JP3064532B2 - Antifungal substance Mer-WF5027-II - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The present invention relates to novel antifungal substances, and more specifically,

[0002]

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[0003] and

[0004]

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The antifungal substance Mer-WF50 represented by
27-IIA and -IIB and methods for their production.

As antifungal substances, substances such as amphotericin B, griseofulvin, polyoxin, and validamycin have been known. However, these known antifungal substances are still insufficient in toxicity, efficacy, drug resistance and the like. I can't say.

The present inventors have now found that certain strains belonging to the genus Aspergillus isolated from soil in Hiratsuka-shi, Kanagawa Prefecture have the antifungal activity and low toxicity described in the above formulas (I) and (II). Novel antifungal substance Mer-WF shown
The present inventors have found that 5027-IIA and -IIB are produced, and have completed the present invention.

The antifungal substance Mer-WF of the above formula (I)
5027-IIA and the antifungal substance Me of the above formula (II)
r-WF5027-IIB is a bond isomer (Vale
have a relationship of nce isome r), in these materials solution state at room temperature, the molar ratio of the Mer-WF5027-IIA and Mer-WF5027-IIB is approximately 5: 1
(Referred to as a result of high performance liquid chromatography) as a mixture of linked isomers (hereinafter, this mixture is referred to as an antifungal substance Mer-WF5027-II).

The antifungal substance Mer-WF502 of the present invention
7-IIA and -IIB can be obtained by, for example, culturing an antifungal substance Mer-WF5027-IIA and / or -IIB-producing bacterium belonging to the genus Aspergillus in a culture medium, and extracting the antifungal substance Mer-WF5027-IIA and / or Or-
It can be produced by collecting IIB. Examples of the antifungal substance Mer-WF5027-IIA and / or -IIB producing bacteria include Aspergillus sp. Me 1207 (Aspergillus sp. Me 1207) which the present inventors isolated from soil in Hiratsuka-shi, Kanagawa. be able to. The mycological properties of this fungus are as follows.

(1) Growth form in various media All cultures were performed at 26 ° C., and the growth form on an agar plate medium was observed.

A) Tuapec agar medium The growth is very fast and grows in a velvety form. However, when matured, the central part becomes fluffy and many conidia are formed, and conidia are formed at the tips. The color tone of the colony surface is initially light yellow to dark yellow, but becomes yellowish green to dark yellow green when matured. The color of the back of the colony is light yellow to dark yellow. Colorless leach droplets are observed, with no sclerotium formation. No formation of milts (ascospore-forming organs) is observed.

B) Malt extract agar medium The growth of velvet-grown colonies is very rapid, and when matured, the confluent pattern is formed with a fluffy tall core when matured, and many conidia are formed. The color of the colony surface is yellowish-green to yellowish-green, and becomes mature yellowish to dark yellowish green when matured. Colorless oozing droplets are observed, with no formation of sclerotium and orchid.

[0013] (2) in culture in the form Characteristics Tsu § Peck agar, length of conidiophores is 2.
It may reach 0-2.5 mm, and conidia are formed at its tip. The surface of the conidiophore is rough and has many small protrusions, and the tip has a spherical to long spherical apex, and the outer side of the conidia has a stalk (5.0 × 8.5μ to 3.0 × 5. 0μ). Conidia (3.0-4.5 μ × 4.0-5.0 μ) originate from the tip of the stalk and spread radially to form a chain of many conidia. The mature conidia at the apex rupture into several dispersing columns.

[0014] From the above properties, this bacterium is a bacterium of Aspergillus (AspergilluS) genus, "The Genus Aspergillus" of its forms a fit Lee par and Fennell (1965) (R a per & Fennell "Th
e Genus Aspergillus "), it was determined to be a kind of koji mold, and it was also examined with reference to" Kojigaku "Japanese brewing association (1986) by Hideya Murakami et al., but the species could not be specified. Did not. Therefore, this bacterium was transformed into Aspergillus sp. Me1207 (A
spergillu sp. Me1207) and deposited on November 9, 1990 with the Research Institute of Microorganisms of the National Institute of Advanced Industrial Science and Technology.
11848).

However, the antifungal substance Mer-WF502
7-IIA and / or -IIB-producing bacteria are not limited to Aspergillus sp. Me1207,
For example, other producers that can be found as follows can be used as well.

