JP2002069075A - New physiologically active substance nk34896b and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new compound having antitumor activity. SOLUTION: The new physiologically active substance NK34896B can be produced by culturing a microorganism belonging to the genus Streptomyces and capable of producing the physiologically active substance NK34896B (compound of formula (1)) and collecting the substance accumulated in the cultured product.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel physiologically active substance NK34896B and a method for producing the same. The compound of the present invention exhibits a cell growth inhibitory effect and is expected as an anticancer substance used as a chemotherapeutic agent for malignant tumors.

[0002]

2. Description of the Related Art Conventionally, as antitumor agents, adriamycin, mitomycin C and the like have been known.

[0003]

However, these are highly toxic and unsatisfactory. The invention of new compounds suitable for these uses is awaited.

[0004]

Means for Solving the Problems As a result of various searches for metabolites of microorganisms, the present inventors have found that one strain belonging to actinomycetes is a physiologically active substance NK34896B having a cell growth inhibitory effect on cancer cells of mammals. Was produced, and it was found that the physiologically active substance NK34896B and a pharmacologically acceptable salt thereof have a cell growth inhibitory effect on cancer cells. Furthermore, the present inventors have conducted intensive studies based on these findings, and have completed the present invention.

That is, the present invention relates to the following items (1) to (5). (1). Equation (1)

[0006]

Embedded image Or a pharmacologically acceptable salt thereof.

(2). A novel physiologically active substance NK34896B having the following physicochemical properties or a pharmacologically acceptable salt thereof. 1) Appearance: colorless liquid 2) Molecular formula: C34H58O9 (measured by high resolution mass spectrometry) 3) Molecular weight: 610 (measured by FAB-MS method ((MH) ⁻ = 609) 4) Hydrogen nuclear magnetic resonance spectrum : 400 in heavy benzene
The spectrum measured at MHz is shown in FIG. 5) Carbon nuclear magnetic resonance spectrum: 100 in heavy benzene
The spectrum measured at MHz is shown in FIG. 6) Solubility: easily soluble in methanol, acetone, ethyl acetate, chloroform and dimethyl sulfoxide. Poorly soluble in water. 7) Color reaction: Positive for phosphoric acid-molybdic acid and sulfuric acid. Negative for Ehrlich, Sakaguchi, ninhydrin, potassium permanganate. 8) Rf value of thin layer chromatography: 0.22 Using a thin layer of silica gel (silica gel 60F254, manufactured by Merck), hexane: acetone (2:
1) was used.

(3). The physiologically active substance NK34896 according to the above (1) or (2), which belongs to the genus Streptomyces.
Culturing a microorganism capable of producing B to produce and accumulate a novel physiologically active substance NK34896B in the culture;
A novel physiologically active substance NK characterized by collecting the same
Production method of 34896B. (4). A microorganism belonging to the genus Streptomyces and having the ability to produce the physiologically active substance NK34896B according to (1) or (2). (5). Streptomyces sp. NA 34896
Strain (FERM P-17984) or a mutant thereof. Hereinafter, the present invention will be described in detail, but the present invention is not limited thereto.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, a novel physiologically active substance NK34896B can be obtained by culturing those producing bacteria belonging to the genus Streptomyces, producing the compound, and collecting the compound from the culture. NK34896B
As typical examples of bacteria producing Streptomyces, for example, Streptomyces sp.
es sp. ) NA 34896 strain. The bacteriological properties of this strain are shown below.

[0010] 1. Morphological properties Observation after 2 weeks at 27 ° C. shows that the aerial hyphae are simply branched, the tip is helical, and no ring-shaped branches are formed. Neither sporangia nor zoospores are observed. The spore surface is smooth, the spores are cylindrical, size is 0.6-0.8x
1.0 to 1.2 μm. In addition, spores are formed in a chain of 10 or more.

