JPH0260596A - Novel material bq180b and use and production thereof - Google Patents

Abstract

PURPOSE:To obtain in high efficiency BQ180B useful for antineoplastic agents by aerobically making a culture of a strain capable of producing BQ180B of specific structural formula classified as Streptomyces to produce the BQ180B. CONSTITUTION:A BQ180B strain capable of producing BQ180B classified as Streptomyces (FERM P-1934) is put to an aerobic culture at 20-37 deg.C in an appropriate medium to produce a cultured product accumulated with BQ180B. When the BQ180B is to be collected from this product, the BQ180B in the culture filtrate is extracted with a solvent such as chloroform, while for the BQ180B within the bacteria, a bacteria aggregate obtained by e.g., filtration is treated with e.g., methanol, thus recovering the BQ180B, respectively. The crude preparation of the BQ180B thus recovered is purification by a combination of extraction, adsorption and gel filtration processes, thereby obtaining the objective compound BQ180B of formula I.

Description

DETAILED DESCRIPTION OF THE INVENTION BACKGROUND OF THE INVENTION Technical Field The present invention relates to a novel substance, and more particularly to the compound BQ180B, which has antitumor properties.

Many prior art 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]

Abstract The present invention meets the above needs.

That is, the novel substance BQ180B according to the present invention is represented by the following general formula (1).

The invention also relates to the use of this substance.

That is, the antitumor agent according to the present invention contains the compound BQ180B represented by the following formula (1) as an active ingredient.

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

That is, BQ18 represented by the following formula (1) according to the present invention
The manufacturing method of 0B belongs to the genus Streptomyces and BQ18
This method is characterized by culturing a bacterial strain capable of producing 0B aerobically in an appropriate medium and obtaining BQ180B from the culture.

[Detailed description of the invention]

New substance BQ180B Chemical structure The new substance BQ180B according to the present invention has the above formula (I)
It has the chemical structure shown below.

These chemical structures were determined as follows.

The chemical structure deduced from the proton nuclear magnetic resonance spectrum (Figure 3) and carbon-13 nuclear magnetic resonance spectrum (Figure 4) of BQ180B is β-rubromycin (β-rubromycin).
roBcin) (Il, Brockmann an
dA, Zeeck: Chem, Ber,, 103.17
09-17213 (1970)). When comparing the physicochemical properties of both, BQ18
The molecular weight of 552 obtained from the FD mass spectrum of 0B is one oxygen atom higher than that of β-lubromycin, and N
BQ180B is β-
It turned out to be the hydroxy form of rubromycin. Furthermore, the substitution position of the hydroxyl group was determined by detailed analysis of NMR data, and the chemical structure of BQ180B was determined as shown in the above formula (1).

Physicochemical properties (1) Appearance Reddish-purple powder (2) Melting point 218-223℃ (turns black at 202-208℃) (3) Solubility Soluble in dimethyl sulfoxide, chloroform, methanol, ethanol, acetone, ethyl acetate Slightly soluble in hexane and water.

(4) Rf value (“Silica gel 60F254” manufactured by Merck & Co., Ltd.)
Used) Chloroform-methanol (20:1) 0,29 Toluene-methanol (10:1) 0,08 (5) FD mass spectrum (m/z) 552 (M) Shown in FIG.

230 (824), 312 (403), 348
(2B2), 362 (208), 512 (118
) (in methanol) (7) Infrared absorption spectrum (KBr disk method) shown in FIG.

(8) Proton nuclear magnetic resonance spectrum (500 MHz, in deuterated chloroform) as shown in FIG.

(9) Carbon-13 nuclear magnetic resonance spectrum (125 MHz, in deuterated dimethyl sulfoxide) is shown in FIG.

