DE1134074B - Process for the production of 16-methylene steroids - Google Patents

Description

Process for the preparation of 16-methylene steroids It has been found that 16-methylene steroids of the glucocorticoid series have interesting pharmacological properties own. US Pat. No. 2,865,808 describes a manufacturing process described for 16-methylene-corticosteroids, however the rework has shown that it is then not possible to produce the desired 16-methylene steroids. In particular, those in Examples 11 and 17, Part 2 of the above United States patent does not reproduce the method described. You don't get the desired 16-methylene steroids dehydrated in the 1 (2) position, but only Decomposition products. Also the zinc dust reductions described in Examples 19 and 20 lead to non-isolable amounts of the products dehalogenated in the 9a-position. Otherwise, according to US Pat. No. 2,865,808, 16cx-hydroxy-hydrocortisone is used as the starting material used, which itself is only available via a large number of reaction stages. So far there is no practically feasible process for the production of 16-methylene corticosteroids.

It has now been found that 16-methylene-corticosteroids, in particular 16-methylene hydrocortisone, 16-methylene cortisone, 16-methylene prednisone, 16-methylene-prednisolone and its 21-ester can be produced if you use it as a starting material 16ß-methyl-16x, 17x-oxido-5-pregnen-3ß-ol-20-one-3-acetate which can be prepared by known methods (I) (cf. the attached reaction scheme) was used.

The invention accordingly provides a process for the preparation of 16-methylene steroids of the general formula A. where X = H, OH (x or ß) or = 0 and Y = a free or esterified hydroxyl group, as well as of their 1-dehydro-derivatives, which consists of 16ß-methyl-16a, 17a-oxido- 5-pregnen-3ß-ol-20-one-3-acetate (I) by heating with catalytic amounts of a strong acid, especially a sulfonic acid, in the presence of an inert solvent, especially benzene, in 16-methylene-5-pregnen-3ß , 17a-diol-20-one-3-acetate (I1) is converted, then the compound II is reacted in a manner known per se with at least 2 molar equivalents of bromine to give the 5,6,21-tribromo-16-methylene-pregnane-3β , 17a-diol-20-one-3-acetate (III) by successive treatment with an iodide and an acetate, in particular sodium iodide and potassium acetate, in 16-methylene-5-pregnen-3ß, 17 x, 21-triol-20- on-3,21-diacetate (IV), this ester is incubated with a culture of Flavobacterium dehydrogenans or an enzyme obtained therefrom and the 16-methylene-4-pregnen-17 # x, 21-x, 21-3, 20-dione (V) by known microbiological 11x- or 11ß-hydroxylation in 16-methylene-4-pregnen-11 (x or ß), 17a, 21-triol-3,20-dione (VII a or b) and optionally the latter Conventional oxidation is converted into 16-methylene-4-pregnen-17x, 21-diol-3,11,20-trione (IX) or the compound V obtained is first microbiologically dehydrated in the 1 (2) position by methods known per se and the 16-methylene-1,4-pregnadiene-17oc, 21-diol-3,20-dione (VI) obtained by known microbiological 11x- or 11ß-hydroxylation in 16-methylene-1,4-pregnadiene-11 (x or ß), 17a, 21-triol-3,20-dione (VIII a or b) and optionally the latter by conventional oxidation in 16-methylene-1,4-pregnadiene-17x, 21-diol-3, 11,20-trione (X) transferred or the 11x- or 11ß-hydroxysteroid VII a or b or the 11-ketosteroid IX obtained is microbiologically dehydrated in a known manner in the 1 (2) position and that, if necessary the hydroxyl group contained in compounds V to X in 2 1-position esterified by methods known per se and, if appropriate, the esters obtained are treated analogously to the corresponding 21-hydroxy compounds V to IX.

According to the method according to the invention, it is thus possible in five or six or seven steps to produce the end products of formula A.

The compounds I to X are not yet in the literature with physical Data described. For splitting the epoxy ring with the formation of the 16-methylene group dissolve the compound I in benzene or another inert solvent and uses catalytic amounts of a strong acid, e.g. B. a sulfonic acid, preferably p-toluenesulfonic acid or benzenesulfonic acid, too. By boiling I in this solution succeeds in the 16x, 17a-Oxidoring in smooth reaction with formation of the connection II split.

The acetate is treated to introduce a 21-hydroxy function 1I in a suitable solvent, e.g. B. in glacial acetic acid-chloroform, with at least 2 molar equivalents, bromine, using a small amount of hydrogen halide as a catalyst clogs. This method gives the tribromo compound III. The raw product this bromination is z. B. treated in acetone solution with sodium iodide, the The 21-position bromine atom was replaced by iodine and the 5 (6) -positioned double bond is regenerated. By boiling with an alkali acetate, preferably potassium acetate, the iodine atom in position 21 is replaced by the acetoxy group. You get in good Yield 16-methylene-5-pregnen-3β, 17a, 21-triol-3,21-diacetate (IV).

