased concentrations of steroid in solvent are had by warming the alcohol or substituting another solvent therefor such as dimethylformamide wherein concentrations of up to 100 mg. and more of steroid are completely dissolved. The concentration of steroid in the total fermentation mixture can be as low as 100 mg. per liter or as high as 2 grams per liter, although a concentration of 500 mg. to 1 g. per liter is preferred. The fermentation mixture is shaken until complete conversion is eliected (e.g. about 6 hours to 96 hours). The conversion of eleven desoxy steroid, such as Substance S to the eleven alpha hydroxylated derivative, 4-pregnene-11a,17a,21-triol-3,20-dione, is determined in the conventional manner by paper chromatography.

Alternatively, instead of employing a one percent inoculum of the vegetative mycelium, a spore suspension may be used directly. However, such a procedure is less preferred since it requires extended periods of time for proper growth of mycelium and conversion of eleven desoxy steroid.

At the conclusion of the fermentation process, the desired ll-hydroxylated steroid, is recovered from the fermentation medium by the following procedure, which describes in particular a ml. fermentation. This is a general procedure and is operative for fermentation of various amounts.

The mixture is extracted (e.g. usually three or more times), each extraction employing, by way of illustration, two volumes of organic solvent per volume of fermentation broth. The solvent, which may be a halogenated organic solvent, e.g. chloroform, methylene chloride, an ester, e.g. ethyl acetate, an alcohol, e.g. butanol, an ether, e.g. diethyl ether, dibutyl ether, an aromatic solvent, toluene or the like, is then cooled and dried over sodium sulfate, or like standard drying agent, such as calcium chloride or magnesium sulfate, and then filtered. The filtrate is evaporated to dryness or to a small volume. This solution is used for characterization of steroid content as described hereinafter.

The foregoing growth and fermentation procedure is generally applicable to a small scale process wherein shake flasks on rotary shakers are employed and may be varied on a larger scale so that the fermentation is carried out in tanks and occurs at a much faster rate. The vegetative mycelium can be added to fresh nutrient at a concentration of from about one percent to ten percent and higher and growth is permitted to occur preferably at about 28 C. for approximately 24 hours. Lower concentration of mycelium are preferred since better aeration, a factor directly associated with more efficient transformation is effected. In this latter instance a submerged inoculum may be employed, into which air is introduced as the oxygen supply. It is generally known that growth mixtures of this type cause the production of large quantities of foam due to the rapid import of air and defoaming agents are therefore usually employed such as, for example, one percent octadecanol or hexadecanol in lard oil, a silicone such as GE. 60 anti-foam (a product of the General Electric Company, Schenectady, New York), a substituted oxazoline which is a non-volatile, amine-type, cationic surface active agent available under the trade name Alkaterge C or the like. In this latter procedure, when the growth period is substantially complete, the steroid substrate, e.g., Substance S, in alcohol, may be added so that its concentration is up to 30 g. of substrate per liter of broth. The fermentation is then allowed to proceed until completion is evidenced by the disappearance of steroid substrate as determined by chromatographic analysis. At the completion of the conversion, the broth is filtered and extracted with a water-immiscible solvent in which the oxygenated steroid reaction products are soluble. Suitable solvents for this purpose are, for

-example, halogenated hydrocarbons, e.g. methylene chloride, acetylene tetrachloride; organic acid esters, e.g. tertiary butyl acetate; aromatic hydrocarbons, e.g. benzene,

toluene; ketones, diethyl ketone, cyclic amines, e.g.

2-methyl-5-ethyl pyridine. The preferred solvents are chloroform and ethyl acetate. The solvent solution containing the product steroid is then evaporated to yield the desired product which can be further purified by recrystallization from acetone, chloroform or other standard sol- .herein and referred to throughout this specification, un-

less otherwise explicitly indicated, is that described by Bush, Journal of Biochemistry, vol. 50, page 370 (1952), as modified by Shull, Paper Chromatography of Steroid Fermentation Products, 126th meeting of the American Chemical Society, September 12 to 17, 1954, New York, New York, Section 9A, Paper No. 24.

As noted above, the lla-hydroxy steroids prepared by the process of this invention are readily converted to the corresponding 11-keto and llfi-hydroxy derivatives by known methods. Thus an illustrative procedure for oxidation of the llu-hydroxy group to the corresponding 11- keto group is described by Peterson, Eppstein et al., in the Journal of the American Chemical Society, vol. 75, pages 412415 (January 20, 1953) wherein, for example, 4-pregnene-l1a,17a,21-triol-3,20-dione is converted to 4-pregnene-17a,21-diol-3,l1,20-trione. The conversion of ll-keto-4-monoenes to the corresponding 1lfi-hydroxy-4- monoenes is desirably accomplished by the procedure of Oliveto, Rausser et al., Journal of the American Chemical Society, vol. 78, pp. 1736-1738 (April 20, 1956). Similarly, llp-hydroxy-lA-dienes are conveniently prepared from the corresponding 11-keto-1,4-dienes by the procedure of Herzog, Payne et al., Journal of the American Chemical Society, vol. 77, pp. 4781-4784 (September 20, 1955).

The following examples are further illustrative of the invention:

EXAMPLE 1 4-pregnene-1 1 a,] 7 a, 21-tri0l-3,20-di0ne Agar slants containing medium No. 1 described above and 1.5% by weight of agar are sterilized for 15 minutes at 121 C. at a pressure of 15 pounds per square inch (p.s.i.). The agar slants are then cooled to about 28 C., slanted and inoculated with a vegetative growth of a culture, Beauveria (ATCC 13144) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 milliliters (ml.) of a similarly sterilized and cooled broth of medium No. 1 is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker (manufactured by the New Brunswick Scientific Company, New Brunswick, New Jersey) set at 280 revolutions per minute (r.p.m.). At the end of this period, 500 mg. of 4-pregnene-17a,21-diol-3,20-dione in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ethyl acetate solvent system, the paper being impregnated with water and acetone, the latter being present as a dispersant which evaporates rapidly. Incubation is continued until chromatography indicates complete transformation of 4-pregnene-17a,21- diol-3,20-dione to 4-pregnene-11a,17a,21-triol-3,20-dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to effect the isolation of the 4-pregnene-11u,l7a,21-triol-3,20-dione from the broth mixture. The chloroform is then evaporated off in vacuo and the residue is recrystallized from acetone whereby 460 mg. of 4-pregnene-11a,17a,21-triol-3,20-dione, melting point (M.P.) 212 C.214 C., identical in all respects with an authentic sample, is obtained.

EXAMPLE 2 4-pregnene-J 1 0a,] 7a,21-tri0l-3,20-di0ne Agar slants containing medium No. 2 described above and 1.5% by weight of agar are sterilized for minutes at 121 C. at a pressure of 15 pounds per square inch (p.s.i.). The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture, Beauveria (ATCC 13144) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 milli-' liters (ml.) of a similarly sterilized and cooled broth of medium No. 2 is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunssolvent system, the paper being impregnated with water,

which is dispersed with a solvent as in Example 1. Incubation is continued until chromatography indicates complete transformation of 4-pregnene-17a,21-diol-3,20- dione to 4-pregnene-11a,17a,21-triol-3,20-dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to etfect the isolation of the 4-pregnene 11a,l7a,21-triol-3,20-dione from the broth mixture. The chloroform is then evaporated off in vacuo and the residue is recrystallized from acetone.-

EXAMPLE 3 4-pregnene-J 1 ot,17a,21-fri0l-3,20-di0ne Agar slants containing medium No. 3 described above and 1.5% agar are sterilized for 15 minutes at 121 C. at a pressure of 15 pounds per square inch (p.s.i.). The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture, Beauveria'lATCC 13144) and incubated at a temperature of 28 C. until heavy sporulation occurs. I

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. '3 is then inoculated with spores from one of the heavily sporulated agar slants and incubated for a period of from 24 hours to 36 hours at 28 C. on a NewBrunswick rotary shaker set up at 280 r.p.m. At "the end of this period 500 mg. of 4-p-regnene-l7u,2l diol-3,20-dione in 5 ml. of ethanol is added. The flaskis replaced on the rotaryshaker and incubation continued for a period of from 24 hours to 30 hours during which latter period 10 ml. samples removed at intervals, from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ethyl acetate solvent system, the paper being impregnated with water and acetone. Incubation is continued until chromatography indicates complete transformation of 4-pregnenel7u,21-diol-3,20-dione to 4-pregnene-11a,17u,21-triol-3, 20-dione. The product is extracted from the broth mix- 1,4-pregnadiener11 a,1 711,21 -tr 1'0 l- 3,20-dione Agar slants containing medium No. 3 described above and 1.5% agar are sterilized for 15 minutes at 121 C. at a pressure of 15 pounds per square inch (p.s.i.). The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture Beauveria (ATCC 13144). and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500ml. of a similarly sterili'zedand cooled broth of medium number 3 is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 r.p.m. At the end of this period 500 'mg. of 1,4-pregnadiene-17a,21-diol-3,20-dione:in 5 ml. of ethanol is added. The flask is replaced on the rotary.

shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period 10 ml.

water and acetone.

