JPH05117298A - Depsipeptide a and b, its production, anti-virus agent and antibacterial agent - Google Patents

Abstract

PURPOSE:To efficiently obtain the subject antibiotics showing an anti-virus effect and an antibacterial effect and useful, e.g. as a drug for improving AIDS and hepatitis by culturing a depsipeptide A, B-producing bacterium belonging to Streptomyces genus and subsequently collecting the products from the cultured material. CONSTITUTION:A depsipeptide A and B-producing bacterium [e.g. Actinomyces RK-1051 strain (FERM P-11624)] belonging to Streptomyces genus is inoculated and cultured in a culture medium in a jar fermenter and the cultured solution is subjected to centrifugation so as to separate the cell bodies and a supernatant. The separated cell bodies are immersed in 70% acetone solution overnight so as to be lysed and the residues are subsequently removed by centrifugation. The acetone solution is concentrated under a reduced pressure and mixed with the supernatant and the resultant mixture is extracted with ethyl acetate. The obtained extract is concentrated under a reduced pressure and treated with silica gel chromatography. The active fraction is then treated with ODS column chromatography to isolate two fractions and the respective isolated fractions are purified by high-performance liquid chromatography, thus obtaining the objective depsipeptide A and B of formulae I (Me is methyl) and II.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antibiotic depsipeptide and a method for producing the same. The present invention also relates to an antiviral agent and an antibacterial agent containing the antibiotic as an active ingredient.

[0002]

2. Description of the Related Art Recently, it has been pointed out that diseases such as AIDS and hepatitis caused by various viruses are increasing. For the treatment of these viral diseases, antiviral agents such as azidothymidine are used, but all of them have problems of insufficient antiviral action and strong toxicity.

[0003]

DISCLOSURE OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION AND MEANS FOR SOLVING THE PROBLEMS The present inventor has aimed to provide an antiviral agent having an excellent antiviral action, and an antibiotic produced by a microorganism isolated from soil. While searching for substances, we found depsipeptides A and B as antibiotics having particularly excellent antiviral action, and completed the present invention. Further, the present inventor has found that the depsipeptides A and B have excellent antibacterial action and antitumor action, and completed the present invention. That is, the present invention relates to the antibiotic depsipeptide A.
And B, a method for producing antibiotic depsipeptides A and B,
The present invention provides an antiviral agent and an antibacterial agent containing the antibiotic depsipeptide A or B as an active ingredient.

The antibiotic depsipeptide A is an antibiotic represented by the following structural formula and has the following physicochemical properties.

[0005]

[Chemical 3]

Properties: Yellow-orange crystal Melting point: 203-220 ° C. Molecular formula: C ₄₇ H ₅₇ N ₇ O ₁₁ Molecular weight (SI-MS, m / z): 896 (MH ⁺ ) Elemental analysis: C: 60.17% H: 6.24 % N: 10.44% Specific rotation: [α] ²² _D = + 270 ° (c 0.45 chloroform) UV absorption spectrum (1% 1,4-dioxane-H ₂ O): λ
_max nm (ε) = 257 (16,110), 354 (sh37,321), 369 (49,
225), 387 (43,855) Infrared absorption spectrum: 3350, 1730, 1620, 1590, 150
0, 1430, 1250, 1100, 1000 Solubility: Easily soluble in chloroform and dimethylsulfoxide Insoluble in methanol Insoluble in water Acid hydrolysis: Alanine, proline, phenylalanine, serine, N-methylalanine, 4-methylproline Substance Depsipeptide B is an antibiotic represented by the following structural formula and has the following physicochemical properties.

[0007]

[Chemical 4]

