JPH07242695A - New substance rk-75, its production and use - Google Patents

Abstract

PURPOSE:To obtain a new substance RK-75, having excellent antitumor actions and useful as an active ingredient of an antitumor agent by culturing a microorganism, belonging to the genus Streptomyces and having the ability to produce the substance RK-75 in a culture medium and providing the substance RK-75 from the resultant culture. CONSTITUTION:This method for producing a new substance RK-75 expressed by the formula is to inoculate a microorganism, belonging to the genus Streptomyces and having the ability to produce the new substance RK-75 [e.g. Streptomyces sp. 91-75 strain (FERM P-14190)] into a culture medium, carry out the shaking culture at 28 deg.C for 96hr, then perform the regular culture at 28 deg.C for 96hr while stirring the culture medium with aeration, centrifuge the culture solution, collect microbial cells, extract the collected microbial cells with acetone, concentrate the extracted solution under reduced pressure, regulate the pH to 7.0, then extract the regulated extracted solution with ethyl acetate, concentrate the obtained extracted solution under reduced pressure, dissolve the resultant syrup in chloroform and purify the syrup with a silica gel column. The resultant new substance RK-75 has excellent antitumor actions and is useful as an active ingredient of an antitumor agent.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel RK-75 substance, a method for producing the same, an antitumor agent containing the substance as an active ingredient, and a microorganism producing the substance.

[0002]

2. Description of the Related Art Many conventional antitumor agents have many compounds that directly act on DNA or act on mitotic apparatus, and have a problem of side effects on normal cells that proliferate in the living body such as bone marrow cells and hair root cells. Met. However, since the mechanism of cell proliferation has been elucidated at the molecular level in recent years, it is becoming possible to develop selective antitumor agents that inhibit the expression of oncogenes and the function of oncogene products. Therefore, in the field of antitumor agents and immunosuppressive agents, development of antitumor agents and immunosuppressive agents having a new mechanism of action and excellent in specificity and effectiveness is desired.

[0003]

An object of the present invention is to provide a novel RK-75 substance which can be an active ingredient of an antitumor agent excellent in specificity and efficacy, a method for producing the same and an antitumor agent. .

[0004]

[Means for Solving the Problems] The present inventors separated microorganisms from a large number of soil samples for the purpose of searching for substances having an antitumor effect, and searched for substances produced by the microorganisms. As a result, Imaichi City, Tochigi Prefecture. The present invention was completed by finding that a specific actinomycete among the strains isolated from the soil collected in 1. produces a novel chemical substance having an antitumor effect.

That is, the present invention has the formula (I)

[0006]

[Chemical 5]

There is a new RK-75 substance represented by Furthermore, the present invention relates to a Streptomyces genus, culturing a microorganism having an ability to produce the RK-75 substance in a medium, and collecting the RK-75 substance from the culture, thereby producing a novel RK-75 substance. Is the way. Furthermore, the present invention is an antitumor agent containing the RK-75 substance as an active ingredient.

Furthermore, the present invention is a Streptomyces sp. Strain 91-75 having the above-mentioned RK-75 substance-producing ability. Hereinafter, the present invention will be described in detail. The present inventors collected soil from Imaichi City, Tochigi Prefecture, found actinomycetes having an antitumor effect in the soil, and isolated this bacterium.

The mycological properties of this isolated strain are as follows. Separated soil: Imaichi City, Tochigi Prefecture Morphological characteristics a) Spore pattern: Straight to gentle waveform b) Surface of spore: Smooth c) Color of aerial mycelium: Gray to dark gray d) Color of basal mycelium: brown e ) Growth temperature: 4 to 33.3 ° C Chemo-taxonomic properties Cell wall composition Main amino acids: L, L-diaminopimelic acid Culture properties Medium aerial hyphae Growth ⁽¹⁾ Backside color Soluble pigment ISP-2 Light gray +++ Brown light brown ISP-3 Dark gray +++ Mustard color-ISP-4 Dark gray brown +++ Yellow brown Light brown ISP-5 Lead color +++ Light yellow light brown ISP-6 Light ocher ++ Colorless-ISP-7 Lead color +++ Light brown-Note 1) +++ : Very good ++: Good Physiological and biochemical properties a) Formation of melanoid pigment: None b) Utilization of carbon source ⁽²⁾ L-arabinose-D-xylose-D-glucose +++ D-fructose- Sucrose-i- Inositol - L- rhamnose - Raffinose - D- mannitol - Note 2) +++: very well be used -: based on the microbiological properties of more that do not use, Bergey's manual of sys
When classified according to tematic bacteriology1986, it was identified as a strain belonging to the genus Streptomyces (Streptomyces sp.). This strain is Streptomyces sp.
(Streptomycessp.) It was named 91-75. This strain has been deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology as FERM P-14190.

