Pregnane derivatives and method of producing the same

There are provided 12-hydroxy-.DELTA..sup.4 or .DELTA..sup.1,4 -pregnan-3-one-20-carbaldehyde and microbial method of producing the same. The compounds are novel and useful as starting materials for the synthesis of corticoids, typically prednisone, prednisolone and hydrocortisone, which have antiinflammatory activity.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to pregnane derivatives of the general formula 
##STR1## 
wherein the dotted line --- represents the possibility of there being a 
double bond and wavy line indicates that the hydroxyl group is either in 
the .alpha.- or in the .beta.-configuration, and a method of producing the 
same with the aid of a microbe. 
2. Description of the Prior Arts 
The pregnane derivatives of the general formula (I) as provided by the 
present invention are novel compounds not yet described in the literature 
buy obtainable by allowing a specific microbe to act upon deoxycholic acid 
and/or a salt thereof. These compounds can be used as starting materials 
for the synthesis of excellent antiinflammatory corticoids represented by 
prednisone, prednisolone, hydrocortisone, etc.. 
The so-far known process for producing prednisone starts with deoxycholic 
acid and involves twenty-odd steps [L. F. Fieser and M. Fieser: Steroids, 
pages 634-647, Reinhold, 1959]. However, as the reagents required are 
expensive and the process is time-consuming, the process is not fully 
satisfactory for industrial application. 
An object of the present invention is to provide novel pregnane derivatives 
which are useful as starting materials for the synthesis of various 
corticoids. 
Another object of the present invention is to provide novel pregnane 
derivatives which are useful as advantageous starting materials for the 
synthesis of prednisone, prednisolone and hydrocortisone. 
A further object of the present invention is to provide a method of 
producing the above-mentioned novel and useful pregnane derivatives. 
These objects as well as other objects and advantages of the present 
invention will become apparent to those skilled in the art from the 
following detailed description. 
SUMMARY OF THE INVENTION 
The present invention provides the novel pregnane derivatives of the 
general formula (I) shown above. 
The present invention also provides a method of producing the pregnane 
derivatives of the general formula (I) which comprises cultivating, in a 
medium containing deoxycholic acid and/or a salt thereof, a microbe 
belonging to the genus Alcaligenes or Pseudomonas and being capable of 
producing the pregnane derivatives of the general formula (I) by utilizing 
deoxycholic acid and/or a salt thereof as the substrate. 
DETAILED DESCRIPTION OF THE INVENTION 
The microbes to be used in the present invention may be wild-type strains 
of the genus Alcaligenes or Pseudomonas or mutants thereof obtained by 
natural mutation or a conventional mutagenic treatment such as x-ray 
irradiation, ultraviolet irradiation, treatment with a chemical mutagen 
such as N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitroquinoline-N-oxide, 
acriflavine or ethyl methanesulfonate, or combination thereof and the 
like. 
Among the microbes obtained by the present inventors and being capable of 
producing the pregnane derivatives of the general formula (I) by utilizing 
deoxycholic acid or a salt thereof as the substrate, the representatives 
have been deposited with the Fermentation Research Institute, Agency of 
Industrial Science and Technology, Japan (hereinafter, referred to as 
FERM). They are the strains Alcaligenes faecalis D4020-K15 (FERM BP-204) 
and Pseudomonas putida D4014-A357 (FERM BP-206). Alcaligenes faecalis 
D4020-K15 is a mutant of Alcaligenes faecalis D4020 (FERM BP-182) which is 
a wild-type strain isolated from soil, and Pseudomonas putida D4014-A357 
is a mutant of Pseudomonas putida D4014 (FERM BP-205) which is a wild-type 
strain isolated from soil. 
The morphological, cultural and physiological characteristics of these 
strains are shown in Table 1 and Table 2. 
TABLE 1 
__________________________________________________________________________ 
Alcaligenes faecalis 
Alcaligenes faecalis 
Taxonomical character D4020 D4020-K15 
__________________________________________________________________________ 
Morphological characteristics 
Form Rods Rods 
Size 0.5 .times. 1.2.about.1.7.mu. 
0.5 .times. 1.0.about.1.7.mu. 
Flagellum Peritrichous flagella 
Peritrichous flagella 
Spore Nil Nil 
Gram stain Negative Negative 
Acid fast stain Nil Nil 
Cultural characteristics 
Bouillon agar plate culture Circular, opaque, convex 
Circular, opaque, convex 
Bouillon agar slant culture Moderate growth, filiform, 
Moderate growth, filiform, 
pigment not produced 
pigment not produced 
Bouillon broth Moderate turbidity, 
Moderate turbidity 
pellicle 
Temperature for growth Growth at 37.degree. C., poor 
Growth at 37.degree. C., poor 
growth at 41.degree. C. 
growth at 41.degree. C. 
Gelatin stab No liquefaction 
No liquefaction 
Litmus milk Alkaline, milk unchanged 
Alkaline, milk unchanged 
BCP milk Alkaline, milk unchanged 
Alkaline, milk unchanged 
Physiological characteristics (Note 1) 
Nitrate reduction + + 
Denitrification - - 
Methyl red test - - 
Voges-Proskauer test - - 
Indole production - - 
Hydrogen sulfide production - - 
Starch hydrolysis - - 
Citrate utilization + + 
Assimilation of inorganic nitrogen sources 
+ + 
Urease .+-. .+-. 
