JP2849460B2 - Novel compound A-70615 substance and production method thereof - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a novel compound A-70615 having trehalase inhibitory activity and a method for producing the same.

(Prior art) Conventionally, as a substance exhibiting trehalase inhibitory activity, an agricultural chemical antibiotic validamycin (Takeda Pharmaceutical Co., Ltd.) used as an anti-smell disease drug has been reported, and the mechanism of action of this validamycin is as follows. Pathogenic bacterium Rhizoctonia. It has been shown that solani inhibits trehalase, an enzyme that converts trehalose, a storage sugar of solani, into glucose, an energy source (N. Asano et. Al. J. Antibiotics Vol. 40, 526 (1987)). ,
M. Ueda et, al. Agric. Biol. Chem. Vol. 49, 3485-3491 (19
85)).

(Means) Trehalase is an enzyme that decomposes trehalose, which is a storage sugar of mold, insects and the like, into glucose, which is an energy source. Therefore, trehalase inhibitors are expected to be useful for applications such as antifungal and insecticidal (insect control agents).

The present inventors searched for a substance that inhibits trehalase as a compound having antifungal and insecticidal activities, and as a result of intensive research, found that a fungus SANK 62390 strain belonging to the genus Micromonospora isolated from soil (Microtechnological Laboratory) No. 11631), a novel compound A-70 having trehalase inhibitory activity
615 were produced, and the present invention was completed.

(Constitution of the Invention) The present invention relates to a novel compound A-70615 and a method for producing the same.

The novel compound A-70615 of the present invention has the following structural formula (I) and physical properties.

1) Structural formula 2) Physical properties 1) Property: Basic white powder 2) Solubility: Soluble in water and methanol. Insoluble in acetone and chloroform.

3) Color test: positive for sulfuric acid 4) Molecular formula: C ₁₃ H ₂₂ N ₂ O ₁₀ 5) Molecular weight: 366 (FAB-mass spectrum) 6) Specific rotation: [α] _D ²⁵ + 99.5 ° (c0. 7) UV absorption spectrum: λ _max nm (E _1cm ^1% ) UV absorption spectrum measured in an aqueous solution is 220 nm
No characteristic absorption is shown above.

8) Infrared absorption spectrum: The infrared absorption spectrum measured on the ν _max ^KBr cm ^-1 KBr disc is the first.
As shown in the figure.

9) ¹ H-nuclear magnetic resonance spectrum: δ ppm ¹ H-nuclear magnetic resonance spectrum (400 MHZ) measured in heavy water using TMS (trimethylsilane) as an external standard is as shown in FIG.

10) ¹³ C-nuclear magnetic resonance spectrum: δ ppm The ¹³ C-nuclear magnetic resonance spectrum is as shown in FIG.

11) High Performance Liquid Chromatography: Separation column; Senshu Pak ODS-H
-2151 (manufactured by Senshu Kagaku Co., Ltd.) 6φ × 150 mm (5 μ) mobile phase; 10% (V / V) acetonitrile-water (containing 0.5% of PIC B8 (manufactured by Waters)) Flow rate: 1.5 ml / min Detection wavelength; A peak was able to be formed at a retention time of 6.9 minutes at a UV 210 nm column temperature of 25 ° C.

 The elution pattern is as shown in FIG.

12) Thin layer chromatography: Rf value; 0.44 adsorbent; silica gel plate Art 5715 manufactured by Merck Co., Ltd. developing solvent; acetonitrile: acetic acid: water = 6: 1: 3 Micromonospora used in the present invention ( Microm
onospora ), for example, Micromonospora . SP.SANK 62390 strain
No.), and the mycological properties of this fungus are as follows.

