JP4500937B2 - MMRC03-0001, a novel epidithiodiketopiperazine antibacterial antibiotic produced by sepedonium chlorinum - Google Patents

Description

  The present invention relates to a novel epidithiodiketopiperazine antibiotic produced by a microorganism belonging to the genus Cepedonium or a pharmacologically acceptable salt thereof, a method for producing the compound, an antibacterial agent comprising the compound as an active ingredient, and the like.

  Conventionally, antibiotics produced by microorganisms include β-lactam antibiotics such as penicillin, cephalosporin, carbapenem, macrolide antibiotics such as erythromycin, josamycin, and rokitamycin, and aminogcoside antibiotics such as kanamycin, gentamicin, and tobramycin Various antibiotics are known. These antibiotics and compounds obtained by improving them by chemical synthesis techniques have been utilized as clinically very useful antibiotics. However, since these antibiotics are frequently used in the treatment of various infectious diseases, pathogenic bacteria that are resistant to these antibiotics have emerged and become problematic. Therefore, in order to solve the problem of resistance of the pathogenic bacteria and to provide an even more effective and safe antibiotic, development of an antibiotic having new antibacterial activity and the like is required.

Among various antibiotics, epidithiodiketopiperazine antibiotics having an epidithio structure are known. For example, gliotoxin having antibacterial activity produced by microorganisms such as Gliocladium virens (Journal of General Microbiology, 134: 2067-2075,1988; JP-A-7-227293), Acetylgliotoxin (J. Amer. Chem. Soc., 75: 2110-2112, 1953) corresponding to the acetyl derivative of gliotoxin produced by microorganisms such as Dicotomomyces cejpii is known. ing. Among the epidithiodiketopiperazine antibiotics, acetylgliotoxin has also been found to have an antitumor activity (WO 98/24926), and substituted derivatives such as alkoxy groups and acyloxy groups of the acetyl groups of the compounds. Is synthesized.
JP-A-7-227293. WO 98/24926.

Non-patent document 1

  Journal of General Microbiology, 134: 2067-2075,1988.

Non-patent document 2

  J. Amer. Chem. Soc., 75: 2110-2112, 1953.

  An object of the present invention is to produce a novel antibiotic having antibacterial activity or a pharmacologically acceptable salt thereof, which is produced by a microorganism and can be used as a pharmaceutical, veterinary, marine, or agricultural or horticultural agent. It is in providing the manufacturing method of this compound, and the antibacterial agent etc. which use this compound as an active ingredient.

  In order to solve the above problems, the present inventor, as a result of earnest search, a strain belonging to the genus Sepedonium, which was newly isolated from the natural world by the present inventor, is a gram-positive bacterium, in particular, Micrococcus luteus (Micrococcus luteus) has been found to produce novel antibiotics with antibacterial activity against Gram-positive bacteria such as ATCC 9343 and has led to the completion of the present invention.

  That is, the present inventor isolated many microorganisms from plant specimens in the natural world and conducted various studies on their products, and as a result, separated from the fruit bodies of Iguchi's fellows collected at Kohoyama, Kanno City, Kanagawa Prefecture. Filamentous fungi have been found to produce new antibacterial antibiotics with antibacterial properties. The novel antibacterial antibiotic was named MMRC03-0001. The MMRC03-0001 antibiotic is an epidithiodiketopiperazine novel antibiotic having an epidithio structure, and the chemical structure of the antibiotic is represented by the following formula (1).

The antibiotic of the present invention comprises a compound represented by the structure and a pharmacologically acceptable salt thereof.

The microorganism that produces the antibiotic MMRC03-0001 of the present invention is a new strain belonging to the genus Sepedonium, and is named Sepedonium chlorinum TAMA73 strain, which is a patent organism of the National Institute of Advanced Industrial Science and Technology. Deposited at the deposit center as FERM P-19617. In order to produce the antibiotic MMRC03-0001 of the present invention, a microorganism having the ability to produce the antibiotic MMRC03-0001 of the present invention is cultured, and the antibiotic MMRC03-0001 is collected from the culture. Can do. The antibiotic of the present invention has a strong antibacterial activity especially against gram-positive bacteria such as micrococcus, and is effective for a wide range of antibacterial agents such as pharmaceuticals, veterinary drugs, marine products, agricultural and horticultural use. It can be used as a component.