Oxidase + + 
Catalase + + 
Require of oxygen Aerobic Aerobic 
Oxidation/Fermentation test Oxidative Oxidative 
Production of acids and gases from carbohydrates (Note 2) 
Production 
Evolution 
Production 
Evolution 
of acids 
of gases 
of acids 
of gases 
(1) L-Arabinose + - + - 
(2) D-Xylose + - + - 
(3) D-Glucose + - + - 
(4) D-Mannose + - + - 
(5) D-Fructose - - - - 
(6) D-Galactose + - + - 
(7) Maltose - - - - 
(8) Sucrose - - - - 
(9) Lactose - - - - 
(10) Trehalose - - - - 
(11) D-Sorbitol - - - - 
(12) D-Mannitol - - - - 
(13) Inositol - - - - 
(14) Glycerol - - - - 
(15) Starch - - - - 
__________________________________________________________________________ 
TABLE 2 
__________________________________________________________________________ 
Pseudomonas putida 
Pseudomonas putida 
Taxonomical character D4014 D4014-A357 
__________________________________________________________________________ 
Morphological characteristics 
Form Rods Rods 
Size 0.5.about.0.6 .times. 1.5.about.2.9.mu. 
0.5 .sup..beta. 1.5.about.2.5.mu. 
4 
Flagellum Polar flagella 
Polar flagella 
Spore Nil Nil 
Gram stain Negative Negative 
Acid fast stain Nil Nil 
Cultural characteristics 
Bouillon agar plate culture Circular, raised, convex, 
Circular, raised, convex, 
smooth, entire 
smooth, entire 
Bouillon agar slant culture Moderate growth, filiform, 
Moderate growth, filiform, 
translucent, fluorescent 
translucent, fluorescent 
Bouillon broth Turbid, pellicle 
Turbid 
Temperature for growth Growth at 37.degree. C. 
Growth at 37.degree. C. 
Gelatin stab No liquefaction 
No liquefaction 
Litmus milk Alkaline, milk unchanged 
Alkaline, milk unchanged 
BCP milk Alkaline, milk unchanged 
Alkaline, milk unchanged 
Physiological characteristics (Note 1) 
Nitrate reduction - - 
Denitrification - - 
Methyl red test + + 
Voges-Proskauer test - - 
Indole production - - 
Hydrogen sulfide production - - 
Starch hydrolysis - - 
Citrate utilization + + 
Assimilation of inorganic nitrogen sources 
+ + 
Urease .+-. .+-. 
Oxidase + + 
Catalase + + 
Arginine dihydrolase + + 
Require of oxygen Aerobic Aerobic 
Oxidation/Fermentation test Oxidative Oxidative 
Production of acids and gases from carbohydrates (Note 2) 
Production 
Evolution 
Production 
Evolution 
of acids 
of gases 
of acids 
of gases 
(1) L-Arabinose + - + - 
(2) D-Xylose + - + - 
(3) D-Glucose + - + - 
(4) D-Mannose + - + - 
(5) D-Fructose - - - - 
(6) D-Galactose + - + - 
(7) Maltose - - - - 
(8) Sucrose - - - - 
(9) Lactose - - - - 
(10) Trehalose - - - - 
(11) D-Sorbitol - - - - 
(12) D-Mannitol - - - - 
(13) Inositol - - - - 
(14) Glycerol - - - - 
(15) Starch - - - - 
__________________________________________________________________________ 
Remarks: 
(Note 1) The symbols used under Physiological characteristics indicate th 
following: 
+: The strain has the corresponding characteristics or produces the 
correspondng product. 
.+-.: It is difficult to determine whether the strain has the 
corresponding characterstics or produces the corresponding product or not 
-: The strain neither has the corresponding characteristics nor produces 
the correspondingproduct. 
(Note 2) By using Hugh and Leifson medium in which each of the 
carbohydrates shown in Table I and Table 2 was used in lieu of the carbon 
source thereof, production of acids and gases by the strain was observed. 
+: An acid or a gas is produced. 
.+-.: It is difficult to determine whether an acid or a gas is produced o 
not. 
-: Neither an acid nor a gas is produced. 
On the basis of these morphological, cultural and physiological 
characteristics, the strains have been classified according to Bergey's 
Manual of Determinative Bacteriology, 7th and 8th Editions. 
The strain Alcaligenes faecalis D4020 has been identified as a strain of 
the genus Alcaligenes based on its morphological characteristics, among 
others, that it is a rod having peritrichous flagella and that it reacts 
negative in Gram staining as well as on the physiological characteristics, 
among others, that it reacts positive in the oxidase and catalase 
reactions, that it is aerobic and that the oxidation/fermentation test 
gives oxidative results, and further identified as a strain of the species 
Alcaligenes faecalis based on the facts that it does not liquefy gelatin, 
that milk becomes alkaline but otherwise remains unchanged and that it 
does not cause denitrification. Generally, a mutant is considered to 
belong to the same species as its parent strain belongs to. Accordingly, 
the strain Alcaligenes faecalis D4020-K15 has been judged as belonging to 
the species Alcaligenes faecalis. 
The strain Pseudomonas putida D4014 has been identified as a strain of the 
genus Pseudomonas based on its morphological characteristics, among 
others, that it is a rod having polar flagella and that Gram staining 
gives negative results as well as on the physiological characteristics, 
among others, that the oxidase and catalase reactions each gives positive 
results, that it is aerobic and that the oxidation/fermentation test 
results are oxidative. Furthermore, the strain Pseudomonas putida D4014 
has been identified as a strain of the species Pseudomonas putida in view 
of the facts, among others, that the slant culture exhibits fluorescence, 
that it does not liquefy gelatin, that it grows at 37.degree. C. and that 
it produces arginin dihydrolase. Since a mutant is generally considered to 
belong to the same strain as the parent strain belongs to, the strain 
Pseudomonas putida D4014-A357 has been judged to belong to the species 
Pseudomonas putida. 
