JPH0249717B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a new antibiotic, mycoplanesin D. Until now, mycoplanesin and its derivatives (Japanese Patent Application Laid-Open Nos. 160302-1982 and 65853-1983), Mycoplanesin B and its derivatives (Japanese Patent Application Laid-Open Nos. 56-16030 and 56-65853),
No. 140957) and mycoplanesin C (Unexamined Japanese Patent Publication No. 140957)
- No. 142250) is known. The present inventors have newly discovered that Actinobacterium strain No. 41042, isolated from the soil of Sumoto City, Hyogo Prefecture, produces mycoplanesin D, a new antibiotic. As a result of further research, the formula It has been discovered that mycoplanesin D, represented by the following formula, has extremely strong antibacterial activity against bacteria, especially Mycobacterium tuberculosis. Therefore, mycoplanesin D is used for the prevention or treatment of human and animal diseases caused by these bacteria. In the above formula, R is α-ketobutyryl-N-methylvalyl group shows. Actinomycetes No.41042 producing mycoplanesin D
The morphological characteristics and physiological properties of the strain are described, for example, in Japanese Patent Application Laid-open No. 160302/1983, and Actinoplanes sp. No. 41042.
It is named. This strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, and its microorganism accession number is FERM BP-1139. As for mycoplanesin D-producing bacteria, it is well known that the properties of actinomycetes are not constant and can easily change naturally or artificially.
Bacterial strains that can be used in the present invention include all strains that produce mycoplanesin D and belong to the genus Actinoplanes. Culture in the method of the present invention is carried out in accordance with the culture method for general actinomycetes, and is preferably carried out by shaking culture in a liquid medium or by aeration stirring culture.
As the medium components, those known as nutritional sources for actinomycetes are used, such as glucose as a carbon source,
Arabinose, galactose, mannose, sucrose, maltose, dextrin, starch, glycerin, etc., as well as vegetable oils such as soybean oil, corn oil, and cottonseed oil, and animal fats and oils such as chicken oil, lard, and fish oil are also used. Nitrogen sources include soybean flour, peanut flour, cottonseed flour, fish meal, corn,
Steep Ritzker, oatmeal, skim milk, peptone, meat extract, fresh yeast, yeast extract, casamino acids, sodium nitrate, ammonium nitrate, ammonium sulfate, etc. Inorganic salts include table salt, potassium chloride, phosphate, magnesium carbonate, calcium carbonate. , calcium chloride is used. Further, if necessary, trace metal salts such as ferrous sulfate, copper sulfate, magnesium sulfate, zinc sulfate, and cobalt chloride are added. During liquid culture, silicone oil, vegetable oil, surfactant, etc. are appropriately used as antifoaming agents. The pH of the medium is around neutral, and the culture temperature is preferably 20 to 30°C, particularly around 28°C. The titer of mycoplanesin D produced in the culture solution as the culture progresses is as follows: Mycobacterium smegmatis
It is quantified by the Cupp assay using ATCC607 strain as the test bacterium. The production amount of mycoplanesin D usually reaches its maximum after 3 to 5 days of culture. Mycoplanesin D is present in both the liquid part and the bacterial cell part of the culture medium. After the cultivation is completed, the bacterial cells and other solid parts are separated from the liquid part by over-operation using diatomaceous earth as an auxiliary agent or by centrifugation. Mycoplanesin D present in the bacterial cell part, liquid, or supernatant can be extracted and purified by utilizing its physicochemical properties. Mycoplanesin D in the bacterial cell portion can be extracted by adding a water-miscible solvent such as methanol, ethanol, isopropanol, acetone, etc. After distilling off the solvent, the extract is extracted into a water-immiscible solvent such as ethyl acetate, methyl isobutyl ketone, chloroform, and methylene chloride. The liquid portion of mycoplanesin D is also extracted with these water-immiscible solvents, combined with the mycoplanesin D extract from the bacterial cell portion, concentrated, and the solvent is distilled off to crudely extract mycoplanesin D. It can be made into a thing. In addition, without separating into bacterial cells and liquid (or supernatant),
It is also possible to extract mycoplanesin D by directly adding the above-mentioned water-miscible solvent to the culture solution, and then distilling off the solvent by concentration, followed by extraction with the above-mentioned water-immiscible solvent. Furthermore, as a method for purifying this mycoplanesin D, all methods used to purify substances having such physical and chemical properties can be applied, including methods using adsorbents and methods using countercurrent distribution. is preferred. As adsorbents, alumina, silica gel, Cephadex, cellulose, etc. are used. Column chromatography using silica gel as a carrier is particularly suitable, and the solvent used is a mixture of methanol, ethyl acetate, chloroform, etc. in an appropriate ratio. In order to obtain mycoplanesin D with even higher purity, high performance liquid column chromatography using silica gel or various carriers for reverse phase column chromatography is effective. Mycoplanesin D thus obtained has the following properties. A. Physicochemical and biological properties of mycoplanesin D (1) Color and shape of substance: white powder (2) Melting point: 160-170℃ (3) Specific rotation: [α] ²¹ _D -60.4° ( c=0.5, chloroform) (4) Elemental analysis values: Actual value; C, 61.41%; H, 8.54%; N,
11.82% (5) Compositional formula: C ₆₂ H ₁₀₄ N ₁₀ O ₁₃ (6) Molecular weight: 1196 (7) Ultraviolet absorption spectrum: As shown in Figure 1, measured at a concentration of 20 μg/ml in 50% methanol solution. Terminal absorption only. (8) Infrared absorption spectrum: The infrared absorption spectrum measured with the KBr disk is shown in Figure 2. (9) Nuclear magnetic resonance spectrum: The nuclear magnetic resonance spectrum measured in deuterated chloroform using tetramethylsilane (TMS) as an internal standard is shown in Figure 3. (10) Solubility: Soluble in methanol, ethanol, ethyl acetate, acetone, chloroform, insoluble in water. (11) Color reaction: On silica gel thin layer chromatography,
Color brown with 50% sulfuric acid, color with iodine, and decolorize with potassium permanganate. Ninhydrin, 2,
4-dinitrophenylhydrazine negative. (12) Amino acid analysis: 1 mol each of proline, glycine, and
Leucine, 2-amino-5-methylhexylic acid, N-methylthreonine, N-methyl-leucine, methylproline, propylproline and 2 moles of N-methylvaline were detected. (13) R _f value (Silica gel thin film chromatography, F ₂₅₄ , 0.25 mm Merck No. 5715) Solvent R _f Ethyl acetate 0.15 Chloroform: Methanol (95:5) 0.63 (14) Antibacterial spectrum Minimum against various microorganisms Inhibitory concentration (MIC)
is shown in Table 1. For Mycobacterium, use the dilution method using Duvos liquid medium supplemented with 10% albumin, and after culturing at 37℃ for one week, for general bacteria, use the agar dilution method using Heart Infusion Agar medium. After 24 hours at 26°C, molds and yeasts were determined after 48 hours at 26°C by the agar dilution method using Sabouraud agar medium.

[Table] (15) Toxicity: When 100 mg/Kg of this substance was administered intraperitoneally to mice, none of the mice died. From the above, mycoplanesin D is used as an antibacterial agent, particularly an antituberculous agent, for various bacterial infectious diseases. When used as an antituberculous agent, it is administered orally or parenterally. Oral administration is usually preferred. Examples of dosage forms include tablets, capsules, powders, and granules. Although the dosage varies depending on the symptoms, age, body weight, etc., it is usually preferable to administer about 200 mg to 2 g per day for adults, either once or in several divided doses. This dosage can be further increased or decreased as necessary. Example 1 Mycoplanesin D-producing bacteria were treated with 1% glucose,
A 500 ml Sakaguchi flask containing 100 ml of a seed medium consisting of 1% glycerin, 0.5% oatmeal, 1% sucrose, 2% soybean flour, 0.5% casamino acids, 1% fresh yeast, and 0.1% CaCO ₃ (PH7.0 before sterilization). 5 flasks were inoculated and cultured at 28°C for 96 hours with reciprocal shaking.The culture solution was inoculated into 8 2-volume Erlenmeyer flasks containing 500 ml of the same medium, 25 ml each, and further 28
Incubate with rotary shaking for 96 hours at °C. The culture solution was mixed with 0.5% glycerin, 2% sucrose, 1% soybean flour,
1% fresh yeast, 0.5% corn steep liquor,
Two 600-capacity tanks containing 300 medium of CoCl ₂ 6H ₂ O 0.001% (PH 7.2 before sterilization) were inoculated with 1.8 in each, and the mixture was heated at 28°C with stirring at 200 revolutions/minute and aeration rate of 300 rpm.
Culture was carried out with aeration for 96 hours. 30 kg of Celite 545 (manufactured by John Manville Product Corporation, USA) was added as a supernatant to the culture solution (PH7.2) at 600 ml, filtered, and divided into 420 ml of liquid and a cake containing 170 kg of bacterial cells. The liquid was extracted with an equal amount of ethyl acetate to extract the fraction containing mycoplanesin, mycoplanesin B, C, and D, and the cake containing bacterial cells was extracted twice with 80% acetone water (400%) to obtain an extract of 750% After distilling off the acetone under reduced pressure,
The residual liquid was extracted with 210 ml of ethyl acetate, and the mixture was combined with the ethyl acetate extract and concentrated under reduced pressure to a volume of 2. The concentrate obtained in this way was mixed with 1 part of 0.05N HCl water,
The mixture was washed successively with 1% NaHCO ₃ water and saturated brine, dried over sodium sulfate, and concentrated to dryness to obtain 400 g of an oily substance. The entire amount of this oil was dissolved in 300 ml of benzene and adsorbed onto a silica gel column (manufactured by Mallinckrodt, USA, 900 g of silica gel) prepared in advance with benzene. After washing the column with benzene, 4 benzene:ethyl acetate 3:1, 500 while developing with a 2:1, 1:1 mixed solvent and then ethyl acetate.
It was fractionated into ml portions and eluted up to fraction 32. Mycoplanesin D was mainly eluted in fractions 15 to 22, and was concentrated to dryness to obtain 15.3 g of oil. 5.0 g of this oil was dissolved in 3 ml of acetonitrile and purified by reverse phase column chromatography. That is, 1 ml of an acetonitrile solution in which the above sample was dissolved was adsorbed onto a column of Merck's Ricroprep RP-8 (Rover Column B), and then developed and eluted with 65% acetonitrile water at a flow rate of 10 ml/min. Mycoplanesin D was eluted between 51 and 57 minutes after sample adsorption, and was found as an active fraction.
60ml was obtained. Mycoplanesin (JP-A-160302) produced by this producing bacterium at the same time is 40 to 50
and mycoplanesin C (Japanese Patent Application Laid-open No.
-142250) was eluted between 60 and 74 minutes. The same operation was repeated twice for the remaining 2 ml of sample to obtain a total of 540 ml of active fraction. After distilling off the acetonitrile under reduced pressure, the extract was extracted with 100 ml of ethyl acetate, and the extract was washed with saturated brine, dehydrated with sodium sulfate, and concentrated to dryness to obtain 56.3 mg of mycoplanesin D as a white powder.

[Brief explanation of the drawing]

Figure 1 shows the ultraviolet absorption spectrum of mycoplanesin D, Figure 2 shows the infrared absorption spectrum of the same substance, and Figure 3 shows the nuclear magnetic resonance spectrum of the same substance.

Claims (1)

[Scope of Claims] 1. Mycoplanesin D-producing bacteria belonging to the genus Actinoplanes are cultured to produce the expression (In the formula, R represents an α-ketobutyryl-N-methylvalyl group.) A method for producing mycoplanesin D, which comprises isolating mycoplanesin D. 2. The production method according to claim 1, wherein the mycoplanesin D-producing bacterium is Actinoplanes sp. No. 41042 (FERM BP-1139).