JPH02233689A - Novel antibiotic pf1015 substance and production thereof - Google Patents

Abstract

NEW MATERIAL:Antibiotic PF1015 substance having the following properties and a salt thereof. Color and shape: white powder. Molecular formula: C46H60O16. Mass spectrum (SI-MS): m/z 869(M+H)<+>. Melting point: 144-146 deg.C. Specific rotatory power: [alpha]D<15>=+24.3 deg.. (C=1.0, MeOH). Solubility: soluble in methanol, slightly soluble in acetone and ethyl acetate and insoluble in water. A weakly acidic substance, etc. USE:An antifungal agent having low toxicity. PREPARATION:A fungus (e.g. FERM P-10510) belonging to asporous Fungi imperfecti, capable of producing antibiotic PF1015 substance is cultured preferably at about 26 deg.C by shaking culture for 2-10 days.

Description

[Detailed Description of the Invention] Industrial Application Field The present invention is... This invention relates to a novel antibiotic PF1015 substance and its production method.

Prior Art Compounds with similar physical and chemical properties to the antibiotic PF1015 substance according to the present invention include pablacandin A, B,
C, D and E (Papulacandins XT
Raxler et al., J. Antibiotics 3
0: 289=296.1977). Chaetiacandin (Komori et al.
J. Antibiotics 38: 455-45
9.1985) etc. are known. Antibiotic PFt
ots substances have different physical and chemical properties from these substances and can be clearly distinguished.

Conventional problem that the invention seeks to solve. A variety of antibiotics produced by microorganisms are known, but not many low-toxic anti-inflammatory antibiotics have been found. There is a constant demand for new anti-inflammatory antibiotics. An object of the present invention is to provide a novel anti-sodium antibiotic PF1015 substance and a method for producing the same.

Means for Solving the Problems The gist of the first invention is to provide a novel antibiotic PF.
1015 substances and their salts. PF1 according to the invention
The physicochemical and biological properties of Substance 015 are as follows.

1. P. Physical and chemical properties of F1015 substance (1) Color and shape: white powder (2) Molecular formula 46H60016 (3) Mass spectrum (SI-MS): m/z
869 (M+H) (4) Melting point: 144-146°C 15. (5) Specific rotation: (g) +24.3° (c
1.0. MeOH) D (6) Ultraviolet absorption spectrum λa+ax nm (E 凰) (MeOH): 206 (574). 215 (s
h 335), 222 (312), (0.1N HC
I-MeOH): 205 (501). 217
(284). 223 (279). 268 (569) (0.1N NaOH-MeOH): 214 (1
074). 265 (565) (7) Infrared absorption spectrum (KBr) (c+i-1): 3400, 2
950. 2920. 2870. 1700, 16
35,1615. 1460, 1380. 1345
．． 1310. 1260, 1210. 1180.
1155. 1075. 1040. 1010,98
0, 870, 850, 775, 740
．． 720(8)'H NMR spectrum (400
MHZ, CD30D) δ (ppa+): 0.8
4 (3H.t). 0.85 (3H, d). 0.
93 (3H, d), 1, lO ~ 1.50 (7H, a
+). 1.88-2.00 (3H.m). 2.30
(2H, m), 3.49 (2H, m). 3.5
1 (1B, m). 3.73 (IH, ddd). 3
．． 79 (IH, br dd), 3.80 (IH, b
rs). 3.95-4.07 (3H, m), 4
．． 20 (18, dd). 4.29 (1B, dd).
4.38 (IH, d). 4.39 (IH, d)
．． 5.00 (IH, br d), 5.04 (IH,
brd). 5.21 (IH, br dd), 5
．． 34 (IH, br dt). 5.44 (IH,
dd), 5.91 (IH, d), 6.19 (IH,
br dt). 6.19 (IH, brd). 6
．． 22 (IH, br d). 6.29(LH, b
r dd), 6.59 (LH, d), 7.30 (
LH, dd). 7.42 (3H, m), 7.6
4(21, +a). 7.76 (LH, d) (9)
l3C NMR spectrum (100 MHz, CD3
0D) δ (ppm): 169.1 s, 168.
3 s. 161-6 S, 154.6 s, 146
．． 7 d, 146.2 d, 145.5 s. 1
42.7 d, 137.5 d, 135.7 s,
131.6 d. 131.5 d. 130.1 d
, 130.1 d, 129.8 d, 129.3
d. 129.3 d. 121.4 d, 1
1g. 8 d, 116.5 s, 112. Os,
105.4 d. 103.1 d, 100.1 d
, 77.8 d. 76.4 d. 75.2 d
．． 74.8 d. 74.7 d, 74. O d
, 73.9 t, 72.6 d, 71.9
d, 70.3 d, 64.9 t, 61.6
t, 39.8 d, 39.3 t, 39
．． 2 d, 33.9 t, 33.9 t, 30
．． 9 t, 28.5 t. 21. Oq. 14
．． 3 q, 12.2 q (lO) Solubility: Soluble in methanol. Hardly soluble in acetone and ethyl acetate. Not soluble in water.

