JPH07324082A - Novel antibiotic DQ112-A, method for producing the same and antitumor agent - Google Patents

Abstract

(57) [Summary] (Correction) [Structure] Antibiotic DQ112- represented by the following formula (1)
A; An antibiotic DQ112-A producing bacterium belonging to the genus Streptomyces is cultured in a medium, and the antibiotic DQ11 is extracted from the culture.
Method for producing antibiotic DQ112-A for collecting 2-A and antibiotic DQ112- represented by the above formula (1)
A and the antibiotic DQ112- represented by the following formula (2)
B An antitumor agent containing as an active ingredient at least one antibiotic selected from the group consisting of: [Effect] The antibiotic DQ112-A is useful as an antitumor agent, and a novel antitumor agent was provided.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel antibiotic, a method for producing the same, and an antitumor agent.

[0002]

2. Description of the Related Art Antibiotics are substances produced by microorganisms and have various useful physiological activities such as antibacterial action, antiviral action, antitumor action and enzyme inhibiting action, and are therefore widely used clinically. For example, antibiotics such as mitomycin C, bleomycin antibiotics, and anthracycline antibiotics have antitumor activity and are generally used as antitumor agents. There is a need for additional novel antibiotics having such antitumor activity.

[0003]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a novel antibiotic and a method for producing the same. Another object of the present invention is to provide an antitumor agent containing the above antibiotic and / or its analog as an active ingredient.

[0004]

As a result of diligent efforts, the present inventors have found that Streptomyces sp. DQ112 (Strep
It was found that certain antibiotics produced by tomyces sp. DQ112) have antitumor activity, and that one of them is a novel compound, which led to the completion of the present invention. That is, the present invention provides the antibiotic DQ1 represented by the following formula (1).
12-A.

[0005]

[Chemical 4]

Further, the present invention is characterized in that an antibiotic DQ112-A-producing bacterium belonging to the genus Streptomyces is cultured in a medium, and the antibiotic DQ112-A is collected from the culture. The present invention provides a method for manufacturing the same. Furthermore, the present invention provides the antibiotic DQ112-A represented by the following formula (1) and

[0007]

[Chemical 5]

The antibiotic DQ11 represented by the following formula (2)
2-B

[0009]

[Chemical 6]

The present invention provides an antitumor agent containing as an active ingredient at least one antibiotic selected from the group consisting of: Hereinafter, the present invention will be described in detail.

The microorganism which produces the antibiotic DQ112-A of the present invention belongs to the genus Streptomyces and belongs to the antibiotic DQ1.
It is a bacterial species capable of producing 12-A. As an example thereof, Streptomyces sp. DQ11
2 (Streptomyces sp. DQ112) (hereinafter "DQ112 strain")
Referred to). Also, natural and artificial mutant strains of the DQ112 strain may be used.

The above-mentioned DQ112 strain was isolated from soil collected in Ajigasawa, Aomori Prefecture, and was deposited at the Institute of Biotechnology, Institute of Industrial Science, Institute of Industrial Science on April 12, 1994, and the microorganism was deposited there. The number is FERM BP-4633.

The DQ112 strain has the following mycological properties.

1. Morphological characteristics DQ112 strain is sucrose / nitrate agar medium, glucose / asparagine agar medium, glycerol / asparagine agar medium, starch / inorganic salt agar medium, tyrosine agar medium, nutrient agar medium, a -Grows well on St. Malt agar medium and Oatmeal agar medium, and also has good aerial mycelial settlement. The aerial hyphae generated from the basal hyphae extend in a simple branching manner, with about 30 to 50 spore chains, and the spore chains are spiral. The spore shape is cylindrical or bale-shaped, and the size is 0.5 to 0.8 × 0.8 to 1.0 μm, and the surface is smooth. In addition, sporangia, flagella spores, sclerotium and other special forms are not recognized.

2. Growth state in various media The DQ112 strain was cultured in the following media at 27 ° C for 3 weeks, and the growth condition in each media was observed.