  Specifically, the present invention specifically relates to the antibiotic MMRC03-0001 represented by the above chemical structural formula [Chemical Formula 1], or a pharmacologically acceptable salt thereof (Claim 1), and the following physicochemical properties: The antibiotic MMRC03-0001 according to claim 1 or a pharmacologically acceptable salt thereof (claim 2).
1) Molecular weight, molecular formula: molecular weight 326, molecular formula (C ₁₃ H ₁₄ N ₂ O _Four S ₂ )
2) Mass spectrum: FAB-MS (NBA); m / z 349 [M + Na] ⁺ , 327 [M + H] ⁺ , 262 [M-2S] ⁺
3) UV absorption spectrum: λ _max  nm (ε) in MeOH; 232 weak shoulder (4270)
4) Infrared absorption spectrum: ν _max  cm ^-1  in KBr; 3360, 1680, 1670, 1400, 1360, 1060, 1020, 875, 740, 620
5) Nuclear magnetic resonance spectrum: ¹ H-NMR (400 MHz, CDCl _Three ) ((ppm): 2.00 (3H, s), 2.83 (1H, m), 3.08 (3H, s), 3.60 (1H, m), 3.65 (1H, m), 4.38 (1H, m), 4.44 ( 1H, m), 4.63 (1H, m), 5.96 (1H, m); ¹³ C-NMR (100.5 MHz, CDCl _Three ) ((ppm): 18.3, 27.6, 37.7, 53.7, 55.9, 56.9, 62.2, 73.6, 74.2, 124.2, 134.4, 163.0, 165.8
6) Optical rotation: [α] _D -240 ((c = 0.38, CHCl _Three )
7) Solubility: Easily soluble in chloroform, ethyl acetate, acetonitrile, acetone, methanol, ethanol, dimethyl sulfoxide, hardly soluble in n-hexane, carbon tetrachloride and water.

  Further, the present invention relates to a pharmaceutical comprising the antibiotic MMRC03-0001 of claim 1 or 2 or a pharmacologically acceptable salt thereof (claim 3), or the antibiotic MMRC03 of claim 1 or 2. -0001, or an antibacterial agent comprising a pharmacologically acceptable salt thereof as an active ingredient (Claim 4) or the antibacterial agent is an antibacterial agent for medicine, veterinary medicine, fisheries, or agriculture and horticulture The antibacterial agent according to claim 4 (Claim 5) and a microorganism belonging to the genus Cepedonium and capable of producing the antibiotic MMRC03-0001, and collecting the antibiotic MMRC03-0001 from the culture The method for producing the antibiotic MMRC03-0001 according to claim 1 or 2 (Claim 6) or the ability to produce the antibiotic MMRC03-0001 belonging to the genus Sepedonium The method for producing the antibiotic MMRC03-0001 (Claim 7) or the antibiotic according to Claim 1 or 2, wherein the organism is Cepedonium chlorinum TAMA73 strain (FERM P-19617). Cepedonium chlorinum TAMA73 strain (FERM P-19617) (Claim 8) having the ability to produce MMRC03-0001.

  The novel antibiotics of the present invention have a strong antibacterial activity especially against gram-positive bacteria such as micrococcus, and are effective for a wide range of medicines, veterinary drugs, fisheries, agricultural and horticultural use. It can be used as an agent. Further, the novel antibiotic of the present invention can be produced by a simple and efficient method using the strain of the present invention.

  The present invention comprises a novel epidithiodiketopiperazine antibiotic MMRC03-0001 having strong antibacterial activity against gram-positive bacteria, and pharmacologically acceptable salts thereof. In order to obtain the antibiotic MMRC03-0001 of the present invention, a microorganism having the ability to produce the antibiotic MMRC03-0001 of the present invention belonging to the genus Sepedonium is cultured, and the antibiotic MMRC03-0001 is collected from the culture Can be done.

(Microorganism used in the present invention)
In the present invention, the microorganisms used for producing the antibiotics of the present invention are isolated from a large number of microorganisms from natural plant specimens, and as a result of various studies on their products, the present invention has antibacterial properties. This is a novel strain obtained by finding that it produces MMRC03-0001, a novel antibacterial antibiotic, and was identified as follows.

