JPH0153676B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel antibiotic 3'-Oxy-Saccharocin and a method for producing the same. The physicochemical properties of the novel amino sugar antibiotic 3'-oxysatucalosin (hereinafter referred to as oxysatucalosin) of the present invention are as follows. Melting point: 230°C or higher (decomposition) [α] ²⁴ _D +162° (C = 0.25, H ₂ O) Elemental analysis (for C ₂₁ H ₄₀ N ₄ O ₁₃・2H ₂ O) Actual value Theoretical value C: 42.07 C: 42.56 H: 7.12 H: 7.48 N: 9.03 N: 9.45 Molecular weight: 556 (from field desorption mass spectrum) Molecular formula: C ₂₁ H ₄₀ N ₄ O ₁₃ Ultraviolet absorption spectrum: 220 ~ in aqueous solution
It does not show the characteristic maximum absorption at 360 nm, but only shows terminal absorption. Infrared absorption spectrum (KBr method) as shown in Figure 1, 3350, 2900, 1585, 1460, 1390,
It has absorption bands at wavelengths around 1345, 1040 ^{, and 1000 -1} _cn . Nuclear magnetic resonance spectrum (hydrogen nuclei): As shown in Figure 2 (in D ₂ O, 100 MHz, internal reference Dss) Nuclear magnetic resonance spectrum (carbon nuclei): 25M in D ₂ O
Measurements were made at Hz with dioxane as an internal reference. No. ppm No. ppm 1 102.6 12 71.1 2 97.0 13 70.4 3 95.9 14 67.4 (dioxane) 4 88.4 15 66.6 5 78.4 16 62.3 6 76.9 17 61.8 7 74.5 18 56.7 8 74 .1 19 51.3 9 73.8 20 50.1 10 72.4 21 36.8 11 72.0 22 33.2 Solubility: Soluble in water, slightly soluble in lower alcohols such as methanol, insoluble in acetone, benzene, ethyl acetate Color reaction Positive for ninhydrin reaction, potassium permanganate decolorization reaction, Elson Morgan reaction,
Sakaguchi reaction was negative. Color property: White powder Acid-base distinction: Basic Thin layer chromatography (silica gel, manufactured by Merck & Co., Ltd., Art. 5735) Chloroform-methanol-28% aqueous ammonia (2:3:2) Rf=0.24 Chloroform-methanol- 14% ammonia water (1:2:1) Rf=0.17 Based on the above physicochemical properties, oxysatsucalosin is recognized as a new amino sugar compound, and its planar structure is predicted to be a new compound with the following structure. It is. In addition, the antibacterial spectrum (indicating the minimum inhibitory concentration) of oxysatsucalosin determined by the agar dilution method is as shown in the table below. [Table] [Table] Furthermore, oxysatsucalosin, which has antibacterial properties of the present invention, especially excellent antibacterial properties against Gram-negative bacteria, can be prepared by adding salts of non-toxic acids such as pharmaceutically acceptable mineral acids and organic acids. For example, hydrochloric acid, hydroiodic acid, sulfuric acid, phosphoric acid, carbonic acid, acetic acid,
It can be used in the form of salts with acids such as fumaric acid, malic acid, citric acid, mandelic acid, succinic acid, ascorbic acid, aspartic acid, and glutamic acid. In addition, the oxysatsucalosin or its non-toxic acid addition salt of the present invention can be used, for example, in a 20 to 100 mg vial or
It is applied as an injection preparation in the form of an ampoule containing 20 to 100 mg, and may also be formulated as a suppository preparation containing 20 to 100 mg per gram. Furthermore, the oxysatsucalosin or non-toxic acid addition salt thereof of the present invention can also be used as an antibacterial therapeutic or prophylactic preparation for livestock, poultry, fish and shellfish, and as a feed composition. In these applications, oxysatsucalosin is highly soluble in water, preferably in the form of an acid addition salt, and is added to the animal's drinking water for use as a solution at an effective concentration for treatment or prophylaxis, growth promotion, or May be added to feed. Furthermore, it is also effective to administer it as an injectable preparation together with an injectable diluent such as water or physiological saline. Furthermore, due to its formulation design, this oxysactucalosin may be used as a prodrug to exert its effects as oxysactucalosin in vivo. The oxysatsucalosin-producing bacterium of the present invention is an actinomycete isolated from field soil in Ato-cho, Abu-gun, Yamaguchi Prefecture.
