US2903398A - Preparation of a steroid compound - Google Patents

Description

United States Patent 2,903,398. PREPARATION OF A STEROID COMPOUND Donald A. Kita, Jackson- Heights, and Gilbert M. Shull, Huntington: Station, N.Y., assignors to Chas. Pfizer & (30., Inc., New York, N.Y., a corporation of Delaware No Drawing. Application February 4, 1954 Serial No. 408,613 6 Claims. (Cl. 195-51);

invention is concerned witha novel process-for the preparation of A -androstadiene-SJ7-dione. More particularly it is concerned with a process. for preparing this compound by subjectingof a variety of steroid compounds to the enzymatic activity of a particular f ngus- This fungus was. received from the Quartermaster Corps and identified by them as a species of Pycnodothis (QM70 2');. A living culture of this; organism has been deposited with the American Type Culture Collection in Washington, DC, and given the number ATCC 1172-1.

It has been discovered that the enzymatic activity of this organism can. produce two types of changes in steroid molecules. The first is, the production of A rings which contain double bonds, at the l and 4 positions, and keto groups at the 3 position. This change is brought about when a steroid containing a double bond at the 4 position and a keto group at the 3 position is subjected to the enzymatic activity of the organism.

Compounds containing the type of A ring shown in the starting material include many well known steroids such as progesterone, testosterone, corticosterone and cortisone. There are also some indications that this type of product results when the starting androstane steroid contains instead a double: bond at the 5 position and an hydroxyl group at the 3 position.

The second type of change is the production of D rings with oxygenated groups at the 17 position. The presence irr the product of a l7'-keto group has definitely been established andthere are indications that a 17- hydroxyl group may instead be introduced in some. of the product molecules. This type of change is produced when the starting steroid contains any of a variety of groups at the 17 position. The original group may be alkyl, including straight and branched hydrocarbon chains containing up to about 10'. carbon atoms, acyl, for example acetyl, benzoyl or other such groups derived from simple acids containing up to about 10; carbon atoms, alkylene, including straight and branched unsaturated hydrocarbon chains containing up to about 10 carbon atoms, COCH OH, COCH Oacyl, COCH O- al'kyl, etc. The starting steroid may also contain a double bond at the 16 position, but this is not necessary.

This product, A -androstadiene-3,17-dione is very valuable. Illustrative of this value is the fact that it may readily be converted in one step, the well-known Inhoffen aromatization reaction, to estrone, a very useful compound.

It is to be understood that it is possible to carry out the processes of this invention, i.,e. subjecting a steroid compound to the enzymatic activity of the fungus ATCC 11721, not only by means of a culture of the living organism, but also by means of asuspension of mycelia from such a culture, orby means of the isolated enzyme systems, either intra-cellular or extra-cellular. These enzymes may be isolated by standard procedures used in enzymol'ogy. It is often more convenient, however, to utilizethe living organism for the carrying out of these processes. In general, it is preferred to grow the organism under conditions of submerged, aerobic fermentation, adding the steroid which is to be treated at either the beginning of the fermentation or during its course. The conditions for carrying out the fermentation may vary considerably. Sterile conditions should be employed throughout, and the temperature is kept between about. 20. and. 35 C. for best results. The composition of the nutrient medium may vary considerably. Ingeneral: a sourceof carbohydrate is required, for example, glucose, sucrose or starch. In addition, a source. of inorganic nitrogen, such as nitrates, ammonia salts, etc. is required. This. may be supplemented or replaced by an organic nitrogen. source, such as protein, hydrolyzate, soybean meal, cottonseed meal, etc. Inorganicsalts, particularly phosphates, potassium. salts, iron salts and magnesium salts, are desirable.

Throughout the fermentation, which on a large scale is run in vessels equipped for submerged, aerobic fermentation, the medium is. agitated and aerated at a rate of at least about /2 volume of air per volume of medium per minute. Occasionally it is necessary touse an anti-foaming agent, many of which are described in the technical literature, in order to prevent excess foaming of the. mixture. In conducting the process of this invention, the steroid compound which is to be treated may be added to the fermentation in finely divided solid form, or as a solution in a suitable nontoxic solvent, such as ethanol, acetone or isopropanol'. Preferably between about 0.01% and about 2% of the steroid by weight of the whole medium may be used. Although greater or lesser amounts may be used, there is no specific advantage. Addition of the compound may be made at the time the fermentation medium is inoculated with the, microorganism, or the; compound may be added after growth of the organism has, been established. Alternatively, the steroid compound may be added. in small portions during the course of the, fermentation; The compound must be sterile in order that the mixture be not contaminated by foreign microorganisms.

A further procedure that; has some value is the utiliza- 0 tion of; the. enzyme. systemsv formed during the growth of A specimen of the fungus ATCC 11721 was inoculated into 100 ml. of a nutrient medium contained in a 300 ml. Erlenmeyer flask. This medium had the following composition:

, Percent Malt extract 5.0 Sucrose 1.0 Dipotassium ortho phosphate 0.1 Sodium nitrate 0.2 Magnesium sulfate heptahydrate 0.05 Potassium chloride 0.05

Ferrous sulfate heptahydrate 0.001 Distilled water, adjusted to pH 7.0

Fifty mg. of progesterone was introduced into the medium. The organism was grown under aseptic, aerobic conditions at 28 C. The flask was constantly agitated by a rotary shaker.

