DE1263766B - Process for the preparation of 9alpha-fluoro-16-methylene-prednisolone or -21-esters - Google Patents

Description

FEDERAL REPUBLIC OF GERMANY

GERMAN

PATENT OFFICE

EDITORIAL

Int. Cl .:

C07c

German class: 12 ο -25/06

Number: 1263 766

File number: M 50789IV b / 12 ο

Filing date: January 31, 1959

Open date: March 21, 1968

It has been found that 9 ″ -fluoro-16-methylene-prednisolone and its 21-ester have interesting pharmacological properties. In the US Pat. No. 2,865,808 describes a production process for 16-methylene-corticosteroids, however, the reworking has shown that it is not then possible to achieve the desired Manufacture 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. The ones in the Examples 9 and 17 of the said USA patent specification therefore have, especially they are not characterized by any physical data and are not to be considered known. Furthermore, according to US Pat. No. 2,865,808, 16a-hydroxy-hydrocortisone is used as the starting material, the itself is only available through a multitude of reaction stages. So far there is no feasible one Process for the preparation of 9a-fluoro-16-methylene-prednisolone and its 21-esters.

It has now been found that 9α-fluoro-16-methylene-prednisolone and 21-esters can be obtained in good yield the same can be produced if the starting material 16 / i-methyl-16a, 17a-oxido-5-pregnen-3 /: i-ol-20-one-3-acetate (I) (see the reaction scheme) is used.

The invention accordingly relates to a process for the preparation of 9 α-fluoro-16-methylene-prednisolone or -21-esters of the general formula A

CH, Y

F = CH,

(Y = free or esterified hydroxyl group) 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, e.g. B. a sulfonic acid, preferably p-toluenesulfonic acid, in an inert solvent such as benzene, in processes for the preparation of
9a-fluoro-l 6-methylene-prednisolone or
-21-esters.

Applicant:

E. Merck Aktiengesellschaft,

6100 Darmstadt, Frankfurter Str. 250

Named as inventor:

Dr. Heinz-Jürgen Mannhardt, 6100 Darmstadt;

Dr. Karl-Heinz Bork, 6103 Griesheim;

Dr. Klaus Brückner,

Dr. Harald Metz, 6100 Darmstadt

16-methylene-5-pregnen-3 / 3,17a-diol-20-one-3-acetate (II) transferred, then in a manner known per se this compound (II) with at least 2 mols equivalent Reacts bromine, the obtained 5,6,2 l-tribromo-16-methylen-pregnan-S / S.na-dioWO-on-S-acetate (III) by sequential treatment with an iodide and an acetate, preferably sodium iodide and potassium acetate, in 16-methylene-5-pregnen-3 / U7a, 21-triol-20-one-3,21-diacetate (IV) transferred, the latter (IV) with a culture of Flavobacterium dehydrogenans or an enzyme obtained therefrom, the 16-methylene-4-pregnen-17a, 21-diol-3,20-dione obtained (V) microbiologically hydroxylated in the Ik or IIß-position, the obtained II (a- or /}) -hydroxy-steroid (VIIa or VIIb) is acetylated in the 21-position, the resulting II (a- or / I) -hydroxy-21-acetate (IXa or IXb) dehydrated in the 9, 11 position, to the 9 (1 continuous double bond of the 9 (II) -dehydro-steroid (XI) HOBr or HOCl formed is added, the 9a-bromine or 9a-chloro-II / J-hydroxy-steroid (XIII) with an alkali acetate or treated with an organic base, the 9β, 11β-oxido-steroid (XV) formed with hydrogen fluoride treated, the obtained 9a-fluoro-16-methylene-4-pregnen-3,20-dione-II / U7a, 21-triol-21-acetate (XVII) by treatment with microorganisms dehydrating in the 1 (2) position in 9a-fluoro-16-methylene-prednisolone-21-acetate or 9a-fluoro-l 6-methylene-prednisolone and converts the latter optionally into 21 position esterified, or that one of the. intermediate compounds obtained according to the method (V, Vila, VIIb, IXa, IXb, XI, XIII or XV) to an

809 519/698

3 4

known microbiological methods in 11 / i-hydroxylation: Curvularia lunata, Cunnigha-

