JPS62294676A - Patulolide and production thereof - Google Patents

Abstract

NEW MATERIAL:Patulolide A expressed by formula I, patulolide B expressed by formula II or patulolide C expressed by formula III. USE:An antifungal agent, antibacterial agent and plant growth inhibitor. PREPARATION:A microorganism, e.g. Penicillium urticae S11R59 strain (FERM P-8168), belonging to the genus Penicillium and having the ability to produce at least one of patulolide A, B and C is cultivated in a culture medium, preferably a liquid culture medium at 6-8 pH and 17-30 deg.C for 1-3 days to collect at least one of patulolide A, B and C produced in the culture fluid.

Description

Detailed Description of the Invention 3. Detailed Description of the Invention Industrial Field of Application The present invention relates to a novel macrocyclic lactone compound, patyurolide, which is useful as an antifungal agent, an antibacterial agent, and a plant growth inhibitor.
Patulol 1de) and its manufacturing method.

Conventional technology Among macrocyclic lactone compounds (macrolides without sugar chains) obtained as fermentation products, 10- to 14-membered ring compounds are derived from Thallatosborium fulvum (Cladosp.
orium fulvum)t'T-113 produced by Cladosporide A (Cladosporide
A, l 2-membered ring) - [Agricultural and Biological Chemistry (, Agricult
LIral and Biological Chemistry
stry), 45.799 (1981)], Cephalosporium lentsei (Cephalosporia
Recifeiolide (R) produced by M rccifei
ec i ['eiol 1de) [Canadian Journal of Chemistry
Journal or Chemistry) 49.
2029 (1971)], Streptomyces pruneogriseus (SLreptomyces brunne)
Alpocycline (A
ibocycle, ! 4-membered a)-4 Chemical and Pharmaceutical Pretain (Ch
chemical and pharmaceutical
Bulletin), 19°649-666 (19
71)] are known.

Problems to be Solved by the Invention In recent years, the large-scale use of antibacterial agents, particularly β-lactam agents, has caused a so-called bacterial replacement phenomenon, resulting in an increase in the number of people suffering from medical mycosis. Also people with immunodeficiency, the elderly.

Antifungal drugs are also considered necessary for young children. Macrolide antibiotics are generally known to have low toxicity and are effective against general bacteria when administered orally, but there are still very few compounds that have antifungal properties in addition to these properties.

Means for Solving the Problems The present inventors cultivated a large number of microorganisms for the purpose of searching for new antibiotics, separated and searched for the products in the culture solution, and found that certain microorganisms were found to be novel antibiotics. We discovered that the isolated antibiotic showed antifungal, antibacterial, and plant growth inhibitory effects, and confirmed from its physicochemical properties that the antibiotic was a new macrocyclic lactone compound. It was designated as nopatuloride A (compound (r)).

The present inventors further investigated the active ingredients in the culture solution and discovered two new ingredients, which they named pachyuroride B (compound (■)) and C (compound (■)).

The present inventors completed the present invention as a result of further research based on these findings.

The present invention provides (1) formula (1), (It) or (
A microorganism having the ability to produce pachylolide A, B, or C represented by I) and (2) at least one of pachylolide A, B, and C belonging to the genus Penicillium is cultured in a medium, and the compound is produced and accumulated in the culture. This is a method for producing at least one of pachyurolides A, B and C, which is characterized by collecting the same.

The bacteria producing patyurolides A, B, and C used in the present invention include Penicillium (Penicillium).
Any microorganism may be used as long as it belongs to the genus i un) and has the ability to produce padurolides A, B, and C. Examples of the microorganisms include microorganisms belonging to the species Venithulium uruteicae,
More specifically, Benithurium uruteicae St1
Strain (hereinafter sometimes abbreviated as rsllllij) 1 Peninrium uruteicae 5llr (59
rslIR59 strain (hereinafter sometimes abbreviated as rslIR59 strain).

Benithurium uruteicae Sll and 5lIR59
The mycological properties of the strain are described in A manual of the Pencilia.
-11ia), No. 53 = l~539 pages, The Wi
The mycological properties of Penicillium uruteicae are the same as those described in LIiams & Wilkins Company (1949). In addition, the Penicillium uruteicae is Penicillium bachurum (P, pa
tlJlum) and Benithurium flexiosum (
Synonymous with P, flexuosum
(see A Manual of the Benicilia above), and Benithurium griseofulvum (Dan, gr.
iseoNvum) and is synonymous with American Type Culture Collection (AmericanTyp).
e Culture Co11ection) Catalog of Strains (Catalogue or 5
trains) I, I 5 thedition
, 1982.