2. Growth in various media Table 1 below shows the growth state of each medium at 27 ° C for 2 weeks.

[0012]

[Table 1] Medium Growth medium hyphae Undergrowth mycelium Soluble pigment Glucose / asparagine good moderate, brown-ash to colorless-none agar medium 5 med.) Bright brown ash light yellow Starch / inorganic salt agar medium good Abundant, brown white to black light yellow to none (ISP 4 med.) Light yellow tea Tyrosine agar medium good rich, brown white to light yellow brown (ISP 7 med.) Light brown ash to light brown nutrient agar medium good moderate, white to bright colorless to brown ash light yellow yeast / malt agar medium good rich, brown to light yellow to slightly (ISP 2 med.) Light brown ash Light yellow brown brownish oatmeal agar medium good moderate, brownish to colorless to none (ISP 3 med.)

[0013] 3. Physiological properties Optimal growth temperature: 27-37 ° C Nitrate reduction: positive Starch hydrolysis (starch / inorganic salt agar medium): positive Coagulation of skim milk: negative Peptoneization of skim milk: negative Liquefaction: Positive Melanin-like pigment formation: Negative Utilization of carbon source (on Prideham-Godrib agar medium) L-arabinose + D-xylose + D-glucose + D-fructoose + sucrose + inositol + L-rhamno -S + Raffinose + D-mannitol +

5. Diaminopimelic acid in cell wall LL-diaminopimelic acid. In summary, this strain has a cell wall of LL-diaminopimelic acid,
The type of menaquinone is mainly MK-9 (H8). Inter
National Streptomyces Project (abbreviated I
According to the method of SP), the spore-forming hypha forms
It belongs to n Spirales, has a smooth spore surface, and has a mature hypha color of Graycolor-series. No melanin-like pigment is produced, and a brown soluble pigment is exhibited in the medium.
In addition, the color of the underlying mycelium changes from light yellow to light yellow brown. L-arabinose, D-xylose, D-
Glucose, D-fructose, sucrose, inositol, L-rhamnose, D-mannitol, raffinose are used.

On the basis of the above properties, Bergey's Manual of Bergey's Manual of Random Bacteriology, edited by Al-Buffanan and N. Gibbons. Determinative Bacteriolog
y) As a result of conducting a search in accordance with the eighth edition, 1974,
The 34896 strain was found to belong to the genus Streptomyces. Therefore, this bacterium was named Streptomyces sp. NA 34896. The strain was sent to the Institute of Biotechnology and Industrial Technology,
Deposited as MP-17984.

[0016] The strain belonging to the genus Streptomyces used in the present invention is liable to change its properties, like the other strains belonging to the genus Streptomyces. For example, it can be easily mutated by an artificial mutating means using ultraviolet rays, X-rays, chemicals and the like. Regardless of the thus obtained mutant strain, any of the mutants capable of producing lysoserine and the novel physiologically active substance NK34896B can be used in the present invention.

In order to produce NK34896B according to the present invention, the above strain is first aerobically cultured in a medium containing nutrients that can be used by actinomycetes. As the nutrient source, known ones conventionally used for cultivation of actinomycetes can be used. For example, as the carbon source, glucose, glycerin,
Galactose, starch syrup, dextrin, sucrose, starch, molasses, animal and vegetable oils and the like can be used. As the nitrogen source, soybean flour, wheat, wheat germ, corn steep liquor, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, urea and the like can be used alone or in combination. In addition, if necessary, it is effective to add an inorganic salt capable of generating sodium, cobalt, chlorine, sulfuric acid, phosphoric acid, and other ions. In addition, organic and inorganic substances that assist the growth of bacteria and promote the production of a novel physiologically active substance NK34896B can be appropriately added.

As a culture method, a liquid culture method, particularly a submerged stirring culture method, is suitable. The temperature suitable for culture is 15 ~ 3
The culture is at 7 ° C, but is often performed at around 26 to 30 ° C. The production of a new physiologically active substance, NK34896B,
Although it depends on the medium and the culturing conditions, the accumulation usually reaches the maximum in 1 to 10 days in both shaking culture and tank culture. When the amount of the physiologically active substance accumulated in the culture reaches the maximum, the culture is stopped and the target substance is isolated from the culture solution.

