JP2706843B2 - Novel anthraquinone derivative, method for producing the same, and antitumor agent containing the derivative - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a novel anthraquinone compound, particularly an anthraquinone derivative having antitumor properties.

[Problems to be solved by conventional technology and invention]

Many new compounds have been synthesized in search of new properties. Particularly, in the field of antitumor agents, many new compounds have been proposed and put to practical use. The present invention is in line with such a series of technological developments, and it is an object of the present invention to provide a novel compound which has not been known so far and has an antitumor activity.

[Means for solving the problem]

The present inventors have studied the antitumor active substance produced by the microorganism and found that the microorganism BY-90 strain produces an antitumor active substance, and then separated and purified the physiologically active substance from the solvent. However, as a result of examining its physicochemical properties, it was confirmed that the substance was a novel substance having a structural formula represented by the following general formula (I). Therefore, a compound in which R is an OH group in the general formula is designated as BY90-OH, and a compound in which R is an H group is also designated as BY90-H.

(In the formula, R represents a hydrogen atom or a hydroxyl group.) The present invention has been completed based on such a new finding, and is a novel substance represented by the general formula (I) BY90-OH and BY90-H, It is intended to provide a method for producing the same and an antitumor agent containing the same as an active ingredient.

The physicochemical properties of BY90-OH and BY90-H are as follows.

Production Method The novel compounds BY90-OH and BY90-H of the present invention can be produced by culturing microorganisms, but can also be produced by synthetic chemical modification of analogous compounds, or can be produced by total synthetic chemistry. .

Examples of strains obtained by culturing microorganisms include Streptomyces atratus.
Those having the ability to produce BY90 compounds belonging to the genus are used. Specifically, the Streptomyces atratus strain isolated by the present inventors can be mentioned, but for other strains, appropriate strains can be isolated from nature by a conventional method for isolating antibiotic-producing bacteria. It is possible. In addition, the production bacteria are subjected to irradiation or other mutation treatment,
There is still room for increasing the productivity of the target compound.

The above-mentioned BY90 strain has the following contents.

1. Origin and Deposit No. BY90 strain is an actinomycete isolated from soil collected in Tomakomai City, Hokkaido, and deposited on October 3, 1990 with the Research Institute of Microbial Industry and Technology (MIC). No. 3121)
(FERM BP-3121).

2. Mycological properties The mycological properties of the BY90 strain are as follows.

1) Morphology The basal hypha of the present strain is often segmented on a synthetic agar medium to exhibit a spore chain shape, and is sometimes divided and zigzag. Also, in rare cases, oval spores may be formed alone or in the form of a chain of two to three. Aerial hyphae consist of 10 to 50 or more numbers on the main axis or branches, forming curved to loop or spiral spore chains. Spores are non-motile, oval or cylindrical, 0.6-0.8
× 0.8 to 1.2 μm, smooth surface. Sclerotia and sporangia are not observed. The cell wall chemical type is type I.

2) Growth state on various media Table 1 shows the culture properties. The flora color of the colony surface is a gray series (light brownish gray), the backside color of the colony is unclear (colorless to pale yellow to light brownish gray to dark brownish gray), pH-insensitive, and the diffusible pigment in the medium is brownish It is white to light brown and pH-insensitive.

3) Physiological properties Table 2 shows the physiological properties. This strain is mesophilic and does not produce melanin pigment.
Casein, egg yolk) degradation and nitrate reduction are both positive.

4) Utilization of carbon sources Table 2 shows the assimilation of carbon sources. L-arabinose, i-inositol and D-mannitol among the diagnostic sugars are not used.

5) Position of classification (identification) Based on the morphological properties and cell wall chemical type of the present strain, the present strain is located in "genus Streptomyces" (hereinafter abbreviated as "genus S"). Based on the above-mentioned properties, a search was performed using the description table of Williams, et al. For the genus S in "Burjay's Bacterial Strain Classification Handbook, Vol. 4," and S. atratus was found as a closely related species. Detected. The same type of standard strain, IFO3897, and this strain were compared and examined in accordance with the method of William et al. For 84 items such as morphological properties, culture properties, and physiological and biochemical properties. As a result, in this strain, the underlying hyphae should be segmented / separated. S. Murinus (S.
no antimicrobial activity against S. murinus, only these two items differed from S. atlatus, and the other 82 items were consistent. That is, the properties of this strain were 97.6% that of S. attratus.
Showed a match. Therefore, this strain was identified as one strain belonging to the genus S and one species of Streptomyces atratus.