The process of the present invention is carried out by cultivating a 
microbe of the genus Alcaligenes or Pseudomonas, which is capable of 
producing the pregnane derivative of the general formula (I) by utilizing 
deoxycholic acid or a salt thereof as the substrate, in a culture medium 
containing said substrate. 
In accordance with the present invention, deoxycholic acid per se can be 
used as the substrate. There can also be used an alkali metal salt of 
deoxycholic acid such as sodium deoxycholate, potassium deoxycholate or 
the like or an alkaline earth metal salt of deoxycholic acid such as 
calcium deoxycholate, magnesium deoxycholate or the like; preferred in a 
alkali metal salt. When a deoxycholate is used, it is dissolved in water 
to prepare an aqueous solution containing the deoxycholate in a 
predetermined concentration. Alternatively, a certain amount of an alkali 
metal compound or an alkaline earth metal compound which forms a salt with 
deoxycholic acid may previously be dissolved in water and thereto is added 
deoxycholic acid to give an aqueous solution containing a deoxycholate is 
a predetermined concentration. 
In general, the concentration of the substrate in a culture medium may be 
varied widely in the range of from about 1 to 200 g/l as deoxycholic acid. 
However, from the viewpoints of the yield of the desired product, 
cultivation conditions and economic efficiency such as operability or 
workability, it is preferable to use the substrate in a concentration of 
about 2 to 50 g/l as deoxycholic acid. 
The cultivation can be carried out according to a known method under 
aerobic conditions and shake or submerged culture using a liquid medium is 
generally employed. 
As the medium, there can be used one containing nutrients which can be 
assimilated by the microbe to be used. The medium may contain deoxycholic 
acid or a salt thereof as the sole carbon source. Optionally, it may 
contain an additional carbon source such as glucose, glycerol, peptone, 
meat extract, yeast extract, etc. or a mixture thereof. Generally, the 
additional carbon source can be added to the medium in a concentration of 
about 0.1 to 20 g/l. As a nitrogen source, there can be used an inorganic 
nitrogen source such as ammonium sulfate, ammonium chloride, ammonium 
phosphate, ammonium nitrate, sodium nitrate, potassium nitrate, etc.; an 
organic nitrogen source such as polypeptone, peptone, meat extract, etc.; 
or a mixture thereof. Generally, the nitrogen source can be added to the 
medium in a concentration of about 0.5 to 5 g/l. In addition, an inorganic 
salt such a dipotassium hydrogen phosphate, potassium dihydrogen 
phosphate, magnesium sulfate, manganese sulfate, zinc sulfate, cobalt 
chloride, sodium molybdate, cuprice sulfate, calcium chloride, sodium 
chloride, etc. or a mixture thereof can be added to the medium. 
The cultivation conditions are not very critical. Generally, the 
cultivation can be carried out in the manner of shake or submerged culture 
at a pH of about 7 to 9 at about 25.degree. to 35.degree. C. for about 10 
hours to 7 days to cause production and accumulation of the pregnane 
derivative of the general formula (I) in the medium. 
When a strain of the genus Alcaligenes is cultivated in accordance with the 
method of the present invention, deoxycholic acid and/or a salt thereof 
used as the substrate is converted to a pregnane derivative of the general 
formula (I) in which the dotted line-marked site involves a double bond, 
namely 12-hydroxypregna-1,4-dien-3-one-20-carbaldehyde. In that case, the 
production of 12.alpha.-hydroxypregna-1,4-dien-3-one-20-carbaldehyde 
generally predominates, whereas 
12.beta.-hydroxypregna-1,4-dien-3-one-20-carbaldehyde is produced rather 
as a byproduct. However, when the cultivation is conducted under 
conditions such that the conversion of deoxycholic acid and/or a slat 
thereof is relatively slow and/or when the cultivation is carried out in a 
short period of time, the yield of 
12.beta.-hydroxypregna-1,4-dien-3-one-20-carbaldehyde increases. When a 
strain of the genus Pseudomonas is used, deoxycholic acid and/or a salt 
thereof is converted to a pregnane derivative of the general formula (I) 
in which no double bond is present at the dotted-line-marked site, namely 
12-hydroxypregna-4-en-3-one-20-carbaldehyde. In this case, 
12.alpha.-hydroxypregna-4-en- 3-one-20-carbaldehyde is obtained 
selectively. 
The pregnane derivative of the general formula (I) as accumulated in the 
culture broth generally precipitates out in the broth, since the 
solubility thereof in water is by far smaller as compared with the 
substrate, namely deoxycholic acid or a salt thereof. To harvest this 
pregnane derivative, the precipitate is separated from the broth 
containing suspended cells either by decanting or by the steps of 
centrifuging the broth at the speed that will not cause sedimentation of 
the suspended cells to additionally obtain a sediment of the pregnane 
derivative and then decanting. From the remaining broth, the cells and 
other insoluble matters are removed by filtration or centrifugation and 
the resulting filtrate or supernatant is made alkaline with an alkali such 
as sodium hydroxide, potassium hydroxide or calcium hydroxide, for 
instance, followed by extraction with a water-immiscible organic solvent 
capable of dissolving said pregnane derivative, e.g. ethyl acetate, 
chloroform or a mixture of chloroform and methanol. The extracts are 
pooled and the solvent is distilled off, whereby the pregnane derivative 
still remaining dissolved in the broth can be recovered. The above 
extraction with an organic solvent can be applied not only to the filtrate 
or supernatant but also to the broth as such. The sediment or extract 
obtained in the above manner is substantially free from residues of the 
substrate deoxycholic acid and/or salt thereof or any byproducts, so that 
a high purity grade of the pregnane derivative of the general formula (I) 
can be easily obtained therefrom, for example by recrystallization from 
aqueous methanol. The separation of a mixture of 
12.alpha.-hydroxypregna-1,4-dien-3-one-20-carbaldehyde and 
12.beta.-hydroxypregna-1,4-dien-3-one-20-carbaldehyde into 
12.alpha.-hydroxypregna-1,4-dien-3-one-20-carbaldehyde and 
12.beta.-hydroxypregna-1,4-dien-3-one-20-carbaldehyde is carried out by 
allowing the above mixture to be adsorbed on a silica gel column followed 
by elution with a mixed solvent composed of chloroform and ethanol. 
