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Патент USA US3033769

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Patented May’ 8, 1962
ether, aqueous methanol, etc., or it is subjected to one of
the following preliminary treatments:
Distribution between petroleum ether and methanol
water (80:20), the products of the process being enriched
Albert Wettstein, Riehen, and Ernst Vischer, Basel, Swit
zerland, assignors to Ciba Pharmaceutical Products
Inc., Silt, NJ.
No Drawing. Filed Feb. 9, 1060, Ser. No. 7,545
Claims priority, application Switzerland Feb. 12, 1959
1 Claim.
in the methanol phase; treatment of 'a solution of the resi‘
due in a suitable solvent such as, for example, methanol,
ethanol, acetone or the like withan absorption agent, such
(Cl. 195-51)
as ‘active carbon or aluminum oxide. In this way, the
lla-hydroxy-derivatives are obtained in pure form.
The starting materials are new. They can be prepared
The object of the present invention is a high-yield
for example in the following manner:
16-a1kyl-11a:17a:21-trihydroxy-pregnene-3:20 - dione and
A1=4-6¢x-?uoro- or -chloro-,l6-alkyl-llu: l7az2l-trihydroxy
pregnadiene-3z20-diones and their functional derivatives 15
by microbiological methods. These compounds are im
A A517(W935:ZO-diacyloxy-IGu-methyI-pregnadiene of
process for the manufacture of A4-6a—?uoro- or -chloro
the formula '
1 v '
portant intermediates for the manufacture of the corre
sponding A4 and Ali4-6az9a-difluoro- or 6az9a-dichloro
16-alkyl-1118: l7az2l-trihydroxy - pregnadiene-3:20-diones
respectively or of the corresponding ll-oxo-compounds 20v
which are distinguished by an antiin?ammatory, glucocor
ticoid, thymolytic, catabolic, antiandrogenic and antioes- ‘
trogenic activity.
is oxidized with a peracid, if desired the isomeric mixture
It is known that A*- ‘and A1’4-3-keto-steroids, e.g. A1";
of 5 : 6, l7 : 20-dioxido-35 : 20-diacyloxy-l6u-methyl-preg
l6cc-methyl-l7azll-dihydroxy - pregnadiene - 3 :20 - dione,
nane'obtained is treated with‘an oxygen-containing min
can be hydroxylated microbiologically in ll-position.
eral acid, the secondary hydroxyl' group in the resulting
However, in the case of the ?-halogenated derivatives, the "
SazGB-dihydroxy compound is esteri?ed with a sulfonic
’ microbiological introduction of hydroxyl into the ll-posi
acid, treatment is then carried out with an alkaline agent,
tion hitherto presented considerable di?iculties.
the resulting 5on6“ - oxido - 17cc - hydroxy - 20 - ketone is
It has now been found that enzymes of the fungus 30 treated with a hydrogen halide and then with bromine;
Aspergilius ochraceus oxidize A1=4~6a-?uoro- or -chloro
the 21-b'romide thus formed is treated with a salt of an
l6-allryl-, in particular A1‘4-6a-?uoro- or -ChlO1‘O-l6a
‘aliphatic car'boxylic acid of low molecular weight; in the
methyl-17a : 2l-dihydroxy-pregnadiene-3 :20-dione, and A4
resulting 3: 17a-dihydroxy-2l-acyloxy-5a26a - oxido - 16oz
6a-?uoro- or -chloro-l6-alkyl-, in particular A4-6w?uoro
methyl-pregnane, before or after oxidizing the 3-hydroxy
or -chloro-16cc-methyl-l7a:2l-dihydroxy - pregnene-3 :20
to the keto group, the 50::6a-OXid0 group is opened to
dione to give the corresponding Ila-hydroxy compounds
form the 5OL-hYdl‘OXY-G?-?UOI‘OhYdI‘lH or Sa-hydroxy-??
in almost quantitative yield. '
chlorohydrin; the 5a-hydroxyl group is eliminated With
The transformation of the above-mentioned starting
formation of the 4:5-double bond; a 6/8-halogeno com
materials by means of the said fungus,that is with the
pound is isomerized to a 6a-halogeno compound; and if
enzymes obtained therefrom, can be carried out by the 40 desired, a double bond is introduced into the 1:2-position
working methods known per _se for microbiological hy
of the resulting A4-3:20-dioxo-17a-hydroxy-2l-acyloxy
droxylation. The starting materials are generally incu
l6oc-m8thYl-6oc-?1101‘0- or 16a-chloro-pregnene; and/ or in a
bated direct with cultures of the said strains growing under
compound the 2l-acyloxy-group is hydrolized.
aerobic conditions. These cultures are advantageously
The invention is described in the following examples.
moved, i.e. shaken or stirred, and contain assimilable car
The temperatures are given in degrees centigrade. '
bon, in particular corn steep liquor or beer wort, and 45
inorganic salts. Thus, natural, synthetic or semisynthetic
nutrient solutions can be used.