1 (2) position dehydrated and the 1-Demella blakesleeana, Mucor griseocyanus, cephalahydro-derivative obtained in this way (VI, Villa, VIIIb, Xa, Xb, XII, XIV thecium sp., Trichothecium sp., Botrytis cinerea, or XVI) if necessary, in an analogous manner Coniathyrium sp., Thamnidium sp., Streptomyces such as the corresponding 5 fradiae, Rhodoseptoria sp., Colletotrichum sp., Do compounds saturated in the 1-position by 11-hydroxylation and / or thichiza sp., Absidia glauca, Pycnosporium sp. 21-acetylation and / or 9,11-dehydration and / 11 «-hydroxylation: Rhizopus (various species),

or HOBr or HOCl addition and / or Dehydro-Aspergillus (various types), Penicillium (halogenation and / or hydrogen fluoride splitting different species), Fusarium (different species), transformed into 9a-fluoro-16-methylene-prednisolone-21-acetate umro Neurospora sitophila, Trichothecium sp., Cephalo and that optionally the 21-Hy thecium roseum, Pestalotia foedans, Dactylium dendroxy group of the compounds obtained (V) to droides, Heliocostylum pyriforme, Thamnidium sp., (VIII) esterifies Eurotium chevalieri, Mucor javanicus and various and optionally the esters obtained analogous to the species, Delacroixia coronata, Absidia glauca, Compounds (V) to (XVII) treated. 15 Cunninghamella echinulata, furthermore other mushrooms of the

By treating compound (I) with the kata order Mucorales.

lytic amounts of a strong acid, e.g. a microbiological 11-hydroxylation is after

Sulfonic acid, preferably p-toluenesulfonic acid, or methods known per se. Special Benzenesulfonic acid, in benzene, or some other successes in 11 a-hydroxylation are achieved inert solvent, the corresponding 20 is obtained when using fungi of the genus Fusarium, 16-methylene steroid (II). being the steroid used as the starting material

To introduce a 21-hydroxyl function, it is completely converted, so that lengthy separation takes place the compound (II) can be avoided in a suitable process.

Solvents, e.g. B. in glacial acetic acid-chloroform, with min- The 11-hydroxysteroids (Vila, VIIb) and (Villa,

at least 2 molar equivalents of bromine, one being 25 VIIIb) after acetylation which is known per se small amount of hydrogen halide as a catalyst for the 21-hydroxy group (formation of the compounds clogs. This method gives the tri-IXa, IXb or Xa, Xb) according to known methods bromine compound (III). The crude product of these bro methods to the 9 (1 ^ -unsaturated steroids (XI) mation is e.g. dehydrated in acetone solution with sodium or (XII). When using the 11 / i-hyiodide treated, whereby the 21-position bromine atom 30 droxysteroide (IXb) or (Xb) are all usual exchanged for iodine and the 5 (6) double trans-dehydrating agents suitable, e.g. B. Phos bond is regenerated. By boiling with a phosphorus oxychloride or thionyl chloride in pyridine. at Alkali acetate, preferably potassium acetate, the use of the 11 «-hydroxysteroids (IXa) or Iodine atom in position 21 through the acetoxy group (Xa) are the usual cis-dehydration methods replaced. One receives in good yield 16-methylene 35 suitable, z. B. Esterification of the hydroxyl group 5-pregnen-3 / il7a, 21-triol-3,21-diacetate (IV). and subsequent basic or thermal

By incubating (IV) with a culture of cleavage of the corresponding acid. Flavobacterium dehydrogenans or one thereof According to known methods, the 9 (11) -stän-

producible enzyme succeeds in the best yield-dige double bond of the thus obtained! 9 (11) -De- and in smooth reaction 16-methylene-Reichsteins- 40 hydrosteroid (XI) or (XII) hypochlorous or substance-S (V) to be produced. Accumulated as a nutrient solution for hypobromous acid. The resulting Flavobacterium dehydrogenans is used z. B. henden ll / ^ - Hydroxy-9 "-chlor- or Mp'-Hydroxy a pH 7.0 buffered solution of a 1% 9a-bromo-steroid (XIII) or (XIV) are - even yeast extract ' in water. After about 10 to 16 hours according to known methods - by treating digem growth at about 28 ° C., compound (VI) is replaced with an alkali acetate, preferably potassium acetate, bacterial culture. The working or an organic base in the corresponding brütung is with aeration for about 6 hours advanced 9 /> \ 11 .beta. Oxidoverbindungen (XV) or (XVI) converted set. The progress of the reaction can be changed through.
Check the measurement of the UV spectrum. By treating the 9 / 5,11 p'-oxido compounds

The compounds (V, Vila, VIIb, IXa, IXb, XI, 5 ° (XV) or (XVI) with hydrogen fluoride are obtained XIII, XV and XVII) can according to the invention ^ «- fluoro-lo-methylene-hydrocortisone-II-acetate (XVII) in a manner known per se in the 1 (2) position micro- or 9a-fluoro-16-methylene-prednisolone-21-acetate be biologically dehydrated. For this z. B. (XVIII). For the compound (XVII) then closes the following microorganisms in question: Bacillus another microbiological l. (2) dehydration in sphaericus, Fusarium solani, Corynebacterium sim- 55 in the manner described above. plex, Alternaria sp., Mycobacterium smegmatis, CaIo- As indicated in the reaction scheme, the

