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Патент USA US3098807

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United States Patent 0 f’ice
16a-hydroxyprednisolone). If a steroid of the andro
stane series is hydroxylated, the l6a-hydroxy derivative is
also formed as exempli?ed by 16a-hydroxy-A4-andro
stene-3,17-dione and 16a-hydroxytestosterone. If a
steroid of the estrane series is hydroxylated, the 160:
hydroxy derivative is also formed as exempli?ed by 160:
Allen I. Laslnin, Somerset, NJ” msignor to Olin Mathis
son Chemical Corporation, New York, N.Y., a cor
poration of Virginia
No Drawing. Filed (Jet. 19, 1961, Ser. No. 146,346
6 Claims. (ill. 1595-51)
hydroxyestrone and 16a-hydroxy estriol.
This invention relates to, and has for its object the
provision of, a method for producing 16-hydroxylated
steroids, and more particularly to a microbial process for
converting a 16-unsubstituted steroid to its 16-hydroxy
Patented July 23, 1963
The action of the enzymes of Streptomyces californicus,
Streptomyces coelicolor, or Strepwmyces vimzceus to pro
duce 16u-hydroxy steroids can be utilized either by in
cluding the steroid in an aerobic culture of the microor
ganism, or by bringing together, in an aqueous medium,
the steroid, air, and enzymes of non-proliferating cells of
the microorganism.
It has been found that steroids having a methylene
In general, the conditions of culturing the Streptomyces
group in the 16-position, especially 3-keto or 3-hydroxy 15 for the purposes of this invention are (except for the in
steroids, or protected derivatives thereof, either of the
clusion of the steroid to be converted) the same as those
androstane (including etiocholane and androstene) series
of culturing various Streptomy-ces for the production of
or pregnane (including allopregnane, pregnene and preg
antibiotics and/ or vitamin B12, i.e., the microorganism is
nadiene) series, the 3,20-diketo steroids of the pregnane
aerobically grown in contact with (in or on) a suitable
series being particularly preferred, can be converted into
fermentation medium. A suitable medium essentially
useful 16a-hydroxy derivatives by subjecting them to the
comprises a source of nitrogenous and growth-promoting
action of enzymes of Streptomyces californicus, Strepto
factors, and an assimilable source of carbon and energy.
myces coelicolor, or Streptomyce‘s vinaceus or to the
action of the organisms themselves, under oxidizing and
preferably aerobic conditions.
Among ‘the steroids which may be oxygenated by the
practice of this invention are those steroids unsubstituted
in the 16-position, which are members of: the andro
stane series, which series includes the androstene and
etiocholane series; the estrane series, which series in
cludes the estratriene series; and the pregnane series,
which series includes the allopregnane, pregnene and
pregnadiene series. Of these, the steroids of the pregnane
The latter may be a carbohydrate and/or the steroid it
self. Preferably, however, the medium includes an assimi
lable source of carbon and energy in addition to the
The nitrogen source materials may be organic (e.g.
soybean meal, cornsteep liquor, meat extract and/ or dis
tillers solubles) or synthetic (i.e., composed of simple,
synthesizable organic or inorganic compounds such as
ammonium salts, alkali nitrates, amino acids, urea or
As to the source material, lipids, especially (1) fatty
series, and more particularly the 3,20-diketo steroids of
acids having at least 14 carbon atoms, (2) fats or (3)’
the pregnane series are preferred. Examples of suitable
mixtures thereof, may be used. Examples of such fats
steroids of the pregnane series include progesterone;
are lard oil, soybean oil, linseed oil, cottonseed oil, pea
pregnenolone; pregnanolone; 95,11?-oxido progesterone;
nut oil, fancy mutton ta-llow, sperm oil, olive oil, tri
95,11,8-oxidodesoxy \corticosterone; 11,13,12?-oxidoproges
stearin, tripalmitin, triolein and trilaurein, and illustra
tive fatty acids include stearic, palmitic, oleic, linoleic
and myristic acids.
