JP2573084B2 - Novel antibiotic PF1032B substance and its production method - Google Patents

Description

The present invention relates to a novel antibiotic PF1032B substance or a salt thereof,
And its manufacturing method.

2. Description of the Related Art The antibiotic PF1032B according to the present invention is considered to be a triterpene or a steroid from the above-mentioned physicochemical properties. -256980), nortriterpene oligoglycoside (JP-A-1-106689), eryloside A (Carmely et al., J. Nat. Prod. 52, No. 1, 167-170,198).
9) etc., but the antibiotic PF1032B is clearly distinguished from these known substances because of its different physicochemical properties.

Problems to be Solved by the Invention Conventionally, various antibiotics produced by microorganisms are known. However, since not many antifungal antibiotics having low toxicity have been found, a new antifungal antibiotic has been developed. Appearance is always desired. The present inventors have found that a PF1032B substance exists in addition to a known PF1032 substance from a culture of a PF1032 substance-producing bacterium, isolated the present invention, and completed the present invention.
It is a further object of the present invention to provide a novel antifungal antibiotic PF1032B
An object of the present invention is to provide a substance and a method for producing the substance.

Means for Solving the Problems The gist of the first invention is to provide a novel antibiotic PF.
1032B substance and its salts. The physicochemical and biological properties of the PF1032B substance according to the present invention are as follows.

1. Physical and chemical properties of PF1032B substance (1) Color and shape: white powder (2) Elemental analysis: C ₃₅ H ₅₄ O ₈ · H ₂ O Measured value C 67.78%, H 9.20% Calculated value C 67.71%, H 9.09% (3) Mass spectrum (FD-MS): m / z 602 (M ⁺ ) (4) Melting point: 192 to 195 ° C. (decomposition) (5) Specific rotation: ▲ [α] ²² _D ▼ = +106.7゜ (cl.0, M
eOH) (6) Ultraviolet absorption spectrum [MeOH]: 206 (125), 226 (sh44) [0.1N HCl-MeOH]: 205 (113), 227 (sh39) [0.1N NaOH-MeOH]: 214 (198) (7) Infrared absorption Spectrum (KBr cm ^-1 ): 3400,2920,2870,1690,1650,1460,1450,14
40,1405,1380,1245,1200,1155,1115,1060,1040,910,86
0,785,720 (8) ¹ H NMR spectrum (400 MHz, CD ₃ OD) δ (ppm): 0.81 (3H, s), 0.91 (3H, s), 0.94 (3H, d),
1.05 (1H, m), 1.35 to 1.52 (5H, m), 1.52 to 1.76 (5H,
m), 1.59 (3H, br s), 1.66 (3H, br s), 1.88 (2H, m),
1.94 (1H, br dd), 1.97 ~ 2.19 (7H, m), 2.26 (1H, m),
2.29 (1H, m), 3.30 (1H, dd), 3.41 (1H, dd), 3.63 (1H,
m), 3.65 (1H, m), 3.73 (1H, m), 3.76 (1H, dd), 4.09
(1H, br dd), 4.66 (1H, br s), 5.00 (1H, d), 5.08 (1
H, br dd), 5.26 (1H, br s) (9) ¹³ C NMR spectrum (100 MHz, CD ₃ OD) δ (ppm): 179.8 s, 151.3 s, 140.1 s, 131.8 s, 129.1 s,
126.1 d, 104.7 t, 96.9 d, 76.7 d, 75.2 d, 74.0 d, 73.6
d, 71.9 d, 63.8 s, 62.7 t, 52.1 d, 48.1 s, 47.8 d, 40.4
s, 37.2 t, 37.0 d, 36.1 t, 32.7 t, 30.2 t, 29.4 t, 29.0
t, 27.7 t, 25.9 q, 25.7 t, 24.4 t, 22.0 t, 19.1 q, 18.6
q, 18.3 q, 17.1 q (10) Solubility: Soluble in methanol, acetone, ethyl acetate, hardly soluble in chloroform, and extremely hardly soluble in water.

(11) Basic, acidic and neutral distinctions: Weakly acidic substances Salts of the PF1032B substance include metal salts, particularly alkali metal salts such as sodium salts and alkaline earth metal salts such as calcium salts. Such as salts with amines.

