JPH01265893A - Novel substance k3619, use and production thereof - Google Patents

Abstract

PURPOSE:To obtain a substance having antitumor action, by aerobically cultivating a strain belonging to the genus Streptomyces, capable of producing K3619. CONSTITUTION:Strain K3619 belonging to the genus Streptomyces having mycologic properties of shape: cylindrical egg-shaped and size of 0.6-0.8X0.8-1.0mum; growth: poor in glucose medium; hydrolysis of starch: negative by 20-30 deg.C culture; using D-glucose and not assimilating D-fructose; etc. is obtained by separation operation of soil collected in Brazil. Then the strain K3619 is cultivated by aerated culture in a medium containing glucose, yeast essence, NaCl, etc. at 20-37 deg.C for about 5 days to give a culture mixture. Then the culture mixture is extracted and purified to recover a substance, a compound shown by the formula. Optionally an antitumor agent comprising the compound as an active ingredient is prepared.

Description

[Detailed description of the invention] Background of the invention The present invention relates to a new substance, more specifically to Streptomyces spp. [36]9 relates to a new substance with antitumor properties.

Many 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 The present invention meets the above needs.

That is, the novel substance K 3 6 1 9 according to the present invention is represented by the following general 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 K 3 6 1 9 represented by the following formula (1) as an active ingredient.

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

That is, K 3 represented by the following formula (I) according to the present invention
The manufacturing method of 6 1 9 belongs to the genus Streptomyces, and K
This method is characterized by culturing a bacterial strain capable of producing 3619 aerobically in an appropriate medium and obtaining 361Cl from the culture.

Novel substance K 3 6 1 9 ]) Chemical structure The new substance 3619 according to the present invention has the above formula (I'
) has the chemical structure shown.

It has the chemical structure shown by (I).

The chemical structure of K3619 was determined as described above by examining in detail the proton nuclear magnetic resonance spectrum, carbon-13 nuclear magnetic resonance spectrum, ultraviolet absorption spectrum, optical external absorption spectrum, and mass spectrometry spectrum.

2) Physicochemical properties (1) Appearance White powder (2) Specific rotation +113 degrees (in chloroform) (cO.5) (3) Solubility Soluble in methanol, ethanol, acetone, ethyl acetate, chloroform, in water Hardly soluble.

(4) Rf value (manufactured by Merck & Co., Ltd. [Silica gel 60F2
54 J) Chloroform-methanol (50:1) 0. 4
5(5) FD-MS spectrum (m/z) 103
5(M+1)” (6) Ultraviolet absorption spectrum Fig. 1 λ (ε, sh) 225 nm (15000) ax (in methanol) (7) Infrared absorption spectrum Fig. 2 (KBr disk method) (8) Proton nuclear magnetism Resonance spectrum Figure 3 (500
(125 megahertz, in deuterochloroform) (9) Carbon-13 nuclear magnetic resonance spectrum Figure 4 (125 mech, in deuterochloroform) (10) Melting point 205°C (]1) Elemental analysis analysis value (%) C H O N58, 1
1 7.88 23.00 10.77 Calculated value C H O N58,
01 7. 98 23. 18 10.
82 (12) Molecular weight (calculated value) 1035.2K
Summary of the production of 3619 Currently, the compound 3619 can only be obtained by culturing microorganisms, but it may also be produced by synthetic chemical modification of related compounds or by total synthetic chemical modification. .

In the case of culturing microorganisms, strains belonging to the genus Streptomyces and having a production ability of 36]9 are used. Specifically, the present inventors have revealed that Streptomyces isolated by the present inventors produces 3619, a strain of 36]9; It is possible to separate suitable things from nature by the conventional method of separation. Furthermore, there is still room to increase the production ability of K 3619 by subjecting Streptomyces strains, including the 3619 strain, to irradiation or other mutational treatments.

Furthermore, now that genetic engineering has developed, K3619
It is also possible to produce K 3619 by genetic engineering techniques using the K 3619 producing gene of the strain.

The 361.9 strain found by the present inventors as a strain of the genus Streptomyces that has the ability to produce 3619 is as follows.

1) Origin and deposit number: Strain 3619 was isolated from soil collected in Brazil, and was deposited with the Institute of Microbiology, Agency of Industrial Science and Technology on March 5, 1985, and designated as the “Feikoken Bacteria Deposit”. No. 9923 J (FERM P-9923).

