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Патент USA US3022236

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United States Patent .0 " 1C@
1
3,@ZZ,ZZ6
Patented Feb. 20, 1962
2
genase is the same as those of culturing microorganisms
3,022,226
PROCESS FOR PREPARING l-DEHYDRO
STEROIDS.
John W. Ross, East Brunswick, NJ., assignor to Olin
Mathieson Chemical Corporation, New York, N.Y., a
corporation of Virginia
N0 Drawing. Filed Nov. 23, 1960, Ser. No. 71,140
6 Claims. (Cl. 195-51)
i
for the production of antibiotics or vitamins. Thus, the
microorganism is grown in contact with (in or on) a
suitable nutrient medium. if an aerobic microorganism
is being grown, an adequate supply of oxygen (air) is
provided during the growth period. A suitable nutrient
medium essentially comprises a source of nitrogenous
factors and an assimilable source of carbon and energy.
The latter may be a carbohydrate, such as sucrose,
This invention relates to a process for preparing steroids 10 molasses, glucose, maltose, starch or dextrin. The source
and, more particularly, to an improved process for the
of nitrogenous factors may be organic (e.g., soybean
microbial l-dehydrogenation of steroids.
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meal, corn steep liquor, meat extract, distiller’s solubles,
With the discovery that the introduction of a double
peptones and/or yeast extract) or synthetic (i.e., com
bond into the 1,2-position of hydrocortisone increased
posed‘ ofsimple, syuthesizable organic and inorganic
the glucocorticoid activity, attention was directed to proc 15 compounds such as ammonium salts, alkali nitrates, amino -'
esses for l-dehydrogenation of steroids of the 3,20-diketo
acids or urea). '
A‘i-pregnene series. Subsequentlyv other l-dehydrogen
In order to induce the formation of the desired 1
ated steroids were found to have'commercial utility as
dehydrogenase enzyme, a 1,2-saturated steroid, such as
glucocorticoid and anti-in?ammatory drugs. Among
progesterone and testosterone,‘ is also added‘to the nu
such steroids can be mentioned triamcinolone and dex
trient‘m‘edium. The steroid is present in suilicient quan
tity to favor optimum formation of the desired enzyme
amethasone. .It was-also found that the desired l-de
hydrogenation could be accomplished either chemically
and preferably is present in a concentration of ‘at least
or microbiologically. Chemical methods, such as by the
0.01% (w./v.) of the nutrient medium.
use of selenium dioxide, however, suffered the disad
After a suitable growth period (at least 24 hours),
vantage of giving a selenium containing by-product which 25 the cells are separated from the nutrient medium in
was di?cult to remove.
Microbial methods, although
the usual manner, such as by ?ltration or centrifugation,
superior, have hithertofor been accompanied by sub
and the separated cells containing the desired l-dehy
stantial reduction of the keto group in the 20-position
to yield an undesired ZO-hydroxy derivative.
drogenase are then mixed with water, an iodoacetate
and the steroid to be l-dehydrogenated, under aerobic
It is an object of this invention, therefore, to provide 30 conditions.
Among the steroids of the 3,20-diketo-A4-pregnene
the 3,20-diketo-A4-pregnene series.
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series which may be converted into useful l-dehydro
It is another object of this invention to provide a
derivatives by the practice of this invention may be
process for l-dehydrogenating a steroid of the 3,20
mentioned monohydroxyprogesterones (e.g., Ila-hy
diketo-n‘i-pregnene series wherein the concomitant reduc
droxyprogesterone, the'9a- and lla-halo-llp-hydroxy
tion of the ZO-keto group to a ZO-hydroxy group is
progesterones, desoxycorticosterone and ‘2l-?uoro-17a
an improved process for l-dehydrogenating a steroid of
minimized or eliminated.
hydroxyprogesterone); the dihydroxyprogesterones (e.g.,
These objects are achieved by the process of this in
corticosterone, the 9a- and 12a-halocorticosterones,
vention which comprises subjecting under aerobic con
Reichsteins’s Compound S, 11p,l7a-dihydroxyprogester
ditions a steroid of the 3,2O-diketo-A4-pregnene series to 40 one, cortisone, the 91x- and 12a-halocor‘tisones, 21-?uoro
the action of a l-dehydrogenase enzyme in the presence
1 l5,17a—dihydroxyprogesterone and 9a,21-di?uoro-1148,
of an iodoacetate compound. For this purpose there’ is
l7a-dihydroxyprogesterone); the trihydroxyprogesterones
used iodoacetic acid itself; a salt thereof, such as an
(e.g., hydrocortisone, A‘i-pregnene-llu,17a,21-triol-3,20
alkali metal salt (e.g., sodium iodoacetate and potas
Idione, the 90:- and 12u-halohydrocortisones, and the 16
sium iodoacetate), an alkaline earth metal salt, the am 45 methy1-9u-halohydrocortisones);. and the tetrahydroxy
monium salt, and an amine salt; or an ester thereof,
progesterones (e.g., 9cz-fluoro-16a-hydroxyhydrocortisone,
such as a lower alkyl ester (e.g., methyl iodoacetate and
6-methyl-9e-?uoro-16a-hydroxyhydrocortisone and the
ethyl iodoacetate) and a monocyclic aralkyl ester.
