US2905592A

US2905592A - Oxidation of steroids by mycobacteria

Info

Publication number: US2905592A
Application number: US524817A
Authority: US
Inventors: Gilbert M Shull; Donald A Kita
Original assignee: Pfizer Inc
Current assignee: Pfizer Inc
Priority date: 1955-07-27
Filing date: 1955-07-27
Publication date: 1959-09-22
Anticipated expiration: 1976-09-22

Description

2,905,592 OXIDATION OF STEROIDS BY MYCOBACTERIA Gilbert M. Shull, Huntington Station, and Donald A. Kita, Jackson Heights, N.Y., assignors to Chas. Pfizer & Co., Inc., New York, N.Y., a corporation of Delaware No Drawing. Application July 27, 1955 Serial No. 524,817

12 Claims. (Cl. 19551) This invention is concerned with the oxidation of certain steroid compounds by microbiological means. In particular, it is concerned with the oxidation of certain 3-keto-4-unsaturated steroid compounds by means of certain microorganisms or oxidizing enzymes produced by these microorganisms.

This application is a continuation in part of applications Serial Number 431,619, filed May 21, 1954; Serial Number 483,842, filed January 24, 1955; and Serial Nlnnber 484,828, filed January 28, 1955, by Gilbert M. Shull et 21. It is also a continuation-in-part of application Serial No. 276,678, filed March 14, 1952, and now abandoned.

As is disclosed in the above mentioned applications, by subjecting a 3-keto-4-unsaturated steroid compound to the action of a species of the genus Mycobacterium, there is produced a compound which is a 3-keto-l,4-unsatu rated steroid. Some nuclear oxygenation also takes place, chiefiy in the 14 position when this position is available. The main product of the oxidation, however, is the dehydrogenation product. It is thus possible by the use of organisms of the genus Mycobacterium to produce compounds having a 3-keto group and unsaturated at the 1 and 4 positions. Certain compounds of this type are extremely valuable because of their biological activity, and it is therefore of great value to have a simple, one step method of dehydrogenation at the 1,2 positions.

This invention is the first case in which dehydrogenation in the A ring of a steroid has been produced by the use of microorganisms without the simultaneous oxidative cleavage of a side chain present in the 17 position. This invention is also the first instance in which an organism of the genus Mycobacterium has been used in a commercial chemical reaction and is indeed believed to be the first time that any use has been found for organisms of the family Mycobacteriaceae.

It has now been found that on contacting a 3-ket0-4-unsaturated steroid compound with the oxidizing activity of a species of the genus Mycobacterium there is produced oxidation of the steroid compound. The chief chemical reaction is one of dehydrogenation in the 1,2 positions, thereby producing a double bond between these positions. Some oxygenation also occurs at the same time, chiefly in the 14 position when this position is available. The term nuclear oxidation includes both dehydrogenation and oxygenation. It is possible that sometimes both types of oxidation, i.e. dehydrogenation and oxygenation, occur in the same molecule, but this is not generally the case to a large extent. The process of this invention, i.e. the contacting of a suitable steroid compound with the oxidizing activity of a species of the genus Mycobacterium, may be carried out by bringing the chosen steroid compound in aqueous solution or suspension into contact with either an actively growing culture of Mycobacterium, with cells of Mycobacterium removed from the growing culture and suspended in a suitable medium, or by contacting the steroid with extracts of oxidizing enzymes obtained from the culture of a species of the genus Mycobacterium.

, oxidation of the steroid nucelus.

2,905,592 Patented Sept. 22, 1959 An object of this invention is a process for the preparation of oxidized steroids, particularly 3-keto-A steroids, from the corresponding 3-keto-4-unsaturated compounds. A further object of this invention is an economical process for this dehydrogenation reaction which may be readily scaled up for large volume production. Further objects of this invention will be apparent from the following discussion.

A variety of 3-keto-4-unsaturated steroid compounds may be used as'starting materials for the reactions of this invention. These include such materials as:

Compound F (hydrocorfisone) 9a-fluoro-Compound F Compound E (cortisone) l4a-hydroxy-Compound F 17a-hydroxyprogesterone 16-dehydroprogesterone ll-ketoprogesterone A9(ll)dehydro-Compou11d S In general this process is most applicable to 3-keto-A steroid compounds having from 18 to 21 atoms in the carbon skeleton. The products of the reaction may be detected by careful comparison of paper chromatograms of the products formed by the reaction of this invention with known steroid compounds. This method has been tested on a variety of compounds and is known to give reliable results. Reports of this method are available in the chemical literature.