The Ac3440 strain was identified as belonging to the genus Saccharopolyspora,
Satsukalopolispora S.P. Ac3440
(Saccharopolyspora sp. Ac3440) [National Institute of Microbiology, Agency of Industrial Science and Technology, contract number: FERMP-6238]. The mycological properties of this bacterium are described below. Morphological properties Starch/inorganic salt agar medium (IPS medium 4)
[Inter.J.System.Bacteriol. 16 :313-340
(1966)], cultured at 37°C for 10 to 14 days, and the following findings were observed. The basal hyphae are curved or linear, elongate with branching, and are divided by hyphal parts or at the late stage of culture, with a diameter of 0.4 to 0.6 μ, and no saplings are attached. Aerial hyphae produced from basal hyphae are curved or straight with simple branches, and have a diameter of 0.5 to 0.7μ.
The tip of the tip may be a loop or a loose spiral wound two or three times, or it may be curved or straight. Aerial hyphae segment to form numerous bead-like chained spores (usually 10 or more), and the spores are often separated by empty hyphae. Spores are oval or short cylindrical, size 0.5
It measures ~0.7 x 0.7 to 1.3μ, and its surface is covered with a shell with many tufts of straight or curved long hair-like material. It does not form sporangia, sclerotia, or flagellated spores in basal hyphae or aerial hyphae. Stainability: Gram staining is positive, acid-fast staining is negative. Bacterial cell composition (1) Method of B. Becker et al. [Appl. Microbiol 12 :
421-423 (1964)], diaminopimelic acid (DAP) was meso-type (meso-type).
type) was detected, and Lechevalier's method [J.
Lab.Clin.Med. 71 :934-944 (1968)], arabinose and galactose were detected. (2) The method of H. Mordarska et al. [J. Gen. Microbiol.
71:77-86 (1972)], lipid LCN-A was not detected, and D.
E. Minnikin et al. [J. Gen. Microbiol. 88 :200−
204 (1975)], no cardomycolic acid or mycolic acid was detected. Culture properties Table 1 shows the findings observed after culturing on various media at 37°C for 14 days. Color display is Color
harmony manual 4th edition 1958 (Container
Corporation of America) color classification. [Table] [Table] Culture was observed at 37°C for 14 days, except for physiological properties [1]. (1) Growth temperature range: 22~53℃ (optimum temperature: 30~
(45℃) [Tested in ISP medium 2] (2) Liquefaction of gelatin: Positive (3) Hydrolysis of starch: Positive (4) Skimmed milk: Peptonization; Positive; Coagulation; Negative (5) Production of melanin-like pigments: Negative on ISP medium 7 and 6 (6) Oxygen requirement: aerobic (7) Hydrogen sulfide production: positive [tested on ISP medium 6 with filter paper containing lead acetate] (8) Nitrate reduction: negative [J .Bacteriol, 73 :
15-27 (1957). ] (9) Resistance to lysozyme: non-resistant [J.Gen.Microbiol, 45 :355−
364 (1961). ] (10) Resistance to sodium chloride: 0-10%
It grows at 11% or higher, but does not grow at 11% or higher. [Basal medium: ISP medium 2] (11) Resistance to antibiotics [J.Antibiot. 32 :
180-186 (1979). [Table] [Table] [Table] [Ecology of soil, edited by TRGGray et al.
bacteriap.293−321, Liverpool University
Press, Liverpool, 1967, J.Gen.Microbiol,
69:33–80 (1971) and J.Gen.Microbiol. 88 :
75-85 (1975). +: Positive, -: Negative〓 [Table] Bad, -〓Negative〓
[Table] I obeyed. +〓Positive, -〓Negative〓
From the above, the characteristic properties of this fungus Ac3440 are (1) Morphology, in which loosely wound spiral, linear, or curved spore chains are formed on aerial hyphae that are generated from fissile basal hyphae. However, the spores are covered with a shell with long hair-like substances, and (2) meso-diaminopimelic acid, arabinose, and galactose were detected in bacterial cell analysis, and lipids
LCN-A, nocardomicolic acid, and mycolic acid were not detected; (3) Gram staining was positive and acid-fast staining was negative; and (4) it was aerobic. Based on these characteristic properties, we searched various documents and found that the genus Saccharopolyspora (Saccharopolyspora Lacey & Goodfellw)
[J.Gen.Microbiol. 88 :75-85 (1975)], the present bacterium Ac3440 was identified as belonging to the genus Satucharopolyspora, and was identified as Satucharopolyspora sp. Ac3440.
