JPH04187632A - New antitumor antibiotic substance resorthiomycin and its production - Google Patents

Abstract

NEW MATERIAL:The compound of formula and its salt. Appearance, colorless or pale yellow oily substance; solubility, easily soluble in methanol, ethanol, ethyl acetate, chloroform and dimethyl sulfoxide and insoluble in water and hexane; specific rotation, [alpha] =-4.34 deg. (C=1.19, methanol). USE:An antitumor agent and an agent for promoting the effect of other antitumor agent by combined use. PREPARATION:A resorthiomycin-producing strain belonging to genus Streptomyces [e.g. Streptomyces collinus 45H-6 (FERM P-10753)] is inoculated in a medium containing nutrient source and cultured under aerobic condition. The objective resorthiomycin is separated from the obtained culture product.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention relates to a novel antitumor antibiotic resolutiomycin obtained from the culture by culturing a microorganism belonging to the genus Streptomyces, and the present invention relates to This invention relates to a method for producing resolutiomycin.

(Prior art and problems to be solved by the invention) Various antitumor antibiotics produced by microorganisms have been known, but antibiotics are useful as effective chemotherapeutic agents to control human cancer. are extremely rare. The present inventors have discovered that a substance that promotes the effects of currently used antitumor agents while exhibiting antitumor effects itself is produced in the culture solution of actinomycetes. The antitumor antibiotic was isolated and named resorthiomycin, and research revealed that it was a derivative of resorcinol, but no antibiotic with a similar structure has yet been known. An object of the present invention is to provide a novel antitumor antibiotic resolutiomycin and a method for producing the same.

(Means for Solving the Problems) The gist of the first invention is that the following formula H・c s
, c, ○ A novel antitumor antibiotic resolutiomycin and its salts.

The properties of the new antibiotic resolutiomycin according to the present invention are as follows.

Resorutiomycin is a colorless to pale yellow oily substance that is easily soluble in methanol, ethanol, ethyl acetate, chloroform, and dimethyl sulfoxide, but insoluble in water and hexane. Specific optical rotation [α]r=-4,34
° (c = 1.19.methanol).

Elemental analysis values are C59.15%, H7.04%, 022
．． 54%, 811.27%, and the theoretical value of C-4H2o04S (C57,68%, H7,10%, 023.23
%, 310.76%), and this molecular formula was verified by EI myspectrum of resortiomycin.

The infrared absorption curve of resolutiomycin measured in chloroform is shown in FIG. 1 of the accompanying drawings. As shown in Figure 2, the ultraviolet and visible absorption curves show an absorption maximum at 285 nm in neutral and acidic methanol, and 0.
350n in methanol containing 0IN sodium hydroxide
The absorption maximum was shown at m. 1 measured in deuterated chloroform
The H nuclear magnetic resonance spectrum showed signals as shown in FIG. 3, and the 13 C nuclear magnetic resonance spectrum (in deuterated chloroform) was as shown in FIG.

The structure of resorutiomycin was determined by the above nuclear magnetic resonance and other physicochemical measurements using the sample obtained by the method shown in Example 2.
It was determined to be 6-dimethyl-5-(3-hydroxybutyl)thiobenzoate. This substance was confirmed to be a new antibiotic.

Resorthiomycin infiltrates Staphylococcus aureus, Bacillus subtilis, and other Gram-positive bacteria in nutrient agar.

Growth of Escherichia coli, Salmonella and other Gram-positive bacteria, and molds such as yeast and Candida at 100μg/mQ
However, as shown in the test example below, the proliferation of mouse leukemia L5178Y cells was inhibited at 15.5 μg/m
Q suppressed it by 50%. Furthermore, 40 μg/mΩ resolutiomycin enhanced the effects of vincristine and actinomycin D on Chinese hamster V79 cells by three times. Therefore, resolutiomycin can be used as an antitumor agent and as an agent for enhancing the effect of antitumor agents when used in combination.

The second gist of the present invention is to provide a method for producing resorutiomycin, which involves culturing resorutiomycin-producing bacteria belonging to the genus Streptomyces to accumulate resorutiomycin, and collecting resorutiomycin from the culture. be.

An example of a resolutiomycin-producing bacterium is an actinomycete strain numbered 458-6, which was isolated from soil collected in Isehara City, Kanagawa Prefecture in 1980.

The mycological properties of this 458-6 are as follows.

1. Form 45H-6 strain forms spiral aerial hyphae that are almost straighter than branched basal hyphae and have small tips, and no whorl waves are observed. Roughly 10 to 20 mature spores are chained together, with the tips forming a small spiral shape. The size of the spore is about 0.5-0.9 x O, 8-1.0 μm, cylindrical, and the surface is smooth.

2. Growth status in each medium (observation after 2 weeks of culturing at 27°C) Aerial mycelium was gray in most of the media, but immature aerial mycelium may be reddish or yellowish. Ta.

The underside of the colony was pale yellow to yellowish brown or grayish yellowish brown.