In carrying out this invention various species of the genus Mycobacterium may be used for bringing about These are available from public culture collections, for example the American Type Culture Collection of Washington, DC. These include known species such as Mycobacterium species 607, M. berolinense, M. lacticola, M. thamnopheos, and the species M. smegmatis ATCC 101. Particularly useful are organisms of the species M. phlei, a living culture of which has been deposited with the American Type Culture Collection of Washington, DC, Where it has been given the number ATCC 354. The various species of Mycobacterium vary considerably in the speed and ease with which dehydrogenation is produced by the cells of the organisms or by products formed during their growth. We have found that certain strains of M. 'phlei and M. smegmatis are particularly active in bringing about the reaction of this invention. The various mycobacteria vary somewhat in their requirements for nutritional media. By a minimum of single testing, however, it is possible to determine the composition of suitable media, as well as the optimum conditions for operation, such as pH, rate of aeration, rate of stirring and so forth. In a like manner, the optimum conditions for the dehydrogenation of a particular steroid may be found.

There are several procedures which may be used in the dehydrogenation of steroid compounds according to this invention. In the first of these, nutrient media are seeded from slants of the selected Mycobacterium. Such a medium may consist, for instance, of a mixture of a standard bacteriological nutrient broth base, together with glycerol. It has been found that a neutral, surface-active agent, such as a polyoxyethylene derivative of a sugar alcohol-fatty acid ester (eg. Tween when added in a minor proportion to the medium, is helpful. The addition of the amino acid asparagine is also helpful. The cultivation of mycobacteria has been described in considerable detail in many publications. The seeded, sterile, nutrient solutions may be grown in shake flasks for two to three days to provide inoculum for larger vessels, and in turn, the larger, stirred, aerated vessels may be used for the inoculation of full production-scale vessels for submerged fermentation. The same medium-of the type described above may be used for the large-scale oxidation of steroidsaccording-to this invention. Considerable variation may, of course, be made in the medium. In general there is required a carbohydrate, a source of organic nitrogen, mineral salts and various trace metals.

As pointedout above, rather than'conducting the oxidation of the selected steroid compound in the presence of the whole fermentation product, cells may be removed fromgrowing cultures and these may be resuspended in a medium -which has been designated the enzyme reaction mixture. Such a reaction mixture may consist, for .instance, of a solution which is 0.01 molar in sodium fumarate or other hydrogen acceptor and in magnesium sulfate and.0.03 molar in sodium citrate. It has been found that the presence of-a certainamount of adenosine triphosphate, e.g.:0.125%, is alsoquite useful. Centrifuged, washed cells of the chosen Mycobacterium may be suspended in this type of reaction mixture, which is adjusted to a pH of about 6, for example with citric acid. After addition of the steroid compound which it is desired to oxidize, the mixture may be incubated at about 37 C., and samples may be removed from time to time to determine the point at which maximum conversion of the steroid has taken place. In general, this occurs after about one to several days. We have found that the cells from about 100 milliliters of the stirred, aerated mycobacteria cultures may be suspended in about 20 milliliters ofan enzyme reaction mixture for suitable results. Considerable variation may be made in these proportions.- The steroidcompound may be used in a proportion of about 25 to about 200 milligrams/100 milliliters of the enzyme reaction mixture. The compound in solid form is merely added to the medium after adjustment of the pH. The flasks are stoppered with cotton so that they are exposed to the air during the incubation. We prefer to-use a small volume compared to the volume of flask, for instance, 20 milliliters in a l25-milliliter Erlenmeyer flask. Alternatively, the mixture may be stirred and aerated. Ingeneral, at least a hydrogen acceptor, a divalent metal particularly magnesium, and a buffer are required in-the medium.

Rather than removing the Mycobacterium cells and carrying out the reaction of this invention in an enzyme reaction mixture, the steroid compoundmay be added directly to a sterilized portion of nutrient medium, such as is described above, and the medium is then seeded with the chosen mycobacteria. Approximately the same proportion of chosen steroid compound may be used in this case also. Samples of the agitated, aerated mixture may be removed at intervals for determination of the conversion of the steroid compound to the oxidized products. The mixture is maintained at between 2537 C. or higher during the growth of the cells and the conversion of the steroid. In general, about twoto seven days is required for maximum production of the oxidized compounds. Alternatively, the growth of the cells may be established before addition of the steroid.

