DE1211638B - Process for the preparation of 6alpha-fluoromethyl-17alpha-hydroxy-1, 4-pregnadiene-3, 20-dione-17-acetate - Google Patents

Description

FEDERAL REPUBLIC OF GERMANY

GERMAN

PATENT OFFICE

EDITORIAL

Int. Cl .:

Number:
File number:
Registration date:
Display day:

C07c

German KL: 12 ο -25/05

1211638
U9313IVb / 12o
October 6, 1962
March 3, 1966

The invention relates to a process for the preparation of 6 ″ -fluoromethyl-l 7 ″ -hydroxy-1,4-pregnadiene-3,20-dione-17-acetate the formula

O =

r- OCOCH ₃

CH ₂ F

This procedure consists in making a compound of the formula

■ - OCOCH ₃

CH ₂ F

in a manner known per se with SeO ₂ , dibenzoyloxyselenoxide. Br ₂ and subsequent dehydrobromination, periodic acid in tert-amyl alcohol or by incubation with a culture of a Fusarium, Nocardia, Septomyxa or Corynebacterium species in the 1 (2) - and 4 (5) position dehydrated and the possibly obtained 17 «- Hydroxy compound reacetylated.

The product of the process has progestational properties when administered parenterally as well as when administered orally Effect. When administered orally, its progestational effect is about 200 times that of that the corresponding compound unsubstituted in the 6-position.

It is suitable for the treatment of functional uterine bleeding and dysmenorrhea and can be given during pregnancy and to regulate fertility. To the therapeutic use it is in the usual dosage forms, such as pills, tablets, capsules, Syrups or elixirs when administered orally, or in processes for the production of 6a-fluoromethyl-

17a-hydroxy-1,4-pregnadiene-3,20-dione-

17-acetate

Applicant:

The Upjohn Company, Kalamazoo, Mich.
(V. St. A.)

Representative:

Dr. W. Beil, A. Hoeppener and Dr. H. J. Wolff,

Lawyers,

Frankfurt / M.-Höchst, Adelonstr. 58

Named as inventor:

J. Allan Campbell,

John Edward Pike, Kalamazoo, Mich. (V. St. A.)

Claimed priority:
V. St. v. America 9 October 1961
(143 567)

taken the form of solutions and suspensions for injections.

The starting material for the process according to the invention is obtained from the known 3β, Πα-ΌΊ- hydroxy-5-pregnen-20-one-3,17-diacylate. This is oxymethylated in the 6-position and the 6-hydroxymethyl-3 ß, 17a-dihydroxy-6α-pregnane-20-one-3,17-diacylate obtained with a halogenating agent, such as triphenylphosphite methoiodide, triphenylphosphite hydrochloride or hydrobromide, to the corresponding 6a- Halomethyl derivative, e.g. B. 6a-iodomethyl-3p ', 1 la - dihydroxy - 5α - pregnan - 3 - one - 3,17 - diacylate, implemented. His treatment with silver fluoride results in a mixture of 6a-fluoromethyl-3 /: Ü7a-dihydroxy-5a-pregnan-20-one-3p \ 17a-diacylate and 6-methylene-3 / j.l7a-dihydroxy-5a-pregnan-20 -on-3 / i, 17a-diacylate, which with strong mineral acid in an alkanol, z. B. with concentrated sulfuric acid in absolute ethanol or concentrated hydrochloric acid in methanol to a mixture of 6u-fluoromethyl-3 / j, 17a-dihydroxy-5a-pregnan-20-one-17-acylate and 6-methylene-3p \ 17u-dihydroxy -5a-pregnan-20-one-17-acylate is hydrolyzed. It can in a known manner, for. B. separated by chromatography and recrystallization

609 510/439

will. The hydroxyl group in the 3-position to the keto group is then oxidized. The oxidation can be done with sodium dichromate in acetic acid, chromic acid in glacial acetic acid or acetone or according to Opp e η a u e r with an aluminum alkoxide, e.g. B. with aluminum isopropylate in the presence of a ketone, such as acetone or cyclohexanone. A mixture of oa-fluoromethyl-na-hydroxy-5a-pregnane-3,20-dione-17-acylate is formed and 6-methylene-17a-hydroxy-5a-pregnane-3,20-dione-17-acylate, which can easily be separated by chromatography. For its production is in the frame protection is not sought for the present invention.

