DK144708B - METHOD FOR PREPARING 21-HYDROXY-20-METHYL PREGNANCY DERIVATIVES - Google Patents

Description

144708

The present invention relates to a process for the preparation of 21-hydroxy-20-methylpregnan derivatives of general formula I

H-zC CHOOH

XXj 2 144708 wherein "the bond ..... means a single bond or a double = bond, by fermentation of zoo or phytosterols with a culture of mycobacterium spec. NRRL B-3683 or NRRL B-3805 or variants or mutants thereof having substantially the same properties as said microorganic pain.

It is known that cultures of mycobacterium spec. NRRL B-3683 and NRRL B-3805 have the ability to form 21-hydroxy-20-methyl-pregnane derivatives of the general formula I from zoo or phytosterols (Applied Microbiology 23, 1972, 72 et seq. And Applied and Enviromental Microbiology 32, 1976, 310 et seq.). These compounds, however, are formed only by trace or in very poor yields, so that the known method is hardly technically applicable.

With the aid of the present invention, which is acceptable by the characterizing part of claim 1, significantly higher yields of 21-hydroxy-20-methyl-pregnane derivatives of the general formula I can be obtained compared to the known process.

Suitable zoo or phytosterols for the fermentation are, for example, the cholesterol, stimasterol, campesterol, brassicasterol or sitosterols, the 3-acyl compounds, such as, for example, the acetates of these sterols or the 3-keto-A obtained from Oppenauer oxidation from these 4 steroids, such as, for example, 4-cholesterol-3-one, 4-stigmasten-3-one, 4-sitosten-3-one or 4 - = - campesten-3-one.

In the process of the invention, the fermentation is carried out in the presence of borations or organic boron compounds. Suitable boron compounds are, for example, boric acid, sodium metaborate, disodium tetraborate, calcium metaborate or triphenylborate. The fermentation is preferably carried out in the presence of 0.1% to 0.5% reagents which supply boron ions. The addition of the reagents supplying borations is preferably carried out 10 to 30 hours after the start of the fermentation.

When adding the reagents which supply borations, care must be taken to adjust the pH of the fermentation culture in the usual manner to a value between 6.0 and 8.0.

Moreover, the fermentation is carried out under the usual conditions. The microorganisms are grown in an appropriate nutrient medium under aeration of submersible cultures. The culture medium is then added to the substrate (dissolved in a suitable solvent and preferably in emulsified form) and fermented until a maximum substrate conversion is achieved (96-160 hours).

Suitable substrate solvents are, for example, methanol, ethanol, glycol monomethyl ether, dime thylformamide or dimethyl sulfoxide. For example, the emulsification of the substrate may be effected by dosing the substrate in micronized form or dissolved in a water miscible solvent (such as methanol, ethanol, acetone, glycol monomethyl ether, dimethylformamide or dimethyl sulfoxide) under heavy turbulence in (preferably decalcified) water. which contains the usual emulsifying aids. Suitable emulsifiers are nonionic emulsifiers, such as, for example, ethylene oxide adducts or fatty acid esters of polyglycols. Suitable emulsifiers include, for example, commercially available wetting agents "Tegin" ®, “Tagat” ®, “Tween” ® and “Span” ®.

In the fermentation, the emulsification of the substrates often allows an increased substrate capacity and thus an increase in the substrate concentration. However, it is of course also possible to use other methods of increasing the substrate capacity in the process according to the invention, as are well known to those skilled in the fermentation field.

The optimum substrate concentration, substrate addition time and fermentation duration depend on the structure of the substrate used. These sizes, as is usually required for microbiological steroid conversions, must in each case be determined by prior experiments, as is well known to those skilled in the art.

Further enhancement of the process products can be achieved when selecting or mutating the mycobacteria species in the usual manner. Thus, for example - appropriately after treatment with mutagens - mycobacterium spec. NRRL B-3805 on blood agar plates and single colonies of different morphological appearance are obtained. If these single colonies are isolated and tested for their ability to form 21-hydroxy-20-methyl-pregnane derivatives of general formula I, one finds that especially among the selection strains forming round colonies, there are strains producing a 1.5 to 3 times higher yield of 21-hydroxy-20-methyl-pregnane derivatives than the unselected strain.

The 21-hydroxy-20-methyl-pregnane derivatives of general formula I are valuable intermediates for the synthesis of pharmacologically active steroids. Thus, for example, it is possible to oxidize these compounds to the corresponding pregnane-20-carboxylic acids of the general formula III, which, by means of that of H. Ruschig et al.

(Chem. Ber. 88, 1955, 883 et seq.) Can be transferred to the corresponding pregnane-3,20-dione derivatives of formula IV.

1 fH3 III

c = o (Y = hydrogen or 17a-hydroxy)

The compounds thus obtained are known, as is well known, by their progestogenic action and are further valuable intermediates for the synthesis of numerous pharmacologically active steroids.

The following examples serve to illustrate the invention.

Example 1 a) A 2 1 Erlenmeyer flask with 500 ml sterile nutrient medium - inside = 5 144708 containing 1% yeast extract 0.45% disodium hydrogen phosphate 0.34% potassium dihydrogen phosphate 0.2% "Tagat" ® 02 - adjusted to pH 6.7 - grafted with a slurry of a dry culture of mycobacterium spec.

NRRL B-3805 and shaken for 3 days at 190 rpm. per minute at 30 ° C.

b) 22 g sitosterol is emulsified with 4.4 g of Tegin® and 430 ml of water at 95 ° C with an ultra "Turrax" ® (companies Jahrike and Kunkel) for 25 minutes and make up to 513 g with water . The emulsion is sterilized for 20 minutes at 120 ° C.

c) A 500 ml Erlenmeyer flask with 65 ml sterile nutrient medium - containing 2 g of corn mold liquid 0.3 g of diammonylhydrogen phosphate 0.25 g of "Tagat" ® 02 - adjusted to pH 6.5 - inoculated with 5 ml of mycobacterium spec, the seed culture. Then, 28 ml of the suspension prepared as indicated in (b) is added (this corresponds to 1.2 g of sitosterol) and after 24 hours 4 ml of 4% aqueous sodium tetraborate solution is added and fermented for an additional 120 hours at 30 ° C. shaking.

After fermentation, the culture broth is extracted twice, each time with 100 ml of ethylene chloride. The combined ethylene chloride extracts are added to 11 g of activated carbon and filtered through a folding filter. The filtrate is then evaporated at 40 ° C on a rotary evaporator and chromatographed on alumina. After chromatography, 135 mg of 21-hydroxy-20-methyl-4-pregnen-3-one is obtained, mp 140-141 ° C (from ethyl acetate).

If the reaction is carried out under the same conditions, but without the addition of sodium tetraborate solution, 45 mg of 21-hydroxy-6UA708 20-methyl-4-pregnan-3-one is obtained.

Example 2 a) 513 g of emulsion is prepared from 22 g of 4-cholesterol-3-one equal to Example 1b.

b) under the conditions of Example 1c, 70 ml of a myco = bacterium spec. NRRL B-3805 culture, 28 ml of the 4-cholesterol-3-one emulsion is added and after 24 hours 4 ml of 4% aqueous sodium tetraborate = solution and further fermented for 120 hours at 30 ° C with shaking.

The fermentation portion is worked up as described in Example 1c and 95 mg of 21-hydroxy-20-methyl-4-pregnen-3-one is obtained, mp 142-144 ° C.

If the reaction is carried out under the same conditions, but without the addition of sodium tetraborate solution, 40 mg of 21-hydroxy-20-methyl-4-pregnen-3-one is obtained.

Example 3 28 ml of the sitosterol emulsion prepared according to Example 1b is fermented as described in Example 1c with 70 ml of a mycobacterium spec, NRRL B-3805 culture, thereby substituting the aqueous one. sodium tetraborate solution uses an addition of 6 ml of k% calcium = cesium metaborate suspension in water.

After working up the fermentation portion as described in Example 1c, 120 mg of 21-hydroxy-20-methyl-4-pregnen-3-one is obtained, mp 141-143 ° C.

Example 4 28 ml of the sitosterol emulsion prepared according to Example 1b is fermented as described in Example 1c with 70 ml of a mycobacterium spec. NRRL-B-3805 culture, using an addition of 5 ml of a k% triphenylborate solution instead of the aqueous sodium tetraborate solution.

7 144708

After working up the fermentation portion as described in Example 1c, 125 mg of 21-hydroxy-20-methyl-4-pregnen-3-one is obtained, mp 142-143 ° C.

Example 5 a) Under the same conditions as in Example 1a a myco = bacterium spec is grown. NRRL-B-3683 culture.

b) 65 ml of the nutrient medium described in Example 1c are added 5 ml of mycobacterium spec. NRRL-B-3683-seed culture. The culture is then added 28 ml of the sitosterol emulsion described in Example 1b and after 24 hours 4 ml of 4% sodium tetraborate solution and fermented for an additional 120 hours. The fermentation amount is worked up as described in Example 1c and 90 mg of 21-hydroxy-20-methyl-1,4-pregnadien-3-one is obtained, mp 180-182 ° C (from ethyl acetate acetone).

Example 6 40 ml of a seed culture of mycobacterium spec. NRRL-B-3805 - prepared according to Example 1a - is centrifuged at 4000 rpm. minute.

The bacterial mass thus obtained is then washed twice with a saline buffered to pH 6 containing 0.5% sodium chloride, 0.012% magnesium sulfate (heptahydrate) and 1.36% potassium dihydrogen = phosphate, suspended in 40 ml of this saline solution and added 10 ml of a 0.5% 1-methyl-3-nitro-1-nitrosoguanidine.

The bacterial suspension is incubated for one hour at 30 ° C, Fracentri = grout the bacteria, wash them twice with the above-mentioned salt = solution and spread on blood agar plates (Oxoid Ltd., London). Of the single colonies formed, those which form round colonies are selected and from these seed graft cultures are prepared as described in Example 1a. The seed cultures serve to carry out the fermentation described in Example 1c with the following result: 8 144708

Number of Yields of 21-Hydroxy-20-Methyl-4 Selection Strains Pregnen-3-One

Without the addition of With the addition of sodium tetraborate sodium tetraborate 19 0 - 200 mg 8 201 - 400 mg 94 401 - 600 mg 35 601 - 800 mg 7 801 - 1,000 mg 1,550 - 2,100 mg 0 1,001 - 1,020 mg 6 1,021 - 1,040 mg 1,900 - 3,000 mg 1 1.041 - 1.060 mg 2.900 mg (150 mg) 0 1.061 - 1.080 mg 1.081 - 2.000 mg 1 (195 mg) 3.450 mg

Claims (2)

9 144708 Patent Claims. A process for the preparation of 21-hydroxy-20-methylpregnan = derivatives of the general formula I h3c ch2oh (i). wherein the bond ..... means a single bond or a double bond, by fermentation of 2OO or phytosterols with a culture of mycobacterium spec. NRRL B-3683 or NRRL B-3805 or variants and mutants thereof having substantially the same characteristics as said microorganisms, characterized in that the fermentation is carried out at a ρΠ-vaxli from 6.0 to 8.0 in the presence of borations or organic boron compounds. A process according to claim 1, characterized in that a zoo or phytosterol of the general formula II is fermented with the formula II 3jC (II) wherein the bonds ..... denote single bonds or double bonds, X represents an oxo group or a hydrogen atom, and R 1 represents a hydrogen atom, a methyl group or an ethyl group. A process according to claim 1 or 2, characterized in that as the reagent which supplies borations, ortho = boric acid, metaboric acid, polyboronic acid or alkali or alkaline earth metal salts thereof are used.