2. Biological activity of PF1032 substance The minimum inhibitory concentrations of the PF1032B substance of the present invention against various fungi and various bacteria are shown in Tables 1 and 2, respectively.
It is shown in the table.

3. Acute toxicity of PF1032B substance Jcl of PF1032B substance: ICR mice and (male, 19-20 g,
In an acute toxicity test by intra-abdominal administration using n = 3), 10
No deaths occurred at the dose of 0 gm / kg.

According to a second aspect of the present invention, an antibiotic PF1032B is produced by culturing an antibiotic PF1032B substance-producing bacterium belonging to the arrhythmic bacteria and collecting the antibiotic PF1032B substance from the culture.
In the method of manufacturing the substance.

Practical examples of microorganisms used for producing the antibiotic PF1032B of the present invention include the strain PF1032 belonging to the genus Neosartria isolated from soil in Nepal.

1. Mycological properties of strain PF1032 I. Growth state in each medium (1) Tuapek agar medium Growth is not so good, and the diameter is 8 in 7 days of culture at 25 ° C.
1010 mm, 22 to 25 mm for 14 days, 18 to 20 mm in diameter for 7 days at 37 ° C., 30 to 35 mm for 14 days. Consisting of white, velvety hyphae, almost no ascocarp and conidia are formed.

(2) Tuapec yeast extract agar medium The growth is fast at 37 ° C., and the diameter reaches 70 to 80 mm in 7 days of culture. It becomes a flat settlement, and the ascocarps grow densely in layers, initially white, but later pale gray brown. Conidia structures do not form much. The backside of the settlement becomes pale yellow-brown. At 25 ° C., the properties are the same as at 37 ° C., but the growth rate is slightly slower at 55 to 60 mm in diameter during 7 days of culture, and the formation of ascocarp is slightly worse.

(3) Malt extract agar medium It grows rapidly at 37 ° C and reaches a diameter of 85 mm or more in 7 days of culture. The properties are almost the same as those of Tuapec yeast extract agar medium, but they form conidia well.

II. Morphological features under microscope (1) Closed cysts Superficial, white to milky white, spherical to subspherical, 90 to 15 in diameter
0 μm, loosely covered with sterile hyphae.

(2) Ascosac 8 spores, colorless, spherical to subspherical, 13 to 16 μm in diameter, vanishing.

(3) Ascospores colorless, lens shape, size is 3.6 ~ 4.0 × 5.0 ~ 6.0μm,
It produces two equatorial ridges and is almost smooth at the level of an optical microscope.

(4) Conidium head Loose cylindrical, white.

(5) Fialide A single row from the upper half of the crown.

(6) Conidia spherical to subspherical, 2.0 to 2.4 μm in diameter.

The mycological properties of this bacterium are as described above, but it forms a closed cyst and the conidium generation is Aspergillus (Aspergillus).
s) Because it belongs to the genus, it belongs to the class of Aspergillus oryzae. Furthermore, it is considered that it belongs to the genus Neosartorya (Aspergillus fumigatus) because it has white to milky white husks, is loosely covered with sterile hyphae, and has a conidium generation of Aspergillus fumigatus. Can be

Therefore, this strain was referred to as the arachnoid fungus strain PF1032. This strain has been deposited with the Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology as Microbial Research Bacteria No. 11036 (FERM P-11036).

The PF1032 strain changes its properties and is cheap as seen in other molds. For example, any mutants (naturally occurring or inducible) derived from the PF1032 strain, transgenes, or transgenics that produce the PF1032B substance can be used in the present invention.

2. Culture method of PF1032B substance producing bacterium The PF1032B substance producing bacterium belonging to the arbuscular bacillus is cultured in a medium containing nutrients that can be used by ordinary microorganisms. As nutrient sources, known nutrients conventionally used for mold culture can be used. For example, glucose, sucrose, starch syrup, dextrin, starch, glycerol, molasses, animal and vegetable oils and the like can be used as the carbonic acid source. Nitrogen sources include soy flour, wheat germ, corn steep,
Liquor, cottonseed meal, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, urea and the like can be used. If necessary, it is effective to add sodium, potassium, calcium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid and other inorganic salts capable of producing ions. It also helps the growth of fungi, PF10
Organic and inorganic substances that promote the production of the 32B substance can be suitably added.

