JPH0432836B2 - - Google Patents

Description

[Detailed description of the invention]

[Object of the invention] <Industrial field of application> The present invention relates to a novel heptaene antibiotic V-28-3. The substance is effective as an antibiotic. <Prior Art> In recent years, bacterial diseases have become easily and effectively treated through the use of a large number of chemotherapeutic agents, mainly antibiotics. However, the use of these chemotherapeutic agents has caused a problem of deep mycosis caused by so-called fungal infections such as mold and yeast. <Problems to be Solved by the Present Invention> The problems to be solved by the present invention are to obtain a new antifungal agent having a new antibacterial activity that has stronger antibacterial activity against fungi. [Structure of the invention] <Means for solving the problem> In view of the above-mentioned circumstances, the present inventors isolated microorganisms from soil for the purpose of searching for a new antifungal agent that has stronger antibacterial activity against fungi. We investigated perilla cultures. As a result, the inventors discovered that Streptomyces arene V-28-3 produces a novel heptaene antibiotic, leading to the completion of the present invention. Heptaene antibiotic V-28-3 of the present invention (hereinafter referred to as
It is abbreviated as V-28-3. ) as microorganisms that produce
Streptomyces arene
arenae) V-28-3 (FERM BP-1537 (FERM
P-8099) is cited as an example. In identifying the mycological properties of Streptomyces arene V-28-3, the experimental method used was "Methods for Characterization of Streptomyces Species" by Shearling and Gottlieb (Int.J.
System . British Patent 719, 230
was used. The mycological properties of the microorganism Streptomyces arene V-28-3 are as follows. a. Color of the bacterial flora: Gray b. Form of spore-forming hyphae: Spiral c. Surface structure of spores: Spiny d. Production of melanin-like pigment. Positive e. Underside of basal hyphae.
Negative, or light yellow to reddish-brownf Diffusion into the medium Negative, or light browng Assimilation of carbon sources Glucose (+) Arabinose (+) Xylose (+) Inositol (+) Mannitol (+) Fructose (+) Rhamnose ( +) Sucrose (+) Raffinose (+) Salicin (+) Cellulose (-) Galactose (+) From the above results, the microorganism is identified as Streptomyces arene. The method of culturing this microorganism is in principle similar to that of general microorganisms, but deep culture using a liquid medium is usually advantageous. As a medium used for culture, antibiotic V
-28-3 It is sufficient that it contains a nutrient source that can be used by the producing bacteria. That is, glucose, fructose, starch, dextrin, etc. are used as carbon sources, and meat extract, casein, gluten, yeast extract, soybean flour, corn stave liquor, urea, ammonium sulfate, ammonium phosphate, etc. are used as nitrogen sources. used. In addition, inorganic salts such as sodium hydrogen phosphate, magnesium sulfate, and calcium carbonate can also be used as necessary. During culture, if foaming is severe, a small amount of silicone compound, higher alcohol, vegetable oil, etc. may be added. The culture temperature is preferably 20 to 35°C, most preferably around 27°C. The culture time is preferably 24 to 36 hours, but it can be changed as appropriate depending on the culture conditions. Since V-28-3 produced by culture accumulates outside and inside the bacterial cells, it is generally used to isolate antibiotics from the culture filtrate and bacterial cells separated by means such as centrifugation or filtration. Separated and purified by For example, solvent extraction using lower alcohols such as methanol and n-butanol, adsorption column chromatography using silica gel, diatomaceous earth, Avicel, alumina, etc., Toyo Pearl HW40
(Gel filtration carrier manufactured by Toyo Soda Co., Ltd.) etc., using octadodecylated (ODS) silica gel as the carrier. It is isolated and purified by performing purification methods such as reverse phase distribution column chromatography and HPLC, countercurrent distribution method, crystallization, recrystallization, etc. sequentially or in an appropriate combination. An example is as follows. Centrifuge the culture solution to collect bacterial cells, and add an appropriate amount of methanol, n