12.beta.-Hydroxypregna-1,4-dien-3-one-20-carbaldehyde is first eluted and 
then 12.alpha.-hydroxypregna-1,4-dien-3-one-20-carbaldehyde is eluted. 
Each aldehyde can be recovered by distilling off the solvent from the 
eluate under reduced pressure. 
The pregnane derivative of the general formula (I) thus obtainable 
according to the method of the present invention, if necessary after 
acylation of the hydroxyl group at position 12, can be derived to a 
12-substituted-.DELTA..sup.4 or .DELTA..sup.1,4 -pregnane-3,20-dione of 
the general formula 
##STR2## 
wherein R is a hydrogen atom or an acyl group, and the dotted line and 
wavy line are as defined above, in a per se conventional manner by 
reacting with a secondary amine such as piperidine or pyrrolidine and 
oxidizing the resulting enamine with ozone or chromium trioxide, for 
instance. The 12-substituted-.DELTA..sup.4 or .DELTA..sup.1,4 
-pregnane-3,20-dione can be converted to a .DELTA..sup.4,11(12) or 
.DELTA..sup.1,4,11(12) -pregnane-3,20-dione of the general formula 
##STR3## 
wherein the dotted line has the same meaning as above, by directed 
elimination of carboxylic acid or by conventional sulfonation, if 
necessary following hydrolysis, and elimination of sulfonic acid. The 
.DELTA..sup.1,4,11(12) -pregnane-3,20-dione is a known compound, whereas 
the .DELTA..sup.4,11(12) -pregnane-3,20-dione can be derived to said 
.DELTA..sup.1,4,11(12) -pregnane-3,20-dione by conventional 
dehydrogenation. The .DELTA..sup.4,11(12) or .DELTA..sup.1,4,11(12) 
-pregnane-3,20-dione represented by the general formula (III) can further 
be derived to prednisone and further to prednisolone or hydrocortisone in 
a conventional manner as shown, for instance, by the following reaction 
scheme: 
##STR4## 
wherein the dotted line has the same meaning as above. 
The following examples and reference examples further illustrate the 
present invention in detail but are not to be construed to limit the scope 
thereof. 
Preparation of mutants 
Preparation of the strain Alcaligenes faecalis D4020-K15 
Alcaligenes faecalis D4020 was grown on a slant of medium 1 (composition: 
0.5% deoxycholic acid, 0.05% sodium hydroxide, 0.5% peptone, 0.5% yeast 
extract, 0.5% sodium chloride and 1.5% agar). A loopful of the microbe so 
grown was used for inoculating 10 ml of medium 2 (composition: 2% 
deoxycholic acid, 0.2% sodium hydroxide, 0.2% ammonium nitrate, 0.1% 
potassium dihydrogen phosphate, 0.6% dipotassium hydrogen phosphate, 0.02% 
magnesium sulfate heptahydrate and 0.02% yeast extract) preliminarily 
prepared in a test tube (200 mm.times.21 mm in diameter), and 
shake-cultured at 30.degree. C. for 8-10 hours. A 0.3-ml-portion o the 
culture was added to 10 ml of medium 3 (composition: 0.5% deoxycholic 
acid, 0.5% sodium hydroxide, 0.1% glucose, 0.2% ammonium nitrate, 0.1% 
potassium dihydrogen phosphate, 0.6% dipotassium hydrogen phosphate, 0.02% 
magnesium sulfate heptahydrate and 0.02% yeast extract) preliminarily 
prepared in a test tube (200 mm.times.21 mm in diameter), followed by 
incubation at 30.degree. C. for 10-15 hours. The cells, which were in the 
logarithmic growth phase, were collected aseptically by filtration using a 
membrane filter (pore size: 0.45.mu.), washed with 20 ml of 0.1M phosphate 
buffer (pH 7.0) and suspended in 25 ml of the same buffer. To the 
suspension was added N-methyl-N'-nitro-N-nitrosoguanidine to a final 
concentration of 20 .mu.g/ml. The mixture was shaken at 30.degree. C. for 
10-15 minutes. The cells so subjected to mutagenic treatment were 
collected by filtration using a membrane filter (pore size: 0.45.mu.), 
washed with 20 ml of 0.1M phosphate buffer (pH 7.0) and suspended in 20 ml 
of the same buffer. The resulting suspension was diluted with sterilized 
physiological saline solution and the dilution was applied to an agar 
plate made of medium 4 (composition: 0.5% deoxycholic acid, 0.05% sodium 
hydroxide, 0.2% ammonium nitrate, 0.1% potassium dihydrogen phosphate, 
0.6% dipotassium hydrogen phosphate, 0.02% magnesium sulfate heptahydrate, 
0.02% yeast extract and 1.5% agar) so that 500 to 1,000 colonies could 
appear on the plate. The incubation was then performed at 30.degree. C. 
for 3-4 days. Among the colonies that had appeared, pin point colonies 
were transferred to a slant made of medium 1, and one loopful thereof was 
used to inoculate 10 ml of medium 5 (composition: 0.2% deoxycholic acid, 
0.02% sodium hydroxide, 0.1% glucose, 0.2% ammonium nitrate, 0.1 % 
potassium dihydrogen phosphate, 0.6% dipotassium hydrogen phosphate, 0.02% 
magnesium sulfate heptahydrate and 0.02% yeast extract) preliminarily 
prepared in a test tube (200 mm.times.21 mm in diameter), followed by 
shake culture at 30.degree. C. for 24 hours. The products in each culture 
obtained in this manner were examined by thin layer chromatography. A 
strain capable of selectively accumulating 
12.alpha.-hydroxypregna-1,4-dien-3-one-20-carbaldehyde under the above 
cultural conditions was found and named Alcaligenes faecalis D4020-K15. 
