JPH0613499B2 - Patchoulide and its manufacturing method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel macrocyclic lactone compound, patiurolide (P), which is useful as an antifungal agent, an antibacterial agent and a plant growth inhibitor.
atulolide) and its manufacturing method.

10 to compounds of the 14-membered ring of the macrocyclic lactone compound was obtained as a prior art fermentation products (no sugar macrolides), Cladosporium, Furubumu (Cladosporium fu
lvum ) FI-113 produced Cladosporium A (Clados
polide A, 12-membered ring)-[Agricultural and Biological Chemistry
Biological Chemistry), 45 , 799 (1981)], Recifeiolide produced by Cephalosporium recifei [Cyanadean
The Journal of Chemistry
Chemistry) 49,2029 (1971)], Streptomyces Bull Neo griseus (Streptomyces brunneogriseus) Aruboshikurin to production, such as (Albocycline, 14-membered ring) -
[Chemical and Pharmaceutical Bulletin, 19 , 649-
666 (1971)] is known.

Problems to be Solved by the Invention In recent years, a large amount of antibacterial agents, particularly β-lactam agents, causes a so-called bacterial alternation phenomenon, and the number of people suffering from medical mycosis is increasing. Antifungal agents may also be needed for people with immunodeficiency, the elderly, and infants. Generally, macrolide antibiotics have low toxicity and are known to be effective by oral administration to general bacteria, but compounds having antifungal properties in addition to these properties are still very few.

Means for Solving the Problems The present inventors cultivated a large number of microorganisms for the purpose of searching for new antibiotics and separated and searched the products in the culture solution, and found that certain microorganisms were novel antibiotics. It was found that the isolated antibiotic has antifungal, antibacterial and plant growth inhibitory effects, and its physicochemical properties confirmed that the antibiotic is a novel macrocyclic lactone compound. Pachiurolide A (Compound (I)) was named.

The present inventors further investigated the active ingredients in the culture medium and found two new ingredients, which were named pachyurolide B (compound (II)) and C (compound (III)).

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

The present invention is (1) formula (I), (II) or (III), respectively. And culturing in a medium a microorganism that has the ability to produce at least one of Patiurolide A, B and C belonging to the genus Penicillium (2)
A method for producing at least one of patiurolides A, B and C, which comprises collecting and accumulating the compound in a culture.

The bacterium producing pachyurolide A, B and C used in the present invention may be any microorganism as long as it belongs to the genus Penicillium and has the ability to produce pachyurolide A, B and C. Examples of the microorganism include, for example, species Penicillium ulteikae ( P. ur
ticae ), more specifically,
Penicillium ultecae S11 strain (in the following,
It may be abbreviated as "S11 strain". ), Penicillium ultecae S11R59 strain (hereinafter, "S1
1R59 strain ". ) Is mentioned.

Penicillium ulteikae S11 and S11R59
The mycological properties of the strain are described in A manual of the Penicillia, pp. 534-539, Th.
The same as the mycological properties of Penicillium ulteicae described in e Williams & Wilkins Company (1949). Incidentally, the Penicillium Uruteikae is Penicillium Pachiyurumu (P. Patulum) and Penicillium Furekusuosumu (P. Flexuosum) in the above formula (synonym) (see above à Maniyuaru Of The Penishiria), also Penicillium Guriseofurubumu Synonymous with ( P. griseoflvum ) [American Type Culture Collection Catalog of Strains I, 15th editi
on, 1982].

One strain of Penicillium ulteikae is Patulin
It has been reported as a ulin-producing bacterium [Neachure (Na
ture) 156, 295, 1945 and Antibiotics and C
hemotherapy) Vol. 1, pp. 573, 1951].

However, the Penicillium ultecae S11
It exhibits patulin non-productivity, and Penicillium ultecae S11R differs in properties in that it exhibits patulin non-productivity and adenine requirement.

The S11 strain is the American Type Culture Collectio.
n, ATCC) under accession number ATCC 48165. The above S11R59 strain was assigned to the Fermentation Research Institute (IFO) on March 19, 1985 under contract number I.
Deposited as FO 31725. Also, the above S1
The 1R59 strain has been deposited under the deposit number FERM P-8168 on March 28, 1985 (FRI) in the Institute of Microbial Technology (FRI), Ministry of International Trade and Industry, Japan.