Properties: Yellow-orange crystal Melting point: 182-190 ° C. Molecular formula: C ₄₆ H ₅₅ N ₇ O ₁₁ Molecular weight (SI-MS, m / z): 882 (MH ⁺ ) Elemental analysis: C: 60.08% H: 6.14 % N: 10.88% Specific rotation: [α] ²² _D = + 211 ° (c 0.45 chloroform) UV absorption spectrum (1% 1,4-dioxane-H ₂ O): λ
_max nm (ε) = 257 (15,417), 354 (sh33,478), 369 (45,
503), 387 (40,526) Infrared absorption spectrum: 3300, 1730, 1650, 1640, 155
0, 1500, 1440, 1350, 1250, 1100, 1000 Solubility: Easily soluble in chloroform and dimethylsulfoxide Insoluble in methanol Insoluble in water Acid hydrolysis: Alanine, proline (2), phenylalanine, serine, N-methylalanine, Antibiotic depsipeptides A and B were isolated from a culture obtained by culturing RK-1051, a microorganism belonging to the genus Streptomyces, which was isolated from soil collected from Tsuruoka City, Yamagata Prefecture. RK-1051 is a microorganism having the following mycological properties. 1. Morphological characteristics This strain was cultivated in a liquid medium containing 1% yeast extract and 1% glucose, and the cells were grown at 110 ° C with 6N hydrochloric acid.
After hydrolysis for 18 hours at 37 ° C., analysis by thin layer chromatography revealed the detection of L, L-diaminopimelic acid. In the micrograph of the product grown on an agar plate medium, the aerial hyphae had a spiral shape. The formation of pseudosporangia was observed. 2. Growth condition on various media (20 days culture at 27 ° C, color harmony manual by US container company) (1) Starch-Yeast agar medium Growth: Good Aeromycelia: Rich Aerobic mycelial color: Ash (5fe) Backside color: Dark olive (1 1 / 2nl) Soluble pigment: None (2) Yeast extract / malt extract agar medium Growth: Good Aerial mycelium: Rich Aerial mycelial thread Color: Covert gray (2fe) Backside color: Dusty yellow (1. 1 / 2gc) Soluble pigment: None (3) Oatmeal agar medium Growth: Good Aerial mycelium: abundant Aerial mycelium color tone: beige gray (3ih) Back color tone: dark brown (4nl) Soluble pigment: none (4) Starch / inorganic salt agar Medium Growth: Normal Aerial mycelia: Low Aerobic mycelia Color: Beige Gray (3ih) Back color: Dark brown (4nl) Soluble pigment: None (5) Sucrose nitrate agar medium Growth Good aerial mycelia: None Aerial mycelial color: − Back side color: colorless Soluble pigment: None (6) Glucose / asparagine agar medium Growth: Good Aerial mycelium: rich Aerial mycelial color: beige gray (3ih) Back side color: dark olive (1. 1 / 2nl) Soluble pigment: None (7) Glycerol-asparagine agar medium Growth: Good aerial mycelium: a lot Airborne hyphae color tone: white Backside color tone: colorless Soluble pigment: none (8) V8 juice agar medium growth: good aerial mycelium: many Aerial mycelia color tone: white Backside color tone: Mustard Gold (2ne) Soluble pigment: None (9) Potato carrot agar medium Growth: Good Aerobic mycelia: a lot of aerial mycelial tone color: White Backside color tone: colorless Soluble pigment: None 3. Sugar Utilization D-Glucose Growth: Good D-Fructose Growth: Normal L-Rhamnose Growth: Not Raffinose Growth: Good D-Xylose Growth: Normal L-A Binosu Growth: Good sucrose Growth: Good inositol Growth: Good D- mannitol Growth: the properties of good above, RK-1051 fungus is clear that a microorganism belonging to Streptomyces (Streptomyces) genus. The RK-1051 strain has been deposited at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology on July 24, 1990 under the accession number Microindustrial Research Institute No. 11624 (FERM P-11624).

The depsipeptides A and B of the present invention are produced by inoculating a nutrient source-containing medium with the above strains and aerobically culturing. The bacteria producing the depsipeptides A and B are not limited to the above strains, and any bacteria belonging to the genus Streptomyces and capable of producing the depsipeptides A and / or B can be used in the present invention. In principle, the method for culturing the above-mentioned microorganism is based on the method for culturing a general microorganism, but it is usually preferable to perform the shaking culture method by liquid culture or the aeration stirring culture method. The medium used for culture may be any medium containing a nutrient source that can be used by microorganisms belonging to the genus Streptomyces, and various synthetic media, semi-synthetic media, natural media and the like can be used.
As a medium composition, glucose as a carbon source, sucrose, fructose, glycerin, dextrin, starch, molasses, corn steep liquor, organic acid and the like can be used alone or in combination. As the nitrogen source, peptone, meat extract, yeast extract, soybean powder, casein, amino acids, organic nitrogen sources such as urea, and inorganic nitrogen sources such as sodium nitrate and ammonium sulfate can be used alone or in combination. Sodium salt, potassium salt, magnesium salt, phosphate, and other heavy metals may be added and used as necessary. When foaming is remarkable during culturing, a known antifoaming agent such as adecanol or silicone oil can be appropriately added to the medium. The pH of the medium is preferably in the optimum pH range of the microorganism, usually around neutral. The medium temperature is a temperature at which microorganisms grow well, usually 20 to 30 ° C, preferably 28 ° C.
It is good to keep it near. The culture time is generally about 3 to 6 days. Although the depsipeptides A and B are accumulated in the medium by the above-mentioned culture, these culture conditions can be appropriately changed depending on the type and characteristics of the microorganism used, external conditions and the like.

