JP2006213662A - New cyclodepsipeptide antibiotic - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new antibiotic which has potent antimycotic activity, shows low cytotoxicity in animals and plants, and can be easily obtained by industrial mass production and to provide an agricultural chemical and an antimycotic drug each containing the same as an active ingredient. <P>SOLUTION: The antibiotic is a compound obtained by culturing bacteria belonging to the Myxococcus group and extracting the obtained culture and represented by formula (I) (wherein R<SP>1</SP>is a halogen atom; R<SP>2</SP>is any one selected from the group consisting of a hydrogen atom, a methyl group, an ethyl group, and an acetyl group; and Me is a methyl group) or a salt of the compound. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to novel compounds, microorganisms producing them and cultures thereof, processes for producing them by microorganisms producing them, and uses thereof. More specifically, the present invention relates to novel antibiotics SMH27-4A, SMH27-4B, SMH27-4C and antifungal agents containing these as active ingredients, and in particular, extracted from a culture in which microorganisms belonging to coastal myxobacteria are cultured. The present invention relates to novel antibiotics SMH27-4A, SMH27-4B, SMH27-4C and agrochemicals and antifungal agents containing this compound as an active ingredient.

  Conventionally, various antifungal agents have been used for fungal diseases caused by fungi such as Candida, Aspergillus, and Trichophyton. Currently, amphotericin, miconazole, griseofulvin and the like are known as such antifungal agents.

  However, existing antifungal drugs also have a problem of drug resistance, and the development of antifungal drugs that are effective against various filamentous fungi is required. In the field of agricultural chemicals, mycosis is an economically important disease, and in addition to the above-mentioned resistance problem, a new agricultural chemical having low toxicity and low persistence in the environment is required. In addition, although conventional antifungal agents have antifungal activity, they are highly cytotoxic to eukaryotic cells such as animal cells in general. Therefore, a sufficient amount cannot be administered at the time of use, and a sufficient therapeutic effect cannot always be achieved. Therefore, there is a demand for a substance having a strong antifungal activity and low cytotoxicity to other animal cells.

In addition, although there are the following reports on antibiotics produced by myxobacteria (for example, see Non-Patent Documents 1 to 3), these antibiotics are different in basic skeleton from the compounds of the present invention. In addition, the bacteria producing the compounds described in these documents require slime bacteria inhabiting the terrestrial environment (soil, decayed plants, etc.) or mucinous bacteria in the marine environment (that is, salt equivalent to seawater for their growth and compound production; Patent Document 4).

H. Reichenbach & G. Hoefle, In: Dworkin, M. & Kaiser, D. (Eds.), Myxobacteria II. American Society for Microbiology, Washington, DC, pp. 347-397 (1993). H. Reichenbach & G. Hoefle, In: Grabley, S. & Thiericke, R. (Eds.). Drug Discovery from Nature.Springer-Verlag, Berlin-Heidelberg. Pp. 149-179 (1999). K. Gerth et.al., Journal of Biotechnology, Vol.106 (2-3), 233-253 (2003) R.Fudou et.al., Journal of the Antibiotics., Vol.54 (2), 149-152 (2001)

  The present invention has been made in view of the above-mentioned points, and has a novel antibacterial activity having a strong antifungal activity, low cytotoxicity to animals and plants, and easy industrial mass production. An object is to provide agricultural chemicals and antifungal agents as ingredients. However, since the compound found by the present invention is a novel compound, the above-mentioned non-patent document merely shows a general technical level as a technical background of the present invention, and is directly related to the problem to be solved by the present invention. Not what you want.

  As a result of continual research to achieve the above object, the present inventors have isolated and cultured bacteria belonging to the genus Eniglomixer from the coastal soil environment, and the novel substance extracted from this culture has a strong antifungal action. The present invention was completed. That is, the present invention is a compound represented by the following general formula (I) or a salt thereof obtained by culturing bacteria belonging to the group of myxobacteria and extracting from the culture.

（式Ｉにおいて、Ｒ^１はハロゲン原子を示し、Ｒ^２は水素原子、メチル基、エチル基、及びアセチル基からなる群より選択される何れかを示し、Ｍｅはメチル基を示す。）

(In Formula I, R ¹ represents a halogen atom, R ² represents any one selected from the group consisting of a hydrogen atom, a methyl group, an ethyl group, and an acetyl group, and Me represents a methyl group.)

