JPS6253519B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel substance DC-11-A-3 and an antitumor agent containing the same. Furthermore, the present invention relates to a method for producing DC-11-A-3 using a fermentation method. In order to find useful antibiotics, the present inventors obtained a large number of naturally occurring microorganisms and conducted research on the production of antibiotics. As a result, when a bacterial strain isolated from soil in Sendai City, Miyagi Prefecture (referred to as KY11091) was cultured in a culture medium, a novel antibiotic, DC-
It was found that 11 were produced. A patent application for DC-11 has already been published by the same applicant. [Patent application No. 53-45916 (Japanese patent application No. 54-138501)
and Japanese Patent Application No. 53-153027 (Japanese Unexamined Patent Publication No. 55-79322)] As a result of further research, the present inventors found that a new substance (DC -11-A-3), and further discovered that this substance exhibits an antitumor effect, thereby completing the present invention. That is, the present invention provides a new substance having the properties described below.
Regarding DC-11-A-3. Furthermore, the present invention involves culturing a microorganism belonging to the genus Micromonospora and having the ability to produce DC-11-A-3 in a nutrient medium, accumulating DC-11-A-3 in the culture, and The present invention relates to a method for producing DC-11-A-3, which comprises collecting DC-11-A-3. The present invention also relates to an antitumor agent containing DC-11-A-3. Since DC-11-A-3 exhibits antibacterial activity against various bacteria, it is expected to have a therapeutic effect on infectious diseases caused by these bacteria. The present invention will be explained in more detail below. The physicochemical properties of the novel substance DC-11-A-3 according to the present invention are as follows. Melting point: 187-190℃ Elemental analysis: H 7.01%, C 59.33%, N
1.90% Infrared absorption spectrum (KBr tablet method): 1st
Figure Ultraviolet absorption spectrum (in 90% methanol): Figure 2 PMR spectrum (in CDCl ₃ , TMS standard):
Figure 3 CMR spectrum (main peaks are as below) (in CDCl ₃ , TMS standard) (δppm)
206.2, 201.3, 192.4, 170.7, 170.0, 166.5,
157.2, 149.4, 141.3, 136.2, 135.9, 126.1,
123.0, 118.2, 100.8, 99.2, 98.7, 96.3,
92.5, 91.9, 91.4, 84.7, 83.9, 81.1, 77.8,
75.2, 71.6, 70.2, 69.2, 68.0, 67.7, 66.4,
64.4, 63.8, 63.3, 54.2, 53.7, 52.8, 51.2,
44.8, 43.2, 41.5, 38.4, 37.0, 35.9, 34.3,
31.1, 30.7, 29.7, 27.3, 26.3, 25.3, 21.9,
20.8, 20.7, 18.8, 18.0, 17.7, 16.9, 16.0,
15.0, 14.3, 13.9 Specific optical rotation [α] ²² _D = -62.5° (c = 1.0, chloroform) Solubility: Soluble in methanol, ethanol, butanol, acetone, ethyl acetate, chloroform, soluble in benzene, water, Insoluble in petroleum ether and n-hexane. Molecular formula: C ₆₉ H ₉₈ N ₂ O ₂₆ Molecular weight: 1371.56 From the above results, DC-11-A-3 has the following formula It turned out to be a new substance expressed by The Rf values of DC-11 and DC-11-A-3 measured by thin layer chromatography are shown in Table 1.

[Table] Next, Table 2 shows the antibacterial activity of DC-11-A-3 against various microorganisms (agar dilution method, PH7.0).

[Table] Acute toxicity (LD ₅₀ ) of antibiotic DC-11-A-3
is 60 mg/Kg when administered intraperitoneally to mice. Next, the antitumor activity of the antibiotic DC-11-A-3 will be shown. (1) Therapeutic effect on Sarcoma 180 ascites-type tumor The results shown in Table 3 were obtained using the same method as described in JP-A-54-138501, except that DC-11-A-3 was used instead of DC-11. I got it.

[Table] (2) Therapeutic effect on Lymphocytosis ryukemia P-388 tumor The same method as described in JP-A-54-138501 except that DC-11-A-3 was used instead of DC-11. The results shown in Table 4 were obtained.