By incubating IV with a culture of Flavobacterium dehydrogenans or an enzyme that can be prepared from it succeeds in the best yield and in smoother Reaction to produce 16-methylene-Reichsteins-Substance-S (V; R1 = H). As a nutrient solution for Flavobacterium dehydrogenans one uses z. B. a buffered to px 7.0 Solution of a 1% yeast extract in water. After about 10 to 16 hours of growth at about 28 ° C., compound VII is added to the bacterial culture. The incubation continued for about 6 hours with ventilation. The progress of the reaction can can be controlled by measuring the UV spectrum.

The compounds V, VII a, VII b and IX or their 21-esters can be used microbiologically dehydrate according to the invention in 1 (2) position. For this z. B. the following microorganisms in question: Bacillus sphaericus, Fusarium solani, Coryne bacterium simplex, Alternaria sp., Mycobacterium smegmatis, Calonectria decora, Mycobacterium lacticola, Ophiobolus sp., Alcaligenes sp., Didymella lycopersici, Protaminobacter sp., Septomyxa affinis, Nocardia sp., Cylindrocarpon radicicola, Streptomyces lavendulae, Bacillus cyclooxydans. The fermentation takes about 4 up to 14 hours, depending on which microorganism is used. Particularly suitable are cultures of Bacillus sphaericus var. fusiformis and Corynebacterium simplex.

To introduce a 11a- or 11ß-hydroxyl group in the The following microorganisms are suitable for compounds V or VI: 11-hydroxylation Curvularia lunata, Cunninghamella blakesleeana, Mucor griseocyanus, Cephalothecium sp., Trichothecium sp., Botrytis cinerea, Coniothyrium sp., Thamnidium sp., Streptomyces fradiae, Rhodoseptoria sp., Colletotrichum sp., Dothichiza sp., Absidia glauca, Pycnosporium sp., L 1 x-hydroxylation Rhizopus (various species), Aspergülus (different types), Penicillium (different types), Fusarium (different types), Neurosparo sitophila, Trichothecium sp., Cephalothecium roseum, Pestalotia foedans, Dactylium dendroides, Heliocostylum pyriforme, Thamnidium sp., Eurotium chevalieri, Mucor javanicus and various species, Delacroixia coronata, Absidia glauca, Cunninghamella echinulata, and further fungi of the order Mucorales.

The microbiological 11-hydroxylation is carried out according to known methods Methods carried out. Particular successes in the IIx-hydroxylation are achieved when using fungi of the genus Fusarium, which is used as the starting material Steroid is completely converted, so that lengthy separation processes are avoided.

The 11-O H group of compounds VII a, VII b and VIII a, VIII b can be obtained by treatment with a mild oxidizing agent, e.g. B. through implementation with chromic acid, a mixture of chromic acid and pyridine or with sub-bromine Oxidize acid to 11-keto group.

The compounds with free hydroxyl groups listed in the reaction scheme in the 21-position can be esterified in the 21-position by methods known per se. As esterifying agents, for. B. the halides or the anhydrides of the following Acids can be used: acetic acid and its higher homologues, e.g. B. tert-butyl acetic acid, Succinic acid and its higher homologues, amino or alkylaminocarboxylic acids, tetrahydrophthalic acid, Aminodicarboxylic acids, e.g. B. aspartic acid, cyclopentylpropionic acid, phosphoric acid or sulfuric acid.

The 16x, 17x-Oxidosteroid I required as starting material can be extracted from 16-methyl-5,16-pregnadien-3ß-ol-20-one-3-acetate using sodium hydroxide and hydrogen peroxide and subsequent acetylation, e.g. B. with a mixture of acetic anhydride and pyridine, getting produced. For the production of the starting material used according to the process Protection is not sought within the scope of the present invention.

