JPH0643435B2 - RK-483A, production method thereof, and antitumor agent and antibacterial agent - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel antibiotic, a method for producing the same, and an antitumor agent and an antibacterial agent containing the antibiotic as an active ingredient.

(Prior Art) It has been reported that anthracycline antibiotics are useful as antitumor agents, and some of them have been put to practical use as anticancer agents.

For example, Daunorubicim (trade name: Daunomycin), Do
xorubicin (trade name: adriacin), Aclarubicinhydr
Examples include ochloride (trade name: for injection of agracinone).

However, the above antibiotics have drawbacks such as large side effects when used clinically, and their clinical use is limited.

(Problems to be Solved by the Invention) Therefore, the present invention has few above-mentioned drawbacks, and an object thereof is to provide a novel anthracycline antibiotic which induces differentiation of cancer cells or suppresses growth of cancer cells. At the same time, it is an object to provide an anticancer agent containing the substance.

(Means for Solving the Problem) The present inventors have found that the novel substance RK-483A, which is an anthracycline antibiotic, is prominent in human leukemia cell K-56.
The present invention has been completed by demonstrating the proliferation inhibitory effect of No. 2 and having a differentiation inducing activity.

Further, the present inventor has clarified that the above substance RK-483A also exhibits antibacterial activity against bacteria, and found that a pharmaceutical composition containing this substance is useful as an antibacterial agent,
The present invention has been completed.

That is, according to the present invention, (1) a novel antibiotic RK-483A, (2) an antibiotic RK-483A-producing bacterium belonging to the genus Streptomyces is cultured, and the antibiotic RK-483A is separated and collected from the culture. A method for producing the antibiotic characterized by the following: (3) an antitumor agent containing the antibiotic RK-483A as an active ingredient, and (4) containing the antibiotic RK-483A as an active ingredient An antibacterial agent characterized by the above.

The antibiotic RK-483A is a novel antibiotic having the following physicochemical properties.

Physicochemical properties of antibiotic RK-483A (1) Property: Yellow powder (2) Melting point: 250 ° C or higher (decomposition point) (3) Molecular formula: C ₅₁ H ₇₂ N ₂ O ₂₀ (4) Mass spectrum (SIMS ): M / z1055 (M + N _a ) ⁺ m / z1033 (M + H) ⁺ (5) Specific rotation: ▲ [α] ²⁰ _D ▼ ＝ + 173 ° (c = 0.30)
3, chloroform-methanol, in 2: 1) (6) UV absorption spectrum (in methanol); (7) Infrared absorption spectrum (KBr); 3410,2910,1675,1630,1580,1410,1255,1095,965cm ^-1 (8) Solubility: Insoluble in dimethyl sulfoxide, ethyl acetate, methanol Insoluble in water (9 ) Color reaction: Positive for iodine and anisaldehyde.

The antibiotic RK-483A was Streptomyces sp. Sp. Isolated from soil collected from Suwa City, Nagano Prefecture. RK-4
It was isolated from the culture obtained by culturing 83 (Streptomyces sp. RK-483). The Streptomyces sp. RK-483
Was deposited on July 24, 1990 at the Institute for Microbial Technology, National Institute of Advanced Industrial Science and Technology, and the deposit number is Microbiology Research Institute No. 11622 (FERMP-11622).

Streptomyces sp. The mycological properties of RK-483 are as follows.

1. Morphological characteristics It was cultured in 1/10 yeast-starch agar medium at 28 ° C for 24 days, and a sample for scanning electron microscope was prepared by a simple drying method and observed.

The spore chain morphology was direct to wavy, the number of spores was more than 50 spores per chain, and the spore surface was smooth.

2. Chemical taxonomic properties (1) Optical isomer of cell wall diaminopimelic acid The freeze-dried microbial cells were hydrolyzed with 6N hydrochloric acid for 20 hours, and after removing the hydrochloric acid, and analyzed by cellulose TLC, diaminopimelic acid was LL type. This is a cell wall type I
Belongs to type.