Some strains of Benithurium uruteicae are paralyzed (P
[Nature, Vol. 156, p. 295,
1945 and Antibiotics &
Chemotherapy (Antibiotics and Ch)
emotherapy) Volume 1.

See page 573, 1951].

However, Penicillium uruteicae S11
Benithurium uruteicae 5IIR has different properties in that it exhibits paralin non-production and adenine auxotrophy.

The American Type Collection (The American T)
ypeCu1ture Co11ection, AT
CC) under accession number ATCC 48165. The above 511R59 strain is the Fermentation Research Institute (
IFO) on March 19, 1985, with accession number rF.

It has been deposited as No. 31725. Also, the above 511R5
The nine strains were deposited with the Microbial Research Institute (FRI), Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan, on March 28, 1985, under the accession number FERM P-8168.

The antibiotic patyurolide A, B and C-producing bacteria belonging to the genus Penicillium, as well as other Penicillium genus,
Its properties are easily changeable, and it can be easily mutated by artificial mutagenic means such as ultraviolet rays, X-rays, and radiation 1 artificial mutating agents. Anything that has the ability to produce C can be used in the method of the present invention.

In the method of the invention, the antibiotic patyurolide A, +3
The medium in which the and C-producing bacteria are cultured may be either liquid or solid, but a liquid medium is more conveniently used, and surface culture and shaking culture methods may also be used, although deep culture methods are more advantageous. It will be done.

The culture medium contains carbon sources that can be assimilated by bacteria producing the antibiotic patyurolides A, B, and C, such as starch, glucose, dextrin, glycerin, sucrose, n-paraffin, alcohols (e.g., methanol), and nitrogen sources such as: For example, corn, steep, and
Liquor, soybean flour, cottonseed flour, 1 peptone, 1 meat extract, etc. As the inorganic nitrogen source, ammonium chloride, ammonium sulfate, ammonium nitrate, urea, etc. can be used. others,
Inorganic salts such as sodium, potassium,
Salts containing magnesium or phosphorus 1 Heavy metal salts such as iron, manganese, zinc, cobalt, copper, nickel, etc. Antifoaming agents such as soybean oil, lard oil, chicken oil, silicone oil, Actol (manufactured by Takeda Pharmaceutical Company Limited) ) etc. may be added as appropriate. For liquid culture, the p of the medium
H is preferably around neutrality, particularly a pn of about 6 to 8. The culture temperature is preferably about 17°C to 30°C and the culture time is preferably about 24 to 72 hours.

The titers of antibiotic pachyurolides A, B, and C produced in the culture solution over the course of the culture are quantified according to the conventional cup method or paper disc method using Candida albicans IF0 0583 as the test bacterium.

Usually, after culturing for about 1 to 3 days, the antibiotic pachyuroride A,
The production of B and C reaches its maximum.

Targeted antibiotic patyurolide A from culture.

To collect B and C, separation means commonly used to collect metabolites produced by microorganisms from cultures of the microorganisms can be used as appropriate. For example, padurolides A, B, and C exhibit neutral fat-soluble properties and are mainly contained in the culture filtrate, so the bacterial cells are removed by first adding a filter aid to the culture solution and filtering it, or directly centrifuging it. do. A water-immiscible organic solvent such as trichloroethane, polytetraform, ethyl acetate, or methyl isobutyl ketone is added to the obtained medium 1 or liquid to extract Baturolito A, B, and C. The extract may be washed with water and concentrated, or the organic solvent layer may be concentrated after washing with a weakly acidic aqueous solution such as hydrochloric acid, sulfuric acid or phosphoric acid and a weakly alkaline aqueous solution such as sodium bicarbonate or sodium carbonate solution.

The a-condensate is further purified by adsorption/partitioning or chromatography utilizing the properties of molecular sieves. That is, a method is advantageously used in which the concentrate is brought into contact with a suitable carrier to adsorb the active ingredient, and then the active ingredient is desorbed using a suitable solvent and then separated and collected. Examples of carriers include silica gel, crystalline cell-A, and adsorbent resins such as Diaion HP-20 (manufactured by Mitsubishi Chemical Industries, Ltd.), Amberlite XAI)-II (manufactured by Rohm and Haas Corporation,
US).

