JPS627905B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing anthracyclinone, a novel anthracyclinone, 2-hydroxyclavinone, and a strain of microorganism used for the production.
galilaeus) MA 144-M1 A method for producing anthracyclinone by culturing a mutant strain derived from FEIGEN BIYORI NO. 2455, FEIGEN BIYORI NO. 5081, or FEIGEN BIYORI NO. The present invention relates to a novel anthracyclinone, 2-hydroxyclavinone, produced thereby, and a strain of the above-mentioned microorganism used for its production. The present invention relates to novel anthracyclinones or their production, and more particularly, to In the formula, R represents a hydrogen atom (H) or a hydroxyl group (OH). The present invention relates to a method for producing anthracyclinone, a novel substance 2-hydroxyclavinone produced thereby, and a microorganism used for its production. In producing a useful anthracycline antibiotic that reduces the side effects of adriamycin and daunomycin, which are widely used clinically as cancer chemotherapeutic agents, and further enhances the accessory cancer activity, we decided to As a result of repeated research, we focused on the fact that anthracyclinone (aglycone part), which is the skeleton part, can be converted by microbial or chemical means to advantageously produce an effective anticancer substance.
The present inventors have discovered that acrapinone and a new 2-hydroxyaclavinone can be most advantageously used as raw materials for anthracycline antibiotics, and have found a method for producing these aglycones in significant amounts by direct fermentation, thereby completing the present invention. In addition, among the above anthracyclinones, 2-
Hydroxyclavinone is a novel substance that has not been reported before, and is the first substance discovered by the present inventors. On the other hand, aclavinone is a known substance that can be chemically obtained by the present inventors as a hydrolysis product of aclacinomycin, but it is not known that it accumulates in significant amounts directly in the culture medium by microorganisms. This was made possible for the first time by an invention. Next, the method for producing 2-hydroxyacrabinone and akravinone of the present invention will be explained. The bacterial strain used in the present invention is Streptomyces
Galilaeus (Streptomyces galilaeus) MA
144-M1, Microtechnical Research Institute No. 2455 with ultraviolet rays or
Bacterial strains obtained by mutation treatment with NTG etc. are used as producing bacteria for both substances, and among them, for example, Streptomyces galilaeus MA 144-ANR-58
and -ANR-635 (hereinafter simply referred to as ANR-58 and ANR
-635) is useful for the production of 2-hydroxyaclavinone and akravinone, respectively. Both strains were entrusted to the National Institute of Microbiology, Agency of Industrial Science and Technology, and the ANR-58 strain was awarded the Microbiological Research Institute No. 5081, and the ANR-635 strain was awarded the Microbiological Research Institute No. 5082. The main mycological properties of the parent strain Streptomyces galilaeus
galilaeus) MA 144−M1 (hereinafter simply MA 144−
It is also listed in comparison with the properties of M1 (abbreviated as M1) (described in Japanese Patent Publication No. 51-34915). Mycological properties of MA 144-M1 (parent strain) and mutant strains ANR-58 and ANR-635 used in the present invention (1) Morphology MA 144-M1, ANR-58, and ANR-635 strains are all Aerial hyphae with a hook-shaped to spiral formation extend from clearly branched basal hyphae, and whorled branches are not observed. MA 144−M1 and ANR−
The mature spore chain of 58 strains was found to be a chain of 10 or more spores, and the spore size was 0.4 to 0.8 x 0.8.
The surface of the spore is smooth at about 1.6 microns.
The ANR-635 strain does not form aerial hyphae on most media, but slightly forms aerial hyphae on yeast extract, maltose extract, and agar media, but no mature spores are observed to adhere to it. (2) Growth status in various media Regarding color descriptions: Standards shown in brackets are Container, Corporation, Of, American Color, Harmony, and Manual (Container-Corporation of America Color).
harmony manual), and also referred to the color standards of the Japan Color Research Institute.

