DE1158063B - Process for the preparation of therapeutically active steroid compounds - Google Patents

Description

Process for the preparation of therapeutically active steroid compounds The invention relates to a process for the preparation of therapeutically active steroid compounds of the general formula in which Y is hydrogen or fluorine, X is keto oxygen or (H, β-OH) and R is hydrogen or the acyl radical of a carboxylic acid having 1 to 12 carbon atoms, characterized in that a compound of the general formula is used in a manner known per se in which X, Y and R have the above meaning, fermentatively hydroxylated with Streptomyces argenteolus ATCC 11009 , optionally acylated the 16- and 21-position hydroxyl group with the halide or anhydride of an organic carboxylic acid with 1 to 12 carbon atoms, optionally the 11-position hydroxyl group oxidized with a chromic acid oxidizing agent and optionally hydrolyzed the 16- and 21-position acylate group of the 11-keto derivative obtained with a base.

It is true that a 16a hydroxyl group has already been introduced into 11-deoxysteroid compounds by means of the actinomycete species ATCC 11009. Due to the extraordinary substrate specificity of microorganisms, however, one could not assume that Streptomyces argenteolus would also introduce a 16a hydroxyl group in steroid compounds containing a 6α-fluorine substituent without at the same time impairing the hydroxyl group in the 11β-position, which is accessible to oxidation .

The compounds that can be prepared according to the invention have stronger glucocorticoids and anti-inflammatory effects as hydrocortisone and cortisone. They also increase the salt and water excretion and are therefore particularly suitable for the treatment of chronic congestive heart disease, cirrhosis of the liver, kidney and adrenal syndromes as well as eclampsia and preeclampsia.

For therapeutic use, they can be in the usual dosage forms administered orally, parenterally and topically. Suitable for oral use pills, tablets and capsules, liquid preparations for parenteral administration, as with natural and synthetic corticosteroid hormones are common, and for topical application ointments, creams, lotions and the like, if necessary Other active ingredients, such as antibiotics, germicides and the like, can also be added.

The introduction of the 16-position hydroxyl group -can under de n in the USA. Pat carried 2,602 769 conditions- given. For this purpose, the Streptomyces argenteolus is grown on a medium which contains assimilable, non-steroidal carbon, e.g. B. carbohydrates such as dextrose, assimilable nitrogen such as soluble or insoluble proteins, peptones or amino acids, and mineral components such as sodium or ammonium phosphate and magnesium sulfate. Before inoculation with the microorganisms, the medium preferably has a pH between about 6.5 and 7.8, but the pH can also be higher or lower. For the growth of the microorganisms, a pH value between about 6.8 and 7.4 is preferred and a temperature range from about 20 to 35 ° C, advantageously from about 20 to 32 ° C.

The growing season prior to the addition of the steroid to be fermented does not seem critical. The steroid can e.g. B. before the sterilization of the Medium at the time of inoculation with the microorganism or later, e.g. B. Added 24 to 48 hours thereafter. The addition of the steroid substrate can in any suitable manner, e.g. B. by dispersing, optionally with a dispersant, or dissolved in an organic solvent. Submerse and surface cultures are equally suitable, but one becomes submerse Culture preferred.

The temperature during fermentation can be the same as it was applied to the growth of the microorganism. You just have to be inside an area in which the microorganism live and grow or that of enzyme produced by it can be effective.

The one required to introduce the 16a hydroxyl group Time depends somewhat on the procedure used. Will the steroid after essential Added growth of the microorganism, e.g. B. after 16 to 24 hours at optimal Temperature, the conversion of the steroid begins immediately and is after 2 to 10 Days practically ended. Usually within 5 days you will get satisfactory results obtain.

After the fermentation is complete, the converted steroid is through Extraction with an organic steroid solvent, e.g. B. methyl isopropyl ketone, Methylene chloride, chloroform, carbon tetrachloride, ethylene chloride, trichlorethylene, Ether, amyl acetate, benzene and the like from the fermentation mixture (fermentation liquid -E- Mycelium). The fermentation liquid and the mycelium can also be separated with suitable Solvents extracted and the extracts .before or after washing with a alkaline solution, e.g. B. sodium bicarbonate solution, are combined. Afterward is z. B. dried over anhydrous sodium sulfate and the steroid by recrystallization or trituration with organic solvents or purified by chromatography-then the 16a and 21 position hydroxyl groups can be esterified and the hydroxyl group in position are oxidized. If. 11-keto compounds with free 16a and 21-position hydroxyl groups are desired, this is followed by another Hydrolysis, expediently with alkali metal carbonate in the absence of oxygen.

