CA1043330A - 16-METHYL-1,4-PREGNADIENE-17.alpha.-OL-3,20 DIONE COMPOUNDS - Google Patents
16-METHYL-1,4-PREGNADIENE-17.alpha.-OL-3,20 DIONE COMPOUNDSInfo
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- CA1043330A CA1043330A CA279,702A CA279702A CA1043330A CA 1043330 A CA1043330 A CA 1043330A CA 279702 A CA279702 A CA 279702A CA 1043330 A CA1043330 A CA 1043330A
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- methyl
- alpha
- dione
- acetate
- diol
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Abstract
ABSTRACT
This invention is concerned with 16.alpha.-methyl cortisone 21-acetate and its preparation. Such compound is a novel steroid compound of value in connection with the treatment of arthritis and related diseases.
This invention is concerned with 16.alpha.-methyl cortisone 21-acetate and its preparation. Such compound is a novel steroid compound of value in connection with the treatment of arthritis and related diseases.
Description
This invention is concerned generally with novel steroid compounds and with processes of preparing the same.
More particularly, it relates to novel 16-met~yl-11-oxygenated-1,4-pregnadiene-17a, 21-diol-3,20-dione compounds, and to the process of preparing these compounds starting with the corresponding 16-methyl-11-oxygenated-4-pregnene-17~,21-diol-3,20-dione compound. These novel l~-methyl-ll-oxygenated-1,4-pregnadiene-17a,21-diol-3,20-dione compounds possess extremely high anti-inflammatory activity, and are especially effective for the treatment of arthritis and related diseases since they can be administered for their cortisone-like action in extremely low dosage thereby minimizing undesired side effects.
These novel 16-methyl-11-oxygenated-1,4-pregnadiene-17a,21-diol-3,20-dione compounds, subject of the present invention, may be chemically represented as follows:
C=O
Z ~ ~ ---OH
~ ---CH
~ ~
~W ' wherein X i~ hydrogen or fluoro, R stands for hydrogen or an acyl radical, and Z is a keto or hydroxy substituent.
These 16-methyl-11-oxygenated-1,4-pregnadiene-l~a,21-diol-3,20-dione compounds are prepared by contacting the corresponding 16-methyl-11-oxygenated-4-pregnene-17~, 21-diol-3,20-dione compound with the dehydrogenating activity of microorganisms of the Class Schizomycetes, which includes microorganisms belonging to the orders Actinomycetales and Eubacteriales. The preferred Eubacteriales include micro-organisms of the genus Acetobacter, the genus Aerobacter, 1~43330 and the genus Bacillus; the preferred Actinomycetales include microorganiSms of the genus Nocardia and the genus Myco~acterium.
I particularly prefer to employ microorganisms of the Class Schizomycetes belonging to the following species, namely:
Acetobacter xylinum, Aerobacter aerogenes, Bacillus spaericus, Nocardia erythropolis, Nocardia blackwellii, Nocardia asteroides, Nocardia minima, Nocardia globerula, Nocardia leishmanii, Nocardia formica, Nocardia convoluta, Nocardia corallina, and Mycobacterium smegmatis, Mycobacterium phlei, Mycobacterium lacticola, and Mycobacterium tuberculois. The species Bacillus sphaericus, as defined in Bergey's Manual for Determinative Bacteriology, Sixty Edition, comprises several varieties such as the rotans variety, the fusiformis variety, - etc. and, in some collections, these varieties are referred to by the species names Bacillus rotans and Bacillus fusiformis.
These microorganisms of the Class Schizomycetes can be obtained from known sources such as the American Type Culture Collection, Washington, D.C., or they may also be isolated from natural sources, such as soil, by known methods.
It is desired to emphasize that for any given species of Schizomycetes, the preferred dehydrogenating strains can be selected by the following test method: A
nutrient medium containing 1 g. of cerelose, 1 g. of edamin, 0.25 ml. of cornsteep liquor, 0.05 g. of yeast extract, and sufficient distilled water to make 50 ml., is adjusted to pH 6.5, sterilized and inoculated with a culture of the par-ticular microorganism strain to be tested for its ~1 de-hydrogenating activity. The resulting culture is incubated for a period of 24 hours at a temperature of 2aC., and a sample of the culture is transferred to a second 50 milliter sample of the aforementioned nutrient medium which has likewise been adjusted to pH 6.5 and sterilized. The resulting inoculated B~ 2 -culture is then incubated at a temperature of 28C., with agitation, for a 24-hour per;od, and to the resulting culture is added a solution containing 10 mg. of hydrocortisone dissolved in 0.1 ml. of dimethylformamide. The culture con-taining the steroid compound is incubated, with agitation, for an additional period of about 10 hours at 28C. The fermentation broth is repeatedly extracted with ethyl acetate, and the ethyl acetate extracts are combined and evaporated to dryness in vacuo. A portion of the residual material is dissolved in acetone and spotted on a paper chromatogram which is developed using formamide as the stationary phase and chloroform as the mobile phase. Two separate bands are ordinarily obtained, the lower band corresponding to unchanged hydrocortisone; the upper band corresponds to the ~ l-dehydro derivative. Both bands are cut off, separately eluted with methanol, and each of the methanol eluants are subjected to ultraviolet absorption analysis. The efficiency of the microorganism strain being tested in effecting L~l-dehydrogenation is indicated by the relative proportions of ~l-dehydro derivative and unchanged hydrocortisone as measured by this ultraviolet absorption analysis.
The 16-methyl-11-oxygenated-4-pregnene-17a,21-diol-3,20-diones used as starting materials in this process are conveniently prepared starting with the known 16-pregnene-3a-ol-11,20-dione 3-acetate in accordance with the following procedure:- 16-pregnene-3a-ol-11,20-dione 3-acetate is reacted with methyl magnesium iodide in the presence of cuprous chloride thereby forming 16~-methyl-pregnane-3a-ol-11,20-dione 3-acetate, which is reacted with aqueous methanolic hydrochloric acid to form l6a- methyl-pregnane-3a-ol-11,20-dione. The latter compound, which is a potent anesthetic, is reacted with acetic anhydride in the presence of p-toluene sulfonic acid catalyst Bj . 1....
lV433;~0 to form a mixture of enol acetates contalning 16a-methyl-17,20-pregnene-3~,20-diol~ one 3,20-diacetate; this mixture, after chromatographic purification to remove any unchanged starting material, is reacted with perbenzoic acid and the resulting 16-methyl-17~,20-epoxy-pregnane-3a,20-diol-11-one 3,20-diacetate is hydrolyzed with methanolic potassium bicarbonate to produce 16a-methyl-pregnane-3~,17~-diol-11,20-dione. The latter compound is reacted with bromine in chloroform to form 21-bromo-16a-methyl-pregnane-3~,17~-diol-11,20-dione which is reacted with sodium iodide in acetone to produce 21-iodo-16~-methyl-pregnane-3~,17~-diol-11,20-dione which is converted without isolation to 16~- methyl-pregnane-3~,17~, 21-triol-11, 20-dione 21-acetate by reaction with anhydrous potassium acetate;
this compound is reacted with chromaum trioxide in pyridine to form 16a-methyl-pregnane-17~,21-diol-3,11,20-trione 21-acetate.
The 16~-methyl-pregnane-17,21-diol-3,11,20-trione 21-acetate is reacted with bromine in glacial acetic acid-chloroform to pro-duce 4-bromo-16~-methyl-pregnane-17a,21-diol-3,11,20-trione, which is then reacted with semicarbazide to form 16~-methyl-4-pregnene-17~,21-diol-3,11,20-trione 3,20-bis-semicarbazone 21-acetate.
This 3,20-bis-semicarbazone is reacted with sodium borohydride to form 16X-methyl-4-pregnene-1~ ,17~,21-triol 3,20-dione 3,20-bis-semicarbazone which is hydrolyzed under acid conditions to form 16x-methyl-4-pregnene-1~ ,17~,21-triol-3,20-dione; this compound is reacted with esterifying agents as for example benzoic anhydride or lower alkanoic anhydrides to form the corresponding 21-ester derivatives.
The 1~ -methyl-4-pregnene-1~ ,21-diol-3,11,20-trione-3,20-bis-semicarbazone 21-acetate is reacted with potas-sium bicarbonate or potassium hydroxide in aqueous methanol to form 1~ -methyl-4-pregnene-17~,21-diol-3,11,20-trione-3,20-bis-semicarbazone whach às then hydrolyzed under acid . ~ 4 ~
i 1t)43330 conditions to produce 16~-meth~1~4-pregnene-17~,21-diol-3,11,20-trione 21-free alcohol; this compound is esterfied using the above-mentioned esterifying agents to produce the corresponding 21-ester derivati~es.
The 16a-methyl-4-pregnene~ ,17~,21-triol-3,20-dione, upon reaction with acetic anhydride in pyridine, gives the corresponding 21-acetate which is reacted with methane sulfonyl chloride followed by potassium acetate, or phos-phorus oxychloride, to produce 16-methyl-4,9(11)-pregnadiene-17a,21-diol-3,20-dione 21-acetate; the latter compound is reacted with hypobromous acid to produce 9-bromo-16~-methyl-4-pregnene-11~,17o721-triol-3,20-dione 21-acetate which is reacted with anhydrous potassium acetate in ethanol to produce 16a-methyl 9,11-epoxy-4-pregnene-17~,21-diol-3,20-dione 21-acetate. This 9,11-epoxide is then reacted with hydrogen fluoride in tetrahydrofuran to produce 16a-methyl-9-fluoro-4-pregnene-11~, 17a,21-triol-3,20-dione 21-acetate; this compound is reacted with a hydrolyzing agent to form 16a-methyl-9-fluoro-4-pregnene-11~,17a,21-triol-3,20-dione 21-free alcohol. This 21-free alcohol is reacted with esterifying agents such as benzoic anhydride, lower alkanoic anhydrides and the like to form the corresponding 21-ester derivatives.
The 16~-methyl-9-fluoro-4-pregnene-11~,17,21-triol-3,20-dione 21-acetate is reacted with chromium trioxide in pyridine to form 16-methyl-9~-fluoro-4-pregnene-17,21-diol-3,11,20-trione 21-acetate which, upon reaction with a hydrolyzing agent, forms 16~-methyl-9a-fluoro-4-pregnene-17a, 21-triol-3,11,20-trione 21-free alcohol. This 21-free alcohol is reacted with esterifying agents such as benzoic anhydride, lower alkanoic anhydrides and the like to form the corresponding 21-ester derivatives.
Thus, these 16-methyl-11-oxygenated-4-pregnene-- 5 ~
~.. , ~, . ~
1~43330 17~,21-diol-3,2Q-dione starting materials include 16o-methyl-4-pregnene-17~,21-diol-3,11,20-trione and 21-esters t~ereof, and particularly 21-lower hydrocarbon carbonyl esters as for example 16~-methyl-4-pregnene-17O,21-diol-3,11,20-trione 21-benzoate; 16~-methyl-4-pregnene-17,21-diol-3,11,20-trione 21-lower alkanoates such as 16a-methyl-4-pregnene-17a,21-diol-3,11,20-trione 21-acetate; 16~-methyl-4-pregnene-17a, 21-diol-3,11,20-trione 21-propionate; 16a-methyl-4-pregnene-11~, 17a,21-triol-3,20-dione and 21-esters thereof, and particularly 21-lower hydrocarbon carbonyl esters as for example 16~-methyl-4-pregnene-11~,17~,21-triol-3,20-dione 21-benzoate; 16~-methyl-4-pregnene-11~,17a,21-triol-3,20-dione 21-lower alkanoates such as 16a-methyl-4-pregnene-11~,17a,21-triol-3,20-dione 21-acetate; 16~-methyl-4-pregnene-11~,17a,21-triol-3,20-dione 21- propionate; 16a-methyl-9~-fluoro-4-pregnene-17a,21-diol-3,11,20-trione and 21-esters thereof, and particularly 21-lower hydrocarbon carbonyl esters as for example 16a-methyl-9a-fluoro-4-pregnene-17a,21-diol-3,11,20-trione 21-benzoate; 16~-methyl-9a-fluoro-4-pregnene-17a,21-diol-3,11, 20-trione 21-lower alkanoates such as 16a-methyl-9a-fluoro-4-pregnene-17a-21-diol-3! 11,20-trione 21-acetate; 16a-methyl-9~-fluoro-4-pregnene-17,21-diol-3,11,20-trione 21-propionate;
16a-methyl-9~-fluoro-4-pregnene-11~,17d,21-triol-3,20-dione and 21-esters thereof, and particularly 21-lower hydrocarbon carbonyl esters thereof, as for example 16a-methyl-9a-fluoro-4-pregnene-11~,17,21-triol-3,20-dione 21-benzoate; 16a-methyl-9a-fluoro-4-pregnene-11~,17a,21-triol-3,20-dione 21-lower alkanoates such as 16a-methyl-9-fluoro-4-pregnene-11~,17a,21-triol-3,20-dione 21-acetate; 16a-methyl-9a-fluoro-4-pregnene-11~,17a, 21-triol-3,20-dione 21-propionate and the like.
In accordance with the present invention and utilizing the preferred strains of Schizomycetes microorganisms, ~'`1 1q)4;~330 the dehydrogenation of the 16-methyl~ oxygenated-4-pregnene 1~ ,21-diol-3,20-dione compound is effected by contacting the steroid compound withthe Schizomycetes microoganisms themselves of, if preferred, with enzyme systems of Schizomycetes micro-organisms whereby the hydrogen attached to the C-l and C-2 carbon atoms is selectively removed to produce the desired a -steroid substantially uncontaminated by unwanted by-products.
When the 16-methyl-11-oxygenated-4-pregnene-17~,21-diol-3,20-dione compound is subjected to the dehydrogenating activity of the preferred dehydrogenating strains of Schizomycetes microorganisms, the corresponding 16-methyl-11-oxygenated-1,4-pregnadiene-17~,21-diol-3,20-dione compound is obtained directly and in high yield.
The presently-invented microbiological dehydro-genation procedure is conducted by contacting the 16-methyl-ll-oxygenated-4-pregnene-17~,21-aiol-3,20-dione compound with the dehydrogenating activity of Schizomycetes microorganisms.
~his can be effected by adding the steroid compound as a solid, or as a solution in a solvent as for example a dialkyl ketone such as acetone, a dialkylformamide such as dimethyl-formamide, and the like, under sterile conditions to a culture of the micro-organism in a nutrient medium and agitating the resulting mixture thereby bringing about growth of the microorganism and dehydrogenation of the steroid compound. The steroid can be added at the time the nutrient medium is inoculated with the culture of Schizomycetes microorganisms or, alternatively may be added to an establishe~ culture. Instead of adding the steroid compound to the established culture in the nutrient medium, the cell growth from such established culture may be filtered from the broth, washed with distilled water, then suspended in buffered aqueous solution containing the 16-methyl-ll-o xygenated-4-pregnenerl7~,21-diol~3,20-dione compound, - ~ 7 ~
and the resulting mixture agitated thPreby effecting de-hydrogenation of the steroid compound to form the corresponding 16-methyl~ oxygenated-1,4-pregnadiene-17~,21-diol-3,20-dione.