The conversion of the 6a-fluoromethyl-17a-hydroxy-5a-pregnane-3,20-dione-17-acylate thus prepared in 6a - fluoromethyl - 17a - hydroxy -1,4 - pregnadiene-3,20-dione-17-acylate can, as mentioned, (1) with selenium dioxide, (2) with selenium dioxide in the presence of a metal of group II or VIII of the periodic Systems such as magnesium, zinc, cadmium or mercury or iron, cobalt or nickel the process of US Pat. No. 2,900,398, (3) with dibenzoyloxyselenoxide according to the process the German patent 1 079 630, (4) by halogenation of the 2- and 4-position with z. B. bromine (2 equivalents) in a hydrobromic acid medium and subsequent elimination of hydrogen halide, z. B. with lithium chloride and dimethylformamide or with γ-collidine according to the method of the USA patent 2,838,531 or (5) with periodic acid according to the method of German patent specification 1,042,580 be performed.

Another possibility is microbiological dehydration, e.g. B. after V i s c h e r and coworkers, Experientia, Vol. IX, p. 371 (1953), with microorganisms of the genus Fusarium, e.g. B. Fusarium solani or Fusarium caucasium.

Furthermore, one of the two double bonds can be microbiological and the introduce others by chemical means.

The reaction with 1 equivalent of bromine and subsequent elimination of hydrogen halide gives z. B. the J! Derivative, which ^{can be converted into the J 1} ^ derivative with microorganisms of the genus Nocardia (cf. S t o udt and coworkers, Arch. Biochem. And Biophys., Vol. 74, p. 280 [ 1958]).

Microorganisms of the genus Septomyxa, on the other hand, have a double bond in the 1 (2) position a (see US Pat. No. 2,902,410), as well as microorganisms belonging to the Corynebacteriaceae family belong, like the genera Corynebacterium, Listeria and Erysipelothrix, in particular Corynebacterium simplex (ATCC 6946) and Corynebacterium hoagii (ATCC 7005). Used in microbiological Dehydration hydrolyzes the 17-position acetoxy group, so it must be done afterwards be reintroduced, e.g. B. with acetic anhydride in the presence of an acid catalyst, such as ρ - Toluenesulfonic acid (see U.S. Patent 2,805,230 and Journ. Amer. Chem. Soc, 74, p. 5394 [1942J).

To isolate the process product, the reaction mixture is first treated with aqueous acetic acid, Hydrochloric or sulfuric acid neutralized, then diluted with water and with one with water not miscible solvents, such as methylene chloride, chloroform, benzene or ether, extracted. the After drying and evaporation, extracts give the crude product, which is obtained from a solvent, such as methanol, ethanol, acetone, hexane hydrocarbons, ethyl acetate or methylene chloride, can be recrystallized to form the pure compound.

The following examples explain the process according to the invention:

example 1

A mixture of 2 g of oa-fluoromethyl-na-hydroxy-5a-pregnane-3,20-dione-17-acetate, 2.6 g of selenium dioxide, 3.1 g of mercury and 5.2 ml of acetic acid was tert in 190 ml. Suspended butyl alcohol and refluxed under nitrogen with stirring for about 48 hours. It was then concentrated to about 75 ml, diluted with methylene chloride and filtered through a pad of diatomaceous earth. The filtrate was washed twice each with fresh ammonium sulfide solution, dilute ammonia and water. Each aqueous phase was extracted with methylene chloride. The combined methylene chloride extracts were dried over magnesium sulfate, filtered, concentrated to about 20 ml and chromatographed over 200 g of synthetic magnesium silicate. The column was successively eluted with 4 liters of a mixture of 8 to 18% acetone and hexane hydrocarbons, fractions of 400 ml each being collected. Fractions 10 and 11 contained a few milligrams of a by-product which was found to be l-dehydro-oa-fluoromethyl-na-hydroxy-5a-pregnane-3.20-dione-17-acetate in the IR analysis. Α ^ ^0Η = 229 ΐημ (ε = 9125). Fractions 14 to 21 contained the main product and were evaporated together. Crystallization and recrystallization from a mixture of acetone and hexane hydrocarbons gave 0.4 g of 6a-fluoromethyl-na-hydroxy-l ^ pregnadiene-S ^ O-dione-n-acetate; F. = 250 to 255 ° C. Another recrystallization from the same solvent mixture gave an analytically pure sample which melted at 252 to 255 ° C; [a] _D = 6 °; * Ό ^Η = 243 ΐημ (ε = 16 250).