Preparation of the strain Pseudomonas putida D4014-A357 
A loopful of Pseudomonas putida D4014 grown on a slant of the 
above-mentioned medium 1 was used for inoculating 10 ml of the 
above-mentioned medium 2 preliminarily prepared in a test tube (200 
mm.times.21 mm in diameter), and shake-cultured at 30.degree. C. for 14-15 
hours. A 0.3 ml portion of the culture was added to 10 ml of the 
above-mentioned medium 3 preliminarily prepared in a test tube (200 
mm.times.21 mm in diameter), followed by incubation at 30.degree. C. for 
8-9 hours. The cells, which were in the logarithmic growth phase, were 
aseptically collected with a membrane filter (pore size: 0.45.mu.), washed 
with 20 ml of 0.1M phosphate buffer (pH 7.0) and suspended in 25 ml of the 
same buffer. To the suspension was added 
N-methyl-N'-nitro-N-nitrosoguanidine to a final concentration of 50 
.mu.g/ml, and the mixture was allowed to stand for 3-4 minutes. The cells, 
which had been subjected to the above mutagenic treatment, were collected 
with a membrane filter (pore size: 0.45.mu.), washed with 20 ml of 0.1M 
phosphate buffer (pH 7.0) and suspended in 20 ml of the same buffer. The 
resulting suspension was diluted with sterilized physiological saline 
solution and the dilution was applied to an agar plate of the 
above-mentioned medium 4 to the extent such that 500-1,000 colonies could 
appear thereon. After incubation at 30.degree. C. for 3-4 days, pin point 
colonies among those that had appeared were isolated and transferred to a 
slant of medium 1 and a loopful thereof was used to inoculate 10 ml of the 
above-mentioned medium 5 preliminarily prepared in a test tube (200 
mm.times.21 mm in diameter). The inoculated medium was shaken at 
30.degree. C. for 24 hours. The products in each culture thus obtained 
were examined by thin layer chromatography. In this manner, a strain 
capable of selectively accumulating 
12.alpha.-hydroxypregna-4-en-3-one-20-carbaldehyde under the above 
cultural conditions was obtained and named Pseudomonas putida D4014-A357.

EXAMPLE 1 
Alcaligenes faecalis D4020-K15 (FERM BP-204) was cultivated in the 
following manner. A medium (pH 8.4) was prepared by adding tap water to 
1.0 g of deoxycholic acid, 0.1 g of glucose, 0.2 g of ammonium nitrate, 
0.12 g of potassium dihydrogen phosphate, 0.61 g of dipotassium hydrogen 
phosphate, 0.02 g of magnesium sulfate heptahydrate, 0.02 g of yeast 
extract and 0.1 g of sodium hydroxide, to a volume of 100 ml. A 10 ml 
portion of this medium was placed in each of 10 test tubes (200 
mm.times.21 mm in diameter) and steam-sterilized at 120.degree. C. for 15 
minutes. Separately and in advance, the above strain was grown in the same 
medium as above on a test tubeshaker for one day, and a 0.5 ml portion of 
the thus obtained seed culture was added to each of the above-mentioned 
test tubes (200 mm.times.21 mm in diameter) and shake-cultured at 
30.degree. C. for 2 days. The pooled culture broth was centrifuged, 
whereby a mixture of the cells and a precipitate which had formed in the 
course of cultivation was separated from a culture supernatant. To said 
mixture was added a 1N aqueous solution of sodium hydroxide to adjust the 
resulting mixture to pH 9, and the mixture was extracted with 200 ml of 
ethyl acetate Separately, the culture supernatant was adjusted to pH 9 by 
adding a 1N aqueous solution of sodium hydroxide, and extracted with 200 
ml of ethyl acetate. This extract and the extract obtained in the above 
extraction were combined and dried over anhydrous sodium sulfate, and the 
ethyl acetate was distilled off using a rotary evaporator to give 750 mg 
of a mixture of 12.beta.-hydroxypregna-1,4-dien-3-one-20-carbaldehyde and 
12.alpha.-hydroxypregna-1,4-dien-3-one-20-carbaldehyde. 
A portion of the thus-obtained mixture was taken, and methanol was added 
thereto to prepare a 1% solution. A 25-.mu.1 portion of this solution was 
injected into a high performance liquid chromatograph equipped with a 
.mu.Bondapak C-18 column (HLC-GPC-244 type manufactured by Waters 
Associates in U.S.A.). A 25:75 (by volume) water-methanol mixture adjusted 
to pH 4.0 was used as the mobile phrase at a flow rate of 1 ml/minute. The 
detection was made by the refractive index method. The areas of the 
chromatographic peaks obtained were measured with an integrator (Shimadzu 
Chromato-Pack C-RIA manufactured by Shimadzu Corporation in Japan) and it 
was indicated that the peak areas for 
12.beta.-hydroxypregna-1,4-dien-3-one-20-carbaldehyde and 
12.alpha.-hydroxypregna-1,4-dien-3-one-20-carbaldehyde accounted for 95% 
of the total peak area. In the above mixture, the ratio between 
12.beta.-hydroxypregna-1,4-dien-3-one-20-carbaldehyde and 
12.alpha.-hydroxypregna-1,4-dien-3-one-20-carbaldehyde was 1/8. 
