JPS6361952B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to DC-52 and its manufacturing method. DC-52 of the present invention is a useful substance that has antibacterial activity against various bacteria and also has antitumor activity. In order to develop new antibiotic substances, the present inventors obtained a large number of microorganisms from nature and investigated their antibiotic productivity. As a result, they discovered that when a bacterial strain isolated from soil in Machida City, Tokyo (referred to as DO-52) was cultured in a culture medium, a novel antibiotic (DC-52) was produced in the culture. The invention will be explained in detail below. The physical and chemical properties of DC-52 according to the present invention are as follows. Elemental analysis value:
C: 65.7% H: 6.5% N: 8.4% Molecular formula: C ₁₈ H ₂₂ N ₂ O ₄ Molecular weight: 330 (field day soap mass spectrometry) Melting point: There is no clear melting point, and it gradually turns brown from around 170°C. Ultraviolet absorption spectrum (in water): Figure 1 Infrared absorption spectrum (KBr tablet method): Figure 2
Figure Specific rotation: [α] ³⁰ _D = -41° (c = 0.35 methanol) PMR spectrum (in D ₂ O): Figure 3 CMR spectrum (dioxane standard in D ₂ O)
(ppm): 180.2, 156.5, 137.1, 128.9, 123.3,
121.2, 110.2, 82.2, 71.9, 69.9, 66.1, 56.3,
54.4, 53.9, 41.6, 40.4, 32.0, 27.6 Solubility: water, methanol, ethanol, n-
Soluble in butanol. Dissolves in ethyl acetate and acetone. Almost insoluble in chloroform, benzene, and n-hexane. DO−52 is DC−52 along with DC−52 in fermentation broth.
Co-produces 52-d. Subsequent studies revealed that DC-52-d can also be obtained by hydrogenating DC-52 in the presence of a palladium catalyst. The physical and chemical properties of DC-52-d are as follows, and its ultraviolet absorption curve is almost the same as that of DC-52. Furthermore, there are many common PMR spectra, indicating that the two are related to each other. Physicochemical properties of DC-52-d Molecular weight: 332 (m/e333 (M+1) was given by field day soap mass spectrometry.) Molecular formula: C ₁₈ H ₂₄ N ₂ O ₄ Melting point: There is no clear melting point, It gradually turns brown from around 190℃. Ultraviolet absorption spectrum (in methanol):
Shows absorption maximum at 280nm and 273nm. PMR spectrum (in D ₂ O): Figure 4 Next, thin layer chromatography (silica gel (trade name) of DC-52 and DC-52-d)
Kieselgel60Art.5721, E.Merck (West Germany),
Develop for 3 hours at room temperature. ] The Rf values are shown in Table 1.

[Table] After development, the spots of DC-52 and DC-52-d were determined by bioassay using Bacillus subtils,
It can be detected using hot sulfuric acid, iodine, nitroprusside reaction, ninhydrin reaction, Ehrlich reagent, Tragendorf reaction, and ultraviolet absorption. DC-52 and DC with the above physical and chemical properties
-52-d is a new substance that has not been described in any literature. Based on the above-mentioned physical and chemical properties and X-ray crystallography studies, it has been found that DC-52 and DC-52-d each have the following structural formulas. Structural formula of DC-52: Structural formula of DC-52-d: Next, Table 2 shows the antibacterial activity of DC-52 and DC-52-d against various bacteria (agar dilution method, pH 7.0).

[Table] Furthermore, the acute toxicity value (LD ₅₀ ) of DC-52 was approximately 180 Kg/Kg when administered intraperitoneally to mice. Next, we will show the antitumor effect of DC-52. (1) Therapeutic effect on Sarcoma 180 ascites-type tumor 5 x 10 ⁶ cells of Sarcoma 180 ascites-type tumor were subcutaneously implanted in the axillary region of 1 group of 6 DDY male mice weighing approximately 20 g. Twenty-four hours after transplantation, 0.2 ml of DC-52 at various concentrations in phosphate buffered saline (PBS) was intraperitoneally administered once. The composition of PBS is NaCl0.8g/
dl, KCl0.02g/dl, Na ₂ HPO ₄ 1.15g/dl,
KH ₂ PO ₄ 0.02g/dl, PH7.2. As a comparative example, a group was provided in which 0.2 ml of PBS containing mitomycin C was intraperitoneally administered 24 hours after tumor cell transplantation. Average tumor volume (mm ³ ) and T/C 10 days after transplantation (T: average tumor volume of test example, C: PBS0.2
The average tumor volume (intraperitoneally administered) was measured, and the results shown in Table 3 were obtained.