The 16-methylene-corticosteroids obtained by the process according to the invention are intended to be used as anti-inflammatory drugs in all possible indications, z. Rheumatoid arthritis, can be used in human medicine. You own can also help fight stubborn allergies. Example 1 Preparation of 16-methylene hydrocortisone (VIIb; R, = H) and its 21-acetate (VII b, R1 = acetyl) a) Preparation of the starting material. 15 g 16-methyl-pregnadienolone-3-acetate (manufactured according to Wettstein, Helvetica Chimica Acta, Vol. 27 [1944], p. 1803) are dissolved in 2.5 liters of methanol. The reaction mixture is cooled to 10 ° C and 30 ml of 4N sodium hydroxide solution and 60 ml of 30% hydrogen peroxide admitted. After standing for 40 hours at about + 5'C, the solution is poured into water, the precipitate which has separated out is filtered off with suction and recrystallized from methanol. That 16β-methyl-16x, 17α-oxido-5-pregnen-3β-ol-20-one obtained (mp. 182 to 184'C, [rx] D -24.6 '(chloroform)) with 120 ml of pyridine and 120 ml of acetic anhydride for 1 hour heated on the steam bath, the solution poured into water and the precipitated 16ß-methyl-16ca, 17a-oxido-5-pregnen-3ß-ol-20-one-3-acetate (I) sucked off. For purification, it is recrystallized from acetone. Yield: 12.9 g (based on 15 g of 16-methyl-pregnadienolone-3-acetate used), melting point 178 to 180 ° C, [.x] D -12 '(chloroform).

b) Epoxy splitting of I. A solution of 9.3 g of as the starting material Serving 16ß-methyl-16x, 17a-oxido-5-pregnen-3ß-ol-20-one-3-acetate (I) in 350m1 anhydrous benzene is after the addition of 630 mg of p-toluenesulfonic acid for 4 hours Refluxed. After cooling, the benzene solution is first with 5% Sodium hydrogen carbonate solution, then washed with water, dried and evaporated. 5.4 g of crystallized 16-methylene-5-pregnen-3β, 17rx-diol-20-one-3-acetate are obtained (II) obtained. Recrystallized from methanol, the compound melts at 196 bis 198'C.

c) bromination of II to III and transesterification to IV. 1.55 g of 16-methylene-5-pregnen-3β, 17a-diol-20-one-3-acetate (II) are dissolved in 60 ml of glacial acetic acid and 15 ml of chloroform and the solution to 5'C cooled down. Within 20 minutes after adding a few drops of a solution of hydrogen bromide in glacial acetic acid while stirring 62 ml of a 2% solution of bromine added dropwise in glacial acetic acid. After the addition has ended, stirring is continued for a further 1/2 hour. The solution is poured into water, extracted with ether and the ethereal solution with washed neutral with an aqueous solution of sodium hydrogen carbonate. The over sodium sulfate dried solution is evaporated in vacuo. The tribromide III remains in oily form, which is further processed without cleaning.

To do this, the residue is dissolved in 60 ml of acetone, 1.5 g of sodium iodide and 6 g of anhydrous potassium acetate are added and the mixture is boiled under reef for 4 hours. After cooling, the inorganic salt is filtered off and the solution is evaporated in vacuo. The residue is dissolved in chloroform, this solution is extracted three times with water, evaporated in vacuo, dissolved with benzene and chromatographed on magnesium silicate, known under the trade name Florisil. The benzene-chloroform (1: 1) eluate gives the diacetate IV on evaporation, which is obtained in pure form by recrystallization from acetone. Yield: 1.05 g, melting point 195 to 197 ° C., [_x] D -65.7- (chloroform).

d) Microbiological saponification and oxidation of IV. A sterilized nutrient solution consisting of 80 g glucose, 50 g yeast extract, 30 g ammonium chloride, made up to 101 with tap water and adjusted to pH 7.0 with 1/30 molar phosphate buffer solution according to Sörensen, is with 200 ml of a shaking culture of Flavobacterium dehydrogenans inoculated. The culture is allowed to grow for 12 hours with stirring and aeration at 28 ° C. After adding a concentrated solution of 12 g of diacetate IV in acetone or acetone-dimethylformamide (1: 1), the culture is allowed to react for 10 hours under unchanged conditions. The solution is extracted several times with chloroform, the chloroform solution is dried and evaporated. The evaporation residue is purified by recrystallization. 8 g of pure 16-methylene-4-pregnen-17a, 21-diol-3,20-dione (V; R1 = H), melting point 205 to 206'C [a] D -I-46.6 '( Chloroform), Amax 240 m # L, Eil 480.

e) Microbiological lß-hydroxylation of V (R1 = H). A nutrient solution of 5 % malt extract, 10 % sucrose, 0.2% sodium nitrate, 0.1% dipotassium phosphate, 0.05% magnesium sulphate, 0.05% potassium chloride is poured into a small fermenter and 0.005% iron (II) sulfate (pH adjusted to 7.0) inoculated with 800 ml of a shaking culture of Curvularia lunata (Wakker) Boadijn and incubated with stirring and strong aeration at 28 ° C. After 24 hours of growth, 5 g of 16-methylene-Reichsteins-Substance-S (V; R1 = H), dissolved in 40 ml of dimethylformamide, are added. The conversion is followed by paper chromatography. If the starting material can no longer be detected in the paper chromatogram, the culture solution is extracted three times with the same volume of chloroform. The combined chloroform solutions. are evaporated and the residue is chromatographed on silica gel to remove the accompanying products. The eluate obtained with chloroform / ethyl acetate (1: 3) contains the desired 16-methylene-hydrocortisone (VIlb; R1 = H), which is obtained therefrom in the usual way. Mp. 224 to 225 ° C, [a] D -69.2 '(dioxane), 2max 241 m # L, El% 466. After further recrystallization from acetone, the compound melts at 232 to 233 ° C.