1) Sucrose / Nitrate agar medium Growth: Good Aerial mycelia: Poor Aerial mycelial color: Light brown Backside color: Dark red brown Soluble pigment: None

2) Glucose / asparagine agar medium Growth: Good Aerial mycelia: Good Aerial mycelial color: Light brown ash Backside color: Dark yellow Soluble pigment: None

3) Glycerol-asparagine agar medium Growth: Good Aerial mycelia: Medium Aerial mycelial color: Light brown ash Backside color: Dark yellow Soluble pigment: None

4) Starch / inorganic salt agar medium Growth: Good Aerial mycelia: Good Aerial mycelial color: Brown ash Backside color: Dark yellow tea Soluble pigment: None

5) Tyrosine agar medium Growth: Good Aerial mycelia: Good Color of aerial mycelia: Bright olive ash Backside color: Red brown Soluble pigment: None

6) Nutrient agar medium Growth: Good Aerial mycelia: Poor Aerial mycelial color: Yellow brown Backside color: Yellow brown Soluble pigment: None

7) Yeast / malt agar medium Growth: Good Aerial mycelium: Good Aerial mycelial color: Brown ash Backside color: Dark yellow Soluble pigment: None

8) Oatmeal agar medium Growth: Good Aerial mycelium: Good Aerial mycelial color: Brown ash Backside color: Dark yellow Soluble pigment: None

3. Physiological properties 1) Growth temperature range 15-37 ℃ 2) Formation of melanin-like pigment Tyrosine agar medium positive Peptone / Yeast / Iron agar medium positive Tryptone / Yeast liquid medium positive 3) Starch hydrolysis negative 4) Gelatin liquefaction positive 5) Negative coagulation of skim milk 6) Negative peptonization of skim milk 7) Nitrate reducing ability is positive 8) Availability of carbon source The availability of carbon source on Pridham-Gotlieve agar medium is shown below.

L-arabinose + raffinose + D-xylose-D-mannitol + D-glucose + galactose + D-fructose + sorbitol-sucrose + D-mannose + inositol + maltose + L-rhamnose + +: Use −: Do not use

4. Chemotaxonomic properties As a result of analysis of diaminopimelic acid which is one of the constituents of the cell wall, it was LL type.

The above-mentioned properties are taken into account by the ISP (international
Streptomyces Project) description (EBShirli
ng and D.Gottlieb: Int.J.Syst.Bact) and Bergey's Manualof Systematic Bacteriolog
By contrast with the description in y), the DQ112 strain was identified as a strain belonging to the genus Streptomyces.

Antibiotics DQ belonging to the genus Streptomyces
The antibiotic DQ112-A of the present invention can be produced by culturing the 112-A producing bacterium in a medium and collecting the antibiotic DQ112-A from the culture. DQ1
The 12-A-producing bacterium can be cultured according to a conventional method, and the form of culture may be liquid culture or solid culture, but aerobic liquid culture is suitable for industrially advantageous culture.

The medium may be any medium containing any nutrient source that can be utilized by the DQ112-A producing bacterium. Specifically, as a carbon source, glucose, sucrose, galactose, glycerol and oils and fats, as a nitrogen source, soybean powder, fish meal, cottonseed meal, dried yeast, yeast extract, and organic matter such as corn steep liquor, In addition, minerals such as ammonium salts or nitrates such as ammonium sulfate, sodium nitrate and ammonium chloride can be used. If necessary, potassium bromide, sodium chloride,
Inorganic salts such as potassium chloride, calcium carbonate, phosphate, and heavy metal salt can also be added. In order to suppress foaming during fermentation, it is also possible to add customary suitable antifoaming agents, for example silicone oils.

The culturing conditions such as culturing temperature and culturing time are selected so as to be suitable for the growth of the bacterium to be used and to maximize the production of DQ112-A. For example, the pH of the medium is p
H6-8 is suitable, pH 6.5-7.5 is preferred, and culture temperature is suitable 15-35 ° C, preferably 25-30 ° C. The stirring speed is suitably 150 to 250 rpm, preferably 180 to 220 rpm, and the culture time is suitably 120 to 216 hours, preferably 144 to 192 hours. However, the culture conditions such as the culture composition, the pH of the medium, the culture temperature, the stirring speed, and the culture time are appropriately adjusted so that the desired result can be obtained according to the type of strain to be used, external conditions, and the like. It goes without saying that it should be done.

In the culture in which DQ112-A is accumulated, DQ112-A is present in the culture filtrate and cells. In order to collect DQ112-A from these, the means usually used for collecting metabolites from the culture can be appropriately used, but the first technique is an extraction operation. Specifically, DQ112-A present in the culture filtrate
Can be extracted with a mixed solvent of CHCl ₃ : MeOH (10: 1), a water immiscible solvent such as ethyl acetate and n-butanol, for example, ethyl acetate. With regard to DQ112-A in the bacterial cells, a bacterial cell aggregate can be obtained by filtration / centrifugation and the like and treated with methanol, ethanol, acetone or the like to obtain a crude preparation of DQ112-A. In the above operation, the separation of the bacterial cells may be omitted and the culture may be subjected to the extraction operation as it is. Countercurrent partitioning with a suitable solvent can also be included in the broader category of extraction.