The latter is more readily recovered from this medium than from the mixture obtained when the steroid is incubated with the microorganism in the original nutrient medium. Alternatively, the 16~methyl-11-oxygenated-4-pregnene-17a,21-diol-3,20-dione compound may be contacted with dehydrogenating enzyme pre-parations fromthe growth of Schizomycetes microorganisms.
The nutrient mediums used in carrying out this bacteriological dehydrogenation are those ordinarily utilized in the propagation of Schizomycetes microorganisms. The usual nutrients include a nitrogen and carbon source, inorganic salts and growth factors when required. The carbon can be provided by compounds such as acetates, lactates, and the like. The nitrogen can be provided by an ammonium salt, amino acids, or proteins such as soy beans, oats, yeast, yeast extracts, tryptic digest of casein, meat extract, blood meal, protein meat and bone scrap, salmon meal, fish meals, fish solubles, distillers solubles, and the like. If desired, the Schizo-mycetes microorganisms can be propagated using protein (or amino acids) without any carbohydrate being present in the medium, in which case the proteins or amino acids serve as the source of both the carbon and nitrogen required by the microorganisms.
While, as noted hereinabove, the dehydrogenation of the 16-methyl-11-oxygenated-4-pregnene-17~,21-diol-3,20-dione compound may be carried out using dehydrogenating enzyme preparations from the growth of Schizomycetes microorganisms, or by contacting the steroid compound with a suspension of an established culture in distilled water, it is ordinarily pre-ferred to add the 16-methyl-11-oxygenated-4-pregnene-17~,21-1C)4;~330 diol-3,20 dione compound to a nutxient medium containing a 24-hour growth of Schizomycetes mic~ooxganisms. The pro-portion of steroid compound which may be added to the medium varies depending upon the particular substrate being de-hydrogenated, but it is ordinarily preferred to employ a concentration of about 0.005~ to 0.2~ of 16-methyl-11-oxygenated-4-pregnene-17~,21-diol-3,20-dione compound, although higher or lower concentrations may be employed, if desired.
The culture containing the added steroid compound is then incubated, preferably with agitation and aeration for an additional period which ordinarily varies between about 10 hours and 50 hours although shorter or longer fermentation times may be advantageous for the dehydrogenation of particular substrates. In view of the fact that prolonged fermentations may result in destruction of a portion of the L~l-dehydrogenated steroid product, it is ordinarily preferred to employ a fermen-tation time of about 10 hours to 24 hours which, depending upon the steroid substrate, has been found to result in maximal yields of the~ dehydrogenated steroid product.
After completion of the dehydrogenation process, the 16-methyl-11-oxygenated-1,4-pregnadiene-17~,21-diol-3,20-dione product is conveniently recovered from the fermented broth by extraction with a water-immiscible solvent as for example a chlorinated hydrocarbon such as chloroform, a ketone such as methyl isobutyl ketone, an alkyl alkanoate such as ethyl acetate, and the like. The extract of ~l-dehydrogenated steroid product and any unreacted starting material which may be present is conveniently purified by chromatography using silica gel, activated alumina, and the like or, if desired by means of paper chromatograms. After separation of the de-hydrogenated product from unreacted starting material, the product can be purified further, if desired by recrystall-1~)43330 ization from a sol~ent such as ethyl acetate, ethyl acetate-petroleum ether, and the like.
In accordance with this microbiological dehydro-genation method, and using the 16-methyl-11-oxygenated-4-pregnene-17,21-dione-3,20-dione starting materials enumerated hereinbelow, there are obtained 16-methyl-11-oxygenated-1, 4-pregnadiene-17a,21-diol-3,20-dione compounds such as 16~-methyl-1,4-pregnadiene-17~,21-diol-3,11,20-trione; 16~-methyl-1,4-pregnadiene-11~,17~,21-triol-3,20-dione; 16~-methyl-9a-fluoro-17,21-diol-3,11,20-trione and 16~-methyl-9~-fluoro-11~, 17~,21-triol-3,20-dione.
Irrespective of whether the 16-methyl-11-oxygenated-4-pregnene-17~,21-diol-3,20-dione starting material employed in this microbiological dehydrogenation reaction is a 21-free alcohol or a 21-ester thereof, the product obtained is the corresponding 16-methyl-11-oxygenated-1,4-pregnadiene-17~,21-diol-3,20-dione 21-free alcohol, since any 21-ester grouping which may be present is hydrolyzed during the microbiological dehydrogenation reaction. These 16-methyl-11-oxygenated 1,4-pregnadiene-17~,21-diol-3,20-dione 21-free alcohols can be converted to the corresponding 21-esters by reaction with an acylating agent e.g. a phosphorylating agent, a lower hydro-carbon carboxylic acid acylating agent such as benzoic anhydride, tertiary butyl acetyl chloride, a lower alkanoic anhydride or lower alkanoyl halide such as acetic anhydride, propionic anhydride, a polybasic acid anhydride such as ~
dimethyl-glutaric anhydride, succinic anhydride, and the like.
In accordance with this acylation procedure there are obtained 16~-methyl-1,4-pregnadiene-17,21-diol-3,11,20-trione 21-esters as, for example, 16-methyl-1,4-pregnadiene-17~,21-diol-3,11,20-trione 21-p~osphate; 16~-methyl-1,4-pregnadiene-17,21-diol-3,11,20-trione 21-lower hydrocarbon ~ ~ lQ ~
iU4~
carbonyl esters $uch as 16~ methyl~1,4~pregnadiene-17~,21-diol-3,11,20^trione 21 ~enzoate; 16~-methyl-1,4-pregnadiene-17a,21-diol-3,11,20-trione 21-tertiary butyl acetate; L16~-methyl-1,4-pregnadiene-17a,21-diol-3,11-20-trione 21-lower alkanoate such as 16a-methyl-1,4-pregnadiene-17a,21-diol-3,11, 20-trione 21-acetate; 16a-methyl-1,4-pregnadiene-17a,21-diol-3,11,20-trione 21-propionate; 16a-methyl-1,4-pregnadiene-11~, 17a,21-triol-3,20-dione 21-esters as, for example, 16~-methyl-1,4-pregnadiene-11~,17~,21-triol-3,20 dione 21-phosphate;
16a-methyl-1,4-pregnadiene-11~,17a,21-triol-3,20-dione 21-lower hydrocarbon carbonyl esters, such as 16a-methyl-1,4-pregnadiene-11~,17a,21-tri~1-3,20-dione 21-benzoate; 16a-methyl-1,4-pregnadiene-11~,17a,21-triol-3,20-dione 21-tertiary butyl acetate; 16a-methyl-1,4-pregnadiene-11~,17a, 21-triol-3,20-dione 21-lower alkanoates such as 16a-methyl-1,4-pregnadiene-11~,17a,21-triol-3,20-dione 21-acetate; 16a-methyl-1,4-pregnadiene-11~,17a,21-triol-3,20-dione 21-propionate;
9a-fluoro-16~-methyl-1,4-pregnadiene-17a,21-diol-3,11,20-trione 21-esters as, for example, 9a-fluoro-16a-methyl-1,4-pregnadiene-17a,21-diol-3,11,20-trione 21-phosphate; 9a-fluoro-16a-methyl-1,4-pregnadiene-17a,21-diol-3,11,20-trione 21-lower hydrocarbon carbonyl esters such as 9a-fluor~16a-methyl-1,4-pregnadiene-17a, 21-diol-3,11,20-trione 21-benzoate; 9a-fluoro-16a-methyl-1,4-pregnadiene-17a,21-diol-3,11,20-trione 21-tertiary butyl acetate;
9a-fluoro-16a-methyl-1,4-pregnadiene-17a,21-diol-3,11,20-trione 21-lower alkanoates such as 9a-fluoro-16~-methyl-1,4-pregnadiene-17a,21-diol-3,11,20-trione 21-acetate; 9a-fluoro-16a-methyl-1,4-pregnadiene-17a,21-diol-3,11,20-trione 21-propionate;
9a-fluoro-16~-methyl-1,4-pregnadiene-17a,21-diol-3,11,20-trione 21-esters as, for example, 9a-fluoro-16a-methyl-1,4-pregnadiene-11~, 17,21-triol-3,20-dione 21-phosphate; 9a-fluoro-16a-methyl-1,4-pregnadiene-11~,17a,21-triol-3,20 dione 21-lower hydrocarbon `, ~ 11 ~
~04;~330 carbonyl esters, such as 9~-fluoro-16o-methyl-1,4-pregnadiene-11~,17~,21-triol-3,20-dione 21-benzoate; 9~-fluoro-l~a-methyl-1,4-pregnadiene-11~,17~,21-triol-3,20-dione 21-tertia~y butyl acetate; 9~-fluoro-16~-methyl-1,4-pregnadiene-11~,17~,21-triol-3,2Q-dione 21-lower alkanoates such as 9~-fluoro-16~-methyl-1,4-pregnadiene-11~,17a,21-triol-3,20-dione 21-acetate;
9~-fluoro-16~-methyl-1,4-pregnadiene -11~,17~,21-triol-3,20-dione-21-propionate, and the like.
Alternatively, instead of the above-mentioned micro-biological dehydrogenation method, the 16-methyl-4-pregnene-17a-ol-3,20-dione compound or, if preferred, the corresponding saturated 16-methyl-pregnane-17~-ol-3,20-dione compound is re-acted with selenium dioxide thereby effecting ring A dehydro-genation to form the corresponding 16-methyl-1,4-pregnadiene-17a-ol-3,20-dione compound. This selenium dioxide dehydro-genation procedure is conveniently conducted by bringing the 16-methyl-4-pregnene-17-ol-3,20-dione compound, or 16-methyl-pregnane-17~-ol-3,20-dione compound, and selenium dioxide to-gether in the presence of an organic solvent such as for example dioxane, and alcohol solvent such as t-butanol, etc., and heat-ing the mixture at an elevated temperature. When t-butanol is used as the solvent, it is ordinarily preferred to carry out this reaction at the boiling point of the solvent, under which conditions the reaction is ordinarily complete in about fifteen hours. The reaction mixture is ordinarily filtered, thereby removing metallic selenium, and the filtered solution is evaporated to dryness in vacuo to give the desired 16-methyl-1,4-pregnadiene-17a-ol-3,20-dione compound. The crude material obtained in this way is conveniently purified by paper strip chromatography in accordance with the procedure outlined hereinabove in connection ~ith the purification of the 16-methyl-1,4-pregnadiene-17a-ol-3,20-dione compounds produced by micro-iO43330 biological dehydrogenation.
The following examples illustrate methods of carrying out the present invention but it is to be understood that these examples are given for purposes of illustration and not of limitation.
Fifty milliliters of a nutrient medium are prepared having the following composition:-Cerelose 1 g.
Edamin 1 g.
Cornsteep liquor a . 25 ml.
Distilled water to make 50 ml.This medium is adjusted to pH 6.5 with KOH, sterilized and inoculated with about 2.5 to 5m~. of a culture of Bacillus sphaericus (ATCC-245) microorganisms, and the inoculated culture is then incubated at a temperature of 28C., with agitation, for a 24-hour period. To the resulting culture is added a solution containing 10 mg. of 16a-methyl-4-pregnene-17R~21-diol-3,11,20-trione dissolved in 0.2 ml. of dimethyl-formamide. The culture containing the steroid compound is incubated, with agitation, for an additional period of about 24 hours at 28C.
The fermentation broth is extracted with four 50 ml.
;~..~. i 104;~330 portions of ethyl acetate, and the ethyl acetate extracts are combined and evaporated in vacuo to a volume of about 5 ml. The concentrated solution is then streaked on paper chromatograms which are developed using dimethylformamide as the stationary phase and 50% benzene-50% chloroform as the mobile phase. After 8 hours development in a descending system, the upper bands for each chromatogram, corresponding to the ~l-dehydro derivative, are cut off, extracted with methanol, and the r.tethanol extracted material is again subjected to streak-paper chromatograph. The upper band is again cut off, thoroughly dried, extracted with methanol, and the methanol extract is evaporated to dryness in vacuo.
The residual material is recrystallized from ethyl acetate-petroleum ether to give 16~-methyl-1,4-pregnadiene-17~_,21-diol-3,11,20-trione.
The 16Q- methyl-1,4-pregnadiene-17Q,21-diol-3,11,20-trione is treated with acetic anhydride and pyridine to give 21-acetyl derivative, which is purified by recrystallization from benzene-petroleum ether to give substantially pure 16~-methyl-1,4-pregnadiene-17a,21-diol-3,11,20-trione 21-acetate.
EX.WLE 2 Fifty milliliters of a nutrient medium are pre-pared having the following composition:-Cerelose 1 g.
Edamin 1 g.
Cornsteep liquor 0.25 ml.
Distilled water to make 50 ml.
This medium is adjusted to pH 6.5 ROH, sterilized and inoculated with about 2.5 to 5 ml. of a culture of Nocardia asteroides ~.-,.
~U43~330 (ATCC 9970~ microorganisms, and the inoculated culture is then incubated at room temperature of 28C., with agitation, for a 24-hour period. To the resulting culture is added a solution containing 10 mg. of 16~-methyl-4-pregnene-11~,17~,21-triol-3,20-dione dissolved in 0.2 ml. of dimethylformamide. The culture containing the steroid compound is incubated, with agitation, for an additional period of about 24 hours at 28C.
The fermentation broth is extracted with four 50 ml.-portions of ethyl acetate; and the ethyl acetate extracts are combined and evaporated in vacuo to a volume of about 5 ml. The concentrated solution is then streaked on paper chromatograms which are developed using dimethylformamide as the stationary phase and 50% benzene-50% chloroform as the mobile phase. The upper bands for each chromatogram corresponding to the ~1 dehydro derivative are cut off, extracted with methanol, and the methanol-extracted material is again subjected to streak-paper chromatography. The upper band is again cut off, thoroughly dried, extracted with methanol, and the methanol extract is evaporated to dryness in vacuo. The residual material is recrystallized from ethyl acetate-petroleum ether to give 16~-methyl-1,4-pregnadiene-11~,17~,21-triol-3,20-dione.
The 16~-methyl-1,4-pregnadiene~ ,17 Q, 21-trio1-3,20-dione is treated with acetic anhydride and pyridine, and the acetylated product recrystallized from benzene-petroleum to give substantially pure 16a-met~yl-1,4-pregnadiene~ ,17d~21-triol-3,20-dione 21-acetate.
., ;
Fifty milliliters of a nutrient medium are prepared having the following composition:
Cerelose 1 g.
Edamin 1 g.