After seven days, the mixture was extracted with 100 ml. of chloroform. The chloroform solution was evaporated to a volume of 2 ml., and a sample of this solution was then subjected to chromatography on alumina-impregnated paper. The developing solvent was a mixture of hexane and ether in the ratio of 15 to 5 by volume. In this way the products were first detected. The technique of paper chromatography which was used has been described by G. M. Shull et al. in Archives of Biochemistry and Biophysics, vol. 37, p. 186 (1952). The system here employed is referred to in that paper as System III.

The final isolation of the products was accomplished by subjecting the above-mentioned concentrated chloro form solution to chromatography on a silica gel-95% ethanol column. The developing solvent was a mixture of 99 volumes of methylene chloride and one volume of 95% ethanol.

Five compounds were isolated. Some unreacted progesterone was first recovered. The next compound isolated was A -androstene-3,17-dione. Then A -androstadiene-3,17-dione was recovered. This compound was present in by far the largest amount. Finally two unidentified steroids were isolated. Although it has not been conclusively established, it is believed that one of these compounds is A -androstadiene-17-hydroxy-3-one. The physical constants of the A -androstadiene-3,l7- dione were as follows: melting point, 139140 C.

sulfuric acid chromogen maxima at 266, 305 and 370 m minima at 288 and 350 m All these values are in excellent agreement with those previously reported in the literature.

Example II The experiment described in Example I was repeated, with no change except the use of 16-dehydroprogesterone in place of progesterone. The products were isolated as in Example I, and once again the main product was A androstadiene-3,l7-dione.

Example III When the same procedure is repeated, using 11-desoxy 'corticosterone in place of progesterone, the main product 1s again A -androstadiene-3,l7-dione.

4 Example IV A -androstadiene-3,17-dione was again produced when this procedure was repeated, using A -androstene-3,17 dione as the starting material. In this example, only the A ring was aflected, the D ring of the starting material being the same as that of the product.

The above examples are given for illustration only and are merely specific embodiments of this invention. Other obvious embodiments include contacting the steroid with a suspension of mycelium obtained from a culture of the organ-ism, or contacting the steroid with the enzymes isolated from the organism. The nutrient medium described above is conveniently used, but it is not a critical feature. Indeed, since many specific embodiments of this invention may be used without departing from the spirit and scope thereof, this invention is to be limited by the appended claims only.

What is claimed is:

l. A process for the preparation of A -androstadiene- 3,17-dione which comprises contacting with the enzymatic activity of the Pycnodothis fungus ATCC 11721 a steroid compound selected from the group consisting of:

R CH where R is a radical selected from the group consisting of where R is a radical selected from the group consisting of alkyl, alkylene, acyl, --COCH OH, COCH Oacyl, and -COCH Oa1kyl.

2. A process as claimed in claim 1, wherein the starting steroid is progesterone.

3. A process as claimed in claim 1, wherein the starting steroid is 16-dehydroprogesterone.

4. A process as claimed in claim 1, wherein the starting steroid is ll-desoxycorticosterone.

5. A process as claimed in claim 1, wherein the starting steroid is A -androstene-3,17-dione.

6. A process for the preparation of A -androstadiene- 3,17-dione which comprises cultivating the Pycnodothis fungus ATCC 11721 under submerged aerobic conditions at between about 20 and 35 C. in the presence of a steroid compound selected from the group consisting of:

R CH3 where R is a radical selected from the group consisting of alkyl, alkylene, acyl, --COCH 0H, -COCH Oacy1, References Cited in the file of this patent -COCHzOa1kY1, km, and hydroxyl; Ann. NY. Acad. Sci. 60, 1, p. 27.

( CH3 Bessey: Morphology and Taxonomy of Fungi, The

R Blakiston Company, Philadelphia, 1950, pp. 15-18. 0113A I 5 Bisby: An Introduction to the Taxonomy and Nomenclature of Fungi, 2d ed., 1953, The Commonwealth Myeological Institute, Kew, Surrey, pp. 3, 4, 29 to 33, 75 and 117.

, Ainsworth & Cowan: Rules of Nomenclature for 10 Fungi and Bacteria, Jour. General Microbiology, 10, 0: 1954, pp. 465-474. Clements et al.: Genera of Fungi, 1931 (2d Printing where R is a radical selected from the group consisting 1954 Hafn p Co 1954, 13 to 15 and 353 of yl, alkylefle, W z 2 YL Ainsworth: A Dictionary of the Fungi, The Commonand -COCH Oa1kyl. 15 wealth Mycological Institute, Kew, Surrey, 1954, p. 301.

Claims (1)

1. A PROCESS FOR THE PREPARATION OF $1,4-ANDROSTADIENE3,17-DIONE WHICH COMPRISES CONTACTING WITH THE ENZYMATIC ACTIVITY OF THE PYCNODOTHIS FUNGUS ATCC 11721 A STEROID COMPOUND SELECTED FROM THE GROUP CONSISTING OF: