Note: Descriptions are shown in the official language in which they were submitted.
1043330
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:
CH2R
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 --
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 -
1043330
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
- 3 -
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 ~
1043330
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-
- 12 -
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.
EXAMPLE 1
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.
- 13 -
;~..~. 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.
., ;
1043330
EXAMPLE 3
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
3Q
- 16 -
,
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.
EXAMPLE 4
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
- 17 -
1043330
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
- 18 -
1043330
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.
EXANPLE 6
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
EXAMPLE 7
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.
EXAMPLE 8
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
- 21 -
1043330
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
- 22 -
.,
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 -
1043330
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
- 24 -
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
- 28 -
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 -
1043330
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.
,~
-- 3 31 -