Overview of the production of BQ180B Compound BQ180B is currently obtained only by culturing microorganisms, but it could also be produced by synthetic chemical modification of related compounds or by total synthetic chemistry. It may also be possible to use genetic engineering techniques. That is, it would be possible to integrate the compound BQ180B-producing gene into a suitable microorganism, culture such a transformed microorganism, and obtain it from this culture.

In the case of culturing microorganisms, for example, a strain belonging to the genus Streptomyces having an ability to produce BQ180B is used. Specifically, the Streptomyces fluvicinis strain 801808 isolated by the present inventors is BQ18
Although the present inventors have shown that 0B is produced, suitable strains of other strains can be isolated from nature by conventional methods for isolating antibiotic-producing bacteria. Furthermore, there remains room to increase BQ180B production ability by subjecting BQ180B producing bacteria, including Streptomyces fulvicinis strain 801808, to irradiation or other mutation treatments. As mentioned above, genetic engineering techniques are also possible.

Strain 80180 B Q 1.80 Strain 80180, which the present inventors have discovered as a Streptomyces strain capable of producing B, has the following content.

■) Origin and Deposit Number Strain 80180 was isolated from soil collected in Thailand, and was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology on July 4, 1988. No. 1934) (
FERM BP-1934) number.

2) Mycological properties The mycological properties of strain 80180 are as follows.

(1) Form 80180 strain grows poorly on glucose-asparagine agar medium, but grows well on other media. Aerial mycelium grows poorly on glucose-asparagine agar and sucrose nitrate agar, and moderately or slightly poorly on oatmeal agar, but generally grows well on other media. Aerial hyphae that originate from substratum hyphae form simple branches and elongate, but spores are relatively difficult to form, with only 5 to 10 spore chains, and most spore chains are linear or hook-shaped. , loop-shaped ones are also seen. The spores are cylindrical or oval in shape and have a size of 0.
6-0.8 μmXQ, 9-1.2 μm, and its surface is smooth.

Specialized forms such as sporangia, flagellated spores, and sclerotia are not observed.

(2) Growth status on various media The results of culturing 80180 strains on various media at 27°C for 3 weeks are shown in Table 1.

(3) Physiological properties The physiological properties of strain 80180 are shown in Table 2.

(4) Carbon source utilization The carbon source utilization of strain 80180 (on Pridham-Gottlieb agar medium) is as shown in Table 3.

(5) Analysis of diaminopimelic acid As a result of analyzing diaminopimelic acid, which is one of the amino acids constituting the cell wall of BQ180, LL-diaminopimelic acid was detected.

From the above mycological properties, strain 80180 was determined to belong to the genus Streptomyces, and has the following characteristics.

(i) Although spore chains are relatively difficult to form, they are mainly linear, hook-shaped, or loop-shaped, and the surface of the spores is smooth.

(if) Aerial mycelium is yellowish white to yellowish brown to light pink to bright olive gray, and the color of the underside is yellowish brown to yellowish red to dark orange to light orange to brown.

(llj) It produces melanin-like pigments in peptone-yeast-iron agar medium and tryptone-yeast liquid medium, but not in tyrosine agar medium.

Based on the above properties, I5P (International Streptomyces Project) is described (E, B).

Shirling and D, Gottlleb: I
nt, J, 5yst, Bact,, 1g.

f39-189, 279-392 (1968) + 19
．． 391-512 (1909); 22285-394
(1972)) and Bersey's Manual of
Deterministic Bacteriology (Bergey'
s Manual oroterm+nativc
When searching for related known bacterial species using the 8th edition (1974) as a reference, Streptomyces
Fulvicimus (StrepLomyces rulv)
:ssimus> is the most similar.

Therefore, when comparing strain 80180 and Streptomyces 0 Fulvicimus, the morphological characteristics of forming spores with linear spore chains and smooth surfaces, the production of melanin-like pigment on peptone, yeast, and iron agar medium, The two also match well in terms of color tone of aerial mycelium, ability to assimilate sugar, and difficulty in forming spore chains on oatmeal medium. The difference is that the 80180 strain does not assimilate fructose.