(11) Basicity. Distinguish between acidic and neutral Weakly acidic substances PF1
Salts of the 015 substance include metal salts, particularly alkali metal salts such as sodium salts, alkaline earth metal salts such as calcium salts, and ammonium salts.

2. Biological Activity of PF1015 Substance The minimum inhibitory concentrations of the PF I O 1 5 substances according to the present invention against various fungi and various bacteria are shown in Tables 1 and 2, respectively.

Table 1 Table 2 Table 3. Acute toxicity of PF1015 substance Jcl of PF1015 substance: ICR? Usu (male.1
9-20g. In an acute toxicity test using intraperitoneal administration (n = 3), there were no deaths at a dose of 150 mg/kg, and all deaths occurred at a dose of 300+++ g/kg.

The second gist of the present invention is to produce an antibiotic PF1015 substance by culturing an antibiotic PF1015 substance-producing bacterium belonging to the apospore-deficient bacterium and collecting the antibiotic PF1015 substance from the culture. It's in the law.

PFI015 strain, which is an example of the microorganism used in the present invention, is a type of mold that was newly isolated from soil in Nagano Prefecture in 1988. Its mycological properties are as follows.

1. Mycological properties of strain PF1015 When cultured on potato dextrose agar (PDA) and malt extract agar (CMEA) media at 25'C, the colony size was 7-10m in 10 days and 16-10m in 20 days. It is 18a+1 and has a velvety appearance. It showed a shape with radial wrinkles and a raised center. The color of the colony is dark gray-green. The back side became dark brown and produced a brown water-soluble pigment. It grew at 15-30℃, but it grew at 37℃.
It didn't grow. Growth was good at pH 5-7. Potato Kyaloft Agar (PCA), Zurbaek
Dox agar (CzA), Miura agar (LcA). YpS
Cultures on commonly used mold media such as S agar and MY20 agar showed almost similar properties.

When observed under a microscope, numerous vacuoles were observed in the hyphae in all media. Characteristic forms such as conidia were not observed.

Place sterilized rice straw or banana leaves on top of the agar to inoculate. It was observed for 1 month at 25°C. In this case as well, no formation of conidia was observed.

Therefore, we decided to call this strain the apospore-deficient strain PFIO15.

Note that this strain has been entrusted to the Institute of Microbial Technology, Agency of Industrial Science and Technology as FERM P-10510.

PFIO: 15 stocks are. Its properties change easily, as seen with other molds. For example, any mutant strain (naturally occurring or induced), transzygote, or genetically modified strain derived from this strain that produces the PF1015 substance can be used in the present invention.

2. Method for culturing PF1015 substance-producing bacteria A PF1015 substance-producing bacteria belonging to the apospore-deficient bacteria is cultured in a medium containing nutrients that can be used by ordinary microorganisms. As the nutrient source, known nutrient sources conventionally used for culturing molds can be used. for example. As a carbon source. glucose. Sucrose, starch syrup, dextrin, starch. Glycerol. Molasses, animal/vegetable oils, etc. can be used. Also,
As a nitrogen source. Soy flour. Wheat germ. Corn steep liquor. Cottonseed meal. Meat extract, beptone. Yeast extract. Ammonium sulfate, sodium nitrate 9M. urine. You can use elementary etc. It is also effective to add, if necessary, inorganic salts capable of producing sodium, potassium, calcium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid, and other ions.゜Also, it is necessary to appropriately add organic and inorganic substances that support the growth of bacteria and promote the production of PFIOI5 substances! Wear.