A third method which is also very useful for the oxidation of the selected steroid compounds involves the use of oxidizing enzymes produced by the mycobacteria. These may be prepared by a variety of methods from the cells of the chosen organisms. These materials may be released from the cells by several different procedures. These include grinding, particularly with abrasive materials such as powdered glass or sand, which serves to break the cell walls and release the essential materials. A second method is by autolysis. The cells may be removed from the medium in which they are grown. They are then washed and suspended in water. The water may be covered with a thin layer of toluene to prevent contamination, and the mixture is allowed to stand at a temperature of from about 20 to about 50 C. The cells disintegrate within one to several days and the cell residue may be removed by filtration, for instance through a Seitz filter or through a sintered glass bacterial filter. A third'method for preparing cell-free elaborationprod- '4 ucts of the mycobacteria useful for the reactions of this invention is by repeated, rapid freezing and thawing of the cellular material. Another method is by the use of ultrasonic energy to rupture the cells. One further meth- 0d of use for the same purpose is by the use of a watermiscible solvent and, in particular, acetone. The cells, when placed in such a solvent, are ruptured and an extract of thefdesired enzymes is obtained. The mycobacteria enzymes may be used for theoxidation of 3-keto-4- unsaturated steroid compounds in media similar to those used with the grown cells that is, one containing ahydrogen acceptor such as fumarate, a buffer and, in some cases, a bivalent metal, particularly magnesium, as well as a minor proportion of adenosine ,triphosphate. The

cell-free oxidizing enzymes of mycobacteria may be used in media indicated above at a temperature of about 20 to about 40 C. In general, the oxidation of the desired steroid compounds is brought about in a period of from a few hours to several days. The optimum time and temperatureand other conditions may readily be determined by a minimum of experimentation. Detailed descriptions of suitabie mediafor both the use of isolated, resuspended cells and of cell-free elaboration products are givenin the textbooks Manometric Technique in Tissue Metabolism, by W. W. Umbreit etalQ, Burgess Publishing Company, Minneapolis (1949), and Respiratory Enzymes, by H. Lardy, Burgess Publishing Company, Minneapolis 1949) The most convenient method for following the course of the oxidation reaction is by means of paper chromatography. Other methods may also be used for this purpose as noted above. In the paper chromatography method, aluminaimpregnated-filter paper is prepared by dippinglarge sheets of thepaper (Whatman No. S t-27" x 22 /2" -sheets-filter paper is particularly useful) in an aluminum sulfate solution (13 grams/ milliliters). The paper is drained, exposed toan atmosphere of am monia for 15 hours and the sheets are then washed continuously for six hours with tap water. The sheets are then drained, ironed at a moderate temperature to obtain a flat surface, and are stored at room temperature for 24 hours prior touse. Various solvent systems are used in the development vof the paper chromatograms. These have-been designated as follows:

SystemI-zHexane System IIHexane-ether (19 volumes to -1 volume) System III=Hexane-ether (16 volumes to 1 volume) System IV-Ether Five microliter quantities of solutions containing the steroids are placed on the paper along aline three centimeters from one edge of the paper and the spots are spaced two centimeters apart. The edge of each paper is stapled to the opposite edge to form cylinders and each paper is placed in a glass jar of appropriate size. They are exposed to the vapors of the solvent system for one hour at 4-8 C. The jars are then covered with transparent plastic lids, which are sealed with high vacuum silicone grease after sufficient solvent has been placed in the bottom of the jar. The solvent is carried up the paper by capillarity and, in passing the spots where the steroids have been deposited the various components of these products are moved up .the paper ata greater or lesser rate depending upon the structure of the individual compound. The development of the sheets is carried out in acold room atabout 48 C. until the solvent front reaches .a point about eight centimeters from the top of the paper :sheet. This generally takes about .four to five hours. The paper cylinders are then removed and dried at room-temperature. V

One further system has been used for-the comparison ofthe various steroid products. Thisis-known as System V. 'It consists of benzene saturated with'water. With this system, however, untreated {Whatman No. 54 filter paper sheets aroused rather than the alumina impregnated sheets, and cylinders of the. papers are prepared of 8" x 18" sheets. Before chromatographing the products, the atmosphere of the jars is saturated with water by means of steam. The benzene saturated with water is poured in the bottom of the jar and the lid is sealed. Development is allowed to occur at room temperature until the solvent front is close to the top of the paper (three to four hours). The paper is then removed and dried at room temperature. The paper chromatography methods of Zaffaroni and of Bush, reported in the literature, are also adaptable for use here.