  The TAMA73 strain producing the antibiotic MMRC03-0001 of the present invention has the following mycological characteristics: growth spreads quickly on malt extract agar, Miura medium, oatmeal agar, etc., 25 ° C., 7 days The colony reaches 60 mm, but the flora is sparse, and when forming fluffy and thick-wall spores, the front and back surfaces are bright yellow. Conidia are formed on aerial hyphae, and phialides are regularly formed into several ring-like shapes, 11.9-70.6 × 2.3-4.0 μm (average 43.4 × 3.0 μm), L / W 3.8-24.7 (average 14.5), the tip is 1.9-2.3 μm in diameter, has an unclear color and forms conidia in a lump shape, the conidia is oval, Various shapes such as cylindrical, inverted egg shape, colorless, smooth, 1.9-13.4 × 1.4-4.5 μm (average 5.6 × 2.1 μm), L / W 1.04-6.18 (average) 2.77), thick-wall spores are formed on colorless rectangular cells lateral to the aerial hyphae or basal hyphae, and fallen oval, boat-shaped or elliptical with a pointed tip, golden color, and a remarkable vertical stripe pattern. 34.7-51.7 × 14.5-21.5 μm (average 42.8 × 17.4 μm), L / W 2.10-3.04 (flat 2.48) and a.

  This strain is obtained from a basidiomycetous fruiting body, grows fast, fluffy, bright yellow, colorless and regularly cyclized Verticillium type fiaro type conidia and bright yellow large allelo type conidia (thick wall) It is clear that it belongs to the genus Sepedonium from the formation of spores. Among the genera known as fungal parasites, the bacterium is easy to produce sepedonium chlorinum (Tul. & C. Tul.) Damon because it produces a thick-walled spore with an inverted egg shape and remarkable stripes. It is understood that it belongs to. Therefore, this strain was identified as Cepedonium chlorinum (Tul. & C. Tul.) Damon TAMA73. This strain has been deposited as FERM P-19617 at the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary.

  The TAMA73 strain has the property of being easily mutated, like general filamentous fungi. For example, all strains producing MMRC03-0001 can be used in the present invention, even the TAMA73 strain or a mutant strain, a zygote strain, or a gene recombinant derived from this strain. In addition, the teleomorph of Sepedonium chlorinum is said to be Hypomyces chlorinigenus Rogerson & Samuels. Therefore, not only cepedonium but also strains of the genus Hipomyces can be used in the present invention.

(Culture of microorganisms of the present invention, collection of antibiotics)
In order to produce the antibiotic MMRC03-0001 in the present invention, the microorganism of the present invention belonging to the genus Sepedonium and capable of producing the antibiotic MMRC03-0001 is cultured, and the antibiotic MMRC03-0001 is produced in the culture. ,accumulate. For the culture of MMRC03-0001 producing bacteria, a medium containing nutrients that can be used by ordinary microorganisms can be used. As the nutrient source, known ones conventionally used for culturing filamentous fungi can be used. For example, glucose, sucrose, glycerin, starch, maltose, lactose, molasses, animal and vegetable oils and fats, dextrin and the like can be used as the carbon source. As the nitrogen source, soybean flour, wheat germ, corn steep liquor, cottonseed meal, meat extract, yeast extract, peptone, nitrate or ammonium nitrogen can be used. If necessary, it is possible to add salts and inorganic salts containing metal ions. Organic or inorganic substances can be added to improve the growth of the producing bacteria or the production of MMRC03-0001.

As a culture method, a culture method under an aerobic condition, a solid stationary culture on a solid substrate, or a deep culture using a liquid medium is possible. A suitable temperature for culturing is 10 to 32 ° C., but usually culturing at 20 to 30 ° C. The number of culture days varies depending on the culture method and the type of medium, but the target substance MMRC03-0001 is produced in several days to several tens of days.
The following method is used for testing antibiotic MMRC03-0001. That is, MMRC03-0001 or purified MMRC03-0001 in an organic solvent crude extract such as n-butanol is detected from a culture solution by a biological method using the pathogenic bacterium Micrococcus luteus ATCC 9343 as a test bacterium. . Alternatively, MMRC03-0001 can also be detected by chemical methods such as high-performance liquid chromatography connected to a C18 reverse layer column and equipped with a photodiode array detector, or a high-performance liquid chromatograph / mass spectrometer directly connected to a mass spectrometer. It can be performed.