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[Table] (3) Physiological properties No differences were observed between the parent strain and both mutant strains in physiological properties such as gelatin decomposition, melanin pigment production, starch decomposition ability, peptonization of skin milk, and carbohydrate utilization. Its properties are as follows. (1) Growth temperature range Maltose, yeast extract agar (maltose
1.0%, yeast extract (oriental) 0.4%,
Using thread agar 3.5% PH6.0), 20℃, 24℃,
As a result of experiments at 27°C, 30°C, 37°C, and 50°C, it grows at all temperatures except 50°C, but the optimum temperature seems to be around 27°C to 37°C. (2) Liquefaction of gelatin (15% simple gelatin, 20℃
Culture; glucose, peptone, gelatin, 27
(°C culture) In the case of simple gelatin, liquefaction is observed from around 14 days after culture, and its effect is weak. In the case of glucose, peptone, and gelatin, liquefaction begins around the seventh day after culturing, and the effect is weak to moderate. (3) Hydrolysis of starch (starch, inorganic salt agar, cultured at 27°C) A slight amount of water decomposition is observed from around the 5th day after culture, but its effect is weak. (4) Coagulation and peptonization of skimmed milk (skimmed milk,
(Culture at 37℃) Peptonization begins around the 5th day after culturing.
It is almost complete by the 17th day, and the effect is moderate to strong. However, only the ANR-635 strain is slightly weak. No coagulation is seen. (5) Production of melanin-like pigments (tryptone, yeast, Pros, ISP-medium 1; peptone, yeast, iron agar, ISP-medium 6; tyrosine agar, ISP-medium 7, all cultured at 27°C) Tryptone, yeast, Production of melanin-like pigments was observed in all of the media: Pros, peptone, yeast, iron agar, and tyrosine agar. (6) Utilization of carbon sources (Pridham-Gottlieb agar, ISP-Medium 9, 27°C culture) It grows well and does not use D-mannitol. (7) Dissolution of malate lime (malate lime agar,
(Cultivated at 27℃) Dissolution of lime malate was observed, and the effect was strong. (8) Nitrate reduction reaction (peptone water containing 1.0% sodium nitrate, ISP-medium 8, cultured at 27°C) Positive. The MA 144-M1 strain, which has the above characteristics, is the closest relative to it, Streptomyces galilaeus.
By comparison with ISP5481 strain, Streptomyces
This strain was identified as Galilaeus MA 144-M1 strain and has been deposited at the National Institute of Microbiology, Agency of Industrial Science and Technology and is kept as deposit number 2455. The mutant strain used in the present invention is subjected to artificial mutagenesis treatments commonly used for the Streptomyces galilaeus MA 144-M1 strain, such as α, β, γ rays and X-rays.
Mutant strains can be induced by irradiation with ionizing radiation such as X-rays or by contact with chemical inducers such as NTG or diepoxybutane.
Separated and cultivated. An example is shown below. () Mutation treatment The mutant strain Streptomyces galilaeus
ANR-58 and -ANR-635 were obtained from the parent strain (Streptmyces galilaeus) MA 144-M1, Kaikoken Bacterium No. 2455, by the following method. Scraped from the surface of one St. galilaeus MA 144-M1 slant culture cultured on YS agar slant medium (yeast extract 0.3%, insoluble starch 1.0%, agar 1.5% PH7.0) at 28℃ for one week. Spores PH7.5
Suspended in 5 ml of 0.2M Tris-malate buffer,
Ultrasonic treatment was performed twice for 15 seconds each.
Distruptor model 1UR-200p type, 20KHz, Tomy Seiko〓). Pass the treated solution through a pre-sterilized cotton filter tube (diameter 0.8 x layer thickness 2 cm),
A solution of N-methyl-N'-nitro-N-nitrosoguanidine (NTG) (10 mg/ml) dissolved in ethanol was added to the spore suspension (4 ml, approximately 5 x 10 ⁸ spores/ml) to a final concentration of 1000 μg/ml. The mixture was shaken at 30°C for 60 minutes in the dark. At this time, the sporicidal effect was 90.6%.
Centrifuge the NTG-treated spore solution at 300 rpm for 10 minutes.
Suspend the spores in 0.85% physiological saline, dilute it appropriately and apply it to a shear plate containing YS agar medium.