terone; 115,12/3-oxidodesoxy corticosterone; hydroxylated
progesterones, such as 2a,6cz,6?,7,8,9,11a,l1{3,12oc,14,15oc,
and IS?-hydroxyprogesterone; halogenated progesterone,
Other carbon-containing materials may also be used;
For example, such materials as glycerol, glucose, fruc
tose, sucrose, lactose, maltose, dextrins, starches, whey,
such as 21-chloroprogesterone; ‘aldosterone; corticoster
one; ll-desoxycorticosterone; 17a-hydroxy-1l-desoxycor
ticosterone (Reichstein’s compound S); hydrocortisone;
prednisolone; 9oc-halohydrocortisones (e.g., 9a-?uorohy
drocortisone); 9a-halo-prednisolones (e.g., 9a-?uoropred—
nisolone); 6,9a-dihalohydrocortisones (e.g., 60¢,9oc-di
fluorohydrocor-tisone); and 6,9a-dihaloprednisolones (e.g.,
-etc., are adequate carbon source ‘materials. These ma
terials may be used either in puri?ed state or as concen
trates,'snch as whey concentrate, corn, wheat or barley
mash; or mixtures of the above may be employed. It is
to be noted, however, that the steroid is added to the fer
60¢,9a-di?uoroprednisolone). As indicated before, how
mentation medium essentially as a precursor and not as
ever, although the preferred starting materials are steroids
of the pregnane series, the process of this invention is a
general one which may be employed to l6a~hydroxylate
an energy source.
other classes of steroids such as those of the androstane
testosterone; and those of the estrane series, as exempli
vention are the l6a-hydroxy derivatives of the pregnane
series, preferably the l6a-hydroxy-3,ZO-diketo-steroids of
series, as exempli?ed by A4-androstene-3,17-dione and 55
?ed by estrone and estradiol.
Among the steroids formed by the process of this in
The following examples are illustrative of the inven
(a) Fermentation
Surface growth from each of ?ve two-week old agar
slant cultures of Streptomyces californicus, WC 3312
the pregnane series. Examples of resulting products of 60 (Culture Collection, Institute of Microbiology, Rutgers
University, New Brunswick, New Jersey), the slant con
the 16or-hydroxy pregnane series include 16a-hydroxy
taining as a nutrient medium (A) :
progesterone; the 16e-hydroxy derivatives of hydroxylated
progesterone; the 16a-hydroxy derivatives of halogen
ated progesterone; 16ix-hydroxyaldosterone; 16a-hydroxy
corticosterone; 16a-hydroxy-1l-desoxycorticosterone; 16a,
17oc-dihydroxy-1l-desoxycorticosterone; 16a - hydroxyhy
K2HPO4 _____ __
drocortisone; l6a-hydroxyprednisolone; 9u-halo-16a-hy
Agar ______________________________________ _..
droxyhydrocortisones (e.g., 9a-?uoro-16ot-hydroxyhydro
cortisone); 9a-halo-16a-hydroxyprednisolones (e.g., tri
amcinolone); 6,90; - dihalo - 16cc - hydroxyhydrocortisones
(e.g., 6a,9¢x-di?uoro-16ot-hydroxyhydrocortisone); and 6,
9u-dihalo-16a-hydroxyprednisolones (e.g., 6a,9oc-di?u0r0—
65 Yeast extract ______________________________ __ 2.5
Distilled water to 1 liter.
is suspended in 2.5 ml. of a 0.01% sodium lauryl sul
phate aqueous solution. One ml. portions of the suspen~
sion are used to inoculate ten 250 m1. conical ?asks, each
containing 50 ml. of the following sterilized medi
Example 6
Following the procedure of Example 1, but substitut
um (B):
ing an equivalent amount of androstenedione for the
Soybean meal ______________________________ __ 20
Soybean oil ________________________________ __ 2.2
CaCO3 ____________________________________ __ 2.5
progesterone, 16a-hydroxy-A4-androstene - 3,17 - dione is
After 72 hours’ incubation at 25° C. with continuous
ing an equivalent amount of prednisolone for the pro
____ __' _____________________________ __
Example 7
Following the procedure of Example 1, but substitut
Distilled water to 1 liter.
rotary agitation (280 cycles per minute; 2 inch radius), 10 gesterone, Mot-prednisolone is obtained.