Cornsteep liquor 0.25 ml.
Distilled water to make 50 ml.
This medium is adjusted to pH 6.5 with KOH, sterilized and inoculated with about 2.5 to 5 ml. of a culture of Mycobacterium smegmatis (NRRL B-1667~ microorganisms, and the inoculated culture is then incubated at a temperature of 28C., with agitation, for a 24-hour period. To the resulting culture is added a solution containing 10 mg. of 16o~-methyl-9~-fluoro-4-pregnene~ ,17~,21-triol-3,20-dione dissolved in 0.2 ml of dimethylformamide. The culture containing the steroid compound is incubated, with agitation, for an additional period of about 24 hours at 28C.
The fermentation broth is extracted with three 50 ml.-portions of ethyl acetate, and the ethyl acetate extracts are combined and evaporated in vacuo to a volume of about 5 ml.
The concentrated solution is then used to prepare streak-paper chromatograms which are developed utilizing dimethylformamide as the stationary liquid phase and 50% benzene-50~ chloroform as the mobile liquid phase. Two bands are secured, one of which (corresponding to the re bile component) shows the ultra-violet adsorption maximum characteristic of the 16o~-methyl-9~-fluoro-4-pregnene-11~,17~,21-triol-3,20-dione starting material and the other ~the less mobile component) shows an ultra-violet adsorption maximum at about 245 mu. The paper chromatogram is , 1043;~30 dried, and the band corresponding to the 245 mu adsorption is cut off and extracted with methanol. The material extracted with methanol is again subjected to streak-paper chromato-graphy, using paper which ~as been extracted for 48 hours with methanol, and employing the chloroform-formamide system previously employed. The resulting chromatogram shows only a trace band corresponding to the starting material with the major band having an ultra-violet adsorption maximum of 245 mu.
The paper chromatogram is thoroughly dried, and the band corresponding to the 245 mu, adsorption maximum is cut off and extracted with methanol. The methanol extract is evaporated to dryness in vacuo to give 16~-methyl-~oL-fluoro-1,4-pregnadiene -11~,17o421-triol-3,20-dione.
The 16~-methyl-9Q-fluoro-1,4-pregnadiene-1 ~,17~,21-triol-3,20-dione is reacted with excess acetic anhydride in pyridine to give the 21-acetyl derivative which is purified by recrystallization from benzene-petroleum ether to give substantially pure 16~-methyl-9~-fluoro-1,4-pregnadiene-1 ~, 17a,21-triol-3,20-dione 21-acetate.
The fermentation procedures of Examples 1, 2, and 3 are repeated but using, in place of the microorganisms and the 16Q-methyl-ll-oxygenated-4-pregnene-17~,21-diol-3,20-dione -~tarting material employed in those examples, the micro-organism strains and steroid starting compounds indicated in the table hereinbelow. The resulting $ermentation broths are treat-ed in accordance with the isolation methods described in ~xamples 1, 2, and 3 to give, for the particular microorganism strain and steroid substrate used, the 16~-methyl-11-oxy-genated-1,4-pregnadiene-17o~21-diol-3,20-dione indicated in the following table:-16-Methyl-ll-oxy-Bacillus genated-1,4-Spaericus Pregnadiene-17a, Expt. Micro- 21-Diol-3,20-No. Substrate organisms Dione Product 1 16~-Methyl-4-pregnene-17~,21-diol-3,11,20-~rione ATCC-7054 16~-Methyl-1,4-pregnadiene-17o421-diol-3,11, 20-trione
More particularly, it relates to novel 16-met~yl-11-oxygenated-1,4-pregnadiene-17a, 21-diol-3,20-dione compounds, and to the process of preparing these compounds starting with the corresponding 16-methyl-11-oxygenated-4-pregnene-17~,21-diol-3,20-dione compound. These novel l~-methyl-ll-oxygenated-1,4-pregnadiene-17a,21-diol-3,20-dione compounds possess extremely high anti-inflammatory activity, and are especially effective for the treatment of arthritis and related diseases since they can be administered for their cortisone-like action in extremely low dosage thereby minimizing undesired side effects.
These novel 16-methyl-11-oxygenated-1,4-pregnadiene-17a,21-diol-3,20-dione compounds, subject of the present invention, may be chemically represented as follows:
C=O
Z ~ ~ ---OH
~ ---CH
~ ~
~W ' wherein X i~ hydrogen or fluoro, R stands for hydrogen or an acyl radical, and Z is a keto or hydroxy substituent.
These 16-methyl-11-oxygenated-1,4-pregnadiene-l~a,21-diol-3,20-dione compounds are prepared by contacting the corresponding 16-methyl-11-oxygenated-4-pregnene-17~, 21-diol-3,20-dione compound with the dehydrogenating activity of microorganisms of the Class Schizomycetes, which includes microorganisms belonging to the orders Actinomycetales and Eubacteriales. The preferred Eubacteriales include micro-organisms of the genus Acetobacter, the genus Aerobacter, 1~43330 and the genus Bacillus; the preferred Actinomycetales include microorganiSms of the genus Nocardia and the genus Myco~acterium.
I particularly prefer to employ microorganisms of the Class Schizomycetes belonging to the following species, namely:
Acetobacter xylinum, Aerobacter aerogenes, Bacillus spaericus, Nocardia erythropolis, Nocardia blackwellii, Nocardia asteroides, Nocardia minima, Nocardia globerula, Nocardia leishmanii, Nocardia formica, Nocardia convoluta, Nocardia corallina, and Mycobacterium smegmatis, Mycobacterium phlei, Mycobacterium lacticola, and Mycobacterium tuberculois. The species Bacillus sphaericus, as defined in Bergey's Manual for Determinative Bacteriology, Sixty Edition, comprises several varieties such as the rotans variety, the fusiformis variety, - etc. and, in some collections, these varieties are referred to by the species names Bacillus rotans and Bacillus fusiformis.
These microorganisms of the Class Schizomycetes can be obtained from known sources such as the American Type Culture Collection, Washington, D.C., or they may also be isolated from natural sources, such as soil, by known methods.
It is desired to emphasize that for any given species of Schizomycetes, the preferred dehydrogenating strains can be selected by the following test method: A
nutrient medium containing 1 g. of cerelose, 1 g. of edamin, 0.25 ml. of cornsteep liquor, 0.05 g. of yeast extract, and sufficient distilled water to make 50 ml., is adjusted to pH 6.5, sterilized and inoculated with a culture of the par-ticular microorganism strain to be tested for its ~1 de-hydrogenating activity. The resulting culture is incubated for a period of 24 hours at a temperature of 2aC., and a sample of the culture is transferred to a second 50 milliter sample of the aforementioned nutrient medium which has likewise been adjusted to pH 6.5 and sterilized. The resulting inoculated B~ 2 -culture is then incubated at a temperature of 28C., with agitation, for a 24-hour per;od, and to the resulting culture is added a solution containing 10 mg. of hydrocortisone dissolved in 0.1 ml. of dimethylformamide. The culture con-taining the steroid compound is incubated, with agitation, for an additional period of about 10 hours at 28C. The fermentation broth is repeatedly extracted with ethyl acetate, and the ethyl acetate extracts are combined and evaporated to dryness in vacuo. A portion of the residual material is dissolved in acetone and spotted on a paper chromatogram which is developed using formamide as the stationary phase and chloroform as the mobile phase. Two separate bands are ordinarily obtained, the lower band corresponding to unchanged hydrocortisone; the upper band corresponds to the ~ l-dehydro derivative. Both bands are cut off, separately eluted with methanol, and each of the methanol eluants are subjected to ultraviolet absorption analysis. The efficiency of the microorganism strain being tested in effecting L~l-dehydrogenation is indicated by the relative proportions of ~l-dehydro derivative and unchanged hydrocortisone as measured by this ultraviolet absorption analysis.
The 16-methyl-11-oxygenated-4-pregnene-17a,21-diol-3,20-diones used as starting materials in this process are conveniently prepared starting with the known 16-pregnene-3a-ol-11,20-dione 3-acetate in accordance with the following procedure:- 16-pregnene-3a-ol-11,20-dione 3-acetate is reacted with methyl magnesium iodide in the presence of cuprous chloride thereby forming 16~-methyl-pregnane-3a-ol-11,20-dione 3-acetate, which is reacted with aqueous methanolic hydrochloric acid to form l6a- methyl-pregnane-3a-ol-11,20-dione. The latter compound, which is a potent anesthetic, is reacted with acetic anhydride in the presence of p-toluene sulfonic acid catalyst Bj . 1....
lV433;~0 to form a mixture of enol acetates contalning 16a-methyl-17,20-pregnene-3~,20-diol~ one 3,20-diacetate; this mixture, after chromatographic purification to remove any unchanged starting material, is reacted with perbenzoic acid and the resulting 16-methyl-17~,20-epoxy-pregnane-3a,20-diol-11-one 3,20-diacetate is hydrolyzed with methanolic potassium bicarbonate to produce 16a-methyl-pregnane-3~,17~-diol-11,20-dione. The latter compound is reacted with bromine in chloroform to form 21-bromo-16a-methyl-pregnane-3~,17~-diol-11,20-dione which is reacted with sodium iodide in acetone to produce 21-iodo-16~-methyl-pregnane-3~,17~-diol-11,20-dione which is converted without isolation to 16~- methyl-pregnane-3~,17~, 21-triol-11, 20-dione 21-acetate by reaction with anhydrous potassium acetate;
this compound is reacted with chromaum trioxide in pyridine to form 16a-methyl-pregnane-17~,21-diol-3,11,20-trione 21-acetate.
The 16~-methyl-pregnane-17,21-diol-3,11,20-trione 21-acetate is reacted with bromine in glacial acetic acid-chloroform to pro-duce 4-bromo-16~-methyl-pregnane-17a,21-diol-3,11,20-trione, which is then reacted with semicarbazide to form 16~-methyl-4-pregnene-17~,21-diol-3,11,20-trione 3,20-bis-semicarbazone 21-acetate.
This 3,20-bis-semicarbazone is reacted with sodium borohydride to form 16X-methyl-4-pregnene-1~ ,17~,21-triol 3,20-dione 3,20-bis-semicarbazone which is hydrolyzed under acid conditions to form 16x-methyl-4-pregnene-1~ ,17~,21-triol-3,20-dione; this compound is reacted with esterifying agents as for example benzoic anhydride or lower alkanoic anhydrides to form the corresponding 21-ester derivatives.
The 1~ -methyl-4-pregnene-1~ ,21-diol-3,11,20-trione-3,20-bis-semicarbazone 21-acetate is reacted with potas-sium bicarbonate or potassium hydroxide in aqueous methanol to form 1~ -methyl-4-pregnene-17~,21-diol-3,11,20-trione-3,20-bis-semicarbazone whach às then hydrolyzed under acid . ~ 4 ~
i 1t)43330 conditions to produce 16~-meth~1~4-pregnene-17~,21-diol-3,11,20-trione 21-free alcohol; this compound is esterfied using the above-mentioned esterifying agents to produce the corresponding 21-ester derivati~es.
The 16a-methyl-4-pregnene~ ,17~,21-triol-3,20-dione, upon reaction with acetic anhydride in pyridine, gives the corresponding 21-acetate which is reacted with methane sulfonyl chloride followed by potassium acetate, or phos-phorus oxychloride, to produce 16-methyl-4,9(11)-pregnadiene-17a,21-diol-3,20-dione 21-acetate; the latter compound is reacted with hypobromous acid to produce 9-bromo-16~-methyl-4-pregnene-11~,17o721-triol-3,20-dione 21-acetate which is reacted with anhydrous potassium acetate in ethanol to produce 16a-methyl 9,11-epoxy-4-pregnene-17~,21-diol-3,20-dione 21-acetate. This 9,11-epoxide is then reacted with hydrogen fluoride in tetrahydrofuran to produce 16a-methyl-9-fluoro-4-pregnene-11~, 17a,21-triol-3,20-dione 21-acetate; this compound is reacted with a hydrolyzing agent to form 16a-methyl-9-fluoro-4-pregnene-11~,17a,21-triol-3,20-dione 21-free alcohol. This 21-free alcohol is reacted with esterifying agents such as benzoic anhydride, lower alkanoic anhydrides and the like to form the corresponding 21-ester derivatives.
The 16~-methyl-9-fluoro-4-pregnene-11~,17,21-triol-3,20-dione 21-acetate is reacted with chromium trioxide in pyridine to form 16-methyl-9~-fluoro-4-pregnene-17,21-diol-3,11,20-trione 21-acetate which, upon reaction with a hydrolyzing agent, forms 16~-methyl-9a-fluoro-4-pregnene-17a, 21-triol-3,11,20-trione 21-free alcohol. This 21-free alcohol is reacted with esterifying agents such as benzoic anhydride, lower alkanoic anhydrides and the like to form the corresponding 21-ester derivatives.
Thus, these 16-methyl-11-oxygenated-4-pregnene-- 5 ~
~.. , ~, . ~
1~43330 17~,21-diol-3,2Q-dione starting materials include 16o-methyl-4-pregnene-17~,21-diol-3,11,20-trione and 21-esters t~ereof, and particularly 21-lower hydrocarbon carbonyl esters as for example 16~-methyl-4-pregnene-17O,21-diol-3,11,20-trione 21-benzoate; 16~-methyl-4-pregnene-17,21-diol-3,11,20-trione 21-lower alkanoates such as 16a-methyl-4-pregnene-17a,21-diol-3,11,20-trione 21-acetate; 16~-methyl-4-pregnene-17a, 21-diol-3,11,20-trione 21-propionate; 16a-methyl-4-pregnene-11~, 17a,21-triol-3,20-dione and 21-esters thereof, and particularly 21-lower hydrocarbon carbonyl esters as for example 16~-methyl-4-pregnene-11~,17~,21-triol-3,20-dione 21-benzoate; 16~-methyl-4-pregnene-11~,17a,21-triol-3,20-dione 21-lower alkanoates such as 16a-methyl-4-pregnene-11~,17a,21-triol-3,20-dione 21-acetate; 16~-methyl-4-pregnene-11~,17a,21-triol-3,20-dione 21- propionate; 16a-methyl-9~-fluoro-4-pregnene-17a,21-diol-3,11,20-trione and 21-esters thereof, and particularly 21-lower hydrocarbon carbonyl esters as for example 16a-methyl-9a-fluoro-4-pregnene-17a,21-diol-3,11,20-trione 21-benzoate; 16~-methyl-9a-fluoro-4-pregnene-17a,21-diol-3,11, 20-trione 21-lower alkanoates such as 16a-methyl-9a-fluoro-4-pregnene-17a-21-diol-3! 11,20-trione 21-acetate; 16a-methyl-9~-fluoro-4-pregnene-17,21-diol-3,11,20-trione 21-propionate;
16a-methyl-9~-fluoro-4-pregnene-11~,17d,21-triol-3,20-dione and 21-esters thereof, and particularly 21-lower hydrocarbon carbonyl esters thereof, as for example 16a-methyl-9a-fluoro-4-pregnene-11~,17,21-triol-3,20-dione 21-benzoate; 16a-methyl-9a-fluoro-4-pregnene-11~,17a,21-triol-3,20-dione 21-lower alkanoates such as 16a-methyl-9-fluoro-4-pregnene-11~,17a,21-triol-3,20-dione 21-acetate; 16a-methyl-9a-fluoro-4-pregnene-11~,17a, 21-triol-3,20-dione 21-propionate and the like.