As described above, although there are some differences, the 80180 strain closely matches those of Streptomyces fluvicimus in its basic characteristics, and therefore it is appropriate to identify it as Streptomyces fluvicimus. Therefore, 8018
0 strains of Streptomyces 0 fluvicimus (Strcpt
omyces fulvissim - Table 1 Table 2 Table 3 +: Utilization - Dual non-utilization culture/Production of BQ180B Compound BQ180B is BQ180B, which belongs to the genus Streptomyces.
It can be produced by culturing Q180B-producing bacteria aerobically 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 BQ180B-producing bacteria. Specifically, for example, glucose, sucrose, maltose, starch, and fats can be used as carbon sources, and organic substances such as soybean flour, cottonseed meal, dried yeast, yeast extract, and cornstarch liquor can be used as nitrogen sources. and inorganic ammonium salts or nitrates such as ammonium sulfate, sodium nitrate and ammonium chloride. Moreover, inorganic salts such as sodium chloride, potassium chloride, phosphates, and heavy metal salts can be added as necessary. In order to suppress foaming during fermentation, suitable antifoaming agents such as silicone oil can also be added according to conventional methods.

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 to 37°C, preferably 25 to 30°C. With this method, the production amount of BQlgOB reaches its maximum within 5 days of culture in both shaking culture and aeration stirring culture.

In this way an enriched culture of BQlgOB is obtained. In this culture, a part of BQlgOB exists in the culture solution, but most of it exists in the bacterial cells.

Any suitable method can be used to harvest BQlgOB from such cultures.

One method is based on the principle of extraction,
Specifically, BQlgOB in the culture solution was mixed with a water-immiscible BQ180B solvent (the BQ180
(Refer to the physicochemical properties section of B), for example, by extraction with chloroform, etc., or for BQlgOB inside the bacterial cells, collect the bacterial mass obtained by filtration, centrifugation, etc., and treat it with methanol, ethanol, acetone, etc. There are methods.

The culture itself can also be subjected 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 BQlgOB from cultures is based on the principle of adsorption, and is obtained by extracting BQ180B-containing substances that are already in liquid form, such as culture furnace liquid, or by performing the extraction operation as described above. A suitable adsorbent, such as silica gel,
The target BQlgOB is adsorbed using activated carbon, Diaion HP-20J (manufactured by Mitsubishi Chemical Corporation), etc., and then eluted with an appropriate solvent to obtain BQlgO.
It is possible to get B. BQ1 obtained in this way
By concentrating the 80B solution to dryness under reduced pressure, a crude sample of BQ180B can be obtained.

In order to further purify the crude sample of BQlgOB 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, high-performance liquid chromatography and countercurrent distribution using adsorbents such as silica gel, column chromatography rYMC back using gel filtration agents such as Sephadex LH-20J (manufactured by Pharmacia) (manufactured by Yamamura Kagaku Co., Ltd.), etc. Laws may be implemented in appropriate combinations. Specifically, for example, a rough sample of BQ180B is
It was applied to an OJ column and chloroform-methanol (1:
1) Elute the active fraction with the mixture and concentrate to dryness to obtain BQ.
A pure product of lgOB is obtained.

Uses of BQlgOB Compound BQ180B according to the present invention is useful in that it has antitumor activity.

Bioactive BQlgOB showed cytostatic activity against tumor cells. For example, 5 x 10 mouse B16 melanoma cells
BQl at various concentrations was suspended in RPM11640 medium containing 10% heat-inactivated calf serum at a concentration of 4 cells/ml.
The 50% growth inhibitory concentration (IC5o value) after culturing with gOB at 37° C. for 2 days was 6.4 μg/ral.

BQlgOB also showed a survival effect on mice inoculated with tumor cells. For example, 1.5.9 BQlgOB was administered intraperitoneally to a BDF1 mouse inoculated intraperitoneally with 8 x 10" mouse B16 melanoma cells.
When administered at a dose of 1.5 g/Kg, survival was prolonged by 127% compared to the control group.

