JP2001247566A - New antimycotic substance and method of producing the substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new antimycotically active substance and provide a method for production of the substance. SOLUTION: The objective compound M-3-A is expressed by general formula (I). The compound is produced by culturing a mold strain M-3 belonging to Ascomycetes in a Potato Dextrose Broth (PD medium, 1/2 nutrition, 50% sea water, pH 6.8) and collecting the produced compound M-3-A from the medium. The antimycotic agent contains the compound M-3-A as an active component.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel antifungal active substance, a method for producing the same using a microorganism, and an antifungal agent containing the same as an active ingredient.

[0002]

2. Description of the Related Art The elucidation of life phenomena is rapidly progressing with the advance of molecular and cell biology. The discovery of biologically active substances and inhibitors that have new effects typified by antibiotics and plant secondary metabolites not only opens the door to the development of chemotherapeutic agents, but also captures biological phenomena from the substance level and explains the mechanism of the mechanism. It is an extremely useful means of obtaining knowledge, and the development of new basic research is expected.

[0003] Natural organic compounds that inhibit the function of microtubules include taxol and vinblasti.
n), griseofulvin, rhizoxin (rhi
Some compounds have been applied as anticancer and antifungal agents such as zoxin), and are very interesting compounds. Recently, microtubule polymerization inhibitors curacin A and Sorangium cellulosum (Myxobacteriu) obtained from the marine cyanobacterium Lyngbya majuscula
As in taxol, epothilones, which promote microtubule polymerization and inhibit depolymerization, have been isolated from m). In the special habitat of the ocean, various marine microorganisms still have great expectations for natural organic compounds having novel biological activities. In general, since the biological activity of a chemical substance largely depends on its chemical structure, it can be said that there is a constant need for a novel compound having biological activity.

[0004]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a novel substance having an antifungal activity, a method for producing the same and an antifungal agent containing the same as an active ingredient. I do.

[0005]

Means for Solving the Problems Accordingly, the present inventors have:
As a result of intensive studies to solve the above problems, seaweed collected from the coast of Kazusaminato, Chiba Prefecture (Susabinori Porphyra ye)
zoensis ) was found to produce a substance having antifungal activity, and the substance was a novel compound. The invention has been completed.

That is, a first aspect of the present invention is the following general formula (I)

Embedded image The present invention relates to a novel compound M-3-A represented by

A second aspect of the present invention belongs to the ascomycetes, and the compound M-3-A of the formula (I) described above (hereinafter, if necessary,
It is referred to as "the compound of the present invention". ) Is cultured in a culture medium to produce and accumulate the compound M-3-A of the above formula (I) in the culture, which is collected from the culture. The compound of formula (I) M-3-, which is characterized above.
It relates to the production method of A.

A third aspect of the present invention relates to an antifungal agent containing the compound M-3-A represented by the above formula (I) as an active ingredient.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.

Structure of the compound of the present invention The compound of the present invention is represented by the following formula (I).

Embedded image

The compound of the present invention has the structure represented by the above formula (I), has the following physicochemical properties as typical examples, and has the following ¹ It has chemical shift values of 1 H NMR and ¹³ C NMR.

The compounds of the present invention have been identified by their physicochemical properties and spectral data.
Its chemical structure consists of one tryptophan unit, one valine residue and one isopentenyl group,
Characteristic is that it has a diketopiperazine attached to the indole ring.

[0013]

[Table 1]

[0014]

[Table 2]

Preparation of the Compound of the Present Invention Next, the preparation of the compound of the formula (I) of the present invention will be described. The compound M-3-A of the formula (I) of the present invention is a compound M-3-A
Can be produced by culturing the microorganism produced in the above in a medium, producing and accumulating it in a culture, and collecting it. As a medium, a PD medium (potato dextrose medium)
ose Broth) can be used. The composition of the medium is preferably nutrition 1/2 and seawater 50%.