[Table] (2) Therapeutic effect on Lymphocyticleukemia P-388 tumor Lymphocyticleukemia P-388 tumor cells 1× were injected into groups of 5 CDF ₁ male mice weighing approximately 22 g.
10 ⁶ were implanted intraperitoneally. DC− 24 hours after transplantation
As a comparative example, a group was prepared in which 0.2 ml of mitomycin C in PBS was intraperitoneally administered 24 hours after tumor cell transplantation. Average survival days and T/C after transplantation (T: average survival days of test cases, C: average survival days of controls)
are shown in Table 4.

[Table] Next, the DC-52 manufacturing method will be explained. DC-52 belongs to the genus Streptomyces, and DC-52 belongs to the genus Streptomyces.
DC-52 can be obtained by culturing a microorganism capable of producing 52 in a medium, allowing DC-52 to accumulate during the culture, and collecting DC-52 from the culture. The microorganism used in the present invention belongs to the genus Streptomyces, and any microorganism can be used as long as it has the ability to produce DC-52. A suitable bacterium is the above-mentioned strain DO-52. Next, the mycological properties of strain DO-52 will be described. (A) Morphological properties Strain DO-52 adheres well to aerial mycelia on a general isolation medium, and its morphology is simple branched and curved (flexible) to helical (spiral). The spores are
10 or more chains, and the surface is smooth. The shape of the spore is cylindrical (approximately 0.4 x 0.6μ). Table 5 shows the growth conditions, colors of the front and back surfaces of colonies, and soluble pigments when the DO-52 strain was grown on various media. Colors are displayed using the Color Harmony Manual
(Container Corporation of America) color classification.

