JPH0662632B2 - Novel antibiotic A1-R2397 substance and its production method - Google Patents

Description

The present invention relates to a new antibiotic Al having antitumor activity and antibacterial activity.
-R2397 substance and its manufacturing method.

2. Description of the Related Art Conventional techniques and problems to be solved by the invention Many antibiotics have been invented and put to practical use in the fields of medicines, veterinary medicines, agricultural chemicals and the like. However,
There are many medical or industrial fields that have not been solved because no effective substances have been found yet. For example, also in the field of cancer chemotherapy, it is always desired to provide a novel antitumor antibiotic having a new action.

The present inventors pay attention to the above points, and provide a novel antitumor antibiotic, and try to solve this by establishing a manufacturing method thereof.

Means for Solving the Problems The inventors of the present invention continued to search for a substance having antitumor activity in order to meet the above-mentioned expectations, and found that mouse leukemia was found in a culture of a strain belonging to the genus Thermomonospora. cell
(P-388) was found to have produced a substance having cytotoxicity and other antitumor and antibacterial activities, and the active substance Al-R2397 was isolated and its physicochemical and biological properties were determined. Then, the present invention was completed by establishing its structure by X-ray crystal diffraction.
Therefore, the first aspect of the present invention provides a novel antitumor antibiotic Al-R2397 substance represented by the following formula (I).

Furthermore, the second present invention is an antibiotic characterized by culturing an antibiotic Al-R2397 substance-producing bacterium belonging to the genus Thermomonospora and collecting the antibiotic Al-R2397 substance from the culture.
A method for producing an Al-R2397 substance is provided.

As a compound with a structure similar to the Al-R2397 substance Yata et al.
(S. Tanida et al. J. Antibiotics, Vol.34, 489 1981), an ansamitocin group compound isolated from metabolites of a microorganism that is considered to be a kind of Nocardia, but Al-R2397 substance is Different from any of the above, it was determined to be a new substance.

An example of the Al-R2397 substance-producing bacterium used in the present invention is Al-R2397 strain newly isolated from the soil of India by the present inventors. The mycological properties of the Al-R2397 strain are as follows.

I. Morphology The basal hyphae are well-extended and branched, and usually do not divide. In general, aerial hyphae do not grow much, and tyrosine agar, glycerol
Slight aerial mycelial colonization is observed on asparagine agar. The aerial mycelium branches are simple branches, and no axle branch is observed.

Spore settlement has been confirmed only under extremely limited conditions. For example, after culturing on aerial hyphae grown on tyrosine agar diluted to 1/4 concentration at 28 ° C for about 1 month, green monospores are observed. Spores were collected and settled. Spore size is 1.0 to 1.2 in diameter
The micron was almost spherical, the surface was smooth, no motility was observed, and the heat resistance of spores was not recognized in the survival test in 100 ° C boiling water. Monospore settlement was also observed in vegetative mycelia. No special structures such as sporangia and sclerotia have been observed.

II. Growth conditions on various media The growth conditions of the Al-R2397 strain on various media are shown in the following table. Regarding the color description, the standard shown in () is for Container C of America.
The one described in "Color Harmony Manual" manufactured by the Corporation of America was used. The observation was performed after culturing at 28 ° C for 14 to 30 days. (37 ° C
The culture properties of were also the same as below. ) III. Physiological properties (1) Growth temperature range: Grows in a temperature range of 28 ° C to 55 ° C,
It grows well at 35 ° C to 45 ° C.

(2) Gelatin liquefaction: Negative (3) Starch hydrolysis: Positive (4) Nitrate reduction: Negative (5) Skim milk peptonization: Positive Skim milk coagulation: Negative (6) Salt tolerance: 1.5% NaCl Grows well on added agar medium.
Grow slightly with 3% NaCl. It does not grow above 4%.

(7) Formation of melanin-like pigment: negative IV. Utilization of carbon source (1) Utilization: D-glucose, D-fructose,
D-xylose, glycerol, L-arabinose, D
-Mannitol, raffinose (2) Not used: i-inositol, L-rhamnose, sucrose V.I. Cell wall composition It has DL-type diaminopimelic acid as an amino acid during whole cell hydrolysis. Arabinose and xylose as sugars are
Almost unrecognized, the presence of majurose is unclear.

As described above, the Al-R2397 strain was identified as Thermomonospora sp. Al-R2397 by judging from morphological properties, physiological properties, results of chemical analysis of bacterial cells and the like.

This strain has been deposited at the Institute of Microbial Science and Technology of the Institute of Industrial Science and Technology as Microorganism Research Institute No. 8802 (FERM P-8802).