In accordance with the present invention and utilizing the preferred strains of Schizomycetes microorganisms, ~'`1 1q)4;~330 the dehydrogenation of the 16-methyl~ oxygenated-4-pregnene 1~ ,21-diol-3,20-dione compound is effected by contacting the steroid compound withthe Schizomycetes microoganisms themselves of, if preferred, with enzyme systems of Schizomycetes micro-organisms whereby the hydrogen attached to the C-l and C-2 carbon atoms is selectively removed to produce the desired a -steroid substantially uncontaminated by unwanted by-products.
When the 16-methyl-11-oxygenated-4-pregnene-17~,21-diol-3,20-dione compound is subjected to the dehydrogenating activity of the preferred dehydrogenating strains of Schizomycetes microorganisms, the corresponding 16-methyl-11-oxygenated-1,4-pregnadiene-17~,21-diol-3,20-dione compound is obtained directly and in high yield.
The presently-invented microbiological dehydro-genation procedure is conducted by contacting the 16-methyl-ll-oxygenated-4-pregnene-17~,21-aiol-3,20-dione compound with the dehydrogenating activity of Schizomycetes microorganisms.
~his can be effected by adding the steroid compound as a solid, or as a solution in a solvent as for example a dialkyl ketone such as acetone, a dialkylformamide such as dimethyl-formamide, and the like, under sterile conditions to a culture of the micro-organism in a nutrient medium and agitating the resulting mixture thereby bringing about growth of the microorganism and dehydrogenation of the steroid compound. The steroid can be added at the time the nutrient medium is inoculated with the culture of Schizomycetes microorganisms or, alternatively may be added to an establishe~ culture. Instead of adding the steroid compound to the established culture in the nutrient medium, the cell growth from such established culture may be filtered from the broth, washed with distilled water, then suspended in buffered aqueous solution containing the 16-methyl-ll-o xygenated-4-pregnenerl7~,21-diol~3,20-dione compound, - ~ 7 ~
and the resulting mixture agitated thPreby effecting de-hydrogenation of the steroid compound to form the corresponding 16-methyl~ oxygenated-1,4-pregnadiene-17~,21-diol-3,20-dione.
The latter is more readily recovered from this medium than from the mixture obtained when the steroid is incubated with the microorganism in the original nutrient medium. Alternatively, the 16~methyl-11-oxygenated-4-pregnene-17a,21-diol-3,20-dione compound may be contacted with dehydrogenating enzyme pre-parations fromthe growth of Schizomycetes microorganisms.
The nutrient mediums used in carrying out this bacteriological dehydrogenation are those ordinarily utilized in the propagation of Schizomycetes microorganisms. The usual nutrients include a nitrogen and carbon source, inorganic salts and growth factors when required. The carbon can be provided by compounds such as acetates, lactates, and the like. The nitrogen can be provided by an ammonium salt, amino acids, or proteins such as soy beans, oats, yeast, yeast extracts, tryptic digest of casein, meat extract, blood meal, protein meat and bone scrap, salmon meal, fish meals, fish solubles, distillers solubles, and the like. If desired, the Schizo-mycetes microorganisms can be propagated using protein (or amino acids) without any carbohydrate being present in the medium, in which case the proteins or amino acids serve as the source of both the carbon and nitrogen required by the microorganisms.
While, as noted hereinabove, the dehydrogenation of the 16-methyl-11-oxygenated-4-pregnene-17~,21-diol-3,20-dione compound may be carried out using dehydrogenating enzyme preparations from the growth of Schizomycetes microorganisms, or by contacting the steroid compound with a suspension of an established culture in distilled water, it is ordinarily pre-ferred to add the 16-methyl-11-oxygenated-4-pregnene-17~,21-1C)4;~330 diol-3,20 dione compound to a nutxient medium containing a 24-hour growth of Schizomycetes mic~ooxganisms. The pro-portion of steroid compound which may be added to the medium varies depending upon the particular substrate being de-hydrogenated, but it is ordinarily preferred to employ a concentration of about 0.005~ to 0.2~ of 16-methyl-11-oxygenated-4-pregnene-17~,21-diol-3,20-dione compound, although higher or lower concentrations may be employed, if desired.
The culture containing the added steroid compound is then incubated, preferably with agitation and aeration for an additional period which ordinarily varies between about 10 hours and 50 hours although shorter or longer fermentation times may be advantageous for the dehydrogenation of particular substrates. In view of the fact that prolonged fermentations may result in destruction of a portion of the L~l-dehydrogenated steroid product, it is ordinarily preferred to employ a fermen-tation time of about 10 hours to 24 hours which, depending upon the steroid substrate, has been found to result in maximal yields of the~ dehydrogenated steroid product.
After completion of the dehydrogenation process, the 16-methyl-11-oxygenated-1,4-pregnadiene-17~,21-diol-3,20-dione product is conveniently recovered from the fermented broth by extraction with a water-immiscible solvent as for example a chlorinated hydrocarbon such as chloroform, a ketone such as methyl isobutyl ketone, an alkyl alkanoate such as ethyl acetate, and the like. The extract of ~l-dehydrogenated steroid product and any unreacted starting material which may be present is conveniently purified by chromatography using silica gel, activated alumina, and the like or, if desired by means of paper chromatograms. After separation of the de-hydrogenated product from unreacted starting material, the product can be purified further, if desired by recrystall-1~)43330 ization from a sol~ent such as ethyl acetate, ethyl acetate-petroleum ether, and the like.
In accordance with this microbiological dehydro-genation method, and using the 16-methyl-11-oxygenated-4-pregnene-17,21-dione-3,20-dione starting materials enumerated hereinbelow, there are obtained 16-methyl-11-oxygenated-1, 4-pregnadiene-17a,21-diol-3,20-dione compounds such as 16~-methyl-1,4-pregnadiene-17~,21-diol-3,11,20-trione; 16~-methyl-1,4-pregnadiene-11~,17~,21-triol-3,20-dione; 16~-methyl-9a-fluoro-17,21-diol-3,11,20-trione and 16~-methyl-9~-fluoro-11~, 17~,21-triol-3,20-dione.
Irrespective of whether the 16-methyl-11-oxygenated-4-pregnene-17~,21-diol-3,20-dione starting material employed in this microbiological dehydrogenation reaction is a 21-free alcohol or a 21-ester thereof, the product obtained is the corresponding 16-methyl-11-oxygenated-1,4-pregnadiene-17~,21-diol-3,20-dione 21-free alcohol, since any 21-ester grouping which may be present is hydrolyzed during the microbiological dehydrogenation reaction. These 16-methyl-11-oxygenated 1,4-pregnadiene-17~,21-diol-3,20-dione 21-free alcohols can be converted to the corresponding 21-esters by reaction with an acylating agent e.g. a phosphorylating agent, a lower hydro-carbon carboxylic acid acylating agent such as benzoic anhydride, tertiary butyl acetyl chloride, a lower alkanoic anhydride or lower alkanoyl halide such as acetic anhydride, propionic anhydride, a polybasic acid anhydride such as ~
dimethyl-glutaric anhydride, succinic anhydride, and the like.
In accordance with this acylation procedure there are obtained 16~-methyl-1,4-pregnadiene-17,21-diol-3,11,20-trione 21-esters as, for example, 16-methyl-1,4-pregnadiene-17~,21-diol-3,11,20-trione 21-p~osphate; 16~-methyl-1,4-pregnadiene-17,21-diol-3,11,20-trione 21-lower hydrocarbon ~ ~ lQ ~
iU4~
carbonyl esters $uch as 16~ methyl~1,4~pregnadiene-17~,21-diol-3,11,20^trione 21 ~enzoate; 16~-methyl-1,4-pregnadiene-17a,21-diol-3,11,20-trione 21-tertiary butyl acetate; L16~-methyl-1,4-pregnadiene-17a,21-diol-3,11-20-trione 21-lower alkanoate such as 16a-methyl-1,4-pregnadiene-17a,21-diol-3,11, 20-trione 21-acetate; 16a-methyl-1,4-pregnadiene-17a,21-diol-3,11,20-trione 21-propionate; 16a-methyl-1,4-pregnadiene-11~, 17a,21-triol-3,20-dione 21-esters as, for example, 16~-methyl-1,4-pregnadiene-11~,17~,21-triol-3,20 dione 21-phosphate;
16a-methyl-1,4-pregnadiene-11~,17a,21-triol-3,20-dione 21-lower hydrocarbon carbonyl esters, such as 16a-methyl-1,4-pregnadiene-11~,17a,21-tri~1-3,20-dione 21-benzoate; 16a-methyl-1,4-pregnadiene-11~,17a,21-triol-3,20-dione 21-tertiary butyl acetate; 16a-methyl-1,4-pregnadiene-11~,17a, 21-triol-3,20-dione 21-lower alkanoates such as 16a-methyl-1,4-pregnadiene-11~,17a,21-triol-3,20-dione 21-acetate; 16a-methyl-1,4-pregnadiene-11~,17a,21-triol-3,20-dione 21-propionate;
9a-fluoro-16~-methyl-1,4-pregnadiene-17a,21-diol-3,11,20-trione 21-esters as, for example, 9a-fluoro-16a-methyl-1,4-pregnadiene-17a,21-diol-3,11,20-trione 21-phosphate; 9a-fluoro-16a-methyl-1,4-pregnadiene-17a,21-diol-3,11,20-trione 21-lower hydrocarbon carbonyl esters such as 9a-fluor~16a-methyl-1,4-pregnadiene-17a, 21-diol-3,11,20-trione 21-benzoate; 9a-fluoro-16a-methyl-1,4-pregnadiene-17a,21-diol-3,11,20-trione 21-tertiary butyl acetate;
9a-fluoro-16a-methyl-1,4-pregnadiene-17a,21-diol-3,11,20-trione 21-lower alkanoates such as 9a-fluoro-16~-methyl-1,4-pregnadiene-17a,21-diol-3,11,20-trione 21-acetate; 9a-fluoro-16a-methyl-1,4-pregnadiene-17a,21-diol-3,11,20-trione 21-propionate;
9a-fluoro-16~-methyl-1,4-pregnadiene-17a,21-diol-3,11,20-trione 21-esters as, for example, 9a-fluoro-16a-methyl-1,4-pregnadiene-11~, 17,21-triol-3,20-dione 21-phosphate; 9a-fluoro-16a-methyl-1,4-pregnadiene-11~,17a,21-triol-3,20 dione 21-lower hydrocarbon `, ~ 11 ~
~04;~330 carbonyl esters, such as 9~-fluoro-16o-methyl-1,4-pregnadiene-11~,17~,21-triol-3,20-dione 21-benzoate; 9~-fluoro-l~a-methyl-1,4-pregnadiene-11~,17~,21-triol-3,20-dione 21-tertia~y butyl acetate; 9~-fluoro-16~-methyl-1,4-pregnadiene-11~,17~,21-triol-3,2Q-dione 21-lower alkanoates such as 9~-fluoro-16~-methyl-1,4-pregnadiene-11~,17a,21-triol-3,20-dione 21-acetate;
9~-fluoro-16~-methyl-1,4-pregnadiene -11~,17~,21-triol-3,20-dione-21-propionate, and the like.
Alternatively, instead of the above-mentioned micro-biological dehydrogenation method, the 16-methyl-4-pregnene-17a-ol-3,20-dione compound or, if preferred, the corresponding saturated 16-methyl-pregnane-17~-ol-3,20-dione compound is re-acted with selenium dioxide thereby effecting ring A dehydro-genation to form the corresponding 16-methyl-1,4-pregnadiene-17a-ol-3,20-dione compound. This selenium dioxide dehydro-genation procedure is conveniently conducted by bringing the 16-methyl-4-pregnene-17-ol-3,20-dione compound, or 16-methyl-pregnane-17~-ol-3,20-dione compound, and selenium dioxide to-gether in the presence of an organic solvent such as for example dioxane, and alcohol solvent such as t-butanol, etc., and heat-ing the mixture at an elevated temperature. When t-butanol is used as the solvent, it is ordinarily preferred to carry out this reaction at the boiling point of the solvent, under which conditions the reaction is ordinarily complete in about fifteen hours. The reaction mixture is ordinarily filtered, thereby removing metallic selenium, and the filtered solution is evaporated to dryness in vacuo to give the desired 16-methyl-1,4-pregnadiene-17a-ol-3,20-dione compound. The crude material obtained in this way is conveniently purified by paper strip chromatography in accordance with the procedure outlined hereinabove in connection ~ith the purification of the 16-methyl-1,4-pregnadiene-17a-ol-3,20-dione compounds produced by micro-iO43330 biological dehydrogenation.
The following examples illustrate methods of carrying out the present invention but it is to be understood that these examples are given for purposes of illustration and not of limitation.
Fifty milliliters of a nutrient medium are prepared having the following composition:-Cerelose 1 g.
Edamin 1 g.
Cornsteep liquor a . 25 ml.
Distilled water to make 50 ml.This medium is adjusted to pH 6.5 with KOH, sterilized and inoculated with about 2.5 to 5m~. of a culture of Bacillus sphaericus (ATCC-245) microorganisms, and the inoculated culture is then incubated at a temperature of 28C., with agitation, for a 24-hour period. To the resulting culture is added a solution containing 10 mg. of 16a-methyl-4-pregnene-17R~21-diol-3,11,20-trione dissolved in 0.2 ml. of dimethyl-formamide. The culture containing the steroid compound is incubated, with agitation, for an additional period of about 24 hours at 28C.
The fermentation broth is extracted with four 50 ml.
;~..~. i 104;~330 portions of ethyl acetate, and the ethyl acetate extracts are combined and evaporated in vacuo to a volume of about 5 ml. The concentrated solution is then streaked on paper chromatograms which are developed using dimethylformamide as the stationary phase and 50% benzene-50% chloroform as the mobile phase. After 8 hours development in a descending system, the upper bands for each chromatogram, corresponding to the ~l-dehydro derivative, are cut off, extracted with methanol, and the r.tethanol extracted material is again subjected to streak-paper chromatograph. The upper band is again cut off, thoroughly dried, extracted with methanol, and the methanol extract is evaporated to dryness in vacuo.
The residual material is recrystallized from ethyl acetate-petroleum ether to give 16~-methyl-1,4-pregnadiene-17~_,21-diol-3,11,20-trione.