Analysis for C24H31FO4:

Calculated .. . C. 7161 H 7 76 F. 4 7? - found .. . C. 71.51, H 7.64, F. 5.15. By S pi el 2

A mixture of 1 g of oa-fluoromethyl-na-hydroxy-5a-pregnane-3,20-dione-17-acetate, 20 ml of tert-butanol and 1.5 g of dibenzoyloxyselenoxide was used a reflux condenser with a water catcher heated for about 12 hours. After cooling it was the selenium compound was filtered off and the filtrate was evaporated in vacuo. The residue was in Ethyl acetate added, the solution washed with aqueous sodium carbonate solution and water, over Dried activated charcoal and chromatographed over 125 g of synthetic magnesium silicate. The pillar was eluted with hexane hydrocarbons containing increasing amounts of acetone. Those Fractions whose UV spectrum showed a maximum at 243 πΐμ and therefore the desired one Containing product were combined and evaporated to dryness. The residue yielded two recrystallization from methanol light-

colored oa-fluoromethyl-na-hydroxy-l ^ -pregnadiene-3,20-dione-17-acetate.

Example 3

A solution of 320 mg of bromine in 15 ml of glacial acetic acid was added with slow stirring to a solution of 410 mgoa-fluoromethyl-na-hydroxy-Sa-pregnan-3,20-dione-17-acetate in 300 ml of acetic acid, the 0.1 ml were added to a 4N hydrogen bromide solution in acetic acid. The mixture was left to stand overnight, then the deposited precipitate was filtered off and dried after washing with water. The crude product was recrystallized from a mixture of ethyl acetate and hexane hydrocarbons, whereupon pure 2,4-dibromo-6 «-fluoromethyl-17a-hydroxy-5a-pregnane-3,20-dione-17-acetate was obtained. 100 mg of this compound in 1 ml of freshly distilled dimethylformamide were heated with 100 g of lithium chloride for about 2 hours. It was then cooled, poured into cold water and the aqueous solution extracted with methylene chloride. The extract was carefully washed with water, dried over sodium sulfate, concentrated and chromatographed over 25 g of synthetic magnesium silicate, eluting with hexane hydrocarbons containing increasing amounts of acetone. Those fractions which had a UV maximum 5 ^ HsOH ₌ 243 _m , j, were combined and evaporated to dryness. After recrystallizing twice from methanol, pure oa-fluoromethyl-na-hydroxy-l ^ -pregnadiene-3,20-dione-17-acetate was obtained.

The elimination of hydrogen bromide can also be achieved in the following way: 100 mg of 2,4-dibromoa-fluoromethyl-na-hydroxy-Sa-pregnane-S ^ O-dione- 17-acetate are dissolved in 1 ml of freshly distilled 7-K0I-lidine dissolved and refluxed for about 40 minutes. The solution is then cold enough Poured dilute sulfuric acid to bind the collidine as sulfate and extracted with methylene chloride. The extract is chromatographed over 25 g of synthetic magnesium silicate and treated with hexane hydrocarbons eluted containing increasing amounts of acetone. Those fractions with a UV maximum at 243 πΐμ are combined and after recrystallization give pure light-colored 6a-fluoromethyl-17a-hydroxy-1,4-pregnadiene-3,20-dione-17-acetate.