When a novel physiologically active substance NK34896B is collected from a culture solution, it can be purified by appropriately combining ordinary separation means utilizing its properties. That is, the culture solution is separated into the filtrate and the cells by a usual filtration method. The obtained cells are suspended in lower alcohol such as methanol or acetone, and filtered to obtain an extract such as methanol. Thereafter, methanol and the like are distilled off under reduced pressure from the filtrate, and the solution is transferred to a common organic solvent that is not mixed with water, for example, ethyl acetate, butyl acetate, chloroform, etc., and the organic solvent is distilled off under reduced pressure and concentrated. Thus, an oil containing NK34896B can be obtained.

The oily substance can be further purified by known methods used for the purification of fat-soluble substances, ie silica gel or OD.
S. It can be purified by a column chromatography using a molecular sieve or a recrystallization method alone or in an appropriate combination.

NK34896 obtained as described above
The physicochemical properties of B are shown below. 1) Appearance: colorless liquid 2) Molecular formula: C34H58O9 (measured by high-resolution mass spectrometry) 3) Molecular weight: 610 (measured by FAB-MS method (M
-H) ^- = 609) 4) Hydrogen nuclear magnetic resonance spectrum: 400 in heavy benzene
The spectrum measured at MHz is shown in FIG. 5) Carbon nuclear magnetic resonance spectrum: 100 in heavy benzene
The spectrum measured at MHz is shown in FIG.

6) Solubility: easily soluble in methanol, acetone, ethyl acetate, chloroform, and dimethyl sulfoxide.
Poorly soluble in water. 7) Color reaction: Positive for phosphoric acid-molybdic acid and sulfuric acid. Negative for Ehrlich, Sakaguchi, ninhydrin, potassium permanganate. 8) Rf value of thin layer chromatography: 0.22 Using a thin layer of silica gel (silica gel 60F254, manufactured by Merck), hexane: acetone (2:
1) was used. And, due to these physicochemical properties, NK34896
The structural formula of B was assumed to be that of the formula (1).

In the present invention, the pharmacologically acceptable salt is not particularly limited as long as it is a salt which can be usually taken, but is a salt with an alkali, for example, a sodium salt, a potassium salt or the like, or a salt with an acid such as a hydrochloride. , Sulfates and phosphates are preferred.

Various methods known in the art can be applied to the preparation and administration of the composition when used as a pharmaceutical. That is, as an administration method, for example, injection, oral or rectal administration can be used. Formulation forms include, for example, injections,
It can take the form of powders, granules, tablets, suppositories or capsules and the like.

Various pharmaceutical additives used for pharmaceuticals, ie, carriers and other auxiliaries, such as stabilizers, as long as they do not adversely affect NK34896B or a pharmacologically acceptable salt thereof during preparation. Preservatives, soothing agents, emulsifiers and the like may be used as necessary. In the formulation, N
The content of K34896 or a pharmacologically acceptable salt thereof can vary widely depending on the form of the preparation and the like, and is generally 0.01 to 100% (weight), preferably 0.1 to 70% (weight). And the rest consists of additives usually used for medicine.

The dose of NK34896B or a pharmacologically acceptable salt thereof varies depending on symptoms, administration method and the like, but is about 0.01 to 800 mg per adult per day.

In the following, test examples are given, and NK34896B
The cell growth inhibitory effect on mammalian cancer cells will be described.

Test Example 1 Cell cycle arrest action Using a DMEM medium supplemented with 10% fetal bovine serum, a human osteogranuloma-derived cancer cell line MG63 was cultured at 37 ° C and 5% C
O ₂ were cultured under. The cells were transferred to serum-free DME
After culturing using the M medium for 1 day, the medium was replaced with a DMEM medium supplemented with 10% fetal bovine serum, and further adjusted to a final concentration of 1, 0.3, 0.1, 0.03 μg / ml. NK34896B was added. After 32 hours, the cells were collected, the chromosomes were stained using FITC, and FACScan was performed.
The content ratio of the cell cycle was identified by measuring the chromosome concentration using the method. The results were analyzed using software and are shown in Table 2.

