JPH03232887A - New substance bk 97, use thereof and production thereof - Google Patents

Abstract

NEW MATERIAL:A compound expressed by the formula having the following physicochemical properties: apprearance, dark blue-colored powder; melting point, 196-197 deg.C (decomposed); solubility, soluble in ethyl acetate, chloroform, dimethylsulfoxide and benzene and slightly soluble in methanol or ethanol and insoluble in hexane and water; Rf value, 0.29 in chloroform-methanol (100:1) and 0.37 in toluene-acetone (4:1), etc. USE:An antiulcer agent. PREPARATION:An actinomyces strain of the genus Streptomyces such as Streptomyces prunicolor 1884-SVT 2 strain (FERM BP-2728) is aerobically cultured, preferably using aerobic liquid culture method at 25-30 deg.C.

Description

DETAILED DESCRIPTION OF THE INVENTION [Background of the Invention] Technical Field The present invention relates to a novel substance, and more particularly to a novel substance BK97 having antitumor properties.

Prior Art A large number of antitumor substances have already been put into practical use as medicines. In general, the physiological activity of a chemical substance largely depends on its chemical structure, so it can be said that there is a constant desire for new compounds with antitumor properties.

[Summary of the invention]

Summary The present invention meets the above needs.

That is, the novel substance BK97 according to the present invention has the following formula (I
).

The invention also relates to the use of this substance.

That is, the antitumor agent according to the present invention contains the compound BK97 represented by the following formula (I) as an active ingredient.

The invention furthermore relates to a method for producing this material.

That is, BK97 represented by the following formula (1) according to the present invention
The production method is characterized by culturing a bacterial strain belonging to the genus Streptomyces and capable of producing BK97 in an appropriate medium aerobically, and obtaining BK97 from the culture.

[Detailed description of the invention]

Novel Substance BK97> (1) Chemical Structure The novel substance BK97 according to the present invention has a chemical structure represented by the above formula (I). These chemical structures were determined as follows.

Analysis of the proton nuclear magnetic resonance spectrum (Figure 3) and carbon-13 nuclear magnetic resonance spectrum (Figure 4) of BK97 revealed that BK97 has partial structures such as a phenazine skeleton, a benzene ring, and a geranyl side chain. Ta.

Furthermore, the presence of a carboxylic acid was revealed by treating BK97 with diazomethane in chloroform to give a methyl ester. Further detailed examination of the spectra revealed the connections between the respective substructures, and the structure of BK97 was determined as shown in Figure (I) from the molecular formula derived from the high-resolution mass spectrum.

2) Physicochemical properties (1) Appearance: Dark blue powder (2) Melting point: 196-197°C (decomposition) (3) Solubility:
Soluble in ethyl acetate, chloroform, dimethyl sulfoxide and benzene, slightly soluble in methanol and ethanol, insoluble in hexane and water.

(4) Rf value (“Silica gel 60F2.4” manufactured by Merck & Co., Ltd.)
Used): Chloroform-methanol 0.29 (100
:1) Toluene-acetone (4:1) 0.37(5)
High resolution mass spectrum (m/z): 493.2071
(M+H)” (calculated value 493.2127C3, H29N204) (
6) Ultraviolet absorption spectrum λff1axnIll(ε)
: Shown in Figure 1.

206 (16300), 247 (14500),
342 (9300), 424 (3700), 62
2 (8600) (in methanol); 206 (171
00), 247 (15300), 344 (940
0), 63g (8100) (in 0.01N hydrochloric acid-methanol); 208 (21300), 247 (
15700), 340 (8700), 364 (6
700), 407 (4000), 428 (400
0), 616 (9200) (0,01 N sodium hydroxide in methanol); (7) Infrared absorption spectrum (KBr disk method): 2nd
As shown in the figure.

(8) Proton nuclear magnetic resonance spectrum (500 MHz, in deuterated chloroform) Two chains shown in Figure 3.

(9) Carbon-13 nuclear magnetic resonance spectrum (125 MHz, in deuterated chloroform): Shown in FIG.