nectria decora, Mycobacterium lacticola, Ophiobolus for the production of 9 α-fluoro-16-methylene-prednisp., Alcaligenes sp., Didymella lycopersici, Protaminosolon necessary microbiological 1 (2) -Dehydriebacter sp., Septomyxa affinis, Nocardia sp., Cylindrication can be carried out on seven different intermediate compounds carpon radicicola, Streptomyces lavendulae, Bacillus 60. Furthermore, the free Hycyclooxydans. The fermentation takes about 4 to 6 hydroxyl groups in the 21-position of the 9 "-fluoro-14 hours obtained, depending on which microorganism 16-methylene-prednisolone is used according to known ones. Cultures methods are particularly suitable to be esterified. As esterifying agents of Bacillus sphaericus var. Fusiformis and Coryne, z. B. the halides or the anhydrides bacterium simplex. ⁶ 5 of the following acids are used: acetic acid and

For the introduction of a Ha- or 1 insistent their higher homologues, z. B. tert-butylacetic acid, Hydroxy group in the compounds (V) or (VI) succinic acid and its higher homologues, aminosind the following microorganisms are suitable: or alkylaminocarboxylic acids, tetrahydrophthalic

5 6

acid, aminodicarboxylic acids, for example aspartic acid, is obtained in pure form. Yield: 1.05 g, m.p. 195 to cyclopentylpropionic acid, phosphoric acid or sulfur 197 C, [u] _D -65.7 (chloroform).

⁶ The starting material required 16 / i-methyl- ^c »microbiological saponification and oxidation

16 «, 17u-oxido-5-pregnen-3 / i-ol-20-one-3-acetate is e.g. 5 A sterilized solution consisting of 80 g of 16-methyl-5,16-pregnadien-3 / -j- ol-20-one-3-acetate kose, 50 g yeast extract, 30 g ammonium chloride, with easily accessible. This in turn can be made up to 10 1 in known tap water and adjusted to pH 7.0 16 (17) double bond of 5,16-pregnadien-3 // - ol- _{with V 30 molar by adding diazomethane to the phosphate buffer solution according to Sörensen} , is with 200 ml of a shaking culture 20-on-3-acetate (this compound is inoculated as technical 10 by Flavobacterium dehydrogenans. Accessible under product from diosgenin) and then stirring and aerating at 28 ° C, the culture is ßende pyrolysis of the result Pyrazoline derivative allowed to grow for 12 hours. After adding one, be prepared (cf. A. Wet t st ei 11, Helvetica concentrated solution of 12 g of the diacetate (IV) Chimica Acta. Vol. 27, p. 1803 [1944]). Then in acetone or acetone-dimethylformamide (1: 1) a reaction with alkaline hydrogen takes place . The solution is with chloroform 3 / - <- - ol-20-one. the extracted several times by conventional acetylation, the chloroform solution dried 3-hydroxy group in the starting compound and evaporated. The evaporation residue is converted into formula I. Recrystallize purified. 8 g of pure are obtained

The 9a-fluoro-16-me-20 16-methylene-4-pregnen-17 «, 21-diol-3,20-dione (V), ethylene-prednisolone and its 21-esters should have a melting point of 205 to 206 C. [u] _D +46.6 '(chloroform), A _11111x anti-inflammatory drugs in all in question 240 nm, EJ "; 480.
coming indications, e.g. B. rheumatic arthritis.,,

tis. be used in human medicine. They are suitable ^d) microbiological 11 ″ hydroxylation

can also help fight stubborn allergies. 25 A suitable nutrient solution (e.g. 5% glucose, 0.1% yeast example 1 extract, 0.05% soy flour, 0.3% NaNO ₃ , 0.05%

a) Epoxvdaufspaltune ^M S ^S0 * ' ^7H 2 ⁰ ' ⁰ ^ KH ₂ PO ₄ , 0.05% KCl, 0.005%

FeSO ₄ -7H ₂ O) with 750 ml of a well grown

A solution of 9.3 g of 16 / i-methyl-16 «.17a-oxido shaking culture of Fusarium sp. (E. Merck Collection 5-pregnen-3 / i-ol-20-one-3-acetate (I) in 350 ml of water no. 2083) inoculated. The culture grows under strong Free benzene is after the addition of 630 mg of p-toluene aeration and stirring at 28 ° C and obtained after sulfonic acid refluxed for 4 hours. After 24 hours an addition of 5 g of 16-methylene-rich demolition The benzene solution is cooled down first steins substance S (V) in 40 ml of dimethylformamide, with 5% sodium hydrogen carbonate solution, then 35 The reaction is confirmed by paper chromatography Washed water, dried and evaporated. follows. If there is no initial Es in the chromatogram 5.4 g more crystallized 16-methylene-5-pregnene substance can be detected, the batch 3,7,17a-diol-20-one-3-acetate (II) was obtained. Aborted from methanol and the culture solution three times with 101 recrystallized, the compound melts at 196 chloroform extracted. The combined chloroform bis 198 C. 40 extracts are evaporated, the residue becomes

b) bromination and transesterification ^{digested with} petroleum ether and recrystallized from acetone

"siert. Pure 16-methylene-II-epi-hydro-

1.55 g of 16-methylene-5-pregnen-3p ', 17a-diol-20-on-cortisone (Vila). M.p. 199-20I = C, [u] _D +41.7 ^; (Di-

3-acetate (II) are dissolved in 60 ml of glacial acetic acid and 15 ml of oxane), A _1110x 241 nm, Ej ^ _n 445.