[0029]

Table 2 Cell cycle arrest effect of NK34896B No treatment 10.3 0.1 0.03 (μg / ml) Content (%) G1 53.9 70.8 50.3 43.8 46.5 S 8.6 16. 5 18.2 8.7 10.5 G2 / M 37.5 12.8 31.5 47.5 43.0

As apparent from the test examples, (NK34896)
B was added at 1 μg / ml and the G1 content was reduced to 70.8%.
%), NK34896B of the present invention has the effect of stopping the cell cycle of cancer cells in the G1 phase.

[0031]

EXAMPLES Examples of the present invention will be described below, but these are merely examples, and do not limit the present invention in any way, and various modifications are possible.

Example 1 Production of NK34896B by Fermentation Method (1) Fermentation As a medium for preparing a frozen bacterium and a seed, glycerin was used.
0%, glucose 1.0%, Sunrich (manufactured by Showa Sangyo) 0.5%, peptone (manufactured by Far East) 0.5%, yeast extract (manufactured by Nippon Pharmaceutical) 0.5%, dipotassium hydrogen phosphate 0 A medium consisting of 0.05%, magnesium sulfate 0.05% and calcium carbonate 0.2% was used. The medium before sterilization was adjusted to pH 7.0 before use.

The above medium was placed in a 500 ml Erlenmeyer flask.
00 ml and sterilized at 120 ° C. for 20 minutes, and then Streptomyces sp. NA 30424 strain (FER
MP-16422) was inoculated with 1 to 2 platinum loops of the slant culture and cultured at 27 ° C. for 2 days on a rotary shaker at 200 rpm. An equal volume of a 30% glycerin aqueous solution was added to the obtained culture, and the mixture was stirred, dispensed in 5 ml portions, and frozen at -80 ° C to obtain frozen bacteria.

The frozen bacterium thus obtained was added to the same medium 100
The inoculation amount is 0.1 ml in a 500 ml Erlenmeyer flask containing 500 ml.
Immediately after thawing, the cells were inoculated so as to have a concentration of 05%, cultured for 2 days under the same conditions, and used as a seed. Further, this seed mother was added to 14 500 ml containing 100 ml of the same medium.
Inoculate the conical flask so that the inoculation volume is 1%,
Culture was performed for 2 days under the same conditions, and this was used as a seed mother.

As the production medium, glucose 1.0%,
Starch syrup 4.0%, corn steep liquor 1.0%, gluten meal 1.0%, yeast extract 0.2%, calcium carbonate 0.2%, iron sulfate 0.00011%, copper sulfate 0.0
0079%, zinc sulfate 0.00015%, manganese chloride 0.00064%, cobalt chloride hexahydrate 0.000%
Dispense 100 ml of 1% medium (pH 7.0)
The solution was autoclaved at 120 ° C. for 20 minutes. This was inoculated with 1 ml of the seed mother and cultured with shaking at 27 ° C. and 220 rpm for 6 days.

(2) Purification The obtained culture (5 L) was separated into cells and supernatant by centrifugation. After adding methanol (1.5 L) to the obtained cells and stirring for 40 minutes, a methanol extract was obtained by filtration. After further adding methanol (1.0 L) to the residue and stirring for 30 minutes, a methanol extract was obtained by filtration. After mixing the obtained methanol extract, methanol was distilled off to obtain a 1.05 L aqueous solution. The aqueous solution was extracted twice with ethyl acetate (1.1 L), and the ethyl acetate phases were combined and concentrated to obtain an oily substance (1.14 g).