(2) Menaquinone composition From freeze-dried cells, chloroform-methanol (2: 1,
vol / vol) to extract the acetone-soluble component with silica gel T
The sample prepared by LC was used as a menaquinone sample. Analysis is H
As a result of using a PLC and a mass spectrometer, menaquinone contained MK-9 (H ₈ ) and MK-9 (H ₆ ) as main components.

3. Culture properties Sealing and Gottlieb (1966) method (IS
P method) was used to examine the culture properties of the bacterial cells.

The color tone is the Color Harmony Manual 4th Edition (Container
Company).

(1) Yeast extract / malt extract agar medium (ISP no.
2) Growth: Abundant substrate Mycelia color tone: Golden tea (3pi) Aerial hyphae: Rich aerial mycelial color tone: Dark ash (5ih) Soluble pigment: None (2) Oatmeal agar medium (ISP no.3) Growth: Abundant substrate Live mycelial color tone: Golden tea (3pg) qi Mycelial thread color: Rich aerial mycelial color tone: Ash (5fe) Soluble pigment: Tea (3) Inorganic salt-starch agar medium Growth: Rich Basic fungal thread color tone: Golden tea (3pg) qi Mycelia: Abundant aerial mycelial color tone: Ash (5fe) Soluble pigment: Tea (4) Glycerol-asparagine agar medium (ISPno.5) Growth: Rich Basic hyphae mycelial color tone: Bamboo color (2gc) Aerial mycelium: Abundant aerial mycelium Color: Ash (5fe) Soluble pigment: None (5) Yeast extract / starch agar medium Growth: Abundant Basic mycelium Color tone: Clove tea (3ni) Aerial mycelium: Abundant aerial mycelial thread Color tone: Ash (5fe) Soluble pigment: Brown (6) Glucose / asparagine agar medium Growth: Good Substrate hyphae color tone: colorless Mycelium: No soluble pigment: No (7) Sucrose nitrate nitrate agar medium (Waksman's no.1) Growth: Good Basic mycelial color: DK Luggagetan (4pg) Aerial mycelium: Good aerial mycelial color: Parchment (1cb) Soluble Pigment: Tea (8) V8 juice agar medium Growth: Abundant substrate mycelial color tone: Golden tea (3pg) Aerial mycelial filament: Rich aerial mycelial color tone: Dark ash (5ih) Soluble pigment: None (9) Potato-carrot agar medium Growth: Good Basal hyphae color tone: Maple (4le) Aerial hyphae: Good aerial hyphae color tone: 5cb Soluble pigment: Tea Aerial hyphae shows a grayish color, and characteristic pigments are not accumulated in the basal hyphae It was Also, a soluble dye of tea type was produced.

4. Physiological and biochemical properties (1) Formation of melanoid pigment Streptomyces spRK-483 strain at 27 ° C on the following medium
After culturing for 20 days, the presence or absence of melanoid pigment secreted in the agar medium was observed.

+ Is positive for pigment production, and-is negative.

Hepton-yeast extract-iron Agar medium (ISP medium 6) ++ Tyrosine agar medium (ISP medium 7) ± Tryptone-yeast extract agar medium (ISP medium 1) ± (2) Utilization of carbon source Only the following sugars or carbohydrates RK- on the Pridham Gottlieb medium (the composition of which is shown below) used as the carbon source of
The 483 strain was cultured at 27 ° C. for 20 days, and the presence or absence of growth was used as the carbon source availability.

Pridham Gottlieb medium composition As the whole composition 1, pH 6.8 to 7 + indicates good growth and availability of carbon source, and-indicates no growth and availability of carbon source.

D-glucose ++ L-arabinose ++ D-xylose + L-inositol-D-mannitol-D-fructose ++ rhamnose-sucrose ++ raffinose ++ From the above mycological properties, the RK-483 strain is a strain belonging to the genus Streptomyces. Identified as present.

