JPS59186989A - Antibiotics: irumanolides i and ii and their preparation - Google Patents

Abstract

NEW MATERIAL:A compound of formula I [R is formula II (IRUMANOLIDE I ) or formula III (IRUMANOLIDE II)]. It has following physical and chemical properties: melting point: I =125 deg.C, II=145 deg.C; elementary analyses in %: I =C 70.65, H 9.37, II=C 68.89, H 9.62; Molecular formula: I =C34H54O7, II=C34H56O8; Molecular weight: I =574.385, II=592.398; Optical rotatory power: [alpha]D<25> (c=1, in CHCl3) I =+140 deg., II=133 deg.. USE:Remedy or preventive for fungal diseases of men, animals and plants, because it is active against fungi, especially phytopathogenic fungi. PREPARATION:Streptomyces subflavus subsp, irumaensis FN-14 (FERM-6925) is aerobically cultivated, e.g., by the shaking culture at 20-40 deg.C for 1-8 days.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel antibiotics ilmanolides I and II and methods for their production. An object of the present invention is to provide novel antibiotics ilmanolides (1) and (2) and methods for their production.

The present invention provides a novel antibiotic represented by the following formula.

Irumanolide I, a novel antibiotic provided by the present invention
and ■ are stable white powders in a purified state, and have the structure shown by the above formula.

Illumanolide ■ and ■ show the physical and chemical properties as shown in Table 1.

Table 1 Illumanolide Illumanolide Melting point 125℃ 145℃ Elemental analysis (chi) C, 70, 65G, 68.89
H,9,37H,9,62 Molecular formula C34H5407C34H5608 Optical rotation chloroform) Ultraviolet absorption maximum wavelength (nm) (in meta-232 terminal absorption nol) Infrared absorption smooth Figure 1 Figure 2 Cuttle (KB
When searching for known antibiotics based on the properties of ilmanolide ■ and ■ in Table 1,
I can't find anything relevant. Relatively similar to venturicidin, a chemically derived aglycone moiety [Brufani et al., Expelliencia et al.
ri*ncia) Volume 27, Page 604, 1971, it is described as compound ■, and its melting point is 135-137.
℃, the molecular formula is CHO34δ6 71, and there is no characteristic absorption in the ultraviolet absorption spectrum]. However, when comparing this compound ■ and ilmanolide ■, ilmanolide I has a strong ultraviolet absorption maximum of 23
2 nm. Also Irumano Ride■
They are clearly distinguished in terms of molecular formula and molecular weight. Comparing the structures, both ilmanolides (1) and (2) have two hydroxyl groups in the 20-membered ring lactone, while the above compound (2) clearly differs in that it has only one hydroxyl group. Therefore, ilmanolide I and ■ are determined to be new substances.

Next, the biological activities of ilmanolide ■ and ■ are shown. The measurement was carried out in a conventional manner using a two-fold dilution method using a potato glucose agar medium, and the minimum inhibitory concentration (MIC) determined after 45 hours at 27°C is shown in Table 2.

Table 2 MIcCttll/ml) Illmanola Illmanola Candida albicans KF-1>100
100 (Candida albiaans) Satsukaromyces salmon KF-26 > 100 > 10
0 (100 (Saecharo@5ake) Aspergillus niger KF-102 > 100 > 10
0 (As100 (Aspsr niger) Soufflerotinia cinerea KF-1812550 (8elero
tinla ein@rsa) Plriaularia oryzae KF-1801,5650 (Plriaularia
oryzae) Botrytis cinerea KF'-1
84100>100(Botrytls cin@r
sa) Microsporum gypseum KF-65>100
>100(Mlcrosploo(gyp*e
um) As is clear from Table 2, ilmanolides ■ and ■ are effective against fungi, especially plant pathogenic fungi, and therefore can be used for the treatment and prevention of human, animal, and plant diseases caused by these microorganisms. It is expected. In addition, Illumano Ride! Alternatively, when ■ was administered intraperitoneally to mice at a concentration of 100■/K5+, all of the mice survived one month later.

Illumanolides I and 1 are useful as starting materials for obtaining, for example, derivatives of the antibiotic AM-3603 (41!F 1989-48996) by chemical and biological means.

