JPH05148230A - New glutarimide-based antibiotic having antiviral activity and its production - Google Patents

Abstract

PURPOSE:To provide the subject new antibiotic, an antiviral antibiotic produced by Stereptomyces microorganisms, giving high antiviral activity against virus such as influnza virus or herpes simplex virus. CONSTITUTION:A new glutarimide-based antiviral abtibiotic of the formula. This antibiotic can be obtained by calturing antiviral antiobiotic AH-135 Y- productive bacteria belonging to Stereptomyces (FERM P-12603) (e.g. Stereptomyces albovinacesous AH-135 strain) and by separating and collecting the objective substance from the resulting caltured product.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a novel antiviral substance A
The present invention relates to H-135Y, a method for producing the same, and an antiviral agent containing the H-135Y as an active ingredient. Substance of the invention AH-135Y
Is a novel antibiotic having an antiviral action which has not been described in the literature, which is isolated and collected from the culture by culturing an antiviral substance AH-135Y-producing bacterium belonging to the genus Streptomyces.

[0002]

Background of the Invention and Problems to be Solved by the Invention The present inventors have extensively studied microbial cultures for the purpose of searching for new antiviral agents, and as a result, Streptomyces (S
Microorganisms belonging to the genus treptomyces Streptomyces albovinaceus strain AH-135 (Streptomyces albovinace
us No. AH-135) containing a novel glutarimide antibiotic (Antiherpe) with strong antiviral activity.
s-135Y, hereinafter referred to as AH-135Y. ) Was produced and accumulated, and its isolation / purification was successful. The present invention has been completed based on this finding.

[0003]

[Constitution of the invention]

<Microorganism used> The microorganism used in the present invention is an actinomycete belonging to the genus Streptomyces and having an ability to produce an antibiotic (AH-135Y) having a novel glutarimide antiviral activity.

[0004] For example, Streptomyces albovinaceus No. A strain AH-135 (Streptomyces albovinaceus No. A)
H-135) (hereinafter referred to as "AH-135 strain") has the above-mentioned characteristics and advantageously produces the antibiotic AH-135Y having the antiviral activity of the present invention. It can be effectively used in the method of the invention.

In addition to natural and artificial mutants of the AH-135 strain, all microorganisms belonging to the genus Streptomyces and having the ability to produce the antibiotic AH-135Y described below are used in the method of the present invention. can do.

The above-mentioned AH-135 strain is a soil actinomycete found in the soil collected in the Yaku-cho, Kumage-gun, Kagoshima Prefecture by the present inventor, and was submitted to the Institute of Microbial Technology, Institute of Industrial Science, November 1991. The microorganism was deposited on the 13th, and the microorganism deposit number is MICROLAB. 12603 (FERMP-1260).
3).

The AH-135 strain has the following mycological properties.

(I) Morphology AH-13 grown on yeast-malt extract agar medium
Five strains were observed with an optical and electron microscope. According to this, the mature aerial hyphae are linear or wavy (Rectus-flexibile
In s), the number of long spores is 50 to 60.
The spore surface was smooth, and no zoospores, sporangia or sclerotia were observed in this strain.

This strain is cultivated in a liquid medium for analyzing cell components (Brain-Heart-Infusion-Bouillon medium),
The cells were separated and hydrolyzed in 6N hydrochloric acid at 105 ° C for 18 hours. When this hydrolyzate was subjected to paper chromatography for diaminopiperic acid analysis to analyze each component, only LL type diaminopiperic acid was detected. Glycine was detected by thin-layer chromatography of amino acids.