<Production of BK97> 1) Overview Compound BK97 is currently obtained only by culturing microorganisms, but it can also be produced by synthetic chemical modification of related compounds or by total synthetic chemical modification. Good morning. It may also be possible to use genetic engineering techniques. That is, it would be possible to introduce the gene involved in the production of compound BK97 into a suitable microorganism, culture such a transformed microorganism, and obtain the compound from this culture.

In the case of culturing microorganisms, for example, those belonging to the genus Streptomyces and having the ability to produce BK97 are used. Specifically, the Streptomyces Φprunicolol 1884-8VT2 strain isolated by the present inventors is BK9
Although the present inventors have revealed that a strain producing antibiotic-producing bacteria No. 7, suitable strains can be isolated from nature using conventional methods for isolating antibiotic-producing bacteria. Also, Streptomyces prunicolol 1
There is also room to increase BK97 production ability by subjecting BK97(7)-producing bacteria, including the 884-8VT2 strain, to radiation irradiation or other mutational treatments.

As mentioned above, genetic engineering techniques are also possible.

2) 1884-SVT2 strain 1884-8VT2 strain, which the present inventors have discovered as a Streptomyces strain capable of producing BK97, is
The contents are as follows.

(1) Origin and deposit number The 1884-8VT2 strain was isolated from soil collected from an apple orchard in Fujisaki Town, Aomori Prefecture, on January 17, 1990.
FERM BP-2728 was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology on
) number has been obtained.

(2) Mycological properties The mycological properties of the 1884-8VT2 strain are as follows.

The basal hyphae of this strain do not divide, but during long-term liquid stationary culture, oval spores may be formed singly or in short chains. The aerial hyphae elongate while branching, and later the entire hyphae segment to form a straight spore chain consisting of 10 to 50 or more spores. Long aerial mycelia are often entangled.
It forms a bird's nest-like hyphal mass, which later becomes a spore mass. Arthrospores are non-motile, oblong or cylindrical, 0.5-0.7
It has a size of ×0.7 to 1.7 μm and a smooth surface. No sclerotia, sporangia, or other special forms are observed. The cell wall chemotype is type I.

The culture properties are shown in Table 1. The bacterial flora on the surface of the colony is in the red series (
It has a pinkish white to light brownish gray color. The color of the underside is light orange or light brown to brown to dark brown or dark brownish gray, and becomes more yellowish when acidic and reddish when basic. The diffusible dye is orange or brown and exhibits pH sensitivity similar to the backside color. Physiological properties are shown in Table 2. This strain is mesophilic, produces melanin pigment, and is positive for both starch and protein degradation (weak) and nitrate reduction. Carbon source assimilation utilizes all of the diagnostic sugars.

Based on the morphological characteristics and cell wall chemotype of this strain, it is placed in the genus A. Based on the above-mentioned properties, we searched for species of the genus S listed in the "Approved List of Bacterial Names, 1980" and the subsequent "List of Valid Names," and selected four closely related species. The recently published “Handbook of Bacterial Systematics Taxonomy 4”
Bergey S Manual of' sy.
Stematic Bacteriology Vol.
4) regarding the genus S.
ams.

According to the description by et al., there are four closely related species (synonyms). Original report by Williams et al. (198
According to 3), standard strains of (species) with approved and valid names of the genus S are treated as "equivalent classification standards for each property" using a numerical classification method, and a large number of strains with high numerical similarity are classified using a numerical classification method. Species are reclassified into clusters (groups). In that report, the four species mentioned above are placed in adjacent clusters. So 4
Diagnostic properties of the species were selected from the same report and compared with the same properties of this strain (Table 3). This strain and S. prunicolol have 19 matching properties and 6 discrepant properties, and with S. tass there are 9 matching properties and 4 discrepant properties. This strain and two species, S-prunicolol and S. fluvissimus, are significantly more closely related than the other two species, with the former being slightly more related than the latter. on the other hand,
According to the description of the International Streptomyces Project (ISP), this strain is more similar to S. fluvissimus than S. fluvissimus.
・It is closely related to Prunicolol, and its morphology is particularly similar to the point where it forms a "bird's nest-like hyphal mass." Therefore, this strain is Sφ
It is positioned as a strain of Prunicolol. For the above reasons, this strain was selected from Streptomyces prunicolol (
StreptoIIlycesprunicolor)
The strain is named 1884-8VT2.