(Saccharopolyspora sp.Ac3440). Next, when carrying out the present invention and obtaining oxysatucalosin, it is convenient and convenient to collect it from a culture using Satucalopolyspora sp. Ac3440 as described above. In addition, as for the bacteria to be used, the above-mentioned bacteria are one example, and in addition to this bacteria, all bacteria belonging to the genus Satucalopolyspora that produce oxysatucalosin can be used in the present invention, such as genus Satucalopolyspora. Examples include oxysatsucalosin-producing bacteria or mutant strains thereof. Next, in order to produce oxysatsucalosin, which is the novel antibiotic of the present invention, for example, the above-mentioned oxysatsucalosin-producing bacteria belonging to the genus Saccharopolyspora are placed in a medium containing medium components used for the cultivation of ordinary microorganisms. It can be obtained by culturing aerobically. As the medium, a solid medium or a liquid medium can be used, and a liquid medium, especially an aqueous medium, is suitable especially for mass production. As the nutrient source for the medium, a wide variety of nutrients commonly used for culturing microorganisms can be used. The carbon source may be any assimilable carbon compound, such as glucose, sucrose, dextrin, starch, and molasses. Any available nitrogen compound may be used as the nitrogen source, for example,
Corn steep liquor, soybean flour, cottonseed flour, peptone, meat extract, yeast extract, ammonium salts, nitrates, etc. are used. In addition, phosphates, carbonates, sulfates, hydrochlorides, calcium,
Salts such as potassium, sodium, copper, iron, zinc, manganese, and cobalt are used. The culture temperature can be changed as appropriate within a range that allows the bacteria to grow and produce oxysatsucalosin, but is particularly preferably 25 to 35°C. Cultivation time varies slightly depending on conditions, but is usually about 72 to 140 hours.
The culture may be terminated at an appropriate time by determining the time when oxysatsucalosin reaches its maximum titer. In the liquid culture of oxysatsucalosin-producing bacteria thus obtained, most of oxysatsucalosin is produced in the liquid portion. Oxysatucalosin is then collected from this culture, and it is convenient to separate and purify it by taking advantage of the fact that oxysatucalosin is a water-soluble basic amino sugar compound. Oxysatsucalosin produced by Bacillus subtilis
Using PCI219 as a test bacterium, the active category can be confirmed and quantified by the usual agar plate method. An example of a means for separating and purifying oxysatsucalosin is as follows. That is, oxysatsucalosin-producing bacteria are cultured as described above, and the solid content is removed from the resulting culture to obtain a culture solution.Since oxysatsucalosin is an amino sugar compound, the culture solution Once the pH of is adjusted to acidic,
It is preferable to neutralize this to obtain a culture solution, and then this solution is adsorbed on a column of cation exchange resin, such as Amberlite IRC-50 (NH ⁺ ₄ type), from which the active substance is extracted. Elute with 1N ammonia water, further concentrate the eluate, adjust its pH, and adsorb it on a column of cation exchange resin, such as CM Sephadex C-25 (NH ⁺ ₄ type), to obtain 0.03N ammonium. By eluting with water, concentrating the eluted active fraction under reduced pressure, and lyophilizing it, a purified white powder of satukalosin can be obtained in the form of a free base. Furthermore, the oxysacchucalosin thus obtained can easily show a single spot in thin layer chromatography. Next, the present invention will be specifically explained with reference to Examples, but the present invention is not limited thereto. Example 1 Dextrin 1%, glucose 1%, casein hydrolyzate 0.5%, yeast extract 0.5%, calcium carbonate
100 ml of a medium containing 0.1% (PH7.0) was dispensed into a 500 ml Erlenmeyer flask and sterilized by heating at 120°C for 20 minutes. In this medium, Saccharopolyspora sp.