[Table] When DC-11-A-3 is used as an antitumor agent, it may be administered intravenously or intraperitoneally as an injection. The dosage is preferably about 0.1-0.5 mg/Kg (human)/day. DC-11-A-3 is not soluble in water, so when preparing an injection, it is best to dissolve it in a solvent or solubilize it using a surfactant.
The latter is usually preferred. For example, solubilization using a surfactant can be performed by converting DC-11-A-3 to DC-11-A-3.
DC-11-A-3 is dissolved in 2500 to 5000 times (weight ratio) ethanol, and a surfactant such as HCO-60 or Tween 80, preferably HCO-60, is added thereto in an amount of about 3 to 5 times that of DC-11-A-3. , the ethanol is removed by suction, an appropriate amount of sterile physiological saline is added to this, and this is used as an injection. Administration is carried out continuously or intermittently depending on the condition of the patient. Next, a method for producing antibiotic DC-11-A-3 will be explained. DC-11-A-3 can be obtained by culturing DC-11-A-3 producing bacteria belonging to the genus Micromonospora in a nutrient medium and collecting DC-11-A-3 from the culture. can. The microorganism used in the present invention belongs to the genus Micromonospora, and any microorganism can be used as long as it has the ability to produce DC-11-A-3. A suitable example of the bacterium is the aforementioned KY11091 strain. Next, the mycological properties of the KY11091 strain are
45916 (Japanese Unexamined Patent Publication No. 54-138501),
It is reproduced below with some corrections and additions. Morphological properties KY11091 does not form true aerial hyphae like those observed in Streptomyces on commonly used agar media, and on agar media with good spore formation, waxy hyphae appear on the agar surface. Forms a shiny, brick-colored spore layer. When cultured in liquid, the color is bright orange in the early stages of growth, but turns brown to dark brown in the later stages, and a large number of spores are observed. When observed under a microscope, the hyphae are approximately 0.5μ in diameter, well elongated, and have no septa. Only one spore forms at the apex of a sporophore (about 0.3 to 1.0 μm in length) that branches from the basal hyphae, and is formed throughout the relatively long hyphae. The diameter of mature spores is approximately 1.0μ, and they are spherical. Growth status on various media Table 4 shows the growth status when KY11091 was grown on various media, the color of the front and back surfaces of colonies, and the soluble pigment. Colors are displayed using the Color Harmony Manual
(Container Corporation of America) according to the color classification.

【table】

[Table] Physiological properties The physiological properties of KY11091 are shown in Table 5. Regarding the effects other than temperature, milk and cellulose, the observation results after 2 weeks at 27°C are shown; for temperature, the results are shown after 5 days, and for the effects on milk and cellulose, the results are shown after 1 month.

【table】

[Table] As is clear from the mycological properties mentioned above,
Strain KY11091 is a mesophilic bacterium that does not form true aerial hyphae on an agar medium but produces a single spore on basal hyphae, and cell wall analysis reveals that it contains meso-diaminopimelic acid (meso-DAP). Therefore, strain KY11091 was identified as a strain belonging to the genus Micromonospora. In addition to the above description, Bergey's Manual of Determinative Bacteriology
Determinative Bacteriology) 8th edition, P846~
849, International Journal of Systematic Bacteriology
Bacteriology) Vol. 21, No. 3, p. 248-253, strain KY11091 was identified as belonging to Micromonospora tyarcea, and this strain will hereinafter be referred to as Micromonospora tyarcea KY11091. This strain Micromonospora charcea
KY11091 (Micromonospora Sp.
KY11091) has been deposited with the Institute of Fine Technology as No. 4458, and has been given the NRRL number.
NRRL11289 has been granted. As seen in the case of known bacterial species belonging to the genus Micromonospora, the properties of this bacterial strain may be mutated by artificial mutagenic means such as ultraviolet rays, X-rays, and chemicals; DC-
Any material capable of producing 11-A-3 substances 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, molasses, etc. are used alone or in combination. Furthermore, depending on the assimilation ability of the bacteria, hydrocarbons, alcohols, organic acids, etc. may also be used. Inorganic and organic nitrogen sources include ammonium chloride, ammonium sulfate, ammonium nitrate, sodium nitrate, and urea; natural nitrogen sources include peptone, meat extract, yeast extract, dried yeast, corn steep liquor, soy flour, and casamino Acids and the like are used alone or in combination. In addition, salt, potassium chloride, magnesium sulfate, calcium carbonate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ferrous sulfate, calcium chloride, manganese sulfate, zinc sulfate,
Add inorganic salts such as copper sulfate. Furthermore, trace components that promote the growth of the bacteria used and the production of DC-11-A-3, such as vitamin B ₁ and biotin, can be appropriately added. As a culture method, a liquid culture method, especially a deep agitation culture method, is suitable. Culture temperature is 25-40
℃, especially 28 to 38℃, is optimal, and the pH of the culture medium is adjusted to PH4 to 4 by adding aqueous ammonia or ammonium carbonate solution.
10, preferably 6 to 8. When culture is carried out in liquid culture for usually 1 to 7 days, the target substance is produced and accumulated in the culture solution. When the production amount in the culture solution reaches the maximum, the culture is stopped, the bacterial cells are separated, and the target product is isolated and purified from the obtained culture solution. Isolation and purification of DC-11-A-3 from the culture solution utilizes separation and purification methods commonly used to isolate metabolic products of microorganisms from the culture solution. For example, the culture product is separated into a culture solution and bacterial cells, and the culture solution is left as is (PH6.0) using a nonionic porous resin (trade name "HP-20" manufactured by Mitsubishi Kasei, etc.)
After the active ingredients are adsorbed, the adsorbed substances are desorbed using methanol, acetone, ethyl acetate, etc. This desorption solution is concentrated to dryness, dissolved in water, and adsorbed onto activated carbon. The active substance is eluted from the activated carbon using an organic solvent such as acetone or ethyl acetate. This eluate is concentrated to dryness, dissolved in chloroform, suspended in chloroform, and then chromatographed using silica gel packed in a column. First, impurity yellow pigment is removed by passing chloroform through the column. Then chloroform:methanol (98:3) (volume ratio)
The active substance can be eluted with a mixture of When this material is concentrated to dryness, DC-11 and DC-11-A-
A mixture of 3 can be obtained. Dissolve this in toluene:acetone (2:1) (volume ratio),
Chromatography is performed using silica gel that has been suspended in the same solvent and packed in a column. First, DC
-11 is followed by DC-11-A-3 which is eluted with toluene:acetone (2:1) (volume ratio). To further complete the separation, repeat the same chromatography as above or use Sephadex LH-20 (Pharmacia Fine Chemicals Inc.).
, Sweden). As a result of the hydrolysis of DC-11-A-3, it was confirmed that DC-11-A-3 contained substances that were identical to amycetose and diquitoxose on TLC. Next, examples will be given to show specific manufacturing methods. In the examples, the trend of DC-11-A-3 was tracked by bioassay using Bacillus subtilis No. 10707. Example 1 Micromonospora charcea as seed fungus
KY11091 was used. This strain has been deposited with the National Institute of Fine Arts and Science as Microtechnical Research Institute Bacteria No. 4458, and has been given the NRRL number NRRL11289. The strain was added to the seed medium [KCl4] in a 2-volume Erlenmeyer flask.
g/, MgSO ₄・7H ₂ O0.5g/, KH ₂ PO ₄ 1.5
g/, ammonium sulfate 5.0 g/, seuclose 20 g/
, fructose 10g/, glucose 10g/
, corn steep liquor 5.0g/, CaCO ₃ 20
g/, PH7.0] and cultured at 30°C for 48 hours with shaking (220 rpm). The seed culture solution obtained in this way was transferred at a rate of 5% (volume) to fermentation medium 15 with the following composition in a 30-volume jar fermenter, and heated at 30°C with aeration stirring method (rotation speed 250 r.pm aeration rate 15/ The culture was carried out by (min). Fermentation medium composition: soluble starch 60g/, soybean meal powder 10g/, peptone 10g/, K ₂ HPO ₄ 0.5
g/, MgSO ₄・7H ₂ O0.5g/, CaCO ₃ 1
g/, adjust the pH to 7.2 (before sterilization) with NaOH. Culture was carried out for 72 hours without controlling the pH of the culture medium. Separate the bacterial cells and precipitate from the culture solution, and
Got 13. First, liquid 13 is passed through a column of nonionic porous resin 1 (trade name "HP-10" Mitsubishi Chemical) to adsorb the active substance, and after washing with water, an additional 30%
(V/V) Wash with acetone aqueous solution to remove impurities. Then elute with acetone. The acetone fraction is concentrated to dryness and dissolved in a 30% (V/V) acetone aqueous solution. This solution is adsorbed onto a column packed with 500 ml of activated carbon. After washing with a 30% (V/V) acetone aqueous solution, the active fraction is eluted with acetone. This operation can remove most of the dyes present as impurities. The active fraction is concentrated to dryness and dissolved in a small amount of chloroform (approximately 10 ml). Silica gel filled with this chloroform solution in advance using chloroform as a solvent (Product name: Silica gel for chromatography (100 mesh or more) Kanto Kagaku, the same applies below)
(500ml), washed thoroughly with chloroform (approximately 2 times), and then eluted with chloroform:methanol (98:3) (volume ratio). DC-11 and DC-11-A- A mixed active fraction of 3 is eluted. This fraction was concentrated to dryness, dissolved in toluene:acetone (2:1, volume ratio), suspended in toluene:acetone (2:1, volume ratio), and filled in a column with silica gel (250
ml) column, and chromatography was performed using the same solvent. First, DC-11 is eluted, followed by DC-11-A-3. This operation was repeated twice, and each active fraction was concentrated to dryness to give 25 mg of DC-11 and DC-11-A.
-3 5 mg was obtained. Physical and chemical properties of DC-11-A-3 obtained here,
The antibacterial activity and anticancer activity are as described above. The above DC-11-A-3 can be obtained as a sample with slightly different secondary physicochemical properties depending on the purification method. For example, silica gel thin layer chromatography using Merck Art5715 plates or Mallinckrodt silica gel (Sillc AR cc-4,
The sample obtained when column chromatography was performed using Co., Ltd. (Mallinckrodt Co., USA) showed a melting point of 206-218°C, and a specific optical rotation of [α] ¹⁷ _D
= -84.4° (C = 1.0, acetone), and as seen in the IR spectrum (Figure 4), it exhibits strong absorption at 1630 cm ^-1 and also near 1400 cm ^-1 . In addition, when performing chromatography using Merck's silica gel (Art7734), the above
It may also indicate intermediate properties of the standard. When the specimen with a melting point of 206-218°C is dissolved in ethyl acetate and washed with dilute hydrochloric acid and then recovered, the DC- with a melting point of 187-190°C obtained above is obtained.
11-A-3 standard sample is obtained. There are two types of melting point amounts obtained as described above.
DC-11-A-3 shows completely the same behavior and data in terms of biological activity, thin layer chromatography, NMR spectrum, UV spectrum, etc., and cannot be distinguished. The IR spectra are also very similar to each other, except for the behavior of the two peaks near 1630 and 1400 cm ^-1 . This difference is not because the two materials of DC-11-A-3 are structurally chemically different; the sample with a higher melting point is the same compound, but it is due to the inclusion of trace elements that cannot be distinguished by elemental analysis. Alternatively, it is presumed that it is due to the formation of salt. Example 2 Culture was carried out in the same manner as in Example 1 except that the composition of the fermentation medium was changed to the following. Fermentation medium composition: soluble starch 40g/, soybean meal powder 30g/, dextrin 5g/, corn steep liquor 5g/, K ₂ HPO ₄ 0.5g/,
MgSO ₄・7H ₂ O0.5g/, CaCO ₃ 1g/, PH
Adjust with NaOH to 7.0 (before sterilization). The culture solution was prepared in the same manner as in Example 1, and DC-11
and DC-11-A-3, 22 mg and 8 mg, respectively, were obtained. Physical and chemical properties of DC-11-A-3, antibacterial activity,
Anticancer activity was obtained in Example 1. Melting point: 187-190
It was in good agreement with ℃. Example 3 Preparation of antitumor injection solution 10 mg of DC-11-A-3 obtained in Reference Example was added to 50 mg
HCO−60 dissolved in ml ethanol
Add 30 mg of Nikkol (manufactured by Nikkol Chemicals), stir, and remove the ethanol by suction. Approximately 10 ml of sterilized physiological saline is added to the residue to make an injection solution.

[Brief explanation of the drawing]

FIG. 1 shows the infrared absorption spectrum of DC-11-A-3. FIG. 2 shows the ultraviolet absorption spectrum of DC-11-A-3. Figure 3 is DC-11-A-
The PMR spectrum of No. 3 is shown. Figure 4 is DC-11-
The infrared absorption spectrum of A-3 (sample with a melting point of 206° to 218°C) is shown.

Claims (1)

[Claims] 1 formula A new substance represented by DC-11-A-3.