JPS5851897A - Biological conversion of steroid and product - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION U.S. Pat.
-Hydroxybisnoric acid] and disclosed the use of Mycobacterium foratum NRRT, B-8119, and filed patent claims for the use thereof. The same microorganism is described in U.S. Patent No. 4.035.
In No. 236, 9-hydroxy-4-androstene-3
, 17-Sion [9-Hydroxyandrostenedione] (HAD), U.S. Pat.
In No. 14,051, 9-hydroxy-3-oxo-4-
It is used to make pregnene-added-carboxylic acid methyl ester [9-hybiloxybisnoric acid methyl ester].

By derivatization of 6-fluoro-Δ'-3-ketone derivatives of strols, such as cholesterol, sitosterol, stigmasterol, etc., useful and medium MC[-fluoro-9α-hydroquino-androst-4-ene-3 ，
A fermentation method for producing 17-sion (6-fluoro-HAD) has been disclosed and is claimed in Patent No. 5-5. See Figure I. This method is carried out using an M. Foretzum NRI B-8119. The present invention relates to the genera of microorganisms disclosed in U.S. Pat. , and the use of mutant strains obtained from Streptomyces. All of these sail microorganisms are well-known sterol-degrading microorganisms. Wild-type strains of these genera contain sterols in non-iA
Alternatively, it is decomposed into low molecular weight compounds, such as C02+H20. The mutant strain is covered by a US patent for $4,029,549.
can be made from these wild types according to the procedure set forth in Example 1 of this issue. This example demonstrates the preparation of M. foretzum NRRL B-8119.

This method is the only known method for making 6α-fluoro-HAD.

A microorganism characterized by the ability to selectively convert 6-fluoro-Δ'-3-ketone derivatives of sterols with 6-l7-alkyl side chains and accumulate 6-fluoro-HAD in the fermentation broth. The mutant strains belong to the following genera: Arthrobacterium, Bacillus, Brevibacterium, Corynebacterium, Micro/4Cterium, Myco/4Cterium, Nocardia, Protaminobacter, Serratia, and Streso2.
obtained by mutating the microorganisms of Seth.

Mycobacterium foretutum NRFtTJB-
8119 is described in US Pat. No. 4,029,549. M.

P. fortutum NRRL B-8119 has been deposited in the permanent stock archive of the U.S. Department of Agriculture Northern Region (iIf Institute), Bioria, Illinois, United States. At the latest, the microorganism has been identified and, after issuance of the above-mentioned U.S. patent, the microorganism has been made available to the public. It should be understood that the availability of a culture medium does not create a patent license to practice the invention in a trench in violation of the patent rights granted with the certificate by government action. .

6-Fluoro-HAD is useful as an intermediate in the synthesis of valuable corticosteroids. For example, as revealed in the steroid literature, with the application of current technology, fluoro-HADum forms can be used pharmacologically as fluocinolone acetonide, fluocinonide, flulandrenolide 9, paramethacin, and fluprednisolone. Can be converted into important steroids.

The following are examples illustrating the fermentation method of the present invention.

These examples are merely illustrative and therefore should not be considered limiting. If there is no other quantity to order,
All percentages are by weight and all proportions of solvent mixtures are by volume.

Example 1 Fermentation bioconversion medium of 6β-cholestenone was prepared in tap water at a concentration of 8.0% Necon per ton. Of,
Serious L/loin5. (1?, NH4C/= 3.Or,
CaCO33,0], Na3[I <citrate]
2H203,017', Tween 802. Of, large V
Powder 1. Of, KH2P 0.0.59, urea 0.57, and 6β-F-cholestenone 2. Contains Off, T) H7
,0. In flasks containing 100 mJ each of this medium,
Inoculate with 10 mA of inoculum culture medium of -8119. This culture medium is mixed with distilled water and Nutriendo Broth 8.0? ,
Glycerol 5. Of, yeast extract 1.07, Tween 80], and grown at 28'C in a medium adjusted to pH 7.0. The culture medium is then incubated for 336 hours on a 4° rotary shaker. After incubation, the mixture is extracted with an equal volume of CH2C42 and the product 6β-fluoro-HAD is isolated from this extract using standard chromatography.

Example 2 In Example 1, 6α-fluorocholestenone was replaced with 6α
- 6α-fluoro-9α-hydroxy-AD75 using fluorocholestenone.

Example 3 By using another sterol in place of 6β-fluorocholestenone in Example 1, such as a β-fluoroketone derivative such as cyclosterol, stigmasterol, 6β-
Fluoro-HAD is obtained.

Either alone or in combination with sterols, purified or 9- can be used in crude form.