In a preferred embodiment, R ¹ and R ² in the general formula (I) are each a bromine atom (Br) and a hydrogen atom (H), a chlorine atom (Cl) and a hydrogen atom (H), or an iodine atom (I). Each of the new antibiotics SMH27-4A, SMH27-4B, or SMH27-4C is provided, which represents hydrogen atom (H). These novel compounds may be in the form of salts.

  From a different point of view, the present invention is an agrochemical or antifungal agent characterized by containing the above compound or a pharmacologically acceptable salt thereof as an active ingredient.

  In another aspect, the method for producing the compound of the present invention comprises culturing myxobacteria belonging to the genus Enigromyxa (genus Enhygromyxa), which can be separated from soil near the coast, in a culture solution to which a halogen compound is added. It is characterized by extracting. Halogen compounds include, but are not limited to, sodium chloride, magnesium chloride, calcium chloride, sodium bromide, potassium bromide, calcium bromide, sodium iodide, magnesium iodide and calcium iodide.

  The compounds of the present invention, including novel antibiotics SMH27-4A, SMH27-4B and SMH27-4C, can be used against various fungi such as filamentous fungi such as phytoftora, absida, lysopus and trichophyton and yeasts such as Candida. Even antibacterial activity. Therefore, the compounds of the present invention can be used as agricultural chemicals and antifungal agents. That is, it is useful as an agrochemical containing the compound of the present invention as an active ingredient and a therapeutic agent for mycosis.

  The compounds of the present invention, including the new antibiotics SMH27-4A, SMH27-4B and SMH27-4C, can be used as pesticides. It can also be used as a therapeutic agent for mycosis administered orally or parenterally.

  Examples of the producing bacteria producing the compound of the present invention including the novel antibiotics SMH27-4A, SMH27-4B and SMH27-4C according to the present invention include, for example, the antibiotics SMH27-4A, SMH27-4B and Microorganisms having the ability to produce SMH27-4C are used, for example, Enhygromyxa sp. AJ-110262 (FERM AP-20376). This fungus belongs to the group of myxobacteria, which are gliding bacteria that form fruiting bodies. Unlike the conventionally known soil-derived myxobacteria, it is isolated from the coastal environment and has a concentration of about 1% for growth. It is a novel microorganism that requires it. The above AJ-110262 shares are registered with the FERM AP-20376 (deposited) at the National Institute of Advanced Industrial Science and Technology, the Patent Biological Depositary Center (Central 1-1, Higashi 1-1-1 Tsukuba, Ibaraki 305-8586). The date is deposited as January 26, 2005).

The taxonomic properties of this bacterium are described below.
(Morphological features)
A diffusible light yellow or grayish white colony is formed on a yeast-1 / 5 artificial seawater medium (Vy2-1 / 5ASW medium). In addition, due to the property of spreading around by a sliding motion while partially eroding the agar quality of the plate medium, the agar surface sinks shallowly to form a circular crater shape. Vegetative cells are rod-shaped and have a length of 1.5 to 8.0 μm and a width of 0.5 to 0.8 μm. Regarding fruit body formation, which is one of the main features of myxobacteria, no clear fruit body was observed for this bacterium as long as it was cultured for 2 to 4 weeks on a Vy2-1 / 5 ASW agar medium.

(Systematic taxonomy)
In order to investigate the phylogenetic position of the AJ-110262 strain, the base sequences of these 16S rRNA genes were examined, and the molecular phylogenetic tree together with the base sequences of the 16S rRNA genes of the bacterial group closest to those base sequences. Was created (see FIG. 1). As a result, it was suggested that AJ-110262 strain may be a microorganism belonging to the order Myxobacterales and suborder Soranginiae. The most closely related genus is the marine mucous bacterium Enhygromyxa salina, which has a low base homology of 93% and was not assigned to any known species. Therefore, the strain was identified as Enhygromyxa sp.