From the product mixture obtained according to the same procedure as 
mentioned above 12.beta.-hydroxypregna-1,4-dien-3-one-20-carbaldehyde and 
12.alpha.-hydroxypregna-1,4-dien-3-one-20-carbaldehyde were respectively 
isolated in the following manner. First, a tubular column, 2.6 cm in 
inside diameter and 70 cm in length, was packed with a suspension of about 
100 g of silica gel in about 200 ml of chloroform. Separately, 1.2 g of the 
above product mixture was dissolved in about 20 ml of chloroform and 
insolubles were removed. The chloroform solution was concentrated to about 
5 ml and then allowed to be adsorbed on the above silica gel column and 
eluted in sequence with chloroform, a 99:1 (by volume) chloroform-ethanol 
mixture and a 97:3 (by volume) chloroform-ethanol mixture. 
12.beta.-Hydroxypregna-1,4-dien-3-one-20-carbaldehyde was eluted in a 
fraction covering about the 250 ml to 280 ml portions of the second 
eluate, i.e. the 99:1 chloroformethanol mixture, and thereafter 
12.alpha.-hydroxypregna-1,4-dien-3-one-20-carbaldehyde was eluted with the 
same eluate in a fraction of about 450 ml to 510 ml. These two aldehydes 
were discriminated from each other based on the facts that, in thin layer 
chromatography using a thin layer plate (silica gel 60, F-254 manufactured 
by Merck in U.S.A.) and an isooctaneethyl acetate-acetic acid mixture 
(10:10:2 by volume) as the developing solvent, 
12.beta.-hydroxypregna-1,4-dien-3-one-20-carbaldehyde gives a spot 
corresponding to R.sub.f =about 0.4 and 
12.alpha.-hydroxypregna-1,4-dien-3-one-20-carbaldehyde gives a spot 
corresponding to R.sub.f =about 0.34. The chloroform and ethanol were 
distilled off from each eluate fraction with a rotary evaporator, and the 
residue was washed with diethyl ether and dried. There were thus obtained 
about 80 mg of 12.beta.-hydroxypregna-1,4-dien-3-one-20-carbaldehyde and 
about 360 mg of 12.alpha.-hydroxypregna-1,4-dien-3-one-20-carbaldehyde. 
The 12.beta.-hydroxypregna-1,4-dien-3-one-20-carbaldehyde and 
12.alpha.-hydroxypregna-1,4-dien-3-one-20-carbaldehyde were identified 
based on the following data: 
12.beta.-Hydroxypregna-1,4-dien-3-one-20-carbaldehyde 
Melting point: 157.degree.-161.degree. C. 
Mass spectrum (m/Z): 342 [M].sup.+, 324 [M--H.sub.2 O].sup.+, 309 
[M--H.sub.2 O--CH.sub.3 ].sup.+ 
The presence of 3-keto-1,4-dien was confirmed by m/Z=121 and 122. 
NMR spectrum (90 MHz) .delta..sub.HMS.sup.DMSO-d.sbsp.6 0.68 (3H, s) 
18-CH.sub.3 0.90 (3H, d, J=6.3 Hz) 21-CH.sub.3 1.16 (3H, s) 19-CH.sub.3 
3.25 (1H, s) 12.beta.-OH 3.40 (1H, m) 12.alpha.-H 5.95 (1H, s) 4-H 6.15 
(1H, dd, J=18 Hz, J=3 Hz) 2-H 7.13 (1H, d, J=12 Hz) 1-H 9.50 (1H, d, J=9 
Hz) 22-CHO 
12.alpha.-Hydroxypregna-1,4-dien-3-one-20-carbaldehyde 
Melting point: 194.degree.-201.degree. C. 
Mass spectrum (m/Z): 342 [M].sup.+, 324 [M-H.sub.2 O].sup.+, 309 [M-H.sub.2 
O-CH.sub.3 ].sup.+ 
The presence of 3-keto-1,4-dien was confirmed by m/Z=121 and 122. 
NMR spectrum (90 MHz) .delta..sub.HMS.sup.DMSO-d.sbsp.6 : 0.71 (3H, s) 
18-CH.sub.3 1.09 (3H, d) 21-CH.sub.3 1.17 (3H, s) 19-CH.sub.3 3.83 (1H, t, 
J=3 Hz) 12.beta.-H 4.33 (1H, d) 12.alpha.-OH 5.95 (1H, s) 4-H 6.08 (1H, d, 
J=10 Hz)2-H 7.08 (1H, d, J=10 Hz)1-H 9.56 (1H, s)22-CHO 
EXAMPLE 2 
Alcaligenes faecalis D4020-K15 (FERM BP-204) was cultivated in the 
following manner. A medium (pH 8.4) was prepared by adding tap water to 
1.0 g of deoxycholic acid, 0.1 g of glucose, 0.2 g of ammonium nitrate, 
0.12 g of potassium dihydrogen phosphate, 0.61 g of dipotassium hydrogen 
phosphate, 0.02 g of magnesium sulfate heptahydrate, 0.02 g of yeast 
extract and 0.1 g of sodium hydroxide to a volume of 100 ml. This medium 
was placed in a 500 ml Sakaguchi flask and steam-sterilized at 120.degree. 