Using a bioassay agar plate with Candida albicans as a test bacterium and a bioassay agar plate with Bacillus subtilis as a test plate, the culture filtrate of the soil-isolated bacteria was assayed by the perforation method, and the former agar was used. The plate is given an inhibition circle, and the latter is searched for soil isolates that give a culture filtrate in which the inhibition circle is almost invisible on the agar plate.
Next, the culture filtrate having the above activity of the soil isolate is filtered through a Millipore filter (DISMC-25cs; Nippon Millipore Limited), and the antifungal substances Mer-WF5027-IIA and / or -IIB are standardized by high performance liquid chromatography. Detected at the same retention time as the sample,
The adsorbing the culture filtrate to a Sep Pack (Nippon Millipore Limited), after deployment of the methanol eluate by thin layer chromatography, if it is possible to detect bio-O over preparative chromatography to test bacteria of Candida albicans, the The bacterium can be said to be an antifungal substance Mer-WF5027-IIA and / or -IIB producing bacterium that can be used in the method of the present invention.

Antifungal substance Mer-WF5027-II
The medium for culturing A and / or -IIB is not particularly limited, and any medium conventionally used for mold cultivation can be used. For example, assimilation of carbohydrates, nitrogen sources, inorganic salts, etc. Media containing the available nutrients can be used. For example, carbohydrates such as glucose, glycerin, maltose, sucrose, molasses, dextrin, starch and the like, and oils and fats such as soybean oil, peanut oil, lard, and fats, carbon sources such as fats; peptone, meat extract, soybean powder, cottonseed powder, dried yeast , Corn steep liquor, yeast extract, skim milk, casein, sodium nitrate, ammonium nitrate,
A medium containing a nitrogen source such as ammonium sulfate; inorganic salts such as dipotassium phosphate, salt, calcium carbonate, and magnesium sulfate can be used, and trace metals such as cobalt and manganese can be added as necessary. Other nutrient sources include the antifungal substance Mer-WF5027-I
As long as the IA and / or -IIB producing bacteria are utilized to produce the antifungal substance Mer-WF5027-IIA and / or -IIB, any of them can be used, and any known mold culture material can be used. . In order to suppress foaming during heat sterilization and during culture, an antifoaming agent such as silicon or vegetable oil may be added.

The cultivation of the production bacterium in such a medium is usually carried out by
It can be carried out aerobically under aeration and stirring at a temperature of about 20 to about 37 ° C and a pH of 4 to 8. Culture time is about 48
It can be about 100 hours.

The antifungal substances Mer-WF5027-IIA and / or -IIB thus accumulated in the culture
Is mainly extracellular, so after culturing,
The cells can be removed by a separation method known per se such as filtration, centrifugation, or extraction, and the cells can be recovered from the filtrate, supernatant, extract, or the like.

The recovery can be effected in various ways known per se, in particular those which are frequently used for the recovery of neutral organic substances. For example, solvent extraction with ethyl acetate, n-butanol, methylene chloride, chloroform, etc .; adsorption with activated carbon, Amberlite XAD (manufactured by Rohm and Haas), Diaion HP-20 (manufactured by Mitsubishi Kasei) and methanol Elution with water, acetone water or the like; gel filtration using Sephadex G-10 (manufactured by Pharmacia), Bio-Gel P-2 (manufactured by Bio-Rad), cellulose, Avicel SF (manufactured by American Viscose), Column method or thin layer chromatography with silica gel, alumina, etc .; normal phase or reverse phase high performance liquid chromatography; forced precipitation by addition of an organic solvent such as acetone; lyophilization method, etc., alone or in combination as appropriate. Some can be separated and purified by repeated use.

The antifungal substance Me provided by the present invention
r-WF5027-II exhibits antibacterial activity against fungi, as is clear from the results of the in vitro test by the liquid medium dilution method shown in Table 1 below.

[0022]

[Table 1]

* Antifungal substance Mer-WF5027-I
After dissolving I in a small amount of methanol, the medium was diluted with yeast nitrogen-based glucose (YNBG),
A two-fold dilution series was prepared with a maximum concentration of 00 μg / ml. 0.1 ml of this in a 96-well microtiter
Was dispensed. The test bacterium is pre-cultured with YNBG, and then YNBG.
Was diluted to 10 ⁵ CFU / ml with 0.1 ml, and 0.1 ml was added to the microtiter. The microtiter plate was cultured at 35 ° C. for 48 hours, and the MIC was defined as the lowest drug concentration at which growth was not visually observed.

As described above, the Mer-WF of the present invention
The 5027-II substance has an antifungal activity and is expected to be used as an antibacterial agent in the fields of medicine, veterinary medicine, agricultural chemicals, and the like. It can also be used as an intermediate for producing pharmaceuticals, agricultural chemicals and the like.

Next, the present invention will be described more specifically with reference to examples.