The antibiotics Pachiurolide A, B and C producing bacteria belonging to the genus Penicillium, like the other genus Penicillium, are likely to change their properties, such as ultraviolet rays, X-rays,
Those that can be easily mutated by artificial mutagenesis using radiation, artificial mutagen, etc., and even if such mutant strains have the ability to produce the antibiotics patiurolide A, B and C, the present invention Can be used for

In the method of the present invention, the medium in which the antibiotics Patiurolide A, B and C-producing bacteria are cultivated may be liquid or solid, but a liquid medium is more conveniently used, and surface culturing and shaking culturing are used. However, the submerged culture method is more advantageously used. In the medium, the antibiotic patiurolide A,
Carbon sources that can be assimilated by B and C-producing bacteria, such as starch, glucose, dextrin, glycerin, sucrose, n-paraffin, alcohols (eg, methanol), and nitrogen sources, for example, corn steep liquor as an organic nitrogen source. , Soybean flour, cottonseed flour, peptone, meat extract, etc., and ammonium chloride, ammonium sulfate, ammonium nitrate, urea, etc. can be used as the inorganic nitrogen source. In addition, if necessary, inorganic salts such as salts containing sodium, potassium, magnesium or phosphorus, heavy metal salts such as iron, manganese, zinc, cobalt, copper and nickel salts, defoaming agents such as soybean oil,
Lard oil, chicken oil, silicone oil, Actol (manufactured by Takeda Pharmaceutical Co., Ltd.), etc. may be added appropriately. In liquid culture, the pH of the medium is near neutral, especially about 6-8.
Is preferred. The culturing temperature is preferably about 17 ° C to 30 ° C, and the culturing time is preferably about 24 to 72 hours.

The titers of the antibiotics pachiurolides A, B and C produced in the culture medium with the progress of the culture are quantified according to the conventional method such as the cup method or the paper disk method using Candeida albicans IFO 0583 as the test bacterium. Usually, the maximum production of the antibiotics patiurolide A, B and C is reached after about 1 to 3 days of culture.

In order to collect the target antibiotics patiurolides A, B and C from the culture, a separation means usually used for collecting the metabolite produced by the microorganism from the culture of the microorganism is appropriately used. For example, patiurolides A, B and C show neutral fat-soluble properties and are mainly contained in the culture filtrate. Therefore, first, a filter aid is added to the culture medium to remove the bacterial cells by filtration or centrifugation as it is. . An organic solvent immiscible with water, such as dichloroethane, chloroform, ethyl acetate or methyl isobutyl ketone, was added to the obtained culture solution to obtain patiurolide A, B and C.
To extract. The extract may be washed with water and then concentrated, but the organic solvent layer may be concentrated after washing with a weak acidic aqueous solution such as dilute hydrochloric acid, sulfuric acid or phosphoric acid and a weak alkaline aqueous solution such as sodium hydrogen carbonate or sodium carbonate solution.

The concentrate is further purified by adsorption, partitioning or chromatography using the properties of molecular sieves. That is, a means for bringing the concentrate into contact with an appropriate carrier to adsorb the active ingredient, then desorbing the active ingredient with an appropriate solvent, and separating and collecting the active ingredient is advantageously used. As the carrier, silica gel, crystalline cellulose, adsorptive resin such as Diaion HP-20 (manufactured by Mitsubishi Kasei Co., Ltd.), Amberlite XAD-II (manufactured by Rohm and Haas Company, USA), Sephadex LH-20 (Farmasia. Huaine Chemicals, Sweden) etc. are used. The elution solvent varies depending on the type of the carrier, but for example, petroleum ether, n-hexane, toluene, chloroform, dichloromethane, ethyl acetate, acetone, alcohols, pyridine, acetic acid, or a mixed solvent thereof is used in an appropriate combination. The eluate containing Patiurolide A, B and C is concentrated to dryness and crystallized from an appropriate solvent such as n-hexane or diethyl ether or powdered by adding petroleum ether or n-hexane to the concentrated dry solid. .