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

Antitumor Agent It was thus revealed that BQlgOB 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, in the case of animals, injection methods such as intraperitoneal injection, subcutaneous injection, intravascular injection into a vein or artery, and local administration are used, and in the case of humans, intravascular injection or injection into a vein or artery. Methods include local administration.

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 appropriate dosage and frequency of administration under certain conditions must be determined by a specialist's testing to determine the appropriate dosage based on the above guidelines. . Specifically, it is about 0.1 to 1 g per day for an adult.

EXPERIMENTAL EXAMPLE Preparation of Seed Mother The medium used was prepared by dissolving the following components in 1 liter of water and adjusting the pH to 7.2.

Glucose malt extract Yeast extract g 0 g g The above medium 1001 was dispensed into a 500 ml Erlenmeyer flask with warts, and after sterilization, Streptomyces fluvicimus strain BQ180 (FERM BP-1934) was seeded with one platinum loop through a slant, This was cultured with shaking at 27°C for 3 days and used as a seed mother.

The culture medium used for the culture was prepared by dissolving the following components in 1 liter of water and adjusting the pH to 7.0.

Glucose 25 g Soy flour 15 g Dried yeast 2 g Calcium carbonate 4 g Dispense 25 liters of the above medium into 50 liter jar fermenters and add 300 ml of the above seed mother to the sterilized ones.
was added and heated at 27°C for 5 days, 20Orpm, 0.4
VVM was cultured with aeration and agitation.

Collection of BQ180B After culturing under the above conditions, the culture solution (50 liters) was filtered, the bacterial cells were extracted with 25 liters of acetone, and the extract was diluted with 50 liters of acetone.
After concentrating to a liter, equal amounts of chloroform-methanol (
Extract with a 10:1) mixture.

Combine this extract with the previous acetone extraction residue extracted with 25 liters of chloroform-methanol (10:1) mixture, wash with 15 liters of water, and add twice the amount of hexane to this. . The resulting precipitate was dissolved in 2 liters of chloroform to remove insoluble materials, and then adsorbed onto a column (5cIIφX50cm) of silica gel (Wako Pure Chemical Industries, Ltd. "Wakogel C-200j") and washed with chloroform-ethanol (100:1). After that, elution is performed with chloroform-ethanol (50:1).The active fraction is concentrated to dryness to obtain a crude powder of BQ180B.

This coarse powder was transferred to a silica gel column (3c+++φ
x20cm) and chloroform-methanol (
50:1). The active fraction is concentrated to dryness and washed four times with 10 Ifil of methanol. The residue is dissolved in a small amount of chloroform-methanol (10:1) mixture and concentrated to dryness to obtain pure BQ180B.

[Brief explanation of the drawing]

FIG. 1 is a reproduction of the ultraviolet absorption spectrum of BQ180B in methanol. FIG. 2 is a reproduction of the infrared absorption spectrum of BQ180B obtained by the KBr disk method. Figure 3 shows the concentration of BQ180B in deuterated chloroform.
00. This is a copy of the megahertz proton nuclear magnetic resonance spectrum. FIG. 4 is a reproduction of the 125 MHz carbon-13 nuclear magnetic resonance spectrum of BQ180B in deuterated dimethyl sulfoxide. Applicant's agent Mr. Sato

Claims (1)

[Claims] 1. Compound BQ180B represented by the following formula (I). ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (I) 2. An antitumor agent whose active ingredient is the compound BQ180B shown by the following formula (I). ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (I) 3. A strain belonging to the genus Streptomyces and capable of producing BQ180B is cultured aerobically in an appropriate medium, and the compound BQ180B is obtained from the culture. A manufacturing method of BQ180B represented by the following formula (I), which is characterized by: ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I)