Process for Preparing Steroids

This application is a continuation-in-part of application Serial No. 382,011, filed May 25, 1982. The invention described herein was made in the course of work under a grant or award from the Department of Health and Human Services. Technical Field

This invention relates to steroid hormones and more particularly to microbiological processes for converting readily available sterols into useful intermediates for the synthesis of more difficultly obtainable steroid hormones. Still more particularly this invention relates to processes for selectively degrading naturally occurring sterols at the C-17 position without concomitant ring cleavage, to obtain intermediates useful in synthesizing steroid hormones. Background Art

Utilization of the soybean sterols, sitosterol, campes- terol and stigmasterol as an economic source of intermediates for steroid manufacture has been long considered for supplementing or supplanting the commercial processes from diosgenin, obtained from the Barbasco root and other sources (R. iechert, Angew. Chem. Int. Ed. Engl. , 9_ _/ 321, 1970). Several microbiological processes are now available for the conversion of phytosterols or modified phytosterols

(19-αxygenated) into useful androstane steroids. (See for example C. K. A. Martin, Adv. in Applied Microbiol. , 22, 28, 1977) .

Disclosure of Invention Broadly the present invention comprises the enhancement of the efficiencies of the cleavage of sterols possessing branched chains at C-24. It has been found that this can be accomplished by the addition of bicarbonate ions to the

fermentation medium. More specifically, it has now been found that the efficiencies of the microbiological degradation of the side chains of sitosterol and campesterol, stigmasterol or their modified derivatives are greatly inproved if an exogenous source of ECO.,- ion is included in the medium.

Examples of ir crcorganisms which respond to HCO_- ion during the cleavage of the phytosterol side chain are those of the genera Nocardia, t^cobacterium, Art-hrobacter and Coryne- bacterium. (See for example, U.S. Patent No. 3,507,749.) Although there is no intention to be bound by the following theoretical considerations, it -is believed that in the fermentation the microorganisms function to degrade the steroid compound initially by oxidation at the C-26 position, introduction of unsaturation at C-24-C-25, incorporation of HCO_- at C-28 before any carbon-carbon breakage of the hydrocarbon side chain takes place. After hydration at C-24, the phytosterol side chain of the sitosterol class is converted into 3-oxo-chol-4-en-24-oic acid and i_wo moles of propionic acid. In the case of campesterol, it is converted into 3-oxo-chol-4-en-24-oic acid accompanied by one mole of acetic acid and one mole of propionic acid. The further transformation of 3-oxo-chol-4-en-24-oic acid into 17-keto steroids are well known. (C. J. Sih et al., Biochemistr , 7, 808, 1968.)

R, =H Campesterol

R ₂ = Coenzyme A

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The above scheme merely emphasizes the need of bicarbonate ion during the degradation. The concentrations of bicarbonate ion present in a normal medium is insufficient for this process to operate at maximum efficiency especially when high concent-rations of phytosterols or their derivatives are present. For the degradation to operate at maximum efficiency an exogenous source of HCO,- ion must be .supplied. This can be supplied in many forms such as NaHCO-, NH HCO-, CaCO,, CO , H_CO_, i.e. a one carbon unit chemical compound which can be converted by the microorganism (the enzyirεs elaborated by the microorganisms) to the bicarbonate ion or CC- . It should be emphasized that the form in which the HCO.,- is supplied is not critical or limiting as long as the form in solution required by the microorganism for incorporation on to the C-24 position is an activated form of bicarbonate ion (e.g., bicarbonate bound to coenzyme) . One of the most effective means for supplying the ion has been found to be the simple expedient of introducing (as by bubbling) C L through the fermentation medium while fermentation is progressing. Microorganisms which are characterized by their ability to degrade the side chain of phytosterols are well known in the art (see Martin, Adv. in Appl. Mic_robiol. , 22, 28, 1977 and U.S. Patent No. 3,507,749) . Any of the genera of microorganisms described therein can be employed in the process of this invention. The sterol starting material can be incorporated in a nutrient medium of standard composition in which such organisms are cultivated and the usual conditions of fermentation can then be employed to effect the sterol conversion. Alternatively, the active principle can be removed from the growing culture of microorgar-isms, for instance, by lysis of the cells to release the enzymes, or by suspension of the cells in a fresh aqueous system, or by iirrnobilization of cells. In any of these techniques the 17-side chain of the sterol will be selectively cleaved, so long as the active principle elaborated by the microorganism