  In collecting the antibiotic MMRC03-0001 from the culture solution, an adsorbent such as activated carbon, alumina and silica gel, a gel filtration agent and the like can be used, but the following method is more effective: By extracting with n-butanol and concentrating the n-butanol layer under reduced pressure, an oily substance containing MMRC03-0001 is obtained. Furthermore, in order to remove substances other than the target product, the following pretreatment can be performed. That is, water is added to the n-butanol concentrate, followed by re-extraction with ethyl acetate, and the resulting ethyl acetate layer is again concentrated under reduced pressure to obtain an oily substance.

  Furthermore, a 90% methanol solution is added to the ethyl acetate concentrate, and an equal volume of n-hexane is added. After stirring, the 90% methanol solution is collected. Further, after adding water so that the 90% methanol solution layer becomes a 75% methanol solution, an equal volume of carbon tetrachloride is added, and after stirring, the 75% methanol solution layer is collected and concentrated under reduced pressure. A solid containing the product is obtained. This is adsorbed on a silica gel column, and the target product is eluted with a mixed solution of organic solvents such as n-hexane, acetone, ethyl acetate, and methanol. The target product is further subjected to thin layer chromatography, low pressure column chromatography, high performance liquid chromatography, etc. by combining various chromatographic carriers and solvents to isolate MMRC03-0001. The structure of the isolated compound is determined by detailed analysis of the various spectral properties of the compound, particularly its nuclear magnetic resonance, mass, ultraviolet and infrared spectra.

(Physicochemical properties of MMRC03-0001)
Antibiotic MMRC03-0001 has the following physicochemical properties:
1) Molecular weight: 326
2) Mass spectrum: FAB-MS (NBA); m / z 349 [M + Na] ⁺ , 327 [M + H] ⁺ , 262 [M-2S] ⁺
Molecular formula (C ₁₃ H ₁₄ N ₂ O ₄ S ₂ )
3) UV absorption spectrum: λ _max nm ( ε ) in MeOH; 232 weak shoulder (4270)
4) Infrared absorption spectrum: ν _max cm ⁻¹ in KBr; 3360, 1680, 1670, 1400, 1360, 1060, 1020, 875, 740, 620
5) Nuclear magnetic resonance spectrum: ¹ H-NMR (400 MHz, CDCl ₃ ) ((ppm): 2.00 (3H, s), 2.83 (1H, m), 3.08 (3H, s), 3.60 (1H, m) , 3.65 (1H, m), 4.38 (1H, m), 4.44 (1H, m), 4.63 (1H, m), 5.96 (1H, m); ¹³ C-NMR (100.5 MHz, CDCl ₃ ) ((ppm ): 18.3, 27.6, 37.7, 53.7, 55.9, 56.9, 62.2, 73.6, 74.2, 124.2, 134.4, 163.0, 165.8
6) Optical rotation: [α] _D -240 ((c = 0.38, CHCl ₃ )
7) Solubility: Soluble in chloroform, ethyl acetate, acetonitrile, acetone, methanol, ethanol, dimethyl sulfoxide, insoluble in n-hexane, carbon tetrachloride, water 8) Chemical structural formula: As described above (MMRC03-0001 Use as an antibacterial agent)
The antibiotic MMRC03-0001 of the present invention can be used as a wide range of antibacterial agents such as pharmaceuticals, veterinary drugs, fisheries, agricultural and horticultural use.
When the antibiotic of the present invention is used as an active ingredient of an antibacterial agent, MMRC03-0001 of the present invention is purified from the culture obtained by culturing the MMRC03-0001 producing strain of the present invention by the above-described means. Can be used. Further, depending on the field of use of the antibacterial agent, it can be used in the state of a roughly purified product.

  When the antibiotic MMRC03-0001 of the present invention is used as a medicine or the like, the compound can be used in the form of a pharmacologically acceptable salt. As such pharmacologically acceptable salts, those known in pharmacology can be used. For example, pharmacologically acceptable metal salts, ammonium salts, containing amine addition salts, amino acid addition salts, and the like can be used. it can. Examples of the metal salt include alkali metal salts such as lithium salt, sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt, zinc salt and the like, and organic amine addition salt includes morpholine, Examples of addition salts such as piperidine and amino acid addition salts include addition salts such as glycine, phenylalanine, aspartic acid, glutamic acid, and lysine. In order to administer the antibiotic MMRC03-0001 of the present invention or a pharmacologically acceptable salt of the compound as a pharmaceutical, the active ingredient is formulated into an appropriate form for pharmacological use, and systemically or locally. In addition, it can be administered orally or parenterally. Examples of the dosage form include tablets, powders, granules, syrups, injections, solutions, emulsions, suspensions, ointments and the like. In the formulation, for example, various excipients, lubricants, binders, disintegrants, suspending agents, auxiliary agents such as emulsifiers can be used.