The compound of the present invention can be produced by filamentous fungi. Preferred examples of the filamentous fungus include the filamentous fungus M-3 isolated from the seaweed ( Porphyra yezoensis ). . Filamentous fungus M
The mycological properties of the -3 strain are as follows.

[0017]

[Table 3]

The nucleotide sequence of the 18S rDNA gene of the filamentous fungus M-3 strain is shown in the following sequence listing. As a result of conducting a homology search based on the results of the nucleotide sequences shown in the above sequence listing, the filamentous fungus M-3 used for producing the compound of the present invention was used.
The strain was found to be a filamentous fungus belonging to the ascomycetes (see Table 3). The strain M-3 has been deposited at the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology as Microbial Deposit No. M-3 (PERM P-17709) (Deposit date: February 2, 2000). Day).

In the production of the compound of the present invention, the method for culturing the above-mentioned strain, filamentous fungus M-3 belonging to the ascomycetes, and collecting the compound M-3-A of the present invention from the culture is specifically described. Is described in Examples described later, and can be generally carried out according to a method for culturing marine filamentous fungi.

The culture conditions are as follows. That is, the cultivation temperature can be appropriately changed within a range in which the microorganism grows and produces the compound M-3-A. Further, it is preferable to maintain the pH during the culturing at around 8.0. The culture may be continued under such conditions to produce and accumulate the target substance in the culture, and when the amount of production reaches the maximum, the culture may be stopped and the target substance may be collected. As the medium, Potato Dextrose Broth (PD medium, 1/2 nutrient, 50% seawater) is preferable.

After completion of the culture, the compound M of the present invention is removed from the culture.
Purification and isolation of -3-A can be carried out by appropriately utilizing means generally used for collecting microbial metabolites. For example, various ion exchange resins, nonionic adsorption resins, gel filtration chromatography or activated carbon,
Chromatography using an adsorbent such as alumina or silica gel and high performance liquid chromatography, or crystallization, concentration under reduced pressure, and lyophilization can be used alone or in appropriate combination. These purification means can be used repeatedly.

Use of the compound of the present invention By utilizing the remarkable antifungal activity of the compound of the formula (I) of the present invention, an antifungal agent containing the compound as an active ingredient can be provided. The composition of the antifungal agent is not particularly limited and can be arbitrarily selected and determined in each field of use. An effective amount of the compound of the present invention, for example, 0.1% to 50% by weight is contained. Just do it.

[0023]

The present invention will be specifically described below with reference to examples. However, the present invention is not limited by the examples and the like.

Example 1 In the present invention, a compound that inhibits or promotes microtubule polymerization is a rice blast fungus ( Pyriculari
a. oryzae P-2b) Morphological changes characteristic of spore germ tubes (curl
ing effect) was used as a primary screening system [H. Kobayashi et. al. J. Anti
biot . 49 , 873-879 (1996)]. Substantially obtained candidate active strain M-3 had only a hyphal germination inhibitory action against rice blast fungus ( P. oryzae P-2b). Compound of the present invention
M-3-A was isolated using the above-mentioned bioassay system as an index.

Potato Dextrose Broth (PD medium, 1/2 nutrition, 50% seawater, pH 6.8) was inoculated with the above strain M-3.
Static culture was performed at 20 ° C. for 1 week. Culture solution (pH 7.8)
3.3 L was extracted with 3 L of 80% aqueous acetone. The aqueous solution obtained after suction filtration and then concentration under reduced pressure was adjusted to pH 7.0, and extraction was repeated three times with 1 L of ethyl acetate. After extraction, all the ethyl acetate portions were combined and concentrated under reduced pressure to obtain 86 mg of a brown syrup. The results of the bioassay indicated that the active ingredient was contained in the ethyl acetate extract.