Dissolved chloroform and the solution removed to 5 ° C.

cools. After the addition, ^e > 21-acetylation occurs within 20 minutes

a few drops of a solution of hydrogen bromide 2 g of 16-methylene-II-epi-hydrocortisone (VIIa)

in glacial acetic acid while stirring 62 ml of a 2% solution dissolved in 10 ml of pyridine and 0.36 g of acetic acid

added dropwise of bromine in glacial acetic acid. When the anhydride was complete, added. After standing for 15 hours

The addition is continued for half an hour. 50 room temperature is poured into water, with

The solution is poured into water, extracted three times with ether and chloroform, the chloroform solution

extracted and the ethereal solution with an aqueous by shaking with sodium hydrogen carbonate solution

Solution of sodium hydrogen carbonate neutralized, dried and evaporated in vacuo,

to wash. The solution, dried over sodium sulfate, The amorphous residue of 16-methylene-II-epi-

is evaporated in vacuo. What remains is the 55 hydrocortisone-21-acetate (IXa) is transferred directly to the

Tribromide (III) in oily form, the following reaction used without purification,
is further processed. To do this, the residue

dissolved in 60 ml of acetone. 1.5 g sodium iodide and 6 g ^l) dehydration

anhydrous potassium acetate was added and 5 g of 16-methylene-11 -epi-hydrocortisone-21-ace-

refluxed. After cooling, the 60 tat (IXa) are dissolved in 25 ml of absolute chloroform

Inorganic salt is filtered off and the solution is dissolved in and 25 ml of pyridine. With cooling to 0 ^c C.

Evaporated in vacuo. The residue is added in chlorine and stirring 7 g of p-toluenesulfochloride

form dissolved, this solution dispensed three times with water. The mixture is still 2 hours at

shaken, evaporated in vacuo, dissolved in benzene 0 ⁰ C continued, then overnight at

and magnesium silicate, known below the commercial 65 room temperature, left to stand in water

named Florisil, chromatographed. The benzene-chloro-poured and extracted several times with chloroform.

roform (1: 1) eluate gives the di- The combined chloroform solutions are neutral

acetate (IV), which siert by recrystallization from acetone and dried. After evaporation, the

Residue of 16-methylene-l l-epi-hydrocortisone-1 l-tosylate-21-acetate recrystallized from methanol. Mp. 160 to 161 C, [«] ₀ +66.6 (chloroform), X _nwx 229 nm, E {°; 433.

The ester obtained in this way (5.9 g) is dissolved in 75 ml of glacial acetic acid and, after addition of 9 g of anhydrous sodium acetate, refluxed for 30 minutes. The mixture is stirred into 500 ml of water and the precipitate of crude 16-methylene-4,9 (11) -pregnadiene-17 «, 21-diol-3,20-dione-21 acetate (XI) is filtered off with suction. For purification, the product is recrystallized from ethyl acetate. Yield: 2.6 g, melting point 215 to 217 ° C., [«] ² S + 5Γ (dioxane), X _max 238.5 nm, E |: ° _m 421.

g) Addition of hypobromous acid

7.8 g of 16-methylene-4,9 (II) -pregnadiene-17 «, 21-diol-3,20-dione-21-acetate (XI) are dissolved in 315 ml of dioxane and 40 ml of water. Then 4.55 g of N-bromosuccinimide and 1.68 ml of 70% perchloric acid added; the reaction mixture is 1 hour at Left to stand at room temperature. It is poured into water, the precipitate is sucked off, washed with water and dried. The crude 16-methylene-9a-bromohydrocortisone-21-acetate (XIII, X = Br) is used directly in the subsequent process stage.

h) elimination of hydrogen bromide

The crude 16-methylene-9a-bromohydrocortisone-21-acetate (XIII) obtained according to Example 1 g) is dissolved in 450 ml of ethanol, 19 g of potassium acetate are added and the solution is refluxed for 2 hours. The mixture is poured into water and the resulting emulsion is extracted several times with chloroform; the combined chloroform solutions are worked up in the usual way. The evaporation residue, crystallized from methanol, gives pure 16-methylene. 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 crude 9u-fluoro-16-methylene-prednisolone (XVIII, R = H) is obtained in pure form by recrystallization from ethanol. Mp. 246 to 248 C, [«] ₀ +26.6 '(dioxane), /,"",. 238.5 nm, EU 410.

