JPH07165761A - New physiologically active substance sf2771 compound and its production - Google Patents

Abstract

PURPOSE:To obtain the subject compound inducing a promotive activity by neutrophils against tumor cells, useful as an immunoenhancing agent and as a material for obviating the interaction between tumor cells and phagocytes such as neutrophils, by taking from a cultured product obtained by culturing specific bacteria belonging to Streptomyces. CONSTITUTION:This compound of the formula is obtained from a cultured product obtained by culturing SF2771 substance-productive bacteria belonging to Streptomyces (FERM P-13884). The physicochemical properties of the compound of the formula are as follows: color an appearance: colorless needlelike crystal; melting point: 178-180 deg.C; molecular formula: C9H12N2O2; molecular weight: 180; specific rotatory power: [alpha]<25>D=109.6 deg. (c=1.0, methanol); solubility: readily soluble in water, methanol, ethyl acetate, acetone and chloroform, sparingly soluble in hexane; color reaction: positive to molybdic acid and iodine, negative to ninhydrin; the Rf value in silica gel thin-layer chromatography: butanol:(acetic acid:water(4:1:2)) 0.85.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a novel bioactive substance SF2771.
The present invention relates to substances and their manufacturing methods. Tumor cells are considered to be eliminated by innate immunity as well as adaptive immunity, and it is known that tumor cells are damaged by neutrophils in vitro. (Morikawa et al .; Cancer R
es.45 3482 (1985))

Attack of neutrophils, macrophages, natural killer cells, etc. on tumor cells is promoted by cytokines such as interleukins and monocyte chemoattractants as well as various immunopotentiators. Elucidation of such interaction between tumor cells and phagocytes is useful for elucidating the mechanism of bacterial infection and inflammatory response, as well as malignant transformation of cancer. In addition, substances that affect the interaction between tumor cells and phagocytes are expected to be applied as medicines for treating malignant tumors, treating bacterial infections, treating inflammation, and improving immune function.

[0003]

2. Description of the Related Art As a system for observing the interaction between tumor cells and neutrophils, the present inventors have established human lung giant cell tumor LU65C and human lung tumor A549 cells and neutrophils. We have established a system for measuring tumor cell damage caused by neutrophils by mixing them, and in the case of human lung giant cell tumor LU65C, secreted cytokines (LUCT / IL-8) were clarified and neutrophils were identified. We report that it acts as a chemotactic factor and its gene structure. (Suzuki et al .; J. Exp.
Med.169 1895 (1989), Hotta et al .; Immunology Lette
r 24 165 (1990))

[0004] The problem to be solved by the present invention is to add various microbial cultures to a mixed culture system using human lung tumor A549 cells as target cells and neutrophils as an effector, to show tumor cell cytotoxic activity by neutrophils. Find new compounds that promote,
In addition to malignant transformation of cancer, it is to provide new materials useful for elucidating the mechanism of bacterial infection and inflammatory response.

[0005]

In order to screen for novel compounds that affect the interaction between tumor cells and phagocytes, the present inventors have performed a human preference for A549 cells, which are human lung tumor cells, on 96-well plates. After the neutrophils were made to adhere, and the culture liquids of various microorganisms were added and cultured, the number of surviving tumor cell colonies was measured by crystal violet staining to determine the effect of the added microorganism culture liquid on the neutrophil tumor cytotoxic activity. evaluated. Human lung tumor cells A549 cells can be obtained from JCRB cell bank of National Institute of Health or RIKEN Cell Bank of RIKEN. Moreover, human neutrophils can be easily prepared by the method of the present inventors. (Suzuki et
al .; Cell Biochem. Func. 3 297 (1985))

The present inventors have added various microbial culture solutions to the above-described mixed culture system of neutrophils adhered to human lung tumor A549 cells, and added neutrophils to the culture solution of actinomycetes belonging to Streptomyces. Was found to have a tumor cell injury-promoting effect, and a novel compound SF2771 was obtained as an active substance from the culture solution.
The material was isolated to complete the present invention. That is, the present invention is
The present invention relates to a novel bioactive substance SF2771 substance produced by actinomycetes belonging to Streptomyces and a method for producing the same. Therefore, the gist of the first aspect of the present invention lies in a novel bioactive substance SF2771 substance having the following physicochemical properties.