As a culture method. Cultivation method under aerobic conditions. In particular, the deep culture method is most suitable. The appropriate temperature for culturing is 15-30℃.
However, in most cases, it is cultured at around 26℃ (-.PF1
Production of 015 substance varies depending on the medium and culture conditions. Shaking culture. In any tank culture, accumulation usually reaches its maximum within 2 to 10 days. When the accumulated amount of the PF1015 substance during culture reaches the maximum, the culture is stopped, and the target substance is isolated and purified from the culture solution.

3. Method for Purifying PF1015 Substance In collecting the PF1015 substance obtained by the present invention from a culture. Ordinary separation methods that take advantage of its properties, such as solvent extraction, ion exchange resin, adsorption or distribution column chromatography, gel filtration, dialysis, and precipitation, can be used alone or in combination as appropriate for extraction and purification. can do. For example, the PF101.5 substance is extracted from cultured bacterial cells using acetone-water, methanol-water, ethyl acetate, or the like. , PFI accumulated in the culture medium
If the 015 substance is extracted with an organic solvent that does not mix with water, such as butanol, ethyl acetate, etc., the PF1015 substance will be extracted into the organic solvent layer.

To further purify the PF I O I 5 substance, use silica gel (Wako Gel C-200, manufactured by Wako Pure Chemical Industries, Ltd., etc.),
Alumina and other suckers and Se7 Adex LH-20 (
7 Almasia). Chromatography using Toyopearl HW-40 (manufactured by Tosoh Corporation) or the like may be performed.

The PF1015 substance thus produced in the culture can be isolated in free form, ie, as the PF1015 substance itself. Alternatively, a solution containing the PF1015 substance or its concentrate may be mixed with a base, that is, an alkali metal compound such as sodium hydroxide or potassium hydroxide,
Alkaline earth metal compounds such as magnesium hydroxide and calcium hydroxide. Extraction and separation using inorganic bases such as ammonium salts, organic bases such as ethanolamine, triethylamine, dicyclohexylamine, etc. Or when treated during each step of purification. The PF1015 substance is converted into its corresponding salt form and isolated. In addition, the salts of the PF1015 substance produced in this way are as follows. It can be converted into the free form by conventional methods. Furthermore, the PF1015 substance obtained in free form can be converted into the corresponding salts using the base in a conventional manner. Therefore, PF1
The 015 substance as well as its salts as described above are intended to be included within the scope of this invention.

Examples of the present invention are shown below, but since the properties of the PF1015 substance have been clarified by the present invention, various methods for producing the PF1015 substance can be devised based on those properties. Therefore, the present invention is not limited to the examples shown below, and the invention can of course be modified. Based on the properties of the PF1015 substance revealed by the present invention, the PF1015 substance is produced by applying known means. concentrated. Includes all extraction and purification methods.

Example seed medium: starch 2.0%, glucose 1.0%
．． Wheat germ 0.6%. Polybeptone 0.5%, soy flour 0
．． 2%, yeast extract 0.3% and calcium carbonate 0.1
A medium consisting of a composition of % was used. Also. As a production medium, glucose 5. θ%, wheat germ 1.0%, dry yeast 1
．． 0%, Corn Steve Liquor 1.5%, Polypeptone 0.5%, Meat Extract 0.5%, Calcium Carbonate 0
．． 3%. Magnesium sulfate (heptahydrate) 0.2% and chloride. A medium having a composition of 0.2% sodium was used. In addition, the pH before sterilization was adjusted to pH 7.0 in all cases.