The antibiotic RK-483A of the present invention is produced by inoculating a nutrient source-containing medium with the above strain and culturing aerobically. The bacterium producing the antibiotic RK-483A is not limited to the above strains, but belongs to the genus Streptomyces, and the antibiotic RK-
Anything capable of producing 483A can be used in the present invention.

In principle, the method for culturing the above-mentioned microorganisms is based on the method for culturing general microorganisms, but it is usually preferable to perform it under aerobic conditions such as a shaking culture method using liquid culture and an aeration-agitation culture method.

The medium used for culture may be any medium containing a nutrient source that can be used by microorganisms belonging to the genus Streptomyces, and various synthetic media, semi-synthetic media, natural media and the like can be used. As a medium composition, glucose as a carbon source, sucrose, fructose, glycerin, dextrin, starch, sugar condensate, corn steve liquor, organic acid, etc. may be used alone or in combination. As the nitrogen source, pharmacological media, peptone, meat extract, yeast extract, soybean powder, casein, amino acids, organic nitrogen sources such as urea, and inorganic nitrogen sources such as sodium nitrate and ammonium sulfate may be used alone or in combination.

Sodium salt, potassium salt, magnesium salt, phosphoric acid
Salt, other heavy metal salts, etc. may be added and used as needed.
obtain. In addition, when foaming is remarkable during culture, ADEKA NOL
, Various well-known antifoam agents such as silicone oil
Although it can be added to the inside, the addition of
It should not adversely affect production. For example
It is preferably used at 0.5% or less.

The pH of the medium is preferably in the optimum pH range of the microorganism, usually around neutral. The medium temperature is preferably maintained at a temperature at which microorganisms grow well, usually 2.0 to 40 ° C, and particularly preferably around 27 ° C. The culture time is generally 1 for liquid culture.
~ 5 days, preferably about 72 hours. By the above culture, the target antibiotic RK-483A is produced and accumulated. Of course, the various culture conditions described above can be appropriately changed according to the type and characteristics of the microorganism used, external conditions, etc.
Further, the optimum condition is selected and adjusted from the above range according to each case.

The isolation of the antibiotic RK-483A produced by the above-mentioned culture is carried out according to a general method of collecting a fermentation product when the accumulation of the antibiotic is maximized, and the solubility difference between RK-483A and impurities is determined. It can be carried out by any of the means of utilizing, the means of utilizing the difference of adsorption affinity, and the means of utilizing the difference of molecular weight, and the respective methods are used singly or in appropriate combination or repeatedly used.

Specifically, since RK-483A is present in the culture filtrate and the bacterial cells, the culture filtrate or the acetone extract of the bacterial cells is subjected to centrifugal liquid partition chromatography, various gel filtration chromatography, adsorption chromatography, separation chromatography. Purification by combination with preparative thin layer chromatography etc.
A purified yellow powder of 483A can be obtained.

An example of the method for producing the antibiotic RK-483A of the present invention will be shown below with reference to Production Examples. However, the antibiotic RK-48 of the present invention is shown.
The manufacturing method of 3A is not limited to these manufacturing examples.

(Production Example) Glucose 2%, soluble starch 1%, meat extract 0.1
%, Dry yeast 0.4%, soybean flour 2.5% sodium chloride 0.2% dipotassium phosphate 0.005%, 181 medium was inoculated with Streptomyces RK-483 strain, and 27
The culture was performed at 72 ° C. for 72 hours with aeration and stirring. The filtrate of this whole culture solution was adjusted to pH 2 and extracted with an equal amount of ethyl acetate. The aqueous layer was adjusted to pH 7.5, extracted again with an equal amount of ethyl acetate, and the ethyl acetate layer was concentrated under reduced pressure to obtain a crude active substance. The crude active substance was subjected to centrifugal liquid-liquid partition chromatography, and the upper layer of ethyl acetate: methanol: 0.01 mol ammonium acetate (pH 7.5) 4: 1: 5 was used as the fixed layer and the lower layer was used as the moving layer, and the reverse layer was used. The active fraction containing RK-483A as the main component was obtained by elution by a downward partitioning method. Finally, the main component of chloroform: methanol: water: acetic acid 80: 20: 6: 14 was scraped off by preparative silica gel thin layer chromatography and eluted with the same solvent system. RK-483A
The fraction containing was concentrated under reduced pressure, the remaining aqueous solution was adjusted to pH 7.5, and then extracted with an equal amount of ethyl acetate. The ethyl acetate layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure to dryness,
About 50 mg of RK-483A was obtained as a yellow powder.