The Streptomyces sp. Strain 91-75 is
Like other Streptomyces actinomycetes, its properties are likely to change, such as ultraviolet rays, X-rays, radiation,
It can be easily mutated by an artificial mutagenesis means using an artificial mutagen, and even if such a mutation has the ability to produce the RK-75 substance having an antitumor effect described later, all of them are It can be used in the method of the invention.

The microorganism used in the present invention may be any microorganism belonging to the genus Streptomyces, which has the ability to produce the RK-75 substance described below having an antitumor effect.
As a medium used for culturing the above-mentioned microorganism, a medium containing nitrogen sources, inorganic salts, vitamins and other nutrient sources may be used. Here, the nitrogen source may be, for example, an inorganic nitrogen source such as ammonium sulfate, ammonium chloride, ammonium nitrate or ammonium phosphate, or an organic nitrogen source such as urea, yeast extract, peptone, glutamic acid or sodium glutamate. The pH of the medium is 5 to 10, preferably 6 to 9, and the culture temperature is usually 10 to 35 ° C, preferably 18 to 29 ° C.

Culturing can be appropriately determined depending on the growth condition of microorganisms and the like, but is usually carried out under aerobic conditions for 3 to 14 days. Isolation / purification of the novel RK-75 substance of the present invention from the culture is carried out by a method commonly used for isolating / purifying a microbial metabolic product from the culture, for example, extraction with an organic solvent, adsorption with an adsorbent and It can be carried out by appropriately combining elution, chromatography and the like.

The substance having an antitumor effect obtained in the examples described below is a compound having the following physicochemical properties and structural formulas. (a) Molecular formula: C ₃₇ H ₅₁ N ₇ O ₇ (b) Molecular weight: 705 (c) Melting point: 180 to 220 ° C. (d) Appearance: White powder

[0014]

(F) Solubility: Easily soluble in dimethyl sulfoxide. Soluble in acetone and methanol. Insoluble in chloroform. Insoluble in water. (g) Color reaction: Positive in color reaction with ninhydrin and I ₂ . (h) Ultraviolet absorption spectrum: λmax (MeOH) (ε) 223 nm (7,760), 300 nm (18,89)
0) (Fig. 1) (i) Infrared absorption spectrum: 3300, 2926, 1670, 1608,
1560, 1541, 1420, 1375, 1254, 1196, 1127, 1001cm ^-1
(FIG. 2) (j) Nuclear magnetic resonance spectrum: ¹ H-NMR result measured in deuterated DMSO ... FIG. 3
As shown in.

Results of ¹³ C-NMR measured in heavy DMSO ... As shown in FIG. As a result of determining the structure of this substance based on the above physicochemical properties, the substance has the formula (I)

[0017]

[Chemical 6]

It is represented by the structural formula: and was named RK-75 substance. This substance was found to be a novel substance with no known chemical match. The administration method when the novel RK-75 substance of the present invention is used as an antitumor agent based on its antitumor action is as follows. When used as an injection, a pH adjusting agent, a buffer, a stabilizer, an excipient and the like may be added to the present active ingredient. Also, lyophilization can be carried out by a conventional method, so that a lyophilized injection can be used. Furthermore, the active ingredient contains a pH adjusting agent, a buffering agent,
By adding a stabilizer, an isotonicity agent, and a local narcotics sugar, it can also be used as a subcutaneous, intramuscular, or intravenous injection in the form of a solution or suspension by a conventional method.

On the other hand, when used as a solid preparation, usual excipients, stabilizers and, if necessary, binders, disintegrating agents, lubricants, coloring agents, flavoring agents, flavoring agents and the like are added to the active ingredient. However, tablets, granules, powders, capsules and the like can be prepared by a conventional method.

[0020]

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to these examples. (Example 1) Medium containing 2% glucose, 1% soluble starch, 0.1% meat extract, 0.4% yeast, 0.2% sodium chloride, 0.005% dipotassium phosphate and 2.5% soybean flour (pH)
7.0) was inoculated with the Actinomyces strain 91-75 and shake-cultured at 28 ° C. for 96 hours. 350 ml of this culture broth was inoculated into 36 L of a medium of the same composition, and agitated at 300 rpm for 28 hours at 28 ° C. with aeration of 7 L / min.

