JPH0279987A - Novel alo81, its use and production thereof - Google Patents

Abstract

NEW MATERIAL:A compound ALO81 having the following physicochemical properties; appearance: white powder; melting point: undefined, beginning changing gradually at 180 deg.C, becoming brown at 185-190 deg.C and decomposing; specific rotation [alpha] =+195 deg. (C=0.5, in methanol); solubility: readily soluble to acetone, chloroform, methanol, ethanol and ethyl acetate, insoluble to water; etc. USE:Antineoplastic agents. PREPARATION:ALO81 matter-productive bacteria classified as Streptomyces [e.g., Streptomyces gannmycicus ALO81 (FERM BP-1998)] is put to culture pref. by an aerobic liquid culture method at 20-30 deg.C for 5 days.

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 AAL081, which has antitumor properties.

PRIOR ART A large number of antitumor substances have been reported, some of which have been put into practical use as anticancer agents. 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 new substance AAL081 according to the present invention has the following (1
) to (8).

<1) Appearance: White powder (2) Melting point: Does not show a clear melting point, changes gradually from 180°C, and browns and decomposes at 185-190°C.

(4) Ultraviolet absorption spectrum: In methanol (Figure 1 30)
9 nm (1500).

(5) Infrared absorption spectrum: The spectrum shown in FIG. 2 is obtained by the KBr disk method.

(6) Proton nuclear magnetic resonance spectrum: The spectrum shown in FIG. 3 is obtained.

(7) Solubility: acetone, chloroform, methanol,
Easily soluble in ethanol and ethyl acetate, insoluble in water.

(8) Rf value: R when developed with chloroform-methanol (10:1) using silica gel thin layer chromatography (Kieselgel 60F254 manufactured by Merck)
The f value is 0.36.

The invention also relates to the use of this substance.

That is, the antitumor agent according to the present invention has the above-mentioned (1) to (8).
) The active ingredient is the compound AAL081, which has the physical and chemical properties of

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

That is, the method for producing compound AAL081 according to the present invention is characterized by culturing AAL081 substance-producing bacteria belonging to the genus Streptomyces and isolating compound AAL081 from the culture solution.

[Detailed description of the invention]

New substance AAL081 AAL081, which is a new substance according to the present invention, is the above-mentioned (
It has the physical and chemical properties of 1) to (8).

Comparing the above physicochemical properties with those of known antibiotics, the ultraviolet absorption spectrum revealed that Compound A according to the present invention
AL081 is hitatimycin (Hlt-achlIIy
cln:S, Omura et al., Tetrahedron
1etters.

23, p. 4713, 1982), Virldenomycin: T, Ilaseg
ava et al., J.

Antiblotlcs, volume 28, page 167,
5acrocyc11c Iaet (1975) etc.
All! This substance is similar to antibiotics, but its properties are different from both, and it was confirmed that this substance is a new substance.

The FD mass spectrum and elemental analysis values of the compound AAL081 according to the present invention are as follows.

FD? Soot vector: m/z555 (M) Elemental analysis: C-73.5, H-6.7, (Analysis value (%)) O-1
7,3,N-2,5 Overview of production of AAL081 Compound AAL081 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. You could do that too. Alternatively, genetic engineering techniques may be used. That is, it is also possible to integrate the compound AAL081-producing gene into a suitable microorganism, culture such a transformed microorganism, and obtain it from this culture.

When culturing a microorganism, for example, a strain belonging to the genus Streptomyces having the ability to produce AAL081 is used. Specifically, the Streptomyces φ gammichicus AL081 strain isolated by the present inventors is AAL08
Although the present inventors have shown that antibiotic-producing strains produce 1, it is possible to isolate suitable strains from nature using conventional methods for isolating antibiotic-producing bacteria. Also, Streptomyces ganmysicus A
There is also room to increase AAL081 production ability by subjecting AAL081 producing bacteria, including the L081 strain, to radiation irradiation or other mutation treatments. As mentioned above, genetic engineering techniques are also possible.

AI, 081 strain The Al2S3 strain, which the present inventors have discovered as a Streptomyces strain capable of producing AAL081, has the following content.

■) Origin and deposit number The Al2S3 strain was isolated from soil collected in Shimogasaki City, Kanagawa Prefecture, and was deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology on August 10, 1986. Joyori 1st
No. 998) (FERMBP-1998).

2) Mycological properties The mycological properties of the Al2S3 strain are as follows.

(1) Morphology The Al2S3 strain grows relatively quickly on the medium used for observation.
Grows well on glycerol-asparagine agar and tyrosine agar. Aerial hyphae adhere well on glycerol-asparagine agar, tyrosine agar, and yeast-malt agar, but poorly on sucrose-nitrate agar, nutrient agar, and oatmeal agar. Aerial hyphae produced from shopkeeper hyphae form simple branches and elongate, forming chains of about 10 to 50 spores. The spore chain is spiral, and the spores are barrel-shaped or oval-shaped, and the size is 0.5-0.7 μm x 0.9-1.3 μm. Its surface is thorn-like, but short thorn-like parts can also be seen in some areas.