[0026]

Example 1 Aspergillus sp Me1207
(FERM P-11848) from a slant medium (potato dextrose agar medium) with a loop of 50 ml of seed medium (potato starch 2%, glucose 1%, soybean powder 2)
%, Potassium phosphate 0.1%, magnesium sulfate 0.0
(5%, no pH adjustment) was inoculated into a 500 ml Erlenmeyer flask, and cultured with shaking at 28 ° C. for 3 days to obtain a seed culture solution. 100 ml of this seed culture was inoculated into 5 l of 10 l jar fermenters containing 5 l of production medium (potato juice: boiled juice of 200 g / l potato, 20 g / l of potato starch). Aeration and agitation culture at 28 ° C. for 88 hours (aeration rate 5 l / min., Agitation 300 r.
p. m. ). After completion of the culture, about 25 l of the culture was filtered to obtain 23 l of the culture filtrate.

The filtrate was applied to a column (8 × 60 cm) packed with Diaion HP-20 (manufactured by Mitsubishi Kasei Kogyo Co., Ltd.), washed with 3 l of ion-exchanged water, and eluted with 5 l of 50% acetone water. Each 300 ml was fractionated. The Mer-WF5027-II substance in the eluted fraction was detected by antibacterial activity measurement (Candida albicans) using a paper disk method. Fraction 3 showing antifungal activity
7 was collected and concentrated to about 1/2 to remove the contained acetone. Unless otherwise specified, the subsequent operations are at 10 ° C.
At low temperature. Next, the above total concentration (about 1 l)
To a QAE-Sephadex column (Pharmacia);
A-25, Cl type, 3.5 × 60 cm). 400
After washing with ml of ion-exchanged water, the flow-through solution and this washing solution were combined. Mer-WF5027-II material is included in this fraction. A column (2.5 × 25 cm) packed with this fraction is packed with Diaion CHP-20 (manufactured by Mitsubishi Kasei Kogyo).
And eluted with 50% acetone water. Antimicrobial activity fractions were collected and concentrated to dryness. Chloroform: methanol (1
00: 1) 3 ml of a chloroform: methanol (25: 1) solution of the above concentrated and dried solid substance was placed on a silica column (Wako Pure Chemical Industries, Ltd .: C-200, 1.8 × 25 cm) packed with a solution.
Dissolved and adsorbed. Next, this column was eluted with a chloroform: methanol (100: 1) solution. 10g
The active components were classified into antibacterial activities and silica thin-layer chromatography (Merck: Art. 5715, developing solvent: chloroform: methanol = 20: 1, detection: ultraviolet absorption 25).
4 nm), and fractions 15 to 21 were collected and concentrated to dryness to obtain 43 mg of Mer-WF5027-II substance.

The antifungal substance Mer-W thus obtained
F5027-II has the following physicochemical properties.

(1) Color and properties: pale yellow oily substance (2) Molecular formula: C ₁₆ H ₂₄ O ₄ (3) Mass spectrum (FAB-MS, matrix;
m-nitrobenzyl alcohol): positive; m / z
303 [(M + Na) ⁺ ], 281 [(M + H) ⁺ ] negative; m / z 279 [(M−H) ⁻ ] (4) Specific rotation: [a] _D ²⁵ = + 54.0 ° (c 0.2
2, chloroform) (5) Ultraviolet absorption spectrum (MeOH) λmax nm (ε) [MeOH] shown in FIG. 1: 304.8 (2
350), 254.8 (6330) [acidic MeOH] 307.0 (2130),
254.6 (6510) [basic MeOH]: 286.4 (sh) (38
00), 256.4 (6120) (6) Infrared absorption spectrum (penetration KBr tablet method) Main absorption peak (cm ^-1 ) shown in FIG. 2: 3385 (br), 29
55, 2930, 2857, 1732, 1645, 15
91, 1462, 1424, 1381, 1304, 12
90,1235,1204,1175,1140,10
73, 1040, 986, 920 (7) ¹ H-NMR spectrum (400 MHz, CDC
l ₃ ) Main peak shown in FIG. 3 δTMS (ppm): 0.89 (6H,
t, J = 6.6), 1.30 (16H, m), 1.56
(4H, m), 1.68 (1H, br m), 1.83
(2H, m), 2.01 (1H, brs), 2.38
(2H, m), 2.50 (2H, m), 2.65 (2H,
m), 3.74 (2H, br m), 3.83 (2H, b
rs), 4.06 (1H, dd, J = 12.8, 5.
5), 4.09 (1H, dd, J = 12.8, 5.5),
4.84 (1H, quint., J = 2.2), 4.9
7 (1H, td, J = 3.7, 1.5), 5.36 (1
H, dd, J = 10.3, 2.6), 5.38 (1H, d
d, J = 9.9, 3.7), 6.56 (1H, dd, J =
10.3, 2.2), 6.60 (1H, d, J = 9.9) (8) ¹³ C-NMR spectrum (100 MHz, CDC
l ₃ ) Main peak shown in FIG. 4 δTMS (ppm): 195.0 s, 1
94.8s, 173.6s, 172.9s, 119.4d,
119.3d, 115.4d, 113.9d, 109.0
s, 108.4s, 81.1d, 80.9d, 74.7d,
73.3d, 70.8d, 31.8t, 31.7t, 31.
5t, 29.2t, 29.1t, 28.8t, 27.0t,
26.9t, 25.6t, 25.4t, 22.5t, 14.
Data on the multiplicity of the 0q signal was obtained by the DEPT test.