C. for 15 minutes. Separately and in advance, the above microbe was grown 
in the same medium as above on a test tube shaker for one day and a 10 ml 
portion of the seed culture was added to the above-mentioned 500-ml 
Sakaguchi flask. The flask was shaken at 30.degree. C. for 2 days. The 
thus-obtained culture broth was centrifuged to separate a mixture of the 
cells and the precipitate which had formed during the incubation from the 
culture supernatant. An aqueous 1 N sodium hydroxide solution was added to 
said mixture in an amount sufficient to adjust the pH to 9, and the 
resultant mixture was extracted with 200 ml of ethyl acetate. On the other 
hand, the culture supernatant was adjusted to pH 9 by adding an aqueous 1N 
sodium hydroxide solution and then extracted with 200 ml of ethyl acetate. 
Both the extracts were combined and dried over anhydrous sodium sulfate, 
and the ethyl acetate was distilled off in a rotary evaporator to give 700 
mg of 12.alpha.-hydroxypregna-1,4-dien-3-one-20-carbaldehyde. 
A portion of the thus-obtained 
12.alpha.-hydroxypregna-1,4-dien-3-one-20-carbaldehyde was dissolved in 
methanol to prepare a 1% solution, and a 25-.mu.l portion of the solution 
was injected into a high performance liquid chromatograph equipped with a 
.mu.Bondapak C-18 column (as mentioned hereinbefore). A 25:75 (by volume) 
water-methanol mixture adjusted to pH 4.0 was used as the mobile phase at 
a flow rate of 1 ml/min. The detection was made by the refractive index 
method. The areas of the chromatographic peaks obtained were measured with 
an integrator (as mentioned hereinbefore). Area ratio indicated that the 
above 12.alpha.-hydroxypregna-1,4-dien-3-one-20-carbaldehyde was 96% pure. 
The identification of the above product as 
12.alpha.-hydroxypregna-1,4-dien-3-one-20-carbaldehyde was carried out in 
the same manner as in Example 1. 
EXAMPLE 3 
Pseudomonas putida D4014-A357 (FERM BP-206) was cultivated in the following 
manner. A medium was prepared by adding 50 ml of tap water to 0.2 g of 
deoxycholic acid, 0.1 g of glucose, 0.2 g of ammonium nitrate, 0.1 g of 
potassium dihydrogen phosphate, 0.6 g of dipotassium hydrogen phosphate, 
0.02 g of magnesium sulfate heptahydrate and 0.02 g of yeast extract, 
adjusting the solution to pH 8.4 with 1N sodium hydroxide and further 
adding water to make the volume 100 ml. This medium was placed in a 500 ml 
Sakaguchi flask and steam-sterilized at 120.degree. C. for 15 minutes. 
Separately and in advance, the above microbe was grown in the same medium 
as above on a test tube shaker for one day and a 10 ml portion of the seed 
culture was added to the above 500 ml Sakaguchi flask, followed by shake 
culture at 30.degree. C. for 2 days. The precipitate which had formed 
during the cultivation and the cells were collected by centrifugation and 
washed with water, and 50 ml of methanol was added thereto to dissolve the 
precipitate to a satisfactory extent. Recentrifugation gave a supernatant 
methanol solution. The methanol was distilled off with a rotary evaporator 
to give 65 mg of 12.alpha.-hydroxypregna-4-en-3-one-20-carbaldehyde. 
A portion of the thus-obtained 
12.alpha.-hydroxypregna-4-en-3-one-20-carbaldehyde. was dissolved in 
methanol in a concentration of 4%, and a 25 .mu.l portion of the solution 
was injected into a high performance liquid chromatograph equipped with a 
.mu.Bondapak C-18 column (as mentioned hereinbefore). A 30:70 (by volume) 
water-methanol mixture adjusted to pH 4.0 was used as the mobile phase at 
a flow rate of 1 ml/min. The refractive index-based detection method was 
employed. The areas of the chromatographic peaks obtained were measured 
with an integrator (as mentioned hereinbefore). The purity of the above 
12.alpha.-hydroxypregna-4-en-3-one-20-carbaldehyde as determined by area 
ratio calculation was 98%. 
The identification of the above product as 
12.alpha.-hydroxypregna-4-en-3-one-20-carbaldehyde was based on the 
following data: 
Melting point: 179.degree.-181.degree. C. 
Mass spectrum (m/Z): 344 [M].sup.+ 326 [M-H.sub.2 O].sup.+ 316 
[M-CO].sup.+. 
The presence of 3-keto-4-ene was confirmed by m/Z=124. 
NMR spectrum (90 MHz) .delta..sub.HMS.sup.CDCl.sbsp.3 : 0.72 (3H, s) 
18-Ch.sub.3 1.10 (3H, d) 21-CH.sub.3 1.15 (3H, s) 19-Ch.sub.3 3.95 (1H, s) 
12.beta.-H 5.70 (1H, s) 4-H 9.40 (1H, d) 22-CHO. 