f) Acetylation of VIIb (R1 = H). 5 g of 16-methylene hydrocortisone (VIIb; R1 = H) are heated with 30 ml of pyridine and 30 ml of acetic anhydride on the steam bath for 1 hour. The mixture is poured into water and the precipitated 16-methylene-hydrocortisone-21-acetate (VIIb, R1 = acetyl) is filtered off with suction. The purification is carried out by recrystallization from acetone. M.p. 217 to 219 ° C. Example 2 Production of 16-methylene-II-epi-hydrocortisone (VII a; R1 = H), its 21-acetate (VII a; R1 = -COCH3) and of 16-methylene-cortisone (IX; R1 = H) and its 21-acetate (IX; R1 = -COC H3) a) Microbiological 1 lx-hydroxylation of V (R1 = H). A suitable nutrient solution (e.g. 5% glucose, 0.1% yeast extract, 0.05% soy flour, 0.3 "/, Na N 03, 0.05" / , Mg S 04 - 7 H2 O, 0.1% K H2 P 04, 0.05% K Cl, 0.001% Fe S 04 - 7 H20) with 750 ml of a well-grown shaking culture of Fusarium sp . (E. Merck Collection No. 2083). The culture grows with vigorous aeration and stirring at 28 ° C. and after 24 hours receives an addition of 5 g of the 16-methylene-Reichstein substance S (V; R1 = H) obtained according to Example 1, d) in 40 ml of dimethylformamide . The conversion is followed by paper chromatography. When the starting substance is no longer detectable in the chromatogram, the batch is terminated and the culture solution is extracted three times with 101 chloroform. The combined chloroform extract is evaporated, the residue is digested with petroleum ether and recrystallized from acetone. Pure 16-methylene-11-epi-hydrocortisone (VIIa; R1 = H) is obtained. Mp. 199 to 201'C, [a] D -I-41.7 '(dioxane), a, max 241 m # L, egg,'. 445.

b) 21-acetylation of VII a (R1 = H). 2 g of 16-methylene-11-epi-hydrocortisone (VII a; R, = H) are dissolved in 10 ml of pyridine and 0.36 g of acetic anhydride are added. After standing for 15 hours at room temperature, it is poured into water with chloroform 3 times extracted the chloroform solution by shaking with sodium hydrogen carbonate solution neutralized, dried and evaporated in vacuo. An amorphous residue is obtained of 16-methylene-11-epi-hydrocortisone-21-acetate (VII a; R1 = acetyl).

cl) The residue of 16-methylene-11-epi-hydrocortisone-21-acetate obtained in Example 2, b) (VII a; R1 = acetyl) is dissolved in 20 ml of pyridine and made into a mixture of 2 g Chromic anhydride and 20 ml of pyridine were added. That soon turned deep dark brown After standing for 8 hours at room temperature, the mixture is poured into 150 ml of warm ethyl acetate poured in, refluxed for 5 minutes and suction filtered. After washing well the voluminous residue with warm ethyl acetate are the combined filtrates washed neutral with dilute sulfuric acid, dried and evaporated in vacuo. The residue of crude 16-methylene-cortisone-21-acetate (IX; R1 = acetyl) is through Recrystallization from methanol obtained in pure form. Yield: 1.15 g, melting point 213 to 214 ° C, [a] D -f-140 ° (dioxane), .1 .. "x 237 m # t, E'1, '409.

c2) 2.7 g of 16-methylene-11-epi-hydrocortisone-21-acetate (VII a; R1 = acetyl), prepared according to Example 2, b), are dissolved in 100 ml of acetone and brought to a temperature between 0 and 10 ° C cooled. With stirring and cooling, 1.92 ml of a solution of aqueous chromosulfuric acid (1 ml = 0.25 g of CrO3) are added dropwise (temperature max. 10 ° C.). When the addition is complete, the mixture is stirred for a further 30 minutes, then poured into water and extracted with chloroform. The chloroform solution is washed neutral, dried and evaporated. The crude residue of 16-methylene-cortisone-21-acetate (IX; R1 = acetyl) is obtained in pure form by recrystallization from ether-acetone. Mp 213-214 ° C, [X] D -I-140 ° (dioxane), Amax 237 m # t, E'11 409.

c3) 10 g of 16-methylene-11-epi-hydrocortisone (VII a, R = H, obtained from Example 2, a), are dissolved in 345 ml of acetone, the solution is then increased Cooled 10 ° C and slowly mixed with 7.05 ml of a chromic acid solution (prepared of 25 g of chromium trioxide, 22 ml of concentrated sulfuric acid and 100 ml of water). The mixture is then stirred for a further 30 minutes at room temperature, then diluted with water and extracted with chloroform. The extract is washed neutral, dried and concentrated. The residue is dissolved in chloroform and over silica gel chromatographed, with increasing additions of ethyl acetate used for the elution will. The 16-methylene cortisone (IX; R1 = H) is isolated from the middle fractions. Crystallized from acetone, it melts at 219 to 220 ° C, [a] D -I-136 '(dioxane); A.ax 237 mp., E'11 453. Example 3 Production of 16-methylene-hydrocortisone (VII b; R, = H) and its 21-acetate (VII b; R1 = -C O C H3) and of 16-methylene-prednisolone (VIIIb; R1 = H) and its 21-acetate (VIIIb; R1 = -C O CH,) a) acetylation of 16-Methylen-Reichsteins-Substance-S (V; Rl = H).

2 g of 16-methylene-4-pregnen-17a, 21-diol-3,20-dione (V; R1 = H, obtained according to Example 1, d) are left to stand for one night at room temperature in 10 ml of pyridine and 10 ml of acetic anhydride . The mixture is then poured into 150 ml of water, the precipitate is filtered off with suction, washed with water, dried and the 16-methylene-4-pregnene-17x, 21-diol-3,20-dione-21-acetate (V; R, = -COC H3) recrystallized from methanol. Mp. 162 to 163 ° C, [_n] D -51.5 ° (dioxane); Amax 240.5 m # t; e = 17,000.

b) Microbiological IIβ-hydroxylation of 16-methylene-Reichsteins-Substance-S-21-acetate (V b; R1 = COCH3).

In a small fermenter 151 nutrient solution (3% / a malt extract, 10%) sucrose, 0.1% yeast extract, 0.2 () /, Na NO 3, 0.10 /, K H2 P 04, 0, 05010 Mg S 04. 7H20, 0.010 /, FeS04 - 7H20, pH 6.5) inoculated with 800m1 submerged culture of Curvularia lunata (Wakker) Boadijn. After about 24 hours of growth at 28 ° C. with vigorous aeration and stirring, the culture is given 7.5 g of 16-methylene-Reichsteins substance S-21 acetate (V, R1 = COC H3) in 300 ml of methanol. The reaction is followed by thin-layer chromatography. After 18 to 24 hours, the original material can no longer be detected. The culture solution is then stirred three times with chloroform each time, and the combined extracts are evaporated. The residue is purified with petroleum ether and recrystallized from ethyl acetate. Pure 16-methylene-hydrocortisone (VII b; R1 = H) is obtained; Mp 232-233 ° C; [a] D -; - 69.2 ° (dioxane); Amaz 241 m # t, Ei1, 466.

The 16-methylene hydrocortisone obtained (VII b; R1 = H) is then as described in Example 1, f), by acetylation in 16-methylene-hydrocortisone-21-acetate (VIIb; R1 = -COCH3) converted.

cl) 1 (2) -Dehydrogenation of 16-methylene-hydrocortisone-21-acetate (VII b; R1 = -COCH3) with Corynebacterium simplex.

A small fermenter with 151 nutrient solution (0.10 /, yeast extract in 1 / 30molar Phosphate buffer solution, pH 6.8) is mixed with 800 ml of a submerged culture of Corynebacterium simplex inoculated. The culture grows at 28'C with strong aeration and stirring and receives after 4 to 8 hours an addition of 5 g of 16-methylene-hydrocortisone-21-acetate (VIl b; R, = C O C H3) in 200 ml of methanol. The dehydration is carried out by thin layer chromatography followed and ended after about 8 hours. The culture solution is used three times each 15l of chloroform stirred, the extract evaporated and purified with petroleum ether. The residue is filtered through silica gel. The 16-methylene-prednisolone-21-acetate obtained (VIII b; R1 = COCH3) is crystallized from acetone; Mp 219 ° C, [a] D -I-30 ° (dioxane).

c2) 1 (2) -Dehydrogenation of 16-methylene-hydrocortisone-21-acetate (VII b; R, = COCH3) with Bacillus sphaericus.

151 of a nutrient solution of 10% yeast extract, pH 6.8, are inoculated in a small fermenter with 600 ml of submerged culture of Bacillus sphaericus. The culture grows with vigorous aeration and stirring at 28 ° C. and after 8 to 10 hours receives an addition of 7.5 g of 16-methylene-hydrocortisone-21-acetate in 300 ml of methanol. After 24 to 28 hours, the dehydration, which is monitored by thin-layer chromatography, has ended. The fermentation solution is stirred three times with 151 chloroform each time, the combined extracts are evaporated and the residue is digested with petroleum ether. The crude 16-methyleneprednisolone (VIII b; R1 = H) obtained in this way is crystallized from acetone; Mp 233-235 ° C; [m] D -I-22 ° (dioxane); At " 243 m # t, E = 15900.

Example 4 Preparation of 16-methylene-prednisolone (VIII b; R1 = H) and its 21-esters a) 1 (2) -dehydration with Bacillus sphaericus.

In a small fermenter 151 of a nutrient solution of 1% yeast extract, px 6.8, are inoculated with 0.5 1 shaking culture of Bacillus sphaericus (E. Merck Collection, No. 1001). The culture grows with constant stirring and vigorous aeration at 28 ° C. and, after about 10 hours, receives an addition of 7.5 g of 16-methylene-hydrocortisone (VII b; R1 = H), prepared according to Example 1, dissolved in 300 ml of methanol. The dehydration is followed by paper chromatography (solvent system B4 according to Bush, ascending) and is complete after 28 to 36 hours. The culture solution is extracted three times with the same volume of chloroform, and the combined chloroform solutions are evaporated. The residue is recrystallized from acetone. The 16-methylene-prednisolone = 16-methylene-1,4-pregnadiene-11ß, 17a, 21-triol-3,20-dione (VIII b; R1 = H) obtained melts at 225 to 226 ° C .; Amax 243 m # t, E = 15900.

b) 1 (2) dehydration with Corynebacterium simplex. In a small fermenter 121 of a nutrient solution of 0.1 % yeast extract are inoculated with 800 ml of submerged culture of Corynebacterium simplex. The culture grows with stirring and strong aeration at 28 ° C. and after 6 to 8 hours receives an addition of 6 g of 16-methylene-hydrocortisone (VII b; R1 = H), prepared according to Example 1, dissolved in 200 ml of methanol (2) -Dehydrogenation is followed by thin-layer chromatography and is complete after 5 to 7 hours. The culture is immediately stirred three times with the same volume of chloroform, and the combined chloroform extracts are evaporated. The residue is washed with petroleum ether and recrystallized from chloroform. 4.4 g of 16-methylene-prednisolone (VIII b; R1 = H) with a melting point of 225 to 226 ° C. are obtained. Recrystallization from acetone increases the melting point to 233 to 235 ° C; A @ nax 243 m # t, s = 15900; @x] D = -I-22 ° (dioxane).

c) Esterification of 16-methylene-prednisolone (VIII b, R1 = H) in 21 -Position.

5 g of 16-methylene-prednisolone (VIII b, R1 = H) are dissolved in 30 ml of pyridine and 30 ml of acetic anhydride heated on the steam bath for 1 hour. The mixture will Poured into water and the precipitated 16-methylene-prednisolone-21-acetate (VIII b; R1 = acetyl) sucked off. The purification is carried out by recrystallization from acetone, Mp 219 ° C, [*] D -I-30 ° (dioxane).

The 16-methylene-prednisolone-21-diethylaminoacetate (VIIIb; R1- (C, H5) 2N-CHZCO-) is obtained analogously at melting point 195 to 196 ° C .; [ml 'D' -f-32 ° (dioxane); Amdx 243m .; Egg l 350 (ethanol). 16-methylene-prednisolone-21-diethylaminoacetate hydrochloride melts at 245 to 246 ° C; [a] δ -I-45 ° (water); Amax 246 to 247 m # t; Egg 1 300 (Ewater).

Phosphorylation gives 16-methylene-prednisolone-21-dihydrog.-nphosphate (VIII b; R1 = -PO (OH) 2), the monosodium salt of which melts at 189 to 190 ° C; Am ,. "246% t; El l 304 (water).

Example 5 Preparation of 16-methylene-prednisone (X; R1 = H) and its 21-acetate (X; R1 = acetyl) a) Microbiological l (2) -Dehydrogenation of 16-methylene-cortisone (IX; R1 = H).

In a small fermenter with a capacity of 201, 151 of a nutrient solution of 0.1 % yeast extract, pA 6.8, are inoculated with 1.5 l shaking culture of Corynebacterium simplex (E. Merck collection, No. 1002). The culture grows with constant stirring and vigorous aeration at 28 ° C. and, after about 4 to 8 hours, receives an addition of 7.5 g of 16-methylene-cortisone (IX; R1 = H), prepared according to Example 2, dissolved in 300 ml of methanol , c3). The dehydration is followed by paper chromatography and is generally complete after 10 to 14 hours. The culture solution is extracted three times with the same volume of chloroform, and the combined chloroform solutions are evaporated. The residue is recrystallized from acetone. The 16-methylene-prednisone = 16-methylene-1,4-pregnadiene-3,11,20-trione-17x, 21-diol (X; R1 = H) obtained in this way is free from by-products. Mp 218-219 ° C; [a] D -f -103 ° (dioxane); 27, t ", 238 m (t; Ei m 434.

b1) Microbiological 1 (2) -Dehydrogenation of 16-Methylen-Reichsteins-Substance-S (V; R1 = H) and subsequent 11a-hydroxylation and 11 -oxidation.

Microbiological l (2) -Dehydrogenation of V (R1 = H) 7.5 g of 16-methylene-Reichsteins-Substance-S (prepared according to Example 1) are in 151 a culture solution of Bacillus sphaericus, as described in Example 4, a) , dehydrated. After working up according to Example 4, a), pure 1-dehydro-16-methylene-Reichsteins-Substance-S (VI; R1 = H) is obtained by recrystallization from acetone. Mp 222-226 ° C; [a] D -16 ° (chloroform); Amax 244 m # x, E '1! L 473.

Microbiological llm-hydroxylation of VI (R1 = H) In a small fermenter, 151 of a nutrient solution of 51110 glucose, 0.20 / 0 yeast extract, 0.301, sodium nitrate, 0.05010 magnesium sulphate, 0.0010 /, iron (II) sulphate and 1/30 molar phosphate buffer according to Sörensen (pg 5.6) with 800 ml shaking culture of Penicillium sp. (E. Merck Collection, No. 2168) inoculated. The culture grows with stirring and vigorous aeration and, after 24 hours, receives an addition of 5 g of 1-dehydro-16-methylene-Reichsteins-Substance-S, dissolved in 40 ml of dimethylformamide. The hydroxylation is followed by paper chromatography. After the reaction has ended, work-up is carried out in accordance with the description in Example 5, a). Pure 16-methylene-II-epi-prednisolone (VIII a; R1 = H) is obtained by recrystallization from acetone. Oxidation of VIIIa (R, = acetyl) Analogously to Beispie12, cl) is 16-methylene-11-epi-prednisolone-21-acetate (VIII a; R, = acetyl), produced by acetylation of 16-methylene-II-epi- Prednisolone analogous to Example 2, b), converted into 16-methylene-prednisone-21-acetate (X; R1 = acetyl) by oxidation with chromic anhydride in pyridine. Mp 214-217 ° C; [4] D -f-123 ° (chloroform); Amax 238 mu., E = 15100. b2) Microbiological 1 (2) -Dehydrogenation of 16-Methylen-Reichsteins-Substance-S-21-Acetate (V; R,. = -COCH3), following IIß-Hydroxyherung, optionally 21- Acetylation, and 11-oxidation. Microbiological 1 (2) -Dehydrogenation of V (R1 = -COC H3) Analogously to Example 3, c1) 16-methylene-Reichsteins-Substance-S-21-acetate (V; R1 = -COCH3) is converted to 16-methylene-1 , 4-pregnadiene-17a, 21-diol-3,20-dione-21-acetate (VI; R1 = -COCH3) dehydrated. Recrystallized from acetone, the compound melts at 192 to 193 ° C; [AD -E-2.6 ° (dioxane); @max 244 mu., E'1 ','. 392. Microbiological IIβ-hydroxylation of VI (R, = -COCH3) 151 nutrient solutions (3% malt extract, 1% sucrose, 0.10 / 0 yeast extract, 0.2% Na N 03, 0.10 / 0 K HZ P 04, 0.05% Mg S 04 '7H20, 0.01% Fe S 04 - 7 H20, pg 6.5) inoculated with 800 ml submerged culture of Curvularia lunata (Wakker) Boadijn. After about 24 hours of growth at 28 ° C with strong aeration and stirring, the culture is given 2.5 g of 16-methylene-1,4-pregnadiene-17a, 21-diol-3,20-dione-21-acetate ( VI; Rz = COCH3) in 150m1 methanol. The reaction is followed by thin-layer chromatography and has ended after about 60 hours. The fermentation solution is stirred three times with 151 chloroform each time and the combined extracts are evaporated. After cleaning with petroleum ether, it is recrystallized from ethyl acetate. The pure 16-methylene-prednisolone (VIIIb; R, = H) is obtained; Mp 233-235 ° C; [oc] D + 22 ° (dioxane); @max 243 mu ,, s = 15900.