Example 4 Mycobacterium foretutum NRE (L.
Using sprawl-degrading signature organisms from the genera Arthrobacterium, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Nocardia, Protaminobacter, Serratia, and Strezotomyces in place of B-August 9, C- A mutant microorganism characterized by the ability to selectively convert 6-fluoro-Δ'-3-ketone conductors of 17-side chain sterols and accumulate 6-fluoroHAD in the product and 1 to 1. can get.

Example 5 Using another sterol in place of 6β-fluorocholestenone in Example 1, such as a 6α-fluoro-Δ4-3-ketone derivative such as sitosterol, stigmasterol, 6α-fluoro-HAD is obtained. The sterols can be used alone or in combination, in purified or crude form.

Example 6 M of Examples 1 and 3, Foretzum NRRLB-8
Using the mutant strain obtained in Example 4 instead of 119, 6β-fluoro-HAD is obtained.

Example 7 M of Examples 2 and 5, Foretzum NRRL B −
Using the mutant strain obtained in Example 4 instead of 8119, 6α-fluoro-HAD is obtained.

Example 8 The sterols 6-fluoro-Δ4-3-
Ketone derivatives are created.

Small force inhalation: y 7 nofu 0 jiyoshi nnnnomany'
1 I [embedding 廿bury± 4/Llq square 1 Yamajinto 1'5. ．． .

(Other 1 nails '1, 5, 11-Continuation of page 1 ■Int, C1, 3 identification code Internal reference number C1
2R1/33 1/365 1/425 1/465 - Inventor: Clifford Niezin Sachs, 80 Newells Lane, Vaultage, Michigan, USA 3 (Old Inventor: Merle Jean Woufcha, Mazur, Michigan, USA)・South Prairie Avenue 145 1 Co. 2- Procedural Amendment (Method) % Formula % ) ) 1. Indication of the case 1982 Patent Application No. 122244
No. 2, Title of the invention Steroid bioconversion method and product 3. Relationship to the amended person's case Patent applicant Kalamazoo, Michigan, United States of America Address Henrietta St. IJ-)3o1 Name The Upjohn Company 4, Agent address 160 2-8 Shinjuku, Shinjuku-ku, Tokyo
No. 1 Shinjuku Seven Building Room 608 (6601 low name 4 Akebono Yaya Sasai, Tabe (phone number 35)
4-1285■～6)

Claims (1)

[Claims] 1. Mycobacterium foratutum NRRL in an aqueous nutrient medium under aerobic conditions in the presence of 1, c-17 (6-fluoro-Δ'-3-ketone derivative of sterol with tlll chain) 2. The sterol is from the group consisting of sitosterol, cholesterol, stigmasterol, camposterol or a mixture of the above sterols. 3. In the presence of a 6-fluoro-Δ'-3-ketone derivative of a sterol with or without a C-17911 chain,
Arthrobafter (A) in an aqueous nutrient medium under aerobic conditions
rthrobacter), Patil y, (Bacchi
llus), Brevibacterium (Bre-viba
cterium), Corynebacterium (Cory-
nθbactθrium), Microbacterium (M
ic-robacterium), Nocardia (
Nocardia), Protaminobacter (Pro
toaminobacter), Serratia (5er
ra, tia), and Streptomyces (Strep
tomyceθ), and this mutant strain is selected from the group consisting of C-
6-Fluoro-Δ'-3 of sterols with 17 side chains
- selectively converting ketone derivatives into 6-fluoro-HA
A process for the fermentative production of a compound as defined in claim 1 characterized by its ability to accumulate D. 4. The method according to claim 3, wherein the sterol is selected from the group consisting of sitosterol, cholesterol, stigmasterol, and hycampesterol or a mixture of sterols. 5. 6-fluoro-sterols with C-17 side chain
A Mycobacterium mutant strain characterized by the ability to selectively convert Δ′-3-ketone derivatives and accumulate 6-fluoro-HAD in the fermentation broth was incubated in the presence of a steroid with a C-17 side button. A process for the fermentative production of a compound as defined in claim 1, comprising culturing it in an aqueous nutrient medium under aerobic conditions. 6. The method according to claim 5, wherein said sterol is selected from the group consisting of sitosterol, cholesterol, stigmasterol, and campesterol or a mixture of sterols. 2. A process according to claim 1, wherein the 6-fluoro-hen'-3-1) derivative of the 1/-care sterol is 6β-fluorocholestenone. Process according to claim 1, wherein the 6-fluoro-Δ'-3-ketone derivative of L sterol is 6α-fluorocholestenone. 9. Things Illustrated Society'Fri 6c <-FIL Saiguchi-Sehi L'・j] A No Acid Ust-4-1> -
3z /'7-;”fn-