The second method for collecting DQ112-A is by the adsorption method. A liquid substance containing DQ112-A, for example, an adsorbent suitable for a culture filtrate or an extract,
For example, silica gel, activated carbon, "Diaion HP20"
(Made by Mitsubishi Kasei Co., Ltd.) to adsorb DQ112-A, elute with an appropriate solvent and concentrate to dryness under reduced pressure to obtain a crude sample of DQ112-A.

In order to further purify the thus-obtained crude product of DQ112-A, in addition to the above-mentioned extraction and adsorption operations, gel filtration, high performance liquid chromatography, etc. may be carried out as many times as necessary. Good. Specifically, column chromatography using an adsorbent such as silica gel, a gel filtration agent such as "Toyopearl HW40" (manufactured by Toyo Soda Co., Ltd.), high performance liquid chromatography using "YMC Pack" (manufactured by Yamamura Scientific Co., Ltd.), etc. It is possible to carry out further purification in combination with the countercurrent distribution method.
Preferably, a crude preparation of DQ112-A is dissolved in a small amount of methanol, applied to a "Toyopearl HW40" column, and the active fraction is eluted with methanol, which is concentrated to dryness to obtain a pure product of DQ112-A. Should get

By a method other than culturing the microorganism as described above,
It would also be possible to manufacture DQ112-A. For example, starting from analogs, DQ112-A may be prepared by synthetic chemical or microbiological modification. Furthermore, even in the case of culturing a microorganism, a gene involved in the production of DQ112-A is incorporated into an appropriate host microorganism, the obtained transformant is cultured, and DQ112 is extracted from this culture.
A genetic engineering technique for obtaining -A may be used.

DQ112-A obtained as described above
The physicochemical properties of are as follows.

Appearance: Yellow powder Melting point: 153-155 ° C (decomposition) Solubility: Soluble in methanol, ethanol, chloroform, acetone and ethyl acetate, insoluble in water and n-hexane Thin layer chromatography: Merck silica gel 60F
₂₅₄ Solvent Chloroform: Methanol = 15: 1 Rf value 0.48 FAB-MS spectrum (m / z): (M + H) ⁺ 713 Ultraviolet absorption spectrum: shown in FIG. λ ^MeOH _max nm (ε) 218 (29200), 318 (4700), 425
(5100) Infrared absorption spectrum (KBr disk method): Shown in FIG. ¹ H-NMR spectrum (500 MHz, in heavy benzene): shown in FIG. ¹³ C-NMR spectrum (125 MHz, in heavy benzene): shown in FIG.

Molecular Formula: C ₃₇ H ₄₄ O _{14 From the} above physicochemical properties, DQ112-A has the following formula:
It was clarified to be a novel compound represented by (1).

[0038]

[Chemical 7]

DQ112-A has physiological activities such as antibacterial activity, antitumor activity, and platelet aggregation inhibitory activity.
It can be used as an antitumor agent, an anticoagulant, or the like. Antibiotic DQ1 of the invention for the treatment of tumors
The compound DQ112-B represented by the following formula (2) can be used for 12-A and its related compounds.

[0040]

[Chemical 8]

DQ112-B is one of the antibiotics produced by the above DQ112 strain.
It can be manufactured by the same method as the manufacturing method of A. For example, a DQ112 strain culture is extracted with a water-immiscible solvent such as ethyl acetate, concentrated, and then applied to an appropriate adsorption column such as a silica gel column to obtain DQ112-A and DQ11.
A fraction of the mixture with 2-B was obtained and concentrated, then
The concentrate is applied to a suitable gel filtration column such as Toyopearl HW40 column, and the active fraction is eluted with a suitable solvent such as methanol to separate DQ112-A and DQ112-B, and further purified to obtain DQ112-A.
Q112-B can be obtained.

The physicochemical properties of DQ112-B thus obtained are as follows.