(II) Growth condition in various media (28 ° C,
Cultivation for 3 weeks) 1. Tuapec agar medium growth: Normal aerial hyphae formation: normal Soluble pigment: Slightly yellowish 2. Glycerol-Tuapek agar medium growth: good Aerobic hyphae formation: rich soluble pigment: brown 3 Glucose / asparagine agar medium Growth: Poor aerial mycelial colonization: Poor soluble pigment: none 4. Glycerol / asparagine agar medium growth: good Aerobic hyphae colonization: rich Soluble pigment: light green brown 5. Nutrient agar medium growth: Poor aerial mycelial epithelium: none Soluble pigment: none 6. Starch inorganic salt agar growth: good Aerial mycelial epithelium: rich Soluble pigment: pale orange 7. Yeast extract agar medium growth: good aerial mycelium agar: rich Color of aerial mycelium: white Soluble pigment: brown

(III) Utilization of carbon source (Prydam Gottlieb agar, 28 ° C culture) 1. D-glucose ++ 2. L-arabinose ++ 3. sucrose + 4. D-xylose ++ 5. L- Inositol -6. D-mannitol ++ 7. D-Fructose ++ 8. Rhamnose -9. Raffinose ＋ 10. Galactose ＋ 11. Salicin ＋ 12. Starch ＋ 13. Cellulose ±

(IV) Other physiological properties (28 ° C. culture) 1. Liquefaction of gelatin (broth-gerlatin medium) Not liquefaction. 2. Nitrate reducing property Adds grease reagent to turn red and reduces nitrate to nitrous acid. 3. Hydrolysis of starch (starch / inorganic salt agar medium) Hydrolyze. 4. Coagulation / peptone of skim milk Peptone, but does not coagulate. 5. Formation of melanin pigment The formation of typical black melanin pigment is not observed in tyrosine agar medium, peptone-yeast-iron agar medium. 6. Growth temperature 12 ℃ -37 ℃ Optimum temperature: 28 ℃

The above characteristics, especially in the cell wall component
Since it has LL-type diaminopiperic acid and glycine is detected as an amino acid, it belongs to a typical Streptomyces genus and has different assimilating behaviors to sucrose, rhamnose, and raffinose, but it is generally Streptomyces arbovinaceus. (Streptomyces albo-vi
naceus) and is named Streptomyces arbobinaceus AH-135 strain.

<Culture and Isolation / Purification> In carrying out the present invention, AH-13 belonging to the genus Streptomyces.
The 5Y-producing bacterium can be cultured by a conventional method for producing an antibiotic. The form of culture may be liquid culture or solid culture, and in order to culture it industrially advantageously, inoculate the culture medium with a spore suspension or culture solution of the above-mentioned producing strain, shake culture, aeration stirring culture, etc. Culture is carried out under dynamic conditions.

The nutrient source of the medium is not particularly limited as long as it contains a carbon source, a nitrogen source and an inorganic salt that can be assimilated by the AH-135Y-producing bacterium, and if necessary, a micronutrient. ..

As the carbon source, for example, glucose, fructose, maltose, starch, dextrin, starch hydrolysates, carbohydrates such as molasses, organic acids such as succinic acid, fumaric acid, acetic acid and alcohols such as glycerin are used. .. Examples of the nitrogen source include ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium nitrate, ammonium salts such as ammonium acetate, nitrates such as sodium nitrate and potassium nitrate, urea, ammonia water, ammonia gas, amino acids, and peptone,
Soy whey, soy flour and their hydrolyzed proteins,
Rice bran is used. In addition, for example, magnesium salts and phosphates are appropriately used as the inorganic salts, and amino acid, vitamins, peptone containing these and peptone, yeast extract, etc. are appropriately used as the organic micronutrients. In addition, an antifoaming agent, plant, mineral oil or the like may be added.

The culturing conditions such as culturing temperature and culturing time are selected so that they are suitable for the growth of the bacteria to be used and the production of the substance AH-135Y is maximized. For example, the pH of the medium is preferably 6.0 to 8.0, and the suitable temperature for culturing is preferably about 20 ° C to 30 ° C. The culture time is preferably about 48 to 96 hours, and the culture is performed under aerobic conditions such as rotary shaking culture and aeration and stirring culture.

However, the culture composition, the liquidity of the medium, the culture temperature, the culture conditions such as stirring conditions are appropriately adjusted and selected so as to obtain preferable results depending on the kind of the strain to be used and external conditions. It goes without saying that it should be done.