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

(2) Growth status on various media The results of culturing the Al2S3 strain on various media at 27°C for 3 weeks are shown in Table 1.

(3) Physiological properties The physiological properties of the Al2S3 strain are as shown in Table 2.

(4) Carbon source utilization The carbon source utilization (on Bridham-Gotlieb agar medium) of the Al2S3 strain 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, LL-diaminopimelic acid was detected.

From the above mycological properties, the ALD 81 strain was determined to belong to the genus Streptomyces, and has the following characteristics.

(1) The spore chain is mainly spiral-shaped, and the surface of the spore is thorn-like, but short thorn-like structures are also observed in some parts.

(ii) The color of aerial mycelium is yellowish white to light olive gray to light brown, and the color of the underside does not show any characteristic color.

(Ill) Tyrosine agar medium, peptone yeast iron agar medium, and tryptone yeast liquid medium all produce melanin-like pigments, but no soluble pigments are produced.

(iv) The upper limit temperature for growth is relatively high; it grows up to 45°C, but does not grow at 55°C.

Based on the above properties, ISP (International Streptomyces Project) description (E, B, Shirl
ing and D, Gottllcb: Int, J;
5yst, Bact,, 1889-189.279-
392 (1988): 19391-512 (1989
): 22265-394 (1972)) and Bergey's Manual of Determinative Bacteriology.
eterminative Bacterio-10g
y) When searching for related known bacterial species using the 8th edition (1974) as a reference, Streptomyces ganmysicus (Streptomyces
tomyces gannmycicus) (Int.
, J, 5yst.

aact, 22.300 (1972)) is the most similar.

Therefore, when comparing the Al0S1 strain and Streptomyces gammysicus, we found that the spores have spiral spore chains and the surface of the spores is thorn-like. The two closely match in terms of the production of similar pigments, the color tone of aerial mycelia, and the ability to assimilate sugars. Differences include that the Al0S1 strain cannot assimilate sucrose on Bridham-Gotlieb agar medium and can grow even at 45°C.

As described above, although there are some differences, the Al2S3 strain closely matches those of Streptomyces gammichicus in terms of basic properties, and therefore it is appropriate to identify it as Streptomyces gammichicus. Therefore, Al2S
Three strains of Streptomyces ganmysicas (Streptomyces
omyces gann+1yctcus)AAL08
Name it 1.

Table 1 Table +: Utilization - Dual non-utilization culture/Produced compound of AAL081 AAL081 is AAL081, which belongs to the genus Streptomyces.
It can be produced by culturing AL081-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 AAL081-producing bacteria. Specifically, for example, glucose, sucrose, maltose, starch, and fats and oils 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; Inorganic ammonium salts or nitrates such as ammonium sulfate, sodium nitrate and ammonium chloride can be utilized. Also,
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 20-45°C, preferably 25-30°C. With this method, the production amount of AAL081 reaches its maximum after 5 days of culture in both shaking culture and aeration stirring culture.

In this way an enriched culture of AAL081 is obtained. In culture, AAL081 is mostly present in bacterial cells.

Any convenient method can be used to harvest AAL081 from such cultures.

One method is based on the principle of extraction,
Specifically, AAL081 in bacterial cells can be extracted using a suitable AAL081 solvent (see above), such as acetone. 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 AAL081 from cultures is based on the principle of adsorption, in which AAL081-containing substances that are already in liquid form, such as culture furnace liquid, or obtained by performing the extraction operation as described above, are available. A suitable adsorbent, such as silica gel,
The target AAL081 is adsorbed using activated carbon, Diaion HP-20J (manufactured by Mitsubishi Chemical Corporation), etc., and then eluted with an appropriate solvent to obtain AAL08.
1 can be obtained. AAL obtained in this way
The AAL081 crude sample is obtained by concentrating the 081 solution to dryness under reduced pressure.

In order to further purify the crude sample of AAL081 obtained in this way, the extraction method and adsorption method described above may be combined with a gel method, high performance liquid chromatography, etc. as necessary, and the procedure may be performed as many times as necessary.

For example, adsorbent such as silica gel, column chromatography rMMC pack using gel or filter 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 AAL081 is dissolved in a small amount of methanol and subjected to semi-preparative high performance liquid chromatography (rYMC Pack D-ODS manufactured by Yamamura Kagaku Co., Ltd.).
-7J) and developed with methanol-water (8:2).

When the active peak is collected and lyophilized, AAL0
81 pure products are obtained.

Uses of AAL081 Compound AAL081 according to the present invention is useful in that it has antitumor activity.

Biologically active anticancer activity AAL081 has no inhibitory effect on murine P388 leukemia.
lvo anticancer activity was observed. Specifically, a suspension of P388 leukemia cells (lx106 cells/mouse) was intraperitoneally transplanted into CDF1 mice, AAL081 was administered 10 days after transplantation, and the survival days of the control group mice that were administered physiological saline were calculated. The effects were expressed in terms of life extension rate (%) set at 100, and were as follows.