The above culture solution was separated into bacterial cells and supernatant by a high speed centrifuge (15000 rpm), and the bacterial cells were extracted with 5 L of 70% acetone. After concentration under reduced pressure, pH of the resulting aqueous solution
It was adjusted to 7.0 and extraction was repeated twice with 5 L of ethyl acetate. After extraction, all ethyl acetate was combined and concentrated under reduced pressure,
0.3 g of brown syrup was obtained. The obtained syrup (300 mg) was dissolved in chloroform (30 ml), and the silica gel column (diameter: 6 cm, length: 90 cm) prepared with chloroform was infiltrated. First, 2 L of chloroform was flowed, and then the chloroform-methanol solution was mixed at different mixing ratios. (50: 1, 10: 1,
5: 1, 1: 1) 1 L each, and finally 2 L methanol.

The activity was eluted in the fraction of chloroform-methanol solution (5: 1 to 1: 1). This was concentrated under reduced pressure to obtain 60 mg of white powder. Furthermore, this white powder was dissolved in a chloroform-methanol solution (1: 1) and prepared in the same solution as a column of Sephadex LH-20 (Pharmacia) (diameter 4.5 cm, length 120 cm).
Elution with chloroform-methanol solution (1: 1). Fractions of 10 ml each were collected, the main active fractions were collected, and 7.2 mg of white powder was obtained from this fraction by concentration under reduced pressure.

Finally, an ODS column (diameter 2 cm, length 25 cm;
High performance liquid chromatography was used for preparative separation. The solvent used was 70% methanol and the flow rate was 6.0 ml / min. As a result, about 3.8 mg of a purified standard product was obtained as a white powder. This white powder is a compound having the aforementioned physicochemical properties and structural formula. (Test Example 1) The activity of the RK-75 substance obtained in Example 1 was measured according to the following method.

Src ^ts -NRK cells prepared by infecting rat kidney cells (NRK) with temperature-sensitive Rous sarcoma virus were prepared,
The src ^ts -NRK cells were cultured at a temperature of 32 ° C. in a culture medium containing RK-75 substance at concentrations of 1, 3.2, 10, 32 and 100 μg / ml, and observed under a microscope. Normally, when src ^ts -NRK cells are cultured at 32 ° C, they show a spherical transform morphology.
When this RK-75 substance was added to the culture medium, the transformation of cells was completely inhibited and the cells showed a normal morphology.
Table 1 shows the inhibition rate of tumor activity at each concentration of the RK-75 substance.

[0025]

[Table 1]

As a result, Streptomyces sp. 91
It was revealed that the novel RK-75 substance of the present invention produced from the -75 strain inhibits cell transformation and has an antitumor effect.

[0027]

INDUSTRIAL APPLICABILITY According to the present invention, a novel RK-75 substance and an antitumor agent containing the substance as an active ingredient can be provided. Furthermore, according to the present invention, it is possible to provide a microorganism that produces a novel RK-75 substance.

[Brief description of drawings]

FIG. 1 is a chart of an ultraviolet absorption spectrum (methanol) of the RK-75 substance of the present invention.

FIG. 2 is a chart of infrared absorption spectrum (KBr) of RK-75 substance of the present invention.

FIG. 3 ¹ H-NMR (DMSO-d ₆ ) of RK-75 substance of the present invention.
It is a chart showing the result of.

FIG. 4 ¹³ C-NMR (DMSO-d ₆ ) of RK-75 substance of the present invention.
It is a chart showing the result of.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location // (C12N 1/20 C12R 1: 465) (C12P 21/04 C12R 1: 465) (72) Inventor Kiyoshi Isono 324-1 Orido, Shimizu City, Shizuoka Prefecture

Claims (4)

[Claims] 1. Formula (I): New RK-75 substance shown by. 2. A member of the genus Streptomyces of the formula (I): A method for producing a novel RK-75 substance, which comprises culturing a microorganism having the ability to produce the novel RK-75 substance in a medium, and collecting the RK-75 substance from the culture. 3. Formula (I): An antitumor agent containing the novel RK-75 substance represented by 4. Formula (I): The Streptomyces sp. Strain 91-75 having the ability to produce the novel RK-75 substance shown in.