JPH03191788A - Novel antibiotic substance nk155141 and its production - Google Patents

Abstract

PURPOSE:To obtain a macrolide antibiotic substance NK155141 useful as an antibacterial agent, antimycotic agent, carcinostatic agent, insecticide, etc., by culturing a microorganism of genus Streptomyces. CONSTITUTION:A microbial strain belonging to genus Streptomyces [e.g. Streptomyces.sp. No.NK155141 (FERM P-11102)] is cultured in a nutrient medium and the objective antibiotic substance NK155141 expressed by formula is separated from the culture product. The antibiotic substance inhibits the growth of Gram-positive bacteria (e.g. Streptococcus.aureus, Bacillus.subtilis, Bacillus.megatherium or Micrococcus.flavus) and phytopathogenic fungi (e.g. pathogen of rice blast or gray mold), suppresses the proliferation of human melanoma cell, human colon cancer cell, human pulmonary cancer cell, human hepatic cancer cell, etc., and exhibits insecticidal effect against mosquito.

Description

Detailed Description of the Invention <Field of Industrial Application> The present invention relates to the antibiotic NK155141 and antibacterial/antifungal agents, anticancer agents, and insecticides containing it as an active ingredient. Used as a pesticide.

<Problems to be solved by conventional technology and inventions rope> Conventionally, various antibiotics have been used as antibacterial agents.

However, new antibiotics are always needed due to problems such as resistance.

<Means for Solving the Problems> The antibiotic NK155141 of the present invention is a novel compound that has not been described in any literature, and is a useful substance as an antibacterial agent, an antifungal agent, an anticancer agent, and an insecticide. The antibiotic NK155141 of the present invention is produced by culturing antibiotic NK155141-producing bacteria belonging to the genus Streptomyces in a nutrient medium.
It can be obtained by producing and accumulating the antibiotic NK155141 and collecting it from the culture.

The antibiotic NK155141-producing bacterium used in the present invention was isolated from the soil of Hanyu City, Saitama Prefecture, and is an actinomycete strain belonging to the genus Streptomyces. Joyori No. 11102 (
FERM P-11102).

For convenience, Streptomyces sp. AN K1551
The mycological properties of this strain, called strain 41, are described below.

1) Culture properties (27°C) (raw version).

2) Physiological properties (culture at 27°C other than growth temperature) Growth temperature range: 10-37°C Optimal temperature: 24-32°C Liquefaction of gelatin Hydrolysis of starch Coagulation of ten-skimmed milk Peptonization of skimmed milk Melanin-like pigment Production tyrosine agar peptone/yeast iron agar + + nitrate reduction + 3) Assimilation of carbon source (27℃) L-arabinos - inositol D-xylose - L-rhamnose D-glucose + raffinose 10-fructose -
D-mannite + sucrose - D-galactose + As a result of observation, this strain forms a spore chain consisting of 20 or more spores, and the cell wall exhibits a square shape, so it belongs to the genus Streptomyces. As a result of comparing the above-mentioned properties of this strain with certain properties listed in Purge's Bacteria Identification Handbook, 8th Edition and ISP, we found that this strain is Streptomyces.
Streptomyces hals
tedii) is most closely related to strain I SP 5068. Comparing the properties of the two, there is only a slight difference in the assimilability of the carbon sources of arabinose and xylose, and most of the properties match well.

For the production of the antibiotic NK155141 according to the present invention, the above-mentioned Streptomyces sp.
Strain fermentation is carried out. The culture of this strain is 20-37
It is carried out under aerobic conditions in a medium containing a water-soluble nutrient source suitable for the culture of conventional actinomycetes at a temperature range of preferably 20-30°C.

The medium used for the purpose of main culture may contain nutritional sources that can be used by actinomycetes, including medium composition, sugars such as sucrose, fructose, and sucrose, carbon sources such as starch and glycerin, and casein, polypeptone, Nitrogen sources such as organic nitrogen such as soybean meal, cottonseed meal, meat extract, dried yeast, and cornstap liquor, or inorganic nitrogen such as ammonium sulfate and ammonium chloride can be used alone or in combination. Furthermore, it is also possible to use sodium chloride, calcium carbonate, iron sulfate, magnesium sulfate, zinc sulfate, various vitamins, etc. for the purpose of growth promotion and regulation.

If necessary, it is also possible to add corn, vegetable oil, or a synthetic antifoaming agent to the medium to prevent foaming.

Streptomyces sp A N K 155141
Various methods can be used to preserve the strain, such as freeze-drying, passage on agar slants, etc.

When producing the antibiotic NK155141, one platinum loop spore is transplanted from an agar slant into a shaker flask and cultured for 2 to 5 days at about 25 to about 30°C to use as a seed. Depending on the scale of the main culture, seed culture can be repeated to obtain a large amount of seed bacteria.

For the main culture, a certain amount of the thus obtained inoculum is inoculated into a fermentation container and incubated at about 20 to about 35°C, preferably at about 25°C.
Aerate and stir at ~30°C for 3-6 days. The cells of the obtained culture solution are collected by means such as centrifugation or filtration, and the target substance is extracted from the cells using a water-miscible solvent such as acetone or methanol. The extraction operation can be repeated as necessary. After obtaining the extract containing the antibiotic NK155141 in this manner, the solvent is removed under reduced pressure.

A water-immiscible solvent such as chloroform or ethyl acetate is added to the thus obtained aqueous solution containing antibiotic NK155141 to extract antibiotic NK155-41 into an organic solvent layer. If necessary, washing the organic solvent layer with a solution such as saturated saline is effective in removing water-soluble impurities. After adding Glauber's salt to the thus obtained solvent layer and dehydrating it,
By distilling off the solvent under reduced pressure, a crude extract containing antibiotic NK155141 can be obtained.

Furthermore, in order to purify the antibiotic NKI55141, ordinary means for purifying fat-soluble low molecular weight substances can be applied. In other words, adsorption chromatography using silica gel as a carrier
Centrifugal liquid chromatography (CPC) and preparative high performance chromatography (HPLC) can be used in appropriate combination. For example, a crude extract is separated using a mixed solvent of hexane, chloroform, and acetonitrile.
Purification is performed by PC, and the active fractions are collected and dried under reduced pressure to obtain a crude material containing the active substance NK155141. After gel filtration of the obtained crude substance, purification was performed by preparative HPLC using 80 thiacetonitrile as a separation solvent, and the antibiotic N
K155141 is isolated as a colorless powder.

The antibiotic NK155141 thus obtained has the physicochemical properties shown below.

(1) Appearance Colorless powder (2) Molecular formula C43H72013 (3) Molecular weight 796 (5) Melting point 130-131℃ (6)
Mass spectrometry value (ZABMS) Theoretical value 819, 487.1 (C43Hy20+a
Na) Actual value 819.4847 (7) Ultraviolet absorption spectrum (measured in methanol)
(Fig. 1) λrnax: 247 nm (Ei! = 496
) nm (Ej!, = 212) (8) Optical external absorption spectrum (measured in KBr) (Figure 2) vcm=: 3500-3400.2970.294
0.1690.1650.1615.1250 (9)
Proton-NMR (measured in deuterated chloroform) (Fig. 3) (to) ``C-NMR (measured in deuterated chloroform (Fig. 4)) M LX;H.

δ (ppm) Multiplicity 7.5q 10.7 q 12.8 9 14, OQ ・ 14.1 q 17.3 q 17.5 q 20.1 q 20.6 q 21.8 q 28.3 d 34 .3 t 36.7 d 38.1 d 38.5 d 38.8 d 39.9 d 41.3 t 46.4 q 55.5 q 58.9 Q 60.2 (1 δ (ppm) 67. 9 69.7 71.7 74.1 74.6 77.0 77.1 81.0 81.3 82.8 92.0 103.0 125.4 127.5 132.7 133.0 133.0 141 .5 142.4 142.8 166.8 (6) Soluble Soluble Chloroform, Ethyl acetate, Acetone, Methanol, Ethanol, DMO Insoluble Hexane, Water QIRf value (Silica gel spray) Merck A37
Measurement by thin layer chromatography using No. 15) Developing solvent system: n-hexane: acetone = 3 = 2 Rf value: 0.40 Next, the biological activity of the antibiotic NK155141 (hereinafter simply referred to as the present compound) will be described.

Test Example 1 Table 1 shows the antibacterial spectrum of this compound on 0.5% peptone agar.

As shown in Table 1, this compound contains Durham-positive bacteria Staphylococcus aureus FDA209P, Bacillus subtilis PCI219, and Bacillus megaterium ATCC14.
It exhibits a strong growth inhibiting effect on Bacillus species such as 945 and Micrococcus frapus ATCC 10240, but does not show this effect on Durham-negative bacteria.

Test example 2 The antifungal effect of this compound was investigated.

Table 2 shows the antifungal activity of this compound on potato sucrose agar medium.

As shown in Table 2, this compound exhibited a growth inhibiting effect on plant pathogenic bacteria Piricularia oryzae (rice blast fungus) and Botrytis cinerea (gray mold fungus).

Table 2 Test Example 3 The growth inhibitory effect of this compound on human melanoma cells (G361) was investigated. 1.0X human melanoma cells
Inoculate a 96-well test plate at a rate of 103 cells/well,
The ridges were cultured for 24 hours at 37°C in a 5% COz incubator, and the compound was added to the culture medium at various concentrations. 72 hours after addition, the number of living cells was measured by a dye method, and the growth inhibition rate of human melanoma cells at various concentrations of the compound was determined.

The results are shown in Table 3. The IC5o value of this compound was 3.08 ng/fnI, indicating a strong growth-inhibiting effect on human melanoma cells.

Table 3 Test Example 4 The growth inhibitory effect of this compound on human colon cancer cells (SW1116) was investigated. 3.0x10 human colon cancer cells
Inoculated into a 96-well test plate at a ratio of 3 cells/well, 37
After culturing for 24 hours at 51°C in a CO2 incubator, the compounds were added to the culture medium at various concentrations. addition 96
After a period of time, the number of living cells was measured by a dye method, and the growth inhibition rate of human colon cancer cells at various concentrations of the present compound was determined.

The results are shown in Table 4. The IC5o value of this compound was 1.54 ng/fnl, indicating a strong growth-inhibiting effect on human colon cancer cells.

Test Example 5 The growth inhibitory effect of this compound on human lung cancer cells (PC-3) was investigated. Human lung cancer cells were inoculated into a 96-well test plate at a rate of 2.0 x 103 cells/well, and incubated at 37°C for 51
After culturing in a CO2 incubator for 24 hours, the compound was added to the culture medium at various concentrations. 72 hours after addition, the number of living cells was measured by a dye method, and the growth inhibition rate of human lung cancer cells at various concentrations of the compound was determined.

The results are shown in Table 5. The IC6 value of this compound was 2.26 ng/ml, indicating a strong growth-inhibiting effect on human lung cancer cells.

Test Example 6 The growth inhibitory effect of this compound on human hepatoma cells (Li 7) was investigated. Human hepatoma cells were inoculated into a 96-well test plate at a rate of 25 x 103 cells/well, and incubated at 37°C with 5%
After culturing in a CO□ incubator for 24 hours, the compound was added to the culture medium at various concentrations. Forty-eight hours after the addition, the number of viable cells was measured by a dye method, and the growth inhibition rate of human hepatoma cells at various concentrations of one compound was determined.

The results are shown in Table 6. The IC5o value of this compound was 1.56 ng/ml, indicating a strong growth-inhibiting effect on human hepatoma cells.

Table 6 Test example 7 The toxicity of this compound to mice was investigated.

A male mouse of the BD F r strain, 6 weeks old, and weighing 23 g was used. The administration route was intravenous administration.

The present compound was administered by suspending it in 5-tyglucose containing 25 ethanol. After administration, the presence or absence of death and general symptoms were observed for 7 days. LDs of this compound
o was between 0.8 mg/kg and 1.6 mg/kg.

Test Example 8 The insecticidal effect of the present compound on the mosquito mosquito was investigated. 10 ml of well water in a small petri dish (capacity 15 m1) and 3
Five instar larvae were added. 10 μl of an ethanol solution of the present compound is added dropwise to a predetermined concentration. After dropping, stir gently, feed, and keep in a constant temperature room (26 ± 1℃) for 3
After a day, the number of live and dead insects was determined. The results are shown in Table 6. This compound has 0. It showed an insecticidal rate of 100 cm at 1 ppm, clearly demonstrating its mosquito-killing effect.

Table 7 As mentioned above, the antibiotic NK155141 has antibacterial effect,
Since it has antifungal, anticancer, and insecticidal effects, it can be used as medicines or agricultural chemicals such as antibacterial agents, antifungal agents, anticancer agents, and insecticides.

When this compound is used as a pharmaceutical, it is administered alone or as an excipient as an injection, oral preparation, suppository, etc.

Any excipient may be used as long as it is pharmaceutically acceptable, and its type and composition are determined by the route and method of administration.

For example, the liquid excipient may be water, alcohol, or an animal or vegetable oil such as soybean oil, peanut oil, sesame oil, mineral oil, or synthetic oil. As solid excipients, saccharides such as maltose and sucrose, various amino acids, cellulose derivatives such as hydroxypropyl cellulose, organic acid salts such as magnesium stearate, etc. can be used.

In the case of injections, preferred excipients include physiological saline, various buffer solutions, saccharide solutions such as glucose, inositol, and mannitol, and glycol solutions such as ethylene glycol and polyethylene glycol. It can also be made into a lyophilized agent with excipients such as sugars such as inositol, mannitol, crucose, mannose, maltose, and sucrose, and amino acids such as phenylalanine, and then dissolved and administered intravenously or intramuscularly.

In the case of oral preparations, tablets, capsules, powders together with the liquid excipient or solid excipient.

It is preferably in the form of granules, liquids, dry syrups, etc. It may also be used as a pellet for local administration such as transdermal or mucosal agents.

The content of this compound in the preparation is usually 0.001 to 1
% by weight, preferably 0.01 to 0.1% by weight. For example, in the case of injections, it is usually 0.01 to 0.0
5% by weight is good. In the case of oral preparations, the amount is 0.005 to 1% by weight, preferably 0.05 to 0.5% by weight, with the remainder being excipients.

The dosage is determined depending on the patient's age, weight, symptoms, therapeutic purpose, etc., but in general, parenteral administration is 0.1 to 5μ.
On day φV, the dose is 0.5 to 30 μg/kg/day by oral administration.

When this compound is used as a medicine, it can be administered either orally or parenterally. The dosage varies depending on the method of administration, but is usually 0.01 to 20
mg/kg/day is preferred.

The compound is administered in the form of a formulation prepared by mixing it with a suitable pharmaceutical carrier. Examples of the formulations used include tablets, granules, fine granules, powders, capsules, suppositories, injections, ointments, and aerosols.

When using this compound as an antifungal agent for agriculture and horticulture, it can be used alone as it is depending on the purpose of use, but in order to enhance or stabilize the effect, it may be mixed with pesticide adjuvants to form a formulation and then used directly. Generally, it is applied by diluting it as necessary. No special conditions are required to formulate the compounds of the present invention, and powders, granules,
Microgranules, irrigation agents, flowable agents, emulsions, microcapsules, oil agents, aerosols, heating fumigants (mosquito coils, electric mosquito repellents, etc.), fumigants such as Phonoking, non-heating fumigants,
It can be used in any formulation form such as poison bait.

