JPS5886096A - Preparation of 12beta-hydroxyandrosta-1,4-diene-3,17-dione - Google Patents

Abstract

PURPOSE:To prepare 12beta-hydroxyandrosta-1,4-diene-3,17-dione, in high selectivity and yield, by culturing microorganisms belonging to Aeromonas genus in a medium containing deoxycholic acid and/or its salt. CONSTITUTION:Microorganism belonging to Aeromonas genus and capable of producing 12beta-hydroxyandrosta-1,4-diene-3,17-dione using deoxycholic acid and/ or its salt as a substrate, e.g. Aeromonas hydrophila D-238 (FERM-P No.6149), is inoculated in a medium containing deoxycholic acid and/or its salt, and cultured at 25-35 deg.C for 1-7 days under aerobic conditions. The objective 12beta- hydroxy androsta-1,4-diene-3,17-dione can be separated from the culture liquid.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing 12β-hydroxyandrosta-1,4-diene-3,17-dione from deoxycholic acid and/or its salts using microorganisms, and more specifically, /or 12β using its salt as a substrate
-hydroxyandro, x, p-1,4-diene-3,
17-dione-producing bacteria belonging to the genus Slearomonas were cultured in a medium containing deoxycholic acid and/or its salts as the main carbon source to produce 12β-hydroxyandroster.
The present invention relates to a method for producing 12β-hydroxyandrosta-1,4-diene-3,17-dione, which comprises producing and collecting 1,4-dinine-3,17-dione.

Conventionally, 12β-
Hydroxyandrosta-1,4-diene-3,17-
The following report has been found as a method for obtaining dione (hereinafter referred to as 12β-hydroxy7ADD). First of all, Barness (
Pseudomonas sp. NCIB 1059010590 (P, J. Barnea) et al.
seudo sp, NCIB 10590) strain has been reported. (J, Chem, Soc
, Chem.

Commun, (1974), pp. 115-116 and Tatrahedron, Vol. 32, pp. 89-93 (1976)]. In this method, Pseudomonas esb. NCIB 10590 strain is aerobically grown for 14 hours in an inorganic salt medium 10j enriched with 0.1% deoxycholic acid, and 128-hydroxyADD960"r and 12α-hydroxy are extracted from deoxycholic acid 10f. Pregna-1,4-dien-3-one-20α-carvone #
Cape 1657 was obtained as the main product, and additionally 12α-hydroxyandrosta-1,4-diene-3°17-dione, 12β-hydroxy-4-androstene-3,17
-dione and 12ξ, 17ξ-dihydroxy-4-a/
Drosten-3-one is obtained as a trace component. This method Fi12β-hydroxy ADD yield is 10
Low points, less than %, 12β-hydroxypre'; f
1,4--), It is not practical because the acid concentration is as low as 0.196. Also, Tenneson (M,,E.

12β-hydroxy ADD and 1 from deoxycholic acid using anaerobic Escherichia coli
Method for obtaining 2α-hydroxypregna-i, 4-dien-3-one-20α-carboxylic acid (Bloch@m.

Soc, Trans, vol. 5, 17513-1760
(1977); Owen (Le, W., O.
w@n) et al.
F 23 (Bacteroid@afragiLi
s XF 23 ) strain iii or Bacteroides fragilis subsb.
59 (Bacteroides fragil)
ia 5ubsp, thetaiotaom
icroni59)@ strain to convert deoxycholic acid into 12β-hydroxyADD, 12α-hydroxydendrostar 1,4-diene-3,17-dione, 12β-
Hydroquine-4-androstene-3,17-dione and 12α-hydroxypregna-1,4-diene-3-
Method for obtaining 1-20α-carboxylic acid (Bioche+
n, 8oc, Trans, vol. 5, 1711-171
3 (1977)], but all of these methods use anaerobic bacteria, the substrate used has a low rIk, the culture requires a long period of time, and the target product There are many problems with industrial fermentation production, such as low yield. As a result of long-term searches for microorganisms that produce 12β-hydroxy ADD using deoxycholic acid and/or its salts as substrates, Person A and others have discovered a specific microorganism belonging to the genus Airmonas. It was discovered that 12β-hydroxyADI)J can be produced with high selectivity and yield using the salt as a substrate, and the present invention was completed.

