JPS62118897A - Hydroxylation of 11beta-position of steroid - Google Patents

Abstract

PURPOSE:To efficiently introduce hydroxyl group into a steroid, by inoculating a microorganism of the genus Nakataea having the ability to hydroxylate hydrogen atom of the steroid, etc., into a culture medium containing the steroid and cultivating the microorganism. CONSTITUTION:A microorganism, e.g. Nakataea sp. MCI2136 or Drechslera sp. MCI2139, etc., belonging to the genus Nakataea or Drechslera and having the ability to hydroxylate hydrogen atom at the 11beta-position of a steroid expressed by the formula (R1 and R2 are hydrogen atoms; R3 and R4 each are hydrogen atoms, hydroxyl or acyloxy groups) is inoculated into a culture medium containing the steroid and aerobically cultivated. Alternatively, oxidase produced by the above-mentioned microorganism is aerobically reacted with the compound expressed by the formula to introduce hydroxyl group into the 11beta-position of the above-mentioned steroid.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention provides pharmaceutically useful noidrocortisone ('/
/β, lliα, 2/−) lyhydroxyderpregnene-3,20-dionno and preddinolone (/β,
77α! The present invention relates to a novel microbiological or enzymatic process for the production of Roxy/, II-pregnagen-3,:lO-dione] or substituted derivatives thereof.

C. Conventional Techniques Conventional methods for producing hydrocortiscin and prednisolone or substituted derivatives thereof include /7α,-nodihydroxyderpregnene-3,coQ-dione (hereinafter referred to as
A method of hydroxylating the 771-position of ``compound day'' or a substituted derivative thereof using a microorganism, and desoxycorticosterone
Various methods are known, such as a method of hydroxylating the 110-position or 9α-position of [co0-dione] using microorganisms and chemically converting it to the //β-position.

Among these, the former method is particularly important.

The microorganisms used in this method include Curvularia-A/Nata N RRL J J 10 (
0urvularialunata NRRLJJff
OJ (, r, jLo, 8..77.71-3°/9
! r! ], Canninghamera Prakisurina H-331t Corticium Sasaki X'FO! r2! It (Oort
iciumliasakii IFOj J & Hiroshi) (Bull, Agr, Ohem, 800°, T
apan, 2/, J de 0. /9 to 7), Streptomyces fradiae 3!3 (8treptomyce
a fradiae 3 j j ! r) (U Japan P!, 4II9゜4IO/), Absidia elegans CAbsidia Rishi Isono, Botry 77. - Minerena (Botrytis c.
inerea) (Folia Microbiol,
6. KO3RI, /9A/), etc. are known.

(Problems to be Solved by the Invention) However, these microorganisms do not necessarily have a high hydroxylation ability, so their reaction rate is slow (and their light absorption is not high either).

Measures for Solving Problems c] Therefore, the present inventors conducted various studies on various microorganisms, and found that
The present invention was completed by discovering that some strains of the genus Nakataea and genus Drechslera have excellent hydroxylation ability.

In other words, the gist of the present invention is based on the following general formula (I) (in the above formula, R1 and R each independently represent a hydrogen atom, an alkyl group, an /SCl gene atom, or a &i water group). , R3
and R4 each independently represent a hydrogen atom, a hydroxyl group or an acyloxy group. Also, the dashed line of Δ Inspector indicates a single bond or a con bond. A microorganism belonging to the genus Nakataea (NeLklLtae&) or the genus Drechslera and having the ability to hydroxylate the hydrogen atom at the 1/β position in the above-mentioned SFCs 4 V class f) was inoculated into a medium containing steroids represented by 7 of the steroid by culturing aerobically, or by aerobically acting on the compound CI) with an intensifying enzyme produced by the bacterium.
The present invention relates to a method for hydroxylating the 111-position of steroids, which is characterized by introducing a hydroxyl group at the 71-position.

The present invention will be explained in detail below.

Nakataea f4 or drek/(L/U() used in the present invention)
It belongs to the genus Drechs1era and is a steroid.
Examples of microorganisms that have the ability to hydroxylate β-position hydrogen atoms include the soil-derived Nakataea Sp. / 3 Is (Nakataea s
p, Me 2/, 7 A) (
IFKRM P-1r/4), Drechalera Fist SP MO Eco 1J9 (Drechalera ap0
MO eco 13de) (IFffiRM P-Pl:/
7).