Sephadex Ll-(-20 (manufactured by Pharmanoa Fine Chemicals, Sweden) or the like may be used.

The elution solvent varies depending on the type of carrier, but examples include petroleum ether, n-hexane, toluene, and chloroform.
Dichloromethane, ethyl acetate, acetone, alcohols, pyrinone, acetic acid, or a mixed solvent thereof may be used in combination as appropriate. The eluate containing patchurolide A, B, and C is concentrated to dryness, and then crystallized from a suitable solvent such as n-hexane or diethyl ether, or powdered by adding petroleum ether or n-hexane to the concentrated and dried product. .

Antibiotic patyuroride A and B obtained in Example I below
The physicochemical properties of and C are as follows.

Pachyuroride A (1) Appearance: colorless crystals (2) Melting point: m, p, 83-84°C (3) Specific light intensity, [α] 25+30. l° (C=0.95. in ethanol) (4) Molecular weight measurement: m/z 210 (M”XE I
-MS method) (5) Elemental analysis values: Actual value, C, 67,84, H, 8,63 calculated value,
C, 68, 54, 1-1, 8, 63, 0, 22, 83
(%) (6) Molecular formula・C1ff1H7803 (7) Ultraviolet absorption (UV) spectrum: λ in methanol
218nm (E1%=778) max 1cm (8) Infrared absorption (IR) spectrum/KBr method (first
Figure) Main absorption (cm-') 3400, 30g0.2940.2g70.1705.
1680.1640°1470, 1450.1440.
1390.1365.1350.1340°1320,
HOo, 1265.1250.1240.1205
．． 1175°1155, 1125.1105.1085
．． 1075.1055.1035゜1025, 1000
．． 980. 950. 910. 885. 850
゜820, 770. 720. 680. 625.
555. 520° (9) l 3 c - Nuclear magnetic resonance (NMR) spectrum, 15 MHz, in deuterochloroform.

20.1(Q), 22.4(t), 24.5(
t), 25.8(t).

25.9(t), 34.9(t), 39.2(
t), 74.9(d).

129.3(d), 141.7(d), 166.6
(s), 202.4(s)I)pm (However, S:
singlet, d: doublet, t: t
rippleL.

Q: Show each quartet. ) Paduloride B (1) Appearance: Colorless crystals (2) Melting point: m, p, 66-67°C (3) Specific light intensity [α] 25-42.4° (C = 2.0
．． (in ethanol) (4) Measured molecular weight: m/z 210 (M”XE r
MS method) (5) Elemental analysis values: Actual value, C, 68, 35; H, 8, 47 Calculated value, C, 68, 54, H, 8, 63, 0, 22, 83
(%) (6) Molecular formula: C,21(+aOs (7) UV spectrum, λ 207 nm in methanol (ε, 8085) max (8) TR spectrum: KBr method (Figure 2) Main absorption (cm- ') 3450, 3070.2960.2870.1740.
1700.1625°1440, 1420.1385.
1350.1340, 1260.1250°1180,
1135.1120.1100.1080.1025.
980°925, 380. 865. 820.
765. 725. 640°590°(9)13(, -NNIR spectrum: 15MIIz,
19.6 (q), 20.4 (t), 23.3 (in heavy chloroform)
t), 24.3(t).

24.9(L), 31.9(t), 40.2(
t), 74.5(d).

125.9(d), 139.7(d), 165
．． 3(s), 202.8(s)I)I)m Pachyuroride C (1) Appearance: Colorless oily substance (2) Specific light intensity: [α] 25-1.89° (C = 2.0
．． (in ethanol) (3) Measured molecular weight: m/z 212 (M”) (E
I-MS method) (4) Elemental analysis values: Actual value, C, 66,98; H, 9,59 Calculated value, C, 67,89: H, 9,50, 0,22,
61 (%) (5) Molecular formula: C+2H2aO* (6) UV spectrum: λ 212 in methanol
nm (ε, 9169) max (7) I R spectrum: KBr method (Figure 3) Main absorption (cm''''1) 3430, 2940.2g60.1700.1640.
1440. H2O.