Subsequently, 86 mg of the above syrup was dissolved in an appropriate amount of methanol, and the solution was separated by preparative thin-layer chromatography.
(L 200 mm, W 200 mm, T 2 mm; Silicagel 60 F2
54; Merck) and hexane-acetone (7:
3) was separated as a developing agent. Biological activity with an R _f value of 0.22
Eluted in a fraction. This was concentrated under reduced pressure to obtain 3.9 mg of a brown oily substance. Then ODS column
(Diameter 20 mm, length 250 mm; Deverosil ODS-HG-5, manufactured by Nomura Chemical Co., Ltd.) and 70% aqueous methanol elution solvent at a flow rate of 9.0 mL / min and a detection wavelength of 210 nm. Purification was performed by chromatography. As a result, 0.8 mg of the compound M-3-A of the present invention was obtained as a colorless solid.

The solubility of M-3-A was soluble in acetone, ethyl acetate, chloroform, methanol and DMSO, and was hardly soluble in water. The biological activity of M-3-A was as follows. That is, the substance of the present invention is a rice blast fungus ( P. oryzae P-2b)
On the other hand, at a concentration of> 36 μM, it showed an inhibitory action on germination of hypha.

[Biological Assay] A culture of the filamentous fungus to which 80% aqueous acetone was added was filtered, and the filtrate was concentrated to remove acetone. This was prepared at eight dilution concentrations from 2 to 256 times, and the rice blast fungus ( P.o.
ryzae P-2b) with spore fluid (containing 0.02% yeast extract)
After culturing at 16 ° C for 16 hours, changes in the germ tube morphology or inhibition of hyphal germination were observed under a microscope. Inhibition of mycelial germination was observed in the culture solution of the M-3 strain up to 128-fold dilution. In addition, rhizoxin and griseofulvin were used as positive controls. In the former, curling at a concentration of> 16 μM is strong against conidia germination
effect, the latter being curling eff at concentrations> 25 μM.
ect.

[0029]

Industrial Applicability According to the present invention, there is provided a novel compound represented by the above formula (I), which exhibits remarkable antifungal activity and has usefulness as an active ingredient of an antifungal agent. Have. Further, the present invention provides a method for producing the compound using a microorganism.

[0030]

[Sequence List] SEQUENCE LISTING <110> Marine Biotechnology Institute Co. Ltd. <120> Antifugal active substance M-3-A and method of producing the same <130> MBI000302 <160> 1 <170> PatentIn Ver. 2.1 <210 > 1 <211> 1731 <212> rDNA <213> Bulgaria Inquinans M-3 <400> 1 caaagattaa gccatgcatg tctaagtata agcaatctat actgtgaaac tgcgaatggc 60 tcattaaatc agttatcgtt tatttgatag taccttacta cttggataac cgtggtaatt 120 ctagagctaa tacatgctaa aaaccccgac ttttggaggg gtgtatttat tagataaaaa 180 accaatgccc ttcggggctc cttggtgatt cataataact taacgaatcg catggccttg 240 tgccggcgat ggttcattca aatttctgcc ctatcaactt tcgatggtta ggtcttggct 300 aaccatggtt tcaacgggta acggggaatt agggttctat tccggagagt gagcctgaga 360 aacggctaac acatccaagg aaggcagcag gcgcgcaaat tacccaatcc cgacacgggg 420 aggtagtgac aataaatact gatccagggc tcttttgggt cttggaattg gaatgagtac 480 aatttaaatc ccttaacgag gaacaattgg agggcaagtc tggtgccagc agccgcggta 540 attccagctc caatagcgta tattaaagtt gttgcagtta aaaagctcgt agttgaacct 600 tgggtctggc tggccggtcc gcctcaccgc gt gtactggt ccggccggac ctttccttct 660 ggggaatcgc atgcccttca ctgggtgtgt cgaggatcca ggacttttac tttgaaaaaa 720 ttagagtgtt caaagcaggc ctatgctcga atacattagc atggaataat agaataggac 780 gtgtggttct attttgttgg tttctaggac cgccgtaatg attaataggg atagtcgggg 840 gcatcagtat tcaattgtca gaggtgaaat tcttggattt attgaagact aactactgcg 900 aaagcatttg ccaaggatgt tttcattaat cagtgaacga aagttagggg atcgaagacg 960 atcagatacc gtcgtagtct taaccataaa ctatgccgac tagggatcgg gcgatgttac 1020 ttttttgact cgctcggcac cttacgagaa atcaaagtct ttgggttctg gggggagtat 1080 ggtcgcaagg ctgaaactta aagaaattga cggaagggca ccaccaggag tggagcctgc 1140 ggcttaattt gactcaacac ggggaaactc accaggtcca gacacaataa ggattgacag 1200 attgagagct ctttcttgat tttgtgggtg gtggtgcatg gccgttctta gttggtggag 1260 tgatttgtct gcttaattgc gataacgaac gagactttga cttttaaata gctaggctag 1320 ctttggctgg tcgctggctt cttagaagga ctatttgctc aagcaaatgg aagtgcgaag 1380 caataacagg tctgtgatgc ccttagatgt tctgggccgc acgcgcgcta cactgacaga 1440 gccaacgagt tcttccttag ccgaaaggtt tgggtaatct tgt taaactc tgtcgtgctg 1500 gggatagagc attgcaatta ttgctcttca acgaggaatt cctagtaagc gcaagtcatc 1560 agcttgcgct gattacgtcc ctgccctttg tacacaccgc ccgtcgctac taccgattga 1620 atgatccagt gaggctttcg gactggccca ggaagagtgg caacactcat ctagggccgg 1680 aaagttgtcc aaacttggtc atttagagga agtaaaagtc gtaacaaggt t 1731