EXAMPLE l6a-methylallopreg ane-11u,1 70:,21 -triol-3,20-di0ne (a) IGa-METHYLALLOPREGNAN-3B-OL-20-ONE (b) 1fia-METHYLALLOPREGNANE-SfLl 7 a-DIOL-20-ONE A solution of 3.5 g. of 16a-methylallopregnane-3,9-01- 20-one in 100 ml. of acetic anhydride containing 2.0 g. of p-toluene-sulfonic acid is kept at 100 C. for 6 hours; during this time about 8 ml. of distillate is removed each half hour by the application of vacuum. The resulting oily residue is dissolved in 50 ml. of benzene and washed three times with water; then with a solution of 1.0 g. of sodium acetate in 15 ml. of water. The benzene layer is thenstirred for 18 hours at room temperature with a mixture of 0.25 g. of sodium acetate in 6 ml. of commercial 40% peracetic acid. The excess peracetic acid is destroyed by the dropwise addition of a solution of 8 g. of sodium sulfite in 25 ml. of water, at a temperature of ca. 10-20". An additional 1 g. of sodium sulfite is then added and the mixture stirred overnight until a starch-iodide test is negative. The benzene layer is separated, washed three times with water and evaporated. The residue is dissolved in 200 ml. of methanol and 30 ml. of water containing 2.7 g. of sodium hydroxide, and the mixture refluxed for minutes. After neutralization with 3 ml. of acetic acid, the solution is concentrated under reduced pressure to a volume of ca. 30 ml., and this is poured into an ice-water mixture. The precipitated solid is removed by filtration and crystallized from methanol-water to give 2.5 g. of 16amethyl-allopregnane3fi,17u-diol-20-dione.

(o) 1ea-METHYLALLOPREGNANE-3B,17a,21-TRIOL-20- ONE ZI-ACETATE A solution of 300 mg. of 16a-methylallopregnane-3p, 17a-diol-20-one in 15 ml. of CF. chloroform (containing a few drops of chloroform previously saturated with hydrogen bromide) is brominated at 20 by the addition (over a two hour period) of 165 mg. of bromine in 10 ml. of chloroform. After removal of the solvent under reduced pressure, 10 ml. of dimethylformamide and one gram of sodium acetate are added. The mixture is stirred at 60-70 for 16 hours, then poured into water and the precipitated solid removed by filtration. Crystallization from acetone-hexane to give 200 mg. of 16ozmethylallopregnane-Bfi,17a,21-triol-20-one 21-acetate.

(d) 16a-METHYLALLOPREGNANE-171,21-DIOL3,20- DIONE ZI-ACETATE A- solution of 3.5 g. of l6a-methylallopregnan- 3fl,17a,2l-triol-20-one in 20 ml. of 80% acetone-water is cooled to 10 C. One drop of concentrated hydrochloric acid is added along with .200 mg. of N-bromoacetamide, and the mixture allowed to stand in the icebox for 20 hours. Excess sodium sulfite solution is then added, and the mixture concentrated under reduced pressure to a small volume to precipitate a crude product. This is crystallized from acetone-hexane to yield 3.0 g. of 16amethylallopregnane-17a,21-diol-3,20-dione 21-acetate.

(e) 16a-METHYLALLOPREGNANE-17 a,21-DIOL- 3,20-DIO NE One gram of 1oa-methylallopregnane-17a,21-diol-3,20- dione 2l-acetate is dissolved in 25 ml. of methanol and 5 ml. of water containing 0.2 g. of potassium bicarbonate. This solution is refluxed for /2 hour, then concentrated under reduced pressure. Water is added to the residue, and the resulting precipitate is filtered and dried. Crystallization from acetone-hexane gives 16a methylallopregnanane-l7a,2l-diol-3,20-dione.

(f) 1(ia-METHYLALIJOPREGNANEJ1a,17a,21 TRIOIr 3,20-DIO NE Agar slants containing meduim No. 1 described above and 1.5% agar are sterilized for 15 minutes and at 121 C. at a pressure of 15 pounds psi. The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture Beauveria (ATCC 13144) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medum No. 1 is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 rpm. At the end of this period 500 mg. of 16a-methylallopregnane-l7a,2l-diol-3,20-dione in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period 10 ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ethyl acetate solvent system, the paper being impregnated with acetone and water. Incubation is continued until chromatography indicates complete transformation of l6u-methylallopregnane-17a,2.1-diol 3,20 dione to 16v: methylallopregnane-l1a,17a,21-triol-3,20-dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to effect the isolation of the ll-hydroxylated steroid from the broth mixture. The chloroform is then evaporated ofi in vacuo and the residue is recrystallized from acetone to yield l6a-methylallopregnane-lla,l7cz,2l-tniol-3,20- dione.

EXAMPLE 6 16,8-methylallopregnane-l 1 0a,] 711,21 -triol-3,20-di0ne (a) 1 GB-METHYLALLOPREGNAN-E!fi-OL-20-ONE In the manner described in Example 5(a), 16fl-methylpregnenolone is reduced to 16B-methylallopregnan-Sfl-ol- 20-one by means of hydrogen and a palladium catalyst.

(b) 16fi-METHYLALLO PREGNANE-3fl,17a-DIOL-20-ONE In the manner described in Example 5(b), 16fi-methylallopregnan-3fl-ol-20-one is enol-acetylated, peroxidized and hydrolyzed to give 16fl-methylallopregnane-3p,17adiol-20-one.

(o) 1fifi-METHYLALLOPREGNANE-35,1711,21-TRIOL- 20-ONE 21-ACETATE In the manner described in Example 5(0), l6/3-methylallopregnane 35,170: diol 20 one is brominated and acetoxylated at (3-21 to give 16fl-methylallopregnane- 3fi,17a,21-triol-20-one 21-acetate.

(ti) 16fl-METHYLALLoPREGNANE-l7a,21-DIOL- 3,20-DIONE 21-ACETATE complete transformation of oxidized and hydrolyzed to (6) IGB-METHYLALLOPREGNANE-17a,21'DIOL- 3,20-DIONE lization from acetone-hexane gives 16,6 methylallopregnanc-17a,21-diol 3,20-dione.

(f) IGfl-METHYLALLOPREGNANE-l1a,17a,21-TRIOL- 3,20-DIONE Agar slants containing medium No. 1 described above and 1.5% agar are sterilized for 15 minutes at 121 C. at a pressure of 15 pounds p.s.i. The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture Beauveria (ATCC 13144) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. 1 is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 rpm. At the end of this period 500 mg. of lSB-methylallopregnane-17a,21-diol-3,20-dione in I111. ofethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period m l.

samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ethyl acetate solvent system, the paper being impregnated with acetone and water. Incubation is continued until chromatography indicates 1 fl-methylallopregnane- 1711,21 diol 3,20 dione to 16,B-methylallopregnane- The broth mixture in the Erlenmeyer flask is then extracted with chloroform to effect the isolation of the ll-hydroxylated steroid from. the broth mixture.

I The chlorofonm is then evaporated off 111 vacuo and the residue is recrystallized from acetone to EXAMPLE 7 I 16a-tert.-butylallopregnane-I 1 11,] 7a,21-tri0l-3,20-di0ne (a) -16a-TERT.-BUTYLALLOPRE GNAN-3fi-OL-20-ON E In the manner described in Example 5(a), 16a-tert.-

butylpregnenolone is reduced to 16 tert.-butylallopregnan-3fi-ol-20-one by means of hydrogen and a'palladium catalyst. p

(b) l6a-TERT.-BUTYLALLOPREGNANE-3B,17a-

DIOL-2O ONE i In the manner described in Example 5 (b), 16a-tert.- butylallopregnane-3 8-ol-20-one is enol acetylated, perpregnane-3 5,17a-diol-20-one.

(0) 1'6d-TERT.BUTYLALLOPREGNANE-lifi,170.,21-TRIOL- 20-ONE ZI-ACETATE In the manner described in Example 5(a), 16a-tert.- butylallopregnane-SB,17a-diol-20-one is brorninated and acetoxylated at C21 to give 16a-tert.-butylallopregnane- 313,17a,21-tnio1-20-one 21-acetate.

(d) i6a-TERT.-BUTYLALLOPREGNANE-17a,21-DIOL- 3,20-DIONE 21-ACETATE In the manner described in Example 5 (d), 16a-tert.- butylallopregnane-3B,17a,21-triol-20-one 21-acetate is oxidized to give 1Ga-tert-butylaIlo regnane-I7a,2l-diol- 3,20-dione Z Lacetate. l

give 16a-tert.-butylallo- (e) I 1'6a-TERT.-BUTYLALLOPREGNANE-l7:1,2LDIOL- 3,20-DIoNn One gram of 1Ga-terL-butylaIlOpregnane-17,21-di0l- 3,2'0-dione 21-acetate is dissolved in 251ml. of methanol and 5 ml. of water containing 0.2 g of potassium bicarbonate. This solution is refluxed for /2 hour, then concentrated under reduced pressure. Water isadded to the residue, and the resulting precipitate is filtered and dried. Crystallization from'acetone-hexane gives 16atert.-butylallopregnane-l7a,2l-diol-3,20-dione.

(f) 16a-TER'I.-BUTYLALLOPREGNANEi-1111,170.21- TRIOL-3,20-DIONE Agar slants containing medium No. '1 described above and 1.5% agar are sterilized for 15 minutes and at 121 C. at a pressure of 15 pounds p.s.i. The agar slants are thencooled to about 28f C. slanted an inoculated with a vegetable growth of a culture Beauveria (ATCC 13144) and incubated at a temperature of 28 C. until heavy sporul-ation occurs. p

A two literErle'nmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. '1 is then inoculated with. spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 rpm. At the end of this period 500 mg; of l6u-tert.butylallopregnane-17a,21-diol-3,20-dione in 5 ml. of ethanol is added. The flask is replaced on the rotaryshaker and incubation continued fora period of from 24 hours to 30 hours during which latter period 10 ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromato graphed on paper (according to the method of'Bush as modified by Shull) employing a toluene/ethyl acetate solvent system, the paper being impregnated with acetone and water. Incubation is continued 'until chromatography indicates complete transformation of l6a-tert'.- butylallopregnane-17ot,21-diol-3,20-dione to l6u-t6lttbutyl-allopregnane-l1a,17a,21 triol 3,20 dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to effect the isolation of the ll hydroxylated steroid from thebroth mixture. The chloroform is then evaporated ofi in vacuo and the residue is recrystallized from acetone to yield l6a-tert.-butylallopregnane-l 1a,17a,2l-lri0l-3,20-di0116.