From the culture thus obtained, AH
To obtain the -135Y substance, the means usually used for collecting metabolites is appropriately used. For example, the means for utilizing the solubility difference between the substance AH-135Y of the present invention and the impurities, the means for utilizing the difference in adsorption affinity, the means for utilizing the difference in ionic bond strength, and the difference in distribution between the organic solvent and Any of the means used may be used alone, in appropriate combination, or repeatedly used.

The AH-135Y of the present invention is present in the culture solution of the compound-producing bacterium and in the microbial cells, but most of it is present in the culture solution. In order to isolate AH-135Y from the culture solution, for example, extraction with an organic solvent such as chloroform and ethyl acetate, ion exchange, adsorption, partition, chromatography such as gel filtration, and high performance liquid chromatography are appropriately combined. For example, the culture filtrate obtained by removing the cells is subjected to adsorption chromatography using a porous column, extracted with an organic solvent such as hydrous methanol, after concentration, extracted with an organic solvent such as ethyl acetate under acidic conditions, Again, adsorption chromatography is carried out using a porous column and eluted with hydrous methanol and the like. If necessary, it is possible to effectively decolorize by performing adsorption chromatography again.

The separated and purified AH-135Y is obtained as a white powder by removing the solvent. Alternatively, the concentrated solution of the compound may be allowed to stand in the presence of acetone or ether to obtain columnar crystals.

The novel glutarimide antiviral substance, AH-135Y, obtained by the above-mentioned method has the following characteristics, and its known antibiotics produced by actinomycetes have the structure Because they are different, they can be clearly distinguished. 1. Inacton Literature R.Paul, S.Tchelitcheff: Bull.Soc.Chim. France
1316 (1955) 2. Actiphenol Reference RJHighet, V.Prelog: Helv.Chim.Acta 42,1523 (19
59) 3. Actiketal literature T. Sonoda, H. Osada, J. Uzawa, K. Isono: J. Antibiot.
44 (2), 160 (1991) 4. Non-Kang 101-G (Nong-Kang 101-G) Reference JC.Hua, YY.Xie: Hua Hsueh Hsueh Pao 38 (3), 275
(1980) (Chinese) 5. Epiderstatin Reference T. Sonoda, H. Osaka, M. Uramoto, J. Uzawa, K. Isono: J.
Antibiot. 42 (11) 1607 (1989)

The physicochemical properties of AH-135Y of the present invention are shown below. Elemental analysis: C: 61.81%, H: 5.98%, N: 4.79%,
O: 27.42% Molecular weight: 291 Molecular formula: C ₁₅ H ₁₇ NO ₅ Melting point: 210-230 ° C. (decomposition) UV absorption spectrum (in methanol): As shown in FIG. 1, λmax nm (E _{1%, 1cm} ) = 218 (634.8) = 259.5 (311.5) = 342.5 (138.4) Infrared absorption spectrum (potassium bromide method): As shown in Fig. 3510, 1715, 1660, 1270cm ^-1 Proton nuclear magnetic resonance spectrum (in heavy dimethyl sulfoxide): As shown in Fig. 2.29 , 2.39, 2.62, 3.19, 4.51, 5.17,7.49, 7.65,10.7
7, 12.2 (ppm) ¹³ C Nuclear magnetic resonance spectrum (in heavy dimethyl sulfoxide)
As shown in Figure 4, 20.1, 25.9, 36.9, 42.3, 57.1, 118.1, 127.2, 128.3,
130.6, 134.8, 156.2, 172.8, 205.3 (ppm) Solubility in solvent: Soluble: Methanol, Ethanol, Acetone, Ethyl acetate, Dimethylsulfoxide Poor solubility: Benzene, ether, hexane, chloroform, water Color reaction: Alcohol chloride The ferric solution gives a dark green color. (Phenol ring) Thin layer chromatography: Kiesel-gel 60F ₂₅₄ (manufactured by Merck), detection was by UV irradiation or iodine color development. A single spot is given under the following conditions. 1. Chloroform: Acetone (3: 1) Rf = 0.38 2. Hexane: Ethyl acetate: Methanol: Acetic acid (10:
5: 0.5; 0.1) Rf = 0.085 Color of substance: White powder Antibacterial activity: The minimum inhibitory concentration of AH-135Y against various microorganisms is shown in Table 1. Antiviral activity: AH-135Y has an action that has not been pointed out so far in the antibiotic group, namely a potent and broad antiviral activity. As for the antiviral activity, it shows an antiviral effect against herpes simplex virus and influenza virus (Table 2).