[Table] (B) Physiological properties The physiological properties of strain DO-52 are shown below. Regarding the effects other than temperature, milk and cellulose, the observation results after 2 weeks at 27℃ are shown.
The temperature results are shown after 5 days, and the effects on milk and cellulose are shown after 1 month. 1 Assimilation of carbon source Carbon source Assimilation D-arabinose ± D-xylose + D-glucose D-fructose Sucrose + Inositol ± L-rhamnose Raffinose + D-mannite − 2 Liquefaction of gelatin ± 3 Effect on milk Liquefaction Coagulation - Peptonization - 4 Decomposition of cellulose Slightly 5 Hydrolysis of starch Yes 6 Optimum growth pH 6.8-7.5 7 Optimum growth temperature 28-38℃ 8 Production of tyrosinase None 9 Production of melanoid pigment None Above Based on its properties, strain DO-52 is classified as an actinomycete belonging to the Streptomyces layer. Regarding the species to which this fungus belongs, E. Ku¨ster (Intern. J. System.
According to the classification by Bacteriol., Vol. 22, No. 3, p. 139, 1972), aerial mycelium is grayish, and the color of the underside of the mycelia is distinct (dark brown to black), and contains melamine pigments. The following four types are listed as those that do not produce spores, have soluble pigments, have curved or spiral-shaped sporophores, and have smooth surfaces. Streptomyces tendae, Streptomyces tendae
Streptomyces nodosus, Streptomyces cyanogenus
cyanogenus), Streptomyces violaceolatus. The shape of the sporophyte is similar to the former two, but they differ in soluble pigments and sugar assimilation. For example DO
In -52, the color does not change on the acidic side, but changes on the alkaline side, whereas the pigments of these target species change color tone on the acidic side. Furthermore, the assimilation properties of raffinose and mannitol are opposite. The latter two have slightly different shapes of sporophores, but their ability to assimilate sugars is very similar; however, the assimilation of mannitol and inositol is different, and their soluble pigments change with acid and alkali. In contrast, the pigment of DO-52 only changes on the alkaline side. (EBShirling and D.Gottlieb.Intern・J.
System.Bacteriol., 18(2)69(′68), InternJ.
System.Bacteriol., 18(4)282(′68), Intern.J.
System. Bacteriol., 19(4)391('69)) Compared to the above four species, the major differences are that the spores are cylindrical (approximately 0.4 x 0.6μ) and that the soluble pigment changes. can give. The DO-52 mauve wine (7 1/2ni) is particularly distinctive.
This is NaOH and cinnamon brown (3g)
Changes to When liquid culture is performed, this black-purple pigment is excreted outside the bacterial cells and accumulates in the medium. From this point of view, DO-52 was determined to be a new species different from any of the target bacterial strains, and was classified as Streptomyces melanobinaceus due to its characteristic pigment.
melanovinaceus DO-52). This bacterium is located at the Institute of Microbial Technology, Agency of Industrial Science and Technology, located at 1-1-3 Higashi, Yatabe-cho, Tsukuba-gun, Ibaraki Prefecture, and at Peoria, Illinois, USA.
They have been deposited with the USD Department of Agriculture and given accession numbers of FEIKEN Article No. 654 and NRRL 12388, respectively. The properties of the strains shown here can sometimes be mutated by artificial mutagenic means such as ultraviolet rays, X-rays, and chemicals, as is the case with known species belonging to the genus Streptomyces. Any mutant strain capable of producing DC-52 can be applied to the present invention. Next, I will talk about the culture method. In the cultivation of the present invention, ordinary methods for culturing actinomycetes are generally used. Various nutrient sources can be used for culturing, as shown below. As the carbon source, glucose, starch, dextrin, mannose, fructose, sucrose, lactose, xylose, arabinose, mannitol, molasses, etc. are used singly or in combination. Furthermore, depending on the assimilation ability of the bacteria, hydrocarbons, alcohols, organic acids, etc. may also be used.
As the nitrogen source, ammonium chloride, ammonium sulfate, ammonium nitrate, sodium nitrate, urea, peptone, meat extract, yeast extract, dried yeast, corn, stave, liquor, soybean flour, casamino acid, etc. are used alone or in combination. In addition, inorganic salts such as table salt, potassium chloride, magnesium sulfate, calcium carbonate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ferrous sulfate, calcium chloride, manganese sulfate, subsulfate, copper sulfate, etc. are added as necessary. .
Furthermore, the growth of the bacteria used, DC-52 and DC-52-d
Trace ingredients that promote the production of vitamin B ₁ ,
Biotin and the like can be appropriately added. As a culture method, a liquid culture method, especially a deep stirring culture method, is suitable. Culture temperature is 25-40
℃, especially 28 to 38℃, and the pH of the medium can be adjusted to PH4 to 4 by adding aqueous ammonia or ammonium carbonate solution.
It is desirable to culture at 10, preferably 6 to 8. When the liquid culture is carried out for usually 1 to 7 days, the target substances (DC-52 and DC-52-d) are produced and accumulated in the culture solution and in the bacterial cells. When the production amount in the culture reaches the maximum, the culture is stopped, the bacterial cells are separated, and the culture solution and the bacterial cells are separated. For isolation and purification of DC-52 and DC-52-d from the culture solution, conventional separation and purification methods used for isolating microbial metabolic products from the culture solution are used. For example, the culture product is separated into a culture solution and bacterial cells, and the culture solution is directly used (PH6.0) with a nonionic porous resin (for example, "Diaion PH-20" manufactured by Mitsubishi Sei Co., Ltd.). After passing through and adsorbing active ingredients, the adsorbed substances are desorbed using methanol, acetone, ethyl acetate, etc. This desorption solution is concentrated to dryness, adsorbed onto Celite powder,
DC-52 and DC-52-d can be desorbed using methanol, acetone, etc. Furthermore, solvent extraction method,
Purification is performed by combining common purification methods such as partition chromatography. Next, an example will be given. In the examples, the trends of DC-52 and DC-52-d were tracked using a bioassay using Bacillus subtilis No. 10707 or the ultraviolet absorption of DC-52 and DC-52-d. Example 1 Streptomyces mereobinaceus DO-52 was used as an inoculum. The strain was grown in a 2-volume Erlenmeyer flask as a seed medium [KCl4g/, _MgSO4 .
7H ₂ O 0.5g/, KH ₂ PO ₄ 1.5g/, Ammonium sulfate 5.0
g/, sucrose 20g/, fructose
10g/, glucose 10g/, corn steep liquor 5.0g/, CaCO ₃ 20g/PH7.0〕300ml
The cells were inoculated and cultured at 30°C for 48 hours with shaking (220 rpm). The seed culture thus obtained was mixed into a fermentation medium 15 with the following composition in a 30-volume jar fermenter.
5% (volume) and cultured at 30° C. using an aeration agitation method (rotation speed 250 rpm, aeration rate 15/min). Fermentation medium composition: glucose 50g/,
KH ₂ PO ₄ 0.3g/, K ₂ HPO ₄ 0.4g/,
MgSO ₄・7H ₂ O0.2g/, powdered soybean meal 20g/,
CaCO ₃ 1g/, adjust pH to 7.0 (before sterilization) with NaOH. Culture is carried out for 72 hours without controlling the pH of the culture medium. Separate the bacterial cells and precipitate from the culture solution, and
Get 13. First, add the solution to the cation exchange resin WK−
20 (manufactured by Mitsubishi Kasei) to adsorb the active substance and wash thoroughly with water. Next, elute with 2M ammonium acetate aqueous solution 3, and leave the eluate as it is.
500ml non-ionic porous resin (Diaion
HP-20, manufactured by Mitsubishi Kasei Co., Ltd.) to adsorb the active substance and wash with 2 parts of water and 20% methanol.
Elute the active substance with 50% methanol 3. After decolorizing the eluate by adding 3 g of activated carbon, the activated carbon is separated. This was concentrated to remove methanol, and then passed through a column of 400 ml of nonionic porous resin (Diaion HP-20, manufactured by Mitsubishi Kasei Corporation) to adsorb it. After washing with water, 30% methanol and methanol
The active fraction is fractionated by linear concentration gradient evacuation. The active fractions were collected and concentrated to dryness, then dissolved in a mixed solvent of ethanol:water (10:1 volume ratio), placed on a column of silica gel (100 g) packed with the same solvent, and developed with the same solvent to give DC-52, DC.
It is eluted in the order of -52-d. These fractions are collected and concentrated, then dissolved in methanol, and ethyl ether is added to obtain a white powder. DC-52 and DC-52-d were each 10 mg
and 2 mg are obtained. The physicochemical properties, antibacterial activity, and anticancer activity of the obtained DC-52 and DC-52-d are as described above. Example 2 The same procedure as in Example 1 was carried out except that the composition of the fermentation medium was changed to the following.
-52 (7 mg) and DC-52-d (1 mg) are obtained. Fermentation medium composition: glucose 50g/,
NaH ₂ PO ₄ 0.2g/, Na ₂ HPO ₄ 0.3g/,
MgSO ₄・7H ₂ O0.3g/, yeast extract 7g/,
CaCO ₃ 1g/, adjust pH to 7.0 (before sterilization) with NaOH. Example 3 Preparation of antitumor injection solution 10 mg of DC-52 obtained in Examples 1 and 2 was
Dissolve in 50 ml of ethanol, stir, and remove the ethanol by suction. Add about 10 ml of sterile physiological saline to the residue to make an injection solution.

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption spectrum of DC-52. Figure 2 shows the infrared absorption spectrum of DC-52. Figure 3 shows the PMR spectrum of DC-52. Figure 4 shows the PMR spectrum of DC-52-d.

Claims (1)

[Claims] 1 formula DC−52 expressed as .