JPH03220196A - Benzanthraquinone compound - Google Patents

Abstract

NEW MATERIAL:The compound of formula having the following physical and chemical properties. Appearance, red powder; molecular weight, 891; melting point, 210-217 deg.C; solubility in solvent, soluble in water and methanol, insoluble in benzene, chloroform and ethyl acetate; classification among basic, acidic and neutral, weakly acidic. USE:The compound inhibits the proliferation of human leukemia cell and is useful as a carcinostatic agent. PREPARATION:Streptomyces griseolus A-6067 (FERM P-11184) separated from the soil of Inami-Village, Minamiaizu District, Fukushima Prefecture, Japan is cultured at pH6-7 and 24-27 deg.C for 4 days under aerobic condition, the cultivated microbial suspension is separated into bacterial cells and supernatant and the cells are extracted with an organic solvent such as a lower alcohol.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to benzanthraquinone compounds useful as anticancer agents.

BACKGROUND OF THE INVENTION Various compounds having a benzanthraquinone skeleton and having similar effects to the compounds of the present invention are known.

Problems to be Solved by the Invention However, the anticancer effects of these compounds were not sufficient.

An object of the present invention is to provide an anticancer agent that is more effective than conventionally known compounds.

Means for Solving the Problems As a result of intensive research, the present inventors discovered that a compound produced by a certain type of bacterial strain achieves the above object, and completed the present invention.

That is, the present invention is based on the formula This is a compound represented by

The strain producing the compound of the present invention is a strain newly isolated by the present inventors from the soil of Inan Village, Minamiaizu District, Fukushima Prefecture,
Name of microorganism Strebtomyces guridanasu A -
6067 (Streptomyces griseo
lus-A-6067 and Microorganism Deposit No. 1 FERM I"11184 (FERM I"11184) J, which has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology.

The mycological properties of this strain are shown below.

[1] Morphotrophic hyphae develop well on synthetic and natural agar media and branch irregularly. In addition, no septa are observed, and spores are well formed at the tips of aerial hyphae extending from vegetative hyphae on starch/inorganic salt agar, yeast/malt extract agar, oatmeal agar, etc. When observed under a microscope, the branching method of spore-forming hyphae is simple branching;
Spores are usually formed in a linear chain at the tip of aerial mycelia, but loop-shaped spores are also observed. The spores are chained in groups of 10 or more and have a smooth surface. The shape of the spore is oval and its size is 1.
．． 3-1.7 μm×0.8-1.2 μm.

No sclerotia, sporangia, or flagellar spores are observed.

[2] Growth status on media Table 1 shows the results of visual observation when cultured on various media at 28°C for 114 days.

Table 1 [3] Physiological properties (1) Growth temperature range Grows well in yeast/malt extract medium in the range of 18 to 30@C. It does not grow at temperatures below 8°C and above 37°C.

(2 Biochemical properties 8) Distinction between aerobic and anaerobic; aerobic b) liquefaction of gelatin; positive C) coagulation of skim milk; positive d) peptonization of skim milk; positive C) hydrolysis of starch; positive f) Melanin-like pigment production; Negative g) Cell wall type; ■Type O) Utilization of carbon source (on Bridham-Godelive agar medium): L
-arabinose, D-glucose.

D-xylose, D-fructose.

Shuu Claus Not used: inositol, L-rhamnose.

From the properties of raffinose and D-mannite, it became clear that this strain belongs to the genus Streptomyces among actinomycetes.The above properties were summarized in 1.
S, P,' The International Streptomyces Project 1. Burge, Manual of Determinative Bacteriology, 8th Edition (197
As a result of comparison with many known bacterial strains reported in "The Actinomycetes," Volume 2 (1961) by John Waxman, the present strain was found to be Strepto-myces griseolus.
) showed the properties closest to that of

Based on the above results, it was determined that this strain is the same species as Streptomyces gnoceolus, and this strain was named Streptomyces griseolus A-6067.

The production of the compounds of the present invention is carried out by culturing the present bacterial strain under aerobic conditions in a medium containing various nutritional substances, roughly in the same manner as in the production of general fermentation products.

The medium used is mainly a liquid medium, and the carbon source used is glucose, sucrose, molasses, starch, etc. alone or in combination. As the nitrogen source, meat extract, oatmeal, yeast extract, soybean flour, polypeptone, etc. are used alone or in combination.