  When the antibiotic MMRC03-0001 of the present invention is used as an antibacterial agent for agricultural or horticultural use, the compound or a salt thereof is formulated with an appropriate auxiliary agent, for example, powder, granule, emulsifier, hydration It can be used in the form of an agent. Further, when the antibiotic MMRC03-0001 of the present invention is used as an antibacterial agent for veterinary drugs, the compound or a salt thereof in an appropriate dosage form, veterinary medicine, or a form in which the drug is mixed with feed or the like Can be administered. Further, when the antibiotic MMRC03-0001 of the present invention is used as an antibacterial agent for fisheries or the like, the compound or a salt thereof is formulated using a conventional diluent, carrier, extender, additive, etc. It can be used for antibacterial treatment such as water for fish farming. In addition, the antibacterial agent can be used by adding to fish feed and the like. Further, the antibacterial agent can be formulated in the form of a coating agent and used for antibacterial treatment of fishing equipment such as fish nets and fishing facilities.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

(Preparation and biological test of antibiotic MMRC03-0001)
Cepedonium chlorinum TAMA73 strain was cultured on a malt extract agar slant medium at 25 ° C. for 10 days. The mycelium obtained by this slope culture was used in a 20 ml production medium (glucose 2%, glycerin 3%, polypeptone (Nippon Pharmaceutical) 0.5%, yeast extract (Nippon Pharmaceutical) 0.2%, salt 0.3%, The cells were inoculated into 50 250 ml Erlenmeyer flasks containing 1% of calcium carbonate, and subjected to rotary shaking culture at 25 ° C. and 220 rpm. The pH of the culture increased with the culturing, and at the same time, the productivity of MMRC03-0001 reached its peak in 12 days. Thus, 1000 ml of a culture solution containing about 21 mg of MMRC03-0001 was obtained. 750 ml of n-butanol was added to the culture on the 12th day, stirred, and stored overnight in a refrigerator (5 ° C.). Thereafter, n-butanol was collected and concentrated under reduced pressure to obtain 2.3 g of an oily substance.

  The same amount of water as 250 ml ethyl acetate was added to the oily substance and stirred, and then the mixture was separated into an ethyl acetate layer and an aqueous layer. Further, 250 ml of ethyl acetate was added to the aqueous layer, and after stirring, the mixture was again divided into an ethyl acetate layer and an aqueous layer. The ethyl acetate layers were combined and concentrated under reduced pressure to obtain 1.9 g of an oily substance. The same amount of n-hexane as 250 ml of 90% methanol solution was added to the oily substance, and after stirring, it was divided into an n-hexane layer and a 90% methanol solution layer. 50 ml of water was added to the obtained 250 ml of 90% methanol solution layer, 300 ml of carbon tetrachloride was further added, and the mixture was divided into a 75% methanol solution layer and carbon tetrachloride. The obtained 300 ml of 75% methanol solution was concentrated under reduced pressure to obtain about 300 mg of a solid. The solid matter was dissolved in a small amount of ethyl acetate, adsorbed on a column (outer diameter 2.5 cm × length 20 cm) previously packed with silica gel, and stepped into ethyl acetate in an n-hexane: ethyl acetate system. The target substance was eluted by elevating it to a high level. As a result, a 400 ml fraction containing MMRC03-0001 was obtained.

  The fraction was concentrated under reduced pressure to obtain 126 mg of a solid containing MMRC03-0001. The obtained solid was dissolved in 2 ml of a mixture of chloroform: methanol = 1: 1, and 1 ml of the solid was connected to a reverse phase column (Shiseido Capsule Pack C18, SG120, 5 μm, outer diameter 20 mm × length 250 mm). Sorting was performed at Elution was performed by using a system of acetonitrile: water as the moving bed solvent and linearly increasing acetonitrile from 5% to 50% in 60 minutes at a flow rate of 7 ml / min. The desired MMRC03-0001 eluted at 41 minutes, yielding 21 ml fractions containing MMRC03-0001. The fraction was concentrated under reduced pressure to obtain 26 mg of a solid.