Table 2 Growth temperature range 10-40 ° C Melanin pigment formation Tyrosine agar-(pink) Peptone yeast, iron agar-Tryptone yeast broth-Starch hydrolysis + Gelatin liquefaction + Skim milk peptone + Skim milk + Coagulation + reduction of nitrate + assimilation of carbon source D-glucose + L-arabinol-D-xylose + D-fructose + sucrose + L-rhamnose + raffinose + i-inositol- D-mannitol- +: yes,-: None Culture / Production of BY90 The compound BY90 can be produced by aerobically culturing a BY90-producing bacterium belonging to the genus Streptomyces atratus in an appropriate medium, and collecting a target substance from the culture.

The culture medium may contain any nutrient sources available to the BY90 producing bacteria. Specifically, for example, glucose as a carbon source, sucrose, maltose, soluble starch, glycerin and oils and the like can be used, soybean flour, fish meal, cottonseed meal, dried yeast, yeast extract and meat extract as a nitrogen source Organics such as corn steep liquor and inorganics such as ammonium salts or nitrates such as ammonium sulfate, sodium nitrate and ammonium chloride can be utilized. If necessary, inorganic salts such as calcium carbonate, sodium chloride, potassium chloride, phosphate, and heavy metal salt can be added. In order to suppress foaming during fermentation, a suitable antifoaming agent such as silicone oil may be added according to a conventional method.

As a culture method, an aerobic liquid culture method is the most suitable, as is the case with a general antibiotic production method.
The culture temperature is suitably from 10 to 40 ° C, preferably from 25 to 30 ° C. By this method, the production amount of BY90 reaches the highest in both shaking culture and aeration-agitation culture in 3 days of culture.

In this way, an accumulated culture of BY90 is obtained.
In culture, BY90 is partially present in the culture filtrate, but mostly in bacterial cells.

Any suitable method for collecting BY90 from such cultures is available. One of the methods is based on the principle of extraction. Specifically, for the BY90 in the culture filtrate, a method for extracting it with a water-immiscible BY90 solvent (see above), for example, chloroform, etc. ,
Alternatively, with respect to BY90 in the cells, there is a method of collecting and collecting the cells collected by filtration, centrifugation, etc., with methanol, ethanol, acetone, or the like. The culture as it is can be subjected to the above extraction operation without separating the cells. Countercurrent distribution using a suitable solvent can also be included in the category of extraction.

Another method of collecting BY90 from cultures is based on the principle of adsorption, which means that BY90 is already in liquid form.
For the contents, for example, the culture filtrate or the extract obtained by performing the extraction operation as described above, a suitable adsorbent, for example, silica gel, activated carbon, "Diaion HP20" (manufactured by Mitsubishi Kasei), etc. Use of purpose
BY90 can be obtained by adsorbing BY90 and then eluting with a suitable solution. The BY90 solution thus obtained is concentrated under reduced pressure to dryness to obtain a crude BY90 product.

In order to further purify the crude sample of BY90 thus obtained, a gel filtration method,
High-performance liquid chromatography or the like may be combined as needed to perform the required number of times. For example, an adsorbent such as silica gel, "Sephadex LH-20" (Pharmacia)
Column chromatography using a gel filtration agent such as
High-performance liquid chromatography using “YCM Pack” (manufactured by Yamamura Kagaku) and a countercurrent distribution method can be combined as appropriate. Specifically, for example, a BY90 crude sample is applied to a “Sephadex LH-20” column, the active fraction is eluted with a chloroform-methanol (1: 1) mixture, and concentrated to dryness to give a pure BY90 product. Is obtained.

Use Compound BY90 according to the invention has antitumor activity.

Biological activity BY90 showed cytostatic activity against tumor cells.
For example, mouse P388 leukemia cells were suspended at 2 × 10 ⁴ cells / ml in RPMI1640 medium containing 10% heat-inactivated calf serum, and at 37 ° C. with various concentrations of BY90-OH and BY90-H.
After culturing for 3 days at 4.5 and 9.8, respectively.
μg / ml.

Thus, the compounds of the present invention have been shown to exhibit antitumor properties against animal tumors.

Therefore, the compound of the present invention can be used as an antitumor agent or a tumor therapeutic agent.

The compounds of the present invention as antitumor agents can be administered by any suitable route of administration and in dosage forms determined by the route of administration employed. The drug is usually in a form diluted with a pharmaceutically acceptable carrier or diluent.

When the compounds of the present invention are actually administered as an antitumor agent, one of the typical methods is to dissolve them in distilled water for injection or physiological saline and inject them. Specifically, in the case of animals, methods such as intraperitoneal injection, subcutaneous injection, intravenous injection into veins or arteries, and local administration by injection, and in humans, methods such as intravenous injection or injection into veins or arteries are used. There are methods such as local administration.