The 16Q- methyl-1,4-pregnadiene-17Q,21-diol-3,11,20-trione is treated with acetic anhydride and pyridine to give 21-acetyl derivative, which is purified by recrystallization from benzene-petroleum ether to give substantially pure 16~-methyl-1,4-pregnadiene-17a,21-diol-3,11,20-trione 21-acetate.
EX.WLE 2 Fifty milliliters of a nutrient medium are pre-pared having the following composition:-Cerelose 1 g.
Edamin 1 g.
Cornsteep liquor 0.25 ml.
Distilled water to make 50 ml.
This medium is adjusted to pH 6.5 ROH, sterilized and inoculated with about 2.5 to 5 ml. of a culture of Nocardia asteroides ~.-,.
~U43~330 (ATCC 9970~ microorganisms, and the inoculated culture is then incubated at room temperature of 28C., with agitation, for a 24-hour period. To the resulting culture is added a solution containing 10 mg. of 16~-methyl-4-pregnene-11~,17~,21-triol-3,20-dione dissolved in 0.2 ml. of dimethylformamide. The culture containing the steroid compound is incubated, with agitation, for an additional period of about 24 hours at 28C.
The fermentation broth is extracted with four 50 ml.-portions of ethyl acetate; and the ethyl acetate extracts are combined and evaporated in vacuo to a volume of about 5 ml. The concentrated solution is then streaked on paper chromatograms which are developed using dimethylformamide as the stationary phase and 50% benzene-50% chloroform as the mobile phase. The upper bands for each chromatogram corresponding to the ~1 dehydro derivative are cut off, extracted with methanol, and the methanol-extracted material is again subjected to streak-paper chromatography. The upper band is again cut off, thoroughly dried, extracted with methanol, and the methanol extract is evaporated to dryness in vacuo. The residual material is recrystallized from ethyl acetate-petroleum ether to give 16~-methyl-1,4-pregnadiene-11~,17~,21-triol-3,20-dione.
The 16~-methyl-1,4-pregnadiene~ ,17 Q, 21-trio1-3,20-dione is treated with acetic anhydride and pyridine, and the acetylated product recrystallized from benzene-petroleum to give substantially pure 16a-met~yl-1,4-pregnadiene~ ,17d~21-triol-3,20-dione 21-acetate.
., ;
Fifty milliliters of a nutrient medium are prepared having the following composition:
Cerelose 1 g.
Edamin 1 g.
Cornsteep liquor 0.25 ml.
Distilled water to make 50 ml.
This medium is adjusted to pH 6.5 with KOH, sterilized and inoculated with about 2.5 to 5 ml. of a culture of Mycobacterium smegmatis (NRRL B-1667~ microorganisms, and the inoculated culture is then incubated at a temperature of 28C., with agitation, for a 24-hour period. To the resulting culture is added a solution containing 10 mg. of 16o~-methyl-9~-fluoro-4-pregnene~ ,17~,21-triol-3,20-dione dissolved in 0.2 ml of dimethylformamide. The culture containing the steroid compound is incubated, with agitation, for an additional period of about 24 hours at 28C.
The fermentation broth is extracted with three 50 ml.-portions of ethyl acetate, and the ethyl acetate extracts are combined and evaporated in vacuo to a volume of about 5 ml.
The concentrated solution is then used to prepare streak-paper chromatograms which are developed utilizing dimethylformamide as the stationary liquid phase and 50% benzene-50~ chloroform as the mobile liquid phase. Two bands are secured, one of which (corresponding to the re bile component) shows the ultra-violet adsorption maximum characteristic of the 16o~-methyl-9~-fluoro-4-pregnene-11~,17~,21-triol-3,20-dione starting material and the other ~the less mobile component) shows an ultra-violet adsorption maximum at about 245 mu. The paper chromatogram is , 1043;~30 dried, and the band corresponding to the 245 mu adsorption is cut off and extracted with methanol. The material extracted with methanol is again subjected to streak-paper chromato-graphy, using paper which ~as been extracted for 48 hours with methanol, and employing the chloroform-formamide system previously employed. The resulting chromatogram shows only a trace band corresponding to the starting material with the major band having an ultra-violet adsorption maximum of 245 mu.
The paper chromatogram is thoroughly dried, and the band corresponding to the 245 mu, adsorption maximum is cut off and extracted with methanol. The methanol extract is evaporated to dryness in vacuo to give 16~-methyl-~oL-fluoro-1,4-pregnadiene -11~,17o421-triol-3,20-dione.
The 16~-methyl-9Q-fluoro-1,4-pregnadiene-1 ~,17~,21-triol-3,20-dione is reacted with excess acetic anhydride in pyridine to give the 21-acetyl derivative which is purified by recrystallization from benzene-petroleum ether to give substantially pure 16~-methyl-9~-fluoro-1,4-pregnadiene-1 ~, 17a,21-triol-3,20-dione 21-acetate.
The fermentation procedures of Examples 1, 2, and 3 are repeated but using, in place of the microorganisms and the 16Q-methyl-ll-oxygenated-4-pregnene-17~,21-diol-3,20-dione -~tarting material employed in those examples, the micro-organism strains and steroid starting compounds indicated in the table hereinbelow. The resulting $ermentation broths are treat-ed in accordance with the isolation methods described in ~xamples 1, 2, and 3 to give, for the particular microorganism strain and steroid substrate used, the 16~-methyl-11-oxy-genated-1,4-pregnadiene-17o~21-diol-3,20-dione indicated in the following table:-16-Methyl-ll-oxy-Bacillus genated-1,4-Spaericus Pregnadiene-17a, Expt. Micro- 21-Diol-3,20-No. Substrate organisms Dione Product 1 16~-Methyl-4-pregnene-17~,21-diol-3,11,20-~rione ATCC-7054 16~-Methyl-1,4-pregnadiene-17o421-diol-3,11, 20-trione
2 16a-Methyl-4-pregnene- ATCC-245 16~-Methyl-1,4-1~,17a,21-triol-3,20- pregnadiene - 11~, dione 17~,21-triol-
3,20-dione 3 16~-Methyl-4-pregnene- ATCC-4525 16~-Methyl-1,4-1 ~,17~,21-triol-3,20- pregnadiene-dione 21-acetate 11~17R~21-triol-3,20-dione
4 16~-Methyl-4-pregnene- ATCC-7055 16Q-Methyl-1,4-17Q,21-diol-3,11,20- pregnadiene-17~, trione 21-diol-3,11,20-trione 16~-Methyl-9~-fluoro- ATCC-7055 16~-Methyl-9~-4-pregnene-17Q,21-diol- fluoro-1,4-3,11,20-trione 21-acetate pregnadiene-17Q,21-diol-3,11,20-trione 2a 6 16~-Methyl-*~-fluoro- ATCC-245 16~-Methyl-~-4-pregnene-11~,17o421- fluoro-1,4-triol-3,20-dione pregnadiene-lL~, 17~,21-triol-3,20-dione 7 16Q-Methyl-9~-fluoro ATCC-7063 16~-Methyl-~-4-pregnene-11~,17~,21- fluoro-1,4-triol-3,20-dione 21- pregnadiene-acetate 1 ~,17~,21-triol-3,20-dione Nocardia Micro-organisms 8 16~-Methyl-4-pregnene- N. blackwellii 16c~-Methyl-1,4-11~,17,-~21-tr;o1-3,20- ATCC-6846 pregnadiene-dione lL~,17~,21-triol-3,20-dione 9 16~-Methyl-4-pregnene- N. globerula 16~=Methyl-1,4-17~,21-diol-3,11,20- ATCC-9356 pregnadiene-dione 17~,21-diol-3,11,20-trione 16~-Methyl-9~-fluoro- N. leishmanii 16Q-Methyl-4-pregnene-l7o~2l-diol- 9~-fluoro-3,11,20-trione 1,4-pregna-diene-17o421-diol-3,11,20-trione 11 1~-Methyl-9c~-fluoro- N. formica 16oL-Methyl-9o-4-pregnene-1~,17cL,21- NRRL-2470 fluoro-1,4-triol-3,20-dione pregnadiene-1~,17o~21-triol-3,20-dione 12 16~-Methyl-4-pregnene- M. phlei 16~-Methyl-11~,17R~21-triol-3,20- ATCC-12,298 1,4-pregnadiene dione 11~,17~,21-triol-3,20-dione 13 16~-Methyl-4-pregnene- M. lacticola 16O-Methyl-17c421-diol-3,11,20- ATCC-12,297 1,4-pregna-trione diene-17o~21-diol-3,11,20-trione 14 l~-Methyl-9~-fluoro- M. tuber- 16o-Methyl-9o-4-pregnene-17Q,21- culosis fluoro-1,4-diol-3,11,20-trione ATCC-12,296 pregnadiene-17o721-diol-3,11,20-trione EXAMPLE S
To a solution of 110 mg. of 16Q-methyl-4-pregnene-17~-21-diol 3,11,20-trione 21-acetate in 6 ml. t-butanol, 0.01 ml. glacial acetic acid and 0.03 ml. of acetic anhydride is added 70 mg.
of selenious acid (H2SeO3~. The mixture is heated to the boiling point overnight, another 50 mg. of selenious acid is added, and the heating is continued for an additional 24 hours.
The solution is decanted from metallic selenium and evaporated to an oil which is then dissolved in ethyl acetate. The ~' 1~43330 ethyl acetate solution is washed with aqueous sodium bicarbonate and then with water until neutral, and dried. The solvent is evaporated from the dried solution to give an oil which is dissolved in benzene and adsorbed from this solvent on acid-washed alumina. The adsorbate is eluted with ether-petroleum ether and then with mixtures of ether and chloroform, increasingly rich in chloroform. The 4:6 ether-chloroform eluates are combined, evaporated to dryness, and the residual material recrystallized from ethyl acetate-ether to give 16~L-methyl-1~4-pregnadiene-l7o~2l-diol-3~ 2o-trione 21-acetate;
M.P. 208-212C.
To a solution of 100 mg. of 16a~methyl-4-pregnene-11~,17~421-triol-3,20-dione 21-acetate in 6 ml. t-butanol, 0.01 ml. glacial acetic acid and 0.03 ml. of acetic anhydride is added 70 mg. of selenious acid. The mixture is heated to the boiling point overnight, another 50 mg. of selenious acid is added, and the heating is continued for an additional 24 hours. The solution is decanted from metallic selenium and evaporated to an oil which is then dissolved in ethyl acetate. The ethyl acetate solution is washed with aqueous sodium bicarbonate and then with water until neutral, and dried. The solvent is evaporated`from the dried solution to give an oil which is dissolved in benzene and adsorbed from this solvent on acid-washed alumina. The adsorbate is eluted with ether-petroleum ether and then with mixtures of ether and chloroform, increasingly rich in chloroform. The 4:6 ether-chloroform eluates are combined, evaporated to dryness, and the residual material recrystallized from ethyl acetate-ether to give 1~-methyl-1,4-pregnadiene-11~,17~,21-triol-3,20-dione 21-acetate.
, - 20 -, 1~43330 In accordance with the selenious acid dehydrogenation procedure of Example 6, but using 9~-fluoro-16~-methyl-4-pregnene-17~,21-diol-3,11,20-trione 21-acetate as the starting material in place of the 16~-methyl-4-pregnene-17a,21-diol 3,11,20-trione 21-acetate utilized in Example 6, there is obtained ~-fluoro-l~-methyl-1,4-pregnadiene-17~,21-diol-3,11,20-trione 21-acetate.
In accordance with the selenious acid dehydrogenation procedure of Example 6, but using 9o=fluoro-1 ~-methyl-4-pregnene-11~,17~,20-triol-3,20-dione 21-acetate as the starting material in place of the 16o-methyl-4-pregnene-17~,21-diol-3,11,20-trione 21-acetate utilized in Example 6, there is obtained 9o-fluoro-16o-methyl-1,4-pregnadiene-11~ ,21-triol-3,20-dione 21-acetate.
EXAMoeLE 9 To a solution of 110 mg. of 16~methyl-pregnane-17o,21-diol-3,11,20-trione 21-acetate in 6 ml. t-butanol, 0.01 ml. glacial acetic acid and 0.03 ml. of acetic anhydride is added 70 mg. of selenious acid. The mixture is heated to the boiling point overnight, another 50 mg. of selenious acid is added, and the heating is continued for an additional 24 hours. The solution is decanted from metallic selenium and evaporated to an oil which is then dissolved in ethyl acetate.
The ethyl acetate solution is washed with aqueous sodium bicarbonate and then with water uhtil neutral, and dried.
The solvent is evaporated from the dried solution to give an oil which is dissolved in benzene and adsorbed from this solvent on acid-washed alumina. The adsorbate is eluted with ether-petroleum ether and then with mixtures of ether and chloroform, increasingly rich in chloroform. The 4:6 ether-chloroform eluates are combined, evaporated to dryness, and the residual material recrystallized from ethyl acetate-ether to give l~-methyl-1,4-pregnadiene-17c421-dio1-3,11,20-trione 21-acetate.
The 16Q-methyl-pregnane-17o421-diol-3,11i2G-trione 21-acetate and 16-methyl-11-ox~genated-4-pregnane, 17~-diol-3,20-dione compounds used as starting materials in Examples 1 to 9 herein above are prepared, starting with the known 16-pregnene-3o=ol-11,20-dione 3-acetate, in accordance wi~h the following procedure:-A solution of 10.22 g. of methyl iodide in50 ml. of ether is added to 1.73 g. of magnesium in 50 ml.
of ether. To the resulting ethereal solution of methyl magnesium iodide, maintained under a nitrogen atmosphere, is added 0.045 g. of anhydrous cu~rous chloride. To this mixture is added, over a period of about one hour, during which period the reaction mixture is stirred vigorously and main-tained at approximately room temperature, a solution of about
To a solution of 110 mg. of 16Q-methyl-4-pregnene-17~-21-diol 3,11,20-trione 21-acetate in 6 ml. t-butanol, 0.01 ml. glacial acetic acid and 0.03 ml. of acetic anhydride is added 70 mg.
of selenious acid (H2SeO3~. The mixture is heated to the boiling point overnight, another 50 mg. of selenious acid is added, and the heating is continued for an additional 24 hours.
The solution is decanted from metallic selenium and evaporated to an oil which is then dissolved in ethyl acetate. The ~' 1~43330 ethyl acetate solution is washed with aqueous sodium bicarbonate and then with water until neutral, and dried. The solvent is evaporated from the dried solution to give an oil which is dissolved in benzene and adsorbed from this solvent on acid-washed alumina. The adsorbate is eluted with ether-petroleum ether and then with mixtures of ether and chloroform, increasingly rich in chloroform. The 4:6 ether-chloroform eluates are combined, evaporated to dryness, and the residual material recrystallized from ethyl acetate-ether to give 16~L-methyl-1~4-pregnadiene-l7o~2l-diol-3~ 2o-trione 21-acetate;
M.P. 208-212C.
To a solution of 100 mg. of 16a~methyl-4-pregnene-11~,17~421-triol-3,20-dione 21-acetate in 6 ml. t-butanol, 0.01 ml. glacial acetic acid and 0.03 ml. of acetic anhydride is added 70 mg. of selenious acid. The mixture is heated to the boiling point overnight, another 50 mg. of selenious acid is added, and the heating is continued for an additional 24 hours. The solution is decanted from metallic selenium and evaporated to an oil which is then dissolved in ethyl acetate. The ethyl acetate solution is washed with aqueous sodium bicarbonate and then with water until neutral, and dried. The solvent is evaporated`from the dried solution to give an oil which is dissolved in benzene and adsorbed from this solvent on acid-washed alumina. The adsorbate is eluted with ether-petroleum ether and then with mixtures of ether and chloroform, increasingly rich in chloroform. The 4:6 ether-chloroform eluates are combined, evaporated to dryness, and the residual material recrystallized from ethyl acetate-ether to give 1~-methyl-1,4-pregnadiene-11~,17~,21-triol-3,20-dione 21-acetate.
, - 20 -, 1~43330 In accordance with the selenious acid dehydrogenation procedure of Example 6, but using 9~-fluoro-16~-methyl-4-pregnene-17~,21-diol-3,11,20-trione 21-acetate as the starting material in place of the 16~-methyl-4-pregnene-17a,21-diol 3,11,20-trione 21-acetate utilized in Example 6, there is obtained ~-fluoro-l~-methyl-1,4-pregnadiene-17~,21-diol-3,11,20-trione 21-acetate.
In accordance with the selenious acid dehydrogenation procedure of Example 6, but using 9o=fluoro-1 ~-methyl-4-pregnene-11~,17~,20-triol-3,20-dione 21-acetate as the starting material in place of the 16o-methyl-4-pregnene-17~,21-diol-3,11,20-trione 21-acetate utilized in Example 6, there is obtained 9o-fluoro-16o-methyl-1,4-pregnadiene-11~ ,21-triol-3,20-dione 21-acetate.
EXAMoeLE 9 To a solution of 110 mg. of 16~methyl-pregnane-17o,21-diol-3,11,20-trione 21-acetate in 6 ml. t-butanol, 0.01 ml. glacial acetic acid and 0.03 ml. of acetic anhydride is added 70 mg. of selenious acid. The mixture is heated to the boiling point overnight, another 50 mg. of selenious acid is added, and the heating is continued for an additional 24 hours. The solution is decanted from metallic selenium and evaporated to an oil which is then dissolved in ethyl acetate.
The ethyl acetate solution is washed with aqueous sodium bicarbonate and then with water uhtil neutral, and dried.
The solvent is evaporated from the dried solution to give an oil which is dissolved in benzene and adsorbed from this solvent on acid-washed alumina. The adsorbate is eluted with ether-petroleum ether and then with mixtures of ether and chloroform, increasingly rich in chloroform. The 4:6 ether-chloroform eluates are combined, evaporated to dryness, and the residual material recrystallized from ethyl acetate-ether to give l~-methyl-1,4-pregnadiene-17c421-dio1-3,11,20-trione 21-acetate.
The 16Q-methyl-pregnane-17o421-diol-3,11i2G-trione 21-acetate and 16-methyl-11-ox~genated-4-pregnane, 17~-diol-3,20-dione compounds used as starting materials in Examples 1 to 9 herein above are prepared, starting with the known 16-pregnene-3o=ol-11,20-dione 3-acetate, in accordance wi~h the following procedure:-A solution of 10.22 g. of methyl iodide in50 ml. of ether is added to 1.73 g. of magnesium in 50 ml.
of ether. To the resulting ethereal solution of methyl magnesium iodide, maintained under a nitrogen atmosphere, is added 0.045 g. of anhydrous cu~rous chloride. To this mixture is added, over a period of about one hour, during which period the reaction mixture is stirred vigorously and main-tained at approximately room temperature, a solution of about
5.6 g. of 16-pregnene-3o~-ol-11,20-dione 3-acetate in 175 ml.
of ether. A white granular solid separates during this addition.
The resulting mixture is heated under gentle reflux for two hours after which the reaction mixture is cooled, and 125 ml.
of saturated, aqueou~ ammonium chloride solution is added follow-ed by 200 ml. of ether. The layers are separated, and the ethereal layer is washed with three 50 ml. portions of water.
The washed ethereal layer is dried, and the solvent evaporated in vacuo to give a brown viscous oil. The latter material is heated for lS minutes at 60-70C. with a mixture of 25 ml.
acetic anhydride and 25 ml. pyridine and the acetylated product is purified by chromatography on acid-washed alumina followed by crystallization from petroleum ether to give approximately ., 1043;~30 1.5 g. of substantially pure 1~-methyl-pregnene-3R-ol-ll, 20-dione 3-acetate.
To a solution of 0.8 g. of 16Q-methyl-pregnane-3G-ol-11,20-dione 3-acetate in 40 ml. of methanol is added 1.5 ml. of concentrated aqueous hydrochloric acid and the resulting solution is stirred overnight at about 25C. The reaction solution is evaporated in vacuo at 25C to a small volume, and the concentrated solution is poured into 50 ml.
of ice water. The white solid which precipitates is re-covered by filtration, washed wlth ~ater and recrystallizedfrom ethyl acetate to give 16~-methyl-pregnane-3~-ol-11,20-dione.
A solution of 22 g. of 16o-methyl-pregnane-3o-ol-11,20-dione 21-acetate and 1 g. of ~-toluene-sulfonic acid in 250 ml. of acetic anhydride is heated at reflux under nitrogen for a period of approximately 3 days. Two grams of potassium acetate tanhydrous~ is added, and the volatile solvents are separated by distillation in vacuo. The residual material is extracted ~ith benzene, and the benzene extract is filtered to remove insoluble material. The benzene extracts are~evaporated to a volume of 100 ml. and petroleum ether is added to the cloud point. The resulting solution is adsorbed on 660 g. of acid-washed alumina; the alumina adsorbate is then washed with 2 liters of petroleum ether. The adsorbate is then eluted with 85:15 petroleum-ether-ether mixture, and the first four liters of eluate is collected, and evaporated to dryness in vacuo to give a mixture of enol acetates-containing 16Q-methyl-17C20~-pregnene-3Q,20-diol-ll-one 3,20-diacetate. This mixture of enolates, weighing approximately 14 g., is dissolved in 50 ml. of benzene and treated with an excess of per-benzoic acid over a 16-hour period. The reaction mixture is shaken with dilute aqueous ~ j - 23 -potassium hydroxide solution until the benzene layer is free of perbenzoic acid; the benzene layer is then washed with water until neutral, dried, and the solvent evaporated in vacuo to give a crystalline material, 16~-methyl-17a,20-epoxy-pregnane-3a,20-diol-11-one 3,20-diacetate. The latter material is dissolved, without purification, in 200 ml. of methanol, 120 ml.
of water and 10 g. of potassium bicarbonate, and the resulting solution is heated at reflux under nitrogen for a period of 16 hours. The methanol is evaporated from the hydrolysis solution in vacuo, and the residual oil is extracted from the resulting aqueous solution with chloroform. The chloroform extract is washed with water to neutrality, dried, and the chloroform is evaporated under reduced pressure. The residual oil is triturated with ether, and the crystalline material thus formed is recrystal-lized from ethyl acetate-petroleum ether to give 16a-methyl-pregnane-3,17-diol-11,20 dione.
To a solution of 7.0 g. of 16a-methyl-pregnane-3,17-diol-11,20-dione in 50 ml. of chloroform is added dropwise with stirring a solution containing 3.36 g. of bromine in 24.2 ml.
of chloroform over a period of about 60 minutes. The reaction mixture is dissolved in 200 ml. of ethyl acetate, and the resulting solution washed with water until neutral, dried, and the solvents evaporated therefrom in vacuo. The residual crude material is dissolved in a minimum quantity of ethyl acetate, the resulting solution is diluted with ether, and the mixture is stirred until crystals form. The crystalline product is recovered by filtration and washed, by slurrying, with 50:50 ether-petroleum ether mixture to give about 5 g.
of 21-bromo-16~-methyl-pregnane-3a,17-diol-11,20-dione.
This 5 g. of 21-bromo-16a-methyl-pregnane-3,17a-diol-11,20-dione is mixed with S.0 g. of anhydrous potassium acetate, 4.0 g. of sodium iodide and 0.03 ml. of glacial acetic acid, and 100 ml. of acetone is added to the resulting mixture.
........ ~7 1~43330 this mixture is then heated at reflux, with stirringl for a period of about 16 hours, and the reaction mixture is cooled, filtered, and the insoluble material is washed with acetone. The filtered solution is evaporated in vacuo thereby removing the solvents, and the residual material is slurried with water, and the aqueous mixture extracted with ethyl acetate. The ethyl acetate extract is washed with water to neutrality, dried, and the solvent is evaporated in vacuo to give an oil. This oil is crystallized from ether, and recrystallized from ethyl acetate-ether to give 16~-methyl-pregnane-3~,17~,21-triol-11,20-dione 21-acetate.
A solution of 400 mg. of 16~-methyl-pregnane-3~-17~, 21-triol-11,20-dione 21-acetate in 4 ml. of pyridine is added to the complex formed by the addition of 400 mg. of chromium trioxide to 4 ml. of pyridine. The mixture is swirled until thoroughly mixed, and then allowed to stand at room temperature overnight. The reaction mixture is poured into water, and the aqueous mixture is extracted with ether, and then twice with ethyl acetate. The combined ether and ethyl acetate extracts are washed with dilute aqueous sulfuric acid at about 0C, and then with water until neutral. The organic solvent layer is then dried, the solvents are evaporated therefrom in vacuo, and the residual crystalline material is purified by the crystallization from ethyl acetate to give 16~-methyl-pregnane-17~,21-diol-3,11,20-trione 21-acetate.
To 100 mg. of 16~-methylpregnane-17~,21-diol-3,11,20-trione 21-acetate dissolved in 2 ml. of chloroform and 2.25 ml.
of glacial acetic acid, at a temperature of -55C., is added two drops of a 0.001 N solution of dry HBr in glacial acetic acid. To about 0.38 ml. of 0.001 N HBr in glacial acetic acid, at -55C., is added 0.43 ml. of a solution containing 40 mg.
of bromine in chloroform, and the resulting solution is added, over about a 10-minute period, to the solution of the steroid, _ _ ~
Bj 25 -1~143330 while maintaining the reaction mixture at about -55C. The reaction mixture is allowed to stand at -55C. for about one-half hour; a solution containing 250 mg. of sodium acetate in 3 ml.
of water is added, and the resulting mixture is stirred for about 5 minutes. Five milliliters of water are then added, and the aqueous mixture is extracted with ethyl acetate. The ethyl acetate extract is washed with aqueous sodium bicarbonate solu-tion to neutrality, then with water, dried, and the solvent is evaporated in vacuo. The residual material is dissolved in 2 ml. of acetone, and to the solution is added 25 mg. of sodium bromide and 1 ml. of water. The resulting mixture is heated under reflux for a period of about 5 hours, the reaction mixture is cooled, and the acetone is evaporated in vacuo. The residual material is extracted into ether, the ether extract is washed with water, dried, and the solvent is evaporated to a volume of about l ml.; petroleum ether is added to this solution, ana the crystalline material which separates is recovered and dried to give approximately 90 mg. of 4-bromo-16a-methyl-pregnane-17a,21-diol-3,11,20-trione 21-acetate.
A mixture of 48 mg. of semicarbazide, 48 mg. of 4-bromo-16a-methyl-pregnane-17a,21-diol-3,11,20-trione 21-acetate 0.6 ml. of ethanol is heated under reflux in contact with a nitrogen atmosphere for a period of about three days, and the reaction solution is evaporated to a small volume, diluted with water and the crystalline material recovered and purified by recrystallization from aqueous methanol to give 16a-methyl-4-pregnene-17a,21-diol-3,11,20-trione-3,20-bis-semicarbazone 21-acetate. Fifty milligrams of 16a-methyl-4-pregnene-17a, 21-diol-3,11,20-trione-3,20-bis-semicarbazone 21-acetate is dissolved in a mixture of 1.0 cc. of benzene and 1.0 cc. of l.l N methanolic potassium hydroxide, and the solution is allowed to stand at room temperature for a period of about lO minutes.
-_ 26 -'1043330 The solution is then acidified with acetic acid, the benzene is evaporated in vacuo, and the residual material is recrystallized from ethyl acetate to give 16~-methyl-4-pregnene-17~,21-diol-3,11,20-trione-3,20-bis-semicarbazone.
A mixture of 60 mg. of 16~-methyl-pregnene-17~,21-diol-3,11,20-trione-3,20-bis-semicarbazone, 0.2 ml. of dimethyl-formamide, 0.6 ml. of chloroform, and 1.5 ml. of 1.0 N aqueous hydrochloric acid is heated under reflux for a period of about three hours. The resulting two-phase system is cooled to a temperature of approximately lSC., and the layers are separated.
The aqueous layer is extracted with chloroform, and the chloro-form extracts are combined with the original chloroform-dimethyl-formamide solution. The combined organic layer is washed with an aqueous solution of sodium bicarbonate, and the chloroform and dimethylformamide in the combined organic layer is replaced with ethyl acetate by evaporation in vacuo. Petroleum ether is added and the resulting solution is subjected to a partition chromatogram using aqueous methanol as the stationary phase and benzene-chloroform as the moving phase to give 16a-methyl-4-pregnene-17~,21-diol-3,11,20-trione.
A solution of 45 mg. of 16~-methyl-4-pregnene-17~21-diol-3,11,20-trione-3,20-bis-semicarbazone 21-acetate, 17 mg.
of sodium borohydride, 1 ml. of tetrahydrofuran and 0.3 ml. of water is maintained at reflux temperature for approximately one hour. The reaction solution is cooled to about 15C., and the excess sodium borohydride decomposed by the addition of a solu-tion of 27 mg. of glacial acetic acid in 0.2 ml. of water. The tetrahydrofuran is evaporated in vacuo, and the residual material is extracted with ethyl acetate. The ethyl acetate extracts are washed with a saturated salt solution, water S~ aqueous sodium bicarbonate solution and again with water. The extracts are dried and the ethyl acetate evaporated in vacuo to give n~
~ - 27 -~i 1~14;~330 16~-methyl~4~pregnene-11~,17~,21-triol-3,20-dione, 3,20-bis-semicarbazone.
A mixture of 60 mg. of 16~-methyl-4-pregnen~ ,17~, 21-triol-3,20-dione 3,20-bis-semicarbazone, 0.2 ml. of dimethyl-formamide, 0.6 ml. of chloroform, and 1.5 ml. of 1.0 N aqueous hydrochloric acid is heated under reflux for a period of about three hours. The resulting two-phase system is cooled to a temperature of approximately 15C., and the layers are separated.