The bacteria belonging to the genus Aeromonas that the present inventors have discovered include Aeromonas noihydrophylla D-238.
(Aeromonas hydrophila D-23
8) 9 strains (aI Koken Sonoyori No. 6149) and Aeromonas punku pp D-298 (Aerononon
a@punctata D-298) strain (II Koken Sonoyori No. 6151) 4R0 One of the Aeromonas noitrophyla D-238 strain and Aeromonas punctata D-2981111 strain obtained by the present inventors. The physical properties are listed in the following table. (Note 1) Medium composition: Potato extract 4.0 t/I, glucose 20.
0 t/J and agar 15. Of/j (Note 2): In physiological properties, shi, ten and - mean the following.

G...The property or the formation of the soil...It is difficult to determine the property or the presence or absence of the formation...The property or the formation is absent Note 3): Hugh and Life The production of enemies from sugars and the generation of gas by noodles were observed in a medium in which the carbon source of the non-medium was replaced with sugars 1 to 15, respectively.

10...'...Acid is generated or gas is generated±...
・It is difficult to determine the presence or absence of acid production or gas production ----No acid production or gas production Based on the bacteriological properties shown in the table above, Aeromonas hygrophila D-238 Bacterial strain and Aeromonas punctata D-2
Identification of 98 bacterial strains using the Purge Aids Manual of Determinative Bacteriology 7th and 8th editions
This was done based on the edition. Aeromonas hydrophila D
-238'1 strain and Aeromonas dunn strain Tata D-2
All 98 strains are bacilli, have polar flagella, and have negative Durham staining.
Both the oxidase reaction and the catalase reaction are positive, and the enzyme is fermentative, producing enzymes from glucose and lactose.
It was identified as a bacterium belonging to the Aeromonas genus based on its physiological properties such as being (ve). These strains differ from the bacteria belonging to the genus Pseudomonas, which is related to Aeromonas and tit, because they have a stimulant for glucose. In addition, both of the stocks are made from fine grains collected from the soil of paddy fields.
Therefore, it is reasonable to say that it belongs to the genus Aeromonas. Furthermore, Aeromonas hydrophylla strain D-238 is a member of the genus Aeromonas due to its physiological properties: it produces acid from glucose and generates gas, it has a positive VP test, and it can utilize glutamate as its only carbon source. It was identified as a bacterium of the species KIs. Aeromonas punctata D-2
Strain 9811i is a strain belonging to the Punctata species of the genus Aeromonas due to its culture findings in the medium, such as liquefying gelatin and acidifying milk, and its physiological properties, such as producing indole and not producing hydrogen sulfide. It was identified that there is.

The production of 12β-hydroxy ADD by the method of the present invention uses deoxycholic acid and/or its salt as a substrate.
Specifically, the salt of deoxycholic acid is obtained by culturing bacteria belonging to the genus Aeromonas that produces 2β-hydroxy ADD in a medium containing deoxycholic acid and/or its salt. Salts of alkali metals such as sodium and potassium, or calcium;
It is a salt of an alkaline earth metal such as magnesium.

The concentration of deoxycholic acid and/or its salt is usually 1~
A range of 200 f/4 may be sufficient, but from an economic standpoint such as the yield of 12β-hydroxy ADD, culture conditions, and operability, a range of about 10 to 100 f/4 is preferable. The method is the same as that used for aerobic culture of general mold organisms, but usually a shaking culture method or aerated agitation culture method using a liquid medium is used.The medium is the above-mentioned deoxycol and/or its salt. As long as it contains a nutrient source that can be assimilated and used by bacteria belonging to the genus Aeromonas that produces 12β-hydroxy ADD using 12β-hydroxyADD as a base carbon source, deoxycholic acid and/or its salt may be used as the sole carbon source. or deoxycholic acid and/or its salt with arabinose; hexoses such as crucose, mannose, fructose, galactose; disaccharides of sucrose and maltose; starch decomposition products; sugar alcohols, Polyhydric alcohols such as glycerin; or polypeptone, peptone, meat extract, malt extract, cornstap liquor, yeast extract, various amino acids, organic acids, etc. may be used in combination.As a nitrogen source, for example, ammonium nitrate,
Inorganic nitrogen sources such as sodium nitrate, potassium nitrate, or organic nitrogen sources such as polypeptone, peptone, meat extract, etc. are used. In addition to this, dipotassium hydrogen phosphate, phosphorus@
Inorganic salts such as potassium dihydrogen and magnesium sulfate are added. There is no particular culture condition, but it is usually 25-35℃.
To separate and collect the 12β-hydroxy ADD accumulated in the culture solution in this way, perform shaking culture or aeration agitation culture for 1 to 7 days.
An extraction method is adopted using an organic solvent that dissolves DD and causes phase separation from water, such as ethyl acetate, chloroform, or a mixture of chloroform and methanol. ? :,0 Extraction with organic solvent is I! This may be carried out on the culture solution containing the body, or it may be carried out on the culture filtrate or supernatant obtained by separating and removing the body and other insoluble components in the culture solution by filtration or centrifugation in advance. .