Example 4

Ig 6 «- fluoromethyl-17a - hydroxy - 5α - pregnane-3,20-dione-17-acetate is dissolved in 65 ml of tert-amyl alcohol and 3 ml of glacial acetic acid, and 400 mg of periodic acid are added while stirring. It is heated to 7O ⁰ C and are at intervals of 30 minutes, four times 400 mg per periodic acid to, wherein the mixture further stirred and maintained at 70 ⁰ C. The mixture is then held at this temperature for a further 25 hours, diluted with 65 ml of water and treated with 3 g of sodium sulfate. After the tert-amyl alcohol has been distilled off in vacuo, the residue is extracted with chloroform. The extract is washed with water, dried over sodium sulfate and evaporated to dryness in vacuo on a water bath. When recrystallized from acetone, the solid obtained gives pure, light-colored 6a-fluoromethyl-17a-hydroxy-1,4-pregnadiene-3,20-dione-17-acetate.

Example 5

Six 100 ml each of a nutrient medium consisting of 1% glucose, 2% corn steep liquor (60% solids) and tap water are adjusted to pH = 4.9 in 250 ml Erlenmeyer flasks and 45 minutes at a pressure of about 1 atm. sterilized. A 1 to 2 day old culture of Fusarium solani ATCC 11712 is then inoculated. The flasks are shaken at about 24 ° C for 3 days. Then the entire 600 ml is used as a vaccine for 101 of the same nutrient medium, which additionally contains 10 ml of an antifoam (mixture of lard oil and octadecanol). The fermentation vessel is placed in a ^{water bath set to 28 °} C. and its contents are stirred (300 rpm) and aerated (0.5 1 air per 10 1 fermentation broth). After 17 hours, during which good growth had developed, 2 g of 6a-fluoromethyl-17a-hydroxy-5a-pregnane-3,20-dione-17-acetate and 1 g 3-Keto-bis-nor-4-cholen-22-al, dissolved in 115 ml of dimethylformamide, was added. It was incubated for a further 48 hours at the same temperature and ventilation rate (final pH 7.9). After filtering off the mycelium, the steroid material was extracted with methylene chloride. The extract was evaporated to dryness and the residue was chromatographed over 200 g of synthetic magnesium silicate. The column was eluted with 400 ml each of methylene chloride, hexane hydrocarbons - acetone in the ratio 9: 1, 8: 2, 7: 3 and 1: 1 and methanol. The eluate obtained with hexane hydrocarbons-acetone in the ratio 7: 3 contained the desired oa-fluoromethyl-na-hydroxy-1 ^ -pregnadiene-S ^ O-dione-17-acetate.

The same compound is obtained if instead of the microorganism ATCC 11712 the microorganism Fusarium solani ATCC 10915 or Fusarium caucasicum No. 5978 of the culture collection of the "Institute for Fermentation of Takeda Pharmaceutical Industries, Ltd.", Osaka, Japan, used.

Example 6

A solution of 160 mg of bromine in 10 ml of glacial acetic acid was added with slow stirring to a solution of 410 mgoa-fluoromethyl-na-hydroxy-Sa-pregnane-3,20-dione-17-acetate in 300 ml of acetic acid, the 0.1 ml 4 N hydrogen bromide solution in acetic acid were added. After allowing to stand overnight, during which a precipitate was formed, the mixture was filtered, the precipitate was washed with water, dried and recrystallized from a mixture of ethyl acetate and hexane hydrocarbons. Light-colored 2-bromo-6a-fluoromethyl-17a-hydroxy-5a-pregnane-3,20-dione-17-acetate was obtained. 100 mg of this compound were heated with 1 ml of freshly distilled dimethylformamide and 100 mg of lithium chloride for about 2 hours. The cooled reaction mixture was poured into cold water and the aqueous solution extracted with methylene chloride. The extract was carefully washed with water, dried over sodium sulfate, concentrated and chromatographed over 25 g of synthetic magnesium silicate. Hexane hydrocarbons containing increasing amounts of acetone were used for elution. The fractions with a UV maximum / l £ ax ⁵ ° ^H = 229 πΐμ (f = 9125) were combined and used

Evaporated to dryness. The residue gave pure after recrystallization twice from methanol l-dehydro-öa-fluoromethyl-lYa-hydroxy-Sa-pregnan-3,20-dione-17-acetate.