1. Morphological properties SANK 62390 strain was grown on an agar medium for strain identification at 28 ° C,
It grows normally or slightly poorly in 14 days of culture. The underlying mycelium elongates and branches favorably, showing a bright orange, orange to dark brownish gray, but no Nocardia strain-like rupture or zigzag elongation is observed. The aerial hyphae are slightly formed in the original trace and show white to brownish white. The spores are observed only on the basal hypha, but are formed one by one at the tip of a relatively short spore stalk and are spherical. The spore surface is smooth. No special organs such as sporangia, sclerotium, and axle branching are found.

2. Properties on various culture media The properties after cultivation on various culture media at 28 ° C for 14 days are as shown in the following table. The color tone display indicates the color chip number of the "Standard Color Table" for the Japan Color Research Institute.

3. Physiological properties The physiological properties of the SANK 62390 strain observed for 2 to 21 days after culturing at 28 ° C. are as follows.

The assimilation of the carbon source of the SANK 62390 strain, which was observed after cultivation at 28 ° C. for 14 days using Prideham Gottlieb agar medium (ISP 9), is as shown in the following table.

4. Cell components The cell wall of the SANK 62390 strain was prepared by the method of B. Becker et al. [B. Becker et al., Applied Microbiology , Vol.
-423 (1984)], as a result, mesodiaminopimelic acid and glycine were detected. Also, SANK 62390
The sugar content in the whole cell wall of the strain was determined by MP Rechevalier method [MPLechevalier, Journal of Laboratory & Cli
nical Medicine, Vol. 71, 834 (1968)], arabinose and xylose were detected. The presence of mycolic acid was not confirmed. The acyl form in the cell wall peptidoglycan was a glycosyl form. MK-10 (H ₆ ), MK-10 (H ₄ ), MK-10
(H ₈₎ has been investigated. From the above, it was found that this strain belongs to the genus Micromonospora among actinomycetes, so that Micromonospora sp.
p.) It was named SANK 62390 (Microtechnical Laboratory No. 11631).

The identification of the SANK 62390 strain was determined by the ISP (The International Streptomyces Project).
ternational Streptomyces Project] standards, Bergey's Manual of Systematic Bac
teriology) Volume 4, The Actin
omycetes) Volume 2 and recent literature on actinomycetes.

As is well known, actinomycetes are susceptible to mutation in nature or by artificial manipulation (eg, ultraviolet irradiation, irradiation, chemical treatment, etc.), and the SANK 62390 of the present invention.
Shares are the same. The SANK 62390 strain referred to in the present invention includes all the mutant strains. Also, among these mutants, genetic methods, such as recombination, transduction,
Also included are those obtained by transformation or the like. That is, the present invention produces A-70615, SANK 62390 strain,
The mutant strains and the strains that are not clearly distinguished therefrom are all included in the SANK 62390 strain.

To obtain the novel compound A-70615 of the present invention, the culture of these microorganisms is performed in a medium such as that used to produce other fermentation products. Such a medium contains a carbon source, a nitrogen source, and inorganic salts that can be assimilated by microorganisms.

Generally, glucose, fructose, maltose, sucrose, mannitol, glycerol, dextrin, oats, rye, corn starch, potato, corn flour, soy flour, cottonseed oil, molasses, citric acid, tartaric acid, etc. Alternatively, they can be used in combination. In general, the amount is varied from 1 to 10% by weight of the medium volume. As the nitrogen source, a protein-containing substance is generally used in the fermentation step.

Suitable nitrogen sources include soy flour, bran, peanut flour,
Cottonseed oil, cottonseed flour, casein hydrolyzate, pharmamine,
Fish meal, corn steep liquor, peptiton, meat extract,
Yeast, yeast extract, malt extract, sodium nitrate, ammonium nitrate, ammonium sulfate and the like.
The nitrogen source is used alone or in combination in the range of 0.2-6% by weight of the medium volume.