' EXAMPLE 8 16B-ethylall0pregnane-11a,1 7ot,2l-tri0l-3,20di0ne a) 1GB-ETHYLALLOPREGNAN-3fl-OL-20-QNE A solution of 3.0 g. of IG-dehydropregnnolone'in'6 ml. of methylene chloride is addedto a solution of about 1 g. of diazoethane in 50ml. of ether at about 0 C. The mixture is kept at this; temperaturev for 6 hours, then allowed to warm to room temperature. Removal of the solvent leaves a residue of the intermediate pyrazoline, which is not further purified, but heated under reduced pressure to ca. 200? C. until the evolution of nitrogen ceases. The resulting oil is crystallized from ether to give 2.0. got 16-ethyl-16-dehydropregnenolone. This is dissolved in 50 ml. of acetic acid, thenreduced with hydrogen and a palladium on charcoal catalyst until 2 moles-of hydrogen have been absorbed. The catalyst is removed .by ,filtration, and the filtrate poured into water. The precipitatedlsolid is removed by filtration and crystallized from methanol-water to yield 1.5g.

. and hydrolyzed to give l6fl-ethylallopregnane 3p,17a

diol-ZO-one.

() 16B-ETHYLALLOBREGNANE-3BJ7a,21-TRIOL-20- ONE 21-ACETATE In the manner described in Example (a), lfifi-ethylallopregnane-BB,17ot-diol--one is brominated and acetoxylated at C-2l to give l6fl-ethylallopregnane-3fi,17a,- 2 l -triol-20-one 21-acetate.

(d) 1GEETHYLALLOPREGNANE-l7a.,21-DIOL-3,20- DIONE ZI-ACETATE In the manner described in Example 5(d), 16p-ethylallopregnane-3B,17a,21-triol-20-one Zl-acetate is oxidized to give l6/3-ethylal1opregnane-l7a,2l-dio1-3,20- dione 2l-acetate.

(6) 16fl-ETHYLALLOPREGNANE-17a,21-DIOL-3,20-DIONE One gram of 16,3-ethylallopregnane-17a,21-diol-3,20- dione 2l-acetate is dissolved in ml. of methanol and 5 ml. of water containing 0.2 g. of potassium bicarbonate. This solution is reflux-ed for /2 hour, then concentrated under reduced pressure. Water is added to the residue, and the resulting precipitate is filtered and dried. Crystallization from acetone-hexane gives l6p-ethy1allopregnane-17a,2l-diol-3,20-dione.

(f) meETHYLALLOPREGNANE-I1a,17a,21-TRIOL- 3,20-DIONE Agar slants containing medium No. 1 described above and 1.5% agar are sterilized for 15 minutes at 121 C. at a pressure of 15 pounds p.s.i. The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture Beauveria (ATCC 13144) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. 1 is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 r.p.m. At the end of this period 500 mg. of 16 3-ethylallopregnane-17a,21-diol-3,20-dione in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to hours during which latter period 10 ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ethyl acetate solvent system, the paper being impregnated with acetone and water. Incubation is continued until chromatography indicates complete transformation of 16B-ethylallopregnane-17a,21-diol-3,20-dione to 16B-ethylallopregnane-lla,17a,2l-triol-3,20-dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to effect the isolation of the ll-hydroxylated steroid from the broth mixture. The chloroform is then evaporated off in vacuo and the residue is recrystallized from acetone to yield 16 8-ethy1allopregnane-11a,17m,21- triol-3,20-dione.

EXAMPLE 9 I 6a-melhyl-I ,4-pregnadiene-11 a,1 7a,21 -triol-3 ,ZO-dione (a) 1Gil-METHYL-l,4-PREGNADIENE-17a.,21-DIOL- 3,20-DIONE 21-ACETATE A solution of 200 mg. of l6amethylallopregnane-lh, 2l-diol-3,20-dione 21-acetate obtained as described in Example 5 (a-d) in 5 m1. of dioxaneis dibrominated in positions 2 and 4 by the rapid addition of 130 mg. of bromine in 1 ml. of dioxane at room temperature. After /2 hour, the solution 'is' poured into water and the precipitated solid removed; by filtration and dried. Without further purification, this is dehydrobrominated by refluxing for 2 hours with 4 niLof dimethylformarnide containing 50 mg. ofv lithium carbonate and 50 mg. of lithium bromide. The mixture is then poured into dilute hydrochloric acid and is extracted with methylene chlo- 16 ride. The organic extracts are evaporated to a residue which is crystallized from methanol-water to yield mg. of IGa-methyl-1,4-pregnadiene-17u,21-diol-3,20-dione 21-acetate.

(b) 1fia-METHYIrlA-PREGNADIENE-l7a,21-DIOL- 3,20-DIONE One gram of 16a-methyl-1,4-pregnadiene-17a,21-diol- 3,20-dione 21-acetate is dissolved in 25 ml. of methanol and 5 m1. of water containing 0.2 g. of potassium bicarbonate. This solution is refluxed for hour, then concentrated under reduced pressure. Water is added to the residue, and the resulting precipitate is filtered and dried. Crystallization from acetone-hexane gives 16a-methyl-l,4- pregnadiene-l7a,2l-diol-3,20-dione.

(c) 16a-METHYL-1,4-PREGNADIENE-l1a,17:1,21-TRI0L- 8,20-DIONE Agar slants containing medium No. 1 described above and 1.5% agar are sterilized for 15 minutes at 121 C. at a pressure of 15 pounds p.s.i. The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture Beauveria (ATCC ,13144) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. l is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 r.p.m. At the end of this period 500 mg. of 16amethyl-l,4-pregnadiene-17a,2l-diol-3,20-dione in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period 10 ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ethyl acetate solvent system, the paper being impregnated with acetone and water. Incubation is continued until chromatography indicates complete transformation of 1Gut-methyl-1,4-pregnadienel7a,2l-diol-3,20-dione to l6a-methyl-1,4-pregnadienel1a,l7a,21-triol-3,20-dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to effect the isolation of the ll-hydroxylated steroid from the broth mixture. The chloroform is then evaporated off in vacuo and the residue is recrystallized from acetone to yield lfia-methyl-1,4-pregnadiene-l la,l7a,2l-triol-3,20- dione.

EXAMPLE 10 I 6 fi-methyl-I ,4-pregnadiene-I 1a,] 711,2 1 -triol-3,20-di0ne (a) 1GB-METHYL-l,4-PREGNADIENE-17a,21-DIOL- 3,20-DIONE 21-ACETATE In the manner described in Example 9(a), l6fi-methylallopregnane-17a,21-diol-3,20-dione ZI-acetate obtained by the procedure described in Example 6(a-d) is dibrominated in positions 2 and 4, then dehydrobrominated to give 16fi-methy1-1,4-pregnadiene-l7a,2l-diol-3,20-dione Zl-acetate.

(b) 1SB-METHYL-l,4-PREGNADIENE-1741,21-DI0L- 3,20-DIONE Agar slants containing medium N0. 1 dSQliibed above 17" and 1.5% agar are sterilizedfor 15 minutes at 121 C. at a pressure of 15 p.s.i. to about 28 C., slanted and inoculated with a vegetative growth of a culture Beauveria (ATCC 13144) and incubated at a temperature of 28 C. untilheavy sporulation' occurs.

Atwo liter Erlenmeyer flask containing 500 ml. of a similarlysterilized and cooled broth of. medium No. 1 is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours. at 28 C. on a New Brunswick rotary shaker set.

at 280 r.p.m. At the end of this period 500mg. of 1613- methyl-1,4-pregnadiene-17a,21-diol-3,20-dione in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to30 hours during which latter period ml. samples'removed at intervalsfrom the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) as noted above employing a toluene/ethyl acetate solvent system, the paper being impregnated with acetone and water. Incubation is continued until chromatography indicates complete transformation of l6/3-methyl-1,4- pregnadiene-17a,21 diol-3,20-dione to l6/8-methyl 1,4- pregnadiene-l'la,17u,21-triol-3,20-dione. The broth mixturein the Erlenmeyer flask is then extracted with chloroform to effect the isolation of the ll-hydroxylated steroid from the broth mixture. The chloroform is then evaporatd off in vacuo and the residue is recrystallized from acetone to yield lfl-methyl-1,4-pregnadiene-l1a,17a,21- triol-3,20-dione.

EXAMPLE 11 lda-terL-butyl-J,4-pregnadiene-11a,17a,21-tri0l-3,20-

' dione (a) 16a 'TERT.BUTYLt'1,4'PREGNADIENE17 a,21DIOL- 3,20-DIONE 21-ACETATE In the manner described in Example 9(a), 16a-ter.- butyla llopregnane-l7a,21-diol-3,20 dione 2l-acetate obtained by the procedure described in Example 7(a-d) is dibrominated in positions 2 and 4, then dehydrobrominated to give 16a-tert.-butyl- 1,4-pregnadiene-17a,21-diol- 3,20-dione21-acetate. p

(b). 1Ga-TERT.-BUTYL-1,4-PREGNADIENE-17a,21DIOL- 3,20-DIONE One gram of l62x-tert.-butyl-1,4-pregnadiene-l7u,21- diol.-3,2'0-dione"21-acetate is dissolved in 25 ml. of methanol and 5 m1. of. water containing 0.2 g. of potassium bicarbonate. This solutionis refluxed for /2 hour, then concentrated under reduced pressure. Water is added to the residue, and the resulting precipitate is filtered and dried." Crystallizationfrom acetone-hexane gives,16atert.-buty1-1,4-pregnadiene-l7ot,21-diol-3,20-dione.