j ₂ ) Microbiological 1 (2) dehydration

A small fermenter with 15 1 nutrient solution (0.1% yeast extract in ¹ Z ₃₀ molar phosphate buffer solution, pH 6.8) is inoculated with 800 ml of a submerged culture of Corynebacterium simplex. The culture grows at 28 ° C. with vigorous aeration and stirring and, after 4 to 8 hours, receives an addition of 5 g of 9 "-fluoro-16-methylene-hydrocortisone-21 acetate (XVII) in 200 ml of methanol. The dehydration is monitored by thin-layer chromatography and is complete after about 8 hours. The culture solution is stirred three times with chloroform each time, the extract is evaporated and purified with petroleum ether. The residue is filtered through silica gel. The 9u-fluoro-16-methylene-prednisoIon-21 -acetat (XVIII, R = acetyl) obtained is crystallized from acetone. Mp. 219 ° C, [«] _D +30 ^c (dioxane).

did (XV). Yield: 4.2 g, mp 210-211 ° C, [a] ₀ -34.7 ° (dioxane), X _max 243 nm, EJ * _m 390th.

i) splitting with hydrogen fluoride

4.2 g of 16 - methylene - 9 / U Eat - oxido - 4 - pregnene-17a, 21-diol-3,20-dione-21-acetate (XV) are dissolved in 42 ml of absolute chloroform and - 6O ⁰ C added to 25 ml of a mixture of 40 ml of tetrahydrofuran, 15 ml of chloroform and 25 g of hydrogen fluoride. The reaction solution is left to stand at -30 ° C. for 4 hours, then at 0 ° C. for a further 4 hours. It is then poured into sodium hydrogen carbonate solution, the steroid extracted with chloroform, the chloroform solution dried and evaporated. The residue is recrystallized from acetone. Yield: 1.9 g 90 ^^ 0 ^ 16-1116 ^^ 1 ^^) 03 ^ 180 ^ 21 acetate (XVH), m.p. 209-211 ° C, [a] _D + 71.4 ° (chloroform ), X _max 238 nm, EJ * 399.

J ₁ ) Microbiological 1 (2) dehydration

In a small fermenter, 15 l of a nutrient solution of 1% yeast extract, pH 6.8, are inoculated with 0.5 l of Bacillus sphaericus shaking culture. 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 9a-fluoro-16-methylene-hydrocortisone-21-acetate (XVII), dissolved in 300 ml of methanol. The dehydration is followed by paper chromatography (solvent system B ₄ according to Bush, ascending) and is complete after 28 to 36 hours.

k) esterification

By conventional esterification of the hydroxyl group in the 21-position of the 9a-fluoro-16-methylene-prednisolone (XVIII, R = H) the following esters are obtained:

9a - fluoro - 16 - methylene - prednisolone - 21 - chloroacetate: mp 232 to 234 ° C, [«] !? +17 ^; (Dioxane), A _1110x 240 nm; EJ * 329;

9a-fluoro-16-methylene-prednisolone-21-trimethylacetate: M.p. 234 to 235 "C;

9α-fluoro-16-methylene-prednisok> 21 n-tert.-butyl acetate: mp 231-232 ⁰ C, [a] _o ^{2 0} + 20 ° (chloroform);. X _max 240 nm, EJl 320;

^ a-fluoro-lo-methylene-prednisolone ^ l-diethylaminoacetate: melting point 216 to 217 ° C, [a] !, ⁰ + 26.6 ° (dioxane); X _max 240 nm, EJ * 320. The hydrochloride of this ester melting at 235 to '236 ⁰ C, [a] I ⁰ + 36.8 ° (water), X _max 240 nm, E \ i 281 °;

9a - fluoro-16-methylene-prednisolone-21-enanthate: mp 231 to 232 ° C; [a] S ¹ + 4.5 ° (chloroform).