Various methods may be used to detect the various steroids on the paper chromatograms. The first of these methods entails no chemical reaction with the separated steroids and may be used before the use of either of the other methods. In this method, the sheets are examined With a fluorescent scanner as described by Haines et al., Federation Proceedings, volume 9, p. 180 (1950). The steroids having an 0a,;3-l11183t1118i6d ketone structure and certain dienic steroids appear as dark spots when viewed through the phosphorescent screen. These may be outlined on the sheets in pencil to establish their position. Standard, known samples of the steroids may be run side by side with material prepared according to this invention. In this way it is possible to distinguish newly produced oxidized steroids. A second method for locating the steroid compounds on the paper sheets is by treatment of the sheets with a atmosphere of chlorine for 20 minutesand then spraying the sheets with a solution of 380 grams of antimony trichloride in 100 milliliters of acetic anhydride. The solution is prepared fresh daily. The sprayed sheets are heated at 90100 C. until dry and immediately examined in a dark room with a sun lamp usinga Corning No. 9863 filter. Spots formed on the paper may be outlined with pencil and the relative position with respect to known samples of the various steroids may then be established.

An alternative method for locating certain of the steroid products is by means of the reagent described by Burton et al., J. Biol. Chem., volume 188, p. 763 (1951), which involves the spraying of the paper with an alcoholic potassium hydroxide solution of triphenyltetrazolium chloride. Steroids having a 2l-hydroxy-20-keto side chain appear as red spots on drying the paper. The spots are immediately outlined in pencil since the contrast with the background soon disappears. By running paper chromatograms with a group of known oxidized steroid compounds, it has been possible to distinguish the products formed by the process of the present invention and prove the oxidation which takes place during the reaction. The various solvent systems have been used to establish, with certainty, the nature of the materials which have been formed. In conducting the paper chromatographic work, a value known as Rf has been used to identify the position of the various steroid compounds on the paper chromatograms. The R value for any given compound using a specific solvent system is the ratio of the distance travelled by the compound from the spot at which it was applied on the paper sheet as compared to the distance travelled by the solvent front under exactly the same conditions. The R value will, of course, vary with different types of compounds, with the different solvent systems, with variations in temperature, and with variations in the nature of the paper used. However, the same compound under the same set of conditions Will give the same Rf and thus serve to indicate the identity of the particular product. A standard table of R, values may be prepared for use with a given solvent system under a specific set of conditions. These values have been listed in a publication of Gilbert M. Shull et a1. (Archives of Biochemistry and Biophysics), vol. 37, p. 186 (1952).

The products of the new method described in this application may be isolated from aqueous solution by extraction with various water-immiscible organic solvents. Lower halogenated hydrocarbons, such'as chloroform,

are particularly useful. After extraction, the solvent may be removed by distillation and the solid product is then isolated. This material may be further purified by recrystallizattion procedures from organic solvents or by chromatography, for instance on alumina columns or on other suitable solid absorbent materials. The use of a silica gel-ethanol column with a 98% to 2% by volume mixture of, methylene chloride and ethanol as a developer has been found particularly advantageous. Methods for the separation of products of this nature have been reported previously in the literature. For some uses the products need not be separated, but the crude mixture may be used as such. It has been found advantageous in some cases to acylate the crude products and work with the resulting esters which are somewhat more stable.

A variety of 3-keto-4-unsaturated steroids are possible starting materials for the reactions of this invention. These include such well-known compounds as testosterone, progesterone, and Reichsteins Compound S. The oxidized products are useful as intermediates in the synthesis of other useful compounds. For example, the dehydrogenated products which contain unsaturation at the 1,2 position, along with the 3-keto group and the 4,5- unsaturation originally present in the starting material, are especially susceptible to Inhoffen aromatization. This gives rise to a group of derivatives of estrone. In the case of the dehydrogenation product of Compound S, side chain cleavage to give a 17-keto group way readily be accomplished by standard means, e.g. oxidation With chromic acid, and when the product of that reaction is aromatized by the Inhoffen reaction, e.g. heated to a high temperature in a hydrocarbon solvent, the very valuable compound estrone is produced. The introduction of oxygen functions into the nuclei of the steroids Which have been oxygenated has imparted to these molecules new centers of chemical activity, and has greatly enhanced their versatility in undergoing further chemical reactions. Very desirable changes in solubility are also effected by the introduction of hydroxyl groups.