The present inventors also found that ilmanolides ① and ② are intermediates in the biosynthesis of antibiotic AM-3603 in the antibiotic AM-3603-producing strain Streptomyces sp. This was confirmed by microbial conversion method in the presence of cerulenin.

The test method used takes advantage of the fact that the antibiotic cerulenin is a specific inhibitor of polyketide biosynthesis, and one of the inventors and his collaborators have already reported that it is a 16-membered ring macrolide antibiotic. It has been applied to research on the biosynthesis of a certain tylosin, and its effectiveness has been confirmed, and details are described in, for example, the following literature.

Omura et al., Chem, Pharm, Bull, Vol. 30, p. 223, 1982.

Omura et al., Biochemical and Biophysical
Research Communication (Biochem, B
iophys, Res, Commun,) No. 107
Volume, 554 pages, 1982.

Using essentially the same method as used above, cerulenin was added to the culture of Streptomyces sp. s o p9/ml
When ilmanolide ■ or ■ was added and culture was continued under conditions in which the biosynthesis of the aglycone moiety of AM-3603 was inhibited by adding it at a rate of Confirmed that it will be converted.

As is clear from this fact, ilmanolide ■ and ■
is also useful for increasing the production of antibiotic AM-3603. The test example described later is an example in which this was confirmed.

Next, the manufacturing method of ilmanolide (1) and (3) will be explained.

Examples of microorganisms used in the present invention include Streptomyces subfrapus, which was isolated by the present inventors.
Subsp. irmaensis strain FN-114 is one suitable example. This strain is Streptomyces sp.
AM-3603 (Feikoken Bacterial Serial No. 5619) strain was subjected to mutation treatment using N-methyl-N'-nitroso-N-nitrosoguanidine by a conventional method, and then each mutant strain was cultured. , was selected as a strain with decreased ability to produce the antibiotic AM-3603.

Regarding the mycological properties of the antibiotic AM-3603 producing strain Streptomyces sp. AM-a 603 (Feikoken Bacterial Serial No. 5619) used as the parent strain, please refer to JP-A-57-48996. Are listed.

The ilmanolide-producing strain Streptomyces subfrapus subsp. ilmanensis strain FN-114 is
It has been deposited with the Institute of Industrial Science and Technology, Department of Microbiology, Agency of Industrial Science and Technology as No. 6925. The mycological properties of this strain include that it substantially lacks the ability to produce the antibiotic AM-3603, and that it has the mycological properties of Streptomyces sp. There is virtually no difference in quality and wear. Antibiotic AM-3603 could not be detected from the culture obtained by the method described in Examples below.

In addition, in the present invention, any microorganisms belonging to the genus Streptomyces and having the ability to produce one or both of ilmanolides (1) and (2) can be used, including wild strains and mutant strains.

The medium used for culturing the microorganisms used in the present invention may be any synthetic medium, natural medium, liquid medium, or solid medium, as long as the strain used can grow and one or both of ilmanolides ■ and ■ can be produced. . The composition of a suitable medium is determined by considering the medium commonly used for the production of antibiotics by actinomycetes. That is, various carbon sources that can be assimilated by the microorganisms used can be used, such as glucose, maltose, sucrose, starch, dextrin, glycerin, animal oil, and vegetable oil.

As the nitrogen source, there can be used various complex nitrogen-containing substances such as yeast extract, peptone, soy flour, and dried yeast, as well as ammonia, urea, and many other substances that can be assimilated by the microorganisms used. In addition, metal salts such as phosphate, magnesium, potassium, sodium, iron, manganese, and cobalt are used as necessary.

For culturing, aerobic conditions such as shaking culture or deep aeration agitation culture tend to give suitable results. Culture temperature is usually 20°
C to 40°C is preferred, but it can be carried out at other temperatures if the strain used grows.

When culturing ilmanolide-producing bacteria, one of magnesium phosphate, natural zeolite, and synthetic zeolite is added to the medium.
By adding more than one, the amount of accumulated ilmanoid may be increased. The production of ilmanolide may also be increased by adding one or more of several amino acids, such as leucine, isoleucine, vali, threonine, serine, alanine, to the medium.