The dose of the compound of the present invention is determined in consideration of the results of animal tests and various situations so that the total dose does not exceed a certain amount when administered continuously or intermittently. It goes without saying that the specific dose varies depending on the administration method, the condition of the patient or the treated animal, for example, age, body weight, sex, sensitivity, diet, administration time, concomitant drug, patient or the degree of the disease. In addition, the appropriate dosage and the number of doses under certain conditions must be determined by a specialist in a dosage determination test based on the above guidelines.
Specifically, it is about 0.1 to 1 g per day for an adult.

EXPERIMENTAL EXAMPLES Example 1) Preparation of Seed Mother The used medium was prepared by dissolving the following components in 1 liter of water.

Soluble starch 10g Polypeptone 10g Molasses 10g Meat extract 10g Dispense 100ml of the above medium into a 500ml Erlenmeyer flask, sterilize, and add 1 streptomyces attratus from slant
The seeds were inoculated with platinum loops and cultured with shaking at 27 ° C for 4 days.

2) Culture The culture medium used was prepared by dissolving the following components in 1 liter of water.

Glycerin 8 g Soluble starch 8 g Soybean flour 3 g Fish meal 8 g Calcium carbonate 2 g To the above medium, 100 ml of each medium was dispensed and sterilized in a 500 ml Erlenmeyer flask, and 2 ml of the above seed was added, followed by shaking culture at 27 ° C. for 3 days.

3) Collection of BY90 After culturing under the above conditions, the culture solution (10 liters) was
The cells were extracted with 5 liters of acetone. This was concentrated under reduced pressure to 0.5 liter, and 0.5 liter of water and 1.0 liter of ethyl acetate were added to perform ethyl acetate extraction. The ethyl acetate extract was concentrated to dryness, redissolved in a small amount of ethyl acetate, adsorbed on a column (5 cmφ × 50 cm) of silica gel (Wako Gel C-200 manufactured by Wako Pure Chemical Industries, Ltd.), washed with ethyl acetate, and then washed with ethyl acetate: methanol. (15: 1). The active fraction was concentrated to dryness to obtain a crude powder of BY90.
This coarse powder was dissolved in a small amount of a mixed solution of chloroform-methanol-ammonia (200: 10: 1), adsorbed again on a silica gel column (3 cmφ × 20 cm), and developed with chloroform-methanol-ammonia (200: 10: 1). The active fraction was concentrated to dryness, redissolved in methanol, and subjected to gel filtration of Toyopearl HW-40 (30 cmφ × 20 cm) with the same solvent.
The active fraction was concentrated again to dryness, and preparative TLC was performed using silica gel 60F ₂₅₄ manufactured by Merck under the conditions of ethyl acetate-methanol (15: 1).
BY90-OH was detected as an active spot near Rf value 0.60 and BY90-H near 0.54 as active spot. Each spot was scraped, eluted with methanol, and the eluate was concentrated to dryness. Further, each spot extract was subjected to gel filtration (3 cmφ × 20 cm) using Sephadex-LH-20 under the condition of chloroform / methanol (1: 1).
10 mg of -OH pure product and 2 mg of BY90-H pure product were obtained as yellow powder.

[Brief description of the drawings]

FIG. 1 is a simulated ultraviolet absorption spectrum of BY90-OH in methanol (solid line) and 0.01N sodium hydroxide-methanol (dashed line). FIG. 2 is a simulated UV absorption spectrum of BY90-H in methanol. FIG. 3 is a simulated infrared absorption spectrum of BY90-OH by the KBr disk method. FIG. 4 is a simulated infrared absorption spectrum of BY90-H by the KBr disk method. FIG. 5 is a copy of a 400 MHz proton nuclear magnetic resonance spectrum of BY90-OH in deuterated methanol. FIG. 6 is a copy of a 400 MHz proton nuclear magnetic resonance spectrum of BY90-H in heavy methanol. FIG. 7 is a simulated 100 MHz carbon 13 nuclear magnetic resonance spectrum of BY90-OH in heavy methanol. FIG. 8 is a simulated 100 MHz 13C nuclear magnetic resonance spectrum of BY90-H in heavy methanol.

Claims (3)

(57) [Claims] 1. Anthraquinone derivative represented by the following general formula (I) (In the formula, R represents a hydrogen atom or a hydroxyl group.) 2. An antitumor agent comprising the anthraquinone derivative represented by the general formula (I) according to claim 1 as an active ingredient. 3. A strain belonging to the genus Streptomyces and capable of producing the anthraquinone derivative represented by the general formula (I) according to claim 1 is aerobically cultured, and the anthraquinone derivative is cultured from the culture. The method for producing an anthraquinone derivative represented by the general formula (I) according to claim (1), wherein