JPH0432816B2 - - Google Patents

Description

[Detailed description of the invention]

(Technical field) The present invention provides a novel compound Deifalanisole A
(Differanisole A) and its manufacturing method. (Background of the Invention) In order to scientifically approach one of the most important problems in biology, which is differentiation and development, the present inventors have conventionally investigated the ability to control endogenous and extrinsic differentiation, particularly the ability to control differentiation. We have been searching for substances with induction ability and conducting scientific research on them. Then, using a simple and highly reproducible differentiation-inducing substance search system previously developed by the present inventors, we searched for differentiation-inducing substances in microbial metabolites, animal tissues, etc., and conducted search, purification, and structural analysis. As a result, we obtained the knowledge that a new substance, which has not yet been described in the literature, exhibits strong differentiation-inducing activity against Friend leukemia cells, is produced and accumulated in cultures of microorganisms belonging to the genus Chaetomium. Successful isolation and purification. (Object of the Invention) An object of the present invention is to provide a differentiation-inducing active substance Deifalanisole A obtained from a culture of a microorganism belonging to the genus Chaetomium, and a method for isolating and collecting the same. (Structure of the Invention) <Microorganisms Used> First, the microorganisms used in the present invention have the ability to produce Deifalanisole A, and are a species belonging to the genus Chaetomium. One example is Ketomium Sp. RB.
−001 (Chaetomium sp. RB−001) (hereinafter referred to as “RB
−001 shares.” ) is a strain having the above-mentioned characteristics, which advantageously produces the deifalanisole A of the present invention, and is mentioned as a microorganism that can be effectively used in the method of the present invention. In addition, not only natural and artificial mutant strains of the above-mentioned RB-001 strain, but also all microorganisms that belong to the genus Chaetomium and have the ability to produce Deifalanisole A of the present invention may be used in the method of the present invention. I can do it. Strain RB-001 is a filamentous fungus belonging to the genus Chaetomium that was discovered by the present inventor in a soil sample collected in Okazaki City, Aichi Prefecture. It was deposited on the 22nd, and its accession number is FERM P-8021. The mycological properties of the RB-001 strain are as follows. 1. Growth status on various agar media (27°C) (1) Potato/glucose agar medium Growth is medium, initially white cotton-like, and after 14 days of culture, cream-colored velvet or hair-like mycelia are formed. Covering the agar, angiospores with yellow-olive apical hairs become scattered. A brown pigment is observed in the agar after about 10 days of culture. (2) Growth from oatmeal agar medium and potato/glucose agar medium is as follows:
It is rather fast, white, fluffy and dense. After 14 days of culture, the hyphae turn from white to brown, and a yellow-brown pigment can be seen in the agar. The epiphytes of the angiosperms are also vigorous, giving the plant an olive color. (3) Sabouraud agar medium The growth of hyphae is extremely slow, the hyphae are white and extremely coarse, and no angiosperm formation is observed even after culturing for one month. (4) Cellulose agar medium It grows quickly, exhibits an olive color after 14 days of culture, and forms numerous angiosperms. The vegetative hyphae are coarse compared to the angiosperm formation. No pigment formation was observed in the agar. 2. Physiological Properties (1) Growth Temperature (Pallet/Glucose Agar Medium) At 10°C, there is almost no mycelial growth, and at 37°C, mycelial growth is very poor. 25-30°C is the optimal temperature for the growth of vegetative hyphae and the formation of angiosperms. (2) Growth PH: Will not grow below PH3.0 and above PH9.0. It grows well at pH 5.5 to 7.0. 3 Morphological observation using a microscope (27°C, Barley