The aqueous layer is extracted with chloroform, and the chloro-form extracts are combined with the original chloroform-dimeth formamide solution. The combined organic layer is washed with an aqueous solution of sodium bicarbonate, and the chloroform and dimethylformamide in the combined organic layer is replaced with ethyl acetate by evaporation in vacuo. Petroleum ether is added and the resulting solution is subjected to a partition chromatogram using aqueous methanol as the stationary phase and benzene-chloroform as the moving phase to give 16~-methyl-4-pregnene-11~,17a,21-triol-3,20-dione. The latter material i~ reacted with an excess of acetic anhydride in pyridine at room temperature for a period of about fifteen hours, and the crude acetylated product is recrystallized from ethyl acetate to give 16-methyl-4-pregnene-11~,17,21-triol-3,20-dione 21-acetate.
To a cooled solution of 600 mg. of 16~-methyl-4-preg~
nene ~ ,17,21-triol-3,20-dione 21-acetate in 5.0 ml. of dry pyridine is added 0.15 ml. of phosphorous oxychloride, and the mixture is allowed to stand at room temperature for a period of approximately 15 hours. The reaction solution is evaporated in vacuo at a temperature of about 20C. to a volume of 2-3 ml.
Seventeen milliliters of water is added slowly to the con-centrated solution, with stirring, and the aqueous mixture is extracted with ethyl acetate. The combined ethyl acetate 1~)43330 extracts are washed with water, then with dilute aqueous hydro-chloric acid solution, again with water, and then with a dilute aqueous sodium bicarbonate solution. The washed ethyl acetate solution is dried, and the solvent is evaporated in vacuo; the residual material is triturated with ether, and the crystalline material is recrystallized from ethyl acetate-ether to give 16a-methyl-4,9(11) pregnadiene-17a,21-diol-3,20-dione 21-acetate. A
suspension of 330 mg. of 16a-methyl-4,9(11)-pregnadiene-17a,21-diol-3,20-dione 21-acetate and 1.8 g. of N-bromo-succinimide in a mixture of 50 ml. of dioxane and 10 ml. of water is cooled to 10C. Then with stirring, 10 ml. of cold 1.0 N aqueous perchloric acid is added. The temperature of the reaction mix-ture is allowed to rise to 15C. and is maintained at this point for about two-one-half hours during which time the solid material slowly dissolves. The resulting yellow solution is treated with 1.0 ml. of allyl alcohol to discharge the color and the remaining N-bromo-succinimide, and the resulting solution is evaporated to a small volume in vacuo. The concentrated solution is diluted with water, and the aqueous mixture is ex-tracted with ethyl acetate The ethyl acetate extracts arewashed, dried and evaporated to dryness, and the residual material is crystallized from ethyl acetate-ether to give 9a-bromo-16a-methyl~4-pregnene-11~,17a,21-triol-3,20-dione 21-acetate.
A solution of 210 mg. of 9a-bromo-16a-methyl-4-Pregnene ~ l7a~2l-triol-3~2o-dione 21-acetate and 240 mg. of potassium acetate in 10 ml. of absolute ethanol is heated under reflux for two hours. The reaction mixture is cooled to room temper-ature, and evaporated in vacuo to a small volume. The con-centrated aqueous mixture is extracted with three portions ofethyl acetate, and the combined ethyl acetate extracts are washed with water, dried, and evaporated to dryness in vacuo.
_~ 1 Ij, - 29 -The residual material is crystallized from ethyl acetate-ether to give 16a-methyl-9,11-epoxy-4-pregnene-17~,21-diol-3,20-dione 21-acetate.
To a solution of 83 mg. of anhydrous hydrogen fluoride in 4.7 ml. of ice-cold alcohol-free chloroform is added an ice-cold solution of 416 mg. of 16~-methyl-9,11-epoxy-4-pregnene-17~,21-diol-3,20-dione 21-acetate. The solution is mixed thoroughly and maintained at 0C. for two hours, at the end of which time 13.0 ml. of ice-cold 20~ aqueous sodium acetate is added, and the resulting mixture agitated vigorously. The layers are separated, and the chloroform layer is washed with water until free of acid, dried, and the chloroform evaporated in vacuo. The residual material is crystallized from acetone-petroleum ether to give 16~-methyl-9~-fluoro-4-pregnene~
17~,21-triol-3,20-dione 21-acetate. Fifty milligrams of 16~-methyl-9~-fluoro-4-pregnene-11~,17a,21-triol-3,20-dione 21-acetate is dissolved in a mixture of 1.0 cc. of benzene and 1.0 cc.
of 1 N methanolic potassium hydroxide, and the solution is allowed to stand at room temperature for a period of about ten minutes.
The solution is then acidified-with acetic acid, the benzene is evaporated in vacuo, and the residual material is crystallized from ethyl acetate-ether to give 16~-methyl-9~-fluoro-4-pregnene-11~,17~,21-triol-3,20-dione.
A solution of 400 mg. of 16~-methyl-9~-fluoro-4-pregnene-11~,17~,21-triol-3,20-dione 21-acetate in 4 ml. of pyridine is added to the complex formed by the addition of 400 mg. of chromium trioxide to 4 ml. of pyridine. The mixture is swirled until thoroughly mixed and then allowed to stand at room temperature overnight. The reaction mixture is poured into water, and the aqueous mixture is extracted with ether and then twice with ethyl acetate. The combined ether and ethyl acetate extracts are washed with dilute aqueous sulfuric acid Bj 30 -. .
1~)43;~30 at about 0C., and then with water until neutral. The organic solvent layer is then dried, the solvents are evaporated therefrom in vacuo, and the residual crystalline material is purified by the crystallization from ethyl acetate-ether to give 16a-methyl-9a-fluoro-4-pregnene-17a,21-diol-3,11,20-trione 21-acetate. Fifty milligrams of 16a-methyl-9a-fluoro-4-pregnene-17~,21-diol-3,11,20-trione 21-acetate is dissolved in a mixture of 1.0 cc. of benzene and 1.0 cc. of 1 N methanolic potassium hydroxide, and the solution is allowed to stand at room temperature for a period of about ten minutes. The solution is then acidified with acetic acid, the benzene is evaporated in vacuo, and the residual material is crystallized from ethyl acetate-ether to give 16a-methyl-9a-fluoro-4-pregnene-17~,21-diol-3,11,20-trione.
Various changes and modifications may be made in carry-ing out the present invention without departing from the spirit and scope thereof. Insofar as these changes and modifications are within the purview of the annexed claims, they are to be considered as part of our invention.
,~
of ether. A white granular solid separates during this addition.
The resulting mixture is heated under gentle reflux for two hours after which the reaction mixture is cooled, and 125 ml.
of saturated, aqueou~ ammonium chloride solution is added follow-ed by 200 ml. of ether. The layers are separated, and the ethereal layer is washed with three 50 ml. portions of water.
The washed ethereal layer is dried, and the solvent evaporated in vacuo to give a brown viscous oil. The latter material is heated for lS minutes at 60-70C. with a mixture of 25 ml.
acetic anhydride and 25 ml. pyridine and the acetylated product is purified by chromatography on acid-washed alumina followed by crystallization from petroleum ether to give approximately ., 1043;~30 1.5 g. of substantially pure 1~-methyl-pregnene-3R-ol-ll, 20-dione 3-acetate.
To a solution of 0.8 g. of 16Q-methyl-pregnane-3G-ol-11,20-dione 3-acetate in 40 ml. of methanol is added 1.5 ml. of concentrated aqueous hydrochloric acid and the resulting solution is stirred overnight at about 25C. The reaction solution is evaporated in vacuo at 25C to a small volume, and the concentrated solution is poured into 50 ml.
of ice water. The white solid which precipitates is re-covered by filtration, washed wlth ~ater and recrystallizedfrom ethyl acetate to give 16~-methyl-pregnane-3~-ol-11,20-dione.
A solution of 22 g. of 16o-methyl-pregnane-3o-ol-11,20-dione 21-acetate and 1 g. of ~-toluene-sulfonic acid in 250 ml. of acetic anhydride is heated at reflux under nitrogen for a period of approximately 3 days. Two grams of potassium acetate tanhydrous~ is added, and the volatile solvents are separated by distillation in vacuo. The residual material is extracted ~ith benzene, and the benzene extract is filtered to remove insoluble material. The benzene extracts are~evaporated to a volume of 100 ml. and petroleum ether is added to the cloud point. The resulting solution is adsorbed on 660 g. of acid-washed alumina; the alumina adsorbate is then washed with 2 liters of petroleum ether. The adsorbate is then eluted with 85:15 petroleum-ether-ether mixture, and the first four liters of eluate is collected, and evaporated to dryness in vacuo to give a mixture of enol acetates-containing 16Q-methyl-17C20~-pregnene-3Q,20-diol-ll-one 3,20-diacetate. This mixture of enolates, weighing approximately 14 g., is dissolved in 50 ml. of benzene and treated with an excess of per-benzoic acid over a 16-hour period. The reaction mixture is shaken with dilute aqueous ~ j - 23 -potassium hydroxide solution until the benzene layer is free of perbenzoic acid; the benzene layer is then washed with water until neutral, dried, and the solvent evaporated in vacuo to give a crystalline material, 16~-methyl-17a,20-epoxy-pregnane-3a,20-diol-11-one 3,20-diacetate. The latter material is dissolved, without purification, in 200 ml. of methanol, 120 ml.
of water and 10 g. of potassium bicarbonate, and the resulting solution is heated at reflux under nitrogen for a period of 16 hours. The methanol is evaporated from the hydrolysis solution in vacuo, and the residual oil is extracted from the resulting aqueous solution with chloroform. The chloroform extract is washed with water to neutrality, dried, and the chloroform is evaporated under reduced pressure. The residual oil is triturated with ether, and the crystalline material thus formed is recrystal-lized from ethyl acetate-petroleum ether to give 16a-methyl-pregnane-3,17-diol-11,20 dione.
To a solution of 7.0 g. of 16a-methyl-pregnane-3,17-diol-11,20-dione in 50 ml. of chloroform is added dropwise with stirring a solution containing 3.36 g. of bromine in 24.2 ml.
of chloroform over a period of about 60 minutes. The reaction mixture is dissolved in 200 ml. of ethyl acetate, and the resulting solution washed with water until neutral, dried, and the solvents evaporated therefrom in vacuo. The residual crude material is dissolved in a minimum quantity of ethyl acetate, the resulting solution is diluted with ether, and the mixture is stirred until crystals form. The crystalline product is recovered by filtration and washed, by slurrying, with 50:50 ether-petroleum ether mixture to give about 5 g.
of 21-bromo-16~-methyl-pregnane-3a,17-diol-11,20-dione.
This 5 g. of 21-bromo-16a-methyl-pregnane-3,17a-diol-11,20-dione is mixed with S.0 g. of anhydrous potassium acetate, 4.0 g. of sodium iodide and 0.03 ml. of glacial acetic acid, and 100 ml. of acetone is added to the resulting mixture.
........ ~7 1~43330 this mixture is then heated at reflux, with stirringl for a period of about 16 hours, and the reaction mixture is cooled, filtered, and the insoluble material is washed with acetone. The filtered solution is evaporated in vacuo thereby removing the solvents, and the residual material is slurried with water, and the aqueous mixture extracted with ethyl acetate. The ethyl acetate extract is washed with water to neutrality, dried, and the solvent is evaporated in vacuo to give an oil. This oil is crystallized from ether, and recrystallized from ethyl acetate-ether to give 16~-methyl-pregnane-3~,17~,21-triol-11,20-dione 21-acetate.
A solution of 400 mg. of 16~-methyl-pregnane-3~-17~, 21-triol-11,20-dione 21-acetate in 4 ml. of pyridine is added to the complex formed by the addition of 400 mg. of chromium trioxide to 4 ml. of pyridine. The mixture is swirled until thoroughly mixed, and then allowed to stand at room temperature overnight. The reaction mixture is poured into water, and the aqueous mixture is extracted with ether, and then twice with ethyl acetate. The combined ether and ethyl acetate extracts are washed with dilute aqueous sulfuric acid at about 0C, and then with water until neutral. The organic solvent layer is then dried, the solvents are evaporated therefrom in vacuo, and the residual crystalline material is purified by the crystallization from ethyl acetate to give 16~-methyl-pregnane-17~,21-diol-3,11,20-trione 21-acetate.
To 100 mg. of 16~-methylpregnane-17~,21-diol-3,11,20-trione 21-acetate dissolved in 2 ml. of chloroform and 2.25 ml.
of glacial acetic acid, at a temperature of -55C., is added two drops of a 0.001 N solution of dry HBr in glacial acetic acid. To about 0.38 ml. of 0.001 N HBr in glacial acetic acid, at -55C., is added 0.43 ml. of a solution containing 40 mg.
of bromine in chloroform, and the resulting solution is added, over about a 10-minute period, to the solution of the steroid, _ _ ~
Bj 25 -1~143330 while maintaining the reaction mixture at about -55C. The reaction mixture is allowed to stand at -55C. for about one-half hour; a solution containing 250 mg. of sodium acetate in 3 ml.
of water is added, and the resulting mixture is stirred for about 5 minutes. Five milliliters of water are then added, and the aqueous mixture is extracted with ethyl acetate. The ethyl acetate extract is washed with aqueous sodium bicarbonate solu-tion to neutrality, then with water, dried, and the solvent is evaporated in vacuo. The residual material is dissolved in 2 ml. of acetone, and to the solution is added 25 mg. of sodium bromide and 1 ml. of water. The resulting mixture is heated under reflux for a period of about 5 hours, the reaction mixture is cooled, and the acetone is evaporated in vacuo. The residual material is extracted into ether, the ether extract is washed with water, dried, and the solvent is evaporated to a volume of about l ml.; petroleum ether is added to this solution, ana the crystalline material which separates is recovered and dried to give approximately 90 mg. of 4-bromo-16a-methyl-pregnane-17a,21-diol-3,11,20-trione 21-acetate.
A mixture of 48 mg. of semicarbazide, 48 mg. of 4-bromo-16a-methyl-pregnane-17a,21-diol-3,11,20-trione 21-acetate 0.6 ml. of ethanol is heated under reflux in contact with a nitrogen atmosphere for a period of about three days, and the reaction solution is evaporated to a small volume, diluted with water and the crystalline material recovered and purified by recrystallization from aqueous methanol to give 16a-methyl-4-pregnene-17a,21-diol-3,11,20-trione-3,20-bis-semicarbazone 21-acetate. Fifty milligrams of 16a-methyl-4-pregnene-17a, 21-diol-3,11,20-trione-3,20-bis-semicarbazone 21-acetate is dissolved in a mixture of 1.0 cc. of benzene and 1.0 cc. of l.l N methanolic potassium hydroxide, and the solution is allowed to stand at room temperature for a period of about lO minutes.