In addition, organic substances and inorganic salts that aid the growth of the present strain and promote the production of the compound of the present invention may be added as necessary. As the antifoaming agent, adecanol, silicone, etc. can be used.

For the culture method, aerobic culture such as shaking culture and aerated agitation culture is suitable, and the pH is 4-8.24-30"C and 3-6[]
The culture is preferably carried out at pH 6 to 7.24 to 27°C for 4 hours.

The compound of the present invention produced by this culture can be isolated at 1° C. according to a general method for collecting fermentation products.

That is, after completion of the culture, the bacterial cells and supernatant are separated by centrifugation or filtration, and the compound of the present invention accumulated in the bacterial cells is extracted with an organic solvent such as a lower alcohol or acetone. The organic solvent is distilled off under reduced pressure, and the residue is combined with the supernatant. Next, the compound of the present invention contained in this solution is adsorbed onto a polystyrene resin, and then eluted with an organic solvent such as a lower alcohol or acetone. A crude fraction can be obtained by distilling off the organic solvent under reduced pressure and freeze-drying the residue.

By dissolving the powder obtained in this operation in water containing an organic solvent such as acetone or methanol, and subjecting it to silica gel column chromatography, gel filtration column chromatography, and high performance liquid column chromatography,
The compounds of the present invention can be purified and isolated.

Effects of the Invention The compounds of the present invention inhibit the proliferation of human leukemia cells (HL-60 cells) and are therefore useful as anticancer agents.

Example Next, the present invention will be specifically explained with reference to Examples.

(Example) (1) Per IOM, Helebutomyces glyseptolaus was added to a sterile liquid medium of pH 7 consisting of 1.5 g of soluble starch, 04 g of yeast extract, 0.05 g of dipotassium hydrogen phosphate, and magnesium sulfate.・A-606
After inoculating 7 strains, they were cultured with shaking at 28°C for 72 hours and used as a seed culture.

Next, using a culture tank with a content of U201, sterile medium 1202 having the same composition as the seed culture was inoculated with the seed culture solution 24, and cultured with stirring and aeration at 28°C for 96 hours.

After the culture was completed, the supernatant and the bacterial cells were separated using a centrifuge. There are 50 bacterial cells! After extraction with acetone, the acetone was distilled off under reduced pressure, and the solution was combined with the supernatant.
P-20 (trade name of polystyrene resin, manufactured by Mitsubishi Chemical Corporation)
column (capacity 6j2), washed with water and eluted with a 50% aqueous acetone solution, and the 1.5 to 2.5 head elution fraction was collected. Acetone was distilled off under reduced pressure, the residue was extracted twice with half the amount of ethyl acetate, and the lower aqueous layer was freeze-dried to obtain 10 g of red powder.

(2> 10g of the red powder obtained in the previous section (1) was dissolved in a mixed solvent of chloroform-methanol-water (3:1:0.1) 2
After adsorption in a column (capacity: 3 fl) filled with silica gel using the above mixed solvent, it was fractionated using the above mixed solvent. The mixture was fractionated into 25M portions, fractions Nos. 5 to 7 were collected, the solvent was distilled off under reduced pressure, and the residue was freeze-dried to obtain 3 g of red powder.

(3) 7r11 3g of red powder obtained in the previous section (2)
500 to 600 mg was dissolved in methanol and subjected to gel filtration with the same solvent using Sephadex LH-20 (trade name, manufactured by Pharmacia) filled with methanol.
By collecting the active fraction eluted, 65 mg of a red powder of the compound of the present invention was obtained.

[Physical and chemical properties] (1) Appearance: Red powder (Elemental analysis values: (as C,, H□0,, N S) Actual values C55,52%, H5,90%, N 1.89%,
33.11% theoretical value C56, 57%, H5, 95%, N 1.57
%, 3 3.59% (3) Molecule it: 891 [
(M + H)” rn/z in FAB mass spectrum
892 (4) Melting point: 210-217°C (S) Ultraviolet absorption spectrum λ-a- (nm) 2215, 280, 470 (measured in water) 204,
286 (sh), 490 (addition of sodium hydroxide) (6) Infrared absorption spectrum ν,,, (am-') 3400.1715,16
22.1510(7) Solubility in solvents Soluble in water and methanol

Claims (1)

[Claims] (1) Formula ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ A compound represented by