Table 2 Physiological properties of strain 1884-8VT2 were observed on the 2nd day for melanin-like pigments, and on the 2nd week for others.
After that, it will be carried out after 3 weeks.

4 Table 5 Comparison of main characteristics and diagnostic characteristics between this strain and related species 5 Table 3 continued +: 90% or more positive, m: 10% or less positive, d: 11% to 89% positive Culture/BK97 Production> Compound BK97 is produced by BK9, which belongs to the genus Streptomyces.
It can be produced by aerobically culturing 7-producing bacteria in an appropriate medium and collecting the target product from the culture.

The medium can contain any nutrient source that can be used by the BK97-producing bacteria. Specifically, for example, glucose, sucrose, maltose, starch, fats and oils can be used as carbon sources, and soybean flour, soybean flour and nitrogen can be used as nitrogen sources.
Organic materials such as cottonseed meal, dried yeast, yeast extract and corn staple liquor and inorganic materials such as ammonium salts or nitrates such as ammonium sulfate, sodium nitrate and ammonium chloride can be utilized. In addition, sodium chloride, potassium chloride, phosphate,
Inorganic salts such as heavy metal salts can be added. In order to suppress foaming during fermentation, a suitable antifoaming agent,
For example, silicone oil can also be added.

The most suitable culture method is the aerobic liquid culture method, which is the same as the commonly used antibiotic production method. The culture temperature is suitably 20-37°C, preferably 25-30°C. With this method, the production amount of BK97 can be determined by shaking culture,
Both the aeration and agitation culture reached the maximum after 2 days of culture.

In this way, a culture enriched with BK97 is obtained. In the culture, a part of BK97 exists in the culture filtrate, but most of it exists in the bacterial cells.

Any convenient method can be used to harvest BK97 from such cultures. One method is based on the principle of extraction, and specifically, for BK97 in the culture filtrate, it is extracted from water-immiscible BK97.
For BK97 in bacterial cells, extract the bacterial mass obtained by filtration, centrifugation, etc. with methanol, ethanol, acetone, etc. There are ways to process and recover it. It is also possible to subject the culture as it is to the above extraction operation without separating the bacterial cells. Countercurrent distribution methods using suitable solvents can also be included in the scope of extraction.

Another method for collecting BK97 from cultures is based on the principle of adsorption, in which BK97 is already in liquid form.
A suitable adsorbent such as silica gel, activated carbon,
[BK97 can be obtained by adsorbing the target BK97 using Diaion HP-20J (manufactured by Mitsubishi Kasei Corporation) or the like, and then eluting it with an appropriate solvent. The BK97 solution thus obtained is concentrated to dryness under reduced pressure to obtain a crude sample of BK97.

In order to further purify the crude sample of BK97 obtained in this way, the extraction method and adsorption method described above may be combined with gel filtration method, high performance liquid chromatography, etc. as necessary, and the procedure may be performed as many times as necessary.

For example, an adsorbent such as silica gel, or a column chromatography rYMC bag using a gel filtration agent such as Sephadex LH-20J (manufactured by Pharmacia).
(manufactured by Yamamura Kagaku Co., Ltd.) or the like and a countercurrent distribution method can be carried out in an appropriate combination. Specifically, for example, a crude sample of BK97 was applied to a Sephadex LH-2OJ column, the active fraction was eluted with a chloroform-methanol (1:1) mixture, and the pure product of BK97 was obtained by concentrating to dryness. can get.

<Applications of BK97> The compound BK97 according to the present invention is useful in that it has antitumor activity.

1) Biologically active BK97 showed cytostatic activity against tumor cells. For example mouse P388 leukemia cells or mouse L1
210 leukemia cells were suspended at 2 x 104 cells/ml in RPM1640 medium containing 10% heat-inactivated beef tallow serum and cultured with various concentrations of BK97 at 37°C for 3 days. The C5o value was as follows: 4.0 and 4 respectively
．． It was 5 μg/ml.