Reference Example 
Synthesis of 12.alpha.-hydroxypregna-1,4-diene-3,20-dione 
12.alpha.-Hydroxypregna-1,4-dien-3-one-20-carbaldehyde (34.2 g) was 
dissolved in 300 ml of methylene chloride. To the solution were added 23.6 
g of acetyl chloride and 27.7 g of pyridine, and the mixture was stirred at 
room temperature for 5 hours. To the reaction mixture was added 300 ml of 
methylene chloride, the resulting solution was washed in sequence with 
diluted hydrochloric acid and water, and dried over anhydrous magnesium 
sulfate. Low-boiling fractions were distilled off from the solution under 
reduced pressure. There was thus obtained crude 
12.alpha.-acetoxypregna-1,4-dien-3-one-20-carbaldehyde as a viscous 
substance. This crude product was dissolved in 300 ml of benzene, 21.3 g 
of piperidine was added to the solution, and the resulting mixture was 
refluxed for 3 hours while removing the byproduct water azeotropically 
with benzene. Low-boiling fractions were distilled off from the 
thus-obtained reaction mixture. There remained crude 
12.alpha.-acetoxy-22-(N-peridyl)bisnor-1,4,20(22)-cholatrien-3-one as a 
viscous substance, which was dissolved in 180 ml of pyridine. To the 
solution was added gradually at room temperature a mixture of 20.0 g of 
chromium trioxide and 250 ml of pyridine. After stirring the resulting 
mixture for one hour, 1 liter of benzene was added to the reaction 
mixture. The solid matter was filtered off, and diluted hydrochloric acid 
was added to the filtrate. After effecting benzene extraction to a 
sufficient extent, the benzene layer was washed in sequence with diluted 
hydrochloric acid and water. Low-boiling fractions were distilled off 
under reduced pressure, and the residue was purified by preparative liquid 
chromatography [column: Waters Associates' Prep LC/System 500, Prep PAK.TM. 
500/SILICA; solvent system: isopropyl alcohol:n-hexan=20:80, v/v] to give 
9.1 g of 12.alpha.-acetoxypregna-1,4-diene-3,20-dione as crystals. It has 
the following physical properties. 
Melting point: 175.degree.-176.degree. C. 
NMR spectrum (90 MHz) .delta..sub.HMS.sup.CDCl.sbsp.3 : 0.76, 1.20, 2.0, 
2.06 (each s, each 3H); 5.10-5.22 (m, 1H); 6.07 (bs, 1H); 6.18, 6.20 (each 
d, 1H); 6.93 (d, 1H). 
To a solution of 2.2 g of potassium hydroxide in 80 ml of methanol was 
added 7.4 g of 12.alpha.-acetoxypregna-1,4-diene-3,20-dione, and the 
mixture was stirred at room temperature for 10 hours. The reaction mixture 
was concentrated to about one tenth the original volume by distilling off 
the methanol under reduced pressure. To the concentrated reaction mixture 
was added 150 ml of benzene, and the solution was washed in sequence with 
water, diluted hydrochloric acid and water and then dried over anhydrous 
magnesium sulfate. Low-boiling fractions were distilled off under reduced 
pressure and the residue was recrystallized from ethyl acetate to give 5.4 
g of 12.alpha.-hydroxypregna-1,4-diene-3,20-dione (Gas chromatographic 
analysis revealed that purity thereof was 90%). It has the following 
physical properties. 
Melting point: 185.degree.-186.degree. C. 
NMR spectrum (90 MHz) .delta..sub.HMS.sup.CDCl.sbsp.3 : 0.69, 1.17, 2.14 
(each s, each 3H); 4.04-4.16 (m, 1H); 6.06 (bs, 1H); 6.16, 6.18 (each d, 
1H); 7.03 (d, 1H). 
Synthesis of pregna-1,4,11(12)-triene-3,20-dione 
12.alpha.-Hydroxypregna-1,4-diene-3,20-dione (3.3 g) was dissolved in 17 ml 
of pyridine. To the solution was added 3.4 g of methanesulfonyl chloride, 
and the mixture was stirred at room temperature for 8 hours. The reaction 
mixture was then poured into 300 ml of diluted hydrochloric acid, and the 
resulting mixture was extracted with three 300 ml portions of benzene. The 
extracts were combined and washed in sequence with diluted hydrochloric 
acid, aqueous sodium bicarbonate solution and water, and dried over 
anhydrous magnesium sulfate. Low-boiling fractions were distilled off 
under reduced pressure from the extract to give 3.8 g of crude 
12.alpha.-mesyloxypregna-1,4-diene-3,20-dione, which was recrystallized 
from ethyl acetate. The physical properties for the thus-purified product 
were as follows: 
Melting point: 185.degree.-186.degree. C. 
NMR spectrum (90 MHz) .delta..sub.HMS.sup.CDCl.sbsp.3 : 0.82, 1.18, 2.10, 
2.96 (each s, each 3H); 5.04-5.16 (m, 1H); 6.05 (bs, 1H); 6.19, 6.21 (each 
d, 1H); 6.95 (d, 1H). 
12.alpha.-Mesyloxypregna-1,4-diene-3,20-dione (3.0 g) was dissolved in 60 
ml of hexamethylphosphoramide. To the solution was added 7.2 g of 
potassium acetate, and the mixture was stirred at 120.degree. C. for 5 
hours. To the reaction mixture was added 300 ml of diluted hydrochloric 
acid, and the whole mixture was extracted with three 200 ml portions of 
benzene. The extracts were combinedly washed in sequence with diluted 
hydrochloric acid and water, and dried over anhydrous magnesium sulfate. 
Low-boiling fractions were distilled off under reduced pressure and the 
residue was purified by silica gel column chromatography (eluent: 
acetone-n-hexane, 4:6 by volume) to give 1.6 g of 
pregna-1,4,11(12)-triene-3,20-dione as crystals. The physical properties 
for the obtained crystal were as follows: 
Melting point: 167.degree.-169.degree. C. 
NMR spectrum (90 MHz) .delta..sub.HMS.sup.CDCl.sbsp.3 : 0.74, 1.17, 2.15 
(each s, each 3H); 5.66 (d, 1H); 6.07-6.40 (m, 3H); 7.12 (d, 1H).