-_ 26 -'1043330 The solution is then acidified with acetic acid, the benzene is evaporated in vacuo, and the residual material is recrystallized from ethyl acetate to give 16~-methyl-4-pregnene-17~,21-diol-3,11,20-trione-3,20-bis-semicarbazone.
A mixture of 60 mg. of 16~-methyl-pregnene-17~,21-diol-3,11,20-trione-3,20-bis-semicarbazone, 0.2 ml. of dimethyl-formamide, 0.6 ml. of chloroform, and 1.5 ml. of 1.0 N aqueous hydrochloric acid is heated under reflux for a period of about three hours. The resulting two-phase system is cooled to a temperature of approximately lSC., and the layers are separated.
The aqueous layer is extracted with chloroform, and the chloro-form extracts are combined with the original chloroform-dimethyl-formamide solution. The combined organic layer is washed with an aqueous solution of sodium bicarbonate, and the chloroform and dimethylformamide in the combined organic layer is replaced with ethyl acetate by evaporation in vacuo. Petroleum ether is added and the resulting solution is subjected to a partition chromatogram using aqueous methanol as the stationary phase and benzene-chloroform as the moving phase to give 16a-methyl-4-pregnene-17~,21-diol-3,11,20-trione.
A solution of 45 mg. of 16~-methyl-4-pregnene-17~21-diol-3,11,20-trione-3,20-bis-semicarbazone 21-acetate, 17 mg.
of sodium borohydride, 1 ml. of tetrahydrofuran and 0.3 ml. of water is maintained at reflux temperature for approximately one hour. The reaction solution is cooled to about 15C., and the excess sodium borohydride decomposed by the addition of a solu-tion of 27 mg. of glacial acetic acid in 0.2 ml. of water. The tetrahydrofuran is evaporated in vacuo, and the residual material is extracted with ethyl acetate. The ethyl acetate extracts are washed with a saturated salt solution, water S~ aqueous sodium bicarbonate solution and again with water. The extracts are dried and the ethyl acetate evaporated in vacuo to give n~
~ - 27 -~i 1~14;~330 16~-methyl~4~pregnene-11~,17~,21-triol-3,20-dione, 3,20-bis-semicarbazone.
A mixture of 60 mg. of 16~-methyl-4-pregnen~ ,17~, 21-triol-3,20-dione 3,20-bis-semicarbazone, 0.2 ml. of dimethyl-formamide, 0.6 ml. of chloroform, and 1.5 ml. of 1.0 N aqueous hydrochloric acid is heated under reflux for a period of about three hours. The resulting two-phase system is cooled to a temperature of approximately 15C., and the layers are separated.
The aqueous layer is extracted with chloroform, and the chloro-form extracts are combined with the original chloroform-dimeth formamide solution. The combined organic layer is washed with an aqueous solution of sodium bicarbonate, and the chloroform and dimethylformamide in the combined organic layer is replaced with ethyl acetate by evaporation in vacuo. Petroleum ether is added and the resulting solution is subjected to a partition chromatogram using aqueous methanol as the stationary phase and benzene-chloroform as the moving phase to give 16~-methyl-4-pregnene-11~,17a,21-triol-3,20-dione. The latter material i~ reacted with an excess of acetic anhydride in pyridine at room temperature for a period of about fifteen hours, and the crude acetylated product is recrystallized from ethyl acetate to give 16-methyl-4-pregnene-11~,17,21-triol-3,20-dione 21-acetate.
To a cooled solution of 600 mg. of 16~-methyl-4-preg~
nene ~ ,17,21-triol-3,20-dione 21-acetate in 5.0 ml. of dry pyridine is added 0.15 ml. of phosphorous oxychloride, and the mixture is allowed to stand at room temperature for a period of approximately 15 hours. The reaction solution is evaporated in vacuo at a temperature of about 20C. to a volume of 2-3 ml.
Seventeen milliliters of water is added slowly to the con-centrated solution, with stirring, and the aqueous mixture is extracted with ethyl acetate. The combined ethyl acetate 1~)43330 extracts are washed with water, then with dilute aqueous hydro-chloric acid solution, again with water, and then with a dilute aqueous sodium bicarbonate solution. The washed ethyl acetate solution is dried, and the solvent is evaporated in vacuo; the residual material is triturated with ether, and the crystalline material is recrystallized from ethyl acetate-ether to give 16a-methyl-4,9(11) pregnadiene-17a,21-diol-3,20-dione 21-acetate. A
suspension of 330 mg. of 16a-methyl-4,9(11)-pregnadiene-17a,21-diol-3,20-dione 21-acetate and 1.8 g. of N-bromo-succinimide in a mixture of 50 ml. of dioxane and 10 ml. of water is cooled to 10C. Then with stirring, 10 ml. of cold 1.0 N aqueous perchloric acid is added. The temperature of the reaction mix-ture is allowed to rise to 15C. and is maintained at this point for about two-one-half hours during which time the solid material slowly dissolves. The resulting yellow solution is treated with 1.0 ml. of allyl alcohol to discharge the color and the remaining N-bromo-succinimide, and the resulting solution is evaporated to a small volume in vacuo. The concentrated solution is diluted with water, and the aqueous mixture is ex-tracted with ethyl acetate The ethyl acetate extracts arewashed, dried and evaporated to dryness, and the residual material is crystallized from ethyl acetate-ether to give 9a-bromo-16a-methyl~4-pregnene-11~,17a,21-triol-3,20-dione 21-acetate.
A solution of 210 mg. of 9a-bromo-16a-methyl-4-Pregnene ~ l7a~2l-triol-3~2o-dione 21-acetate and 240 mg. of potassium acetate in 10 ml. of absolute ethanol is heated under reflux for two hours. The reaction mixture is cooled to room temper-ature, and evaporated in vacuo to a small volume. The con-centrated aqueous mixture is extracted with three portions ofethyl acetate, and the combined ethyl acetate extracts are washed with water, dried, and evaporated to dryness in vacuo.
_~ 1 Ij, - 29 -The residual material is crystallized from ethyl acetate-ether to give 16a-methyl-9,11-epoxy-4-pregnene-17~,21-diol-3,20-dione 21-acetate.
To a solution of 83 mg. of anhydrous hydrogen fluoride in 4.7 ml. of ice-cold alcohol-free chloroform is added an ice-cold solution of 416 mg. of 16~-methyl-9,11-epoxy-4-pregnene-17~,21-diol-3,20-dione 21-acetate. The solution is mixed thoroughly and maintained at 0C. for two hours, at the end of which time 13.0 ml. of ice-cold 20~ aqueous sodium acetate is added, and the resulting mixture agitated vigorously. The layers are separated, and the chloroform layer is washed with water until free of acid, dried, and the chloroform evaporated in vacuo. The residual material is crystallized from acetone-petroleum ether to give 16~-methyl-9~-fluoro-4-pregnene~
17~,21-triol-3,20-dione 21-acetate. Fifty milligrams of 16~-methyl-9~-fluoro-4-pregnene-11~,17a,21-triol-3,20-dione 21-acetate is dissolved in a mixture of 1.0 cc. of benzene and 1.0 cc.
of 1 N methanolic potassium hydroxide, and the solution is allowed to stand at room temperature for a period of about ten minutes.
The solution is then acidified-with acetic acid, the benzene is evaporated in vacuo, and the residual material is crystallized from ethyl acetate-ether to give 16~-methyl-9~-fluoro-4-pregnene-11~,17~,21-triol-3,20-dione.
A solution of 400 mg. of 16~-methyl-9~-fluoro-4-pregnene-11~,17~,21-triol-3,20-dione 21-acetate in 4 ml. of pyridine is added to the complex formed by the addition of 400 mg. of chromium trioxide to 4 ml. of pyridine. The mixture is swirled until thoroughly mixed and then allowed to stand at room temperature overnight. The reaction mixture is poured into water, and the aqueous mixture is extracted with ether and then twice with ethyl acetate. The combined ether and ethyl acetate extracts are washed with dilute aqueous sulfuric acid Bj 30 -. .
1~)43;~30 at about 0C., and then with water until neutral. The organic solvent layer is then dried, the solvents are evaporated therefrom in vacuo, and the residual crystalline material is purified by the crystallization from ethyl acetate-ether to give 16a-methyl-9a-fluoro-4-pregnene-17a,21-diol-3,11,20-trione 21-acetate. Fifty milligrams of 16a-methyl-9a-fluoro-4-pregnene-17~,21-diol-3,11,20-trione 21-acetate is dissolved in a mixture of 1.0 cc. of benzene and 1.0 cc. of 1 N methanolic potassium hydroxide, and the solution is allowed to stand at room temperature for a period of about ten minutes. The solution is then acidified with acetic acid, the benzene is evaporated in vacuo, and the residual material is crystallized from ethyl acetate-ether to give 16a-methyl-9a-fluoro-4-pregnene-17~,21-diol-3,11,20-trione.
Various changes and modifications may be made in carry-ing out the present invention without departing from the spirit and scope thereof. Insofar as these changes and modifications are within the purview of the annexed claims, they are to be considered as part of our invention.
,~
Claims (18)
- The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:
The process of preparing wherein R is hydrogen or acetyl which comprises adding one atom of bromine at position 4 of the corresponding ring-A saturated compound followed by dehydrobromination. - 2. The process of preparing 16.alpha.-methyl cortisone which comprises hydrolyzing the 21-acetate thereof.
- 3. The process of preparing 16.alpha.-methyl hydro-cortisone which comprises reacting 16.alpha.-methyl-cortisone 21-acetate 3,20 bissemicarbazone with an alkali metal borohydride and hydrolyzing the product so formed.
- 4. The process of preparing 16.alpha.-methyl hydro-cortisone 21-acetate which comprises reacting 16.alpha.-methyl-hydro cortisone with acetic anhydride.
- 5. The process of preparing 16.alpha.-methyl-pregnane-17.alpha.,21-diol-3,11,20-trione 21-acetate which comprises reacting 16.alpha.-methyl-pregnane-3.alpha.,17.alpha.,21-triol-11,20-dione 21-acetate with chromium trioxide in pyridine.
- 6. The process of preparing 16.alpha.-methyl-pregnane-3.alpha.,17.alpha.,21-triol-11,20-dione 21-acetate which comprises reacting 21-bromo-16.alpha.-methyl-pregnane-3.alpha.,17.alpha.-diol-11,20-dione with potassium acetate and sodium iodide in acetone containing glacial acetic acid.
- 7. The process which comprises heating 16.alpha.-methyl-pregnane-3.alpha.-ol-11,20-dione 3-acetate with acetic anhydride containing p-toluene sulfonic acid catalyst to form 16.alpha.-methyl-17(20)-pregnene-3.alpha.,20-diol-11-one 3,20-diacetate.
- 8. The process of preparing 16.alpha.-methyl-pregnane-3.alpha.,17.alpha.-diol-11,20-dione which comprises reacting 16.alpha.-methyl-17(20)pregnane-3.alpha.,20-diol-11-one 3,20-diacetate with perbenzoic acid followed by hydrolysis of the resulting 17.alpha.,20-epoxide.
- 9. The process of preparing 16.alpha.-methyl-3-acetoxy-pregnane-11,20-dione which comprises reacting 16-pregnene-3.alpha.-ol-11,20-dione 3-acetate with methyl magnesium iodide.
- 10. 16.alpha.-Methyl cortisone 21-acetate whenever prepared or produced by the process of claim 1 or an obvious chemical equivalent thereof.
- 11. 16.alpha.-Methyl cortisone whenever prepared or produced by the process of claims 1 or 2 or an obvious chemical equivalent thereof.
- 12. 16.alpha.-Methyl hydrocortisone whenever prepared or produced by the process of claim 3 or an obvious chemical equivalent thereof.
- 13. 16.alpha.-Methyl hydrocortisone 21-acetate when-ever prepared or produced by the process of claim 4 or an obvious chemical equivalent thereof.
- 14. 16.alpha.-Methyl-pregnane-17.alpha.,21-diol-3,11,20-dione 21-acetate whenever prepared or produced by the process of claim 5 or an obvious chemical equivalent thereof.
- 15. 16.alpha.-Methyl-pregnane-3.alpha.,17.alpha.,21-triol-11,20-dione 21-acetate whenever prepared or produced by the process of claim 6 or an obvious chemical equivalent thereof.
- 16. 16.alpha.-Methyl-17(20)-pregnane-3.alpha.,20-diol-11-one 3,20 diacetate whenever prepared or produced by the process of claim 7 or an obvious chemical equivalent thereof.
- 17. 16.alpha.-Methyl-pregnane-3.alpha.,17.alpha.,diol-11,20-dione whenever prepared or produced by the process of claim 8 or an obvious chemical equivalent thereof.
- 18. 16.alpha.-Methyl-3-acetoxy-pregnane-11,20-dione when-ever prepared or produced by the process of claim 9 or an obvious chemical equivalent thereof.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US64265557A | 1957-02-27 | 1957-02-27 | |
| CA154,365A CA1011730A (en) | 1957-02-27 | 1972-10-20 | 16-METHYL-1,4-PREGNADIENE-17.alpha.-OL-3,20-DIONE COMPOUNDS |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1043330A true CA1043330A (en) | 1978-11-28 |
Family
ID=25667133
Family Applications (3)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA279,702A Expired CA1043330A (en) | 1957-02-27 | 1977-06-02 | 16-METHYL-1,4-PREGNADIENE-17.alpha.-OL-3,20 DIONE COMPOUNDS |
| CA279,703A Expired CA1041444A (en) | 1957-02-27 | 1977-06-02 | 16-METHYL-1,4-PREGNADIENE-17.alpha.-OL-3,20 DIONE COMPOUNDS |
| CA279,704A Expired CA1043331A (en) | 1957-02-27 | 1977-06-02 | 16-METHYL-1,4-PREGNADIENE-17.alpha.-OL-3,20 DIONE COMPOUNDS |
Family Applications After (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA279,703A Expired CA1041444A (en) | 1957-02-27 | 1977-06-02 | 16-METHYL-1,4-PREGNADIENE-17.alpha.-OL-3,20 DIONE COMPOUNDS |
| CA279,704A Expired CA1043331A (en) | 1957-02-27 | 1977-06-02 | 16-METHYL-1,4-PREGNADIENE-17.alpha.-OL-3,20 DIONE COMPOUNDS |
Country Status (1)
| Country | Link |
|---|---|
| CA (3) | CA1043330A (en) |
-
1977
- 1977-06-02 CA CA279,702A patent/CA1043330A/en not_active Expired
- 1977-06-02 CA CA279,703A patent/CA1041444A/en not_active Expired
- 1977-06-02 CA CA279,704A patent/CA1043331A/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| CA1043331A (en) | 1978-11-28 |
| CA1041444A (en) | 1978-10-31 |
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