The nutrient inorganic salts to be taken into the medium are ordinary salts from which ions such as sodium, ammonium, calcium, phosphate, sulfate, chloride and carbonate can be obtained. Also, potassium, calcium,
Includes trace metals such as cobalt, manganese, iron and magnesium. At the time of liquid culture, silicone oil, vegetable oil, surfactant and the like are used as an antifoaming agent.

Cultured Micromonospora sp. SANK 62390 strain,
The pH of the medium producing A-70615 can vary from 5.0-8.0.

The growth of the bacterium is preferably in the range of 22 ° C to 38 ° C.
Temperatures between 22 ° C and 28 ° C are preferred for the production of -70615. A-706
15 can be obtained by aerobic culturing, and aerobic culturing methods usually used, for example, a solid culturing method, a shaking culturing method, aeration stirring culturing and the like are used.

In a small-scale culture, it is preferable to perform shaking culture at 28 ° C. for several days.

The culture is started in a conical flask with baffles (water flow control walls) by a 1-2 stage seed development process. The medium at the seed development stage can use a carbon source and a nitrogen source together. Seed flasks are shaken in a constant temperature incubator at 28 ° C. for 7 days or until fully grown. The grown seed is used to inoculate a second seed or production medium. If an intermediate development step is used, it is used in part to grow and inoculate the production medium in essentially the same way. The inoculated flask is shaken at a constant temperature for several days, and after incubation the contents of the flask are centrifuged or filtered.

In the case of mass culture, it is preferable to culture in a suitable tank equipped with a stirrer and aeration device. According to this method,
A nutrient medium can be made in the tank. Culture medium at 125 ° C
Heat to sterilize, cool and inoculate a sterile medium with seeds that have been previously grown. Culture is performed at 28 ° C. with aeration and stirring. This method is suitable for obtaining large amounts of compounds.

The time-dependent change in the amount of A-70615 produced as the culture proceeds can be determined by measuring the trehalase inhibitory activity. Normally, the production of A-70615 reaches its maximum value after 72 to 150 hours of culture.

After completion of the cultivation, A-70615 present in the liquid part (and the cells) in the culture solution is separated from the cells and other solid parts by a filtration operation using diatomaceous earth as a filter aid or by centrifugation. It is obtained by extracting and purifying A-70615 present in the supernatant utilizing its physicochemical properties.

For example, A-70615 present in the filtrate or supernatant is passed through a layer of an ion exchange resin, for example, Amberlite IRC-50, CG-50, Dowex 50W × 4, Dowex SBR-P to cause impurities to adhere. Or by adsorbing A-70615 and eluting with aqueous ammonia. Alternatively, as an adsorbent, for example, activated carbon or Amberlite XAD-2, XAD-4 (ROHM
And Haas), Diaion HP-10, HP-2
0, CHP20, HP-50, etc. (manufactured by Mitsubishi Chemical Industries, Ltd.) are used. The liquid containing A-70615 is passed through a layer of the adsorbent as described above to absorb and remove impurities, or
Is adsorbed and eluted with methanol water, acetone water or the like.

A-70615 obtained in this manner is further subjected to adsorption column chromatography using a carrier such as silica gel or florisil, Avicel (manufactured by Asahi Kasei Corporation), Sephadex LH-20 (manufactured by Pharmacia) or the like. It can be purified by conventional partition column chromatography and high-performance liquid chromatography using normal-phase and reverse-phase columns.

When the compound A-70615 of the present invention is used as an agricultural or horticultural medicine, it is prepared and used in various forms, for example, powders, coarse powders, granules, granules, wettable powders, emulsions, liquids and the like.

Next, the present invention will be described in more detail with reference to Examples and Test Examples, but the present invention is not limited to these methods as well as the type of culture medium, culture conditions, collection, purification method, etc. It is needless to say that can be variously changed.

(Example) Purification of compound A-70615 (A) Culture Micromonospora. One strain of a loop of SP.SANK 62390 was inoculated into three 500 ml Erlenmeyer flasks (with baffles) containing 80 ml of the medium represented by Medium Composition-1 and cultured at 28 ° C. for 216 hours using a 220 rpm rotary shaking incubator.

40 ml of this culture was mixed with 800 ml of the medium represented by Medium Composition-2.
Was inoculated into four Erlenmeyer flasks containing 2 and cultured at 28 ° C. for 96 hours using a rotary shaking incubator at 220 rpm. Then, the medium represented by Medium Composition-2 was added to two 30-volume jar fermenters.
15 and heat sterilized at 120 ° C for 35 minutes and cooled to 28 ° C, inoculated with seed culture solution 1.5, and rotated at 100 rpm at an aeration rate of 15
Per minute at 28 ° C for 96 hours.

(B) Isolation Celite 545 was added to the obtained culture solution 30 as a filter aid.
(Manufactured by Johns Manville Product Corporation) (3 kg), and the mixture was filtered to obtain a filtrate 29.
Dow filtrate X SBR-P (Cl ^-) passed through a column of (Dow Chemical) 6, Dowex 50 W × 4 by adjusting the pH of the effluent to 5.0 (H ⁺⁾ (manufactured by Dow Chemical Company) 6
A-70615 was adsorbed through the column. After washing with 20 ionized water, elute with 0.5N aqueous ammonia 30 and elute active fraction
I got 13. The obtained eluate 13 was concentrated under reduced pressure and freeze-dried to obtain 43.4 g of a crude powder containing A-70615. This coarse powder was dissolved in 2 of 10 mM ammonium formate buffer, pH 6.0, adsorbed on a Dowex 50W × 4, 1.5 column previously buffered with 20 mM, pH 6.0 ammonium formate buffer, and adsorbed on the column. After washing with a buffer solution, the plate was washed with deionized water (2) and eluted with 0.2N aqueous ammonia. 500 ml eluate
When fractionated, A-70615 fractions 2 to 4
This fraction was collected, concentrated under reduced pressure, and freeze-dried to obtain 2.55 g of a crude powder.

Next, this powder was adsorbed on a 200 ml Avicel (Asahi Kasei Kogyo Co., Ltd.) column previously filled with 80% acetonitrile-water, washed with 700 ml of 80% acetonitrile-water, and then washed with 500 ml of 75% acetonitrile-water and 700 ml of water. When the eluate eluted sequentially with 70% acetonitrile-water was fractionated in 19 ml portions, A-70615 was eluted in fractions 35 to 50. This fraction was collected, concentrated under reduced pressure, freeze-dried,
8 mg of powder were obtained. This powder was further dissolved in 150 ml of water,
After adjusting to 6.0, 300 of Amberlite CG-50 (H ⁺ : NH ₄ ⁺² : 3)
0.1 ml after adsorption on a 500 ml column and washing with 500 ml of deionized water.
When the eluate eluted with N ammonia water was fractionated in 20 ml increments, A-70615 was eluted in fractions 92 to 121, and the fractions were collected, concentrated under reduced pressure, and lyophilized to obtain 102.8 mg of a powder. . Next, this powder was dissolved in 10 ml of 2 mM ammonium formate buffer, pH 6.0, adsorbed on a 400 ml column of DIAION CHP20P (manufactured by Mitsubishi Kasei Kogyo Co., Ltd.) filled with the buffer, and eluted with the buffer in 5 ml increments. A-70615 was eluted in fractions 59 to 73, and the fractions were collected, concentrated under reduced pressure, and lyophilized to obtain 18 mg of a powder. This powder was purified again by a Diaion CHP20P column to obtain 6.2 mg of a white powder.