The elimination of hydrogen bromide can also be achieved by adding 100 mg of 2-bromo-6a-fluoromethyl-na-hydroxy-Sa-pregnane-S ^ O-dione-n-acetate Dissolve in 1 ml of freshly distilled yCollidin and reflux for about 40 minutes. The solution is then poured into sufficiently cold dilute sulfuric acid to bind the collidine as sulfate. Then it is extracted with methylene chloride and the extract over 25 g of synthetic magnesium silicate chromatographed, eluting with hexane hydrocarbons, the increasing amounts of acetone contain. The fractions with a UV maximum at 229 πΐμ (ε = 9125) are combined. You surrender when recrystallizing pure 1-dehydro-oa-fluoromethyl-lYa-hydroxy-Sa-pregnan-S ^ O-dione-n-acetate. The methods described in Example 7, a) below, i. H. by bromination and Splitting off of hydrogen bromide or by microbiological means with microorganisms of the genus Nocardia can become dehydrated in the 4 (5) position.

Example 7

a) oa-fluoromethyl-na-hydroxy ^ pregnen-3,20-dione-17-acetate

410 mg of oa-fluoromethyl-na-hydroxy-Sa-pregnane-3,20-dione-17-acetate in 10 ml of dioxane were acidified with a drop of a 4N hydrogen bromide solution in dioxane, whereupon 320 mg of bromine were added over the course of one minute . After standing at room temperature for about 1 hour, excess sodium bicarbonate solution was added to the reaction mixture. The precipitated 2,4-dibromo derivative desoa-fluoromethyl-na-hydroxy-Sa-pregnan-S ^ O-dione-17-acetate was heated for 2 ^ ¹ hours under reflux with 0.9 g of sodium iodide in 15 ml acetone containing bromoacetone. After adding 0.3 g of oxalic acid, the mixture was heated for a further hour. The mixture was then cooled, ethyl acetate was added and the solution was filtered. The filtrate was washed with water and sodium bicarbonate solution, dried over sodium sulfate, stirred with 500 mg of zinc dust in 2 ml of acetic acid for about 1 hour and then filtered again. The organic layer was washed successively with water and sodium bicarbonate solution, dried over sodium sulfate and evaporated. The crude α, β-unsaturated ketone was obtained which, after purification with Girard's reagent and crystallization, gave pure 6α-fluoromethyl-17α-hydroxy-4-pregnene-3,20-dione-17-acetate. It can also be obtained by chromatography on synthetic magnesium silicate using hexane hydrocarbons containing increasing amounts of acetone and subsequent recrystallization.

The same compound is obtained when a nutrient medium consisting of 1% dextrose hydrate, 2% corn steep liquor (60% solids content) and tap water is adjusted to pH = 4.9 with sodium hydroxide, at about 1 atm. sterilized with steam for about 30 minutes and then inoculated with a 24 hour old culture of spores of Nocardia blackwellii NCTC 630 (Medical Research Council of the Lister Institute, London). The medium is stirred and aerated with sterile air at a rate of ¹ part by volume of air per part by volume of nutrient medium per minute. After fermentation for 24 hours at room temperature, the pH is around 7.4. A solution of 5 parts of 6a-fluoromethyl-na-hydroxy-Sa-pregnane-S ^ O-dione-n-acetate in 100 parts of dimethylformamide is then added in an amount of about 10 ml per liter of nutrient medium. After about 6 hours of fermentation, the mycelium and fermentation solution are carefully extracted with methylene chloride. The extract is washed with sodium bicarbonate solution and then with water and dried. On evaporation it gives oa-fluoromethyl-nct-hydroxy-4-pregnen-3,20-dione. The microorganism Nocardia convoluta ATCC 4275, Nocardia gardneri ATCC 9604 or Norcardia coeliaca NRRL B-1365 can also be used with the same success for the dehydration of the 4 (5) position.