When the latter extraction method is adopted, the insoluble components that are separated and removed by filtration or centrifugation as necessary are subjected to the extraction operation using the organic solvent described above, thereby removing the precipitated or precipitated components in the culture solution. 12β-hydroxyAD
D can be recovered. By collecting the extract obtained by this pressure and distilling off the solvent, almost all of the target 12β-hydroxy ADD can be recovered.6 The obtained extract contains 12β- In addition to hydroxy ADD, the remaining substrate deoxycol and/or its salts and byproducts are included, and 12β-hydroxy ADD can be obtained by adsorbing the extract onto a silica gel column and eluting with a mixture of chloroform and ethanol. can be obtained.

12β-hydroxy ADD obtained by the method of the present invention
Fi, by partially hydrogenating its human ring and chemically removing the hydroxyl group at the 12-position, it is converted into 4-androstene-3,17-dione, which is a raw material for the synthesis of steroids such as spironolactone, which is useful as a diuretic. can do. For example, by partially hydrogenating 12β-hydroxy ADD with tris(triphenylphosphine) chlororhodium, 4-androste in 12β-hydro:
/-3,17-dione was obtained ((, DJ@rassi
and J.

Gutzwill@r, J, Am, Ch@m, B
oa,, g vol. 53, 4538-4539ji (19
6a)], by reacting this 12β-hydroxy-4-androstene-3,17-dione with 2-methyl-2-ethyl-1,3-dioxolane, the keto groups at the 3- and 17-positions are preserved. 12β-hydroxy-5-
Androstene-3゜17-thion-3.17-bis(
ethylene acetal) (G, Rosenkr
anz et al., J. Am. Chem.

8oc, vol. 75, pp. 5024-5026 (1954
0 This acetal was tosylated with tp-toluenesulfonyl chloride, reduced with lithium triethylborohydride to eliminate the sil group in the hydro at the 12th position, and then mineral acid/
4-androstene-3°17-dione can be obtained by treating with a7tone to remove the binding groups at the 3- and 17-positions.

The present invention will be explained in more detail below with reference to Examples.

Example 1 Aeromonas no hydrophylla D-238 strain (Koukou Kenzonoyori No. 6149) was cultured by the following method. Deogysicol 93 fs ammonium nitrate 0.12f
, phosphorus [2 hydrogen power IJ l) A 0.06 f, dipotassium hydrogen phosphate 0.3F, magnesium sulfate 7 hydrate 0.012F, yeast extract 0.024f and water-enriched sodium 0.3t, tap water Add to increase the capacity to 6. The culture medium was adjusted to OsJ and used as a medium. Pour 500g of this medium into a 1-volume Slope flask and heat at 120°C for 15 minutes.

Steam sterilization was performed. The 1Od of the inoculum grown for 2 days in a test tube shaker in the same medium as above was
00d was added to a Yosakaro flask and cultured with shaking at 30°C for 5 days. After culturing, the culture solution was collected and the bacterial cells were removed using a centrifugal separator. The resulting culture supernatant was extracted with 300 g of a mixture of chloroform/methanol at a volume ratio of 271, and then dissolved in chloroform/methanol using a rotary evaporator. By distilling off the methanol mixture, an oxidation product of i and sr deoxycholic acid and a mixture of unconverted deoxycholic acid and cholic acid were obtained.