This powder was then purified by preparative TLC. Dissolve 6.2 mg of the powder with a small amount of water, adsorb to three silica gel plates Art5715 (20 × 20 cm) manufactured by Merck, develop 15 cm with acetonitrile: acetic acid: water: (6: 1: 3), and apply Rf 0.42
And fill the column with silica gel powder.
Elution was performed with 00 ml of deionized water. The eluate is Dowex 50
A-70615 was adsorbed through a column of 5 ml of W × 4 (H ⁺ ),
The column was washed with deionized water and eluted with 50 ml of 0.5N aqueous ammonia, and the eluate was concentrated under reduced pressure and freeze-dried to give a white powder of A-70615 showing a single peak by high performance liquid chromatography. mg was obtained.

(Test Example) Inhibitory activity of compound A-70615 on silkworm trehalase In 10 ml (44) g of the fifth instar larva of silkworm, 120 ml of a 20 mM citric acid-40 mM disodium phosphate buffer (pH 5.6), polytron was added under ice cooling. The mixture was homogenized for 2 minutes, and centrifuged at 6,000 rpm for 10 minutes. To 120 ml of the supernatant, 240 ml of acetone was added with stirring under ice-cooling. This was centrifuged at 9,000 rpm for 20 minutes, the precipitate was dissolved in water, freeze-dried, and the crude enzyme 2.0 g
I got

 Deoxynojirimycin was purchased from Sigma.

Hereinafter, 20 mM citric acid-40 mM disodium phosphate buffer (pH 5.6) was used for silkworm trehalase activity measurement. In a test tube, 130 μl of a buffer solution, 50 μl of a sample solution, and 50 μl of a 4 mg / ml silkworm enzyme solution were added, and the mixture was shaken in a 37 ° C. water bath for 15 minutes. The reaction solution was allowed to stand on boiling water for 3 minutes, cooled with ice water, centrifuged at 3,000 rpm for 10 minutes to remove precipitates, and the glucose concentration in the solution was quantified. This reaction was performed using glucose C-Test Wako, a product of Wako Pure Chemical Industries, and adding a 10-fold amount of a sample solution by a standard operation method.
The inhibition rates were calculated as 0% and 100% inhibition, respectively, when the buffer solution was used as the sample solution during the enzyme reaction and when the buffer solution was used instead of the substrate solution.
The 0% inhibitory concentration was calculated. Table 1 shows the results.

(Effect of the Invention) As described above, the compound A-70615 of the present invention has a remarkable trehalase inhibitory activity, and is useful as, for example, an antifungal agent, an insecticide and the like.

[Brief description of the drawings]

FIG. 1 shows an infrared absorption spectrum of compound A-70615, FIG. 2 shows a ¹ H-nuclear magnetic resonance spectrum of the same substance, FIG. 3 shows a ¹³ C-nuclear magnetic resonance spectrum of the same substance, FIG. 4 shows the elution pattern of the same substance in high performance liquid chromatography.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI (C12P 19/28 C12R 1:29) (C12N 1/20 C12R 1:29) (72) Inventor Akio Torigata Hiroshi Shinagawa-ku, Tokyo 1-58, Sancho-cho, Sankyo Co., Ltd. (72) Inventor Hideyuki Haruyama 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 1-58 Sankyo Co., Ltd. (72) Takeshi Kinoshita, 1-2-2 Hiromachi, Shinagawa-ku, Tokyo No. 58 Sankyo Co., Ltd. (72) Inventor Nao Okazaki 33 Miyukigaoka, Tsukuba, Ibaraki Sankyo Co., Ltd.In-house (58) Field surveyed (Int.Cl. ⁶ , DB name) C07H 17/00 C12P 19/28 CA (STN) REGISTRY (STN)

Claims (3)

(57) [Claims] (1) Formula (I) A-70615 represented by the following formula: 2. An A-70615 compound producing bacterium belonging to the genus Micromonospora is cultured, and the compound A-70615 is isolated from the culture.
A method for producing A-70615. 3. An A-70615 compound producing bacterium belonging to the genus Micromonospora. SP SANK 62390
3. The method according to claim 2, wherein the strain is a strain (Microtechnical Laboratory No. 11631).