The Al-R2397 strain is likely to change its properties as seen in other actinomycetes. For example, the Al-R2397 strain, or a mutant strain (natural or inducible) derived from this strain,
Antibiotics, whether transzygous or recombinant
Anything that produces Al-R2397 material can be used in the present invention. In the method of the present invention, the above-mentioned bacterium is cultivated in a medium containing nutrients that can be utilized by ordinary microorganisms. As a nutrient source, glucose, starch syrup, dextrin, sucrose, starch, molasses, animal / vegetable oil, etc. can be used. As the nitrogen source, soybean flour, wheat germ, corn steep liquor, cottonseed meal, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, urea and the like can be used. In addition, it is effective to add inorganic salts capable of generating sodium, potassium, calcium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid, and other ions, if necessary. It also helps the growth of bacteria
Organic and inorganic substances which promote the production of Al-R2397 material can be added appropriately. As the culturing method, the culturing method under aerobic conditions, especially the submerged culturing method is most suitable.
A suitable temperature for culturing is 28-55 ° C, but in most cases 28
Incubate at ~ 37 ° C. The production of the antibiotic Al-R2397 varies depending on the medium and culture conditions, but the accumulation generally reaches optimum within 3 to 10 days in both shaking culture and tank culture. When the accumulated amount of the antibiotic Al-R2397 substance in the culture reaches the maximum, the culture is stopped, and the target substance is isolated and purified from the culture solution.

In the assay of the Al-R2397 substance of the present invention, Cryptococcus neoformans M9010 (Cryptoco) as a measurement and assay strain for cytotoxicity against mouse leukemia cells (P-388)
ccus neoformans M9010) is used. When the diameter of the growth inhibition circle on the agar medium is 30 to 300 μg / ml, it shows a linear relationship with the logarithm of the concentration and gives an inhibition circle of 14 to 25 mm.

Since the Al-R2397 substance obtained from the present invention is a neutral fat-soluble substance, its characteristics can be used for the isolation and purification of the Al-R2397 substance from the culture. That is, synthetic adsorbents such as Amberlite XAD-2 (made by Rohm and Haas), Diaion HP-20 (made by Mitsubishi Kasei), Sephadex LH-20 (made by Pharmacia),
Toyopearl HW-40 (manufactured by Toyo Soda Co., Ltd.), gel filter, silica gel, silica gel chromatography with alumina, solvent extraction method with ether acetate, chloroform, etc.
Further, a crystallization method using methanol or the like as a solvent is effective.

A highly pure Al-R2397 substance can be obtained by the method described above or by appropriately combining them. The physicochemical properties of the obtained Al-R2397 substance are as follows.

(A) Molecular formula and elemental analysis value: C ₃₁ H ₄₁ N ₂ O ₈ Cl carbon 61.19% hydrogen 6.80% nitrogen 4.53% chlorine 6.03% (b) molecular weight: 604 (M / Z605 (M + 1) in FD-MS (C) Melting point: 208 to 211 ° C. (D) Specific rotation: [α] _D ²³ = -69.7 ° (c0.1, methanol) (V) UV absorption spectrum: spectrum measured in methanol solution Is shown in FIG. 221nm (ε37,730), 240nm (sh.ε28,790), 283nm (ε
The absorption maximum was observed at 2,400).

(F) Infrared absorption spectrum: The spectrum measured with a potassium bromide tablet is shown in FIG.

(G) Hydrogen nuclear magnetic resonance spectrum (heavy dimethyl sulfoxide, 400MHz) ppm: 0.94 (3H, d), 1.07 (6H, d), 1.55 (3H, brs), 1.84 (3H, s), 3.22 (3H, s), 3.88 (3H, s), 5.28 (1H, dd), others (h) Solubility: Soluble in dimethyl sulfoxide, methanol; sparingly soluble in acetone, ethyl acetate, chloroform; insoluble in water, n-hexane ( I) Color reaction: Potassium permanganate, Greig Reebuck reagent, 10
% Sulfuric acid, Molybdic acid reagent positive; Ninhydrin reagent negative (N) Thin layer chromatography (Merck silica gel plate, 0.25 mm); a) Developing solvent: Chloroform-methanol (10: 1) Rf0.69 b) Developing solvent: Toluene-acetone (2: 1) Rf0.29 (L) Appearance: colorless plate crystals Effect of the invention The Al-R2397 substance according to the present invention exhibits antitumor activity and its 50% inhibition against mouse leukemia cells (P-388) Concentration is 4pg / ml
It was below. The life prolonging effect of this substance is shown in the test examples described below. The Al-R2397 substance exhibits antifungal activity, and the antifungal spectrum against various fungi is as shown in Table 1. 20 μl of each concentration of the Al-R2397 substance in methanol was soaked in a paper disk, and the diameter of the inhibition circle was measured by the agar plate method.

As is clear from the above results, the Al-R2397 substance exhibits an antibacterial action against fungi and is expected to be useful as a fungal therapeutic agent.

Examples Examples of the present invention will be shown below, but these are merely examples and do not limit the present invention. Of course, many modified or modified means not exemplified here can be used.

Examples As a seed medium, a medium containing 2.0% starch, 1.0% glucose, 0.6% wheat germ, 0.5% polypeptone, 0.3% yeast extract, 0.2% soybean flour, and 0.1% calcium carbonate was used.

As a production medium, starch syrup 1.0%, starch 1.0%, soybean flour 1.5%, sun grain F2 1.0%, calcium carbonate 0.2
%, Magnesium sulfate (heptahydrate) 0.1%, ferrous sulfate (heptahydrate) 0.0005%, cobalt chloride (hexahydrate) 0.0005
%, Was used.