The minimum inhibitory concentration of the antibiotic RK-483A obtained as described above against various microorganisms is shown in Table 1 below.

Since the above antibiotic RK-483A exhibits a cell growth suppressing effect and an antibacterial effect, a composition containing RK-483A is useful as an anticancer agent and an antibacterial agent.

Test Example 1 (Cancer cell growth inhibition test and differentiation induction activity test of antibiotic RK-483A) Human leukemia cells K-562 were mixed with RPMI 1640 medium (10
% Fetal calf serum). To this was added a series of dilution series RK-483A. After culturing for 96 hours,
After observing the growth under an inverted microscope, the benzidine solution was observed for its differentiation-inducing activity. The results are shown in Table 2.

The above-mentioned antibiotic RK-483A can be prepared into tablets, powders, capsules, injections, inhalants, external preparations and the like by a conventional method, and is orally or parenterally administered to clinically used as a carcinostatic agent or antibacterial agent. It The dose depends on the symptoms to be treated and the administration method, but when administered as an anti-cancer agent, 1 mg to 1,000 mg per day for an adult
When administered as an antibacterial agent, it is 10 mg to 1,000 mg per day for an adult.

Test Example 2 (Acute Toxicity Test of Antibiotic RK-483A in Mice) As an animal to be used, one group of SIC-ICR male mice of 5 weeks old (body weight: 23.0 to 27.0 g) was used.

As a method of administration, the sample was 0.2% M. After C suspension preparation,
It was administered intraperitoneally.

The acute toxicity test result showed that LD ₅₀ was 37.5 mg / kg.

[Brief description of drawings]

FIG. 1 is an ultraviolet absorption spectrum of the antibiotic RK-483A, FIG. 2 is an infrared absorption spectrum (KBr), FIG. 3 is a 400 MHz ¹ H-NMR spectrum (in CD ₃ OD), and FIG. The figure shows 400 MHz ¹³ C-NMR spectrum (CD ₃ OD
Middle). In Figure 1, Is in 100% methanol; Is in 100% methanol / 0.1N hydrochloric acid; Indicates the result measured in 100% methanol / 0.1N NaOH.

Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location // (C12P 19/56 C12R 1: 465)

Claims (5)

[Claims] 1. An antibiotic RK having the following physicochemical properties:
-483A. Properties: Yellow powder Melting point: 250 ° C or higher (decomposition point) Molecular formula: C ₅₁ H ₇₂ N ₂ O ₂₀ mass spectrum (SIMS): m / z 1055 (M + N _a ) ⁺ m / z 1033 (M + H) ⁺ Specific rotation: ▲ [α] ²⁰ _D ▼ = + 173 ° (c = 0.303, chloroform-methanol, in 2: 1) Ultraviolet absorption spectrum (in methanol); Infrared absorption spectrum (KBr); 3410,2910,1675,1630,1580,1410,1255,1095,965cm ^-1 Solubility: Easily soluble in dimethyl sulfoxide, ethyl acetate, chloroform, methanol Insoluble in hexane Color reaction: iodine, Positive for anisaldehyde 2. A method for producing an antibiotic RK-483A, which comprises culturing an antibiotic RK-483A-producing bacterium belonging to the genus Streptomyces and separating and collecting the antibiotic RK-483A from the culture. . 3. An antibiotic RK-483A producing bacterium is Streptomyces sp. RK-483 (Streptomyces sp. RK-
483), The manufacturing method of Claim (2). 4. An antitumor agent comprising an antibiotic RK-483A as an active ingredient. 5. An antibacterial agent comprising the antibiotic RK-483A as an active ingredient.