The physicochemical properties of the antibiotics patiurolides A, B and C obtained in Example 1 described below are as follows. Pachiyurolide A (1) Appearance: colorless crystals (2) Melting point: mp83-84 ° C (3) Specific optical rotation: [α] ²⁵ _D + 30.1 ° (C = 0.95, in ethanol) (4) Measured molecular weight: m / z210 (M ⁺ ) (EI-MS method) (5) Elemental analysis value: measured value, C, 67.84; H, 8.63 calculated value, C, 68.54; H, 8.63; O, 22.83 (%) (6) molecular formula : C ₁₂ H ₁₈ O ₃ (7) Ultraviolet absorption (UV) spectrum: in methanol (8) Infrared absorption (IR) spectrum: KBr method (Fig. 1) Main absorption (cm ^-1 ) 3400,3080,2940,2870,1705,1680,1640,1470,1450,1440,1390,1365, 1350,1340, 1320,1300,1265,1250,1240,1205,1175, 1155,1125,1105,1085,1075,1055,1035, 1025,1000, 980, 950, 910, 885, 850, 820, 770, 720, 680, 625, 555, 520. (9) ¹³ 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), 129.8 (d), 141.7 (d ), 166.6 (s), 202.4 (s) ppm (however, s: singlet, d: doublet, t: triplet,
q: indicates a quartet, respectively. ) Pachiurolide B (1) Appearance: colorless crystals (2) Melting point: mp 66-67 ° C (3) Specific light rotation: [α] ²⁵ _D -42.4 ° (C = 2.0 in ethanol) (4) Measured molecular weight: m / z210 (M ⁺ ) (EI-MS method) (5) Elemental analysis value: measured value, C, 68.35; H, 8.47 calculated value, C, 68.54; H, 8.63; O, 22.83 (%) (6) molecular formula : C ₁₂ H ₁₈ O ₃ (7) UV spectrum: λ _max 207 nm (ε, 8085) in methanol (8) IR spectrum: KBr method (Fig. 2) Main absorption (cm ^-1 ) 3450, 3070, 2960, 2870,1740,1700,1625, 1440,1420,1385,1350,1340,1260,1250, 1180,1135,1120,1100,1080,1025, 980, 925, 880, 865, 820, 765, 725, 640, 590. (9) ¹³ C-NMR spectrum: 15 MHz, 19.6 (q), 20.4 (t), 23.3 (t), 24.3 (t), 24.9 (t), 31.9 (t), 40.2 (t) in deuterochloroform. , 74.5 (d), 125.9 (d), 139.7 (d), 165.3 (s), 202.8 (s) ppm Patiurolide C (1) Appearance: colorless oily substance (2) Specific illuminance: [α] ²⁵ _D −1.89゜ (C = 2.0, in ethanol) (3) minutes The amount ^{measurement: m / z212 (M +)} (EI-MS method) (4) Elemental analysis: Found, C, 66.98; H, 9.59 Calculated, C, 67.89; H, 9.50 ; O, 22.61 (% ) (5) Molecular formula: C ₁₂ H ₂₀ O ₃ (6) UV spectrum: λ _{max in} methanol 212 nm (ε, 9169) (7) IR spectrum: KBr method (Fig. 3) Main absorption (cm ^-1 ) 3430 , 2940,2860,1700,1640,1440,1350, 1265,1155,1100, 985, 870, 825, 700 (8) ¹³ C-NMR spectrum: 15 MHz in deuterochloroform. The following signals are shown (ppm) 19.4, 20.8, 22.4, 27.7, 28.3, 33.1, 36.0, 70.7, 73.2, 121.2, 150.4, 168.2 In addition, the behavior of Pachiurolide A, B and C on thin layer chromatography is as follows. It is as follows.

Carrier: Silica gel G (manufactured by Merck & Co., West Germany) Detection method: potassium permanganate color development method, UV (254 nm) From the physicochemical properties described above, the structural formulas of the pachieurolides A, B and C are respectively the above formula (I). ), (II) and (I
It is represented by II).

From the properties described above, patiurolides A, B and C are novel compounds belonging to the macrocyclic lactone system represented by the above formulas (I), (II) and (III), respectively.

Table 1 shows the antibacterial spectra of patiurolide A, B and C against various microorganisms.

The antifungal spectra of patiurolide A, B and C are shown in Tables 2 and 3, respectively.

Moreover, the acute toxicity (LD ₅₀ > 400 mg / kg) of oral administration of patiurolide A and B in mice is low.

As described above in detail, Pachiyurorido A, B and C are in vi
In the tro test, A and B show a broad antibacterial activity against Gram-positive and negative bacteria and phytopathogenic and medicinal fungi, and C shows an antibacterial activity against medicinal fungi and have low toxicity. Therefore, Pachiurolides A and B are used in warm-blooded mammals [eg, mice, rats, dogs, cats, cows, pigs,
It can be used as a therapeutic agent for bacterial infections or a therapeutic agent for fungal infections for the purpose of preventing and treating bacterial infections of sheep and humans] and fungal infections. Further, C can be used as a fungal infection treatment agent for the purpose of preventing and treating fungal infections of warm-blooded mammals.