6,9-dihalo-16a-hydroXyhyd-rocortisones); as well as the
The l-dehydrogenase enzyme utilized in the process
Zl-ester derivatives of those steroids containing a 21
of this invention is preferably prepared separately 'in an 50 hydroxyl group (e.g., Compound S acetate, hydro
initial step wherein a microorganism known to effect ~
cortisone acetate, 9u4?uorohydrocortisone acetate and
l-dehydrogenation of steroids is grown in a suitable
9a-?uorocortisone acetate). The preferred 21-esters are
aqueous nutrient medium containing a substance which
those of hydrocarbon carboxylic acids having less than
induces the formation of the desired l-dehydrogenase
ten carbon atoms as exempli?ed by the lower fatty, acids
enzyme. Suitable inducing substances include steroids 55 (e.g., acetic and propionic acids), the monocyclic aryl
saturated in the 1,2-position. Although any such steroid
carboxylic acids (e.g., benzoic and a-toluic acids), the
may be used, because of their low cost testosterone and
monocyclic aryl lower alkanoic acids (e.g., phenacetic
progesterone are particularly preferred for this purpose.
and ?-phenylpropionic acids), the cycloalkane carboxylic
Suitable microorganisms include those known to effect
acids and the cycloalkene carboxylic acids.
.l-dehydrogenation of steroids as exempli?ed by mem
Although the process of this invention is being de
bers of the genera: Corynebacterium (e.g., C. simplex), 60 scribed as one employing separated cells, the process
Nocardia (e.g., N. aurantia and N. asteroides), Bacterium
can also be carried out using either isolated l-dehydro
(e.g., B. cyclooxydans), Mycobacterium (e.g., M. rhodo
genase enzyme or the entire nutrient medium containing
chrous), Bacillus (e.g., B. sphaericus), Septomyxa (e.g.,
the desired microorganism. However, because of the
S. a?i‘nis), Didymella (e.g., D. Iycopersici), Calonectria 65 extra cost involved in isolating the former and the diffi
(e.g., C. decora), Fusarium (e.g., F. solzmi), Cylindro—
culty of inhibiting undesired enzyme action in the latter,
carpon (e.g., C. radicicola), Pseudomonas (e.g., P. testos
the preferred method is, as hereinbefore stated, one
teroni), Streptomyces (e.g., S. lavena'alae), and also se
where the desired microorganism is ?rst cultured and the
lected species of the genera Protaminobacter, Alcaligenes,
separated cells containing l-dehydrogenase enzyme are
Alternaria, Ophio'oolus and Pyinodithis.
then used to e?ect l-dehydrogenation.
In general, the conditions of culturing the micro 70
To carry out the process of this invention the sepa
organisms for the purpose of preparing the l-dehydro
rated cells are suspended in an aqueous medium and
3,022,226
.
3
4
.
tent by paper chromatography which reveals a 77%
the iodoacetate compound and steroid to be converted
are added tothe medium, the steroid preferably being
added either after or with the iodoacetate compound,
and the iodoacetate compound being added in a preferred
concentration of about 0.001 molar to about 0.1 molar
and optimally about 0.005 molar to about 0.02 molar.
An adequate supply of oxygen is also provided, prefer
I ably either by aerating or'agitating the mixture, or both.
The following examples are illustrative of the inven
tion (all temperatures being in centigrade):
yield of triamcinolone.
.
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'By way of contrast, if the sodium iodoacetate is
omitted in step (b) of Example 1, the yield of tri
amcinolone is decreased from 77% to 68% and the
yield of total triamcinolone and starting steroid (i.e.,
9a-?uoro-l6a-hydroxyhydrocortisone) recovered de
creased from 97% to 73%.
