JPH01290673A - Novel substance k3543r1, its use and production - Google Patents

Abstract

NEW MATERIAL: A new material K3543R1 represented by the formula or an acid adduct thereof.

USE: An antitumoral.

PREPARATION: A strain belonging to the genus Streptomyces and having the ability to produce K3543R1, Streptomyces. tanasiensis K3543 strain (FERM-P 9922) is aerobically cultured in a suitable medium (e.g. glucose, malt extract or yeast extract), and K3543R1 is obtained from the culture.

COPYRIGHT: (C)1989,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION [Background of the Invention] The present invention relates to a novel substance, and more particularly to 3543R1, a novel substance having antitumor properties produced by the 3543 strain belonging to Strebtomyces.

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.

Zunai) Among the new substances according to the present invention, 3543R1 is
It is represented by the following general formula (I).

The invention also relates to this acid addition salt.

The invention also relates to the use of this substance.

That is, the antitumor agent according to the present invention contains 3543R1 or an acid addition salt thereof as an H-active ingredient in the compound represented by the following formula (I).

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

That is, according to the present invention, as shown by the following formula (I), 354
The production method for 3R1 is based on K35, which belongs to the genus Streptomyces.
This method is characterized in that a strain exhibiting the ability to produce 43R1 is aerobically cultured in an appropriate medium, and 3543R1 is obtained from the culture.

[Detailed description of the invention]

3543R1 as a new substance 1) Chemical structure 3543R1 as a new substance according to the present invention has the above formula (I
) is the chemical structure shown by H. These chemical structures are
It was determined as follows.

Proton nuclear magnetic resonance spectrum of K3543R1 (3rd
Figure 4) and carbon-13 nuclear magnetic resonance spectra (Figure 4) of lactoquinomycin
in) (N, Tanaka et
al,, J, Ant4bioties3B,
1327-1338 (I985)). From a more detailed examination of the spectrum and the molecular formula derived from the mass spectrum and elemental analysis, 354
The structure of 3R1 was determined as shown in Figure (I).

2) Physicochemical properties (I) Appearance Red powder (2) Melting point 105-110℃ (decomposition) (3) Solubility
Soluble in methanol, ethanol, acetone, ethyl acetate, chloroform, sparingly soluble in hexane, insoluble in water.

(4) Rf value (manufactured by Merck [Silica gel 60F254]
Use) Chloroform-methanol (I0: 1) 0. 25
Chloroform-methanol-concentrated ammonia water (20+2
:0.1) 0.56(5)FD7
Soot vector (m/z) 414 (M+H)” is shown in FIG.

264 (310), 323 (I01), 425
(I06), 494 (I26) (in methanol) 26
8 (294), 320 (I11), 424 (9
3), 498 (I22) (0.01N hydrochloric acid in methanol) 268 (308), 330 (I07), 531
(I22) (0.01N sodium hydroxide in methanol) (7) Infrared absorption spectrum (KBr disk method) shown in FIG.

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

(9) Carbon-13 nuclear magnetic resonance spectrum (I25 MHz, in deuterated chloroform) is shown in FIG.

The compound according to the present invention has a basic amino group in the molecule.
Therefore, it can exist in the form of acid addition salts. Examples of acids that can be used to form such acid addition salts include:
Inorganic acids such as hydrochloric acid, sulfuric acid, and phosphoric acid, and organic acids such as acetic acid, tartaric acid, propionic acid, citric acid, and succinic acid are included.

Overview of the production of K3543R1 The compound 3543R1 has currently been obtained only by microorganisms, but it may be produced by synthetic chemical modification of related compounds or by total synthetic chemistry.

When culturing a microorganism, a strain belonging to the genus Streptomyces and having the ability to produce 3543R1 is used. Specifically, 3543 strain Kani 3543R1 was added to Streptomyces thanashensis isolated by the present inventors.
Although the present inventors have shown that antibiotic-producing strains produce antibiotics, it is possible to isolate suitable strains from nature using conventional methods for isolating antibiotic-producing bacteria. Furthermore, there is still room to increase the K3543R1 production ability by subjecting 3543R1 producing bacteria, including the 3543 strain of Streptomyces thanashensis, to irradiation or other mutational treatments. Furthermore, now that genetic engineering has developed, 3543R1 can be produced using genetic engineering techniques that utilize the 3543R1 production gene of the K3543 strain.
It is also possible to produce

The K3543 strain found by the present inventors as a Streptomyces strain having the ability to produce 3543R1 is as follows.

1゜ Strain 3543 in terms of origin and deposit number was isolated from soil collected in the People's Republic of China, and was deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology on March 5B, 1986. No. 992
It has a number of No. 2 J (FERM P-9922).

2. Mycological properties The mycological properties of the 3543 strain are as follows.

1) Morphology: Under a microscope, strain 3543 forms straight aerial hyphae that are relatively longer than branched basal hyphae, and no whorling pattern is observed. A mature spore chain is an almost straight chain of 20 or more spores, and the spore size is 0.4 to 0.6X.
The surface of the spore is smooth, measuring 1.0 to 1.3 micrometers.