Example 1
The method which is
4 liters of a nutrient solution containing the following
simplest from the practical point of view is described
additions to llliter of tap water: 10 grams of crude
hereinafter, but it is not intended that the invention be
50 glucose, 10 grams of distillers solubles, 5 grams of sodium
limited by these details:
chloride, 1 gram of sodium nitrate and 10 grams of cal
The organisms are cultivated in apparatus under con~
cium carbonate, are prepared. The pH is adjusted to
ditions similar to those known in the manufacture of anti
7.5 and the solution is sterilized in a shaking vesselfor
biotics as the so-called deep tank process. The tempera
30 minutes at 1.1 atmospheres gauge pressure. After
ture is preferably maintained at 24°—27° C. and under
55 cooling, the nutrient solution is inoculated with a culture
these conditions the cultures are fully developed after
1-2 days. The starting material is then added under
of Aspergillus ochmceus (Ciba 924), after which the
vessel is shaken at a temperature of 25° C. After 2 days,
a solution of 1 gram of A4-6a-?uoro-l6a-methyl-170::21
ample in methanol, ethanol, acetone, dioxane or propyl
dihydroxy-pregnene-3:20-dione-2l-acetate in 25 cc. of
ene glycol, either in a single operation or in stages, and
methanol is added to the well-developed culture under
incubation is continued. Finally, the culture ?ltrate is
sterile conditions and shaking is continued for a further
separated from the mycelium, the mycelium is extracted
48, hours at the same temperature. The mycelium is then
if required, for example with methanol or acetone, and
separated, suspended for one hour in 800cc. of Warm
the concentrated extract is added to the culture ?ltrate.
acetone and once more ?ltered off. The acetone ?ltrate
This is extracted with a suitable organic solvent, such as
65 is thereupon strongly concentrated in vacuo and thenv
ethyl acetate, chloroform, ethylene chloride or methylene
combined with the culture ?ltrate. The combined ?ltrates
chloride, the extract is washed with dilute sodium hydro
are extracted three times with 0.8 liter of acetic ester in
gen carbonate solution and with water and ?nally it is
each case and the extracts are then washed with a 2%
sterile conditions in ?ne dispersion or solution, for ex
evaporated. The oxidation product can be isolated from
solution of sodium hydrogen carbonate and with water,
the residue in one of the following Ways: Either the resi 70 dried over sodium sulfate, ?ltered and evaporated to dry
ness in vacuo at a temperature of 30°—35° C. 1.4 grams
due is crystallized direct from a suitable solvent, such as,
for instance, ‘acetone-petroleum ether, methylene chloride
of an oily residue are obtained, the steroid constituent of
7 4
which consistsbaccordingt to, evaluation by paper chroma
tography, almost exclusively of A4-6ot-?uoro-16ot-methy1
Example 3
11a:17a:2l-trihydroXy-pregnene-3:ZO-dione. This 'resi
duais. dissolved in sun, of methanol and-100 cc. oi
0.27 gram of magnesium carbonate, 0.17 gram of am
monium sulfate and 50 grams of crude glucose: to l-liter
of tap Water is'employed, excellent transformation of
of petroleum ether; in each case and after the ‘petroleum
etherphase has been separated oil isevaporated at a tem
perature of 30°—35° in vacuo. "980 mg. of a partially
from aniacetone-‘ether mixture >A4-éa-?uororl6m-methyl
11cc: 1474x722-letrihydrexy-pregneneé :ZO-dione is
monium tartrate, 2.6 grams of tartaric acid, 0.4 gram of
potassium carbonate, 0.4 gram of ammonium phosphate,
petroleum ether audj20 cc. of Water areradded to the solu-.
tion; and the mixture is shaken. ' The methanol phase is
thereupon separated o?, shaken up twice more with 375 cc.
crystalline jresidueare obtained. vBy
V _
If a nutrient solution containing 2.6‘ grams of am~
3 :ZO-dione and of the ‘corresponding 21-acet'ateinto 44-61:
?uoro' - 16oz .- methyl -i 11a: 171x521 - trihydroxy ‘- pregnene
3:2;0-dione is obtained when the remainder of the proce
dure detailed in Examplel isjfollowedq .
; 'What
7 Process for the manufacture of oxygenated steroids by
i ; In similar-manner there isobitained from ‘A1=4-‘6¢§£-flu0ro-v ' 15 microbiological 'l-l-hydroxylation,v wherein ‘a compound
l6u-methyl-l7uz2l - dihydroky-pregnadiene- 3yz20-dione
selected from the group’consisting of A4-6éx'-?u0ro,-16a
ZI-acetate, by crystallization of the crude oxidation prod.
alkyl- 17bcz2l- dihydroxy- pregnene l3 :ZO-diones, A4-6a~
net obtained fromv an acetone-ether mixture, 'AP4-6a- ’ ChlOl‘O-IGoL-HIKYL1706Z21-dlhydl‘GXy-3ZZO-diOIleS, their" 1
?LlOI‘OJGoc-DlethYI-lla:171x221 - trihydroxy - pregnadiene!
3:20-dione in colorless crystals. '
dehydro-derivatives and 21-esters is treated with an
20 enzyme of the fungus Aspergillus ochraceus.
r’ A 4-liter culture of Aspergillus ochraceus (Ciba 924)
' References
the. ?le of this patent
' is prepared as described in Example 1 and a solution of
1 'gram of A4r60L-v?UOI‘O-160L-1I16thY1-170tI21-dlhYdfOXY
25 ’ 2,649,402
pregnene-BzZO-dione in 275 cc. of methanol is added there;
2,905,593’ -
to under sterile conditions. After incubation and work
ing up as indicated‘ in. Examplel'l, homogeneous A4760;
?uoro - 16a - methyl ’-7 11a: 170::21? - trihydroxy'é pregnene
.~.3':20-dione is obtained.‘
so I:
Murray et al __________ __ Aug. 18, 1953
Dulaney et a1. ________ .. Aug. 13, 1957
Dulaneyet a1. -_..; ____ -4- Sept. 22, 1959
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