In addition to the utility mentioned above, many of the compounds produced by this reaction are extremely valuable because of their great biological activity. For example, when hydrocortisone is treated With mycobacteria according to the process of this present invention, the compound formed is prednisolone, which is of great utility and has advantages over hydrocortisone in the treatment of rheumatoid arthritis. When cortisone is treated with mycobacteria, there is produced the compound known as prednisone which also has great utility in the treatment of rheumatoid arthritis. It has also been found that other 3-keto-A steroids possess great activity as adrenocortical hormones and are useful for the same type of therapy as hydrocortisone. Many naturally occurring steroids, and steroids readily prepared from naturally occurring ones, have a 3-keto-A structure, but no 3-keto-A compounds are readily available as raw materials. For this reason, the process of this invention whereby it is possible to transform a 3-keto-A compound.

having from 18 to 21 atoms in the carbon skeleton into a 3-keto-A compound in one single step in large scale commercial production is of tremendous value.

The following examples are given by way of illustration and are not to be considered as limitations of this invention, since as many apparently Widely different embodiments 0f the present invention may be made without departing from the spirit and scope hereof.

A living culture of the organism employed in Examples I through IV hereinafter, namely Mycobacterium smegmazis (Pfizer Culture Collection No. 2), has been deposited with the American Type Culture Collection, 2029 M Street, NW., Washington, DC, and added to its permanent collection of microorganisms as ATCC 12,549..

7 EXAMPLE I A culture of Mycobacteriumsmegm'atis' (Pfizer- Cultu're Collection No: 2)- grown on solid agar medium, was

added to 50 milliliters of nutrient solution having the The medium was sterilizedbefore' seeding. This mixture was placed in a" 250-milliliter Erlenmeyer flask and the flaskwas shaken at about 27 C. for three days. The contents-of one shake flask were used to inoculate tlwo liters of the same sterilized medium inc. 4 liter vessel equipped for conducting submerged fermentation. The medium was seeded and the mixture was stirred and aerated-under sterile conditions.- The stirrer was operated at aspeed of 1750 r.p.m. and aeration was continued throughout 48 hours at' a rate of one volume of sterile air per'volume of medium per minute. The cells that had formed in the medium were centrifuged, and washed with water.

An enzyme reaction mixture wasprepared containing 0.01 molar sodium fumarate, 0.01 molar magnesium sulfate, 0.03' molar sodium citrate and 0.125% adenosine triphosphate; Twenty milliliters of this medium was placed in a 125-milliliter Erlenmeyer flask. After the pH had been adjusted to 6.0 with citric acid, to the reaction-mixture was added 25 milligrams of Compound S acetate. The reaction mixture was seeded with the cells'from 100 milliliters of the submerged fermentation' and the mixture was incubated at 37 C. Samples were removed at intervals, and the steroids present were extracted into chloroform. After separation of: the organic phase, the solvent was removed and the product was dissolved'in ethanol; Samples ofthe ethanol solution wereplace'd on paper sheets using the chromatographic technique described above. of six hours some oxidation of the nucleus of the Com pound S acetate had occurred. The reaction proceeded quite-rapidly and in 24 hours appreciable conversion had occurred; At the end of 48' hours, a maximum oxidation of the steroid nucleus had taken place. Paper chromatograms run using solvent SystemV gave the following results'. The figures given in the table are R values and the names in parentheses are the steroid compounds to which each of the spots on the paper chromatogram conforms. These tmts were made 48 hours after inoculation of the medium with cells.

R ValueS on System V:

(Compound S acetate) 1.0 (Compound S) 0.58 (A -dehydro-Compound S and l4a-hydroxy- Compound'S) 0.30 0.20

(Not yet identified) EXAMPLE H To a four liter Pyrex glass vessel equipped for conducting submerged aerated fermentation was added two liters of the following medium (known as Turfitts medium):

Ferrous sulfate heptahydrate 0.00001 Calcium carbonate 0.5

The aqueous medium was sterilized and with 0.25 gram of Compound S acetate. The mixture was then: seeded with 100 mls. of a culture of Mycobacte'rium smegmatis (Pfizer Culture Collection No. 2)

grown in a shake flask on-nutrient broth. The mixture- It was" found that atthe end then treated by means of paper chromatograms using three=dififerent systems. The following table indicates the R; valuesof thevarious systems. The compoundsv to which these spots correspond are designated at theside of the' tables Rt Values System Steroid Compounds 0 III IV Compound S Compounds acetate." Compound-S; A -dehydro-Clompound S and 14m-hydroxy-Compound S Another system of paper chromatography gave better results, especially in the separation of the A -dehydro- Compound S and l4 t-hydroxy-Compound S, each of which had the R; value of 0.45 when System V was employed. This other systemwasemployed ina descending'chro'matograph' instead'of an ascending one as those previously described. The paper was impregnated with a mixture of -volumes of methanol and 20 volumes of water. The atmosphere in the jar was saturated with' the vapor of toluene which had been saturated with water. Developing was accomplished by means of a mixture of l00'volun1es of toluene and 20 volumes of ethyl alcohol;

By use of this system, the following results were obtamed.