Example 2

a) According to Examples 1 a) to 1 c), 16-methylene-Reichsteins-Substance-S is used manufactured.

b) Microbiological 11 / i-hydroxylation

In a small fermenter, 15 l of a nutrient solution from 5% malt extract, 1% sucrose, 0.2% sodium nitrate, 0.1% dipotassium phosphate, 0.05% magnesium sulfate, 0.05% potassium chloride and 0.005% iron (II) sulfate (pH adjusted to 7.0) in 800 ml a shaking culture of Curvularia lunata (Wacker) Boadijn inoculated and with stirring and stronger Aeration incubated at 28 ° C. After 24 hours of growth, 5 g of 16-methylene-Reichsteins-Substance-S, dissolved in 40 ml of dimethylformamide, added. The conversion is followed by paper chromatography. When the starting material is no longer detectable in the paper chromatogram, the culture solution becomes extracted three times with the same volume of chloroform. The combined chloroform solutions are evaporated and the residue for removal

the by-products are chroraatographed on silica gel. The eluate obtained with chloroform-ethyl acetate (1: 3) contains the desired 16-methylene-hydrocortisone (VII b), which is obtained therefrom in the usual way. Mp. 224 to 225 ° C, [α] ₀ + 69.2 ° (dioxane), X _max 241 min. E | * _ra 466. After further recrystallization from acetone, the compound melts at 232 to 233 ° C.

c) acetylation

5 g of 16-methylene hydrocortisone (VIIb) 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 (IXb) is filtered off with suction. The purification is carried out by recrystallization from acetone. Mp. 217 to 219 ⁰ C.

d) elimination of water

1 g of lo-methylene-hydrocortisone- ^ l-acetate (IXb) is mixed with 0.150 ml of thionyl chloride in 10 ml of pyridine and the reaction mixture is then heated ^{to 100 ° C. for 15 minutes.} After cooling, the mixture is poured into 75 ml of water and the precipitated crystals are filtered off with suction. This gives 16 - methylene - 4,9 (11) - pregnadiene - 17a, 21 - diol-3,20-dione-21-acetate (XI), which melts after recrystallization from ethyl acetate at 215 to 217 ° C, [α] ?? + 51 ° (dioxane), X _max 238.5 nm, E} * 421.

e) The 16-methylene-4,9 (II) -pregnadiene-17ci, 21-diol-3,20-dione-21-acetate (XI) obtained is described as in Examples 1 g) to 1 J ₁ ) , converted to '9a-fluoromethylene-prednisolone (XVIII, R = H).

Example 3
a) Microbiological 1 (2) dehydration

In a small fermenter, 15 l of a nutrient solution of 1% yeast extract, pH 6.8, are inoculated with 0.5 l of Bacillus sphaerieus shaking culture. 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-Reichsteins-Substance-S (V), prepared according to example 1 c), dissolved in 300 ml Methanol. The dehydration is followed by paper chromatography (solvent system B ₄ 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. Pure l-dehydro-16-methylene-Reichsteins-Substance-S (VI), melting point 222 to 226 ° C., [α] ?? -16 ° (chloroform), X _max 244 nm, E} * 473.

b) Microbiological Ila hydroxylation

A nutrient solution of 5% glucose, 0.2% yeast extract, 0.3% sodium nitrate, 0.05% magnesium sulphate, 0.001% iron (II) sulphate and V ₃₀ mol of Sörensen phosphate buffer (pH 5.6 ) with 800ml shaking culture of Penicilliüm 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-Reichstein-Substanz-S, dissolved in 40 ml of dimethylformamide. The hydroxylation is followed by paper chromatography. After the end of the reaction, as indicated in Example 3 a), worked up. Pure 16-methylene-11-epi-prednisolone (Villa) is obtained by recrystallization from acetone.

c) elimination of water

Analogously to Example If), 16-methylene-II-epiprednisolone is converted (via the 21-acetate and the 11-tosylate-21 - acetate) 16 - methylene -1,4,9 (11) - pregnatrien-17a, 21-diol-3,20-dione-21-acetate (XII) shown.

d) Addition of hypobromous acid

Analogously to Example 1 g), the 9a- bromo-16-methylene-prednisolone-21-acetate ( XIV, X = Br).

e) elimination of hydrogen bromide

Analogously to example lh) is made from 9a-bromo-16-methy-

len-prednisolone-21-acetate (XIV, X = Br) 16-methylene-9ß, l Iß - oxido -1,4 - pregnadiene - 17a, 21 - diol-3,20-dione-21-acetate (XVI) shown. Mp. 216 to 220 ° C, [a] f -13.3 ° (dioxane), X _max 249 nm, E} * _m 357.

f) splitting with hydrogen fluoride

Analogously to Example Ii), 16-methylene-9 / ?, ll /? - oxido-1,4-pregnadiene-17a, 21-diol-3,20-dione 21-acetate (XVI) 9a-fluoro-16-methylene-prednisolone-21-acetate (XVIII, R = acetyl). M.p. 223 to 224 ° C,

, E !? _m 379.