1. Physicochemical properties of SF2771 substance (1) Color and properties Colorless needle crystals (2) Melting point 178-180 ° C (3) Molecular formula C ₉ H ₁₂ N ₂ O ₂ (4) Molecular weight EI-MS (m / z) 180 [M] ⁺ SI-MS (m / z) 181 [M + H] ⁺ HREI-MS (m / z) Calculated value 180.0926 Experimental value 180.0912 (5) Specific rotation [α] _D ²⁵ = + 109.6 ° ( c = 1.O, methanol) (6) Ultraviolet absorption spectrum λmax = 225.8 nm (in methanol)

(7) Infrared absorption spectrum KB
The spectrum measured with r-lock

FIG. 1 shows. (8) Nuclear magnetic resonance spectrum Hydrogen nuclear magnetic resonance spectrum in deuterated dimethyl sulfoxide

Fig. 2 shows the carbon nuclear magnetic resonance spectrum

FIG. 3 shows. (9) Solubility water, methanol,
Easily soluble in ethyl acetate, acetone, chloroform, sparingly soluble in hexane. (10) Color reaction Molybdic acid, positive for iodine, negative for ninhydrin. (11) Rf value of silica gel thin layer chromatography Butanol: acetic acid: water (4: 1: 2) 0.85

From the physicochemical properties of the above-mentioned SF2771 substance and various spectral data, it is presumed that the SF2771 substance has a molecular formula of C ₉ H ₁₂ N ₂ O ₂ and has a diketopiperazine structure represented by the following structural formula. It

[0010]

[Chemical 2] However, since the structure is different from the compounds having a diketopiperazine structure found so far, SF
2771 substances were determined to be new substances.

2. SF2771 substance-producing bacterium As an example of the novel physiologically active substance SF2771 substance-producing bacterium used in the present invention, there is Streptomyces sp. SF2771 strain which is isolated from soil in Tokyo and belongs to the genus Streptomyces. The mycological properties of Streptomyces sp. SF2771 strain are as follows.

I. Morphology Basal hyphae are elongated, well branched, and do not divide under normal conditions. Aerial aerial hyphae abundantly grow on starch agar, oatmeal agar, yeast / malt agar, etc. and have good spore formation. There are relatively few aerial mycelium branches, and no axle branches are seen. The spore chain at the tip of the aerial hyphae is spiral or curled. The spores were oval-shaped by observation with an electron microscope.
Cylindrical type, with a size of 0.5-0.7 x 0.6-1.3 μm,
The surface is smooth and usually 30 to 50 chains are linked. No sporangia, motile spores, sclerotia, etc. were observed.

II. Growth conditions on various media The growth conditions of the SF2771 strain on various media are shown in Table 1 below. Regarding the description of colors, the standard shown in () is the Container Corporation of America (Containe
The product described in “Color Harmony Manual” manufactured by R Corporation of America was used. The observation was performed after culturing at 28 ° C for 14 to 21 days.

[0014]

[Table 1] Cultivation Growth (color on the back side) Aerial mycelium Soluble pigment Sucrose / Nitrate agar Weak, None Poor, None Glucose / Asparagine Agar Weak, None Poor, None Glycerol / Asparagine Agar Good, None Rich, White (a) None Starch agar good, brown rich, gray peach (a-5dc) None Oatmeal agar Normal, none Rich, gray peach (5dc) None Yeast / malt agar good Brown rich, gray peach (5ba) None Tyrosine agar good, brown rich, White (13ba) None Nutrient agar good, none Rich Gray peach (5ba) None Malic acid / calcium agar Normal, none Rich, none Bennett agar Good, none Rich, none

III. Physiological properties (1) Growth temperature range: In yeast-starch agar
It grows in the temperature range of 20-37 ℃, and grows well around 25-30 ℃. (2) Liquefaction of gelatin: Negative (3) Hydrolysis of starch: Positive (4) Reduction of nitrate; Positive (5) Peptonization of skim milk: Negative Coagulation of skim milk: Negative (6) Salt tolerance: 7% NaCl It grows in the medium containing but not 10%. (7) Formation of melanin-like pigment: negative

IV. Utilization of carbon source (using ISP-9 medium) (1) Utilization: D-glucose, D-fructose,
D-xylose, glycerol, L-arabinose, D
-Mannitol, myo-inositol, raffinose
L-rhamnose (2) Not used: sucrose, V.I. Cell analysis Becker et al. (Appl. Microbiol. 13: 2
36, 1965), diaminopimelic acid in the whole cell hydrolyzate was LL type.

From the above properties, the SF2771 strain belongs to the genus Streptomyces among actinomycetes, the aerial mycelia color tone is Red series, the aerial mycelia tip is spiral to curled, the spore surface is smooth and the backside color tone is Are colorless to dark brown and are summarized as strains that do not produce significant soluble pigment. We have SF27
71 shares of Streptomyces sp. SF2771 (Stre
ptomyces sp. SF2771). This strain has been deposited as FERM P-13884 at the Institute of Biotechnology, Institute of Biotechnology, AIST.