Glucose agar medium) The angiosperm is spherical, close to oval, approximately 300 x 250 microns in size, open at the apex, and has numerous apical and lateral hairs. The apical hairs are long and spirally intertwined, with septa, and the lateral hairs are almost linear.
Asci are octosporous, club-shaped, and colorless; ascospores are lemon-shaped and size 9.0-12.0 x 7.0-9.0.
It has an olive color in micron size. As a result of comparing the above properties with those of known bacterial strains, LMAmes: A monograph of the
Chaetomiaceae.USArmy Res.Dev.Ser.No.2
(1961) and Shunichi Udagawa et al., 1978, Illustrated Guide to Sterility, published by Kodansha, etc., strain RB-001 was identified as a strain belonging to the phylum R. fungi, family Chaetomycota, genus Chaetomium. The properties of the strain belonging to the genus Chaetomium and having the ability to produce Deifalanisole A used in the present invention are not constant, and are exposed to natural or commonly used treatments such as ultraviolet irradiation, radiation irradiation, and chemical mutagens. Bacterial strains that can be mutated by the artificial mutation means used in the present invention belong to the genus Chaetomium and include all strains that produce Deifalanisole A. <Culture and Separation/Purification> The culturing method in the present invention can be carried out according to the usual culturing method for microorganisms. As the medium component, for example, as a carbon source, glycose, maltose, sucrose, molasses, glycerin, cellulose, powder, wrinkles, dextrin, starch, soybean oil, cottonseed oil, etc. can be used.
In addition, soybean flour, peanut flour, cottonseed flour, fish meal, corn steep liquor, peptone, meat extract, dried yeast, yeast extract, casein, ammonium sulfate, ammonium nitrate, ammonium chloride, sodium nitrate, etc. can be used as the nitrogen source. In addition to sulfates, hydrochlorides, phosphates of magnesium, potassium, sodium, calcium, etc., trace metal salts and the like can also be used as necessary. The medium is usually preferably a liquid medium, and the culture is preferably carried out under aerobic conditions. Conditions such as culture temperature and culture time are selected to be suitable for the growth of the bacteria used and to maximize the production of deifalanisole A. For example, the pH of the medium is slightly acidic to neutral, preferably PH6-7, and the culture temperature is 25-30.
°C is preferred. However, these culture conditions, such as the culture composition, hydrogen ion concentration of the medium, culture temperature, and stirring conditions, should be adjusted as appropriate to obtain favorable results depending on the type of bacterial strain used and external conditions. Needless to say, it is. In order to obtain deifalanisole A from the culture obtained in this manner, means commonly used for collecting metabolites can be appropriately used. For example, any of the means utilizing the solubility difference between Deifalanisole A and impurities and the means utilizing the difference in adsorption affinity may be used alone, in combination, or repeatedly. Specifically, most of Deifalanisole A is present in the culture solution, but from this it can be separated and purified, for example, by the following steps.