  The method of studying the evolution of organisms and genes based on molecular phylogenetic trees has been established as molecular taxonomy (eg, Kimura Shigeo, Introduction to Molecular Evolution Chemistry (Baifukan), pages 164-184, (See "How to make a seven-molecule phylogenetic tree and its evaluation"). The molecular phylogenetic tree based on the base sequence of the 16S rRNA gene multiplexes the base sequence of the 16S rRNA gene of the target microorganism together with the base sequence of the 16S rRNA gene of a known microorganism presumed to be the same or similar to the same microorganism from the mycological properties. It can be obtained by calculating alignment and evolutionary distance and creating a phylogenetic tree based on the obtained values. The base sequence of the 16S rRNA gene of a known microorganism used to create a molecular phylogenetic tree can also be obtained by homology search of an existing database. Here, the evolution distance refers to the total number of mutations per locus (sequence length) between certain genes. Multiple alignments and evolutionary distances are included, for example, in commercially available software (CLUSTALW; program collection “Phylogeny Programs” (available from http://evolution.genetics.washington.edu/phylip/software.html). Thompson, DJ, et al., Nucleic Acids Res., 22, 4673-4680 (1994)). Phylogenetic trees are also included in the publicly available software (TreeView (Tree drawing software for Apple Machintosh: by Roderic D., Page 1995, Institute of Biomedical and Life sciences, University of Glasgow, UK), Phylogeny Programs). Can be created). Specifically, the result calculated by CLUSTALW may be output as PHLYP format data and read by TreeView. PHLYP (Felsenstein J. (1995) Phylogenetic inference package, version 3.5.7., Department of Genetics, University of Washington, Seatle WA, USA) is also included in the Phylogeny Programs.

(Growth characteristics)
This bacterium required salt for growth, and the optimum concentration was 0.5-1.0%. Such a property requiring a low concentration of salt is a unique property that is not found in the conventionally known terrestrial or marine myxobacteria. In addition to salt (sodium), the main feature of this bacterium is that it uses inorganic ions such as calcium and magnesium as essential growth factors.

  Such a novel antibiotic SMH27-4-producing bacterium can be cultured using a general microorganism culture method. As a medium used for culture, a medium containing a nutrient source that can be used by a novel antibiotic SMH27-4-producing bacterium can be used. For example, various proteins such as casein, gluten, soybean flour, yeast extract, and amino acid mixtures are suitable as the carbon source and nitrogen source. In addition, yeast or bacterial cells used for fermentation production or carbohydrates such as glucose, starch, dextrin, and inorganic nitrogen sources such as urea, ammonium sulfate, and ammonium phosphate can be used. Moreover, the culture medium can contain inorganic salts such as calcium carbonate, sodium phosphate, and magnesium sulfate as necessary. However, sodium chloride or the like of about 0.5 to 1% (weight basis) is essential for any medium. Usually, artificial seawater (for example, manufactured by Rohto, Tokyo) is preferably added to about 1/5 times the predetermined amount. The medium can be cultured at a pH of 6 to 9, but is preferably cultured at 7.0 to 8.5. In addition, for the purpose of increasing the yield of the product, various types of adsorption resins can be added to the medium and cultured.

  The producing bacteria can be cultured at 10 ° C to 38 ° C, but it is most desirable to culture at 25 ° C to 30 ° C. The culture time is usually 5 to 14 days, but can be appropriately changed depending on the culture conditions.

  Novel antibiotics SMH27-4 produced by culture accumulate mainly in the culture. In order to obtain novel antibiotics SMH27-4 from the culture solution of the producing bacteria, it is possible to use a method usually used for collecting microbial metabolites. For example, a method using the solubility of novel antibiotics SMH27-4 and other substances contained in the culture, a method using the difference from the ionic binding force, a method using the difference in adsorption affinity, and a difference in molecular weight. The methods to be used can be used alone or in appropriate combination or repeatedly. Specifically, for example, a novel antibiotic SMH27-4 is produced by purifying a culture of a novel antibiotic SMH27-4-producing bacterium and an extract of bacterial cells using a combination of gel filtration chromatography, adsorption chromatography, liquid chromatography and the like. A fraction containing -4 and other active ingredients is obtained. A novel antibiotic SMH27-4 can be obtained by further developing and purifying the solid obtained by concentrating this fraction under reduced pressure using high performance liquid chromatography.

