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Патент USA US3031484

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iillnite States Patent
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3,031,477
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benzoic and 0-, m- or p-toluic acid) can be used in lieu
of known salt-retaining steroids, and may be administered
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A4-PREGENE-7ot,17oc,21-TRI0L-3,20-DI0NE
AND ESTERS THEREOF
Patented Apr. 24, 1962
2
1
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3,031,477
ice
parenterally in the treatment of Addison’s disease, being
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Richard W. Thoma, Somerville, and-Josef Fried, Prince
ton, N.J., assignors to Olin Mathieson Chemical Cor
poration, New York, N.Y., a corporation of Virginia
No Drawing. Original application Feb. 24, 1956,.Ser.
_. formulated for such administration in the same type of
preparations as desoxycorticosterone acetate, for example,
with concentration and/ or dosage based on the activity
of‘the particular steroid.
The esters of A4-pregnene-7a,l7u,21-triol-3,20-dione
No. 567,478, now Patent No. 2,960,513, dated Nov.
are prepared in the usual manner, as by treatment with
15, 1960. Divided and this application Apr. 5, 1960,
10 the desired acid anhydride or acyl halide in an organic
Ser. No. 20,036
3 Claims. (Cl. 260-39747)
solvent (preferably an organic base such as pyridine)
to yield either the 2l-mono ester or the 7a,21-diester de
This application is a division of Serial No. 567,478,
pending on the ratio of acylating agent to steroid present
?led February 24, 1956, now Patent No. 2,960,513.
in the reaction mixture. A4-pregnene-7a,17a,2l-triol
This invention relates to, and has for its object,vthe
provision of the following steroids: A4-pregnene-7a,l7a, 15 3,20-dione can also, if desired, be ‘dehydrated in the
usual manner, as by treatment with a base (e.g., meth
2l-triol-3,20-dione (7a-hydr0xy-Compound S) and its
esters, particularly esters thereof with organic hydrocar
anolic potassium hydroxide) to give A‘lrs-pregnadiene
17a,21-diol-3,20-dione, which can then, if desired, be
bon carboxylic acids of less than ten carbon atoms, and
tothe preparation of M-CompoundS (i.e., Abs-pregna
diene-l7u,21-diol-3 ,20-dione) .
A4-pregnene-7a,17a,21-triol-3,20~dione, useful as an in- '
esteri?ed to the 2.1-ester. Atrs-pregnadiene-17a,21-diol
20 3,20-dione and its 21-esters are also useful as mineralocor
ticoids. Hence, they can be used in lieu of known salt
retaining steroids and may be administered parenterally
termediate in the preparation of other steroids, is prepared
'in the treatment of Addison’s disease.
The following examples are illustrative of the inven
by subjecting A4-pregnene-17a,21-diol-3,20-dione (Com
pound S) to the action of enzymes of the microorganism
Diplodia natalensis under oxidizing conditions.
This 25
tion:
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oxidation can best be e?ected by either including the
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‘EXAMPLE, 1
steroid in an aerobic culture of the microorganism, or by
A4-Pregnene-7a,1
7a,21 -Tri0l-3,20-Di0ne
bringing together, in an aqueous medium, the steroid, air
(a) Fermentation.—-A fermentation medium of the
and enzymes of non-proliferating cells of the microorga
30 following composition is prepared:
'nisrn.
G.
In general, the conditions of culturing Diplodia na
talensis for the purposes of this invention are (except
Dextrose ___
10
Corn steep liquor
___
6
for the inclusion of the steroid to be converted) the
same as those of culturing various other molds for the
production of antibiotics and/ or ribo?avin, i.e., the micro 35
organism is aerobically grown in contact with (in or on)
a suitable fermentation medium. A suitable medium
NH4H2PO4 _____________________________ -_'__..__
3
CaCO3 ____________________________________ __ 2.5
Yeast
extract
Soybean oil
_____
_ 2.5
______ .. 2.2
essentially comprises a source of carbon and energy.
The latter may be a carbohydrate (such as sucrose,
Distilled water to make one liter.
distillers solubies) or synthetic (i.e., composed of simple,
synthesizable organic or inorganic compounds such as
of water (with 0.0l%-Duponol as wetting agent) to
suspend the sporulated growth of a 3 month old
Sabouraud dextrose agar slant '(4 parts dextrose, 1 part
molasses, glucose, maltose, starch or dextrin), a fatty 40 The pH of the medium is adjusted to 70:01 with
acid, a fat and/or the steroid itself. Preferably, how
2 N NaOH solution, and 50 ml. portions of the medium
ever, the medium includes an assirnilable source of car
are distributed in six 250 ml. ?asks, the ?asks plugged
with cotton and sterilized by autoclaving for 30 minutes
bon and energy in addition to the steroid.
The source of nitrogenous factors may be organic (e.g.,
at 120° C. When cool, each of the ?asks is inoculated
soybean meal, corn steep liquor, meat extract and/or 45 with 1.0 ml. of a suspension prepared by usingv7.0 ml.
ammonium salts, alkali nitrates, amino acids or urea).
An adequate sterile-air supply should be maintained
' neopeptone and 1.5 parts agar to 100 parts water) cul
during fermentation, for example bythe conventional
50 ture of Diplodia natalensis ATCC No. 9055. or derived
methods of exposing a large surface of the medium to
strains; the parent organism is obtainable, inter alia, from
the American Type Culture Collection, Washington, DC.
air or by utilizing submerged aerated culture. The steroid
may be added to the culture during the incubation period,
or included in the medium prior to sterilization or inocula
The ?asks are then mechanically shaken for 69 hours
at 25° C. on a 280 cycle per minute rotary shaker, after
tion. The preferred (but not limiting) ,range of con 55 which about 9% (v./v.) is transferred to each of 51
centration of the steroid in the culture is about 0.01 to
?asks containing 50 ml. of the same medium. After 48
0.10%. The culture period may vary considerably, the
hours’ incubation, a total of 638 mg. of At-pregnene-lh;
range of about 6 to 96 hours being feasible, but not
21-diol-3,20-dione in 25.5 ml. of methanol (to give 0.25
mg. of steroid per ml. in the fermentation vessel) is
limiting.