  The solid matter was dissolved in 1 ml of a mixture of chloroform: methanol = 1: 1 and re-sorted by a high performance liquid chromatograph connected with a reverse phase column (Shiseido Capsule Pack C18, SG120, 5 μm, outer diameter 20 mm × length 250 mm). went. As the moving bed solvent, acetonitrile: water system was used, and acetonitrile was linearly increased from 5% to 30% in 60 minutes at a flow rate of 7 ml / min, held at 30% for 30 minutes, and then from 30% to 50 in 30 minutes. Elution was carried out with a linear increase to%. The desired MMRC03-0001 eluted at about 58 minutes, yielding a 21 ml fraction containing MMRC03-0001. The fraction was concentrated under reduced pressure to obtain 8 mg of amorphous white powder.

In assay medium 1ml containing various amounts of the resulting MMRC03-0001, inoculated with 10 ⁴ cells of Micrococcus luteus ATCC9343, after 2 days of culture at 37 ° C., by measuring the respective turbidity, micro The minimum inhibitory concentration (MIC ₁₀₀ ) for Coccus luteus ATCC 9343 was determined. As a result, MIC ₁₀₀ against Micrococcus luteus ATCC9343 of MMRC03-0001 was 2.5 [mu] g / ml. Thus, since the antibacterial agent MMRC03-0001 according to the present invention exhibits excellent antibacterial activity against Gram-positive bacteria, particularly Micrococcus luteus ATCC 9343, effective therapeutic agents, disinfectants, and animals for Gram-positive bacterial infections It becomes medicine, marine chemicals, and agricultural chemicals.

(Production of antibiotic MMRC03-0001)
Well-grown Cepedonium chlorinum TAMA73 strain malt extract agar slope culture 1 platinum ear mycelium in 70 ml seed medium (glycerin 2%, glucose 0.5%, soy flour 1%, yeast extract 0.2%, salt Five 500 ml Erlenmeyer flasks containing 0.25% and calcium carbonate 0.4%) were inoculated and cultured at 25 ° C. and 220 rpm for 3 days on a rotary shaker. 350 ml of the obtained seed culture was used as a seed mother for 50 L jar culture. Jar culture is 30L of main culture medium (glycerin 2%, glucose 0.5%, soybean powder 2%, yeast extract 0.2%, salt 0.25%, calcium carbonate 0.4%, antifoam (CC- Inoculated into one 50L jar fermenter containing 438, Japanese fat and oil) 0.03%), 27 ° C, air flow rate 15L / min (0.5VVM), pressure 0.5kg / cm ² · G, initial stirring Incubation was started at 200 rpm, and stirring was adjusted to a dissolved oxygen content of 10% or more between 200 and 600 rpm. Although the pH immediately after the start of the culture was around 7, it reached 8.0 after 72 hours, and the amount of cells was almost in a steady state. After culturing for 100 hours, the pH became 8.5, and 27 L of a culture solution containing 710 mg of MMRC03-0001 was obtained.

  The obtained culture solution was filtered under reduced pressure using a Buchner funnel, and divided into about 5 kg of cake and 27 L of filtrate. The obtained cake was extracted with 10 L of methanol and separated into a methanol solution and cells. On the other hand, the culture broth was extracted with 27 L of ethyl acetate. The methanol extract of the obtained cake was concentrated under reduced pressure, 10 L of ethyl acetate in the same amount of water was added and stirred, and then the mixture was separated into an ethyl acetate layer and an aqueous layer. The obtained ethyl acetate layer was combined with the ethyl acetate extract of the culture broth and concentrated under reduced pressure to obtain about 63 g of an oily substance. The same amount of n-hexane as 7.5 L of 90% methanol solution was added to the oily substance, and after stirring, it was divided into an n-hexane layer and a 90% methanol solution layer. 1.5 L of water was added to the obtained 7.5 L of 90% methanol solution layer, and 9 L of carbon tetrachloride was further added to separate into 75% methanol solution layer and carbon tetrachloride.