R values:

(Compound S) 1.00

These R values were obtained by assigning a value of 1.00 to the distance traveled by Compound S, and to' each of the other compounds a value equal to the ratio of the distance traveled by-that' compound to the distance traveled by Compound S; Both the solvent front and Compound 8* acetate went over the entire length of the sheet and off the paper.

The mixture was finally separated by successive applications to silica gel-ethanol columns. When fermentation was completed, the fermentation mixture was extracted with" chloroform, and evaporated to a volume of about 20 ml. This concentrated extract was then applied to a silica gel-ethanol column, and eluted with methylene chloride-alcohol mixture until most of the n -dehydro-Compound S had been removed. The column was then eluted with methanol" to remove all the'remaining' steroids. This procedure was repeated several times, each time starting with the extract of a fermentation conducted as described above. These methanol eluates' were thencombined, evaporated to dryness and dissolved in20' ml; of chloroform. This chloroform solution was then applied to a silica gel-ethanol column and eluted with the methylene chloride-alcohol mixture.

Fractions of 50 ml. volume were collected about every two hours, and found to contain the. following:

Fractions l-8 (Nothing).

Fractions 9-17 (Compound S).

Fractions 18-19 (n- -d'ehydro-Compound S and l4u-hydroxy-Compound S);

Fractions 22-33 (l4a-hydroxy-Compound'S) The'fractions were evaporated to dryness and the' crystals recovered; The n -dehydro-Compound S was twice recrystallized from ethyl acetate, and the l4a-hydroxy-Compound S was twice recrystallized from. ethyl alcohol. I i

n -dehydro-Compound S',. which may also be called .9 l7a,2l-dihydroxy-A -pregnadien-3,20-dione, has the following physical constants: M. P. 235236 C., optical rotation am w 71.9

ultraviolet absorption maximum at 245 mn.

l4a-hydroxy-Compound S, which may also be called 14a,17a,2l-trihydroxy-A -pregnene-3,20-dione, is a white crystalline compound and has the following physical constants: M.P. 226228 C., and optical rotation Analysis.C, 69.64; H, 8.30. Calculated for C H O C, 69.57; H, 8.35.

EXAMPLE III An experiment was run exactly as outlined in the example above except that 0.25 gram of desoxycorticosterone acetate Was used as the starting material. After incubation for four days at 27 C. under otherwise the same conditions the product was recovered and analyzed by means of paper chromatograms. The following table indicates the results:

Rf Values-System Steroid Compounds Desoxycorticosterone acetate Desoxyccrticosterone 0. 97 A -dehydrodesoxycorticosteron 0.72 A monohydroxy nuclear substitution prodnot of desoxycorfim terone 0. 44

Again in this example the products are formed by the nuclear oxidation of the steroid starting material, one of them in each example being formed by oxygenation, and one in each example being formed by dehydrogenation at the 1,2 positions. The exact position of the hydroxyl group in the monohydroxy nuclear substitution product here obtained is not yet certainly known, except that it is in a different position from that of the nuclear hydroxyl group in corticosterone, and it is believed to be the 14 position.

EXAMPLE IV EXAMPLE V Submerged, aerated fermentation using progesterone as the starting material was run with M. smegmatis ATCC. 278 in 4 liter fermenters for 96 hours. The products obtained were A -dehydroprogesterone, and a product with a saturated A ring, along with some unreacted progesterone. The mixture was separated by countercurrent distribution. The physical constants for the A dehydroprogesterone obtained here check with those reported in the literature.

EXAMPLE VI A culture of Mycobacterium smegmatis ATCC. 12,051, grown on solid agar medium was rinsed into a sterile nutrient solution having the following composition:

Solid, standard nutrient broth grams/liter 8 Glycerol mls./1iter 20 Tween 80 mls./liter 0.2 Asparagine grams/liter One liter of inoculated medium was placed ineach of two Fernbach shake flasks. After three days, one-half gram of hydrocortisone was added to each flask. The reaction was continued for four days at the end of which time the contents of the flasks were combined and extracted 3 times, each time with an equal volume of chloroform. The combined chloroform extracts were concentrated to a volume of approximately 150 ml. A small amount of activated carbon (Nuchar) was added and the mixture was gently heated and stirred for a few minutes. The Nuchar was removed by filtration and the filtrate was put on a silica gel-ethanol chromatography column. The column was developed, using a mixture of 97% by volume ethylene chloride and 3% ethanol. 50 ml. fractions were collected. The first 37 contained no steroids. In fractions 38 through 66, unreacted hydro cortisone was recovered. Fractions 67 through 82 contained hydrocortisone and delta l-dehydrohydrocortisone. Fractions 83 through 115 contained delta l-dehydrohydrocortisone. Fractions 116 through 269 contained no steroid. Fractions 270 through 332 contained A preg nen-l 113, l 7u,20,B,21-tetrol-3 -one.