As a culture method, a culture method under aerobic conditions, particularly a submerged culture method is most suitable. A suitable temperature for culturing is 15 to 40 ° C, but the culturing is often performed at around 26 ° C. The production of the PF1032B substance varies depending on the culture medium and culture conditions, but usually reaches the maximum in both shaking culture and tank culture in 2 to 10 days. When the accumulated amount of the PF1032B substance in the culture reaches the maximum, the culture is stopped, and the target substance is isolated and purified from the culture solution.

3.Purification method of PF1032B substance In collecting the PF1032B substance obtained by the present invention from a culture, a usual separation method utilizing its properties, for example, a solvent extraction method, an ion exchange resin method, an adsorption or distribution column chromatography method , Gel filtration, dialysis, precipitation, etc., alone or in combination as appropriate. For example, the PF1032B substance is extracted from the cultured cells with acetone-water, methanol-water, ethyl acetate, or the like. Also, the PF1032B substance accumulated in the culture solution is
Extraction with an organic solvent immiscible with water, for example, butanol, ethyl acetate, etc., will extract the PF1032B substance into the organic solvent layer.

To further purify the PF1032B substance, use silica gel (Wakogel C-300, manufactured by Wako Pure Chemical Industries, Ltd.), adsorbent such as alumina, Sephadex LH-20 (manufactured by Pharmacia), Toyopearl HW-40 (Tosoh Corporation) It is preferable to carry out chromatography using, for example, the following method.

The PF1032B substance thus produced in the culture can be separated in free form, i.e., as the PF1032B substance itself, and the solution containing the PF1032B substance or its concentrated solution can be treated with a base, i.e. sodium hydroxide, for example.
Operation of each step using an alkali metal compound such as potassium hydroxide, an alkaline earth metal compound such as calcium hydroxide or magnesium hydroxide, an inorganic base such as an ammonium salt, or an organic base such as ethanolamine, triethylamine or dicyclohexylamine. During processing, for example during the operation of each step of extraction, separation or purification, the PF1032B substance changes to its corresponding salt form and is separated. Alternatively, the salts of the PF1032B substance thus produced can be converted into a free form, the PF1032B substance itself, by a conventional method. Further, the PF1032B substance obtained in the free form may be converted into the corresponding salts by a conventional method using the base. Accordingly, salts thereof as described above, as well as the PF1032B material, are intended to be included within the scope of the present invention.

Examples Examples of the present invention will be described below. Since the properties of the PF1032B substance have been clarified by the present invention, various methods for producing the PF1032B substance can be devised based on those properties. Therefore, the present invention is not limited to the examples, and it is possible to produce, concentrate, and extract the PF1032B substance by applying known means based on the properties of the PF1032B substance revealed by the present invention, as well as the modification means of the examples. , Encompasses all purification methods.

As a seed medium, starch 2.0%, glucose 1.0%, polypeptone 0.5%, wheat germ 0.6%, yeast extract 0.3%,
A medium having a composition of soybean meal 0.2% and calcium carbonate 0.2% was used. In addition, as a production medium, 2.0% syrup, soybean oil
0.3%, soybean meal 1.2%, wheat germ 1.2%, sodium sulfate
0.02%, iron sulfate (heptahydrate) 0.0005%, cobalt chloride (7
A medium having a composition of (water salt) 0.0005% and calcium carbonate 0.1% was used. The pH before sterilization was all adjusted to pH 7.0 before use.

A 100 ml Erlenmeyer flask to which the above seed medium (20 ml) was dispensed was sterilized at 120 ° C. for 15 minutes, and Neosartorya sp.
One platinum loop of the slant agar culture of the strain (FERM P-11036) was inoculated and shake-cultured at 26 ° C for 2 days to obtain a seed culture. Then
A 500-ml Erlenmeyer flask (200 tubes) into which the production medium (100 ml) was dispensed was sterilized at 120 ° C. for 15 minutes, inoculated with the seed culture (2 ml each), and cultured with shaking at 26 ° C. for 4 days. After the completion of the culture, diatomaceous earth was added as a filter aid, followed by filtration to obtain a filtrate and cells.