The culture period is usually 1 to 8 days, and one or both of ilmanolides (1) and (2) are produced and accumulated inside and outside the bacterial cells.

After completion of the culture, ilmanolide is recovered from the culture by a known method. In this case, various extraction methods and concentration methods used for recovering neutral and fat-soluble substances are used. For example, the culture is separated into bacterial cells and R solution by centrifugation, and the P solution is extracted with an organic solvent such as ethyl acetate or benzene and concentrated. The bacterial body part is extracted with aqueous methanol or acetone and concentrated. Thereafter, ilmanolide I and (2) are purified and separated by a known purification method. For example, silica gel column chromatography, alumina column chromatography, silica gel high performance liquid chromatography, preparative thin layer chromatography, etc. can be used.

Next, examples will be shown. These are merely examples and do not limit the invention.

Example One platinum loop was collected from a slant culture of Streptomyces subfrapus subsp.
Seed medium (glycerin 1.0%, glucose 0.2%
, soybean flour 1.0%, salt 0.3%, pH 7.0) was inoculated into a 5001 volume Sakaro flask, and cultured with shaking at 27°C for 2 days to obtain a seed culture. 200 g of this seed culture solution was mixed with 20 g of fermentation medium (glycerin 2.0%, glucose 0.4%, soybean flour 1.0%, salt 0.3%, pi(y,
o) into a 301-volume jar fermentor, and cultured with aeration at 270G for 3 days (aeration rate 101/m2).
Stirring was performed for 1 ns at 250 r, p, m, ). Culture solution (18)) was adjusted to pH 7.0 with caustic soda, and
! Extracted with benzene. The benzene layer was concentrated under reduced pressure to give a yellow oil 21.5. ! I got il. This oily substance at 100mA! After washing with normal hexane, 6.15 g of pale yellow crude powder was obtained.

The above coarse powder was added to the top of a 5001 Lt column prepared with silica gel (Merck, Art 7734.25Og) suspended in benzene/acetone (5/1.600 mA). The column was eluted with benzene/acetone (5/1.3 J) and fractionated into 15 to 20 particles. Silica gel thin layer chromatography (Merck, Kieselgel 60 F254, developing solvent benzene/acetone =
5/2), and the fractions exhibiting an Rf of about 0.5/ were collected and concentrated under reduced pressure to isolate ilmanolide 1750 as a white powder. Fractions exhibiting an Rf of approximately 0.4 were collected and concentrated under reduced pressure to isolate ilmanolide 1.75II as a white powder. Irmano2id I obtained in this way
The physical and chemical properties of the river are as described above.

Test example Streptomyces sp. AM-360 as a Mycobacterium
3 (Feikoken Bibori No. 5619) strain was used, and this strain was mixed with antibiotic AM-3603 production medium (2% glycerin, 0.4% glucose).

1 tch soybean flour, 0.3% salt, pH 7.0) 100ml
Cultured in a 5001 Yosaka flask containing antibiotic AM
When producing -3603, ilmanolide (■) or (■) was added at a concentration of 200 μg/111 on the first day of culture. As a result, AM-36 was found in the culture solution on the fourth day.
03 are 700 μI/vrl and 560 μV1, respectively.
Generated and accumulated. The amount of AM-3603 produced in the culture solution of a control (no addition) conducted at the same time was 360 μl//d.

[Brief explanation of drawings]

In the accompanying drawings, FIG. 1 shows an infrared absorption spectrum (KBr method) of ilmanolide I. Figure 2 shows the infrared absorption spectrum CK of ilmanolide H.
Br method). Patent applicant Kitasato Research Institute (incorporated association) (15)

Claims (2)

[Claims] (1) Antibiotics ilmanolide ■ and ■ represented by the formula manolide ■). (2) By aerobically cultivating a microorganism belonging to the genus Streptomyces and having the ability to produce one or both of the antibiotic ilmanolide I and (■) represented by formula (I), the antibiotic ilmanolide The method for producing the antibiotic ilmanolide I or (2) according to claim 1, which comprises accumulating one or both of (1) and (2) inside and outside the bacterial cells and recovering them.