Microbiological synthesis of 17β-hydroxyandrosta-1,4-dien-3-one

Full text of DOI:10.1023/A:1010470215607

Pharmaceutical Chemistry Journal
Vol. 35, No. 3, 2001
MICROBIOLOGICAL SYNTHESIS OF
17b-HYDROXYANDROSTA-1,4-DIEN-3-ONE
E. V. Popova,1 K. N. Gabinskaya,1 S. D. Shuvalova,1 S. A. Dovzhenko,2
and V. A. Andryushina1
Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 35, No. 3, pp. 53 – 54, March, 2001.
Original article submitted September 21, 2000.
Many drugs possessing steroidal structures are obtained
from sterols, which are first microbiologically transformed
lated to the simultaneous reduction of 17-keto groups and
1,2-double bonds by the Myc. sp. NRRL B 3683 culture [5].
Investigation of the analogous process with Myc. sp. BKM
Ac 1815D showed the absence of 17b-reductase activity with
respect to 1,2-reduced steroids (e.g., AD) and the presence of
such activity with respect to 1,2-dehydrosteroids (such as
ADD) [3].
into
androst-4-en-3,17-dione
(I,
AD)
and
androsta-1,4-dien-3,17-dione (II, ADD) and then chemically
converted into target compounds (e.g., III and IV).
O
OH
We report on the results of investigation of the process of
AD and ADD reduction by Myc. vaccae and Myc. sp. No. 22
cultures, which transform sterols into AD and ADD. We
have established that these strains only weakly modify AD:
98% of the initial amount was isolated after a 24 h incuba-
tion.
;
O
O
IV
II (ADD)
OH
In contrast to the cases described in [3, 5], Myc. vaccae
and Myc. sp. No. 22 cultures only reduce 17-keto groups in
ADD, while not modifying the 1,2-double bond. As a result,
we obtained 1,2-dehydrotestosterone (IV) with a yield of
80% (relative to the cultural liquid) for both strains. In order
to evaluate the transformation results, the cultural liquid (to-
gether with biomass) was extracted with ethyl acetate. Then
ethyl acetate was evaporated and the residue was analyzed by
HPLC. Dehydrotestosterone IV was isolated by chromatog-
raphy and identified based on the results of spectroscopic
measurements and published data.
O
III
The process of microbiological degradation of sterols
was studied in sufficient detail [1]. It should be noted that in-
vestigations of the synthesis of compounds I and II (selecting
strains, media, biotransformation conditions, etc.) were ac-
companied by studying the chemical stability of these prod-
ucts and the possibility of converting them microbiologically
into some other compounds [2, 3].
During the experiments, we selected optimum conditions
for the microbiological transformation process. It was found
that optimum conditions for Myc. sp. No. 22 are provided by
medium 1 (%): glucose, 1; microbe fermentation agent
(MFA), 2; potassium dihydrophosphate, 0.5; ammonium
hydrophosphate, 0.3; magnesium sulfate, 0.02 (pH 7.4). For
Myc. vaccae, the optimum conditions are provided by me-
dium 4 (%): glucose, 1; soybean meal, 1; citric acid, 0.22;
ammonium hydrophosphate, 0.15; magnesium sulfate, 0.02;
iron sulfate, 0.005 (pH 7.0). Somewhat less favorable were
medium 2 (glucose, 0.3; MFA, 1; peptone, 0.3; pH 6.4) and
medium 3 (glucose, 0.3; MFA, 1; potassium hydrophosphate,
0.18; sodium hydrophosphate, 0.4; sodium chloride, 0.2;
pH 7.3). The results of our experiments are summarized in
The early papers reported that cultures of Myc. sp. NRRL
B 3805 and Myc. sp. NRRL 3683 transformed AD, albeit in
insignificant amounts, into 6a-hydroxy-AD; moreover, the
Myc. sp. NRRL 3683 culture produced an estrone as well. At
present, the fact of reduction of the 17-keto groups in
3,17-diketones by Myc. sp. is reliably established [3 – 5].
The process of the 17-keto group reduction was found to de-
pend on the presence of a 1,2-double bond in the initial sub-
strate. The formation of testosterone (III) from ADD is re-
1
Bioengineering Center, Russian Academy of Sciences, Moscow, Russia.
State Scientific Center for Antibiotics, Russian Academy of Medical Sci-
ences, Moscow, Russia.
2
174
0091-150X/01/3503-0174$25.00 © 2001 Plenum Publishing Corporation