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is present in the medium. Of course, the temperature, time and pressure conditions under which the contact of the phytosterol derivative with the degradative principle is carried out are interdependent as will be apparent to those skilled in this art. For instance, with gentle heating and at atmospheric pressure the time required to effect the sterol conversion will be less than at room temperature under conditions otherwise the same. Of course, neither temperature nor pressure nor time should be so great that the sterol is thereby degraded. Where a growing culture of the organism is used, the process conditions should also be sufficiently gentle so the organism is not killed prematurely, i.e. before it elaborate sufficient enzymes. Generally speaking, the temperature can range from about 10°C to about 37°C and the time from about 12 hours to about 12 days.

Best Mode for Carrying Out the Invention

The following examples are given to illustrate this invention but are not to be construed in any way to limit its scope. EXAMPLE I

A) Fermentation - Surface growth from a one week old agar slant of Mycobacterium sp. NRR B-3805 grown on agar of the following composition:

Glucose-Agar -Solid Medium Cms

Agar 20

Glucose 10

Yeast extract 2.5

K_HP0. 1

2 4 Distilled water, q.s. 1 liter

(Sterilized 15 minutes at 20 p.s.i.) was suspended in 5 ml of an 0.85% saline solution. One ml portions of this suspension were used to inoculate a 250 ml Erlenmeyer flask (F-l stage) each conta__ning 60 ml of the soybean-dextrose medium (SDM) :

OMH

Soybean Dextrose Medium (SDM)

Gms

Soybean meal 5

Dextrose 20 Yeast extract 5

K_HP0..7H»0 8.7

2 4 2

Sitosterol (containing campesterol) 0.1

Tap water, q.s. 1 liter, adjusted to pH 7.8 (Sterilized 20 iron at 30 p.s.i.) The flasks were incubated at 25°C on a rotary shaker (200 cycles/min - 2" radius) for 48 hours, after which a 10% by volume transfer was made to a series of 250 ml Erlen- ireyer flasks (F-2 stage) each containing 60 ml of the above medium. After 48 hours, 120 mg of sitosterol, sus- pended in 5 ml of 1% Tween 80, was added to each flask resulting in a final substrate concentration of 2 mg/ml. At the same time various amounts of NaHC0 ₃ powder was added to each flask to give the indicated HCO_- concen¬ trations. The flasks were incubated under the same conditions as the F-l stage flasks. The pH was main¬ tained at 7.6 ± 0.1 by the addition of sterile IM KH-PO. solution each day. B) Quantative analysis of androst-4-ene-3,17-dione - Forty- -eight hours after the addition of the sitosterol, each flask was extracted separately with ethyl acetate (50 x 3) . The combined extracts were washed with water, dried over Na_S0 , and evaporated to dryness in vacuo. A por¬ tion of the residue was dissolved in acetone and'chroma- tographed over a 10 x 20 cm thin-layer plate [EM. - 0.5 mm thickness silica gel containing PF254 indicator, Brink- ann] and developed in a system which consisted of: ethyl acetate-hexane (1:1) . Sitosterol has R- = 0.49; androst-4-ene-3,17-dione has R,. = 0.27. The TV absorbing band corresponding to androst-4-ene-3,17-dione was eluted

exhaustively with ethyl acetate and the TJV absorbancy at 240 πm (β= 15,000) was measured. The results were as follows:

Concentration of m moles of androst-4-

HCO.,- (rrM) ene -3,: 17-dione

0 55 .7 25 99 .0

50 91. .1 75 94, .2

^• EXAMPLE 2

A) Fermentation - Surface growth from a one-week old agar slant of Mycobacterium sp. NRRL-3683, grown on the glucose-agar solid medium, was suspended in 5 ml of an 0.85% saline solution. One ml portions of this suspension were used to inoculate a 250 ml Erlenmeyer flask (F-l stage) each containing 60 ml of the soybean- dextrose medium (SDM) . The flasks were incubated at 25°C on a rotary shaker (200 cycles/min - 2" radius) for 48 hours, after which a 10% by volume transfer was made to a series of 250 ml Erlenmeyer flasks (F-2 stage) each con¬ taining 60 ml of the SDM medium. After 48 hours of incu¬ bation (same as F-l stage) , 120 mg of sitosterol (con¬ taining campestero) , suspended in 5 ml of 1% Tween 80 was added to each flask. At the same time various quantities of NaHCO_ powder was added to each flask to give the desired HCO.,- concentration. The flasks were incubated on the rotary shaker (same as F-l stage) and at 48 and 116 hours, samples were analyzed for androsta-1,4-diene- 3,17-dione. B) Quantitative analysis of androsta-1,4-diene-3,17-dione (ADD) . At the indicated time intervals (48 hrs and 116 hrs) , each flask was adjusted to pH 3 and extracted separately with ethyl acetate (50 ml x 3) . The combined extracts were washed with water, dried over Na-SO., and

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evaporated to dryness in vacuo. A portion of the residue was dissolved in acetone and chromatographed over a 10 x 20 cm thin layer plate [EM - 0.5 thickness silica gel containing PF254 indicator, Brinkmann] and developed in a system consisting of ethyl acetate-hexane (1:1) . Sito¬ sterol has P = 0.49; androsta-1,4-diene-3,17-dione {ADD) has Rp = 0.16. The UV absorbing band corresponding to androsta-1,4-diene-3,17-dione was eluted exhaustively with ethyl acetate and the UV absorbancy at 244 nm (£.= 15,000) was measured. The results were as follows: m moles of ADD HC0 ₃ -- (irM) 48 hrs 116 hrs

0 39.4 78.6

25 56.0 105.8

50 142.1

75 51.6 102.8

100 59.7 100.9

EXAMPLE 3 Fermentation. Surface from a one-week old agar slant of Mycobacterium phlei NREL-B-15050, grown on the glucose agar solid medium, was suspended in 5 ml of an 0.85% saline solution. One ml portions of this suspension were used to inoulate a 250 ml Erlenmeyer flask (F-l stage) each contai ing 60 ml of the following medium: GlycerOl-Medium A

Gms

Nutrient broth (Difco) 8

Yeast extract 1

Glycerol 5

K 3P0..7H-0 2 4 2 8.7

Sitosterol (containing campesterol) 0.1 Tap water, q.s. 1 liter, adjusted to pH 7.8 (Sterilized 20 min at 30 p.s.i.)

After 48 hours of incubation (same as F-l stage) , 120 mg of sitosterol (containing campesterol) suspended in 5 ml of 1% Tween 80 was added to each flask. At the same time various quantities of NaHCO- powder was added to each flask to give the desired HC0 ₃ ~ concentrations. The flasks were incubated on the rotary shaker (same as F-l stage) and at 48 and 96 hours, samples were analyzed for androsta-1,4-diene-3,17-dione (ADD) . m moles of ADD HD0_- ( M) 48 hrs 96 hrs

0 18.4 40.8 25 42.7 67.3 50 46.0 69.4

75 42.4 64.8 In the foregoing Examples bicarbonate ion concentrations as high as 200 irM can be used with comparable results, although the preferred range is from 25 mM to 150 irM. In any event, concentrations should be used which will enhance the efficiencies of the selective cleavage of the 17-side chains of sterols possessing branched chains at C-24 but which will not adversely affect such cleavage.

It is to be understood that any of the .rάcroorganisms of the genera Nocardia, Mycobacterium, Arthrobacter and Coryne- bacterium (U.S. Patent No. 3,507,749) which respond to the ECO-.- ion during cleavage of the phytosterol side chain can be readily substituted for those microorganisms specified in the foregoing specific Examples and that comparable production of the desired product androsta-1,4-diene-3,17-dione or androst- 4-ene-3,17-dione will be obtained.