(c) 1Ga-TER'L-BUTYL-l,4PPREGNADIENE-110.,17a,

. zi-mnroL-azo-nronn Agar slants containing mediunr No. 1 described above and 1.5% agar are sterilized for minutes at 121 C. at a pressure of 15 pounds p.s.i. Theagar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture Beauveria (ATCC 13144) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. lis then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at-28 C. ona New Brunswick rotary shaker set at 280 r.p.m. At the endjof this period 500. mg. of 160: tert.-butyl%-1,4-pregnadiene-l7bt,2l-diol-3,20-dione in 5 The agar slants are then cooled.

18 extracted withchloroform are then chromatographed on paper (according to the method of Bush as modifiedby Shull) employing a toluene/ethyl acetate solvent'systern, the paper being impregnated with acetone and water.

, Incubation is continued until chromatography indicates (a) lfifl-ETHYL-l,4-PREGNADIENE-l7a,21-DIOL-3,20- DIONE 21-ACETATE In the manner described in Example 9(a), 16l9-eth* ylallo pregnane 17a,21-diol-3,20-dione 21-acetate obtained by the procedure described in Example 8(a-d) is dibrominated in positions 2 and 4, then dehydrobrominated to give 16 3-ethyl-1,4-pregnadiene-l7a,2l-diol3,20-di one 2l-acetate.

(b) 1Gfi-ETHYL-l,4-PREGNADIENE47a,21-DIOL- 3,20-DIONE One gram of 16 8-ethyl-1,4-pregnadiene-l7a,2ldiol- 3,20-dlone 21-acetate is dissolved in 25 ml. of methanol and 5 ml. of water containing 0.2 g. of potassium bicar bonate. This solution is refluxed for /2 hour, then concentrated under reduced pressure. Water is added to the residue, and the resulting precipitate is filtered and dried. Crystallization from acetone-hexane gives 16/3-ethyl-1,4- pregnadiene-l7ot,21-diol-3,20-clione.

(c) ltlfl-ETHYL-l,4-PREGNADIENE-11a,17a,21-TRIOL- 3,20-DIONE Agar slants containing medium No. 1 describediabove and 1.5% agar are sterilized for 15 minutes at 121 C. at a pressure of 15 pounds p.s.i. The agar slants are then cooled to, about 28 C. slanted and inoculated with a vegetative growth of a culture Beauveria (ATCC.

13144) and incubated at a temperatureof 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. 1 is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hoursto 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 r.p.m. At the end'of this period 500 mg. of l6B-ethyl-1,4-pregnadiene-l7u,21-diol-3,20-dione in 5 ml; of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of'from 24 hours to 30 hours during which latter period 10 ml. same ples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ethyl acetate solvent system; the paper being impregnated with acetone and water: In cubation is continued until chromatography indicates complete transformation of 1 SB-ethyl-1,4-pregnadiene-17 ot,2 1-

diol-3,20-dione to l6fi-ethyl-1,4-pregnadiene 1-1dc, 1701,21

H EXAMPLE s Agar slants containing medium No. 1 described above. and. 1.5 by weight agar are sterilized for15.minutes."'at

The chloroform isthen evaporated off in.

19 121 C. at a pressure of 15 pounds psi. The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture Beauveria (ATCC 13144) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. 1 is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 r.p.m. At the end of this period 500 mg. of 16cx-methylallopregnane-17a,21-diol-3,20-dione, prepared as described in Example 5(a-e), in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ethyl acetate solvent system, the paper being impregnated with acetone and 'Water. Incubation is continued until chromatography indicates complete transformation of l6a-methylallopregnanel7a,2l diol 3,20 dione to 160: methylallopregnane- 1l0t,17cc,21t1'iOl-3,2O-dl0l16. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to effect the isolation of the ll-hydroxylated steroid from the broth mixture. The chloroform is then evaporated off in vacuo and the residue is recrystallized from acetonehexane, to yield 16a-methylallopregnane-1la,l7 x,2l-triol- 3,20-dione.

EXAMPLE 14 16fl-methyIall0pregnane-1 10c, l'7a,21-tri0l-3,20-dione Agar slants containing medium No. 1 described above and 1.5% by weight agar are sterilized for 15 minutes at 121 C. at a pressure of 15 pounds p.s.i. The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture Beauveria (ATCC 13144) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. 1 is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 r.p.m. At the end of this period 500 mg. of 168- methylallopregnane-l7u,2l-diol-3,20-dione, prepared as described in Example 6(ae), in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period 10 ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ethyl acetate solvent system, the paper being impregnated with acetone and water. Incubation is continued until chromatography indicates complete transformation of 16fi-methylallopregnane-17u,21-diol-3,20- dione to 16B-methylallopreganne-l1a,l7a,21-triol 3,20- dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to effect the isolation of the ll-hydroxylated steroid from the broth mixture. The chloroform is then evaporated OH in vacuo and the residue is recrystallized from acetone-hexane to yield 16fi-methylallopregnane-l 1a,17oz,21-trl0l-3,20-dl011.

EXAM PLE l5 I6a-tert.-butylalloprcgizane-I1 05,1 7 41,21 -tri0l-3,20-dione Agar slants containing medium No. 1 described above and 1.5% agar are sterilized for 15 minutes at 121 C. at a pressure of 15 pounds p.s.i. The agar slants are then cooled to about 28 C., slanted and inoculated with a vegetative growth of a culture Beauveria (ATCC 13144,)

20 and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. 1 is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 r.p.m. At the end of this period 500 mg. of 16a tert.-butylallopregnane-17a,21-diol-3,20-dione, prepared as described in Example 7 (a-e), in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period 10 ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ethyl acetate solvent system, the paper being impregnated with acetone and water. Incubation is continued until chromatography indicates complete transformation of 16a-tert.-butylallopregnane- 17a,21 diol-3,20-dione to 16a-tert.-buty1allopregnane- 11a,17a,21-triol-3,20-dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to effect the isolation of the ll-hydroxylated steroid from the broth mixture. The chloroform is then evaporated off in vacuo and the residue is recrystallized from acetone-hexane to yield 16a-tert.-butylallopregnane-l1a,17a,- 2 1-triol-3 ,20-dione.

EXAMPLE 16 1 6 fl-ethylallopregnane-I 1 11,1 7a,21-tri0l3,20-dione Agar slants containing medium No. 1 described above and 1.5% agar are sterilized for 15 minutes at 121 C. at a pressure of 15 pounds p.s.i. The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture Beauveria (ATCC 13144) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. 1 is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 r.p.m. At the end of this period 500 mg. of 16p-ethylallopregnane 17a,2l-diOl-3,20-di0116, prepared as described in Example 8(a-e), in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period 10 ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ ethyl acetate solvent system, the paper being impregnated with acetone and water. Incubation is continued until chromatography indicates complete transformation of 1dfl-ethylallopregnane-l7m,- 2l-diol-3.20-di0ne to 16,8-ethylallopregnane 1101,1711,- 21-trol-3,20-dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to eifect the isolation of the ll-hydroxylated steroid from the broth mixture. The chloroform is then evaporated otf. in vacuo and the residue is recrystallized from acetone-hexane to yield -ethylallopregnane-11a,17a,21-triol-3.20 -.dione.

EXAMPLE 17 1 6a-methyl-1 ,4-pregnadiene-I I 01,1 71x21 -tri0l-3,20-dzone Agar slants containing medium No. 1 described above and 1.5 by weight agar are sterilized for 15 minutes at 121 C. at a pressure of 15 pounds p.s.i. The agar slants are thencooled to about 28 C. slanted and inoculated with a vegetative growth of a culture Beauveria (ATCC 13144) and incubated at a temperature of 28 C. until heavy sporulation occurs. 7

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No. 1 is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on'aNew Brunswick rotary shaker set at 280 r.p.m. At the end of this period 500 mg. of l6ot-methyl 1,4-pregnadiene-l7a,2l-diol-3,20-dione, prepared as described in Example 9(a, b), in ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to- 30 hours during which latter period 10 ml. samples removed at intervals from the Erlenmeyer flask and extracted With chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ethyl acetate solvent system, the paper being impregnated with acetone and water. Incubation is continued until chromatography indicates complete transformation of l6a-methyl-1,4-pregnadiene- 170:,21 diol-3,20-dione to l6a-metl1yl-1,4-pregnadienellor,17a,2l-triol-3,20-dione. The broth mixture in the Erlenmeyerfias k is then extracted with chloroform to effect the isolation of the ll-hydroxylated steroid from the broth mixture. The chloroform is then evaporated ofi in vacuo and the residue is recrystallized from acetone to yield l6ot-methyl-l,4-pregnadiene-11u,l7a,21 triol 3,20-dione.