AH-135Y is a novel substance having the above-mentioned physicochemical properties, is ineffective against Gram-positive and negative bacteria shown in Table 1, is slightly effective against Saccharomyces cerevisiae yeast, and has antifungal activity. Was only shown. However, it surprisingly showed excellent antiviral activity (see Table 2).

[0025]

[0026]

Hereinafter, the present invention will be described in detail with reference to specific examples and test examples, but the present invention is not limited thereto. .

[0028]

EXAMPLES Example 1 A culture medium containing 20 g of glucose, 30 g of starch, 5 g of peptone, 10 g of corn steep liquor, 10 g of soybean flour, 3 g of sodium chloride and 5 g of calcium carbonate per 1 liter of water was adjusted to pH 7.0, Dispense 50 ml each into 8 200 ml Erlenmeyer flasks, 120 ℃
And sterilized for 15 minutes. From this slant medium of Streptomyces arbobinaceus AH-135 strain, 1
A platinum loop was inoculated, and shaking culture (180 times / minute) was performed at 28 ° C. for 48 hours.

400 ml of this preculture liquid was added to 5.0 l of the above medium.
Transfer to 2 batches of 10 liter glass jar fermenter containing, and send 4 liters of sterile air per minute at 28 ° C, 400
The main culture was carried out by rotation stirring. The culture was stopped for 96 hours, and the culture solution (the pH of the culture solution was almost neutral) was centrifuged to remove the bacterial cells, to obtain 7.2 l of the supernatant. 1,8 of this supernatant
The 00-fold dilution reduced plaque formation by 50%.

7.2 l of this supernatant was added to p with 2N hydrochloric acid.
After adjusting to H3-4, extraction is performed with 4.8 l of ethyl acetate. The ethyl acetate layer was concentrated under reduced pressure, and after completely removing ethyl acetate, water was added to 200 ml, and this was washed thoroughly with water in advance to Diaion HP-10 (manufactured by Mitsubishi Kasei) 3.2 cm x 25 cm. Pour into the column and add 1 column
After washing with l of water, stepwise elution was carried out with 500 ml of 20%, 40%, 60% and 80% methanol, and 600 ml of the eluent of 40%, 60% and 80% methanol, which was the active fraction, was concentrated under reduced pressure. , Methanol was completely removed, and water was added to make up to 400 ml.

This active fraction was washed thoroughly with water beforehand, D
iaion HP-10 (manufactured by Mitsubishi Kasei) 1.8 cm x 16 cm
Pour into the column and wash with 200 ml of water, 40%, 60
Elute with 200 ml each of 80% and 80% methanol, and concentrate the active fraction under reduced pressure. This operation is repeated until the purity by HPLC becomes 50 to 60%. Diaion
HP-10 (manufactured by Mitsubishi Kasei) column chromatography yielded 50 ml of the active fraction, 33,000 of this solution.
The 0-fold dilution reduced plaque formation by 50%.

This active fraction was dried under reduced pressure, dissolved in 50 ml of methanol, and washed with methanol to obtain a small amount of Dia.
Ion HP-20ss (manufactured by Mitsubishi Kasei) was added, and the mixture was dried under reduced pressure using a 300 ml eggplant-shaped flask to obtain a slightly yellow powder. This powder was thoroughly washed with water Diaion HP-
The solution was overlaid on a 1.8 cm × 18 cm column of 20 ss (manufactured by Mitsubishi Kasei), washed with 200 ml of water and 200 ml of 40% methanol, and then eluted with 60% methanol to obtain 100 ml of an active fraction. This operation was repeated until the dye disappeared, and the active fraction having a purity of 95% or more by HPLC was 50% or more.
ml was obtained and a 30,000-fold dilution of this solution reduced plaque formation by 50%.