2) Mycological properties The mycological properties of the 3619 strain are as follows.

(1) Regarding the morphology, strain 3619 grew slowly and poorly on the medium used for observation;
Grows moderately on malt agar.

Aerial mycelia adhere well on starch inorganic salt agar medium and yeast/malt agar medium, but do not adhere or adhere poorly on other media. Aerial hyphae produced from basal hyphae are curved and elongate with simple branches, forming chains of 10 to 50 spores. Spores are curved, often hook-shaped or loop-shaped, but do not form spirals. In addition, the shape of the spores is cylindrical or oval, and the size is 0.66 to 0.8
μm×018 to 1.0 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 K3619 strain on various media at 27° C. for 4 weeks are shown in Table 1.

(3) Physiological properties K The physiological properties of strain K3619 are as shown in Table 2.

(4) Carbon source utilization The carbon source utilization of strain 3619 (on Pridham-Gotlieb 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, LL-diaminopimelic acid was detected.

From the above mycological properties, strain K3619 was determined to belong to the genus Streptomyces, and has the following characteristics.

(1) The spore chain has a curved shape and the surface of the spore is smooth.

(2) Aerial mycelium is white to yellowish white to pale yellow or bright olive gray.

(3) The color of the back side is light yellowish brown to yellow.

(4) Melanin-like pigments or other soluble pigments are not produced.

Based on the above properties, IS'P (International Society of Streptomyces Project) describes (E, B).

Shirling and D, GoLtlieb:
InL, J, 5yst, Bact, 1869-189
．． 279-392 (1968), Su 391-512
(1969), Competition 285-394 (1972)) and Bersey's Manual of Bacteriology (
BergeY″s Manual of Determi
native Bacteriology) 8th edition (1
974) and searched for related known bacterial species, Streptomyces citreus (Streptomyces citreus) was found.
citreus ) (Int, J, 5yst, Bac
t, 1941B (1969)) is the closest approximation.

Therefore, when strain K 3619 and Streptomyces citreus are compared, they match in the morphological characteristics of forming spores with curved spore chains and smooth surfaces, as well as the color tone of aerial hyphae. The difference is that K 361
The 9 strains generally have poor growth compared to Streptomyces citreus, and their sugar utilization is particularly poor on Prudham-Gotlieb agar, and they do not utilize arabinose, mannitol, or fructose. You can get points.

As mentioned above, although there are some differences, the 3619 strain closely matches those of Streptomyces citreus in terms of its basic characteristics. 9 strains were Streptomyces citreus (StrepLorrlyces c
itreus) K3619.

Table 2 Table 3 Table 10: Utilized -: Not utilized Cultivation/Produced compound K of 3619 3619 is K belonging to the genus Streptomyces.
It can be produced by culturing 3619-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 utilized by the K3619-producing bacteria. Specifically, for example, glucose, maltose, starch, 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, as well as ammonium salts or Inorganic nitrates such as ammonium sulfate, sodium nitrate and ammonium chloride can be utilized. Also, if necessary,
Inorganic salts such as sodium chloride, potassium chloride, phosphates, heavy metal salts, etc. can be added. In order to suppress foaming during fermentation, suitable antifoaming agents, such as silicone oil, can also be added as usual.

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. In this method, the production amount of K 3619 reaches its maximum after 5 days of culture in both shaking culture and aeration stirring culture.

In this way an enriched culture of K 361.9 is obtained. In the culture, a part of K3619 exists in the bacterial cells, but most of it exists in the culture filtrate.

Any convenient method can be used to harvest K 3619 from such cultures. One method is based on the principle of extraction, and specifically, K 3619 in the culture filtrate is extracted with a suitable water-immiscible solvent for K 3619, such as ethyl acetate. and so on. 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 K 3'619 from cultures is based on the principle of adsorption, in which the 3619-containing material is already in liquid form, such as culture filtrate, or subjected to the extraction procedure as described above. A suitable adsorbent, such as silica gel,
K 3619 of 1 was adsorbed using activated carbon, such as ``Diaion HP-20J (manufactured by Mitsubishi Kasei Corporation)'', and then 361 was eluted with an appropriate solvent.
It is possible to get a 9. K3 obtained in this way
6: If the l-9 solution is concentrated to dryness under reduced pressure, K 361.
9 You can get a rough sample.