2) Growth status on various media (+) Sucrose/nitric acid agar medium Growth is moderate. Slightly young, pale yellow powdery aerial mycelium, with no soluble pigment observed.

(2) Growth on glucose-asparagine agar medium is slight. A moderate amount of pale yellow powdery aerial mycelium is observed, and no soluble pigment is observed.

(3) Glycerin-asparagine agar medium (ISP-medium 5) Growth is moderate. It has a good epiphyte of gray velvety aerial mycelium, and yellowish brown soluble pigment is observed.

(4) Growth on starch/inorganic salt agar medium (ISP-medium 4) is good. It has a good epiphyte of yellow-brown-gray velvety aerial mycelium, and dark yellow-brown soluble pigment is observed.

(5) Growth on tyrosine agar medium (ISP-medium 7) is good. It is covered with abundant brownish-gray hair-like aerial mycelium, and dark brownish-gray soluble pigment is observed.

(8) Growth on nutrient agar medium is slight. Slightly young, pale yellow powdery aerial mycelia with dark brownish-gray soluble pigments are observed.

(7) Growth on yeast malt agar medium (ISP-medium 2) is good. It grows well with gray velvety aerial mycelium, and has a night vision gray soluble pigment.

3) Physiological properties (I) Liquefaction of gelatin Glucose-peptone-gelatin medium (liquefaction of gelatin is observed at 27°C).

(2) Hydrolysis of starch Hydrolysis of starch is observed on starch/inorganic salt agar medium (cultured at 27°C).

(3) Coagulation and peptonization of skim milk (skim milk, 27℃
Culture) No obvious coagulation was observed, and peptonization was observed.

(4) Production of melanin-like pigments The production of melanin-like pigments is observed in peptone-yeast-iron agar medium and tryptone-yeast liquid medium.

(5) Utilization of carbon source (Pritonom Gotlieb agar medium 1sP-medium 9.27°C culture) D-glucose, L-arabinose, D-xylose,
It grows using galactose and salicin, but hardly uses sucrose, D-fructose, raffinose, rhamnose, inositol, and D-mannitol.

(8) It grows at a growth temperature of 10 to 37°C, and the optimum growth temperature is 27 to 30°C (yeast/malt medium).

It grows at 20-37°C, and the optimum temperature is 20-30°C (oatmeal medium).

To summarize the above characteristics, strain K3543 belongs to the genus Streptomyces, and all bacterial cells of this strain contain L-type diaminopimelic acid (LL).
-DAP) was detected, the aerial hyphae were straight and slightly curved, and the spore chains were also straight and slightly curved. No whorling is observed and the surface of the spores is smooth. Aerial mycelia are often gray in color and produce melanin-like pigments in certain media. Regarding the self-resolution ability, the coagulation of skim milk is extremely weak, but the peptonization of skim milk and the liquefaction of gelatin are moderate. D-glucose, L-arabinose, D-xylose, galactose, and salicin are used, but sucrose, D-fructose, raffinose, rhamnose, inositol, and D-manitol are hardly used.

Judging from the above properties, this strain is StrepLomyces Lanashensis.
hien-sls), the present inventors identified this strain as Streptomyces tanashensis (st.
repto-syces tanashiensis)
It was named strain K3543.

This bacterial strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, and its microbiological laboratory accession number is FEMP-9922.

Culture/K 3543R1 production compound 3543R1 is produced by culturing 3543R1-producing bacteria belonging to the genus Streptomyces aerobically in an appropriate medium,
It can be produced by collecting the target product from a culture.

The medium can contain any nutrient source that can be utilized by the K3543R1 producing bacteria. Specifically, for example, glucose, sucrose, maltose, starch, and oils can be used as carbon sources, and soybean flour, cottonseed meal, dried yeast, yeast extract, cornstarch liquor, etc. can be used as nitrogen sources. Organic and inorganic substances such as ammonium salts or nitrates such as ammonium sulfate, sodium nitrate and ammonium chloride are available.

In addition, if necessary, sodium chloride, potassium chloride,
Inorganic salts such as phosphates and heavy metal salts can be added. 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. A suitable culture temperature is 20-37°C, preferably 25-30°C. In this method, the production amount of 3543R1 reaches its maximum after 5 days of culture in both the shaking culture and aerated agitation culture.

In this way, an enriched culture of K3543R1 is obtained. In the culture, a part of K3543R1 exists in the bacterial cells, but most of it exists in the culture solution.

To collect 3543R1 from such a culture,
Any suitable method can be used.

One method is based on the principle of extraction, specifically, for K 3543 R1 in the culture solution, it is extracted with a water-immiscible solvent for 3543 R1 (see above), such as ethyl acetate. Alternatively, for 3543R1 inside the bacterial cells, there are methods of recovering bacterial cell aggregates obtained by filtration, centrifugation, etc., and treating them with methanol, ethanol, acetone, etc. 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 extracting 3543R1 from cultures is based on the principle of adsorption, in which the 3543R1-containing material, which is already in liquid form, is extracted from the culture tank liquid or by the extraction procedure as described above. 3543R1 is adsorbed on the extract obtained by using a suitable adsorbent such as silica gel, activated carbon, Diaion HP-20J (manufactured by Mitsubishi Kasei Corporation), and then a suitable solvent is used to adsorb 3543R1. 3543R1 can be obtained by elution with K35.If the 3543R1 solution thus obtained is concentrated to dryness under reduced pressure, K35
43R1 cheap sample is the best.