The isolation of the depsipeptides A and B produced by the above-mentioned culture is carried out by collecting the fermentation product after the accumulation of the antibiotic depsipeptides A and / or B in the medium, preferably when the accumulation becomes maximum. According to the general method described above, various means such as salting-out method, extraction method, various gel chromatography, ion-exchange chromatography, adsorption chromatography and the like may be used alone or in combination. The accumulation of depsipeptides A and / or B may be detected by a bioassay method or the like. More specifically,
Since the antibiotic depsipeptides A and B produced by the above-mentioned culture are present in both the microbial cells and the medium, after the culture filtrate and microbial cell solids are separated by means such as filtration and centrifugation, The depsipeptides A and / or B may be extracted with a solvent such as methanol or acetone, and the aqueous solution from which the organic solvent has been removed may be extracted with ethyl acetate, chloroform or the like to obtain an extract containing the desired product. Furthermore, an extract containing the desired product may be obtained from the culture broth by a method such as extraction with ethyl acetate. Then, the extract containing the desired product may be concentrated and then purified by silica gel column chromatography, chromatography using a Sephadex LH-20 column or the like. Moreover, the separation of the depsipeptides A and B can be efficiently performed by a method such as silica gel column chromatography or HPLC.

Acute toxicity of depsipeptides A and B (LD
₅₀ , intraperitoneal administration) is 200 mg / kg when using ICR mice, and toxicity is low. Further, since the depsipeptides A and B have antiviral and antibacterial actions, the pharmaceutical composition containing the depsipeptide A or B as an active ingredient is useful as an antiviral agent and an antibacterial agent. Examples of the pharmaceutical composition include tablets, capsules, powders as a pharmaceutical composition for oral administration, and injections, suppositories, external preparations, etc. as a pharmaceutical composition for parenteral administration. When manufacturing pharmaceutical compositions, excipients (lactose, D-mannitol, starch, crystalline cellulose, etc.), disintegrants (carboxymethylcellulose, carboxymethylcellulose calcium, etc.), binders (hydroxyl) are used for oral agents and suppositories. Formulation components such as propylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, etc., lubricants (magnesium stearate, talc, etc.) coating agents (hydroxypropylmethylcellulose, sucrose, etc.), bases (polyethylene glycol, hard fat, etc.), For injections, eye drops, and ear drops, solubilizers or solubilizers (distilled water for injection, physiological saline, propylene glycol, etc.), pH adjusters (inorganic or organic acids or bases), isotonic agents For external preparations, formulation ingredients such as Formulation components (white petrolatum, macrogol,
In general, in the case of oral administration as depsipeptide A or B in which glycerin etc.) is used, a daily dose of 1 to 5,000 mg, preferably 10 to 1,000 mg may be administered in 2 to 5 divided doses per day. The dose should be adjusted according to the route of administration, the disease to be treated, the symptoms, etc.

[0012]

EXAMPLES The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. Example 1 Production of Depsipeptide A Using Actinomycete RK-1051 Strain Actinomycete RK-1051 strain was used in a 30-liter jar fermenter in a medium of 18 liters (glucose 20 g, starch 10 g, meat extract 1 g, dry yeast 4 g, salt 2 g,
After culturing with 25 g of soybean powder, 1 liter of water, pH 7.3), the cells were separated by centrifugation into culture supernatant. The cells were immersed in 10 liters of 70% acetone solution overnight to lyse them, and the residue was removed by centrifugation. The acetone solution was concentrated under reduced pressure to give an aqueous solution, which was then combined with the culture supernatant. When this was extracted with an equal amount of ethyl acetate, the active ingredient was recovered in the ethyl acetate layer. The solvent was concentrated under reduced pressure and then subjected to silica gel column chromatography (column diameter 1
2 cm x 60 cm long). Chloroform / methanol (2
After washing with a solvent system of 0: 1), the ratio of methanol was sequentially increased to 20: 1 → 10: 1 → 5: 1. The obtained active ingredient was concentrated and then subjected to ODS column chromatography (column diameter 8 cm × length 40 cm). The active fraction was eluted when the ratio of methanol was increased from a solvent system of 60% methanol to 100%. From the fraction eluted with 80% of methanol, about 25 mg of crude crystals of depsipeptide A was obtained.