Combined fractions 83 through 115 were evaporated to dryness on a steam bath. The residue was dissolved in ethyl acetate and treated with a small amount of activated carbon. The carbon was removed by filtration, and the filtrate concentrated to a small volume and refrigerated. Crystals which formed after refrigeration were redissolved in the minimum amount of acetone. An equal volume of hexane was then added, followed by the addition of cyclohexane to incipient turbidity. On addition al refrigeration, white crystals formed. These have been identified as being delta l-dehydrohydrocortisone, which may also be called prednisolone or A -pregnadien-llfl, 17a,2l-t1i01-3,20-di0ne. The crystals had a melting point of l96198 C., a specific rotation of [M +108.2 in ethanol and EXAMPLE VII The procedure of Example VI was repeated except that cortisone was used in place of hydrocortisone. The product, A -dehydrocortisone, which may also be called prednisone or A -pregnadien-17a,21-di0l-3,11,20-trione, was isolated in a similar fashion.

EXAMPLE VIII To a four liter Pyrex glass vessel equipped for conducting submerged aerated fermentation was added two liters of Turfitts medium. The aqueous medium was sterilized and then inoculated with ml. of a culture of Mycobacterium smegmatis ATCC 12,051, grown in a shake flask on nutrient broth. After three days, 0.25 gram of hydrocortisone was added. The mixture was agitated and aerated with sterile air. Four days after the addition of the steroid, the whole mixture was extracted with approximately 2 liters of chloroform.

Chromatography proved the presence of A -dehydrohydrocortisone in the product.

EXAMPLE IX The procedure of Example VIII was repeated except that cortisone was used in place of hydrocortisone. In an identical manner, the product A -dehydrocortisone was prepared.

9 EXAMPLE X 5 7,5 scribed in corresponding application Serial Number 476,-

11 556,@filed-;on;December 20, 1954, by G. Mushull et al.,

was added; The mixture was agitated and aerated with sterile air-. Four days after the addition of the steroidflche wh'ole mixture was extracted with approximately 2 liters of chloroform; The chloroform extract was treated with--activated carbon, filtered and reduced to a smallvolume by evaporation, and: then placed on a silica gel ethanol chromatography column. The column was eluted withmixtures of ethylene chloride and 95% ethanol, starting with mixtures containing about 2% by volume ethanol, and gradually increasing the percentage of ethanol. The compound A -pregnadien-11fl,14a,17u,21- tetrol-3, 'dione was recovered in this fashion. The physical. constants of this novel compound are as follows: M.P. 227229 0., [0519 +1035 (dioxane),

Infrared 1% in: 14811 pellet) most prominent absorption peaks at 2.92, 5.78, 6.03, and 9.65 microns. This compound possesses great activity as. an adrenocortical a nutrient agar slant to-a Fernbach flask containing 1000 cc. of the following medium:

Nutrient broth (Difco) g 8.0 Glycerol cc 20.0 Tween 80 (Atlas Powder Co.) cc 0.2

Distilled H O to make 1000.0 cc.

After 2 days shaking at 28", 100 cc. of the resulting broth wasus'ed to inoculate each-of four ferme'ntors containing-'2000'cc.-of Medium" CC.

Medium co, g. mane, 1.0 Kano, 0.25 MgSO -7H O 0.25 NaCl 0.005 resource 0.0001. CaCO 5.0-

Distilled H O to 1000 cc.

Another 8' mg. was obtained as a second crop. Comparison of this compound with corticosterone in the liver glycogen and thymus involution assays for glucocorticoids showed that the new compound was much more active than corticosterone. The compound was identified as A -pregnadiene-llflgZl-diol-B,20-dione, the expected transformation product.