The 17-position acetoxy group split off during the microbiological dehydration of the 4 (5) position is reintroduced by adding a solution of 1 g of oa-fluoromethyl-na-hydroxy-4-pregnen-3,20-dione in 2.5 ml of distilled acetic anhydride, 250 mg of p-toluenesulfonic acid and 2.5 ml Stir acetic acid for about 90 minutes and then pour into water with vigorous stirring. The failing one Solid is filtered off, dried, using hexane hydrocarbons, the increasing Containing amounts of acetone, chromatographed on synthetic magnesium silicate and extracted from ethyl acetate recrystallized. Light-colored 6a-fluoromethyl-17a-hydroxy-4-pregnen-3,20-dione-17-acetate is obtained.

b) oct-fluoromethyl-na-hydroxy-M-pregnadiene-3,20-dione-17-acetate

Six 100 ml each of a nutrient medium consisting of 1% glucose, 2% corn steep liquor (60% solids content) and tap water were adjusted to pH 4.9 in 250 ml Erlenmeyer flasks. The nutrient medium was at about 1 atm for 45 minutes. sterilized and inoculated with a 1 to 2 day old culture of Septomyxa affinis ATCC 6737. The flasks were shaken at about 24 ° C for 8 days. Then the entire 600 ml was used as a vaccine for 10 liters of the same medium, which additionally contained 10 ml of an anti-foaming agent (mixture of lard oil and octadecanol). The fermentation vessel was placed in a ^{water bath set to 28 °} C. and its contents were stirred (300 rpm) and aerated (0.5 l of air per 101 of fermentation broth). After 17 hours of incubation, after good growth had developed and the pH had risen to 6.7, there were 2 g of 6a-fluoromethyl-17a-hydroxy-4-pregnen-3,20-dione-17-acetate and 1 g 3-Keto-bis-nor-4-cholen-22-al, dissolved in 115 ml of dimethylformamide, was added, and the incubation was continued for a further 24 hours at the same temperature and ventilation rate (final pH value 7.9). The mycelium was then filtered off and the steroid material extracted with methylene chloride. The residue obtained on evaporating the extracts was chromatographed over 200 g of synthetic magnesium silicate. The column was developed with 400 ml each of methylene chloride, hexane hydrocarbon-acetone mixtures in a ratio of 9: 1, 8: 2, 7: 3 and 1: 1 and methanol. The eluate obtained with hexane hydrocarbons and acetone in a ratio of 7: 3 resulted after evaporation and recrystallization

ίο

Acetone the desired oa-fluoromethyl-na-hydroxyl, 4-pregnadiene-3,20-dione-17-acetate. Instead of the Septomyxa affinis ATCC 6737, the 1 (2) position can also be used for microbiological dehydration Microorganism Corynebacterium simplex ATCC 6946 can be used.

It is also possible to chemically change the 1 (2) position with z. B. to dehydrate selenium dioxide. For this purpose, a mixture of 70 mg 6a-fluoromethyl-17a-hydroxy-4-pregnen-3,20-dione-17-acetate, 4.5 ml of tert-butyl alcohol, 0.45 ml of acetic acid and 24 g of selenium dioxide were heated to 75 ° C. for 24 hours while stirring. Then another 24 mg Selenium dioxide is added, and the mixture is further heated to 75 ° C. and stirred. Then it is cooled by selenium dioxide filtered off and evaporated. Contained on the basis of a paper chromatographic analysis the residue about 50 to 55% 6a-fluoromethyl-17a-hydroxy-1,4-pregnadiene-3,20-dione-l7-acetate, which was obtained pure by recrystallization and chromatography.

Claims (1)

Claim: Process for the preparation of 6a-fluoromethyl-17a-hydroxy-1,4-pregnadiene-3,20-dione-17-acetate of formula CH ₃ O = * CH ₂ F characterized in that a compound of the formula CH ₃ CH ₂ F in a manner known per se by reaction with SeO ₂ , dibenzoyloxyselenoxide, Br ₂ and subsequent dehydrobromination, periodic acid in tert-amyl alcohol or by incubation with a culture of a Fusarium, Nocardia, Septomyxa or Corynebacterium species in 1 (2) and 4 (5) position is dehydrated and the optionally obtained free 17α-hydroxy compound is reacetylated. 609 510/439 2.66 © Bundesdruckerei Berlin