Appearance: Yellow powder Melting point: 143-145 ° C (decomposition) Solubility: Soluble in methanol, ethanol, chloroform, acetone and ethyl acetate, insoluble in water and n-hexane Thin layer chromatography: Merck silica gel 60F.
₂₅₄ solvent Chloroform: methanol = 15: 1 Rf value 0.48 FAB-MS spectrum (m / z): (M + H) ⁺ 711 UV absorption spectrum: shown in FIG. λ ^MeOH _max nm (ε) 218 (31000), 318 (4500), 425
(5000) Infrared absorption spectrum (KBr disk method): Shown in FIG. ¹ H-NMR spectrum (500 MHz, in heavy benzene): shown in FIG. 7. ¹³ C-NMR spectrum (125 MHz, in heavy benzene): shown in FIG. Molecular formula: C ₃₇ H ₄₂ O ₁₄

Antibiotics DQ112-A and Antibiotics D
The antitumor agent containing at least one antibiotic selected from the group consisting of Q112-B as an active ingredient can be used by any of the oral and parenteral administration routes.
Specifically, in the case of animals, it can be administered by methods such as intraperitoneal administration, subcutaneous administration, intravascular administration to veins or arteries, and local administration by injection, and in the case of humans, intravenous administration. , Intraarterial administration, local administration by injection,
It can be administered by methods such as intraperitoneal, thoracic administration, oral administration, subcutaneous administration, intramuscular administration, sublingual administration, transdermal absorption, or rectal administration.

The above-mentioned antitumor agent is in an appropriate dosage form depending on the administration method and administration purpose, for example, injections, drops, suspensions, tablets, granules, powders, capsules, fine granules, ointments. , A cream or the like. In order to produce these preparations, in addition to pharmaceutically acceptable carriers or diluents, solubilizers, binders, disintegrants, lubricants, stabilizers, isotonic agents, preservatives, antioxidants, etc. Can be added.

As the carrier or diluent, any form of liquid, gel and solid can be used.
Water, liquid materials such as physiological saline, lactose, starch, crystalline cellulose, mannitol, maltose, calcium hydrogen phosphate, light anhydrous silicic acid, solid materials such as calcium carbonate, and fat-free ointment, gel base, lotion,
Examples thereof include gel materials such as FAPG base. In addition, as a solubilizer, ethanol, polysorbate, etc., as a binder, starch, polyvinylpyrrolidone, hydroxypropyl cellulose, ethyl cellulose, carboxymethyl cellulose, gum arabic, etc.,
As the disintegrant, starch, carboxymethylcellulose calcium, etc., as the lubricant, magnesium stearate, talc, hydrogenated oil, etc., as the stabilizer, lactose, mannitol, maltose, polysorbates, macrogol, polyoxy Ethylene hydrogenated castor oil,
As a tonicity agent, sodium chloride, glucose, citrate, acetate, phosphate, etc., and as a preservative, benzoic acid, paraoxybenzoate, dehydroacetic acid, boric acid, chlorobutanol, benzyl alcohol, etc. As an oxidant,
Examples include tocophenol, ascorbic acid, citric acid and the like. In addition, glycerin, dimethylacetamide, 70% sodium lactate, a surfactant, and a basic substance (for example, ethylenediamine, ethanolamine, sodium carbonate, arginine, meglumine, trisaminomethane) can be added if necessary.

The dose of the antibiotic, which is the active ingredient, may be any amount that is sufficient to effectively treat the target tumor, but the method of administration, the age, weight, sex and sensitivity of the patient or treated animal Needless to say, it varies depending on the time of meal (diet) administration, the drug used in combination, the degree of illness and the like. Generally, a dose of about 0.01 to 500 mg per day for an adult is suitable, and a dose of about 0.1 to 100 mg is preferable.

DQ112-A and DQ using mouse
When the acute toxicity test of 112-B was conducted, DQ112
-A has a toxicity value of 6.25 to 12.5 mg / Kg by intravenous injection and DQ
The toxicity value of 112-B was 6.25 to 12.5 mg / Kg by intravenous injection. Hereinafter, the present invention will be specifically described with reference to Examples.
The scope of the present invention is not limited to these.

[0049]

【Example】

[Production Example] DQ112-A and DQ112- from DQ112 strain
Production of B

(1) Preparation of Seed Mother A medium having the following composition was dissolved in 1 liter of water to prepare a medium. Glucose 25.0 g Soybean meal 15.0 g Dry yeast 2.5 g Calcium carbonate 4.0 g pH 6.2 100 ml of the above medium was dispensed into a 500 ml Erlenmeyer flask with a wart, and sterilized. , 27 ° C for 7 days, 20
The culture was performed with shaking at 0 rpm.