The pH before sterilization was adjusted to pH 7.0 and used. A 100 ml Erlenmeyer flask into which 20 ml of the seed medium was dispensed was placed at 120 ° C.
Sterilize for 30 minutes and add to it Thermo Monospora Al-R2397 (FERM
P-8802) Slope culture 3-4 platinum loops were inoculated and incubated at 28 ° C for 4
The culture was performed for 1 day with shaking to obtain a first seed culture. Then 80 ml of seed medium
The 500 ml Erlenmeyer flask into which was dispensed was sterilized at 120 ° C. for 30 minutes, 5 ml of the first seed culture was inoculated, and the mixture was shake-cultured at 28 ° C. for 2 days, and this was designated as the second seed culture. Furthermore, the 5-volume Erlenmeyer flask into which the seed medium 1 was dispensed was sterilized at 120 ° C. for 30 minutes, 50 ml of the second seed culture was inoculated, and the mixture was shake-cultured at 28 ° C. for 1 day, and this was designated as the third seed culture.

Includes 35 production media sterilized at 120 ° C for 30 minutes, 50
A jar fermenter was inoculated with the above-mentioned third seed culture 1, aerated at 28 ° C. for 5 days (20 / min), and initially stirred at 250 rpm, 41
After the time, 350 rpm). After the completion of the culture, diatomaceous earth was added as a filter aid and the mixture was filtered. The obtained culture filtrate 85,
The microbial cells were stirred with 50% acetone water 40 at room temperature for 1 hour, filtered to remove the microbial cells, and the microbial cell extract concentrate was combined with 12 to obtain DIAION HP-20 ( The active ingredient was adsorbed by passing through a column packed with 4.5 (manufactured by Mitsubishi Kasei). Subsequently, the column was washed with deionized water 70 and the active ingredient was eluted with 50% acetone water.

The active fractions were collected, and acetone was distilled off under reduced pressure to 15 and this was extracted with ethyl acetate 15. The ethyl acetate layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 16 g.
Of oily substance was obtained. The obtained oily substance was sprinkled with 16 g of diatomaceous earth, dried overnight under reduced pressure, and placed on a 700 ml column of silica gel C-200 (manufactured by Wako Pure Chemical Industries, Ltd.) filled with chloroform, followed by chloroform and then a mixture of chloroform and methanol. After washing with (100: 1), chloroform-methanol mixture
Chromatography was performed at (50: 1). The developing solution is silica gel thin film chromatography (Merck)
60F254 5714, developing solvent: chloroform-methanol (1
0: 1)) was performed to collect fractions having an Rf value of 0.69 and an ultraviolet absorption spectrum as shown in FIG. 1, and concentrated and dried under reduced pressure to obtain 933 mg of an oily substance. . This oily substance was dissolved in a small amount of methanol, placed on a Sephadex LH-20 (Pharmacia) column 1 filled with methanol, and developed with methanol for chromatography. The active fraction was concentrated to dryness under reduced pressure to obtain 335 mg of an oily substance. Dissolve this oily substance in a small amount of methanol,
When left at room temperature for 16 hours, colorless crystals of Al-R2397 substance
94.1 mg was obtained. Of this, 20 mg is hot methanol 20 ml
After removing methanol under reduced pressure, ethanol was added to the residue, and the mixture was allowed to stand at room temperature and recrystallized to obtain 18 mg of a colorless plate crystal of Al-R2397 substance. The substance has the physicochemical properties described above. Further, this crystal was subjected to X-ray crystal diffraction,
The structure of Al-R2397 material was established.

Test Example (Anti-tumor effect) Al-R2397 was administered to mice in which P-388 tumor cells were intraperitoneally transplanted.
The substance was administered intraperitoneally once a day for 2 consecutive days. This life-prolonging effect is shown in Table 2 as a T / C% value.

[Brief description of drawings]

Figure 1 shows the ultraviolet absorption spectrum of Al-R2397 substance in methanol solution at a concentration of 20 μg / ml, and Figure 2 shows Al-R2397.
The infrared absorption spectrum of the substance in potassium bromide tablet is shown.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location (C12P 17/18 C12R 1:01) (C12N 1/20 C12R 1:01) (72) Inventor Mieko Nagasawa 760 Meiji Seika Co., Ltd., Meiji Seika Co., Ltd., 760 Shimooka-cho, Kohoku-ku, Yokohama, Kanagawa Prefecture (72) Inventor Takashi Shomura 760, Shimooka-cho, Kohoku-ku, Yokohama-shi, Kanagawa Meiji Seika Co., Ltd. (72) Inventor Tadashi Sezaki Next 760 Meiji Seika Co., Ltd. Pharmaceutical Research Laboratory, 760 Shimooka-cho, Kohoku-ku, Yokohama-shi, Kanagawa Prefecture

Claims (2)

[Claims] 1. A novel antibiotic A having the following chemical structural formula:
1-R2397 substances 2. An antibiotic A1 belonging to the genus Thermomonospora.
-A method for producing a new antibiotic A1-R2397 substance, which comprises culturing a bacterium producing the R2397 substance and collecting the A1-R2397 substance from the culture.