(9) Solubility: soluble in ethyl acetate, chloroform, toluene and methanol. Does not dissolve in water.

(10) Thin-layer chromatography As shown in FIG. 5, silica gel plate Art. 5715 used,
Phosphomolybdic acid color development Rf value is as follows: developing solvent system Mer-WF5027-IIA Mer-WF5027-IIB ethyl acetate 0.7 0.5 chloroform: methanol (20: 1) 0.4 0.3 (11) High-speed liquid Chromatography The measurement conditions shown in FIG. 6 are as follows. Column: YMC-Pack A-312 ODS (6φ ×
Column temperature: constant temperature of 20 to 25 ° C. Mobile phase: 80% (v / v) methanol / 0.02 M phosphate buffer (pH 6.8) Flow rate: 1.0 ml / min Detection: UV 254 nm Mer-WF5027-IIB: 5.0 min Mer-WF5027-IIA: 5.7 min The abundance ratio is as follows Mer-WF5027-IIA: Mer-WF5027
-IIB ≒ 5: 1 (molar ratio)

[Brief description of the drawings]

FIG. 1 shows the antifungal substance Mer-WF5027-I.
The ultraviolet absorption spectrum of I in methanol (solid line), acidic methanol (dotted line), and basic methanol (dashed line) is shown.

FIG. 2 shows the antifungal substance Mer-WF5027-I.
1 shows the infrared absorption spectrum of I in the osmotic potassium bromide tablet method.

FIG. 3 shows the antifungal substance Mer-WF5027-I.
¹ shows a 400 MHz ¹ H-NMR spectrum of I in deuterated chloroform.

FIG. 4 shows the antifungal substance Mer-WF5027-I.
1 shows a 100 MHz ¹³ C-NMR spectrum of I in deuterated chloroform.

FIG. 5 shows the antifungal substance Mer-WF5027-I.
1 shows a thin layer chromatograph of I.

FIG. 6 shows the antifungal substance Mer-WF5027-I.
1 shows a high performance liquid chromatograph of I.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI // (C12P 17/06 C12R 1:66) (72) Inventor Takeo Yoshioka 1959 Kamizuna, Ayase City, Kanagawa Prefecture Green Heights 3-3102 (72 Inventor Toshihiko Kumamoto 1-9-12 Kugenuma Sakuragaoka, Fujisawa City, Kanagawa Prefecture (72) Inventor Hiroshi Nishida 568, Tsukui, Yokosuka City, Kanagawa Prefecture Green Heights 11-3-503 (72) Inventor Rokuro Okamoto 2-18 Flower Tree, Fujisawa City, Kanagawa Prefecture (58) Fields investigated (Int. Cl. ⁷ , DB name) C07D 311/74 C12P 17/00-17/08 CA (STN) REGISTRY (STN) WPI (DIALOG) BIOSIS (DIALOG)

Claims (4)

(57) [Claims] (1) Formula (1) An antifungal substance Mer-WF5027-II represented by
A. 2. The formula: An antifungal substance Mer-WF5027-II represented by
B. 3. The formula: And a compound represented by the formula: An antifungal substance Mer-WF5027-II consisting of a mixture of the binding isomers with the compound represented by the formula: 4. A compound belonging to the genus Aspergillus and having the formula: An antifungal substance Mer-WF5027-IIA represented by
And / or the formula An antifungal substance Mer-WF5027-IIB represented by
A strain having the ability to produce E. coli is cultured in a medium, and the antifungal substances Mer-WF5027-IIA and / or -IIB are collected from the culture, and the above formula (I) and / or (II) is used. Antifungal substance shown Mer-WF
Method for producing 5027-IIA and / or -IIB.