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The process yields, inter alia, A4-pregnene-7a,l7a,21
triol-3,20-dione, a steroid useful not only as an inter
mediate in the preparation of other steroids, but also in
common with its mono and diester derivatives, as a
60 added.
The ?asks are then incubated an additional 24
hours, after which the ?asks are harvested, and the con
tents ?ltered through a Seitz pad and washed with water
give a ?nal volume of ?ltrate and washings of 2480 ml.
([2) Isolation of A4 - pregnene - 7ot,17oc,21 - triol - 3,20
mineralocorticoid (i.e., an agent causing the retention of 65
di0ne.--The culture ?ltrate (2480 ml.) is extracted with
sodium and excretion of potassium). Hence, A4-pregnene
three 1500 ml. portions of chloroform and the combined
7u,l7u,2l-triol-3,20-dione and esters thereof, particularly
extracts evaporated to dryness in vacuo. The crystalline
esters with organic hydrocarbon carboxylic acids of less
residue (about 438 mg.) is Washed with hexane and
than ten carbon atoms (e.g., the lower alkanoic acids as
exempli?ed by acetic, propionic and enanthic acid, the 70 recrystallized from 95% alcohol. 125 mg. of A‘i-preg
nene-7a,17a,2l-triol—3,20-dione is obtained, which melts
aralkanoic acids as exempli?ed by oc-toluic and ?-phenyl
at about 245-247 ° C. and which after additional recrys
propionic, and the aromatic acids as exempli?ed by
sham???‘
tallization, melts at about 248+250° C.; M1132? +97°
4
A‘i-pregnene-h,17a,217triolf3,20-dione can be .dehy(
drated as illustrated by the following example:
( c., 0.3 in 95% alcohol);
Age, 241 m,‘ (€=16,00_Q) ; x9135?‘ 2.90”, 3.00,‘ and 3.08“
EXAMPLE 3
(OH), 5.91,. (20-lreto), 6.10”, 6.20“ (A4-3-keto)
AQ-yPrpegnadiena-l 79;,21 -D fol-3,20-Dgig0ne
Analysis.-—Calcd. for 021113005 (362.45): C, 69.58; H,
8.34. Found: C, 69.76; H, 8.41.
A solution of 50 mg. of A47pregnene-7u,17a,21-triol
3,20-dione-in 2 ml. of 2.5% methanolic potassium hy
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A4-pregnene-7a,17a,21-triol-3,2O-dione can be esteri?ed
as illustrated by the following example:
droxide is allowed to stand at room temperature for 24
hours. During this period of time, the ultraviolet maxi
mum at 241 mu gradually decreases'and gives way to a
‘maximum at 285'mp characteristic of the A4'6-3-ketone
A4-Pregnene-7u,1 7 a,21EXAMPLE
-Triol-3,20-Dione 7a,,21_-Diacetat_e
grouping. 'The'isolutioin is neutralized with dilute acetic
A solution of 20 mg. of A4-pregnene-7a,17¢,21-triol
acid, water is added and the methanol is evaporated in
3,20-dione in 0.5 ml. of pyridine and 0.5 ml. of'acetic
vacuo. .The resulting suspension is extracted with chloro
anhydride is allowed to stand overnight at room tem 15 form, the chloroform removed in vacuo and the resulting
perature. Removal of the excess reagents in vacuo leaves
residue crystallized from acetone-hexane to give pure
A4’6-pregnadiene-17a,21-diol-3,20-dione.
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a crystalline residue (about 26 mg.) which after two re
crystallizations from acetone-hexane furnishes the pure
The invention may be variously otherwise embodied
A4-pregnene-7a,17a,21-triol-3,20~dione diacetate having
the following properties: MP. about 180—182° C; [@1323 20
+39° (_c., 0.40 in CHCl3);'
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What is claimed is:
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Found: C, 67.63; H, 7.70.
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Similarly by substituting other acid anhydrides, such
2. A4 - pregnene - 7a,17a,21 - triol - 3,20 - dione 70;,21
diacetate.
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duced. Furthermore, if only one mole equivalent of
acetic anhydride is used in the procedure of'Example 2,
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3. A compound selectedvfrom the group consisting of
I the 7oc-monoesters, 21-monoesters and 7a,2l-diesters of
A4-pregnene-7a,17a,21-tri01-3,2O-dione, wherein the ester
is derived from an organic hydrocarbon carboxylic acid
of less than ten carbon atoms.
as propionic anhydride, or acyl halides, such as benzoyl
chloride, for the acetic anhydride in the procedure of 30’
References Cited in the ?le of this patent
Example 2, the corresponding ester derivatives are pro
the 21-monoacetate is obtained.
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1. A4-pregnene-7a,117u,2l-triol-3,20-dione.
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A313}, 238 mil (e=15,500); $31?‘ 2.8811 (OH); 575p, 5.79;‘;
(aoetyl and ZO-keto), 6.00M,'6.18/-L (A4-3-keto)
Analysis.-Calcd. for C25H3407 (446.52)’: C, 67.24; H,
7.68.
within the scope of the appended claims.
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UNITED STATES PATENTS
2,753,290
2,783,255
Fried et al. ___________ __ July 3, 1956
)Shull et al. ..________ _____ Feb. ‘26, 1957
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