(2) DQ112-A and DQ112-
Collection of B After culturing under the above conditions, the culture solution (1 liter) was subjected to Buchner filtration to obtain bacterial cells. The cells were extracted with 0.5 liter of acetone. After concentrating the extract under reduced pressure to 0.2 liter,
It was combined with the culture filtrate and extracted twice with an equal volume of ethyl acetate.
Anhydrous sodium sulfate was added to the extract for dehydration and filtration, and then the filtrate was concentrated to obtain 150 mg of a concentrate. This concentrate was equilibrated with chloroform-methanol (100: 1) on silica gel (Wako Gel C-20 manufactured by Wako Pure Chemical Industries, Ltd.).
0]) column (3 cmφ × 30 cm) and developed with 300 ml of the same composition solution. DQ112-A and DQ11
2-B eluted in exactly the same fraction as the mixture. This fraction (65 mg) was collected, concentrated under reduced pressure, dissolved in methanol, and then dissolved in a Toyopearl HW40 column (3 cmφ x
50 cm) and the active fraction was eluted with methanol.
By this column chromatography, DQ112-A and DQ1
12-B was separated, and each fraction was concentrated to dryness to obtain 25 mg of purified DQ112-A and purified DQ112-A.
20 mg of Q112-B was obtained.

Ultraviolet absorption spectrum (in methanol), infrared absorption spectrum (KBr disk method), ¹ H-NMR spectrum (500 MHz, in heavy benzene) and ¹³ C-NMR spectrum (125 MHz, heavy benzene) of purified DQ112-A. Middle) are shown in FIGS. 1, 2, 3 and 4, respectively. Further, the ultraviolet absorption spectrum (in methanol), infrared absorption spectrum (KBr disk method), ¹ H-NMR spectrum (500 MHz, in heavy benzene) and ¹³ C-NMR spectrum (125 MHz, in heavy benzene) of purified DQ112-B were used. ) Are shown in FIGS. 5, 6, 7 and 8, respectively.

[Test Example] Measurement of antitumor activity of DQ112-A and DQ112-B In a U-bottom 96-well microplate, RPMI 1640 was added to fetal bovine serum (10%), penicillin (100 U / ml), streptomycin (100 μg). / Ml) and 2-mercaptoethanol (5.0 x 10 ^-5 M) in a medium to be tested DQ
112-A and DQ112-B are 1.0 to
Serially dilute to 0.01 μg / ml, and add 5 × 10 ³ / well of L1210 cells, which are mouse tumor cells, to 5% CO ₂
After incubating at 37 ° C for 2 days in the incubator,
MTT method (Michael C. Alley et al., Cancer
Res., 48 589-601 (1988)). Since the substance to be tested is poorly soluble in water, it was dissolved in methanol and then diluted with the above medium. As a control, the above operation was repeated except that the test substance was not added.

[0054] The reduced concentration cell numbers to 50% of control as IC ₅₀ (μg / ml), were measured IC ₅₀ of DQ112-A and DQ112-B. As a result, DQ
The IC _{50 of} 112-A is 0.16 μg / ml, and DQ11
The IC _{50 for} 2-B was 0.16 μg / ml.

[0055]

According to the present invention, the novel antibiotic DQ112
-A and a method for producing the same are provided. Antibiotic DQ1
12-A has antitumor activity and is therefore useful as an antitumor agent. Further, the present invention provides an effective antitumor agent.

[Brief description of drawings]

FIG. 1 is a diagram showing an ultraviolet absorption spectrum (in methanol) of an antibiotic DQ112-A of the present invention.

FIG. 2 is a diagram showing an infrared absorption spectrum (KBr disk method) of antibiotic DQ112-A.

FIG. 3 shows ¹ H-NM of antibiotic DQ112-A.
It is a figure which shows R spectrum (500 MHz, in heavy benzene).

FIG. 4 shows ¹³ C-NM of antibiotic DQ112-A.
It is a figure which shows R spectrum (125 MHz, in heavy benzene).

FIG. 5 is a diagram showing an ultraviolet absorption spectrum (in methanol) of antibiotic DQ112-B.

FIG. 6 is a diagram showing an infrared absorption spectrum (KBr disk method) of antibiotic DQ112-B.

FIG. 7 shows ¹ H-NM of antibiotic DQ112-B.
It is a figure which shows R spectrum (500 MHz, in heavy benzene).

FIG. 8: ¹³ C-NM of antibiotic DQ112-B
It is a figure which shows R spectrum (125 MHz, in heavy benzene).

Claims (3)

[Claims] 1. An antibiotic DQ1 represented by the following formula (1):
12-A. [Chemical 1] 2. An antibiotic D belonging to the genus Streptomyces
A method for producing an antibiotic DQ112-A, comprising culturing a Q112-A producing bacterium in a medium and collecting the antibiotic DQ112-A from the culture. 3. An antibiotic DQ1 represented by the following formula (1):
12-A and Antibiotic DQ112-B represented by the following formula (2): An antitumor agent containing as an active ingredient at least one antibiotic selected from the group consisting of: