JPH0422917B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to a novel physiologically active substance SF2547 and a method for producing the same. Background Art and Problems to be Solved by the Invention In the past, many physiologically active substances have been invented and put into practical use in the fields of pharmaceuticals, veterinary drugs, agricultural chemicals, and the like. However, since no effective substance has been found yet, there remain many unsolved medical and industrial fields. The present inventors have focused on the above-mentioned points, and have attempted to solve the problems by providing a novel physiologically active substance and establishing a method for producing the same. Means for Solving the Problems As a result of continuing the search for a new useful physiologically active substance, the present inventors discovered a novel physiologically active substance that has excellent antitumor activity in a culture of a certain strain belonging to the genus Streptomyces. They discovered that SF2547 substance is being produced and completed the present invention. That is, the present invention provides novel physiologically active substances.
The present invention provides an SF2547 substance and a method for producing the same. The SF2547 substance and its manufacturing method will be specifically explained below. (1) Producing bacteria and mycological properties of the producing bacteria The SF2547 substance producing bacteria used in the method of the present invention must be prepared in a sufficient amount to be collected in the culture.
Any strain may be used as long as it has the ability to produce the SF2547 substance; an example of such a strain is the SF2547 strain newly isolated by the present inventors from soil in Shiga Prefecture. SF2547
The mycological properties of the strain are as follows. Morphology The basal hyphae are elongated, well branched, and do not divide under normal conditions. Aerial mycelial attachment is extremely poor, with only a small amount of attachment occurring on sucrose/nitrate agar, and spore formation is barely observed. The branching of aerial hyphae is simple, and no axle branching is observed. The spore chain at the tip of the aerial hyphae is mainly linear, but wavy and loop shapes are also observed. When observed using an electron microscope, the spores are cylindrical and 0.5−0.7×0.9−
It has a size of 1.4 μm and a smooth surface. Spores usually form chains of 10 or more spores. No spore settlement, sporangia, motile spores, or sclerotia were observed on the basal hyphae. Growth status on various media The growth status of SF2547 strain on various media is as shown in Table 1. Regarding color descriptions: Standards shown in parentheses are those of Container Corporation of America.
The one described in the "Color Harmony Manual" manufactured by America) was used. Observations were made after culturing at 28°C for 14-21 days.

[Table] Physiological properties (1) Growth temperature range: in yeast and malt agar
It grows in the temperature range of 15-34℃ and grows well at 25-30℃. (2) Liquefaction of gelatin: Negative (3) Hydrolysis of starch: Negative (4) Reduction of nitrate: Negative (5) Peptonization of skim milk: Negative Coagulation of skim milk: Negative (6) Salt tolerance: 3% It grows in a medium containing NaCl, but does not grow in a medium containing NaCl above 4%. (7) Production of melanin-like pigment: Negative Carbon source utilization (ISP No. 9 medium used) (1) Used: D-glucose, glycerol (2) Not used: D-xylose, L-arabinose, raffinose, D-mannitol, sucrose (3) Use questionable: D-fructose, myo-
Inositol, L-rhamnose Cell wall composition The method of Becker et al. (Appl.
Microbiol. 13 :236, 1965).
Diaminopimelic acid in the cell wall composition was of the LL type. Based on the above characteristics, it is reasonable to consider that strain SF2547 belongs to the genus Streptomyces among actinomycetes. The present inventors identified the SF2547 strain as Streptomyces sp.
SF2547). In addition, strain SF2547 has been entrusted to the Institute of Microbial Technology, Agency of Industrial Science and Technology as FERM P-9531. The SF2547 strain is susceptible to changes in its properties, as seen in the case of other actinomycetes. For example, SF2547
Any strain or mutant strain (naturally occurring or induced) derived from this strain, trait zygote, or genetically modified strain that produces the new physiologically active substance SF2547 can be used in the present invention. (2) Cultivation method In the method of the present invention, the above-mentioned bacteria are cultured in a medium containing nutrients that can be used by ordinary microorganisms. As the nutrient source, known nutrient sources conventionally used for culturing actinomycetes can be used. For example, glucose, starch syrup, dextrin, sucrose, molasses, animal/vegetable oil, etc. can be used as the carbon source. Further, as a nitrogen source, soybean flour, wheat germ, cornstarch liquor, cottonseed meal, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, urea, etc. can be used. Others, as necessary.
It is useful to add inorganic salts capable of producing sodium, potassium, calcium, magnesium, cobalt, chlorine, phosphate, sulfate, and other ions. In addition, organic and inorganic substances that aid the growth of bacteria and promote the production of the new physiologically active substance SF2547 can be appropriately added. The most suitable culture method is a culture method under aerobic conditions, especially a deep culture method. The appropriate temperature for culturing is 15-34°C, but in many cases it is cultured at around 28°C. Production of the new physiologically active substance SF2547 differs depending on the medium and culture conditions, but its accumulation usually reaches its maximum within 2 to 7 days in both shaking culture and tank culture. When the accumulated amount of the new physiologically active substance SF2547 during culture reaches its maximum, the culture is stopped, and the target substance is isolated and purified from the culture solution. (3) Purification When collecting the SF2547 substance obtained according to the present invention from a culture, conventional separation methods utilizing its properties, such as solvent extraction method, ion exchange resin method, adsorption or distribution column, can be used. Extraction and purification can be carried out by chromatography, gel filtration, dialysis, precipitation, etc. alone or in appropriate combinations. for example,
The SF2547 substance is extracted from the cultured bacterial cells with acetone-water or methanol-water. Furthermore, if extraction is performed using an organic solvent that does not mix with water, such as ethyl acetate or chloroform, the SF2547 substance will be extracted into the organic solvent layer. To further purify the SF2547 substance, adsorbents such as silica gel (Wako Gel C-300, manufactured by Wako Pure Chemical Industries, Ltd.), alumina, and Cephadex LH can be used.
Chromatography may be performed using -20 (manufactured by Pharmacia) or the like. (4) Physicochemical properties New physiologically active substance obtained according to the above production method
SF2547 substance has the following physicochemical properties. Color and properties of substance: Colorless prismatic crystal Melting point: 227-228℃ (decomposition) Elemental analysis: Carbon 55.62%, Hydrogen 8.15%, Nitrogen 11.45% Specific optical rotation: [α] ²⁴ _D +138° (c0.5, (Chloroform) Ultraviolet absorption spectrum: The spectrum measured in a methanol solution with a concentration of 150 μg/ml is as shown in Figure 1.
Its absorption value (λmax(MeOH)) is 210 nm. Infrared absorption spectrum: The spectrum measured in the potassium bromide tablet is as shown in Figure 2, and its absorption values (cm ^-1 ) are as follows. 3350, 3250, 2950, 2920, 1740, 1630, 1490,
1440, 1350, 1320, 1300, 1255, 1220, 1200,
1185, 1145, 1120, 1090, 1045, 1020, 1000,
970, 930, 910, 880, 860, 835, 810, 765,
690, 655, 590, 525, 490 ¹ H NMR spectrum (400MHz): As shown in Figure 3, measured in a deuterated chloroform solution using TMS as a reference material, and its δ
The values (ppm) are as follows. 9.77 (1H, s), 8.31 (1H, s), 8.26 (1H, d),
6.89 (1H, d), 6.51 (1H, s), 5.97 (1H, m),
5.91 (1H, s), 5.42 (2H, m), 5.18 (1H, d),
5.06 (1H, d), 4.81-4.90 (4H, m), 4.34
(1H, d), 3.61 (2H, m), 3.49 (1H, s),
3.24 (1H, m), 3.09 (2H, m), 3.03 (1H, m),
2.92 (1H, m), 2.80 (1H, m), 2.46 (1H, m),
2.36 (1H, m), 2.22 (1H, m), 2.17 (1H, m),
2.01 (1H, m), 1.90 (1H, m), 1.85 (1H, m),
1.70~1.78 (3H, m), 1.67 (1H, m), 1.51~
1.59 (3H, m), 1.47 (3H, s), 1.46 (3H, s),
1.41 (1H, m), 1.33-1.42 (5H, m), 1.25
(1H, m), 1.22 (2H, m), 1.00~1.16 (8H,
m), 0.96 (3H, d), 0.91 (3H, d), 0.86 (3H,
t), 0.81 (3H, d), 0.80 (3H, t) ¹³ C NMR spectrum (100MHz): As shown in Figure 4, measured in a deuterated chloroform solution using TMS as a reference substance, and its δ
The values (ppm) are as follows. 179.6 (s), 177.4 (s), 171.4 (s), 170.7 (s)
，
169.4 (s), 168.1 (s), 165.9 (s), 99.0 (s)
，
78.4(s), 76.8(d), 75.7(d), 68.1(d), 62
.6
(d), 59.0 (d), 55.9 (d), 54.3 (t), 50.9
(d), 49.9(d), 47.7(d), 46.6(t), 45.9
(d), 45.9 (t), 38.1 (t), 37.3 (t), 35.8
(d), 31.0 (d), 30.9 (t), 29.4 (d), 29.4
(t), 29.1 (d), 27.8 (t), 27.3 (q), 24.8
(t), 24.8 (t), 23.9 (t), 20.6 (t), 20.4
(q), 19.9 (q), 19.6 (q), 19.3 (q), 18.6
(q), 15.5 (q), 14.5 (q), 11.6 (q), 8.7 (
q) Solubility: Soluble in chloroform and ethyl acetate, slightly soluble in methanol and acetone, insoluble in water and hexane. Color reaction: Greig-Leaback
Positive for reagent and 10% sulfuric acid reagent. Thin layer chromatography: Support: Silica gel plate (manufactured by Merck & Co.)

[Table] (5) Antitumor effect Table 2 shows the 50% growth inhibitory concentration (IC ₅₀ ) of the new physiologically active substance SF2547 against various cancer cells.

[Table] (6) Antibacterial activity No antibacterial activity at a concentration of 1 mg/ml (paper disc method). (7) Acute toxicity Acute toxicity was tested in ICR mice (male, 5 weeks old) with 3 mice per group, and the LD ₅₀ was 4.7 after intraperitoneal administration.
It was mg/Kg. Conventionally, various physiologically active substances produced by microorganisms have been known, but Azinothricin [J.
Antibiotics Vol. 39, 17-25 (1986)]. However, ¹ H NMR of SF2547 substance and
The ¹³ C NMR spectrum is different from that of azinothricin, and although azinothricin has a strong antibacterial effect against Gram-positive bacteria,
SF2547 substance does not show antibacterial activity (1 mg/ml,
paper disk method) and other physical and chemical properties are clearly different from azinothricin, and are clearly distinguishable. Therefore, this SF2547 substance is a new substance. Examples Examples of the present invention are shown below, but these are merely examples and do not limit the present invention.
Of course, many variations and modifications not exemplified here may be used. Example 1 Starch 1.0%, glucose 2.0% as seed medium
%, wheat embryo 0.6%, polypeptone 0.5%, yeast extract 0.3%, soy flour 0.2%, calcium carbonate 0.1%
A medium containing the following was used. In addition, as a production medium, glucose 4.0%, wheat embryo 2.0%, sungrain F2
(manufactured by Sungrain) 0.5%, yeast extract 0.5%,
A medium containing 0.1% magnesium sulfate (heptahydrate) and 0.2% calcium carbonate was used. In addition, the pH before sterilization
All were adjusted to 7.0. A 100 ml Erlenmeyer flask containing 20 ml of the seed medium was sterilized at 120°C for 30 minutes, and Streptomyces sp. SF2547 (FERM P-9531) was added to the flask.
Platinum loops 1-2 of the slant culture were inoculated and cultured with shaking at 28°C for 3 days to form the first type culture. Next, the 500 ml Erlenmeyer flask containing 80 ml of the seed medium was heated at 120°C.
Sterilize for 30 minutes, inoculate with 2 ml of the above type 1 culture and incubate at 28°C.
The cells were cultured with shaking for 2 days, and this was used as a second type culture. Two 50L jar fermenters containing 35L of production medium previously sterilized at 120℃ for 30 minutes were inoculated with 160ml each of the above type 2 culture, and cultured at 28℃ for 6 days with aeration (20L/min) and stirring (250rpm). did. After culturing, diatomaceous earth is added as a filter aid and filtered.
A solid containing 40 L of filtrate and bacterial cells with a compressed volume of 10 L was obtained. Example 2 40L of 50% acetone water was added to the bacterial cells obtained in Example 1 to extract the active ingredient. After filtering the bacterial cells, concentrate the bacterial cell extract to 10L, and add 4L of Diaion HP-20 (manufactured by Mitsubishi Kasei Corporation) along with 40L of culture filtrate.
column. The active ingredient was extracted from this pass-through with 20 L of ethyl acetate. The extracted ethyl acetate solution was concentrated under reduced pressure to obtain 2.07 g of an oily substance. 2.07 g of this oily substance was sprinkled on 15 g of silica gel, dried under reduced pressure, and then packed onto the top of a 300 ml silica gel column that had been filled with chloroform in advance. When 1000 ml of chloroform, 600 ml of chloroform-methanol (200:1), and 1200 ml of chloroform-methanol (100:1) were developed in order, fractions No. 140-157 (15 ml/fraction) contained SF2547.
An active fraction containing mainly the substance was obtained. This fraction was concentrated under reduced pressure, subjected to column chromatography using Sephadex LH-20 (800 ml) filled with methanol in advance, developed with methanol, active fractions were collected, and concentrated to dryness under reduced pressure. Colorless prismatic crystals (36 mg) of SF2547 substance were obtained by crystallization from a mixed solvent of chloroform-methanol.
was gotten.

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption spectrum of SF2547 substance (150 μg/ml in methanol solution). Second
The figure is an infrared absorption spectrum diagram of SF2547 substance (potassium bromide tablet). Figure 3 shows the SF2547 substance.
^1H NMR spectrum (in deuterated chloroform solution,
400MHz). Figure 4 shows ^13C of SF2547 substance.
NMR spectrum (in deuterated chloroform solution,
100MHz).

Claims (1)

[Claims] 1. A new physiologically active substance having the following physical and chemical properties:
SF2547 substance Color and properties of substance: Colorless prismatic crystal Melting point: 227-228℃ (decomposed) Elemental analysis: Carbon 55.62%, hydrogen 8.15%, nitrogen 11.45% Specific rotation: [α] ²⁴ _D +138° (c0. 5, Chloroform) Ultraviolet absorption spectrum: The spectrum measured in a methanol solution with a concentration of 150 μg/ml is as shown in Figure 1.
Its absorption value (λmax(MeOH)) is 210 nm. Infrared absorption spectrum: The spectrum measured in the potassium bromide tablet is as shown in Figure 2, and its absorption values (cm ^-1 ) are as follows. 3350, 3250, 2950, 2920, 1740, 1630, 1490,
1440, 1350, 1320, 1300, 1255, 1220, 1200,
1185, 1145, 1120, 1090, 1045, 1020, 1000,
970, 930, 910, 880, 860, 835, 810, 765,
690, 655, 590, 525, 490 ¹ H NMR spectrum (400MHz): The spectrum measured in a deuterated chloroform solution using TMS as a reference material is as shown in Figure 3, and its δ value (ppm) is as follows: It is. 9.77 (1H, s), 8.31 (1H, s), 8.26 (1H, d),
6.89 (1H, d), 6.51 (1H, s), 5.97 (1H, m),
5.91 (1H, s), 5.42 (2H, m), 5.18 (1H, d),
5.06 (1H, d), 4.81-4.90 (4H, m), 4.34
(1H, d), 3.61 (2H, m), 3.49 (1H, s),
3.24 (1H, m), 3.09 (2H, m), 3.03 (1H, m),
2.92 (1H, m), 2.80 (1H, m), 2.46 (1H, m),
2.36 (1H, m), 2.22 (1H, m), 2.17 (1H, m),
2.01 (1H, m), 1.90 (1H, m), 1.85 (1H, m),
1.70~1.78 (3H, m), 1.67 (1H, m), 1.51~
1.59 (3H, m), 1.47 (3H, s), 1.46 (3H, s),
1.41 (1H, m), 1.33-1.42 (5H, m), 1.25
(1H, m), 1.22 (2H, m), 1.00~1.16 (8H,
m), 0.96 (3H, d), 0.91 (3H, d), 0.86 (3H,
t), 0.81 (3H, d), 0.80 (3H, t) ¹³ C NMR spectrum (100MHz): The spectrum measured in a deuterated chloroform solution using TMS as a reference material is as shown in Figure 4, and its δ value (ppm) is as follows. 179.6 (s), 177.4 (s), 171.4 (s), 170.7 (s)
，
169.4 (s), 168.1 (s), 165.9 (s), 99.0 (s)
，
78.4(s), 76.8(d), 75.7(d), 68.1(d), 62
.6
(d), 59.0 (d), 55.9 (d), 54.3 (t), 50.9
(d), 49.9(d), 47.7(d), 46.6(t), 45.9
(d), 45.9 (t), 38.1 (t), 37.3 (t), 35.8
(d), 31.0 (d), 30.9 (t), 29.4 (d), 29.4
(t), 29.1 (d), 27.8 (t), 27.3 (q), 24.8
(t), 24.8 (t), 23.9 (t), 20.6 (t), 20.4
(q), 19.9 (q), 19.6 (q), 19.3 (q), 18.6
(q), 15.5 (q), 14.5 (q), 11.6 (q), 8.7 (
q) Solubility: Soluble in chloroform and ethyl acetate, slightly soluble in methanol and acetone, insoluble in water and hexane. Color reaction: Greig-Leaback
Positive for reagent and 10% sulfuric acid reagent. Thin layer chromatography: Carrier: Silica gel plate (manufactured by Merck & Co.) [Table] 2 Characterized by culturing SF2547 substance-producing bacteria belonging to the genus Streptomyces in a medium, and collecting SF2547 substance from the culture. Production method of SF2547 substance. 3 The producing bacteria belonging to the genus Streptomyces is Streptomyces sp.
The manufacturing method according to claim 2, which is SF2547 (Feikoken Kyoiyori No. 9531).