PROCEDE POUR LA PRODUCTION DE LA 4-ANDROSTENE-3,17-DIONE ET DE LA 1,4-ANDROSTARDIENE-3,17-DIONE

PROCESS FOR PRODUCING 4-ANDROSTENE -3,17-DIONE AND 1,4-ANDROSTARDIENE -3,17-DIONE

Abrégé anglais

ABSTRACT OF THE DISCLOSURE
A process for producing 4-androstene-3,17-dione and
1,4-androstadiene-3,17-dione having the general formula
<IMG>
wherein <IMG> symbolizes a single bond or double bond, in which
.alpha.-sitosterol
<IMG>
is fermented with a culture of a microorganism strain capable
of side-chain degradation of sterols.

Revendications

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for producing 4-androstene-3,17-dione
and 1,4-androstadiene-3,17-dione having the general formula
<IMG>
wherein <IMG> symbolizes a single bond or a double bond, in
which .alpha.-sitosterol
<IMG>
is fermented with a culture of a microorganism strain capable
of side-chain degradation of sterols.
2. A process according to claim 1 in which the fer-
mentation is effected with a microorgansim of the species
Arthrobacter, Brevibacterium, Microbacterium, Protaminobacter,
Streptomyces or Mycobacterium.
3. A process according to claim 1 in which .alpha.-
sitosterol is fermented with a microorganism strain of the
species Micobacterium or Norcardia which is capable of side-
chain degradation of sterols.
4. A process according to claim 1 in which .alpha.-
sitosterol is fermented with Mycobacterium spec. NRRL B-=3805,
Mycobacterium spec. NRRL B-3683, Microbacterium pheli NRRL B-
8154 or Micobacterium fortuitum NRRL B-8153.

Description

1302923
The present invention relates to a process for pro-
ducing 4-androstene-3,17-dione and 1,4-androstadiene-3,17-
dione.
According to the present invention there is provided
a process for producing 4-androstene-3,17-dione and 1,4-
androstadiene-3,17-dione having the general formula
,~"~~
0~
wherein ' symboIizes a single bond or a double bond, in
which ~-sitosterol ~ I
/C~' ~
110 ~
~l3H
is fermented with a culture of a microorganism strain capable
of side-chain degradation of sterols.
It is known that numerous microorganisms (as for
example, those of the species Arthrobacter, Brevibacterium,
Microbacterium, Protamlnobacter, Bacill~s, Norcardia, Strepto-
myces and particularly Mycobacterium) have the natural capa-
bility of degrading naturally occurring 3 ~ ~hydroxy- ~ 5-
sterols (such as cholesterol or sitosterol) to carbon dioxide
and water and that 4-androstene-3,17-dione and 1,4-andros-
tadiene-3,17-dione are intermediately formed in this degrada-
tion.
It is also known that by adding inhibitors or
mutated microorganisms it~is possible to so control the degra-
dation of the sterols that a degradation of the 4-androstene-
3,17-dione or 1,4-androstadiene-3,17-dione is avoided (see
- 1 - ~k

i;~O29~3
German Offenlegungsschriften 15 43 269 and 15 9~ 327 and ~.S.
Patent 3 684 657).
For a person skilled in the art it is surprising
that under conventional conditions the side-chain of ~ -
sitosterol is also degraded since it is known that the side-
chain degradation of sterols is caused by a very complex
fermentation system and it could not be expected that all the
enzymes cooperating in the side-chain degradation of natural
steroids also have the capability of causing the side-chain
degradation of this compound. Furthermore, it could not be
predicted that in this degradation the ~ 7 double bond of the
~-sitosterol hydrogenates and that the methyl group in the 4
position is split.
Apart from the use of other starting compounds the
process according to the present invention is carried under
the same fermentation conditions as those used in conventional
microbiological side-chain degradation reactions of sterols.
According to the present invention the fermentation
is,carried out by using the microorganisms culture usually
applied for the side-chain degradation of sterols. Suitable
cultures are, for example, bacteria cultures capable of side-
chain degradation of sterols, i.e., of the species Arthrobac-
ter, Brevibacterium, Microbacterium, Protaminobacter, Strepto-
myces or particularly of the species Mycobacterium. The fol-
lowing microorganisms are suitable: Microbacterium lactum IAM-
1640, Protaminobacter alboflavus IAM-1040, Bacillus roseus
IAM-1257, Bacillus spharicus ATTC-7055, Norcardia gardneri
IAM-105, Norcardia minima IAM-374, Norcardia corallina IFO-
3338, Streptomyces rubescens IAM-74 or particularly the
microorganisms Mycobacterium avium IFO-3082, Mycobacterium
pheli IFO-3158, Mycobacterium pheli (Institut fur Gesund-
heitswesen, Budapest No. 29), Mycobacterium pheli ATCC-354,