The novel antibiotics SMH27-4 thus obtained have the following physicochemical properties.
(SMH27-4A)
White powder.
IR (KBr): 3448, 2936, 1697, 1684, 1636, 1508, 1256, 1223, 1119 cm ^-1
UV (MeOH): λ _max 203 (ε 49000), 276 (ε 11000) nm.
[α] ²⁴ _D : + 70 (c 0.17, MeOH)
MS (ESI-TOF) (rel.int.): M / z 684.2 and 686.2 (49:60) [M + H] ⁺ , 706.2 and 708.2 (96: 100) [M + Na] ⁺
HRMS (ESI, positive): found m / z 684.2278 [M + H] ⁺ ,
calcd for C ₃₄ H ₄₃ N ₃ O ₇ ⁷⁹ Br 684.2279.
¹ H-NMR (CDCl ₃ , 600 MHz): δ 7.31 (m, 1H), 7.28 (d, J = 1.2 Hz, 1H), 7.19 (m, 5H), 7.06 (s, 1H), 6.96 (dd, J = 1.8,8.4 Hz, 1H), 6.88 (d, J = 8.4 Hz, 1H), 5.72 (s, 1H), 5.11 (m, 1H), 5.07 (m, 2H), 4.79 (m, 1H), 3.49 (s, 3H), 3.24 (dd, J = 10.8, 13.2 Hz, 1H), 2.86 (s, 3H), 2.61 (dd, J = 4.8, 13.2 Hz, 1H), 2.32 (ddd, J = 8.4, 8.4, 13.2 Hz, 1H), 2.18 (m, 1H), 2.14 (m, 1H), 2.08 (m, 2H), 1.97 (m, 1H), 1.87 (m, 1H), 1.82 (m, 1H), 1.70 (m, 1H), 1.64 (m, 1H), 1.60 (s, 3H), 1.33 (d, J = 6.6 Hz, 3H), 1.29 (d, J = 6.0 Hz, 3H).
¹³ C-NMR (CDCl ₃ , 150 MHz): δ 173.4 (s), 172.8 (s), 168.8 (s), 165.8 (s), 165.0 (s), 151.1 (s), 134.7 (s), 132.5 ( d), 131.7 (s), 130.4 (s), 130.1 (d), 130.0 (d), 128.6 (d, 2C), 128.2 (d, 2C), 125.4 (d), 116.1 (d), 111.1 (s ), 110.0 (s), 70.2 (d), 58.5 (q), 56.7 (d), 46.1 (d), 35.0 (t), 34.9 (t), 33.3 (t), 31.8 (t), 30.2 (q ), 26.5 (t), 25.9 (t), 19.7 (q), 17.2 (q), 16.3 (q).
(SMH27-4B)
MS (ESI-TOF) (rel.int.): M / z 640 and 642 (3: 1) [M + H] ⁺ , 662 and 664 (3: 1) [M + Na] ⁺
(SMH27-4C)
MS (ESI-TOF) (rel. Int.): M / z 732 [M + H] ⁺ , 754 [M + Na] ⁺

  Further, the novel antibiotic SMH27-4, or a pharmacologically acceptable salt thereof, can be used as a pharmaceutical, for example, an antifungal agent for treating fungal infections. It can be prepared using an agent, preservative, buffer, antioxidant, fragrance and the like. Furthermore, the compounds of the present invention can be used as agricultural chemicals and are usually used together with adjuvants commonly used in the pharmaceutical field. The compounds of the present invention can be obtained by known methods, for example, emulsion stock solution, sprayable paste, sprayable or dilutable solution, diluted emulsion, wettable powder, water solvent, powder, granule, flowable, dry flowable, smoke Formulated into capsules, fumigants, and capsules, eg with polymeric substances.

Examples of the present invention will be described below, but the present invention is not limited thereto.
1. Production of novel antibiotics SMH27-4;
A yeast agar medium for seeds having the following composition was prepared, sterilized by heating with steam at 120 ° C. for 20 minutes, and dispensed in 20 ml portions. This was inoculated with an agar piece (5 mm × 10 mm, in 10% glycerin) of AJ-110262 strain (FERM AP-20376) stored at −80 ° C. at a low temperature and cultured at 28 ° C. for 14 to 18 days.

  Next, a production medium having the following composition was prepared, and 100 ml thereof was poured into a 500 ml Erlenmeyer flask and sterilized by heating steam (120 ° C., 20 minutes). A spatula that sterilizes the cells of the fan-shaped portion corresponding to the area of 1/5 to 1/4 of the circular colony when the colony grows to a diameter of about 6 to 7 cm on the above-mentioned seed agar medium. The whole agar was cut out using a medium and the like, cut into a size of about 5 mm square, and aseptically inoculated into the production medium. The culture temperature was set to 28 ° C., and swirling culture (180 rpm) was performed for 7 to 10 days.