The agrochemical auxiliaries mentioned herein include carriers (diluents) and other auxiliaries such as spreading agents, emulsifiers, wetting agents, dispersants, fixing agents, and disintegrants.

The amount used varies depending on the dosage form, method of application, timing, and other conditions, but agricultural and horticultural agents usually contain 10 to 300 g of active ingredient per 10 ares, preferably 15 to 20 g.
0g is used.

The method for producing the compound of the present invention will be illustrated below with reference to Examples.

Example 1 Soluble starch 2%, glucose 0.5%, peptone 05%
, meat extract 0.5 t, soybean flour 0.5 t, K2PO40,
0.05%, MgSO40.05 medium at pH 7,
After adjusting the temperature to 2, add 302% CaCO and
Dispense into 1 500ml Erlenmeyer flask,
After sterilization Streptomyces sp. AN K1551
One platinum loopful of 41 strains was inoculated and cultured at 27°C for 48 hours on a rotary shaker to serve as the inoculum for the main culture.

Next, dextrin 5%, soy flour 3.5%, CaCO3
The above-mentioned inoculum was added at a ratio of 2% to 100 m1 Ersinmeyer flasks, and cultured on a rotary shaker at 27°C for 5 days. Next, the culture solution was collected and centrifuged to obtain 820 g of wet bacterial cells.

3 tons of acetone was added to the obtained bacterial cells, stirred, left for 5 hours, and filtered to obtain an extract. Acetone was distilled off under reduced pressure.
The resulting concentrated solution was adjusted to pH 2 with 4N-MCI and then extracted with ethyl acetate. The ethyl acetate layer was dried over Na2SO4 and concentrated under reduced pressure to obtain 1.5 g of an oil. The obtained oil is hexane:chloroform:acetonitrile=
Using centrifugal liquid-liquid partition chromatography (manufactured by Miki Engineering Co., Ltd.) with the upper layer of the 5:1°4 solution as a moving layer and the lower layer as a fixed layer, the antibiotic NK1 was purified by the ascending method.
960 mg of crude material containing 55141 was obtained. The obtained crude substance was dissolved in acetone and purified using a Toyopearl HW-40 column equilibrated with the same solvent, and the active fractions were collected and concentrated to dryness to obtain 380 mg of a crude powder. This was subjected to preparative high performance liquid chromatography (column: 0DS-5301-N (20φ
X300mm) Eluent: 80% (v/v) aqueous acetonate) IJl, flow rate: 10 mlzfnin, detection: UV detector). The portion corresponding to the peak around the retention time of 36 minutes was collected and concentrated under reduced pressure to obtain antibiotic NK155141.
7.8 mg of colorless powder was obtained.

[Brief explanation of drawings]

FIG. 1 shows the ultraviolet absorption spectrum of a 25 μg/frII methanol solution of substance NK155141. Figure 2 shows NK15514 measured as potassium bromide tablets.
1 infrared spectrum. FIG. 3 is a hydrogen nuclear magnetic resonance spectrum of NK155141 measured in deuterated chloroform. FIG. 4 is a carbon nuclear magnetic resonance spectrum of NK155141 measured in deuterated chloroform.

Claims (1)

[Claims] 1) Antibiotic NK155141 represented by the formula ▲There are mathematical formulas, chemical formulas, tables, etc.▼. 2) Antibacterial agent containing the antibiotic NK155141 as an active ingredient.
Antifungal agent 3) Anticancer agent containing antibiotic NK155141 as an active ingredient 4) Insecticide containing antibiotic NK155141 as an active ingredient 5) Antibiotic NK1551 belonging to the genus Streptomyces
41-producing bacteria were cultured in a nutrient medium to produce antibiotic NK1551.
41 was produced and accumulated, and the antibiotic NK1 was produced from the culture.
Antibiotic NK1 characterized by collecting 55141
55141 manufacturing method. 6) Streptomyces sp. no. NK15514
1 (Feikoken Bacillus No. 11102) strain