50% acetone water (16 L) was added to the cells, and after stirring for 1 hour, the cells were filtered off to obtain a cell extract. The cell extract is
Acetone was distilled off under reduced pressure to obtain a 4 L concentrated solution. Ethyl acetate (20L) was obtained from the mixture of this concentrated solution and culture filtrate (16L).
The ethyl acetate layer was concentrated to give an oily substance (3.82 g). This oily substance was placed on the top of a silica gel column (400 g), washed with chloroform-methanol (10: 1, 800 ml), and then chloroform-methanol (5: 8).
Chromatography was performed using 1) as a developing solvent, and the eluate was fractionated in 20 ml portions. The fraction containing the PF1032B substance (fraction numbers 81 to 90) was concentrated to dryness to give a pale yellow powder (3
2.3 mg) were obtained. Next, this pale yellow powder was separated with Sephadex LH-20 (140 ml) using methanol as a developing solvent.
Purification was performed by column chromatography (1 fraction, 6.5 ml), and fractions 15 to 19 were concentrated to dryness to give pure
The PF1032B material was obtained as a white powder (25.8mg). Similarly, in the first silica gel column chromatography, the fraction containing the PF1032 substance (fraction numbers 92 to 13)
6) was concentrated to dryness to give a pale yellow powder (214 mg).
Then, the pale yellow powder was purified by column chromatography (1 fraction, 13 ml) using Sephadex LH-20 (560 ml) using methanol as a developing solvent, and fractions 34 to 42 were concentrated to dryness to give pure PF1032. Material was obtained as a white powder (189 mg).

Effect of the Invention The PF1032B substance of the present invention is characterized by having antifungal activity as shown in Table 1 and having lower toxicity than conventional antifungal antibiotics. Based on these properties, the PF of the present invention
The 1032B substance can be used as an antifungal agent or a material for conversion to it.

[Brief description of the drawings]

FIG. 1: UV absorption spectra of the PF1032B substance in methanol (100 μg / ml, solid line), acidic methanol (100 μg / ml, broken line) and basic methanol (100 μg / ml, dashed line). FIG. 2 shows an infrared absorption spectrum of a potassium bromide tablet of the PF1032B substance. Figure 3: PF1032B 400MHz ¹ in heavy methanol solution of a substance
1 shows a 1 H NMR spectrum. Figure 4: 100MHz in PF1032B substance in heavy methanol solution
¹³ shows a ¹³ C NMR spectrum.

Continued on the front page (72) Inventor Chieko Moriyama 760, Shiokaokacho, Kohoku-ku, Yokohama, Kanagawa Prefecture Inside the Pharmaceutical Research Institute (72) Inventor Tomoko Yugami 760, Shikaokacho, Kohoku-ku, Yokohama-shi, Kanagawa Meiji Seika Co., Ltd. Inside the Research Institute (72) Inventor Masao Koyama 760 Meijiokacho, Kohoku-ku, Yokohama-shi, Kanagawa Prefecture Meiji Seika Co., Ltd.

Claims (2)

(57) [Claims] 1. Antibiotic PF1032B substance and its salt having the following properties: (1) Color and shape: white powder (2) Elemental analysis: measured values C 67.78%, H 9.20% (3) Mass spectrum (FD-MS) ): M / z602 (4) Melting point: 192 to 195 ° C (decomposition) (5) Specific rotation: ▲ [α] ²² _D ▼ = +106.7 ゜ (cl.0, M
eOH) (6) Ultraviolet absorption spectrum: shown in FIG. (7) Infrared external absorption spectrum: shown in FIG. (8) ¹ H NMR spectrum: shown in FIG. (9) ¹³ C NMR spectrum: shown in FIG. (10) Solubility: soluble in methanol, acetone, ethyl acetate, hardly soluble in chloroform, and extremely hardly soluble in water. (11) Basic, acidic, and neutral: reverse acidic substances 2. A method for producing an antibiotic PF1032B, comprising culturing an antibiotic PF1032B substance-producing bacterium belonging to the arachnoid bacterium (genus Neosartorya) and collecting the antibiotic PF1032B from the culture. .