The SF2771 strain, as found in other actinomycetes,
Its properties are likely to change. For example, the SF2771 strain, or a mutant strain (spontaneous or inducible) derived from this strain,
Any zygote or gene recombinant that produces the physiologically active substance SF2771 can be used in the present invention.
In the method of the present invention, the above-mentioned bacterium is cultivated in a medium containing nutrients that can be utilized by ordinary microorganisms. As a nutrient source,
Known substances conventionally used for culturing actinomycetes can be used. For example, as the carbon source, glucose, starch syrup,
Dextrin, starch, molasses, animal and vegetable oils, etc. may be used. As the nitrogen source, soybean meal, wheat germ, corn steep liquor, cottonseed meal, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, urea and the like can be used. In addition, if necessary, sodium, potassium, calcium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid,
And it is effective to add inorganic salts capable of producing other ions. In addition, organic and inorganic substances that help the growth of bacteria and promote the production of the physiologically active substance SF2771 can be added appropriately.

As the culture method, a culture method under aerobic conditions,
Particularly, the deep culture method is most suitable. Suitable temperature for culturing
The temperature is 25 to 30 ° C, but in most cases, the culture is performed at around 28 ° C.
The production of the novel physiologically active substance, SF2771 substance, varies depending on the medium and culture conditions, but in both shaking culture and tank culture, the maximum accumulation is usually reached within 2 to 7 days. When the accumulated amount of the novel bioactive substance SF2771 in the culture reaches the maximum, the culture is stopped, and the target substance is isolated and purified from the culture solution.

Examples of the present invention will be shown below, but these are merely examples and do not limit the present invention. Of course, many modified or modified means not exemplified here can be used.

3. Method for Purifying SF2771 Substance Since the SF2771 substance thus produced has the above-mentioned physicochemical properties, it can be purified from the culture broth according to the properties, but in particular, it can be efficiently purified by the following method. it can.

That is, the solid matter is separated from the culture containing the active ingredient by filtration to separate the filtrate and the solid matter. The filtrate is treated with activated carbon to adsorb the active substance on the resin. Then, the solvent is distilled off by elution with a suitable solvent such as acetone, and the eluate is concentrated under reduced pressure to give an aqueous solution. This aqueous solution is treated with the adsorption resin Diaion HP20 or SepaBeads SP207 (manufactured by Mitsubishi Kasei) to adsorb the active substance on the resin. Then, it is eluted with a suitable solvent such as methanol, and the eluate is concentrated under reduced pressure to obtain an active crude powder. The crude powder is subjected to gel filtration using Sephadex G-10 (Pharmacia), and the obtained active solution is concentrated and then crystallized to isolate the SF2771 substance.

In the assay of the SF2771 substance, a biological assay method for measuring the ability of this substance to enhance the cytotoxic activity of neutrophils against tumor cells and a chemical assay method using silica gel thin layer chromatography or HPLC can be used. it can. Examples of the present invention will be shown below, but these are merely examples and do not limit the present invention. It goes without saying that many modified or modified means not exemplified here can be used.

[0024]

Example: Starch 2.0%, glucose as seed medium
1.0%, polypeptone 0.5%, wheat germ 0.6%, yeast extract 0.3%, soybean meal 0.2% and calcium carbonate 0.2
%, (PH 7.0 before sterilization) was used as the medium. As a production medium, starch syrup 2.0%, soybean oil 0.15%, soybean meal 1.0%, sun grains 0.25%, pharma media
0.5%, ferrous sulfate 0.001%, nickel chloride 0.0001%,
A medium having a composition of 0.0001% cobalt chloride and 0.15% calcium carbonate (pH 7.0 before sterilization) was used.

A 100 ml Erlenmeyer flask, to which 20 ml of the seed medium was dispensed, was sterilized at 121 ° C. for 20 minutes, and 2 to 3 platinum of the agar culture of Streptomyces sp. SF2771 strain (FERM P-13884) was sterilized. The ears were inoculated and cultured with shaking at 28 ° C. for 24 hours to prepare a first seed culture. Then, a 500 ml Erlenmeyer flask into which 80 ml of the seed medium was dispensed was sterilized at 121 ° C. for 30 minutes, 4 ml of the first seed culture was inoculated, and the mixture was shake-cultured at 28 ° C. for 24 hours, and this was designated as the second seed culture.

300 ml of the above-mentioned second seed culture was inoculated into a 10 L jar fermenter containing 6 L of production medium sterilized at 121 ° C. for 30 minutes in advance, and aeration (17.5 L /
Min) and agitated at 300 rpm. After the completion of the culture, diatomaceous earth was added as a filter aid and the mixture was filtered.