EXAMPLE 18 16,8-methyl-1,4-pregnadiene-11 a,1.7a,21-triol-3,20-dione Agar slants containing medium No. 1 described above and 1.5% by weight agar are sterilized for 15 minutes at 121 C. at a pressure of 15 pounds p.s.i. The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture Beauveria (ATCC 13144) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized and cooled broth of medium No.- l is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 r.p.1n. At the end of this period 500 mg. of 16}3- methyl 1,4-pregnadiene-17or,2l-diol-3,20-dione, prepared as described in Example 10(a, b), in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period 10 ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ethyl acetate solvent system, the paper being impregnated with acetone and water. Incubation is continued until chromatography indicates complete transformation of l6 S-meth'yl-l,4-pregnadiene- 17a,21-diol-3,20-dione to 16B-methyl-l,4-pregadiene-l1a,- 17a,21-triol-3,20-dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to-efiect the isolation of the-l l-hydroxylated steroid frorn'the broth mixture. The chloroform is then-evaporated off in vacuo and the residue is recrystallized from acetone to-yield' 1 6 a-tern-b utyl-I ,4-pregnadiene-JJ ,1 70:,21 -rri0l- 3,20-dione Agar slants containing medium No. 1 described above and 1.5% agar are sterilized for 15' minutes at 121 C. at a pressure of 15 pounds p.s.i. The agar, slants are then cooled to about 28 C., slanted and inoculated with a vegetative growth of a culture Beauveria '(ATCC 13144) andincubated at a temperature. of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized andcooled broth of medium No; 1

at 280 rpm. At: the end otthis period.-500=-mg;- of

160: tert.-butyl-1,4-pregnadiene-17a,211-diol-3,20-dione, prepared as described in Example 1 1(a,-b), in 5 ml. of ethanol is added. The flask isreplaced on the rotary shaker. and incubation. continued for a period of from 24 hours to 30' hours during which latter period 10' ml.

samples removed at intervals from the Erlenmeyer flask} and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ethyl acetate solvent system, the paper, being impregnated with acetone and water. Incubation is continued until chromatography indicates complete transformation of 16a-tert.-butyl-lg4 pregnadiene-l7a,21-diol-3,20-dione to 16a-tert.-butyl-1,4-pregnadiene-l1u,17a,21-t1iol-3;20-dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform" to effect the isolation of the llrhydroxylated steroid from the broth mixture. The chloroform is then evaporated off in vacuo' and the residue is recrystallized from acetone to yield. 16u-tert.-butyl-1,4-pregnadiene-11a,17.a,21-triol- 3,20-dione.

EXAMPLE 20 16flethyl-1,4-pregnudiene llon]7u,21-triol-3,20ra'ione Agar slants containing'medium No. 1 described above and 1.5% agar are sterilized for 15 minutes at 121 C; at a pressure of 15 pounds psi The agar slants are then cooled to about 28 C., slanted and inoculated with a vegetative growth of a culture Beauveria (ATCC 13144) and incubated at a temperature of 28 C. until heavy sporulation occurs. p

A two liter Erlenmeyerflask containingSOO'mIK'of a" similarly sterilized and cooled broth of medium No. 1' is then inoculated with spores from one of the heavily sporula'ted agar slants and 10 ml. of anti-form, Larex, (1 percent octadecanol in lard oil) addedto the culture medium and incubated 'from 24 hours; to 36 hours ,at 28 C. on a New Brunswick'rotary shaker set at 280"r.pimi At the end of this period500 mg. of- 16,8-ethyl-1-,4-preg-. nadiene-17u,21-dio1-3,20-dione, prepared as described in Example 12(a, b), in 5 ml. of ethanol isadded. The

flask is replacedv on the rotary shaker and incubation residue is recrystallized from acetoneahexane to yield 16,6-ethyl-1;4apr'egnadiene-l1u;17d,21-triol-3,20 dione;

' EXAMPLE 21 (a) ma-METHYL-aeDIoHLoao-pnnonnnuaeon' gnomes-ACETATE ml. of, carbon tetrachlorideis cooled to"23' C.

To this. stirred solution is added .di'opwise over a ten minute period 1.16 g. 0.016 mole) of chlorine con";

tained in 20 ml. of carbon tetrachloride. Thereaction solution is then allowed to warm to approximately 15 C while maintaining the stirring. Methylene chloride (70 ml.) is then added and the solution-is washed'suc cessively with" dilute. hydrochloric acid, water, aqueo sodium bicarbonate, and their water.) The organic iph is dried ever magnesium sulfatepfilter'ed, an the filtrate evaporated to near dryness, and"treat'ed with methanol;

zaa-methymre nae-zro rmala ial-sudden" 23 whereupon there is obtained l6a-methyl-5,6-dichloro pregnane-3/3-ol-20-one-3-acetate, M.P. 195-196.

(b) lfia-METHYL-Ei,S-DICHLORO-PREGNANE-SJ'M- DIOL-20-ONE-3-ACETATE To 125 m1..of acetic anhydride is added 5.0g. (0.0111 mole) of 16m-methyl-5,6-dichloro-pregnane-3,8rol-20-one- B-acetate and 1.0 g. (0.0053 mole) of p-toluene sulfonic acid monohydrate. The solution is refluxed for 6 hours while maintaining a constant distillation rate so that there is collected during this time 100 ml. of distillate. The reaction solution is cooled and poured into 400 ml. of water and the mixture stirred in order to facilitate the hydrolysis of the acetic anhydride. The aqueous mixture is extracted two times with 100 ml. portions of benzene. The combined benzene solution is washed two times with 50 ml. portions of water and one time with 50 ml. of a 2% solution of sodium acetate in water. The organic phase is dried over magnesium sulfate and filtered. The filtrate is concentrated under vacuum to a volume of 70 ml. of benzene and then stirred for 19 hours with a mixture of 0.52 g. of sodium acetate in 12 ml. of commercial 40% peracetic acid. Excess peracetic acid is then destroyed by the dropwise addition of a solution of 15.6 g. of sodium sulfite in 53 ml. of water, while maintaining the temperature between 1020 C. An additional 1.7 g. of sodium sulfite is then added, and the mixture is stirred until a starch-iodide test is negative. The benzene layer is separated, washed three times with water, and evaporated. To the residue is added 200 ml. of methanol and 20 ml. of water containing 6.2 g. of potassium bicarbonate. The reaction solution is refluxed for 2 hours, and after the addition of 4 ml. of acetic acid, is concentrated under vacuum to a volume of 40 ml., which is distilled with 20 volumes of water. Separation of the resultant precipitate and crystallization from ethyl acetate affords 16a-methyl-5,6-dichloro-3fi,17a-diol-20- one, M.P. 216-217 C.

(c) 16a-METHYL5,G-DICHLORO-PREGNANr 20-0NE- To a solution containing 1.0 g. (0.0024 mole) of 16amethyl 5,6-dichloro-pregnane-20-one-3,3,17e-diol-20-one in 50 ml. of CF. chloroform (containing a few drops of chloroform previously saturated with hydrogen bromide) maintained at 20 C. is added (over a 20 minute-period) 0.396 g. of bromine in 6 ml. of chloroform. The solution is stirred an additional 20 minutes and then washed 3 times with water, dried over magnesium sulfate, and filtered. The filtrate is concentrated under vacuum to 20 ml. and stirred at 45 C. with 20 ml. of methanol and 0.72 g. (0.0048 mole) of sodium iodide for 1 hour and thirty minutes. Water is added and the mixture extracted with chloroform. The combined chloroform extracts are washed with water, dried over magnesium sulfate, and filtered. The filtrate is evaporated to dryness and the residue dissolved in 40 ml. of acetone and 2 ml. of water containing 0.72 g. (0.0063 mole) of potassium acetate. The solution is refluxed for 18 hours, evaporated to near dryness and water added. The resultant precipitate is filtered, washed with water, and then crystallized from isopropanol to obtain 16e-methyl-5,6-dichloro-pregnane-20-one-313,17,21-triol 21-acetate.

(d) 16a-METHYL-5,GDICHLOROPREGNANE-17a,21-

- DIOL-3,20-DIONE To a stirred solution of 1.5 g. of 16a-methyl-5,6-dichloropregnane-20-one-3f3,17a,21-triol-2i1-acetate in 40 ml. of acetic acid and 4 ml. of water maintained at 10 C. is first added over a 20 minute-period a solution containing 0.34 g. of chromium trioxide in ml. of acetic acid and 0.5 ml. of water, and then overa four-minute period 0.19ml. of concentrated sulfuric'acid. Thereaction mixture is stirred for two hours, then diluted with water, and'extracted with chloroform. Thecombine'd chloroform extracts (150' ml.) are washed successively with water (70 ml.) three times with a 3% sodium bi- 24 carbonate solution and finally with 60 ml. of water. The chloroform solution is dried over magnesium sulfate and evaporated to dryness. The residue upon crystallization from acetone-hexane affords 16e-methyl-5,6-dichloropregnane-l7tz,21-diol-3,20-dione.

(e) 16e-METHYL-4-PREGNENE-l7a,21-DIOL-3,20-DIONE 2l-ACETATE To a stirred solution of 1.5 g. of 16e-methyl-5,6-dichloropregnanel7a,21-diol-3,20-dione 21-acetate in 100 ml. of acetic acid maintained at 75 C. there is added 1g. of zinc dust, followed after 45 minutes by an additional gram of zinc dust. After another 45 minutes at 75 the reaction solution is filtered to remove the insoluble zinc. The filtrate is air evaporated to approximately 5 ml. and then diluted with 30 ml. of water. The resultant precipitate is filtered, washed with water, and after crystallization from acetone-hexane affords 16amethyl-4-pregnene-17a,21-cliol-3,20-dione 21-acetate.

(f) 1(ia-METHYL-l-PREGNENE-17a,21-DIOL-3,20 DIONE Method I.One gram of 16a-methyl-4-pregnene-17a, 21-diol-3,20-dione 21-acetate is dissolved in 25 ml. of methanol and 5 ml. of water containing 0.19 g. of potassium bicarbonate. This solution is refluxed for 35 minutes and then after the addition of 0.12 ml. of acetic acid is concentrated under vacuum to a residue, to which 15 ml. of water is added. The resulting precipitate is filtered and dried. Crystallization from acetonehexane gives 16u-methyl-4-pregnene-17a,2l-diol-3,20-dione.