A 100 mQ Erlenmeyer flask into which the seed medium 20a+Q was dispensed was sterilized at 120°C for 15 minutes, and then sterilized with the apospore-deficient bacteria PF 1 0 1 5 strain (FERM p-1051).
Inoculate 2 to 3 platinum loops of slant agar culture of 0). The culture was cultured with shaking at 26° C. for 7 days to obtain a first type culture. Next. One 500aQ Erlenmeyer flask containing the seed medium (80mg)
The cells were sterilized at 20°C for 15 minutes, inoculated with the first type culture (4 mU), and cultured with shaking at 26°C for 3 days, which was used as a second type culture.

Two 50 L jar fermenters containing 35 L of production medium that had been previously sterilized at 120° C. for 30 minutes were inoculated with 400 microliters each of the second type culture. Aerated at 26℃ for 4 days (2
OL/min). Culture was carried out with stirring (initial 250 rpm, 400 rpm after 41 hours). After the culture was completed, diatomaceous earth was added as a filter aid and the mixture was filtered to obtain a filtrate and bacterial cells.

Add 85% acetone water (64 L) to the bacterial cells. After stirring for 1 hour, the bacterial cells were filtered to obtain a bacterial cell extract. The bacterial cell extract is. Acetone was distilled off under reduced pressure to obtain 13 L of a concentrated liquid.

From this concentrated solution, PF101 was prepared using ethyl acetate (18Lx2).
Extract 5 substances. The ethyl acetate layer was concentrated to give an oil (16 g). This oil was placed on top of a silica gel column (400 g) and chloroform-methanol-50:1 (IL). 25: 1 (l L)
．． lO:l(2L). Chromatography was performed using 5:1 (2 L) as a developing solvent, and both fractions containing the PF1015 substance were concentrated to dryness to obtain a brown oily substance (1.5 g). This oily substance was then subjected to a silica gel force column (10
0g) on top of chloroform-methanol-10
Chromatography was performed using :1 as a developing solvent. The obtained crude PF1015 substance (680 mg) was further added to Sephadex LH-20 (2
L) When purified by column chromatography,
A pale yellow powder (555 mg) was obtained. This pale yellow powder was dissolved in acetone-methanol (5:1) (6d), hexane (25m(+) was added thereto, the precipitate was filtered and dried, and the pure PF1015 substance was turned into a white powder (3
73 mg) I: . Effects of the Invention The PF1015 substance of the present invention has antifungal activity as shown in Table I. It is also characterized by lower toxicity than conventional antifungal antibiotics. Based on these properties, the PF1015 substance of the present invention can be used as an antifungal agent or a material for conversion thereto.

[Brief explanation of the drawing]

Figure l: PF1015 substance in methanol (20μg7
mQ, solid line), <20 p g/mQ in acidic methanol,
The ultraviolet absorption spectra in (dashed line) and basic methanol (20 μg/mQ, one-dot chain line) are shown. Figure 2: Shows the infrared absorption spectrum of the PF1015 substance in potassium bromide tablets. Figure 3: 4 of PF1015 substance in heavy methanol solution
00MHz 'H NMR spectrum is shown. Figure 4: 1 of PF1015 substance in heavy methanol solution
00MHz l3C NMR spectrum is shown.

Claims (1)

[Claims] 1. Antibiotic PF1015 substance and its salts having the following properties (1) Color and shape: white powder (2) Molecular formula: C_4_6H_6_0O_1_6 (3)
Mass spectrum (SI-MS): m/z869 (M+H
) (4) Melting point: 144-146℃ (5) Specific rotation: [α]^1^5_D=+24.3°(
c1.0, MeOH) (6) Ultraviolet absorption spectrum: Shown in FIG. (7) Infrared absorption spectrum: shown in Figure 2. (8)^1H NMR spectrum: Shown in Figure 3. (9)^1^3CNMR spectrum: Shown in Figure 4. (10) Solubility: Soluble in methanol, poorly soluble in acetone and ethyl acetate, and insoluble in water. (11) Distinction between basic, acidic, and neutral: Weakly acidic substance 2, characterized by culturing antibiotic PF1015 substance-producing bacteria belonging to apospore-deficient bacteria and collecting antibiotic PF1015 substance from the culture. A method for producing an antibiotic PF1015 substance.