-It is extracted from the bacterial cells when extracted with lower alcohol such as butanol. In this case, extraction is easier with water-containing methanol, and alkaline water-containing methanol with aqueous ammonia is particularly desirable.On the other hand, V-28-3 in the culture supernatant is easily extracted with n-butanol. is extracted. When the solvent extract thus obtained is concentrated under reduced pressure, V-28-3 is precipitated as crude crystals. The crude crystals were dissolved in aqueous tetrahydrofuran, diluted with a 50% methanol solution to an appropriate concentration, and passed through an HP-20 column (Mitsubishi Chemical Industries, adsorption resin) equilibrated with 50% methanol. is adsorbed and eluted with 80% methanol solution containing 0.05% aqueous ammonia.
When the antibacterial active fractions against Candeida and Albicans HUT7501 are collected and concentrated under reduced pressure, yellow-brown crude crystals are precipitated. This precipitate was suspended in chloroform, and Hyflo Supercell (John-
Manville Co., diatomaceous earth) column (5 x 10
cm). After washing the column with a mixture of chloroform:methanol=1:1, acetonitrile:0.05M ammonium acetate (PH9.0)=1:1
Elute with a solution of When the antibacterial active fraction is collected and the solvent is removed under reduced pressure, V-28-3 becomes insoluble in water and precipitates. The precipitate was collected by centrifugation, dissolved in a small amount of aqueous tetrahydrofuran solution, and loaded onto a Whatmann LRP-1 (Whatmann C-18 reverse phase chromatography carrier) column. .0)=
When eluted with a 4.5:5.5 mixture, the antibacterial activity is separated into two parts and eluted. The active fraction eluted later is collected, concentrated under reduced pressure, and the solvent is gradually removed until the liquid becomes slightly cloudy. When this is left overnight at a low temperature, yellow crystals are precipitated. V-28-3 is isolated as yellow crystals by collecting yellow crystals by centrifugation, washing with water and acetone, and drying under reduced pressure. The physical and chemical properties of V-28-3 obtained by recrystallization are as follows. (1) Molecular weight: 1112 (MS) (2) Optical rotation [α] _D ²⁶ : +335° (C = 0.1%, DMSO) (3) Melting point: Browning at 170°C (4) UV spectrum: As shown in Figure 1 (Indicated by a solid line) (5) IR spectrum: As shown in Figure 2 (6) Solubility in solvents Insoluble in water, ethyl ether, hexane, chloroform, slightly soluble in methanol, ethanol, n-butanol, dimethyl sulfoxide , soluble in dimethylformamide. (7) Color reaction: Phenol sulfate: positive, ferric chloride: negative, ninhydrin: positive, concentrated sulfuric acid: positive,
Iodine: Positive. (8) ¹ H-NMR spectrum: As shown in Figure 3 (9) ¹³ C-NMR spectrum: As shown in Figure 4 (10) UVλ _MAX ^MoOH (ε): 404 (150, 380), 380
(130, 700), 361 (82, 900), 342 (44, 440) (11) Rf value: 0.23 (TLC plate: Watmann
PLKC18F, developing solvent: acetonitrile: 0.05M
Ammonium acetate = 4.5:5.5) Based on the above results, V-28-3 is a new substance represented by the following chemical structural formula. In addition, V
It was isolated and purified using the same method as -28-3, and its physicochemical properties were investigated. MW: 1113 (measured by MS), Rf value in TLC 0.34, retention time in HPLC 7.71 minutes, Particulate B (J.antibiotics 35 ,
No. 8, 997-1012 (1982)). Furthermore, since the ¹ H-NMR spectra of the two were completely identical, this product was identified as partolysin B. V-28-3, which has the above physical and chemical properties, is the first
It is clear from the UV spectrum etc. shown in the figure that this is an antifungal agent belonging to the heptaene antibiotics having seven conjugated double bonds. In addition to the above-mentioned particin B, heptaene antibiotics include particin A (MW: 1127) and amphotericin B (MW:
924), perimycin (MW: 1096), 67-121-A
(MW: 1128) 67-121-C (MW: 1218), Tricomycin A (MW: 1230), Candidein (MW: 921), Candidein (MW: 1167),
Mycoheptin A (MW: 939) and Reporin A ₂
(1108), etc., but V-28-3 differs from these heptaenes other than partolysin B in molecular weight. Comparing the physical and chemical properties of V-28-3 and Partolysin B, which have the same molecular weight, the first
As shown in the table, the Rf value in TLC, the retention time (RT) in HPLC, the value of UVλ _MAX , the value of [α], and the antibacterial activity against fungi described below are clearly different.