After this solution was concentrated to dryness under reduced pressure, a very small amount of methanol was added to dissolve it, a small amount of acetone was added, and when it became cloudy, ether was added to obtain a white precipitate. After removing the organic solvent by decantation,
The mixture was dried under reduced pressure and left in a desiccator under reduced pressure for 1 hour or more to obtain about 20 mg of white powder.

Test Example 1 The antiviral activity was measured using the purified sample. The purified sample was suspended in Eagle MEM medium at a concentration of 1 mg / ml and serially diluted with the same medium to prepare 200 PFU.
Of herpes simplex virus were added to Vero cells infected. Similarly, MDCK cells infected with 200 PFU of influenza virus were cultured in a medium containing a serially diluted preparation. The number of plaques was counted 3 days after infection and compared with the number of plaques in a control to which the standard solution was not added. As controls, acyclovir was used for herpes simplex and amantadine for influenza virus.

[0035]

[Brief description of drawings]

FIG. 1 shows an ultraviolet absorption spectrum of AH-135Y in a methanol and acidic methanol solution, in which the vertical axis represents absorbance and the horizontal axis represents wavelength (nm). FIG. 2 shows an infrared absorption spectrum measured by mixing AH-135Y with potassium bromide. The vertical axis shows the transmittance (%), and the horizontal axis shows the wavelength (cm ^-1 ). Figure 3
Shows the nuclear magnetic resonance spectrum of the hydrogen nucleus ( ¹ H) in the heavy dimethyl sulfoxide solution of AH-135Y, the vertical axis shows the peak intensity, and the horizontal axis shows the chemical shift (ppm). Figure 4 is AH-
The nuclear magnetic resonance spectrum of carbon nucleus ( ¹³ C) of 135Y is shown, the vertical axis shows peak intensity, and the horizontal axis shows chemical shift (ppm).

Claims (4)

[Claims] 1. An antiviral substance AH-135Y represented by the formula (I), which has the following chemical properties. [Chemical 1] Elemental analysis: C: 61.81%, H: 5.98%, N: 4.79%,
O: 27.42% Molecular weight: 291 Molecular formula: C ₁₅ H ₁₇ NO ₅ mp: 210 to 230 ° C. (decomposition) ultraviolet absorption spectrum (in _{methanol): λmax nm (E 1%} , 1cm) = 218 (634.8) = 259.5 (311.5 ) = 342.5 (138.4) Infrared absorption spectrum (potassium bromide method): 3510, 1715, 1660, 1270 cm ^-1 Proton nuclear magnetic resonance spectrum (in heavy dimethyl sulfoxide): ppm 2.29, 2.39, 2.62, 3.19, 4.51, 5.17 , 7.49, 7.65,10.7
7, 12.2 ¹³ C nuclear magnetic resonance spectrum (in heavy dimethyl sulfoxide): ppm 20.1, 25.9, 36.9, 42.3, 57.1, 118.1, 127.2, 128.3,
130.6, 134.8, 156.2, 172.8, 205.3 Solubility in solvent: Soluble: Methanol, Ethanol, Acetone, Ethyl acetate, Dimethyl sulfoxide Poor solubility: Benzene, Ether, Hexane, Chloroform, Water Color reaction: Alcoholic ferric chloride It turns dark green in solution. (Phenolation ring) Thin layer chromatography: Kiesel gel 60F ₂₅₄ (Merck), detection was by UV irradiation or iodine color. A single spot is given under the following conditions. 1. Chloroform: Acetone (3: 1) Rf = 0.38 2. Hexane: Ethyl acetate: Methanol: Acetic acid (10:
5: 0.5; 0.1) Rf = 0.085 Color of substance: White powder 2. An antiviral substance AH-135Y producing bacterium belonging to the genus Streptomyces is cultivated,
The antiviral substance AH-135Y was produced and accumulated in the culture, and the antiviral substance AH-135Y was produced from the culture.
Antiviral substance A characterized in that Y is separated and collected
Manufacturing method of H-135Y. 3. An antiviral substance producing AH-135Y is Streptomyces arbobinaceus AH-135 strain.
(Streptomyces albovinaceus No. AH-135). 4. An antiviral agent comprising an antiviral substance AH-135Y as an active ingredient.