AL081 511,3±1.37 123 Control 9.2±0.42 100 Antitumor agent Thus, it is clear that AAL081 of the present invention exhibits antitumor properties against animal tumors, particularly malignant tumors. was made into

Therefore, the compounds of the present invention can be used as antitumor agents 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 compound of the present invention as an antitumor agent, one typical method is to dissolve the compound in distilled water for injection or physiological saline and inject it. Specifically, in the case of animals, methods such as intraperitoneal injection, subcutaneous injection, intravascular injection into a vein or artery, and local administration by injection, and in humans, intravascular injection or injection into a vein or artery. There are methods such as 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, it goes without saying that the appropriate amount and frequency of administration under certain conditions will vary depending on age, weight, sex, sensitivity, diet, administration time, concomitant drugs, patient and degree of disease. The appropriate dosage must be determined through a test conducted by a specialist based on the above guidelines. Specifically, it is about 0.1 to 0.5 g per day for adults.

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 4g Malto extract 10g Yeast extract 4g The above medium 1001 was dispensed into a 5001 Erlenmeyer flask with warts, and after sterilization, one platinum loop of Streptomyces ganmysicus strain AAL081 (FERM BP-1998) was inoculated from 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 7.2:A.

Glucose 25.0g Soybean flour 15.0g Yeast extract 2.0g Calcium carbonate 4.0g 25 liters of the above medium was dispensed into 50 liter jar fermenters and sterilized, the above seed mother 3001 was added and the mixture was heated at 27°C. for 5 days, 20 Orpm, 0.4V
VM was cultured with aeration and stirring.

Collection of HachiL081 After culturing under the above conditions, 50 liters of the culture solution was filtered to obtain bacterial cells. This was extracted with 25 liters of acetone, concentrated, and then extracted with 5 liters of ethyl acetate, and the solvent layer was concentrated. The concentrate was dissolved in chloroform to remove insoluble materials to obtain 20 g of AAL081 crude sample. The crude sample thus obtained was subjected to semi-preparative high performance liquid chromatography (rYMC pack D-ODS-7J, manufactured by Yamamura Kagaku Co., Ltd., 2 cmφx
30 am) and methanol-water (8:2)
, and was developed at a flow rate of 2017 m1n. A peak with a retention time of 11.7 minutes is collected, concentrated, and then freeze-dried. By performing this operation 5 times, 110ff of AAL081 refined product can be obtained.
Get 1.

[Brief explanation of the drawing]

FIG. 1 is a reproduction of the ultraviolet absorption spectrum of AAL081 in methanol. FIG. 2 is a reproduction of the infrared absorption spectrum of AAL081 obtained by the KBr disk method. Figure 3 shows the concentration of AAL081 at 50% in deuterated chloroform.
This is a copy of the 0 MHz proton nuclear magnetic resonance spectrum. Applicant's representative: Display of the Sato-Yu case (1) [(Analysis value...2゜5) on page 6, line 8 of the specification
'', add the following content on a new line. "Also, this compound has a 500% concentration in deuterated chloroform.
The megahertz two-dimensional proton-proton nuclear magnetic resonance spectrum (two-dimensional correlation spectrum) is as shown in FIG. From these physical and chemical properties, the compound AAL according to the present invention
081 has a chemical structure represented by the following formula (1). ``Subject of number of claims to be amended by amendment''. (2) On the last line of page 22 of the same book, after the line ``This is a copy of the original.'', there is a line break and ``Figure 4 shows the 500 MHz two-dimensional proton-proton magnetic resonance spectrum (two-dimensional correlation spectrum) in deuterium chloroform. ) is added. (3) Figure 4 is additionally attached as attached.

Claims (1)

[Claims] 1. Compound AL081 with the following physical and chemical properties (1) Appearance: White powder (2) Melting point: Does not show a clear melting point, gradually changes from 180°C and browns and decomposes at 185-190°C do. (3) Specific rotation: [α]^2^5_D=+195°(C
= 0.5 in methanol) (4) Ultraviolet absorption spectrum: The spectrum shown in Figure 1 was obtained in methanol, and λ_m_a_x(E^1^%_
1_cm)=309 nm (1500). (5) Infrared absorption spectrum: The spectrum shown in FIG. 2 is obtained by the KBr disk method. (6) Proton nuclear magnetic resonance spectrum: The spectrum shown in Figure 3 is obtained in deuterated chloroform at 500 MHz. (7) Solubility: acetone, chloroform, methanol,
Easily soluble in ethanol and ethyl acetate, insoluble in water. (8) Rf value: Rf when developed with chloroform-methanol (10:1) using silica gel thin layer chromatography (Kieselgel 60F254 manufactured by Merck)
The value is 0.36. 2. Compound AL having the physicochemical properties according to claim 1
An antitumor agent containing 081 as an active ingredient. 3. A method for producing the compound AL081 having the physical and chemical properties according to claim 1, which comprises culturing an AL081 substance-producing bacterium belonging to the genus Streptomyces and isolating the compound AL081 from the culture solution.