【table】

[Table] Furthermore, when the ¹ H-NMR spectrum of V-28-3 is compared with that of Partolysin B, there is good overall agreement, but a clear difference is seen between 5.5 and 6.6 ppm. <Effect> The antibacterial activity (MIC) of V-28-3 is shown in Table 2, and the MIC against Candida albicancans HUT7501 is extremely strong at 0.005 μg/ml, which is much higher than that of amphotericin B and particin B. Shows strong antibacterial activity.

[Table] In the experiment shown in Table 2, a suspension of the test bacteria was smeared on a Saburaud agar plate, cultured at 27°C for 2 to 4 days, and the growth inhibitory concentration was measured. (Mold: 27℃, 4
Judging from these results, V-28-3 is a heptaene antibiotic with a chemical structure similar to Partolysin B, and is similar to Partolysin B. is presumed to be a substance with different optical or steric structure. <Effects of the Invention> As shown in Table 3, V-28-3 has extremely strong antibacterial activity against yeasts such as Candeida, Cryptocacotucus, Rhodotorula and Satucharomyces, as well as filamentous fungi of Aspergillus, Penicillium, Mucor and Trichophyton. It can be used as a new antifungal agent. Hereinafter, the present invention will be explained in detail with reference to Examples. Example 100 ml of a medium having the composition shown in Table 4 was dispensed into a 500 ml flask and sterilized by heating at 120° C. for 10 minutes. In addition, Streptomyces arene V-28-3 (FERM BP-
1537 (FERM P-8099)) was inoculated into a platinum loop at 27°C.
Shaking culture was performed for 3 days. Table 4 Medium composition * Ingredient content (in 1.0) Soluble starch 30.0g Malt extract 6.0g Yeast extract 6.0g Polypeptone 12.0g Salt 5.0g MgSO ₄・7H ₂ O 0.5g *(PH: 7.0) On the other hand, a medium with the same composition 20 (antifoaming agent, silicone
KM-75 (supplemented with 10 ml) was poured into a 30-volume jar fermentor and sterilized by heating at 120°C for 10 minutes, then inoculated with the above seed culture solution and incubated at 27°C for 24 hours.
Time aeration agitation culture was performed. (Airflow rate 1/2V.V.
M.; stirring number 200 rpm). The culture solution was centrifuged (4000 rpm for 10 minutes), and 1.6 kg of bacterial cell cake was collected. 80% methanol to 500g of bacterial cake
After adjusting the pH to 9.5 by adding aqueous ammonia, extraction was performed at room temperature for 3 hours with gentle stirring. After lowering the pH of the extract to 7.0 and adding 1.5 of water, use Diaion HP-20 (Mitsubishi Kasei, adsorption carrier).
A column (10 x 30 cm) was loaded. After washing this column with a small amount of 50% methanol, it was eluted with 80% methanol containing 0.45% aqueous ammonia. Quillandida. The fractions having antibacterial activity against C. albicans HUT7501 were collected and concentrated under reduced pressure, and the resulting yellow-brown precipitate was collected by centrifugation, washed with cold acetone, and dried under reduced pressure to produce 520 mg of yellow-green powder.
I got mg. Suspend this powder in 100ml of chloroform and place it on a Hyflow Super Cellcam (5 x 20cm).
was loaded. Fill the column with 100ml of chloroform and 100ml of a mixture of chloroform:methanol = 1:1.
After sequentially washing with water, elution was performed with an eluent of acetonitrile:0.05M ammonium acetate (PH9.5) = 1:1. The active fraction was collected, the solvent was removed under reduced pressure, and the resulting precipitate was collected by centrifugation, washed with acetone, and dried under reduced pressure to obtain 420 mg of yellow powder.
Of this, 60 mg was taken and dissolved in 5.0 ml of a 66% aqueous tetrahydrofuran solution. This was loaded onto an LRP-1 column (Whatmann's C-18 reverse phase chromatography carrier) and eluted with an eluent of acetonitrile:0.05M ammonium acetate (PH9.0)=4.5:5.5. The antibacterial active substance was separated into two parts and eluted (the active substance eluted first was the ¹ H-
It was identified as the same substance as particin B from the NMR spectrum. ) The antibacterial active fraction eluted later was collected, the solvent was gradually removed under reduced pressure until a slight cloudiness occurred, and the mixture was stored at 50°C overnight to precipitate yellow crystals. These yellow crystals are collected by centrifugation,
After washing with cold water and cold acetone, dry under reduced pressure.
30 mg of yellow crystals were obtained. The UV spectrum, IR spectrum, ¹ H-NMR spectrum and ¹³ C-NMR spectrum of this yellow crystal are shown in FIGS. 1 to 4, respectively. Based on the above results, V-28
-3 was a new substance represented by the following chemical structural formula.

[Brief explanation of drawings]

In Figure 1 UV spectrum diagram, the solid line is V-28-3.
UV spectrum, dashed line indicates particin B.
Figure 2 shows the IR spectrum diagram of V-28-3,
Figures 3 and 4 are ¹ H-NMR and ¹³ C-NMR spectra of V-28-3 measured at 400 MHz. In the figure, the vertical axis represents the absorption intensity, and the horizontal axis represents the absorption intensity.
Chemical shift expressed in ppm.

Claims (1)

[Claims] 1. A novel substance V-28-3 represented by the following chemical structural formula.