Process for the production of B The last "about 25% from the fraction eluted with 80% methanol.
Crude crystals of mg of Depsipeptide A were obtained. After that, a fraction containing depsipeptide B was continuously obtained in that fraction, but since depsipeptide A also coexists in this fraction, both fractions were subjected to HPLC (column: Senshupack N solvent: methanol / acetonitrile).・ Water ・ 0.1 M diethylamine buffer (adjusted to pH 7.0 with formic acid) = 7: 0.5: 1.
Separated 5: 1).

The total ratio of depsipeptides A and B obtained from 18 liters of culture is about 20: 1.
106 mg and B 5 mg were obtained. Example 2 Antiphage activity of Depsipeptide A The following experiment was carried out using Phage B, which lyses the actinomycete Streptomyces griseus, as a bacteriophage that is a virus that proliferates using Pacteria as a host. Experimental method: 18 g of ordinary broth (manufactured by Eiken), 2 g of yeast extract (manufactured by Difco), 1 agar per liter of water
An agar plate containing 5 g of the component was prepared, and Streptomyces griseus and phage B were layered on the upper layer in the same medium. The assay sample was impregnated in an 8 mm diameter paper disk and placed on an agar plate. After incubating at 28 ° C. for 48 hours, the presence or absence of growth of actinomycetes that blocked phage plaque around the paper disc was determined. Results: Concentration of depsipeptide A (μg / disc) 160 80 40 20 10 5 2.5 ++++++++++++++ indicates strength of antiphage activity ++++: Good growth of actinomycetes, no plaques +: Plaques -: No antiphage activity Example 3 Antiphage activity of Depsipeptide B 160 80 40 20 10 5 2.5 +++++++++++++ ± -Example 4 Antibacterial activity of Depsipeptide A and B Experimental method: Gandida -Albicans was cultured in Sabouraud medium, Pyricularia oryzae was cultured in potato sucrose medium, and bacteria (Escherichia coli, Staphylococcus aureus Pseudomonas aeruginosa) were cultured in nutrient medium.

A dilution series was prepared in each medium containing depsipeptide A or B and used as an agar plate. Apply fungi and bacteria to this, fungus at 28 ℃ for 48 hours, bacteria at 37 ℃ 2
After culturing for 4 hours, the presence or absence of bacterial growth was determined to determine the minimum inhibitory concentration (MIC, μg / ml). Results: Bacteria Species Dept. 100 50 Staphylococcus pyogenes Cook 3.13 100 0.2 Streptococcus faecalis 1373 25 ＞ 100 25 Escherichia coli NIHJ JC-2 ＞ 100 ＞ 100 0.2 Escherichia coli ＞ 4 100 0.1 Pseudomonas aeruginosa PAO-1>100> 100 0.78 Pyricularia oryzae IFO 5994>100>100> 100 Candida albicans IFO 1594>100>100> 100

[Brief description of drawings]

FIG. 1 is a diagram showing an infrared absorption spectrum of depsipeptide A.

FIG. 2 is a diagram showing an infrared absorption spectrum of depsipeptide B.

FIG. 3 is a diagram showing an ultraviolet absorption spectrum of depsipeptide A.

FIG. 4 is a diagram showing an ultraviolet absorption spectrum of depsipeptide B.

FIG. 5 is a diagram showing an NMR spectrum of depsipeptide A.

FIG. 6 is a diagram showing an NMR spectrum of depsipeptide B.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display area C07K 99:00

Claims (8)

[Claims] 1. An antibiotic depsipeptide A represented by the following formula: [Chemical 1] 2. An antibiotic depsipeptide B represented by the following formula: [Chemical 2] 3. The method for producing depsipeptide A according to claim 1, which comprises culturing a depsipeptide A-producing bacterium belonging to the genus Streptomyces and collecting the depsipeptide A from the culture. 4. The method for producing depsipeptide B according to claim 2, which comprises culturing a depsipeptide B-producing bacterium belonging to the genus Streptomyces and collecting the depsipeptide B from the culture. 5. An antiviral agent containing the depsipeptide A according to claim 1 as an active ingredient. 6. An antiviral agent containing the depsipeptide B according to claim 2 as an active ingredient. 7. An antibacterial agent containing the depsipeptide A according to claim 1 as an active ingredient. 8. An antibacterial agent containing the depsipeptide B according to claim 2 as an active ingredient.