As mentioned above, it was revealed that BK97 of the present invention exhibits antitumor properties. Therefore, BK97 of the present invention can be used as an antitumor agent.

2) Antitumor agent It has thus been revealed that BK97 of the present invention exhibits antitumor properties against animal tumors, particularly malignant tumors.

Therefore, the compounds of the present invention can be used as antitumor or tumor therapeutic agents.

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

When actually administering the compounds of the present invention as antitumor agents, one typical method is to dissolve them in distilled water for injection or physiological saline and inject them.

Specifically, methods include intraperitoneal injection, subcutaneous injection, intravascular injection into a vein or artery, and local administration by injection.

The dosage of the compound of the present invention is determined in consideration of the results of animal tests and various situations so that the total dosage does not exceed a certain amount when administered continuously or intermittently. The specific dosage depends on the administration method, the situation of the patient or treated animal,
For example, age, weight, gender, sensitivity, diet, time of administration,
It goes without saying that this will vary depending on the concomitant drugs, the patient, and the severity of the disease, and the excursion and number of doses under certain conditions must be determined by a specialist's appropriate dosage determination test based on the above guidelines. . Specifically, it is about 0.1 to 1 g per day for an adult.

Experimental Examples ■) Cultivation The medium used was prepared by dissolving the following components in 1 liter of water and adjusting the pH to 7.0.

Starch 25g Soy flour 15g Dried yeast 2g 2 Calcium carbonate 4g 100ml of the above medium was dispensed into sterilized 500ml Erlenmeyer flasks with warts, and Streptomyces Q prunicolol 1884-3 VT2 strain was inoculated at 27°C for 2 days. , rotational culture was performed at 20 rpm.

2) Collection of BK97 After culturing under the above conditions, filter the culture solution (5 liters),
Extract the bacterial cells with 1 liter of acetone.

After concentrating the extract, it is extracted three times with 200 ml of ethyl acetate, and concentrated to dryness. This was dissolved in 10 ml of chloroform, and silica gel (Wako Pure Chemical Industries, Ltd. [Wako Gel C200J]
) and eluted with chloroform. The active fraction was concentrated to dryness and coated on a Toyopearl HW-40F column (manufactured by Tosoh Corporation) (2,
'5cmφ×50cm) and eluted with methanol to obtain the crude side of BK97. This was further adsorbed onto a silica gel column (3.5 φ x 25 cm), and chloroform-methanol-28% aqueous ammonia (200:2
Expand with 0:1)3. The blue band was collected and concentrated, and Sephadex LH-20 (manufactured by Pharmacia) (20mφX55
cm) and chloroform-methanol (1:1)
When the blue fraction was concentrated to dryness, 5 mg
Obtain a pure BK97 product.

[Brief explanation of drawings]

Figure 1 shows BK97 in methanol (solid line), 0.01
This is a reproduction of the ultraviolet absorption spectrum of BK97 in normal hydrochloric acid-methanol (dotted line) and in 0.01N sodium hydroxide-methanol (broken line). FIG. 2 is a reproduction of the infrared absorption spectrum of BK97 obtained by the KBr disk method. Figure 3 shows the concentration of BK97 at 500% in deuterated chloroform.
This is a copy of the megahertz proton nuclear magnetic resonance spectrum. Figure 4 shows the concentration of 125 BK97 in deuterated chloroform.
This is a reproduction of a megahertz carbon-13 nuclear magnetic resonance spectrum. JP-A-3-232887 (8) JP-A-3-232887 (9)

Claims (1)

[Claims] 1. Compound BK97 or a base addition salt thereof represented by the following formula (I). ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (I) 2. An antitumor agent containing the compound BK97 shown by the following formula (I) or its base addition salt as an active ingredient. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (I) 3. A strain belonging to the genus Streptomyces and capable of producing BK97 is cultured aerobically in an appropriate medium, and the compound BK97 is obtained from the culture. The following formula (I
) The manufacturing method of BK97 represented by ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I)