10
EXAMPLE 2
Following the procedure of Example 1, step (b) but
' EXAMPLE 1
adjusting the initial mixture to pH 8.8,‘ the temperature
Triamcinolone
to 35° and the sodium iodoacetate to 0.25 mmole in
50 ml. of ?nal volume, triamicinolone is obtained in a
(a) Preparation of cellsx?gurface growth ‘from an agar
slant (beef extract, 1.5 g.; yeast extract, 3 g.; peptone,
6 g.; dextrose, 1 'g.; agar, 20 g; distilled water to 1
78% yield.‘
,
If the sodium iodoacetate is omitted in Example 2,
the yield of triamcinolone is'decreased from 78% to 62%
liter) culture of Bacterium cyclo-oxydans. (A.T.C.C. No.
and the yield of total triamcinolone and starting steroid
12,673) ‘is used to inoculate 50. ml. portions of the
following medium (A) contained in three 250 ml. Erlen- ‘
recovered from 99% to 67%..
r
‘
EXAMPLE 3
.
Peptone
3.
_
Following the procedure of Example 1, step (b) but
1
Yeast extract _________________ __' ___________ -_ 0.25.
KH2PO4
Prednz'solone
Percent
Glucose __________________________________ __
_____ __
0.1
‘substituting 10 mg. of'hydrocortisone for the 9a-?uoro~
25
16a-hydroxyhydrocortisone, prednisolone is obtained.
The invention may be variously otherwise embodied
Ucon antifoam (a polyalkylene glycol) ________ __ 0.01
within the scope of the appended claims.
in distilled water, pH adjusted to 7.0 before autoclaving
What is claimed is:
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at 121° for 30 minutes.
1. A process for preparing a. l-dehydro steroid which
The inoculated ?asks are incubated at 25° with rotary 30 comprises subjecting under aerobic conditions a steroid
‘shaking’ at 280 cycles per minute in a radius of about
of the 3,20-diketo-A4-pregnene series to the action of a
one'inch. After 48 hours, each ?ask is emptied into ,
l-dehydrogenase enzyme in the presence of an iodoacetate
a four liter ?ask containing one liter of the same medium
compound selected from the group consisting of iodoacetic
acid, salts thereof and esters thereof, said iodoacetate
48 hours after which the contents of the three ?asks are 35 compound being present in a molar concentration of
(A). Incubation is continued as described above for
used to inoculate 20 litersof the same medium (A)
in a 30 liter stainless steel ba?ied tank. To this tank
is also added 6 g. of progesterone dissolved in 120 ml.
of chloroform. The tank medium is agitated by an ?
impeller running at 220 r.p.m. and is aerated at the
rate of 2.3 cubic feet per minute. vAfter 48 hours in-.
cubation, the cells are harvested by centrifugation and
the cell pastc'obtained is frozen. These cells have ,1
dehydrogenase activity;
-
g
V
pound is sodium iodoacetate.
mmoles'of Na2HPO4, 0.8 mmole of sodium iodoacetate
and sufficient water to make 30 ml; in a 250 ml. Erlen—
meyer' ?ask is adjusted to pH 7.8 with N sulfuric acid
10.8 mg. of 9.1-, ‘
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drocortisone.
6. The process of'claim l'wherein the Il-dehydrogenase
enzyme is prepared. by vculturing a l-dehydrogenating
strain of a microorganism in the presence of a 1,2-satu
rated steroid."
2,844,513
two hours while shaking at 250 c.p.m. on a rotary shaker
' 2,951,016
with a three-quarter inch stroke. 5 ml. of the resulting 55
. .
I
References Cited in the ?le of this patent
UNITED STATES PATENTS
?uoro-l6a-hydroxyhydrocortisone, dissolved in 0.176 ml.
of dimethyl formamide plus 20 ml. of water is then
added. The resulting mixture .is incubated at 30° for
ketone. The extract is then analyzed for steroid con
.
4. The process of claim 1 wherein the steroid is 90:
5. The process of claim 1 wherein the steroid is hy
‘A '
(b) Steroid dehydrogenazi0n.—A mixture of l g. of 45
mixture is removed, treated with 0.2 ml. of 10 N sul
furic acid and extracted with 2 ml. of methyl isobutyl
'
2. The process of claim 1 wherein the iodoacetate
compound is an alkali metal iodoacetate.
3. The process of claim 1 wherein the iodoacetate com
'?uoro-l6u-hydroxyhydrocortisone.
the cell paste (wet weight) obtained in step (a), 10
and incubated at 30° for 20 minutes.
at least about 0.001.
Wettstein et a1. ______ __ July 22, 1958
Charney ____________ .. Aug. 30, 1960
OTHER REFERENCES
Bergey’s Manual, 7thedition, The Williams and Wil
kins Co., Baltimore, Md, 1957, page 1018', POSL,
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