(1) Nakataea lip, Molko/,
14 colonies initially appear white and later dark brown on potato dextrose agar medium. Conidiophores solitary, usually unbranched, rarely branched, septated, brown, smooth,
Goo 3 - Leaves a clear separation mark (5 car J).

Conidia are symbogeo-shaped conidia, sickle-shaped to S-shaped curved, with J-septata. The intermediate cells are larger than the terminal cells;
The cells at both ends of the cell are light brownish and narrow, pointed, and colorless, 10-/4tμm. Eco/36) is /) The conidiation mode shows the symbogeotype, the sword conidia are sickle-shaped to S-shaped, have 3-septate walls, and the 3-noconidial intermediate cells are larger than the cells at both ends. , light brown (L Deku/J' H7phOm7
013tee″N', gAr3-g!j? By Deimadacious and Ellis (/? 7/) ' Dema
tiaceous Hyphomy Seven Deaths Hyphomycetes” P, 2/9~!,
2/ (IIJ Drechsle
ra sp, Me engineering J/,? ? Colonies appear gray to black on potato dextrose agar. The hyphae are stiff and elongate radially. The conidiophore is succulent;
Rigid, erect or bent; often zigzag, light brown to brown, smooth, up to 7-70 μm wide and 140 μm long. Conidia are bolo-shaped conidia, cylindrical, with 3-9 pseudoseptates, olive-brown in color, eco,! −
/! r, 4 μm, X39-! Da μm.

Growth temperature 2 S℃~30℃ Growth pant-i.

This strain (Moeco 13?) shows a υboro-type conidia formation mode, and the 2 conidia have pseudoseptates.
a Hyphomycetes” P, '103~ Just in case (matches).

The nutrient medium for these bacteria preferably contains carbon and nitrogen sources that can be assimilated by the bacteria, as well as inorganic salts necessary for their growth. Carbon sources include glucose, lactose, sucrose, dextrin, starch, glycerin, and other substances that can be assimilated by bacteria, which are commonly used for this purpose; nitrogen sources include, for example, peptone, meat extract, casein, edamine, and corn steep liquor. , yeast or yeast extract, nitrogen-containing organic substances such as soybean products, nitrogen-containing organic compounds such as urea, amino acids, ammonium salts of organic acids, and inorganic nitrogen compounds such as ammonium nitrate, ammonium phosphate, ammonium sulfate, sodium nitrate, as well as inorganic salts. A desired medium can be prepared by appropriately adding inorganic salts necessary for the growth of bacteria, such as potassium phosphate, sodium chloride, magnesium sulfate, and iron sulfate.

An aqueous medium having a composition as described above has a pFi of about pFi before sterilization.
p~? is adjusted to

As a method for culturing bacteria, it is possible to use static culture, but since the bacteria themselves are aerobic, culture methods carried out under aerobic conditions, such as shaking culture or deep cultivation using agitation and aeration, are recommended. Cultivation methods and the like are advantageous in practice.

Further, the raw material steroids used in the present invention are represented by the above general formula (I).

The acyloxy group is generally an aliphatic oxy group such as an acetoquine group or a propionyloxy group.

The starting time of the addition of the raw material steroid compound is selected at the beginning of the culture or at an appropriate time during the course of the culture.

The raw material steroid compound can be used as it is in the form of a fine powder, or for example, methanol, ethanol, n-propanol, isopropanol, n-butanol, ethyl acetate,
As a solution or suspension in a suitable solvent such as acetone, dioxane, dimethylformamide, etc., or as a solution or suspension in which a surfactant, dispersant, etc. are added, either all at once or continuously over a certain period of time. Or, it is intermittent. In particular, methanol and ethanol can have a good effect on the reaction when added to the reaction solution in an amount of 0.1 A-10.

The acidity of the medium, reaction temperature (culture temperature), reaction time (culture time), and other conditions in the reaction vary depending on the raw material steroid compound, the selected bacteria, the substrate concentration, the medium composition, etc. It is desirable to select suitable conditions for implementation. Therefore, the reaction is generally carried out at an initial pH of 4 (-7) at a temperature of -0-JθC for about 10 hours to 10 days, but the reaction is not limited thereto.

In addition, in the reaction, the above-mentioned proliferating microbial cells may be used as other germ-static cells, immobilized microbial cells, and treated microbial cells.