This oily substance was applied to a silica gel column (Wakogel C-200, manufactured by Wako Pure Chemical Industries, 110 g) previously filled with a mixed solvent of hexane: acetone (20: 1) and developed with the same mixed solvent (440 ml). Then, increase the ratio of acetone sequentially (10: 1, 660 ml;
5: 1, 660 ml; 1: 1, 660 ml), and the fraction containing NK34896B was collected and concentrated to dryness to obtain a brown oil (85 mg). A silica gel column (BW-700, manufactured by Fuji Silysia Chemical Ltd.),
g) and eluted with the same solvent (275 ml), eluted with 275 ml of each with a mixed solvent of chloroform: methanol (50: 1, 25: 1), collected a fraction containing NK34896B, and further packed a silica gel column (hexane). BW-700, manufactured by Fuji Silysia Science Ltd., 30
g), elute with the same solvent (220 ml), elute with 220 ml each of a mixed solvent of hexane: acetone (20: 1, 10: 1, 5: 1), collect the fraction containing NK34896B, and concentrate to dryness This gave a yellow oil (30 mg).

This oily substance was separated by silica gel thin layer chromatography to obtain NK34896B as a colorless oily substance (16 mg).

[0039]

According to the present invention, there has been provided a physiologically active substance NK34896 or a pharmacologically acceptable salt thereof, which exhibits a cell growth inhibitory action. These can be used as chemotherapeutic agents for malignant tumors and the like.

[Brief description of the drawings]

FIG. 1. Measured in heavy benzene of NK34896B4
FIG. 1 shows the 00 MHz hydrogen nuclear magnetic resonance spectrum.

FIG. 2: 1 measured in heavy benzene of NK34896B
FIG. 2 shows the 00 MHz carbon nuclear magnetic resonance spectrum.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // (C12N 1/20 (C12N 1/20 A C12R 1: 465) C12R 1: 465) (C12P 17 / 18 (C12P 17/18 D C12R 1: 465) C12R 1: 465) F term (reference) 4B064 AE59 BA07 BE07 BE09 BE12 BE14 BE19 BG01 BG08 BH04 BH05 BH08 BH10 CA04 DA05 4B065 AA50X AC14 BA22 CA18 CA44 4C071 AA03 BB01 CC GG01 GG03 JJ06 KK17 LL01 4C086 AA02 AA03 AA04 CB40 NA14 ZB26

Claims (5)

[Claims] (1) Formula (1) Or a pharmacologically acceptable salt thereof. 2. A novel physiologically active substance NK34896B having the following physicochemical properties or a pharmacologically acceptable salt thereof. 1) Appearance: colorless liquid 2) Molecular formula: C34H58O9 (measured by high resolution mass spectrometry) 3) Molecular weight: 610 (measured by FAB-MS method ((MH) ⁻ = 609) 4) Hydrogen nuclear magnetic resonance spectrum : 400 in heavy benzene
The spectrum measured at MHz is shown in FIG. 5) Carbon nuclear magnetic resonance spectrum: 100 in heavy benzene
The spectrum measured at MHz is shown in FIG. 6) Solubility: easily soluble in methanol, acetone, ethyl acetate, chloroform and dimethyl sulfoxide. Poorly soluble in water. 7) Color reaction: Positive for phosphoric acid-molybdic acid and sulfuric acid. Negative for Ehrlich, Sakaguchi, ninhydrin, potassium permanganate. 8) Rf value of thin layer chromatography: 0.22 Using a thin layer of silica gel (silica gel 60F254, manufactured by Merck), hexane: acetone (2:
1) was used. 3. A microorganism belonging to the genus Streptomyces and having the ability to produce the physiologically active substance NK34896B according to claim 1 or 2 is cultured, and a new physiologically active substance NK34896B is produced and accumulated in the culture, and collected. A method for producing a novel physiologically active substance NK34896B. 4. A microorganism belonging to the genus Streptomyces and having the ability to produce the physiologically active substance NK34896B according to claim 1 or 2. 5. Streptomyces sp. NA 348
96 strains (FERM P-17984) or mutants thereof