B e i s ρ i e 1 4.

a) Microbiological 1 (2) dehydration

Analogously to Example Ij ₁ ), 7.5 g of 16-methylene hydrocortisone (VlIb) are dehydrated to 16-methylene-prednisolone (VIIIb) by the action of Bacillus sphaerieus. Mp. 225 to 226 ° C. Recrystallization from acetone increases the melting point to 233 to 235 ° C; X _max 243 ηΐμ, ε = 15,900, [α] ₀ + 22 ° (dioxane).

b) 21-acetylation

5 g of 16-methylene-prednisolone (VIIIb) are heated in 30 ml of pyridine and 30 ml of acetic anhydride for 1 hour on the steam bath. The mixture is poured into water and the precipitated 16-methylene-prednisolone-21-acetate (Xb) is filtered off with suction. The purification is carried out by recrystallization from acetone, melting point 219 ° C., [a] _D + 30 ° (dioxane).

c) elimination of water

Analogously to Example 2d), 16-methylene-prednisolone-21-acetate is used (Xb) with thionyl chloride-pyridine in 16-methylene 1,4,9 (II) -pregnatriene-3,20-dione-17a, 21-diol-21-acetate (XII) converted.

d) The 16-methylene-1,4,9 (ll) -pregnatriene-3,20-dione-17a, 21-diol-21-acetate (XII) is described, as in Examples 3d) to 3f), in 9a-fluoro-16-methyleneprednisolone-21-acetate (XVIII, R = acetyl) converted. Mp. 223-224 ° C, X _max 238 nm, E} 1,379.

Example 5

a) Analogously to Example 2 c), 4 g of 16-methylene-4-pregnen-17a, 21-diol-3,20-dione (V) are treated with acetic anhydride and pyridine. After the usual work-up, lo-methylene ^ -pregnen-Ha ^ l-diol-3,20-dione-21-acetate (V) is obtained. Mp. 160 to 161 ⁰ C, {_ _a y _D ⁰ + 72 ° (chloroform); X _max 240 nm, EJ * 424.

809 519/698

b) 1! ^ - hydroxylation

Analogously to Example 2 b), 5 g of 16-methylene-4-pregnen-17a, 21-diol-3,20-dione-21-acetate are obtained (V) exposed to a submerged culture of Curvularia lunata. The oily crude product is put on silica gel chromatographed to remove by-products formed, in particular VIIb formed by saponification. to separate. The eluate obtained with chloroform contains the desired 16-methylene-hydrocortisone-21-acetate (Kb), which is recrystallized from acetone after isolation. Mp. 216-219 ° C.

c) The 16-methylene-hydrocortisone-21-acetate obtained is, as described in Examples 2 d) and Ig) to Ij ₂ ), in 9a- fluoro-16-methylene-prednisolone-21-acetate (XVIII, R = Acetyl). Mp. 219 ° C.

Example 6 a) 1,2-dehydrogenation

Analogously to Example Ij ₂ ), 5 g of 16-methylene-4-pregnen-17a, 21-diol-3,20-dione-21-acetate (V) are inoculated with Corynebacterium simplex. The crude product obtained is purified by chromatography on silica gel and the benzene-chloroform eluate (1: 1) is concentrated in vacuo. The residue is recrystallized from acetone and gives 16-methylene-1,4-pregnadiene-17a, 21-diol-3,20-dione-21-acetate (VI '). Mp. 189 to 190 ⁰ C, [α]? + 2.6 ° (dioxane), X _max 244 nm, E} 392.

30 b) Microbiological IIβ-hydroxylation

Analogously to Example 2 b), 3 g of 16-methylene-1,4-pregnadiene-17a, 21-diol-3,20-dione-21-acetate are obtained (VI ') inoculated with Curvularia lunata. The oily crude product is separated by chromatography on silica gel, where the chloroform-ethyl acetate eluate (1: 1) which contains 16-methylene-prednisolone-21-acetate (Xb), which is recrystallized after isolation from acetone. Mp. 217 to 219 ° C, [a] l ° + 30 ° (dioxane).

c) The 16-methylene-prednisolone-21-acetate (Xb) obtained is, as described in Examples 4c) and 4d), in 9a- fluoro-16-methylene-prednisolone-21-acetate (XVIII, R = acetyl ) converted. Mp. 223 to 224 ° C.

Example 7

45

a) The 16-methylene hydrocortisone (VIIb) prepared according to Example 2 b) is used in a conventional manner converted into the following esters (IXb):

16-methylene-hydrocortisone-21-trimethylacetate, m.p. 195-197 ° C;

16 - methylene - hydrocortisone - 21 - tert. - butyl acetate, mp 196-197 ⁰ C, [α] !?. + ^T 73 ° (chloroform); ' X _max 240 nm, EU 382.

b) The esters (IXb) obtained according to Example 7 a) are converted into the corresponding 9a-fluoro compounds analogously to Examples 2d) and 2e), 9a-fluoro-16-meth'ylen-prednisolone-21-trimethylacetate, mp. 234-235 ° C, and 9a-fluoro-16-methylene-prednisolone-21-tert.-butyl acetate, mp 231-232 ⁰ C, _{[a] D} ² ° + 20 ° (chloroform). % _max 240nm, E ¹ Z 320 receives.