1302923
Mycobacterium smegmatis IF0-3084, Mycobacterium smegmatis
ATCC-20, Mycobacterium smegmatis (Institut fur Gesundheitswe-
sen, Budapest No. 27), Mycobacterium smegmatis ATCC-19979 and
Mycobacterium fortuitum CBS-49566.
Particularly preferred microorganisms are Mycobac-
terium spec. NRRL B-3805, Mycobacterium spec. B-3683, Mycobac-
terium pheli NRRL B-8154 and mycobacterium fortuitum NRRL s-
8153. With the aid of these microorganisms, the fermentation
of c~-sitosterol can be carried out without using additional
agents for inhibiting the 9-hydroxylation.
Under the culture conditions conventionally used for
these microorganisms submerse cultures are grown in a suitable
nutrient medium while aerating. The substrate (dissolved in a
suitable solvent or preferably in the emulsified form) is then
added to the cultures and the fermentation is carried on until
a maximal conversion of the substrate is attained.
Suitable substrate solvents are, for example,
methanol, ethanol, glycol monomethyl ether, dimethyl formamide
or dimethyl sulphoxide. The emulsification of the substrate
can be carried out, for example, in that it is in;ected in a
micronized form or dissolved in solvent miscible with water
(such as methanol, ethanol, acetone, glycol monomethyl ether,
dimethyl formamide or dimethyl sulphoxide) with intense turbu-
lence in (preferably decalcified) water contalning the usual
emulsifying aids. Non-ionogenic emulsifiers, as for example,
ethyleneoxy adducts or fatty esters of polyglycols, are suit-
able emulsifying aids. The commercial wetting agents
Tegin(R), Tween(R) and Span(R) are examples of suitable emul-
sifiers.
The optimal substrate concentration, the time of
substrate addition and the fermentation time depend on the
structure of the substrate used on the type of microorganism

13029~3
used. AS generally required in microbiological steroid con-
versions these parameters must be determlned in the individual
case by preliminary tests with which the person skilled in the
art is familiar.
As is well known the 4-androstene-3,17-dione deriva-
tives having the general formula I and producible by means of
the process according to the present invention are valuable
intermediate products which are today used for the synthesis
of pharmacologically active steroids.
The process according to the present invention will
be illustrated by the following Examples.
Example l
A 2-litre Erlenmeyer flask with 500 ml of sterile
nutrient medium containing 1% of yeast extract, 0.45% of
disodium hydrogen phosphate, 0.34~ of potassium dihydrogen
phosphate and 0.2% of Tween(R)80 - adjusted to pH 6.7 - is
inoculated with a suspension of Mycobacterium spec. NRRL B-
3805 dry culture and shaken for 3 days at 30C with l9O revo-
lutions per minute.
Ten Erlenmeyer flasks (500 ml) each with 100 ml of
nutrient medium containing 2.5% of cornsteep liquor, 0.3% of
diammonium hydrogen phosphate, 0.25% of soybean mean and 0.25%
of Tween~R)80 - adjusted to p~ 7.0 - are inoculated with 5 ml
of Myobacterium spec. growth culture and shaken for 24 hours
at 30C with 220 revolutions per minute, whereupon 100 mg of
~ -sitosterol dissolved in 3.0 ml of dimethyl formamide are
added to each culture. The fermentation is then carried on
for further 96 hours at 30C.
The combined cultures are extracted with ethylene
chloride, the extract is concentrated in vacuo and the residue
is chromatographed via a silica gel column. On recrystalliz-
ing from diisopropyl ether 160 mg of 4-androstene-3,l7-dione

1302923
are obtained.
In addltion 385 mg of non-reacted ~-sitosterol are
recovered.
Example 2
Under the conditions of Example 1 but using
Mycobecterium spec. NRRL B-3683 a total of 1000 mg of ~-
sitosterol is reacted and further treated. 140 mg of 1~4-
androstadiene-3,17-dione are obtained in addition to 630 mg of
non-reacted q-sitosterol.