Method II.Alternatively, =16a-methyl-4-pregnene-17a, 21-diol-3,20-dione is prepared from l6a-methyl-5,6-dichloro-pregnane-ZO-one 319,170:,2l-iflOl-21-3C0i3i6 as follows:

To a solution of 1.0 g. of l6u-methy1-5,6-dichloropregnane-20-one-3B,17u,21-triol 21-acetate in 150 ml. acetone under an atmosphere of carbon dioxide there is added a solution of chromous chloride (prepared from 40 g. of amalgamated zinc dust, 8 ml. concentrated bydrochloric acid, ml. water and 20 g. chromic chloride). After standing at room temperature for two hours, 20 ml. of water is added and the solution is evaporated to approximately 10 ml., diluted with 30 ml. water, and extracted 3 times with 30 ml. portions of methylene chloride. The combined methylene chloride extracts are washed with water and then dried over magnesium sulfate and filtered. The filtrate is evaporated to a crystalline residue, which upon treatment with acetonehexane gives 16a methyl-5pregnene-20-one-3 [3,17e,21-triol-21- acct-ate, which is then employed in the following procedure:

A medium having a composition of 10 grams of yeast extract (Difco), 4.5 g. of potassium dihydrogen phosphate and 4.7 grams of disodium hydrogen phosphate monohydrate is diluted to 1 liter with tap water, dispersed in aliquots of 100 ml. into 300 ml. Erlenmeyer flasks and sterilized for 20 minutes at 15 pounds steam pressure. The pH after sterilization is 6.8.

The sterile medium in the flasks is inoculated with agar slant of Flavobacferium dehydrogenans var. hydrolyticum or with 1% by volume of a 24-hour broth culture. The inoculated flask is placed in a shaking machine set at 248 strokes per minute, in an incubator kept at 30 C. The shake cultures are subjected to continuous illumination.

Twelve to twenty-four hours later, 200 mg. of 16amethyl-S-pregnene-20-one-3 3,17a,21-triol 2l-acetate dissolved in 5 ml. of ethanol is added to each flask. The pH is'now 7.27.4.

After 60 hours of shaking, the fermentation is stopped. The final pH is 7.5-7.8. The pH is then adjusted to 3.5 with hydrochloric acid and the fermentation liquors autoclaved for 15 minutes at 15 pounds steam pressure. After cooling, the broth is filtered with the aid of 2% of Filter-Gel (i.e. diatomaceous earth). Both the filtrate arid the filter cake are extracted thoroughly with chloroform and the combined extracts evaporated to dryness in vacuo. The combined residual solid from the ten flasks is crystallized from acetone-hexane to give l6ot-methyl-4-pregnene-17a,21-diol-3,20-dione.

Alternatively, 16oz methyl--pregnene-20-one-3B,17a, 2-1-triol-2l-acetate (1.9 g.) is dissolved in 200 m1. of acetone (distilled from permanganate) andcooled to under an atmosphere of nitrogen. To this stirred solution is added rapidly, but dropwise, 1.4 ml. of standard chromium trioxide reagent (prepared from 13.36 g. of chromium trioxide in 11.5 ml. of concentrated sulfuric acid diluted with water to a volume of 50 ml.). After 5 minutes, water is added and the resulting precipitate was washed well with water. In this manner there is obtained 160:. methyl-S-pregnene-17a,2|1-diol-3,20-dione. When 1.0 g. of this latter substance is treated with 0.3 g. of potassium bicarbonate in 40 ml. of methanol and 4 ml. of water at reflux for 35 minutes under a nitrogen atmosphere, there is obtained, after the usual work-up, the desired 16a-methyl-4-pregnene-l7a,2l-diol-3,20-dione.

( g) 16a.-METHYL-4-PREGNENE'-11a,17a,21-TRIOL- 3,20-DIONE Agar slants containing medium No. 1 described above and 1.5% agar are sterilized for 15 minutes at 121 C. at a pressure of 15 pounds p.s.i. The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture Beauveri a (ATCC 13144) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer flask containing 500 ml. of a similarly sterilized'and cooled broth of medium No. 1 is then inoculated with spores from one of the heavily sporul-ated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 r.p.m. At the end of this period 500 mg. of 16ot-methyl-4pregnene-17a,21-diol-3,20-dione in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from .24 hours to 30 hours during which latter period 10 ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ ethyl acetate solvent system, the paper being impregnated with acetone as a dispersant and water. Incubation is continued until chromatography indicates complete transformation of l6u-methyl-4pregnene-17u,21-diol-3,20-dione to l6a-methyl-4-pregnene- 11a,17a,21-triol-3,20-di0ne. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to elfect the isolation of the ll-hydroxylated steroid from the broth mixture. The chloroform is then evaporated off in vacuo and the residue is recrystallized from acetone to yield 16a-methyl4-pregnene-11a,17a,21-triol- 3,20-dione.

I EXAIWPLE 22 16/3-methyl-4-pregnene-l 1 a,1 7a .21-triol-3,20-dione (a) 1GB-METHYL-l6a.,17a-EPOXY-5-PREGNEN-3B- OL--ONE To a stirred solution of 395 g. (1. 2 moles) of 16 methyl-5,16pregnadien-Bfi-ol-ZO-one in a mixture of 1200 ml. of chloroform and 3 000-ml. of :methanol was added 7 5.4-ml..of 50% sodium hydroxide, 300 ml. of water and 540 ml. of. 30% hydrogen peroxide. After stirring for 72 hours at room temperature, 1500 ml. of water were added and the mixture was. then neutralized with acetic acid. The organic solvents were removed by steam distil- (b) 3B-ACETOXY-1BBMETHYL-IG IYa-EPOXY-E PREGNENE 20-ONEi A solution of 657 g. of the crude epoxy compound in 2000 ml. of dry pyridine and 657 ml. of acetic anhydride was kept at 60 C. for 4 hours. After approximately one I hour, crystals of the acetate appeared. The acetylation mixture was poured into ice water and the product fil tered and dried .to give a yield of 712 g., M.P. 163174'. Recrystallization from acetone gave a product without ultravioletadsorption; yield 557 g. (75.5 MP. 180-- 182, 16.7 (1% dioxane).

(c) 35-ACETOXY-16-METHYLENE-5-PREGNEN-17aoL-20-0NE To a stirred solution of 275g. (0.71 mole) of 3B- acetoxy 16p methyl-l6a,17a-epoxy pregnene-ZO-one in 6000 ml. of glacial acetic acid at 27 C. was added 6.05 g. of HBr (0.075 mole) in 100 ml. of glacial acetic acid; Within one minute crystallization of the product ensued. After stirring an additional 5 minutes, the methylene compound was filtered, washed with aqueous acetic acid and finally with Water. The productwas dried at 110 C. under vacuum to break the acetic acid solvate; yield 207 g. (76.4%, MP. 200 Kofler hot bench, [M l09 (1% dioxane). The acetic acid mother liquor, not including the water wash, was treated with 8 g. of potassium acetate to neutralize the HBr'present. The volume was reduced to approximately one liter and cooled to give an additional 34 g. of product, melting at 200 (Kofier hot bench) [aJ (1% dioxane). This was an overall yield of 88.9% onthe two fractions of 3B-acetoxy-l6-methylene 5 pregnene-l7u-ol-20-one. The sample for analysis was recrystallized from acetone, M.P. 199201 0., 200 C. (Kofler hot bench) .[a] l10 (1% dioxane).

a ea-Aon'rzoxr-ieaMnrHYL-ePREGNEN-Ma- O L20-0NE A solution of 367 g. (0.95 mole) of the 3B-acetoxy-16- methylene-S-pregnene-l7or-ol-20-one in 4.671. 'of tetrahydrofuran containing 59.7 ml. of triethylamine, was hydrogenated under 10 pounds pressure at 23 C. in the presence of 184 g. of 5% palladium on carbon catalyst; After minutes, the absorption of hydrogen stopped at 1 mole. The sterol solution, after removal of the catalyst, was concentrated under reduced pressureto a heavy slurry of crystals, and then 2.5 l. of hexane was added. The pure product, 3,8-acetoxy-l6B-methyl-5-pregnene-17a-ol-20-one was filtered and washed with hexane to give 315 g. (85.7%), M.P. 168 C. '(Kofler hot bench) -l7.'9- (1% dioxane)-.' The mother liquor upon concentration gave an additional 34. g. of pure product; M.P. 169 (Kofler hot bench), [M '16.5 (1% dioxane). This was an overall yield of 94.5%.

The analytical sample was recrystallized from acetonehexane, M.P. 169-170 C. [(1113 ---20.6 (1% dioxane).

Analysis.-"-Found: C, 74.06; H, 9.2. Calcd.: C, 74.30,; H, 9.15. a

(e) 1err-METHYL-5-PREGNENESeHa-DIOLQO O'ATETT To a refluxing solution of'50 g. of. 3,8-acetoxya-l6flmethyl-S-pregnene-17ot-ol 2=0-one in 2500 m1. of methanol, was added 25 g. of sodium hydroxide dissolved in 250 ml. water.

minutes andthen acidified with 40 ml. of acetic acid.