  The novel antibiotic SMH27-4 was isolated from the obtained culture medium of the production medium by the following procedure. That is, 17.9 L of this culture solution was centrifuged to remove the supernatant, and wet cells and an adsorbent resin were obtained. About 10 L of acetone was added thereto, and the mixture was stirred for 2 hours at room temperature to extract the active substance. This was repeated again. Subsequently, the acetone solution from which the bacterial cells and the adsorbent resin were removed by filtration was concentrated under reduced pressure using an evaporator until a small amount of water remained. 100 ml of water was added to this, then extracted twice with 100 ml of ethyl acetate and concentrated to obtain an ethyl acetate soluble part (812 mg). This was subjected to a silica gel column (Wakogel C-300, 40 g; manufactured by Wako). Elution was performed stepwise using a hexane-ethyl acetate mixed solution (5: 5, 4: 6, 3: 7, 2: 8 (v / v), 240 ml each) as an eluent. Of these, the active fraction (all the fraction eluted with hexane-ethyl acetate (3: 7) and 80 ml before and after that) was collected (37.3 mg), and this was then added to a reverse phase HPLC column (Develosil ODS UG-5 ( Nomura Chemical) and an inner diameter of 10 mm × 250 mm). Pure SMH27-4A (2.6 mg) was obtained by fractionating the fraction eluted at a retention time of 19.6 minutes using a 70% methanol solution (4 ml / min) as the eluent. Similarly, by fractionating a fraction that elutes at a retention time of 18.0 minutes, pure SMH27-4B (<0.5 mg) is fractionated, and by fractionating a fraction that elutes at a retention time of 22.4 minutes. Pure SMH27-4C (<0.5 mg) was obtained. The HPLC chart at this time is shown in FIG.

2. Antibacterial activity test The antifungal activity of the novel antibiotic SMH27-4A was determined using the dilution plate method.
The novel antibiotic SMH27-4A was dissolved in a small amount of acetone, and then an equal dilution series was prepared with a yeast nitrogen base medium (Difco). This was dispensed into 24-well plates or 96-well plates (Corning). The test bacteria, Candida and Rhodotorula, were cultured overnight in a yeast nitrogen base medium, and then diluted with a medium so as to be about 104 / ml. Aspergillus, Absidia, and Rhizopus spp. Were grown on yeast nitrogen base agar and then collected spores and diluted to about 104/103, 103 / ml, respectively. did. For other filamentous fungi, an agar piece (about 1 mm × 1 mm) at the tip of a colony grown on the same agar medium was cut out and used as an inoculation source. The test bacteria thus prepared were inoculated into the antibiotic-containing medium and cultured at 25 ° C. for 40 hours to determine the minimum concentration (MIC) at which no growth was observed. The results are shown in Table 1 below.

表１から明らかなように新規抗生物質ＳＭＨ２７−４Ａは優れた抗真菌活性を有することが分かった。なお、ＳＭＨ２７−４Ｂ及びＳＭＨ２７−４Ｃについては、被検菌として少なくとも、フィトフトラ・カプシッシ(Phytophthora capsici)に対して生育阻害を示すことが分かっている。

As is clear from Table 1, the novel antibiotic SMH27-4A was found to have excellent antifungal activity. In addition, about SMH27-4B and SMH27-4C, it turns out that growth inhibition is shown at least to Phytophthora capsici (Phytophthora capsici) as a test microbe.

The molecular phylogenetic tree of AJ-110262 strain which concerns on this invention is shown. 1 is a preparative chromatogram of novel antibiotics SMH27-4A (Br), SMH27-4B (Cl) and SMH27-4C (I) of the present invention.

Claims (6)

A compound represented by the following general formula (I) or a salt thereof:

(In Formula I, R ¹ represents a halogen atom, R ² represents any one selected from the group consisting of a hydrogen atom, a methyl group, an ethyl group, and an acetyl group, and Me represents a methyl group.) The compound or a salt thereof according to claim 1, wherein, in the formula I, R ¹ represents a bromine atom (Br) and R ² represents a hydrogen atom. The compound or a salt thereof according to claim 1, wherein, in the formula I, R ¹ represents a chlorine atom (Cl) and R ² represents a hydrogen atom. The compound or a salt thereof according to claim 1, wherein, in the formula I, R ¹ represents an iodine atom (I) and R ² represents a hydrogen atom.   An agrochemical or antifungal agent comprising the compound according to any one of claims 1 to 4 or a pharmacologically acceptable salt thereof as an active ingredient. The slime bacterium belonging to the genus Enhygromyxa (genus Enhygromyxa) that can be separated from the soil in the vicinity of the coast is cultured in a culture solution to which a halogen compound is added, and extracted from the culture. A method for producing the compound described

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