(1) Growth on sucrose/nitrate agar medium is slight. A moderate amount of light brownish-gray aerial mycelium grows on the plant, and no soluble pigments are observed.

(2) Growth on glucose-asparagine agar medium is moderate. Light brown aerial mycelia are well established, and light yellowish brown soluble pigment is observed.

(3) Glycerin-asparagine agar medium (ISP-Medium 5) Growth is moderate. A moderate amount of brownish-gray aerial mycelium grows on it, and pale orange soluble pigment is observed.

(4) Growth on starch/inorganic salt agar medium (ISP-medium 4) is good. It is covered with abundant brownish-gray aerial mycelia and has light brown soluble pigment.

(5) Tyrosine agar medium (ISP-Medium 7) Growth is moderate. Brown aerial mycelium is well established, and no soluble pigments are observed.

(6) Growth on nutrient agar medium is slight. No aerial mycelium is produced, and no soluble pigments are observed.

(7) Growth on yeast/malt agar medium (ISP-medium 2) is good. Abundant brown-gray aerial mycelia are attached, and no soluble pigments are observed.

(8) Growth is good on oatmeal agar medium (15P-medium 3). It has a good epiphyte of brownish-gray aerial mycelium, and pale yellowish-brown soluble pigment is observed.

3. Physiological and biochemical properties (1) Liquefaction of gelatin Liquefaction of gelatin is observed in glucose-peptone-gelatin medium.

(2) Hydrolysis of starch Hydrolysis of starch is observed on starch/inorganic salt agar medium.

(3) Coagulation and peptonization of skim milk No obvious coagulation was observed, and peptonization was observed.

(4) Production of melanin-like pigments The production of melanin-like pigments is observed in tyrosine yeast extract medium, rosin agar medium, and peptone yeast extract iron agar medium.

(5) Utilization of carbon source Assimilation of carbon source is determined by Pridham-Gotlieb's basal medium N.
A9 was cultured at 27°C for 14 days, and the determination was made. D
-Glucose, leaf xylose, L~arabinose, Shi-
They grew well using rhamnose, D-fructose, raffinose, D-mannitol, and inositol, but
Sucrose was not used.

In addition, cell wall components were analyzed using whole bacterial cells.
LL-diaminopimelic acid was inspected.

(6) Growth temperature: Grows in the range of 20 to 40°C on yeast/malt agar medium and oatmeal agar medium, and the optimal temperature is 20°C on the former.
7-37°C, the latter 27-30°C.

As described above, the 45H-6 strain belongs to the genus Streptomyces based on its morphological characteristics and cell wall type. Furthermore, the form,
From the characteristics such as culture properties, physiological and biochemical properties, 45H
-6 strain is determined to be Streptomyces corinus.

The 45H-6 strain was applied for deposit with the Institute of Microbial Technology, Agency of Industrial Science and Technology on May 31, 1989, and the accession number is Microbiological Research Institute No. 10753.

The 45H-6 strain, as seen in the case of many other strains of actinomycetes, is susceptible to changes in its properties, such as exposure to ultraviolet light,
Although they can be mutated by artificial mutagenesis methods using X-rays, radiation, chemicals, etc., any mutant strain that has the ability to produce the antibiotic resolutiomycin can be used in the method of the present invention. can.

In carrying out the method for producing resorutiomycin of the present invention, resorutiomycin-producing bacteria, such as Streptomyces corinus 45H-6 strain, are inoculated into a nutrient-containing medium and grown by aerobic cultivation. A culture containing resolutiomycin is obtained. As the nutrient source in the medium used, any known nutrient source used as a nutrient source for actinomycetes can be used. For example, commercially available peptone, meat extract.

Nitrogen sources such as corn steep liquor, cottonseed flour, peanut flour, soybean flour, yeast extract, NZ-amine, thawed casein, fish meal, sodium nitrate, ammonium nitrate, ammonium sulfate, and commercially available glycerin, sucrose,
carbohydrates such as starch, glucose, maltose, and molasses;
Alternatively, carbon sources such as fats can be used. Also, salt,
Phosphate.

Inorganic salts such as calcium carbonate and magnesium sulfate can be added. In addition, trace amounts of metal salts can also be added if necessary. These materials can be used as long as they are useful for the production of resortiomycin and can be used by the producing bacteria, and all known culture materials for actinomycetes can be used.

When collecting resolutiomycin from the culture obtained according to the present invention, for example, from the culture filtrate, conventional separation methods that utilize the properties of resolutiomycin, such as solvent extraction, ion exchange resin, adsorption or partition chromatography, can be used. Extraction and purification can be carried out using operations such as , precipitation, etc. alone or in appropriate combinations. Alternatively, a solution of resolutiomycin obtained in the free form can be treated with a base, such as an alkali metal compound such as sodium hydroxide or potassium hydroxide, an alkaline earth metal compound such as calcium hydroxide or magnesium hydroxide, an ammonium salt, etc. When treated with an inorganic base such as ethanolamine or an organic base such as ethanolamine, resorutiomycin can be separated in the form of the salts of the base used.