Culture was performed at ℃ for 5 days to form colonies. () Method for isolating mutant strains Colonies formed on the YS agar plate medium as described above were picked up on a YS slant medium and cultured at 28°C for one week. Add 1 platinum loop from each slant to 4ml of YS liquid medium.
The whole amount was inoculated into a 250 ml Erlenmeyer flask containing 25 ml of sterilized fermentation medium (as shown in Example 1 below) for 48 hours. Culture on a rotary shaker (220 rpm). Sample 3 ml of culture fluid, add 0.5 ml of 0.1 M Tris buffer (PH7.5) and 1 ml of toluene, stir and extract with a mixer, and completely separate the toluene extracted layer by centrifugation. , 10μ of the solution was spotted on a thin layer of silica gel (Merck F254) and developed with chloroform-methanol (20:1).
Spots showing the same Rf value as the standard akravinone were detected using a Shimadzu TLC chromatography scanner (CS-910).
Search for strains that produce a significant amount of akravinone, and find useful mutant strains such as ANR-635.
In addition to isolating a bacterial strain, we searched for a strain that provides a new aglycone spot that has not been previously observed.
ANR producing hydroxyclavinone-
58 bacteria were obtained. To culture both strains, first, the strains stored at 6-7°C in a YS agar slant culture medium as a seed medium are inoculated into a normal liquid medium containing, for example, starch, glucose, an organic nitrogen source, and inorganic salts. A seed mother is prepared by culturing with shaking at ℃ for 1 to 2 days. Next, 1-3% of the above seed mother was inoculated into a normal liquid medium such as sucrose, glucose, soybean flour, and inorganic salts, and the seed mother was incubated at 25-32°C for 36-100 min.
Perform shaking culture for hours. The culture solution is separated into bacterial cells and liquid, and the pigments are extracted and purified from each. Acetone, chloroform, methanol, and an acidic buffer are usually used for extraction from bacterial cells; the solution is extracted with chloroform, toluene, methanol, and an acidic buffer. Sephadex for purification
LH 20 (cross-linked dextran gel)
linked dextran gels）Pharmacia Fine
Chemical AB), silica gel (for columns,
Wakogel C-200 (Wako Pure Chemical Industries)) and for thin layer chromatography, Kieselgel 60PF254
Gel filtration, adsorption chromatography, thin layer chromatography, etc. such as those manufactured by Merck (Merck) are advantageously used. The obtained compounds were identified by ultraviolet, visible light absorption spectra (CV), infrared absorption spectra (IR), 100MHz high-resolution proton nuclear magnetic resonance spectroscopy (PMR), and mass spectrometry analysis, and were found to be derived from the ANR-58 strain. The desired 2-hydroxyaclavinone can be obtained from the culture solution, and akravinone can be obtained from the culture solution derived from the ANR-635 strain. The 2-hydroxyclavinone thus obtained is useful as a synthetic intermediate or a precursor for microbiological conversion of anthracycline antibiotics, as described above. For example, “Topics in Antibiotics Chemistry,” edited by P.G.
Antibiotic Chemistry”ed.by PGSammes,
Vol.2, Ellis Horwood, Chichester,
England), 99-239 (1978) to give anthracycline glycosides, and as is clear from the examples described in that document, such glycoside derivatives have a saturated A ring. Due to its structural feature of naphthacenedione bone core, it is highly likely to be an excellent anticancer agent. Examples of the present invention are shown below. Example 1 Production of 2-hydroxyaclavinone Soluble starch 1.0%, glucose 1.0%, meat (soybean flour, manufactured by Ajinomoto Co., Ltd.) 1.0%, KH ₂ PO ₄ 0.1%,
_MgSO4・_7H2O 0.1%, _CuSO4・_5H2O 0.0007
%, _FeSO4・_7H2O 0.0001%, _MnCl2・_4H2O
Transfer a medium consisting of 0.0008% ZnSO ₄ 7H ₂ O 0.0002% (PH 7.4) to a 500 ml Erlenmeyer flask.