1265, 1155.1100. 935. 870.
825. 700 (8)" C-N to IR spectrum: 15 MHz, in deuterochloroform, showing the following signals (ppm) 19.4.20.8.22.4.27.7.28.3.
33.1.36.0°70.7.73.2. t21.
2.150.4.168.2 Also, pachyuroride A, B
and C thin layer chroma carrier Nijirica Gel G (manufactured by Merck & Co., West Germany) Detection method: Potassium permanganate coloring method.

tJV (254nm) From the physicochemical properties described above, padurolide A, I3
The structural formula of and C is the above formula (I).

It is represented by (II) and (II[).

Based on the properties described above, Pachyurolide A, B and C are new compounds belonging to the macrocyclic lactone system represented by the above formulas (0, (ff) and (III)), and are antibacterial against various microorganisms. The spectra are shown in Table 1.

1' Medium/Bact/Antihiotic Medium 3
(manufactured by Difco, USA): 17.5 g, Bacto Yeast Extract (Difco I); 5 g.

Bacto agar (manufactured by Difco); 20 g, diaminopimelic acid; 20 mg, distilled water; Iσ, pi headless.

Amount of inoculated bacteria: One loopful of approximately +08/+nl bacterial solution was used.

Furthermore, the antifungal spectra of Patiyurolide A, B, and C are shown in Tables 2 and 3, respectively.

Talatosborium cucumerinum 23 A
IFo 637G Diapornonotori IFO917025A Rhizoctonia solani KIIG-225A Pyricularia oryzae IFO527923A Penicillium chrysogenum
IFO462620A Candida albicans IP
0058326 A Satucharomyces cerevisiae IFO02093+ A Trichophyton mentagrophyi 20 B Tes IFO7522 Cryptococcus neoforma 15 I
A vapor disk with a diameter of 8 mm containing 3 Oμe was used.

``A: Modified Betuffer-CPreffer) Agar Medium B Nigelcose Nutrient Agar Medium Table 3 Minimum Inhibitory Concentration Test Bacteria (μg/m I
) ” Medium IC Alternaria quicutiana IF07515 50
>1.00 A Cladosporium cucumerinum 50 100 AIPO6370 Diaporscinto'J IFO917012,55
OA Resocto=7-/Sea 1- KIIG-212,5
50A Pili Clari 7-Oryzae IFO527912,5
5OA Penicillium chrysogenum IFO462625
>10111 A Candida albicans IF
OO51(35[) >100 A Satsukaromyces cerevisiae 1FOQ209 50 >joo
A' Agar dilution method A + modified Pferfer agar medium B
Nigelcose Nutrient Agar Medium Also, the acute toxicity (LDs. > 400 mg/kg) of badurolides A and B by oral administration in mice is low.

As detailed above, patyurolides A, B and C are i
n In vitro tests, A and B exhibit a wide range of antibacterial activity against Gram-positive and -negative bacteria as well as plant pathogenic and medical fungi, while C exhibits antibacterial activity against medical fungi and each has low toxicity. Therefore, patyuloride A and B can be used for the prevention and treatment of bacterial infections and fungal infections in warm-blooded mammals [e.g., mice, rats, dogs, cats, cows, pigs, sheep, and humans]. It can be used as a therapeutic agent or a therapeutic agent for fungal infections. Further, C can be used as a therapeutic agent for fungal infections for the purpose of preventing and treating fungal infections in warm-blooded mammals.

When administering pachyuloride A or B, it may be administered to the warm blood as described above, for example, as an ointment containing vaseline, carbowax, etc. as a base and containing pachyuloride B at a concentration of 0.1 to 1% (W/V). It is administered by applying it to the skin of the hands and feet of mammals.

Furthermore, pachyurolides A and B were found to have an inhibitory effect on plant growth. Table 4 shows the effects of pachyurolides A and B on the growth of Japanese millet.

Note: 10 individuals in 1 area, average value of 3 replicates As shown above, Pachyurolides A and B are useful as plant growth inhibitors.

Furthermore, pachyurolides A, B, and C are extremely promising compounds as intermediates for the synthesis of new pharmaceuticals and agricultural chemicals.

The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited thereto.

Note that percentages (%) in the culture medium represent weight/volume percentages unless otherwise specified.

Example 1 Penicillium uruteicae 511R59 (IFO31725, FERM P-
8168) strain at 2% glucose.