In order to further purify the crude sample of 3543R1 obtained in this way, it is necessary to combine the above-mentioned extraction method and dilution method with gel filtration method, fast liquid chromatography, etc. as many times as necessary. good. For example, an adsorbent such as silica gel, column chromatography using a gel filtration agent such as Sephadex LH-20J (manufactured by Pharmacia), rYMc bag (manufactured by Yamamura Kagaku)
High-performance liquid chromatography using, for example, and a countercurrent distribution method can be carried out in appropriate combination. Specifically, for example, a crude sample of K3543R1 was applied to a Se7adex LH-2OJ column, the active fraction was eluted with a chloroform-methanol (I:1) mixture, and when concentrated to dryness, pure 3543R1 was obtained. Goods can be obtained.

Uses of K3543R1 3543R1 is a useful compound according to the present invention in that it has anti-U tumor activity.

Regarding biological activity, 3543R1 showed cytostatic activity against Ml tumor cells. For example, mouse B16 melanoma cells were
], the IC5o value was 0.6 μg/ml after being suspended in RPMI 1640 medium containing 10% heat-inactivated calf serum and incubated with various concentrations of 3543R1 at 37°C for 2 days. It was ml.

As mentioned above, it was revealed that the drug 3543R of the present invention exhibits antitumor properties. Therefore, in the present invention, 35
43R1 can be used as an anti-tumor agent 17.

Antitumor agent Thus, 3543R1 of the present invention can be used to treat animal tumors,
It has been revealed that it exhibits antitumor properties, especially against malignant tumors.

Therefore, the compounds of the present invention can be used as anti-M1 tumor agents or tumor therapeutic agents.

The compound of the present invention as an antitumor agent 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, 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 into a vein or artery or local administration. There are injection methods such as

The dosage of the compound of the present invention is determined in consideration of the results of animal tests and the weighing conditions 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 1.5 g of free base per day for an adult.

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

Example 1) Preparation of Seed Mother The medium used was prepared by dissolving the following components in 1 liter of water and adjusting the PU to 7.2.

Glucose 0.4g Malt extract 1.0g Yeast extract 0.4g 100ml of the above medium was dispensed into a 500ml Erlenmeyer flask with warts, and after killing, 3543 strain (FERM P-9922) was added to Streptomyces thanashensis from a slant. The seed mother was inoculated with a platinum loop and cultured with shaking at 27°C for 2 days.

2) The culture medium used was prepared by dissolving the following components in 1 liter of water and adjusting the pH to 7.4.

Glycerol 3. Og fishmeal 2. OK Calcium carbonate 0.2g Dispense 25 liters of the above medium into 50 liter jar fermenters and sterilize them, add 300ml of the above seed mother
was added and heated at 27° C. for 5 days, 20 Orpm, 0.4.
VVM was cultured with aeration and agitation.

3) Collection of K3543R1 After culturing under the above conditions, the culture solution (50 liters) is filtered, and the fermentation solution is extracted with an equal volume of ethyl acetate at pH 8.5.

After reverse extraction of the extract with an equal volume of 0.01N hydrochloric acid, the aqueous layer was adjusted to pH 8.5, and an equal volume of ethyl acetate was extracted and concentrated to dryness.

After dissolving this in 100 ml of chloroform and removing insoluble matter, silica gel (Wako Pure Chemical's "Wako Gel C200"
J) column (4 cmφ x 20 cm) and elute with chloroform-methanol (20:1). The active fraction was concentrated to dryness to obtain 47 mg of red powder of 3543R1.

[Brief explanation of the drawing]

Figure 1 shows K3543R1 in methanol (solid line), 0
．． 01 N hydrochloric acid in methanol (dotted line) and 0.01
3 in normal sodium hydroxide-methanol (dashed line)
This is a reproduction of the ultraviolet absorption spectrum of 543R1. FIG. 2 is a reproduction of the infrared absorption spectrum of K3543R1 obtained by the KBr disk method. FIG. 3 is a reproduction of the 500 MHz proton nuclear magnetic resonance spectrum of K3543R1 in deuterated chloroform. FIG. 4 is a reproduction of the 125 MHz charcoal 1g13 nuclear magnetic resonance spectrum of K3543R1 in deuterated chloroform. Applicant's agent: Sae-O

Claims (1)

[Claims] 1. A novel substance K3543R1 represented by the following formula (I) or an acid addition salt thereof. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (I) 2. An antitumor agent containing the compound K3543R1 shown by the following formula (I) or its acid addition salt as an active ingredient. ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ (I) 3. A strain belonging to the genus Streptomyces and capable of producing K3543R1 is cultured aerobically in an appropriate medium, and K3543R1 is obtained from the culture. , the following formula (I
) Production method of K3543R1 shown in ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I)