In order to further purify the crude sample of K 3619 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, chromatography using adsorbents such as silica gel, gel filtration agents such as Sephadex LH-20J (manufactured by Pharmacia), rYMC Pack (
High performance liquid chromatography (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, K 3619 crude sample was dissolved in a small amount of methanol, [Sephadex LH-2OJ
The active fraction is eluted with methanol from the column and concentrated to dryness to obtain pure K 3619.

Uses of K 3619/Antineoplastic Agent The compound K 3619 according to the present invention is useful in that it has anti-+1ff tumor activity.

Biological activity]) Cytostatic activity Mouse B 16 melanoma to 5X 1'O' solids/ml] RPM116 containing 0% heat-inactivated calf serum
The IC5o value after suspension in 40 medium and incubation with various concentrations of K 361.9 at 37°C for 20 minutes was 0.1.
μz/ml.

2) Antitumor activity K3619 was found to have antitumor activity against mouse P388 leukemia. Specifically, a suspension of P388 leukemia cells (1 x 10 cells/mouse) was intraperitoneally transplanted into CDFI mice, and administered 1.5 days after transplantation. ．． The effects were shown below in terms of life extension rate (%) based on OO.

Sample Dose Survival days Life extension rate (mg
/kg) (Mean=!=SD) (%)K36
19 211.7±1.03123Control
9.5±0.53100 As mentioned above, it was revealed that 3619 of the present invention exhibits antitumor activity. Therefore, K 3619 of the present invention can be used as an antitumor agent.

Anti-tumor agent When using this substance as an anti-tumor agent, the dosage varies depending on the type of cancer and the severity of the patient's symptoms, and there is no particular restriction, but it is usually about 10-100 mg per mouth for adults. IL is administered orally or parenterally (for example, by intravenous injection) about 11 times.

Examples of dosage forms include powders, fine granules, granules, tablets, capsules, and injections. When formulating the formulation, it can be produced by a conventional method using a conventional pharmaceutical carrier. The acute toxicity of this substance to mice when administered intraperitoneally is 2 mg/kg or more and 20 mg/kg or less.

Experimental example In the following, "%" means "rw/v%".

1) 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 Malt extract 10g Yeast extract 4g Dispense 100ml of the above medium into a 500ml wart τj Erlenmeyer flask, sterilize it, and Streptomyces K361.
One platinum loop of 9 strains was inoculated from a slant and cultured with shaking at 27°C for 3 days, which was used as a seed mother.

2) The culture medium used was prepared by dissolving the following components in 1 liter of water. The pH was adjusted to 7.2.

Glucose 25. 0g soy flour
15.0g Yeast extract 2.0g Calcium carbonate 4.0g 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, 0.4V
VM was cultured with aeration and agitation.

3) Collection of K3619 After culturing under the above conditions, the culture solution was filtered to obtain a filtrate. After extracting this with 25 liters of ethyl acetate and concentrating,
It was adsorbed on a column (4c + nφ x 20 cm) of silica gel (Wako Gel C200J manufactured by Wako Pure Chemical Industries, Ltd.), washed with chloroform, and then chloroform-methanol (50: 1).
Elutes with The eluate was concentrated to dryness and K3619 crude sample 2
00mg was obtained. Dissolve 200 μm of this crude sample in a small amount of methanol and use it on a Sephadex LH-20 column (4
0 cm) and developed with methanol.

Concentrate and dry K 3619 to obtain K 3619 purified product 1
00 mg was obtained.

[Brief Description of the Drawings] Figure 1 is a reproduction of the ultraviolet absorption spectrum of Ni 3619 in methanol. FIG. 2 is a reproduction of the infrared absorption spectrum of K3619 obtained by the KBr disk method. Figure 3 shows K 3619 at 50% in deuterated chloroform.
This is a copy of the 0 MHz proton nuclear magnetic resonance spectrum. Figure 4 shows 125 of 3619 in deuterated chloroform.
This is a reproduction of a megahertz carbon-13 nuclear magnetic resonance spectrum. Applicant's agent Sato -Ofu Kaihei 1- Zbib893 (9) Soto

Claims (1)

[Claims] 1. A novel substance K3619 represented by the following formula (I). ▲There are mathematical formulas, chemical formulas, tables, etc.▼ 2. An antitumor agent whose active ingredient is compound K3619 represented 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 K3619 is cultured aerobically in an appropriate medium, and the culture is expressed by the following formula (I). A method for producing K3619, characterized in that K3619 is obtained. ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I)