The following examples explain the process according to the invention. Example 1 a) 6a-Fluor-IIß, 16a, 17a, 21-tetraoxy-4-pregnen-3,20-dione 100m1 2% corn steep liquor, solids content 60% were adjusted to pH 6.8 to 7.4 with sodium hydroxide solution and 30 Minutes at a pressure of about 1 kg (cm2). Then a similarly sterilized solution of 2 g of technical dextrose, which is known under the trade name "Cerelose", in 4 ml of water was added. The sterile medium was mixed with a suspension of Spores and mycelium of Streptomyces argenteolus ATCC 11009 were inoculated and placed on a rotary shaker for 24 hours, during which time a good culture of the microorganism developed -pregnen-3,20-dione (6a-fluorohydrocortisone), dissolved in 0.2 ml of dimethylformamide, was added. The steroid was incubated with agitation for 5 days. The pH was then 8.6. Mycelia and The mycelium was first separated twice with each 25 ml of acetone and then extracted four times with 25 ml of methyl isopropyl ketone each time. The fermentation liquid was extracted four times with 25 ml of methyl isopropyl ketone each time. All extracts were combined, washed with 20/0 aqueous sodium bicarbonate solution and then with water, dried with anhydrous sodium sulfate and evaporated to dryness. The residue, which on the basis of a paper chromatographic analysis contained the 6a-fluoro-l1ß, 16a, 17a, 21-tetraoxy-4-pregnen-3,20-dione, was determined by chromatography over synthetic magnesium silicate, known under the trade name "Florisil" is purified, and crystallization from acetone. F. = 234 to 236 ° C.

b) 6a-fluoro-11ß, 16a, 17a, 21-tetraoxy-4-pregnen-3,20-dione-16,21-diacetate A solution of 1.2 g of 6a-fluoro-1 1ß, 16a, 17a, 21-tetraoxy-4-pregnen-3,20-dione-in 20m1 pyridine and 20m1 acetic anhydride were added for 17 hours at room temperature (approx 25 ° C) and then poured into 200 ml of ice water. The received The mixture was extracted with methylene chloride and the extract successively with dilute Hydrochloric acid, dilute sodium bicarbonate and water. After drying the solution over anhydrous sodium sulfate, the solvent was evaporated and the residue by chromatography over synthetic magnesium silicate and crystallization purified from acetone. 6a-fluoro-113, 16a, 17a, 21-tetraoxy-4-pregnene-3,20-dione-16,21-diacetate were obtained from melting point 187 to 192 ° C. Example 2 Using the im The procedure described in Example 1, a) was obtained from 6a, 9a-Difluorllß, 17a, 21-trihydroxy-4-pregnen-3,20-dione the corresponding 16a-hydroxy derivative of melting point 242 to 248'C, which after the Method of Example 1, b) converted into 6a, 9a-difluoro-11ß, 16a, 17a, 21-tetraoxy-4-pregnen-3,20-dione-16a, 21-diacetate became. F. = 182 to 185'C.

Claims (2)

PATENT CLAIMS: 1. Process for the preparation of therapeutically active steroid compounds of the general formula in which Y denotes hydrogen or fluorine, X denotes keto oxygen or (H, β - OH) and R denotes hydrogen or the acyl radical of a carboxylic acid having 1 to 12 carbon atoms, characterized in that a compound of the general formula is used in a manner known per se in which X, Y and R have the above meaning, fermentatively hydroxylated with Streptomyces argenteolus ATCC 11009, optionally acylated the 16- and 21-position hydroxyl group with the halide or anhydride of an organic carboxylic acid having 1 to 12 carbon atoms, optionally the 11-position hydroxyl group oxidized with a chromic acid oxidizing agent and optionally hydrolyzed the 16- and 21-position acylate group of the 11-keto derivative obtained with a base. 2. The method according to claim 1, characterized characterized in that the starting material is 6a-fluoro-11 ß, 17a, 21-trioxy-4-pregnen-3,20-dione or 6a, 9a-Difluor-11ß, 17a, 21-trioxy-4-pregnen-3,20-dione used. Into consideration drawn publications: J. Amer. Chem. Soc., 74, p. 2126 (1952); Journ. Bacteriol., 69, p. 347 (1955); Rec. Progr. Horm. Res., 11, pp. 149-180.