Dispense in ml portions, sterilize, Streptomyces galilaeus MA 144-ANR-58 (Feikoken Bacterial Collection No. 5081)
One platinum loop was inoculated from the slant culture of No. 1 into three of the above-mentioned medium, and cultured with shaking at 28°C for 48 hours to prepare a seed mother. Soluble starch 1.5%, glucose 1.0%, meat (soybean flour, manufactured by Ajinomoto Co.) 3.0%, K ₂ HPO ₄ 0.1%,
_MgSO4・_7H2O 0.1%, _CuSO4・_5H2O 0.0007
%, _FeSO4・_7H2O 0.0001%, _MnCl2・_4H2O
0.0008% ZnSO ₄ 7H ₂ O 0.0002% (PH 7.4) was dispensed into 300 500 ml Erlenmeyer flasks in 50 ml portions and sterilized, the above seed mother was inoculated with 1 ml per flask, and the rotary shaker ( Number of revolutions
220 rpm) for 48 hours. obtained 14
The culture solution was separated into bacterial cells and liquid by filtration, and the substance was extracted and purified from the bacterial cell fraction as follows.
The total amount of bacterial cells was suspended in 2.5 parts of acetone, stirred and extracted, and the acetone extract obtained separately was concentrated under reduced pressure, and the target substance was extracted into the chloroform layer with a total volume of 1 part of chloroform, and concentrated. 1.8g
An oily substance was obtained. Dissolve in 30 ml of chloroform and add to a silica gel column (3.5 cm diameter x 30 cm height).
Elution was performed with a chloroform-methanol (50:1) mixture. The first eluted yellow pigment fraction was collected, concentrated to dryness (180 mg), dissolved in ethyl acetate, spotted on a silica gel plate for preparation, and developed with ether for further purification. On a silica gel plate (F254, Merck & Co.), the main section giving an Rf value of 0.77 was scraped off with the solvent system chloroform and methanol (30:1), eluted with ethyl vinegar, concentrated to dryness, and crystallized in acetone-n-hexane. 218 mg of a purified sample exhibiting a horizontal columnar shape was obtained. This substance is

[Table] Reference Example Manufacture of Acravinone Streptomyces galilaeus MA 144-ANR-635 (Feikoken Bacterial Serial No. 5082) was cultured in the same manner as shown in Example 1. The production amount of the substance in the culture solution immediately after fermentation is determined by adding 1 ml of the culture solution to 4 ml of acetone, stirring, centrifuging to separate the supernatant, concentrating, and extracting with 2 ml of toluene.
was spotted on a thin layer silica gel plate (F254 Merck) and mixed with chloroform-methanol (50:
1) Develop with the solvent system and use a chromatography scanner (Shimadzu TLC chromatography scanner (CS-910 model)).
Approximately 600μ as determined by Shimadzu Corporation)
g/ml. The 14 culture fluids thus obtained were separated into bacterial cells and liquid by filtration, and the substance was extracted and purified from the bacterial cells as follows. Total bacterial mass to 2.0
After suspending in acetone and extracting with stirring, the acetone extract obtained separately was concentrated under reduced pressure, and the target substance was extracted into the chloroform layer with a total amount of 1 chloroform and concentrated to obtain 9.2 g of oily extract. . Chloroform-methanol (1:10) mixture 70
ml, and after removing insoluble matter by centrifugation,
Sephadex LH20 column (5cm diameter x 50cm
high), as well as chloroform-methanol (1:
10) Elution was performed with a mixed solution. Concentrate and dry the main elution fraction containing the substance, dissolve in 200 ml of hot toluene,
The mixture was left at 4° C. overnight to obtain 4.8 g of purified specimen as yellow needle-like crystals. The physicochemical properties of this substance are as shown below, which are similar to the previously reported akravinone Tetrahedron Letter 8 , 28-34 (1964) Chem.Ber. 98 , 1260-1269 (1965) Tetrahedron 30 , 3787 ~3791 (1974).

【table】 [Brief explanation of the drawing]

Figure 1 is an infrared absorption spectrum of 2-hydroxyclavinone of the present invention (KBr tablet), Figure 2 is an NMR spectrum of the same (100MHz CDCl ₃ :CD ₃ OD)
=1:1).

Claims (1)

[Claims] 1 formula 2-Hydroxyaclavinone represented by