The slurry was poured-into 4.1. of ice water, filtered, and

washed neutral to give 43 g. (97%) of crude product melting with decomposition at 253-26'0 (Kofler hot bench), [M 16.8. Recrystallization from methanol onemelting at 260 (Kofler hot bench).

f sazl-nmonroxr ie -MET YLe-PREGNENEL A solution at 163g. of bromine (1.02 moles) it 500' Crystallization took place almost immediately; The reaction was stirredat reflux for 101 ml. of chloroform was added dropwise over a 25 minute period to 194.3 g., 0.5 mole, of 16,8-methyl--pregnene- 3fi,17a-diol-20-one-3 acetate in 1750 ml. of chloroform with stirring at 2530 C. Stirring was continued for an additional 5 minutes when decolorization was complete. The solution was neutralized with 100 g. of solid sodium bicarbonate and filtered. The chloroform solution of the 5,6,21-tribromo compound was concentrated to 1400 ml. under reduced pressure below 40 C. Methanol (1250 ml.) and 375 g. of sodium iodide were added and the mixture was stirred for one hour at 43 to 48 C. The reaction mixture was cooled to C. by the addition of 3750 ml. of ice water and 125 g. of sodium bicarbonate was added. With good mechanical agitation 8.5% hydrazine hydrate solution was added dropwise until the iodine color was discharged. The hydrazine hydrate required (140 ml.) calculated to be 92% of the theoretical amount. The chloroform layer was separated from the aqueous phase and concentrated to almost dryness under reduced pressure below 40 C. The crude 21-iodo compound was then stirred 17 hours at refluxing temperatures with 1250 ml. of acetone, 250 ml. of water and 100 g. of potassium acetate. The acetone was then removed by steam distillation and the resulting crystalline product filtered and dried. The crude product was dissolved in 7 l. of ethyl ether, treated with decolorizing charcoal, and the product was finally crystallized from a mixture of ether-hexane. The product, 3p,2l-diacetoxy-16 3-methyl- 5-pregnene-17a'ol-20-one was filtered and dried at 95 C. under vacuum to give 127 g. (57%) M.P. 170 (Kofler hot bench) [a] (1% dioxane).

(g) lGfi-METHYL-l7a.,21-DIHYDROXY-4-PREGNENE- 3,20-DIONE A medium having a composition of 10 g. of yeast extract (Difco), 45 g. of potassium dihydrogen phosphate and 4.7 g. of disodium hydrogen phosphate monohydrate is dissolved in 1 liter of tap water, dispersed in aliquots of 100 ml. into 300 ml. Erlenmeyer flasks and sterilized for minutes at 15 pounds steam pressure. The pH after sterilization is 6.8.

The sterile medium in the'flasks is inoculated with agar slant of F Iavobacterium dehydrogenans var. hydrolyticum (Rutgers Collection No. 130) or with 1% by volume of a 24-hour broth culture. The inoculated flask is placed in a shaking machine set at 248 strokes per minute, in an incubator kept at 30 C. The shake cultures are subject to continuous illumination.

Twelve to twenty-four hours later, 200 mg. of 165- methyl-35,21-diacetoxy 17a hydroxy 5 pregnen-20- one dissolved in 5 ml. of 95% ethanol is added to each flask. The pH is now 7.2-7.4.

After 60 hours of shaking, the fermentation is stopped. Reaction was followed by specific rotation until complete and the sterol extracted from the fermentation broth with ethyl acetate. The extracts were evaporated to dryness and sludged in 10 volumes of ethyl ether to remove impurities and unconverted starting material to give a substantially pure IGfJ-methyl-l701,2l-dihydroxy-4-pregnene- 3,20-dione, M.P. 214 C. Recrystallization from acetoneether gives a pure product, M.P. 219 C.-220 C., +130 C., (c.=1% dioxane).

(h) 16fl-METHYL-1111,17a,21-TRIHYDROXY-4PREGNENE- 3,20-DIONE Agar slants containing medium No. 1 described above and 1.5% agar are sterilized for 15 minutes at 121 C. at a pressure of 15 pounds p.s.i. The agar slants are then cooled to about 28 C. slanted and inoculated with a vegetative growth of a culture Beauveria (ATCC 13144) and incubated at a temperature of 28 C. until heavy sporulation occurs.

A two liter Erlenmeyer fiask containing 500 ml. of a similarlysterilized and cooled broth of medium No. 1 is then inoculated with spores from one of the heavily sporulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswick rotary shaker set at 280 r.p.m. At the end of this period 500 mg. of 16 3-methyl- 170:,21-dihydroxy-4-pregnene-3,20-dione in 5 ml. of ethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 hours to 30 hours during which latter period 10 ml. samples removed at intervals from the'Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull), employing a toluene/ethyl acetate solvent system, the paper being impregnated with acetone and water. Incubation is continued until chromatography indicates complete transformation of l6fi-methyl-17a,21-dihydroxy-4- pregnene-3,20-dione to l6{3-methyl-l la,17a,2l-trihydroxy- 4-pregnene-3,20-dione. The broth mixture in the Erlenmeyer flask is then extracted with chloroform to effect the isolation of the ll-hydroxylated steroid from the broth mixture. The chloroform is then evaporated off in vacuo and the residue is recrystallized from acetone to yield pure 16 '3-methyl-1la,17a,21 trihydroxy 4 pregnene 3,20- dione.

This latter compound can be converted to l6fi-methyl- 11e,17a,2l-trihydroxy1,4-pregnadiene-3,20-dione by reaction with Corynebacterium simplex by the described procedure in United States Patent 2,837,464, issued June 3, 1958, to Arthur Nobile.

EXAMPLE 23 I6}8-mezhyl-4-pregnene-11a,]7a,21-tri0l-3,20-dione (a) 21-ACETOXY-16fi-1\IETHYI.r5-PREGN ENE-35,170.- DIOL-ZO-ON E A solution of 16.3 g. of bromine (0.102 mole) in 50 ml. of chloroform was added dropwise over a 30 minute period to 17.33 g. (0.05 mole) of l6B-methyl-5-pregnene- 3B,17a-dihydroxy-20-one in 1900 ml. of chloroform with stirring at 2530 C. A small amount of hydrogen bromide gas was introduced at the start of the bromine addition to catalyse the bromination. Stirring was continued for an additional 10 minutes when decolorization was complete. The solution was neutralized with 50 g. of solid sodium bicarbonate and filtered. The chloroform solution of the 5,6,2l-tribromo compound was concentrated to 1400 ml. under reduced pressure below 40 C. Methanol (125 ml.) and 37.5 g. sodium iodide were added and the mixture was stirred for one hour at 43 to 48 C. The reaction mixture was cooled to 10 C. by the addition of 375 ml. of ice water and 125 g. sodium bicarbonate was added. With good mechanical agitation 8.5% hydrazine hydrate solution was added dropwise until the iodine color was discharged. The hydrazine hydrate required (14 ml.) calculated to be of the theoretical amount for 0.05 mole of iodine. The chloroform layer was separated from the aqueous phase and concentrated to almost dryness under reduced pressure below 40 C. The crude 21-iodo compound was then stirred 17 hours at refluxing temperatures with ml. acetone, 10 ml. of water and 10 g. of potassium acetate. The acetone was removed by steam distillation and the resulting crystalline product filtered. The wet filter cake was dissolved in methylene chloride, the water separated from the organic phase and the product was crystallized from a methylene chloride-hexane mixture. The yield of compound, 21-acetoxy 16fl-methyl- 5-pregnene-3B,17a-dio1-20-one, was 16.0 g. (79.2%), M.P. (Kofier hot bench). After recrystallization from acetic acid and acetone-water, the pure compound melted at -176 C.

(b) 1 GB-METHYL-l7wHYDROXY-21-ACETOXY-4- PREGNENE-3,20-DIONE Seven grams of 16B-methyl-5-pregnene-3fl,17a-diol-21- acetoxy-ZO-one was dissolved in 650 ml. of acetone, cooled to 10 C. and reacted with 5 ml. of an aqueous solution of 1.33 g. chromium trioxide and 1.15 ml. of concentrated sulfuric acid. The chromium trioxide solution was added dropwise and the reaction blanketed with nitrogen.

The reaction was stirred for an additional five minutes. and then poured into 3.0 l. of ice water. After. stirring for minutes, the precipitate was" filtered, washed neutral with water and dried at 60 C. to give a yieldof. 6.0. gramS, M.P. below 100 C., of 3-keto-A compound. This was dissolved in tetrahydrofuran (50 ml.) and treated with5 m1. of 6 N hydrochloric acid for two hours. I The solvent isremoved under reduced pressure and the res.- idue crystallized from acetone-hexane to givethe 16 3- methyl-l7a-hydroxy 21-acetoxy-4-pregnene-3,20-dione.

3,20-DIONE Agar slants containing medium No. 1 described above and 1.5% agar are sterilized for minutes at 121 C. at a pressure of 15 pounds p.s.i. The agar slant is-then cooled to about28 C.,.slanted*and inoculated with a vegetative growth of a. culture Beauveria (ATCC 13144) and incubated at a temperature of 28 C. 'until heavy sporulation occurs.

A two liter Erlenmyer flask containingSOO ml. of a similarly sterilized and cooled broth of medium No. 1 is then inoculated with spores from oneof the heavily spor'ulated agar slants and incubated from 24 hours to 36 hours at 28 C. on a New Brunswickrotary shaker set at 280 r.p.m.. At the end of this period 500 mg; of 16/3- methyl-4-pregnene-17m21-diol-3,20=dione 21-acetate in 5 ml. ofethanol is added. The flask is replaced on the rotary shaker and incubation continued for a period of from 24 I hours to 30 hours during which latter period 10 ml. samples removed at intervals from the Erlenmeyer flask and extracted with chloroform are then chromatographed on paper (according to the method of Bush as modified by Shull) employing a toluene/ethyl acetate solvent system, the paper being impregnated with water together with acetone as a dispersant. Incubation is continued until chromatography indicates complete transformation of l6 8'-'methyl-4 pregnene-17a,21-diol-3,20*dione to 16fl-methyl-4-pregnen'e-11 a,17a,21-triol 3,20 dione; The broth mixture in the Erlenmeyer flask is then extracted with chloroform to effect the isolation of the 11- hydroxylated steroid from the broth mixture. The chloroform is then evaporated off in vacuo and the residue is recrystallized from acetone-hexane to yield 16,6-methyl-4- pregnene-l1a,17a,21-triol-3,20-dione.