[Table] ↓

[Physicochemical and biological properties of Deifalanisole A]

(1) Melting point: 128℃ (2) High-resolution mass spectrometry: (molecular weight) 278 (molecular formula) C ₁₁ H ₁₂ O ₄ Cl ₂ (3) Ultraviolet absorption spectrum: (as shown in Figure 1) In methanol, the following wavelengths shows maximum absorption. (nm) (ε) 318 2700 257 (shoulder) 4060 (4) Infrared absorption spectrum: (as shown in Figure 2) 3450, 2995, 2895, 1675, 1635, which has a characteristic absorption band at the middle wave number of KBr . 1560,
1463, 1415, 1365, 1260, 1225, 1197,
1120, 1085, 1025, 955, 900, 887, 865,
825, 778, 758, 720 cm ^-1 (5) Nuclear magnetic resonance spectra (protons, CDCl ₃ ,
400MHz): As shown in Figure 3. (6) Nuclear magnetic resonance spectrum ( ^13C , _CD3OD ,
100MHz): As shown in Figure 4. (7) Solubility: Soluble in water, methanol, acetone, dimethyl sulfoxide, and dimethyl formamide. Insoluble in n-hexane, petroleum ether. (8) Color reaction: Iodine, potassium permanganate reaction...Positive Fehring's solution, 2,4-dinitrophenylhydrazine...Negative (9) Biological activity: At concentrations of 5 μg/ml or higher, erythroid leukemia ( B8) Shows a differentiation-inducing effect on cells,
At 20 μg/ml, approximately 40% hemoglobin is induced in B8 cells. Deifalanisole A of the present invention was identified as a novel substance having the following structural formula from the above-mentioned physicochemical properties and X-ray crystal analysis. <Structural formula of Deifalanisole A> Deifalanisole A is expected to be used as an antitumor agent because it exhibits a differentiation-inducing effect on mouse erythroid leukemia cells and an effect on inducing normal cells. The present invention will be explained in detail below by way of examples. Examples (Medium used) Sugar 2% Corn steep liquor 1% Peptone 0.2% Yeast 0.05% Salt 0.1% K ₂ HPO ₄ 0.02% MgSO _{4.7H 2} O 0.05% (PH6.0) <Flask culture> Above 100 ml of the culture medium was dispensed into a 500 ml flat-bottomed flask and steam sterilized at 120°C for 30 minutes, then inoculated with RB-001 strain (Feikoken Bacteria No. 8021) and cultured at 27°C for 3 days with reciprocal shaking. This was used as the seed culture solution. Dispense 100 ml of the same medium into a 500 ml Erlenmeyer flask, steam sterilize it at 120°C for 30 minutes, inoculate it with 5 ml of the seed culture, culture it with rotary shaking at 27°C for 5 to 7 days, and use it as the main culture. do. <Jafer Armenter (30) Culture> Add the above medium 15, steam sterilize at 120°C for 30 minutes, then inoculate with flask culture seed culture solution 1.
Culture with aeration at 28°C for 4 to 6 days (rotation speed:
After passing through the culture solution, the solution (13) was extracted twice with ethyl acetate (9) under acidic conditions (PH2.5). Next, 0.05M-Tris-HCl buffer (PH8.5)
The mixture was extracted with ethyl acetate under acidic conditions (PH2.5). The ethyl acetate layer was concentrated to obtain about 10 ml of a concentrated solution. The obtained concentrate is
Silica gel column chromatography (3.1〓×40
_Friend leukemia cells ₍ mouse erythroid
The active fraction was collected while checking using an assay method using leukemia cell (B8cell). This was further subjected to silica gel chromatography in the same manner using a 3.1×30 cm column, and the active fraction was fractionated. The obtained active fractions were collected and subjected to gel filtration (carrier;
Sephadex LH-20, column: 1.5φ x 30cm, developing solvent: 0.05M acetate buffer: methanol = 3:
7) was carried out, and the active fraction was fractionated while being checked by the above-mentioned assay method. The obtained active fraction was concentrated and extracted with ethyl acetate under acidic conditions (PH2.5). The ethyl acetate layer was concentrated to obtain crystals, which were further recrystallized from methanol-water (7:3) to obtain colorless needle-like crystals of Deifalanisole A (17.0%).
I got mg.

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption spectrum of Deifalanisole A of the present invention, Figure 2 shows the infrared absorption spectrum of Deifalanisole A, and Figure 3 shows the infrared absorption spectrum of Deifalanisole A of the present invention.
FIG. 4 is a diagram showing the nuclear magnetic resonance spectrum (proton) of deifalanisole A, and FIG. 4 shows the nuclear magnetic resonance spectrum ( ¹³ C) of deifalanisole A.

Claims (1)

[Claims] 1 Structural formula: Differanisole denoted by
A. 2. A method for producing deifalanisol A, which comprises culturing deifalanisole A-producing bacteria belonging to the genus Chaetomium, and separating and collecting deifalanisol A from the culture. 3 Deifalanisole A belonging to the genus Chaetomium
The producing bacterium is Chaetomium sp. RB-001
(Chaetomium sp.RB-001) (FERM P-8021)
The manufacturing method according to claim 2.