Take a very small portion of the above mixture, add methanol to it to make a superior solution, and apply 20μ4 of this solution to a high performance liquid chromatography (HLC-GPC, manufactured by Waters Corporation, USA, equipped with a Micro Bondervac C-18 column). -244
all)&'C was injected. Adjust to 4.0 as mobile phase 1. When a mixed solution of water/methanol with a volume ratio of 25/75 was flowed at a flow rate of 11 L/min and detection was performed using the refractive index method, the chromatogram shown in Figure 1 was obtained. A% Beak B1 Beak C
1 Beak D1 Beak E1 Beak F and Beak G were determined by comparing with the standard product beak.
, 4-dien-3-one-20α-carboxylic acid, 12α
-Hydroxy-4-pregnen-3-one-20α-carboxylic acid, 12α-hydroxy-3-keto-4-choline, 12α-hydroxy-3-keto-5β-cholanic acid and deoxycholic acid. .

The above mixture 1.81 was dissolved in a small amount of chloroform and adsorbed on a silica gel column of about 30 f.9. Chloroform 100117 was passed through this column, and then 200d of a mixed solution with a volume ratio of chloroform/ethanol of 99.510.5, 200-d of a mixed solution with a volume ratio of 99/1, and 300d of a mixed solution with a volume ratio of 9872.

Mixed liquid 4QQs+j with a volume ratio of 97/3, a volume ratio of 95
A mixed solution 250114 of 15 and a mixed solution 300- with a volume ratio of 90/10 were run in this order, the eluate was fractionated into a fraction tube destined for 20-, and both fractions were separated to give a single spot by thin layer chromatography. The solvent was distilled off using a rotary volator and then eluted with a mixture of chloroform/ethanol in a volume ratio of 99.5/0.5 - 12β-hydroxy-
30'lt of 4-androstene-3,17-dione,
220"f of 12β-hydroxy ADD was obtained from the fraction eluted with the mixture at a volume ratio of 99/1, and 12α-hydroxy-3-keto-
4-cholenic acid to 30”F and 12α-hydroquine-3-
2 osql of keto-5β-cholanic acid! friend. IZa-hydroxy-4-pregnen-3-one-20α-carboxylic acid was extracted from the eluted fraction with a mixture of chloroform/ethanol at a ratio of 97/3 vol. 7011v of on-20α-carboxylic acid was obtained. Further, 450"lf of deoxycholic acid was recovered from the elution fraction with a mixed solution of chloroform/ethanol 09515 to 90/10 volume. Each of the obtained compounds was disclosed by the following method.

12β-hydroxy ADD Melting point = 213°C Mass hect/l/m/z: 300(M)”285
(M-CHa)”.

282CM-H20)”.

Furthermore, the presence of 3-keto-1,4-diene was confirmed from m/Z''=122.

N M Rx Hector (90MHz) δCDα3゜) IMS' 0.96 (・3H,a) 18-CHsl, 21(3H
, I) 19-CH3 2,30-2.50 (2H, m) 16-CHa3
．． 72 (IH, dd, J=5Hz and 10Hz)
1=2 a-H6,06(IH,!I)4-H6,20(tfi,d,J=toHz)z-H6,98
(IH, d, J=10H Tsuta) 1-H12β-hydroquine-4-androstene-3,17-diemma xxbe/
/I/m/z: 302 (Ml”287 (M-C
Although the presence of 3-keto-4-ene was confirmed by m/Z=124, the NMR spectrum k(90MHz) δI)MSO-46°HMS' 0.81 (3H, s) 18-CHal, 10(3
H, 5) 19-CHa 2.20-2.30 (2H, m) 16-CH23,50
(IH, dd, J=5Hz and 10H1) 12a-H
4,19 (IH, d) 12β-0H 5,60 (IH, 5) 4-H 12α-hydroxypregna-1,4-dien-3-one-2oα monocarbon enemy mass spectrum m/z −= 358 (ME “34
0 (M-HzO'J" 322 [M-2H20]""' Also, the presence of 3-keto-1,4-diene was confirmed from m/Z = 122, and the presence of 10H-COOH was confirmed from 1m/z = 74. Confirmed existence of OHs NMR spelling (90MHz) δDMSO-d5゜E
MS' 0.65 (3H,8) 18-CH51,05(3)
1. d) 21-CHs 1, 13(3H,a) 19-CHa 3.74(1)1. t, J=3Hri12β-H4,33
(IH, d) 12α-0H 5,93 (IH, 5) 4-H 6,03 (IH, d, J=IQHz) 2-H 7,08 (
IH, d, J = 10Hz) l-H12α-Hydroxypregnane-1,4-dien-3-one-20α-carboxylic acid methyl ester melting point = 237°C 12α-hydroxy-4-pregnen-3-one-20α -Carbonne mass spectrum m/z: 360 (M)”345
(M-CHa) “342 CM-H20)” “324 (M-HiO-CHa)”.