Microbiological Synthesis of 17b-Hydroxyandrosta-1,4-dien-3-one
175
TABLE 1. Microbiological Synthesis of 17b-Hydroxyandrosta-
1,4-dien-3-one Using Myc. vaccae and Myc. sp. No. 22 Cultures
agar. The biomass was grown on media 1 – 4 with composi-
tions indicated above.
Medium
(growth
Cultural liquid composition,
% (HPLC data)
The transformation products from the Myc. sp. No. 22
culture were isolated as follows: medium 1 was loaded with
2.0 g/liter II and incubated for 24 h. After transformation,
the content of six flasks (0.25 ´ 6 = 1.5 g) was combined.
The biomass was separated from cultural liquid and treated
with acetone. Then acetone was evaporated and the oily resi-
due triturated with ether, which yielded 0.90 g of
17b-hydroxyandrosta-1,4-dien-3-one (IV) with an admixture
(~5%) of III; yield, 60%. The cultural liquid contains II with
an admixture of I, which can be isolated by extracting with
ethyl acetate.
Substrate
load,
Culture
and trans-
formation)
g/liter
I (AD) II (ADD)
III
IV
Myc.
vaccae
4
2
1.5
1.5
1
8.0
4.0
6.2
5.3
80.0
34.4
0.8
Myc. sp
No. 22
3
1
2
2.0
2.0
2.5
0.8
0.3
3.0
6.0
6.2
8.3
8.6
52.8
80.3
64.0
11.8
10.0
For identification of the individual products, the above
experiment (6 ´ 250 ml) was used to obtain 2.2 g of the
product. According to HPLC data, this product contains 5%
II, 51% IV, 0.7% I, 4.25% III, and 39% biomass. This corre-
sponds to the product (IV) obtained with a yield of 80% (rel-
ative to the cultural liquid). The product was purified by
chromatography on 50 g silica gel eluted stepwise with ben-
zene and benzene – ether (5 and 10%) mixtures. Insignificant
amounts of I and III, as well as of residual II, allows the
product to be identified by TLC and HPLC with reference to
the standard samples [7 – 9].
Table 1. The low content of steroids observed after ADD
transformation by Myc. vaccae in medium No. 2 can be ex-
plained by the degradation of compound IV.
Optimum media for the growth of transforming cultures
are the same as those used for the transformation process.
The amount of inoculated material in the medium must
amount to 30%. We have also studied the role of the method
of substrate introduction and its concentration in the cultural
liquid (see Table 1), as well as of the aeration conditions and
transformation duration. It was established that a useful fac-
tor is an additive of 10% calcium chloride in methanol
(0.6 – 1 ml per 100 ml of substrate). The optimum time of
transformation is 24 h; a further increase in the time is ac-
companied by the reverse process, whereby product IV is ox-
idized to ADD.
The main target product is obtained from the silica gel
column in an amount of 0.5 g; m.p., 172 – 173°C; lmax
,
243 nm (e, 15,700); IR spectrum in chloroform (nmax, cm – 1):
3616 (OH), 1662 (C=O), 1636, 1602 (C=C). Published data
[7]: m.p., 167 – 168°C; lmax, 243 nm (e, 16,100).
17b-Hydroxyandrosta-1,4-dien-3-one (IV) obtained
from II using Myc. vaccae culture was identified by a similar
method.
Thus, our investigation determined optimum conditions
for the microbiological synthesis of dehydrotestosterone us-
ing Myc. vaccae and Myc. sp. No. 22 cultures with a yield of
up to 60 – 65%. Dehydrotestosterone and its esters are used
in medicine as anabolic preparations. These products are
available from foreign companies under the trade names of
boldenone, vebonol, parenabol, and boldenone undecylate.
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