In administering Patiurolide A or B, for example, as an ointment containing Vaseline, Carbowax or the like as a base, and Patiurolide B in a concentration of about 0.1 to 1% (W / V), the above warm-blooded mammal It is administered by applying it to the skin of hands and feet.

In addition, pachiurolides A and B were found to have an action of inhibiting plant growth. Table 4 shows the effects of Pachiurolide A and B on the growth of Taenia cinerea.

Thus, patiurolide A and B are useful as plant growth inhibitors.

In addition, patiurolide A, B and C are extremely promising compounds as synthetic intermediates for new drugs and agricultural chemicals.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.
The percentage (%) in the medium represents weight / volume percentage unless otherwise specified.

Example 1 Penicillium ultecae S11R59 (IFO 31725, FERM P- grown on a nutrient agar slope)
8168), glucose 2%, maltose 3%, raw soybean powder 1.5%, corn staple liquor 1.0%, polypeptone (manufactured by Daigo Nutrition Chemical Co., Ltd., Japan) 0.5%, yeast extract (Daigo Nutrition Chemistry stock) Inoculated into a 2-volume Sakaguchi flask containing 500 ml of a medium containing 0.3% (manufactured by Japan, Japan) and 0.3% sodium chloride (pH 6.0), and reciprocally shake-cultured at 28 ° C. for 48 hours. The whole amount of this culture solution was inoculated into a 50-volume fermenter containing a medium 30 containing 0.05% of an antifoaming agent ACT COL (Takeda Pharmaceutical Co., Ltd., Japan) added to the above medium.
The cells were cultured at 28 ° C. for 48 hours under the conditions of aeration rate of 30 / min and 280 revolutions / min. This culture solution 6 was added with glucose 1.5
%, Soluble starch 4.5%, proflo (Traders Protein, manufactured by BATKY CELLULOSE INC., USA) 1.5%, corn staple liquor 0.5% (pH 7.0)
200 volumes containing 120 medium supplemented with Actol 0.05%
(2 groups) were inoculated and cultured at 24 ° C. for 66 hours under the conditions of an aeration rate of 120 / min and 140 revolutions / min.

Hyflo Super Cell (manufactured by Jyon's Manville Co., USA) was added to the culture solution (200) from the mentor and filtered. The filtrate (193) was adjusted to pH 6.5 and extracted with ethyl acetate. The extract (104) was washed with 2% aqueous sodium hydrogen carbonate and then with water, and the ethyl acetate layer was concentrated. Concentrated residue (71g) is silica gel 60 (Merck & Co., West Germany) (300g)
Column chromatography, and fractionated and eluted with dichloromethane.

The fraction containing the active substance was concentrated to obtain a mixed crude substance (39.1 g) of patiurolide A, B and C. Dissolve the crude material in ethyl acetate, and use it for preparative high performance liquid chromatography (HPL
C). The carrier used was silica gel G (Merck, West Germany), and the developing solvent was n-hexane: ethyl acetate (8.
5: 1.5) was used. The three peaks containing the active substance were collected, the eluates were concentrated, and n-hexane was added to the concentrated liquid.
Crystals precipitated upon standing. The crystals were collected by filtration to obtain pachiurolide B (6.39 g) and A (3.61 g).

The third fraction in which crystals did not precipitate contained Patiurolide C, and this crude Patiurolide C was again subjected to preparative HPLC and eluted with n-hexane: isopropyl ether: isopropanol (8: 2: 1). Purified patiurolide C (6.29 g) was obtained as an oily substance.

EFFECTS OF THE INVENTION Since the patiurolides A and B produced by the present invention have excellent antifungal, antibacterial and plant growth inhibitory effects, they can be used as therapeutic agents for fungal infections and bacterial infections and as plant growth inhibitors. Since Patiurolide C has an excellent antifungal activity, it can be used as a therapeutic agent for fungal infections.

[Brief description of drawings]

1, 2 and 3 show the infrared absorption spectra of the patiurolides A, B and C obtained in Example 1, respectively.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location (C12P 17/08 C12R 1:80)

Claims (2)

[Claims] 1. A formula I, II or III, respectively. Pachiurolide A, B or C represented by. 2. A penicillium genus of formula I, II or III, respectively. A patieurolide A, characterized in that a microorganism having the ability to produce at least one of patiurolides A, B and C represented by the formula is cultured in a medium, the compound is produced and accumulated in the culture, and this is collected.
At least one production method of B and C.