Also, from m/z=124, the presence of 3-keto-4-ene is 1
Iuiii.

The presence of -CH-COOH was confirmed from m/z=74.

CHa NMR spectrum (90Mfiz)δDM80
-d6゜HMS' 0.62 (3H, 5) 18-CHa l, 08 (6H, m) 21-CHa and 19-CHa3
．． 75 (1)1. t, J=3Hz) 12β-
H4, 28 (1) 1. d) t2α-0H 5,57(1)1. a) 4-H λ2α-hydroquine-3-keto-4-cholenic acid Max Spectrum m/z: 388 (M co".

370 (M-H5I(J) "355 CM-HgO-CHa)" Also, the presence of 3-keto-4-ene was confirmed from m/z = 124, and the presence of -CH2-C0OH was confirmed from m/Z = 60. Amed.

NMR spectrum (90MH2) δDMSO-d6°)
(MS' 0.60 (3H,a) 18-CHso, 86(3
H, d) 21-CHs l, 07 (3) 1, s) 19-CH53,7
5(114,t, J=3Hz)12β-H5.56(I
H, auto) 4-H 12α-hydroxy-3-keto-5β-cholanic acid mass spectrum m/m: 390 (M)”.

372 (M-H2O)".

354(M-2HaO)”.

339 (M-2H 0-CHs)" t*m, /z=6o j t) -CHx -COOH
f) Existence dllllWtLfiney.

NMR2 vector (90MHz) aDMSO”HMS
' 0.57 (3M, 11) 18-CHa o, 83 (3H, d) 21-CHa o, 89 (3H, a) t9-CI (a 3.76 (IH, t, , y=anz) 12β -H
Integrating the area ratio of each peak in the chromatogram shown in Figure 1 with an integrating needle (Takafusa Chromato Pack C-RIA, manufactured by Takafuji Seisakusho)
The yield of the oxidation product of deoxycholic acid obtained from this area ratio and the remaining amount of unconverted deoxycholic acid are calculated as shown in the following table.

Example 2 The same method as in Example 1 was repeated except that Aeromonas hydrophila D-23811a (i. A mixture of its oxidation product and unconverted deoxycholic acid 1.02F was obtained from Cole@31.

Take a very small portion of the mixture, add methanol to it to make a solution, and transfer this solution (20 μm) to a high performance liquid chromatography machine equipped with a Microbondapak C-18 column (manufactured by Waters, USA, 1 (LG)). -GPC-244
11i). As the mobile phase...a 25/75 volume mixture of water/methanol adjusted to 4.0 was used at a flow rate of 1.
g//min and detection was performed. Hereinafter, the yield of the oxidation product of deoxycholic acid obtained by the same method as in Example 1 and the remaining amount of unconverted deoxycholic acid were determined. The results are shown in the table below.

[Brief explanation of the drawing]

FIG. 1 shows a liquid chromatogram of the deoxycholic acid oxidation product and unconverted deoxycholic acid obtained in Example 1. 0 3 6 q 1215 /2r 21
Procedural amendment (1 letter) January 11, 1980 Mr. Haruki Shimada, Commissioner of the Japan Patent Office ■, Indication of the case 1981 Patent Application No. 181783 2, Name of the invention 12β-Hydroxyandros ll- 1,4-diene-
3,17-Dione manufacturing method RiRa Co., Ltd. 1621 Sakazu (108), Kurashiki City Representative Director: Tsuguo Okabayashi 4, Agent: Kuraray Patent Department Telephone, Tokyo 03 (277) 31826, Contents of the amendment (1) The statements (claims) on page 1, lines 6 to 14 of the specification are corrected as shown in the attached sheet. (2) Delete "as the main carbon source" in the second line of page 2 of the specification. (8) [M-H2O at the bottom of the 19th line of the specification
-Correct CHa J to l M-2H20J. Above (attached sheet)

Claims (1)

[Claims] 12β using deoxycholic acid and/or its salt as a substrate
-Hydroxyandrosta-1,4-diene-3,1
Bacteria belonging to the genus Aeromonas that produce 7-dione,
12β-hydroxyandrosta-1 was cultured in a medium containing deoxycholic acid and/or its salt as the main carbon source.
, 4-diene-3,17-dione is produced, grown, and collected.