Various separation means are used to separate the oxidation products that have accumulated in the reaction medium. For example, there is an adsorption method in which the target substance is adsorbed on an appropriate adsorbent such as alumina, 70 lysyl (synthetic magnesium silicate, activated carbon, etc.) and then eluted with an appropriate monoprotic solvent such as methanol or ethanol, or an adsorption method in which the target substance is adsorbed on an appropriate adsorbent such as alumina, 70 lysyl (synthetic magnesium silicate, activated carbon, etc.), and then eluted with an appropriate monoprotic solvent such as methanol or ethanol, or chloroform, methylene chloride, ethylene, etc. Direct extraction using a halogenated hydrocarbon such as chloride or an organic solvent that can form a co-liquid phase with water such as acetic acid esters, or a method that utilizes the difference in distribution likelihood between the co-liquid phases through countercurrent distribution, or Based on the same principle, a chromatographic method using a suitable carrier such as alumina, silica gel, or cellulose bulb, or a method that utilizes the difference in solubility, or a solution containing the target substance once extracted, is used to extract trimethylammonium acetate hydrazide, pyridinium, etc. A variety of separation methods are used to separate the target substance, such as a means of separately collecting the target substance as its functional conductor using a hydrazide region such as acetic acid hydrazide or an acylating agent using a lower fatty anhydride and a deoxidizing agent, and then returning it to its original form. They are appropriately selected and used depending on the structure and functional group of the target product.

The product compound obtained by the method of the present invention is useful, for example, as an adrenocortical hormone active substance or a synthetic intermediate thereof.

(Examples) Hereinafter, the present invention will be explained in more detail with reference to Examples.
The present invention is not limited to the following examples unless it exceeds the gist thereof.

Example 1 Nakataea (NakataeaJ 8p, MOL2/
JA 500r containing 100ml of medium with the following composition
Seed culture was performed using a rotary shaker at 17° C. for 27 hours using an Erlenmeyer flask with a hemlock.

Medium Glucose IOg/i00 Gopeptone Coconut Staple Liquor □,! 1 twin 30″
0.3 [J manufactured by Kao Atlas Co., Ltd. [Surfactant] (PHj, θ) Next, this culture solution 1 was added with the same composition as the free culture, 10θ. ; 00ml Erlenmeyer flask with hemlock Kfn was cultured for 1 day and then reacted.

After the reaction, the bacteria were removed by centrifuging the methyl alcohol at 100 ml, and the amount of hydrocortisone produced was determined. The production yield was 30.3 mol.

Analysis was performed by high performance liquid chromatography.

Analysis conditions Power Ram II “Waters Radial Pack CG”
(Waters Inc.) Eluent: Water/Methyl Alcohol = Da/6th stream i:
/ ml/min Detection: UV
The reaction was carried out in the same manner as in Example 1 except that 1Me eco/, 79 was used.

The production yield of hydrocortiscin was 41 mol%.

Example 3 The reaction was carried out in the same manner as in Example v12 except that the number of days for the reaction was changed to 5 days. )・Idrocortisone production yield is ko/,
It was in charge of 1 mole.

(Effect of the invention) According to the method of the present invention, the effectiveness of ink (, /lβ of the steroid region
A hydroxyl group is introduced at this position.

Claims (3)

[Claims] (1) The following general formula (I) ▲Mathematical formulas, chemical formulas, tables, etc.▼(I) (In the above formula, R_1 and R_2 each independently represent a hydrogen atom, an alkyl group, a halogen atom, or a hydroxyl group, R_3 and R_4 each independently represent a hydrogen atom, a hydroxyl group, or an acyloxy group. Also, the broken lines in parts △^1^ and ^2 indicate a single bond or a double bond.) A medium containing steroids represented by In the genus Nakataea (￥Nakataea￥) or Drechslera (
A microorganism that belongs to the genus A method for hydroxylating the 11β-position of steroids, which comprises introducing a hydroxyl group at the 11β-position of the steroid by aerobically using an oxidase to oxidize the steroid. (2) The method according to claim 1, wherein R_1 and R_2 of the compound (I) are hydrogen atoms, and R_3 and R_4 are each independently a hydroxyl group or an acyloxy group. (3) The method according to claim 1, wherein R_3 and R_4 of the compound (I) are hydroxyl groups.