10% (vol/vol.) transfers are made to 67 2-50 ml. coni
Example 8
cal ?asks each containing 50 m1. of fresh sterilized medi
Following the procedure of Example 1, but substitut
um B plus 300 micrograms/ml. of progesterone. The
ing an equivalent amount of 9a-?uoroprednisolone for
steroid is added by supplementing each ?ask with 0.25
ml. of a sterile solution of the steroid in N,N-dimethyl 15 the progesterone, triamcinolone is obtained.
formamide containing 60 mg./ml. ‘of steroid. After 48
Example 9
hours of further incubation, the contents of the ?asks are
pooled and ?ltered through a Seitz clarifying pad. The
Following the procedure of Exampde 1, but substitut
?asks, mycelium and pads are washed with successive 50
ing an equivalent amount of 9a-?uorol1ydrocortisone for
ml. portions of warm water.
20 the progesterone, 9a-?uoro-16a-hydroxyhydrocortisone is
(b) Isolation of J6a-Hydroxyprogester0ne
Example 10
The combined ?ltrate and washings are extracted with
Following the procedure of Example 1, but substitut
chloroform. The chloroform extracts are then washed
with water and evaporated to dryness in vacuo. The re 25 ing an equivalent amount of estrone for the progesterone,
16a-hydroxyestrone is obtained.
Similarly, any other l6-unsubstituted steroid of the
sulting crude product is crystallized from acetone-hexane
to give 16a-hydroxyprogesterone.
pregnane, androstane or estrane series can be converted
Example 2
by Streptomyces californicus, Streptomyces coelz'color or
Following the procedure of Example 1, but substitut 30 Streptomyces vinaceus to its 16a-l1ydroxy derivative.
This invention may be variously otherwise embodied
within the scope of the appended claims.
What is claimed is:
ing a culture of Streptomyces coelicolor, WC 3593, for
the Streptomyces californicus in part a, l6a-hydroxy
progesterone is isolated.
l. The method ‘of converting a steroid into a 16a
Example 3
Following the procedure of Example 1, but substitut
ing a culture of Streptomyces vinaceus, ATCC 11861
(American Type Culture Collection, Washington, D.C.),
hydroxy derivative thereof, which ‘comprises subjecting a
steroid selected from the group consisting of a steroid
of the pregnane series, a steroid of the androstane series
> and a steroid of the estrane series to the action of enzymes
of a microorganism of the class consisting of Streptom'yces
for the Streptomyces calij’ornicus in part a, 116oz-hydroxy 40 californicus, Streptomyces coelicolor and Streptomyces
progesterone is isolated.
Example 4
Following the procedure of Example 1, but substitut
ing an equivalent amount of Reichstein’s compound S ace
tate for the progesterone, 16a-hydroxy compound S is
vinaceus, under oxidizing conditions, and recovering the
IGa-hydroxy steroid formed.
2. The method of claim 1 wherein the steroid is of the
pregnane series.
3. The method of claim 1 wherein ‘the steroid is of the
androstane series.
4. The method of claim 1 wherein the steroid is of the
estrane series.
Example 5
5. The method of claim 1 wherein the steroid is a 3
Following the procedure of Example 1, but substitut 50 ketosteroid.
‘ing an equivalent amount of desoxycorticosterone acetate
for the progesterone, 16whydroxy-desoxycorticosterone
is obtained.
Similarly, S. ycoelicolor WC 3593 and S. vinaceus
ATCC 11861 convert desoxycortioosterone acetate to
6. The method of claim 1 wherein the steroid is proge
References Cited in the ?le of this patent
Fried et al. ____________ __ Oct. 7, 19-58
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