Examples will be shown below, but since the properties and chemical structure of resorutiomycin have been clarified by the present invention, various methods for producing resorutiomycin can be devised based on the properties. Therefore, the present invention is not limited to the examples, and can be used to produce resolutiomycin by applying known means based on the properties of resolutiomycin revealed by the present invention, as well as by modifying the examples. Concentration, extraction.

It covers all methods of purification.

Example 1 Streptomyces strain 45H-6 (Feikoken Bacteria No. 10753) cultured on an agar slant of oatmeal agar medium (20 g of oatmeal, 1 g of yeast extract, 15 g of agar in 1 liter of medium) with the same composition. liquid medium 100
It was inoculated into a Sakaro flask containing mQ, and cultured with shaking at 27°C for 4 days. After separating 4 liters of culture solution into supernatant and bacterial cells by centrifugation (10,000 revolutions per minute, 15 minutes), 400 m of Diaion HP-20 resin (manufactured by Mitsubishi Kasei Corporation) was added to the supernatant.
After that, the resin was washed with water, eluted with 4 liters of methanol, the eluted solution was concentrated under reduced pressure, and then dissolved in water. This was adjusted to pH 3 with IN hydrochloric acid, and ethyl acetate 6
After extraction with 40 mQ in three portions, the ethyl acetate layer was concentrated to dryness under reduced pressure, dissolved in the chloroform layer, and subjected to silica gel column chromatography. The active ingredient obtained by elution with chloroform-methanol (20 (1: l)) was concentrated to dryness under reduced pressure, then dissolved in methanol and subjected to ODS reversed-phase high performance liquid chromatography. A peak appeared. When the active fractions were collected and concentrated to dryness under reduced pressure, 2.5 μm of resolutiomycin was obtained. [α)6' = -4,34° (C = 1
．． 19. methanol).

Example 2 In order to obtain a large amount of resortiomycin, two jar culture vessels were each filled with three doses of oatmeal yeast extract medium.
Add 0 liters of seed culture solution (culture using Sakaro flask)
600 mR each was added and cultured at 27° C. for 120 hours (200 revolutions per minute, aeration rate 10 liters per minute). After adding Celite (manufactured by Johns-Manville) to 60 liters of the culture solution and suction filtration, the supernatant was adjusted to pH 3.
This was extracted with 100 liters of ethyl acetate. Thereafter, separation and purification was carried out in the same manner as in Example 1, and 39.1 μm of resoltiomycin was obtained by fractionation using high performance liquid chromatography.

Next, test examples illustrate that resolutiomycin according to the present invention has the effect of suppressing the proliferation of cancer cells (Test Example 1) and the effect of enhancing the effect of anticancer drugs (Test Example 2). .

Test Example 1 When mouse leukemia L5178Y cells were cultured at 37° C. for 3 days in RPM11640 medium containing 10% fetal bovine serum, the cells increased from 15,000 cells/mQ to 980,000 cells/mQ. At this time, when resolutiomycin is added to the medium at various concentrations, cell proliferation is inhibited depending on the concentration, and a resolutiomycin concentration of 15.5 μg/vnQ inhibits cell proliferation by 50% ( IC5°) was observed.

Test Example 2 Chinese hamster V79 cells were grown at 200-300 cells/in Eagle MEM medium containing 10% calf serum.
After 20 hours, vincristine, which is known as an antitumor agent, was added to the culture medium and incubated at 37°C for 7 to 70 minutes.
It was cultured for 8 days. After that, the cells on the plate were fixed with 10% formalin solution, stained with crystal violet, and the number of colonies was counted.
, , was 17.3 ng/mΩ, but when 40 μg/n+Q of resortiomycin was added together with vincristine, the IC5o of vincristine became 5.1 ng/−Q, and the anticancer effect of vincristine was enhanced by 3.4 times.

In a similar experiment, resolutiomycin 40 μg/mQ enhanced the effect of actinomycin D by 3.3 times.

[Brief explanation of the drawing]

FIG. 1 of the accompanying drawings shows the infrared absorption curve of resolutiomycin measured in chloroform. FIG. 2 shows the ultraviolet and visible absorption curves of resolutiomycin measured in neutral/acidic methanol and in methanol containing 0.01 N sodium hydroxide. Figure 3 shows the 1H nuclear magnetic resonance spectrum (5001'1Hz) of resortiomycin measured in deuterated chloroform, and Figure 4 shows the 13
This is a C nuclear nuclear resonance spectrum (100M7). Rimei visit

Claims (1)

[Claims] 1. An antitumor antibiotic resolutiomycin and its salts represented by the following formula ▲ Numerical formula, chemical formula, table, etc. ▼. 2. A method for producing resolutiomycin, which comprises culturing a resolutiomycin-producing bacterium belonging to the genus Streptomyces in a medium containing a nutrient source, and collecting resolutiomycin from the culture.