What is claimed is:

1. A process which comprises subjecting an ll-desoxy steroid to the oxygenating fermentative activity of the microorganism Beauveria (ATCC 13144) to cause the formation of the corresponding ll-hydoxylated derivative thereof.

2. A process which comprises subjecting an ll-desoxy steroid to the oxygenating fermentative'activity of the microorganism Beauveria (ATCC 13144) to produce the corresponding llrx-hydroxylated derivative thereof.

3. A process which compirses subjecting an ll-desoxy steroid selected from the group consisting of a 3-ketopregnane, a 3-ketoallopregnane, and an unsaturated analogue thereof to the action of an oxygenating enzyme of the microorganism Beauveria (ATCC 13144) to produce the corresponding l1ot-hydroxylated steroid derivative thereof.

4. The process of claim 1 wherein the ll-desoxy steroid is subjected to oxygenation under aerobic conditions.

5. A process which comprises subjecting a member selected from the group consisting of 4-pregnene-17a,21- diol-3,20-dione, 1,4-pregnadiene-17a,21-diol-3,2O dione and a C-21-acylate thereof to the oxygenating fermentative action of the microorganism Beauveria (ATCC 13144) to cause the formation of the corresponding lla-hydroxylated derivative thereof. I

6. A process which comprises subjecting 4-pregnene- 17oz,21-di0l-3,20*-di0116 to the oxygenating fermentative action of the microorganism, Beauveria (ATCC 13144) to cause the formation of the corresponding Ila-hydroxylated derivative thereof.

21-acetate I 7. A process which comprises. subjecting; the Z'Irace'tate of.4-pregnene-17bt,21-diolr3..20 dione to the. oxygenating fermentative action. of the microorganism Beauveria (ATCC 13144) to cause the formation of.the correspond ingllu-hydroxylated derivative thereof.

8. A. process which cornprises subjecting, 1,4-pregnadiene-17a,21-diol-3,20 dione totheoitygenating fermentative action of the microorganism Beauveria. (ATCC 13144) to cause theformation of- 1,4-pregnadien e-11m,

17a,21-triol-3,20-dione.

9. A process which. comprises subjecting the C 21- acetate of 1,4-pregnadiene-17a,21-diol 3,20 dione to the I oxygenating fermentative. action of the. microorganism Beauveria (ATCC 13144). to cause the formation of 1,4-prcgnadie'ne-1 1a,17ot,21-t1'i01-3,20-di01116.

10. A process which. comprises subjecting a membe selected-from the group consistingof 16-alkyl al1opreg-. nane-17a,21-diol-3,20-dione and a=C-21 acylate thereofto the oxygenating fermentative action ofthe. microorganism Beauveria (ATCC 13144) to. cause the. formation of the corresponding ll-a-hydroxylated derivative thereof;

11'. A process which comprisessubjecting. Moe-methyl a11opregnane-17a,21 diol-3,20 dione to the oxygenating fermentative action of the microorganism Beauveria (ATCC 13144) to. cause the formation" of lowmethylallopregnane-l 1a,17a,2l-triol-3',20-dione;

12. A process which comprises subjecting the 0-21- acetate of 16a-methylallopregnane-1704-21-diol-3,2'0-dione tothe oxygenatingfermentative action of the microorgam ism Beauveria (ATCC 13144)-to cause-the-forrnation of 16a-methylallopregnane 11a,170:;21 trio1 3 ,20-dione.

' 13. A process which comprises subjecting lop-methyl allopregnane 17d,21-diol-3,2'0-dione toflthe oxygenating fermentative action of the -rnicroorganism Beauveria (ATCC 13144) to-cause the formation" of 16B-methy1 allopregnane-l1a,17a 21 tri0l-3;20 dione. a

14. A process which comprises subjecting'the 0-21- acetate of' 16fl-methylallopre'gnane-17oz,21+diol-3,20-dione tothe oxygenatingfermentativeaction of the microorgan ism Beauveria (ATCC 13144) to cause the formation of 16p-methyla1lo'pregnane-11a,17,21-t-riol-3,20-dione.

15. A process which comprises subjecting 16a-tert.- butylallopregnane-17a,21-diol-3,20-dione to the oxygenating fermentative action of the microorganism Beauveria (ATCC 13144) to cause the formation of 16u-tert.-butylallopregnane-l 1a,17zx,21-1IriOl-3 ,20-dione.

16. A process which comprises subjecting 16B-ethylallopregnane-17a,21-diol-3.20 dione to the oxygenating fermentative action of the microorganism Beauveria (ATCC 13144) to cause the formation of IGfl-ethylallo- 1 pregnane-l1a,17a,21-triol-3,20-dione.

17,. A process which comprises subjecting a member selected from the group cnsisting of -16-alkyl-1,4-pregnadiene 17m,21-diol-3,2O dione and a C-21 acylate thereof to the oxygenating fermentative action of the A microorganism Beauveria (ATCC 13144) to cause the 7 20. A process which comprises the steps of inoculating a nutrient medium containing assimilable carbon, nitrogen and mineral salts and an ll-desoxy steroid selected from J the group consisting of a pregnane, allopregnane, andan unsaturated analogue thereof with the microorganism Beauveria (ATCC 13144) and 'permittingthe fermenta tion to proceed until a substantial amount of the 110:- hydroxylated derivative thereof has been formed.

21. A process which comprises subjecting '16 3-methyl- 1,4-pregnadiene-17a,21-diol-3,20-dione to the oxygenating fermentative action of a species of fungus of the genus Beauveria (ATCC 13144) to cause the formation of 16,6- methyl-1,4-pregnadiene 1 1oz,17a,21-triOl-3,20-di0nc.

22. A process which comprises subjecting the C21- acetate of 1618-methyl-1,4-pregnadiene 17a,21-diOl-3,20 dione to the oxyg'enating fermentative action of a species of fungus of the genus Beauveria (ATCC 13144) to cause the formation of 16 3-methyl-1,4-pregnadiene-11a,17a,21- triol-3,20-dione. 23. A process which comprises subjecting 16u-tert.- butylallopregnane 1,4 pregnadiene 17a,21-di0l-3,20- dione to the oxygenating fermentative action of a species of fungus of the genus Beauveria (ATCC 13144) to cause the formation of 16a tert.-butylallopregnane-1,4-pregnadiene-l1a,17u,21-triol-3,20-dione.

24. A process which comprises subjecting 16fi-ethyl- 1,4-pregnadiene-17a,21-diol-3,20-dione to the oxygenating fermentative action of a species of fungus of the genus Beauveria (ATCC 13144) to cause the formation of 168- ethyl-1,4-pregnadiene-1 1a,17a,21-tri01-3 ,20dione.

25. A process which comprises subjecting a member selected from the group consisting of a 16-alkyl-4-pregnene-17a,21-diol-3,20-dione and a 21-acylate thereof to the oxygenating fermentative action of the microorganism Beauveria (ATCC 13144) to cause the formation of the corresponding lla-hydroxylated derivative thereof.

26. A process which comprises subjecting 16a-methyl- 4-pregnene-17a,21-diol-3,20 dione to the oxygenating fermentative action of a species of fungus of the genus Beauveria (ATCC 13144) to cause the formation of 16amethyl-4-pregnane-11a,17a,21-triol-3,20-dione.

27. A process which comprises subjecting the C 21- acetate of 16a methyl-4pregneue-17a,21-diol-3,20-dione to the oxygenating fermentative action of a species of fungus of the genus Beauveria (ATCC 13144) to cause the formation of 16a methyl-4'pregnane-1la,17a,21- triol-3,20-dione.

28. A process which comprises subjecting 16/3-methyl- 4-pregnene 17a,21-diol-3,20-dione to the oxygenating fermentative action of a species of fungus of the genus Beauveria (ATCC 13144) to cause the formation of 165- methyl-4-pregnane-1 1oz,17a,21-t1'i013,20-di0116.

29. A process which comprises subjecting the 0-21- acetate of 16/3-methyl-4-pregnene-17a,21-diol-3,20-dione to the oxygenating fermentative action of a species of fungus of the genus Beauveria (ATCC 13144) to cause the formation of 1613 methyl-4-pregnane-11a,17a,21- triol-3,20-dione.

30. A process which comprises the steps of subjecting an ll-desoxy steroid selected from the group consisting of a pregnane, allopregnane, and an unsaturated analogue thereof, to the enzymatic action of the fungus Beauveria (ATCC 13144) to cause the formation of the Ila-hydroxylated derivative of said steroid and recovering said hydroxylated derivatives therefrom.

References Cited in the file of this patent UNITED STATES PATENTS 2,649,402 Murray et al. Aug. 18, 1953 2,802,775 Dulaney et al. Aug. 13, 1957 2,823,170 Muir Feb. 11, 1958 2,844,513 Wettstein July 22, 1958 OTHER REFERENCES Ainsworth and Cowan: Rules of Nomenclature for Fungi and Bacteria, Journ. Gen. Microbiology, 10, 1954, pages 465-474.

Bisby: An Introduction to the Taxonomy and Nomenclature of Fungi, 2nd Ed., 1953, The Commonwealth Mycological Institute of Kew, Surrey, pages 3, 4, 29 to 33, and 117.

Clements et al.: General of Fungi, 1931 (2nd printing 1954), Hofner Pub. Co., 1954, pages 13-15.

Thom: New York Acad. Sci., 60, 1, page 27.

Claims

1. A PROCESS WHICH COMPRISES SUBJECTING AN 11-DESOXY STEROID OF THE OXYGENATING FERMENTATIVE ACTIVITY OF THE MICROORGANISM BEAUVERIA (ATCC 13144) TO CAUSE THE FORMATION OF THE CORRESPONDING 11-HYDROXYLATED DERIVATIVE THEREOF.