I EXAMPLE Mycbliacte rium smegmat is ATCC 361' wastransferred from, anutrient: agar slant. to seven-,Fernbach flasks eacl of: which contained: 1000: cc. of the-following medium:

- G1 Malt extract (Difco) 510 Dextrose hydrate 30.0 NaNO 2.0.- K-H PO 1.0 KCL I 0.5 MgSO JI-I Q 0.5- FeSO -7H 0 0.5 pH 6.7. Tap H O to 1000 cc.

After 5 days shaking at 28, 250 mg. of 14a-hydroxy- Compound S was added to each flask. After an additional 3 days, the fermentations were stopped and the broth extracted with CHClg. The combined extracts were chromatographed on silica gel to yield 33 mg. of a white crystalline product, M.PL 217-218, [ml (fl i);

xfig zss', 315-, 468, 52mm The compound has been identified as A '-pregnadiene- 14a,17a,2'1=triol-3,20-dione.

EXAMPLE XIV A scriesot experiments were run using the procedures described in-th'e above examples, and using the following.

species of Mycobaoteria, all of which are available from.

public culture collections:

M. phlei ATCC 10,142 M. thamnopheos M. ranae ATCC M. lacticola M-. butyricum M. friea'manni ATCC 114 M. berolinense ATCC M. tuberculosis other used steroids included the following:

A androstadiendione' 1 15,17a-hydroxyprogesterorie A -dehydro-Compound S H Androstenedione 19-nortestosterone l7a-hydroxyprogesterone 1 6 dehydropro'g'esterone l'l -ketoprogesterone l' lmfiu-epoxiddCompound S (This compound may be obtained by the method describedin copending application Serial Number 459,848, filed October 1, 19541) 14h,-1 5u-epoxido-Compound F (This compound may be obtained" by the method described in oopendi'ng application. Serial Number 432,621, filed May 26, 1954".)

In-eachcase the products were recovered from the reaction mixture by extraction and were subjected to evaluation by the paper chromatography method. In each case, it was found that dehydrogenation at the 1- positi'on had occurred; What is claimed is; 1.1. Auprocess forthe prep'arationofa 3 ket0-A -stcroid compound which. process comprises contacting a- 3-keto- At-steroid' compound having' from 18" to- 2 1 atoms inthe carbon.- skeletonwithihe oxidizing enzymes of an organism of.theigenus Mycobacterium;

2. A process for'the preparation of'a 3'-lreto-A '-steroid compound which process comprises subjecting a 3-keto- A -steroid having from- 18- to- 21. atoms in the carbon skeleton to submerged, aerated fermentation with a living culture ofan organism of the genus Mycobacterium;

3 A processas claimed in claim 1 wherein the organ ism is-of thefisp'ecies-Mycobacterium phlei.

4. A processasclaimed inclaim 1 wherein theorganism is of the-species Mycobacterium smegmaris.

5. A process-as claimed in claim 2 wherein the organism is of; thespecies M ycqbacterium phlei.

6. A process as claimed in claim 2 wherein the organism is of the species Mycobacterium smegmatis.

7. A process for the preparation of A -pregnadiem 11,8,17oz,21-tri013,20-di011e which process comprises contacting A -pregnene-11,8,17a,2-trio1-3,20-dione with the oxidizing enzymes of an organism of the genus Mycobacterium.

8. A process for the preparation of A -pregnadien- 17oc,21-d.i01-3,11,20-t1i0ne which process comprises contacting A -pregnene-17a,21-diol-3,11,20-trione with the oxidizing enzymes of an organism of the genus Mycobacterium.

9. A process for the preparation of A -pregnadien- 17a,21-dio1-3,20-dione which process comprises contacting A -pregnene-17u,21-dio1-3,20-dione with the oxidizing enzymes of an organism of the genus Mycobacterium.

10. A process for the preparation of A -pregnadien- 115,14a,17a,21-tetrol-3,20-dione which process comprises contacting A pregnene 115,14u,17a,21t6t1'01-3,20-di0116 with the oxidizing enzymes of an organism of the genus Mycobacterium.

11. A process for the preparation of A -pregnadien- 14a,17oc,21t1i01-3,11,20-13110116 which process comprises contacting A pregnene 14a,170c,21 trio1-3,11,20-tri0ne with the oxidizing enzymes of an organism of the genus Mycobacterium.

12. A process for the preparation of A -pregnadien- 11/8,17a,21-trio1-3,20-dione which process comprises contacting M-pregnene-l1,8,17a,21-trio1-3,20-dione with the oxidizing enzymes of Mycobacterium phlei.

References Cited in the file of this patent UNITED STATES PATENTS Murray et a1 July 8, 1952 OTHER REFERENCES