[Brief description of the drawings]

FIG. 1 shows an infrared absorption spectrum of compound M-3-A of Example 1.

FIG. 2 shows an ultraviolet absorption spectrum of compound M-3-A of Example 1.

FIG. 3 shows ¹ H NMR (CDCl) of compound M-3-A of Example 1.
₃ ) Show the spectrum.

FIG. 4 ¹³ C NMR (CDC) of compound M-3-A of Example 1
l ₃ ) Show the spectrum.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12P 17/16 C12N 1/14 B // C12N 1/14 (C12P 1/02 (C12P 1/02 C12R 1 : 645) C12R 1: 645) (C12P 17/16 (C12P 17/16 C12R 1: 645) C12R 1: 645) (C12N 1/14 (C12N 1/14 C12R 1: 645) C12R 1: 645) C12N 15 / 00 ZNAA (72) Inventor Masaki Mochizuki 1900 Sodeshim-cho, Shimizu City, Shizuoka Prefecture Inside the Kaiyo Biotechnology Laboratory Co., Ltd. Utilization Research Center (72) Inventor Yoshikazu Shisato 1900 Sodesoshi-cho, Shimizu-shi, Shizuoka Pref. In-house F-term (reference) 4B024 AA07 BA67 CA11 GA19 GA27 4B064 AE49 AE54 BA04 BD01 BE07 BE09 BE14 BG01 BH04 BH05 BJ03 BJ06 BJ13 CA05 CE08 CE10 DA12 4B065 AA58X AC13 AC14 BA22 BB15 BB26 BD16 CA01 A34 CA01 A34 AA03

Claims (3)

[Claims] 1. A compound represented by the following formula (I): A compound represented by the formula: 2. A filamentous fungus strain M-3 belonging to Ascomycetes and having the ability to produce the compound according to claim 1, is cultured in a medium, and the compound according to claim 1 is produced and accumulated in the culture. The method for producing a compound according to claim 1, wherein the compound according to claim 1 is collected from a culture. 3. The compound according to claim 2, wherein the filamentous fungus strain M-3 has a rDNA gene having a nucleotide sequence represented by SEQ ID NO: 1 or a nucleotide sequence substantially identical to the nucleotide sequence. Manufacturing method.