The 16-methylene-prednisolone (VIII b; R, = H) obtained can optionally, as described in Example 4, c), by acetylation in 16-methylene-prednisolone-21-acetate (VIII b; R1 = -COCH3). 11-oxidation of 16-methylene-prednisolone (VIIIb; R, = H) 10 g of 16-methylene-prednisolone (VIII b; R1 = H) are dissolved in 350 ml of acetone dissolved, the solution is then cooled to 10 ° C and slowly with stirring with 7.05 ml of a chromic acid solution (made from 25 g of chromium trioxide, 22 ml of concentrated Sulfuric acid and 100 ml of water). The mixture is then used for another 30 minutes stirred at room temperature, then diluted with water and with chloroform extracted. The extract is washed neutral, dried and concentrated and the The residue was taken up in chloroform and chromatographed on silica gel, with increasing Additions of ethyl acetate can be used for elution. From the middle factions 16-methylene-prednisone (X; R,. = H) is isolated. Recrystallized from acetone, the compound melts at 218 to 219 ° C; [x] D -E -103 ° (dioxane); .lm "" 238 mu ,, E'11 434.

11-oxidation of 16-methylene-prednisolone-21-acetate VIIIb (R1 = COCH3) 2.4g 16-methylene-prednisolone-21-acetate (VIII b; R1 = acetyl), prepared according to the example 4, are dissolved in 24 ml of pyridine and add to a previously prepared mixture of 2.4 g chromic anhydride and 24 ml pyridine added. The mixture is left to stand overnight and then, as described in Example 2, c,), worked up. It will be 1.6 g of 16-methylene-prednisone-21-acetate (X; R1 = acetyl) were obtained.

The oxidation can also be carried out with good yields according to the example 2, cD described procedures can be carried out.

Claims (2)

PATENT CLAIMS: 1. Process for the preparation of 16-methylene steroids of the general formula X = H, OH (a or β) or = O, Y = a free or esterified hydroxyl group, as well as 1-dehydro-derivatives thereof, characterized in that 16β-methyl-16ca, 17a-oxido-5-pregnen -3ß-ol-20-one-3-acetate (I) by heating with catalytic amounts of a strong acid, especially a sulfonic acid, in the presence of an inert solvent, especially benzene, in 16-methylene-5-pregnen-3ß, 17a- diol-20-one-3-acetate (II) converts, then in a manner known per se the compound II is reacted with at least 2 molar equivalents of bromine, the resulting 5,6,21-tribromo-16-methylene-pregnane-3ß, 17a diol-20-one-3-acetate (III) by successive treatment with an iodide and an acetate, in particular sodium iodide and potassium acetate, in 16-methylene-5-pregnen-3ß, 17a-21-triol-20-one-3, 21-diacetate (IV) transferred, this ester is incubated with a culture of Flavobacterium dehydrogenans or an enzyme obtained therefrom, and the 16-methylene-4-pregnen-17a, 21-diol-3,20-dione (V) obtained in this way is through known nth microbiological 11a or 11ß hydroxylation in 16-methylene-4-pregnen-11 (a or ß), 17a, 21-triol-3,20-dione (VII a or VII b) and optionally the latter by conventional oxidation in 16-methylene-4-pregnen-17a, 21-diol-3,11,20-trione (IX) or that the compound V obtained is first microbiologically dehydrated by methods known per se in the 1 (2) position and that 16-methylene-1,4-pregnadiene-17a, 21-diol-3,20-dione (VI) obtained by known microbiological 11a or 11ß-hydroxylation in 16-methylene-1,4-pregnadiene-11 (a or .ß), 17a, 21-triol-3,20-dione (VIII a or VIII b) and possibly the latter by conventional oxidation in 16-methylene -1,4 - pregnadiene -17x, 21-diol - 3,11, 20-trione (X) transferred or the 11a or 11ß-hydroxysteroid VII a or VII b obtained or the 11-keto steroid IX obtained is microbiologically dehydrated in a manner known per se in the 1 (2) position and that if appropriate, the hydroxyl group contained in the compounds V to X in 21-position is esterified by methods known per se and, if appropriate, the esters obtained are treated analogously to the corresponding 21-hydroxy compounds V to IX. 2. The method according to claim 1, characterized in that one the conversion of the compound I into the compound II by treatment with catalytic effective amounts of p-toluenesulfonic acid in benzene solution. Into consideration printed publications: Journ. At the. Chem. Soc., Vol. 72 (1950), pp. 5145 ff.