  The obtained 9 L of 75% methanol solution was concentrated under reduced pressure to obtain about 11 g of a solid. The obtained solid was dissolved in 40 ml of ethyl acetate, adsorbed on a column (outer diameter 8 cm × length 34 cm) previously packed with silica gel, and stepped with ethyl acetate in a system of n-hexane: ethyl acetate. And the target substance was eluted. As a result, a 8.5 L fraction containing MMRC03-0001 was obtained. The fraction was concentrated under reduced pressure to obtain about 3.8 g of a solid containing MMRC03-0001.

The obtained solid was dissolved in 43 ml of a mixture of chloroform: methanol = 1: 1, and the solution was adsorbed on a column filled with 500 g Wakogel 50C18 with a 10% acetonitrile aqueous solution. Of MMRC03-0001 was eluted. As a result, a 0.6 L fraction containing MMRC03-0001 was obtained. The fraction was concentrated under reduced pressure to obtain about 1.6 g of a solid containing MMRC03-0001. The obtained solid was dissolved in 10 ml of a mixture of chloroform: methanol = 1: 1, and the solution was adsorbed onto a column filled with 200 g Wakogel 50C18 with a 10% acetonitrile aqueous solution, and acetonitrile was added to 10% in 8 hours at a flow rate of 8 ml / min. The target substance MMRC03-0001 was eluted by linearly increasing from 50 to 50%. As a result, 240 ml of a fraction containing MMRC03-0001 was obtained. The fraction was concentrated under reduced pressure to obtain 524 mg of amorphous white powder of MMRC03-0001.

Claims (8)

Formula (1)

Or an pharmacologically acceptable salt thereof. The antibiotic MMRC03-0001 or a pharmacologically acceptable salt thereof according to claim 1, which has the following physicochemical properties.
1) Molecular weight, molecular formula: Molecular weight 326, molecular formula _{(C 13 H 14 N 2 O} 4 S 2)
2) Mass spectrum: FAB-MS (NBA); m / z 349 [M + Na] ⁺ , 327 [M + H] ⁺ , 262 [M-2S] ⁺
3) UV absorption spectrum: λ _max nm ( ε ) in MeOH; 232 weak shoulder (4270)
4) Infrared absorption spectrum: ν _max cm ⁻¹ in KBr; 3360, 1680, 1670, 1400, 1360, 1060, 1020, 875, 740, 620
5) Nuclear magnetic resonance spectrum: ¹ H-NMR (400 MHz, CDCl ₃ ) ((ppm): 2.00 (3H, s), 2.83 (1H, m), 3.08 (3H, s), 3.60 (1H, m) , 3.65 (1H, m), 4.38 (1H, m), 4.44 (1H, m), 4.63 (1H, m), 5.96 (1H, m); ¹³ C-NMR (100.5 MHz, CDCl ₃ ) ((ppm ): 18.3, 27.6, 37.7, 53.7, 55.9, 56.9, 62.2, 73.6, 74.2, 124.2, 134.4, 163.0, 165.8
6) Optical rotation: [α] _D -240 ((c = 0.38, CHCl ₃ )
7) Solubility: Easily soluble in chloroform, ethyl acetate, acetonitrile, acetone, methanol, ethanol, dimethyl sulfoxide, hardly soluble in n-hexane, carbon tetrachloride, water   A pharmaceutical comprising the antibiotic MMRC03-0001 according to claim 1 or 2 or a pharmacologically acceptable salt thereof as an active ingredient.   The antibacterial agent which uses the antibiotic MMRC03-0001 of Claim 1 or 2 or its pharmacologically acceptable salt as an active ingredient.   The antibacterial agent according to claim 4, wherein the antibacterial agent is an antibacterial agent for medicine, veterinary medicine, fisheries, or agriculture and horticulture. An antibiotic MMRC03-0001, wherein the microorganism MMRC03-0001 belongs to the genus Cepedonium and has the ability to produce the antibiotic MMRC03-0001 according to claim 1 or 2, and the antibiotic MMRC03-0001 is collected from the culture. Manufacturing method. The antibiotic according to claim 6, wherein the microorganism belonging to the genus Sepedonium and having the ability to produce the antibiotic MMRC03-0001 according to claim 1 or 2 is Cepedonium chlorinum TAMA73 strain (FERM P-19617). Production method of the substance MMRC03-0001. Cepedonium chlorinum TAMA73 strain (FERM P-19617) having the ability to produce the antibiotic MMRC03-0001 according to claim 1 or 2 .