50 L of the filtrate thus obtained was added with 5 L of activated carbon.
After adsorbing the active ingredient on the above column and washing with 30 L of water, the active ingredient was eluted with 15 L of 50% acetone water (pH 2.5). Further, 10 L of an aqueous layer obtained by distilling acetone off from the eluate by concentration under reduced pressure was applied to a 3 L column of Diaion HP20 (manufactured by Mitsubishi Kasei) to adsorb the active ingredient, and then 10 L of water was adsorbed.
After washing with, the active ingredient was eluted with 5 L of 50% acetone water (pH 2.5). Furthermore, 3 L of an aqueous layer obtained by distilling acetone off from the eluate by vacuum concentration was put on a 500 ml Sepabeads SP207 (manufactured by Mitsubishi Kasei) column to adsorb the active ingredient, and then water.
After washing 4 L, the active ingredient was eluted with 2 L of 50% acetone water. The SF2771 substance in the eluted fraction was analyzed by HPLC (column; Radial Pack 5C18 (Waters), developing solvent).
Detected with 20% methanol water, flow rate; 1 ml / min, detection; 250 nm).

The obtained active fractions were combined, concentrated under reduced pressure and freeze-dried to obtain 4.6 g of active crude powder. This powder was dissolved in a small amount of water and filled with water Sephadex G-
10 (manufactured by Pharmacia) was placed in a 1800 ml tower and developed with water to obtain an active fraction. Concentrate this active fraction to a small amount,
From this concentrated liquid, 620 mg of SF2771 substance was isolated as crude crystals. Further, the crude crystals were recrystallized from water to obtain SF2771 substance as colorless needle-shaped pure crystals.

[0029]

[Test Example] The collected SF2771 substance was added to a mixed culture system in which neutrophils were adhered to human lung tumor A549 cells, and the influence on the interaction of phagocytes with tumor cells was examined. That is, human lung tumor A549 cells cultured in MEM medium containing 10% fetal bovine serum were seeded at 10 ⁴ / well on a 96-well plate and cultured for 2 days.

[0030] To this cell layer, 2 x 10 ⁵ neutrophils prepared by the method of Suzuki et al. Were dispensed by 2 x 105 to adhere the neutrophils, and then each concentration of SF2771 substance was added to the cell in a CO ₂ incubator. After culturing at 37 ℃ for 4 hours, trypsin treatment is performed.
0 A549 cells were seeded on another 96-well plate and the culture was continued for 4 days in a CO ₂ incubator at 37 ° C. The number of remaining colonies of tumor cells was determined by the OD method using crystal violet (54
6 nm). As shown in Table 2 below, it is clear that the SF2771 substance does not show tumor cytotoxicity by itself, but promotes the tumor cytotoxicity activity of neutrophils.

[0031]

[Table 2] Addition amount of SF2771 Residual amount of tumor cells OD 546nm (% survival) (μg / ml) No neutrophil addition No neutrophil addition 0.407 (100) 0.414 (100) 0.073 0.311 (76) 0.364 (88) ) 0.73 0.384 (94) 0.329 (79) 7.3 0.279 (69) 0.101 (24) 22 0.324 (80) 0.096 (23) 66 0.363 (89) 0.069 (17)

[0032]

INDUSTRIAL APPLICABILITY Since the novel physiologically active substance SF2771 substance according to the present invention has an action of promoting neutrophil tumor cell-damaging activity, it is expected to have an effect as an immunopotentiator, tumor cells and neutrophils, etc. It is expected to be used as a valuable material in clarifying the interaction of erythrocytes with phagocytes.

[Brief description of drawings]

FIG. 1 shows an infrared absorption spectrum of SF2771 substance in a KBr tablet.

FIG. 2 shows a hydrogen nuclear magnetic resonance spectrum of SF2771 substance measured in deuterated dimethyl sulfoxide.

FIG. 3 shows a carbon nuclear magnetic resonance spectrum of SF2771 substance measured in deuterated dimethyl sulfoxide.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) From the inventor Masahiro, 760 Shimooka-cho, Kohoku-ku, Yokohama-shi, Kanagawa Meiji Seika Co., Ltd., Pharmaceutical Research Laboratory (72) Akira Shimizu 760, Shimooka-cho, Kohoku-ku, Yokohama-shi, Kanagawa Meiji Seika Co., Ltd., Pharmaceutical Research Laboratory (72) Inventor Shoichi Amano, 760, Shimooka-cho, Kohoku-ku, Yokohama-shi, Kanagawa Meiji Seika Co., Ltd., Pharmaceutical Research Institute (72) Satoshi Imai, Shimooka-cho, Kohoku-ku, Yokohama, Kanagawa Prefecture 760 Meiji Confectionery Co., Ltd. Pharmaceutical Research Laboratory (72) Inventor Seiji Shibahara 760 Shimooka-cho, Kohoku-ku, Yokohama-shi, Kanagawa Meiji Confectionery Research Institute

Claims (2)

[Claims] 1. The following structural formula: Compound shown by 2. An SF271 substance-producing bacterium belonging to the genus Streptomyces is cultured, and the SF27 according to claim 1 is obtained from the culture.
A method for producing SF2771 substance, which comprises collecting 71 substances.