One platinum loop was inoculated from a slant culture of Ac3440 strain, and 30
The culture was cultured with shaking at ℃ for 72 hours, and this was used as a seed culture. Separately, glucose 0.2%, glycerin 4%, peptone 5%, starch 0.2%, defatted soybean flour 0.5%,
Dried yeast 0.5%, salt 0.5%, calcium carbonate 0.2%
(PH7.0) was dispensed into 500 ml flasks, and after heat sterilization, 3% of the above seed culture was transplanted into 100 bottles of the medium. Culture at 30℃96
After incubation with shaking for an hour, these were merged to approx.
Ten cultures were obtained. Example 2 The culture obtained in Example 1 was adjusted to pH 2.0 with 12N sulfuric acid aqueous solution, stirred for 10 minutes, adjusted to pH 7.0 with concentrated aqueous ammonia, and concentrated by centrifugation. Clear liquid 9 was obtained. The obtained supernatant was transferred to Amberlite IRC-50 (NH ⁺ ₄ type) (Roam & Co., Ltd.).
The column was charged to a column packed with 1 (manufactured by Haas), washed with water, and eluted with 1N ammonia water 2 to obtain the entire eluate, which was concentrated under reduced pressure to 30 ml. Next, this concentrated solution was adjusted to pH 7.0 with 6N sulfuric acid, and then added to CM Sephadex C-25 (manufactured by Pharmacia Fine Chemicals) (NH ⁺ ₄ type).
It was charged into a column (diameter 3 cm) packed with 300 ml and adsorbed. Thereafter, it was eluted with 0.03N ammonia water, and the eluate was fractionated into 20ml portions. Each fraction was subjected to thin layer chromatography using chloroform-methanol-concentrated aqueous ammonia (2:3:2) as a developing solvent, and the target product was confirmed by ninhydrin coloring. As a result, the 98th fraction
109 fractions contained only oxysatsucalosin. The fractions were then collected, combined, concentrated under reduced pressure, and then lyophilized to obtain a white powder, which was further dried under reduced pressure at 40°C for 48 hours in the presence of phosphorus pentoxide to obtain a purified white powder of oxysatsucalosin. (Free base) 30 mg was obtained.

[Brief explanation of drawings]

FIG. 1 shows the infrared absorption spectrum of oxysatsucalosin, and FIG. 2 shows the nuclear magnetic resonance spectrum (hydrogen nuclei) of oxysatsucalosin.

Claims (1)

[Scope of Claims] 1. An amino sugar antibiotic 3'-oxysatsucalosin or its non-toxic acid addition salt having the following physicochemical properties. Melting point: 230°C or higher (decomposition) [α] ²⁴ _D : +162° (C = 0.25, H ₂ O) Elemental analysis (for C ₂₁ H ₄₀ N ₄ O ₁₃・2H ₂ O) Actual value C: 42.07, H: 7.12, N: 9.03 Theoretical value C: 42.56, H: 7.48, N: 9.45 Molecular weight: 556 (from mass spectrum) Molecular formula: C ₂₁ H ₄₀ N ₄ O ₁₃ Ultraviolet absorption spectrum: Terminal absorption infrared absorption spectrum: 3350, 2900, 1585,
Has absorption bands at each wavelength around 1460, 1390, 1345, 1040, and 1000 cm ^-1 (KBr method) Nuclear magnetic resonance spectrum (carbon nuclei) No. ppm No. ppm 1 102.6 12 71.1 2 97.0 13 70.4 3 95.9 14 67.4 (Dioxane) 4 88.4 15 66.6 5 78.4 16 62.3 6 76.9 17 61.8 7 74.5 18 56.7 8 74.1 19 51.3 9 73.8 20 50.1 10 72.4 21 36.8 11 72.0 22 3 3.2 Solubility: Soluble in water, acetone, benzene, ethyl acetate Insoluble color reaction: ninhydrin reaction, potassium permanganate decolorization reaction positive, Elson Morgan reaction,
The Sakaguchi reaction is negative Color property: White powder Acid-base distinction: Basic 2 A bacterium that produces amino sugar antibiotic 3'-oxysatucalosin belonging to the genus Satucalopolyspora is cultured in a medium, and 3'-oxy A method for producing 3′-oxysatsucalosin, which comprises collecting satsukalosin. 3 The amino sugar antibiotic 3'-oxysatsucalosin-producing bacterium belonging to the genus Satucalopolyspora is Satucalopolyspora SP AC3440FERMP-
6238, the manufacturing method according to claim 2.