B e i s ρ i e 1 8

'a) The 16-methyleneprednisolone prepared according to Example 4 a) (VIIIb) is esterified in a conventional manner, the following 21-esters (Xb) receives:

16 - methylene - prednisolone - 21 - trimethylacetate, m.p. 224 to 226 ° C, [a] f + 19 ° (chloroform); X _max nm, EJ * 339;

16 - methylene - prednisolone - 21 - tert. - butyl acetate, melting point 217 to 218 ° C, [α] !? + 32 ° (dioxane); X _max 242 nm, E ₁ ¹ * 344;

lo-methylene-prednisolone ^ l-enanthate, m.p. 199 to 200 ° C, [a] ² S + 18 ° (chloroform); X _max 242 nm, EJ * _m 333;

lo-methylene-prednisolone ^ l-p-tert-butyl benzoate, Mp. 202 to 204 ° C, [α] ?? + 96 ° (chloroform);

16 - methylene - prednisolone - 21a - naphthyl acetate, mp 238-240 ⁰ C, [α] ί, ^α + 2.0 (chloroform)..

b) The esters (Xb) obtained according to Example 8 a) are converted into the corresponding 9a-fluoro derivatives converted, the following compounds being obtained:

9a - fluorine -16 - methylene - prednisolone - 21 - trimethylacetate, Mp 234-235 ° C;

^ a-fluoro-lo-methylene-prednisolone ^ l-tert-butyl acetate, Mp 231-232 ° C;

9a-fluoro-16-methylene-prednisolone-21 -önan that, Mp 231-232 ° C, [α] ff + 4.5 ° (chloroform).

Claims (1)

Claim: Process for the preparation of 9a-fluoro-16-methylene-prednisolone or -21-esters of the general Formula A. CH ₂ Y HO / OH F = CH, (Y = free or esterified hydroxyl group) characterized in that 16 / i-methyl-16a, l 7a-oxido-5-pregnen-3 /? - ol-20-one-3-acetate (I) by heating with catalytic amounts of a strong acid, especially a sulfonic acid, such as p-toluenesulfonic acid, in an inert solvent, especially benzene, in 16-methylene-5-pregnen-3 / i, 17a-dipl-20-on-3-acetate (II) transferred, then in a manner known per se. this compound (II) reacts with at least 2 molar equivalents of bromine, the resulting 5,6,21-tribromo -16-methylene-pregnane- 3ß, 1 la- diol-20-on-3-acetate (III) by successive treatment with a Iodide and an acetate - in particular with sodium iodide and potassium acetate, converted into 16 - methylene - 5 - pregnen - 3j?, 17a, 21 - triol - 20 - one-3,21-diacetate (IV), this ester (IV) with a culture of Flavobacterium dehydrogenans or an enzyme obtained therefrom is incubated, the obtained 16-methylene-4-pregnen-17a, 21-diol-3,20-dione (V) is microbiologically hydroxylated in Uo- or conversion, the obtained ll (a- or /?) hydroxy steroid (VIIa) or (VIIb) acetylated in the 21 position, the 1 l (a- or / J) -hydroxy-21-acetate (Ka) or (Kb) obtained in 9,11-position dehydrated, to the 9 (1 continuous double bond of the 9 (1 l) -dehydro-steroid (XI) HOBr or HQCl added, the obtained 9a-bromine or 9α-chlorine- lift- hydroxy-steroid (XIII) treated with an alkali acetate or an organic base, the formed 9 / Ul / i-oxidosteroid (XV) treated with hydrogen fluoride, the obtained 9a-fluorine -16 - methylene - 4 - pregnen-3,20-dione-II / U7a, 21-triol-21-acetate (XVII) by treatment with microorganisms which dehydrate in the 1 (2) position in 9 α-fluoro-16-methylene-prednisolone ^ l-acetate or ^ a-fluoro-lo-methylene-prednisolone converts and the latter optionally esterified in the 21-position, or that one of the intermediate compounds obtained according to the process (V, Vila, VIIb, IXa, IXb, XI, XIII or XV) after known microbiological measurement methods dehydrated in the 1 (2) position and the 1-dehydro-derivative obtained in this way (VI, Villa, VIIIb, Xa, Xb, XII, XIV or XVI) if necessary, in an analogous manner by 11-hydroxylation and / or 21-acetylation and / or 9,11-dehydration and / or HOBr or HOCl addition and / or dehydrohalogenation and / or splitting of hydrogen fluoride converts into 9a-fluoro-16-methylenepednisolone-21-acetate and that one optionally the 21-hydroxy group of the compounds (V) to (VIII) obtained per se known methods esterified and optionally the esters obtained analogously to the compounds (V) to (XVII) treated. 1 sheet of drawings 809 519/698 3. 68 © Bundesdruckerei Berlin