3% maltose, 1.5% raw soybean flour, 1.0% corn steep liquor, 0.5% polypeptone (9 Japan manufactured by Daigo Nutritional Chemical Co., Ltd.), yeast extract (1 manufactured by Taigo Nutritional Chemical Co., Ltd.) Japan) 0.3%, sodium chloride 0.3% (
The cells were inoculated into a 2σ volume Slope flask containing 500 ml of a medium (pH 6.0), and cultured with reciprocal shaking at 28°C for 48 hours. The entire amount of this culture solution was inoculated into a 50Q fermenter containing 3N of a medium prepared by adding 0.05% of the antifoaming agent Actol (Takeda Pharmaceutical Co., Ltd., Japan), and heated at 28°C with aeration. The cells were cultured for 48 hours at a rate of 30 Q/min and 280 revolutions/min. This culture solution 6C was mixed with 15% glucose, 4.5% soluble starch, 1.5% Proflo (Traders Protein, manufactured by Basocky Cellulose, USA), and 0.5% corn staple liquor (
Actocol 0.05% in a medium consisting of pH 7.0)
It was inoculated into 200g fermenters (2 units) containing 120σ medium supplemented with
/min, t4o rotations/min) for 66 hours.

Hyflo Super Cell (manufactured by Johns Manville, USA) was added to the culture solution (200 g) from the fermenter and filtered. The filtrate (193υ was adjusted to p++a, s and extracted with ethyl acetate. The extract (104ρ) was diluted with 2%
The mixture was washed with aqueous sodium hydrogen carbonate and then with water, and the ethyl acetate layer was concentrated. The concentrated residue (71 g) was subjected to column chromatography on silica gel 60 (manufactured by Merck & Co., West Germany, 8300 g) and fractionated and eluted with dichloromethane.

The fraction containing the active substance was concentrated and extracted with pachyurolide A.

A mixed mixture of materials B and C (39.1 g) was obtained. The combined material was dissolved in ethyl acetate and subjected to preparative high performance liquid chromatography (HPLC). The carrier used was silica gel G (manufactured by Merck & Co., West Germany), and the developing solvent was n-hexane/ethyl acetate (8.5:1.5). Three peaks containing the active substance were separated, each eluate was concentrated, n-hexane was added to the concentrated solution, and when it was left to stand, crystals precipitated.

When the crystals were collected by filtration, Pachyurolide B (6.39 g) and Pachyuroride A (3.61 g) were obtained.

The third fraction, in which no crystals were formed, contained batchuroli FC, and this crude batchurolide C was again subjected to preparative HPLC and treated with n-hexane:isopropyl ether, isopropanol (8:2:l). Purified pachyurolide c (6.29 g) was obtained as an oily substance.

Effects of the Invention Pachyuroride A and B produced according to the present invention have excellent antifungal, antibacterial, and plant growth inhibitory effects, so they can be used as therapeutic agents for fungal infections and bacterial infections, and as plant growth inhibitors. In addition, since pachyuroride C has an excellent antifungal effect, it can be used as a therapeutic agent for fungal infections.

[Brief explanation of the drawing]

1.2 and 3 show the optical external absorption spectra of pachyurolides A, B and C obtained in Example 1, respectively. Town - Stomach w 3 Teoroju ``1↑Sho4 [Method] June 26, 1985 l Case description 1988 Patent Application No. 479.10 2 Name of the invention Badurolide and its manufacturing method 3 Amendment Relationship with the case of a person who does
93) Takeda Pharmaceutical Co., Ltd. Representative Junmasa Kiki 4 Agent (]:, Address: 2-17-85 Jusanhonmachi, Yodoyo-ku, Inusaka-shi Tokyo Contact information (Special 9-cho Laws and Regulations Division) Telephone: 27 g-2218.
22196, Column 7 of the title of the invention in the specification subject to amendment, contents of the amendment (1) The title of the invention stated on page 1 of the specification, "Paduloride and its manufacturing method" is corrected to "Paduloride and its manufacturing method" . that's all

Claims (2)

[Claims] (1) Formula I, II or III respectively ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (I) ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (II) ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (III) Pachyuroride A, B or C as represented. (2) Pachyurolide, which is characterized in that a microorganism belonging to the genus Penicillium and having the ability to produce at least one of Pachyurolide A, B, and C is cultured in a medium, the compound is produced and accumulated in the culture, and the compound is collected. A method for producing at least one of A, B and C.