JPH10273497A - Substance having antibacterial action - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new antibacterial substance consisting of a specific antibacterial substance originated from a microorganism belonging to the genus Actinomadura, exhibiting excellent antibacterial power against methicillin resistant Staphylococcus aureus and useful as a medicine. SOLUTION: This new antibacterial substance is expressed by the formula (R1 is methyl or ethyl; R2 and R3 are each H or Cl; R4 is H or methyl). It has excellent antibacterial power against methicillin-resistant Staphylococcus aureus and is useful e.g. as a medicine having improved antibacterial action. The antibacterial substance can be produced by using Actinomadura sp.TA-0327 (FERM P-16135) newly separated from the natural world, inoculating and culturing the strain on a culture medium, separating the cultured liquid into supernatant and bacterial cells by centrifugal separation, bringing the supernatant into contact with an adsorptive resin, eluting the adsorbed component with acetone, extracting the bacterial cell with acetone, concentrating the eluted liquid and extracted liquid and separating and purifying the product by high- performance liquid chromatography.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel compound having an antibacterial action.

[0002]

2. Description of the Related Art At present, various antibiotics are used as medicaments, but their effects are not always sufficient, and development of new compounds having more excellent antibacterial activity is desired.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to find a novel compound having an antibacterial action from microorganisms isolated from the natural world.

[0004]

Means for Solving the Problems The present inventors have conducted extensive research to obtain a novel compound having antibacterial activity from soil isolates, and as a result, the specific microorganisms found by the present inventors have become The present inventors have found that a novel bioactive substance having an action is produced, and completed the present invention.

That is, the present invention provides

[0006]

Embedded image

Wherein R ₁ is a methyl group or an ethyl group, R ₂ and R ₃ are each a hydrogen atom or a chlorine atom, and R ₄ is a hydrogen atom or a methyl group. An active substance (hereinafter, AEA0060 when R ₁ is an ethyl group, R ₂ is a chlorine atom, and R ₃ and R ₄ are hydrogen atoms,
When R ₁ is an ethyl group and R ₂ , R ₃ and R ₄ are hydrogen atoms, A
EA0061, where R ₁ is a methyl group, R ₂ is a chlorine atom, and R ₃ and R ₄ are hydrogen atoms AEA0062, where R ₁ is an ethyl group, R ₂ and R ₃ are chlorine atoms, and R ₄ is a hydrogen atom A
EA0063, R ₁ is ethyl, R ₂ is a chlorine atom, R ₃ is a hydrogen atom, a case where R ₄ is a methyl group AEA0064, R ₁
Is an ethyl group, R ₂ and R ₃ are chlorine atoms, and R ₄ is a methyl group. ).

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The antibacterial substance AEA00 of the present invention
60, AEA0061, AEA0062, AEA006
3. The strains producing AEA0064 and AEA0065 are strains newly isolated from the natural world by the present inventors, and have the industrial name of microorganism " Actinomadura sp. TA-0327" and microorganism deposit number "FERM P-16135". Deposited at the Institute of Biotechnology and Industrial Technology.

The mycological properties of this strain are shown below. A. Morphological characteristics The basal hypha grows well and branches, but no division is observed. The formation of aerial hyphae tends to be poor and is found only on some agar media. Spores form moderately on yeast-malt agar, but are absent or negligible on other agars. No sporulation is observed in the agar medium. Three to four spore chains consisting of 4 to 10 spores are formed in the aerial hyphae of the main shaft. The spore chain is curved or looped at the tip. The spores are 0.9-1.1 μm in length, 0.6-1.1 μm in width, subspherical to elliptical, and wart-shaped on the surface. No sporangia or sclerotium formation or spore motility is observed.

B. Cultural Properties on Various Media The following Table 1 shows the results of macroscopic observations when the cells were cultured on various media at 28 ° C. for 28 days. In addition, the color display quoted the system color name of the Japan Standards Association and JIS color name book (1985).

[0011]

[Table 1]

C. Physiological Properties The growth temperature test was observed for 1 week and the other tests were observed after 3 weeks of culture. Growth temperature range: 20-39 ° C Optimum growth temperature: 31-35 ° C Gelatin liquefaction: Positive Skim milk coagulation: Negative Skim milk peptone: Negative Melanin-like pigment production: Negative Starch hydrolysis: Negative Carbon source Availability (on Prideham Gottlieb agar medium) L-arabinose:-L-lamulose: + D-xylose:-raffinose:-D-glucose: + inositol:-D-fructose:-D-mannitol:-sucrose:- (+: Grows-: hardly grows).

D. Chemical classification properties Presence / absence of diaminopimelic acid and optical isomer: Meso-type diaminopimelic acid was detected. Amino acid composition of cell wall: Alanine and glutamic acid were clearly detected. Glycine was detected in very small amounts. Types of reducing sugars in all cells: ribose, madulose, galactose and glucose were detected. Menaquinone: MK-9 (H ₆ ) was detected as the main component. MK-9 (H ₈₎ were detected in small amounts. Phospholipids: phosphatidylinositol and diphosphatidylglycerol were detected. No unknown glucosamine-containing phospholipids were detected.

The TA-0327 strain was used as described above 1).
Diaminopimelic acid is detected in the meso form and does not contain glycine for amino acids. 2) Contains madulose as reducing sugar. (The cell wall composition is IIIB
Type) 3) Menaquinone contains MK-9 (H ₆ ) as a main component. 4) The phospholipid is PI type. From these results, it was clarified that this strain is a species of Actinomadura genus of actinomycetes. Therefore, this strain was transformed into Actinomadura s
It was named p.TA-0327.

AEA0060, AEA0061, AEA
0062, AEA0063, AEA0064 and AEA
0065 is produced in the same manner as producing general fermentation products, and in a medium containing various nutrients, Actinomadura s.
p.TA-0327 strain is cultured under aerobic conditions. The medium is mainly a liquid medium, carbon source, nitrogen source,
Consisting of inorganic salts, vitamins, precursors,
An antifoam can be added and the pH is adjusted around 7.
As the carbon source, for example, glucose, maltose, dextrin, glycerin, starch or the like is used alone or as a mixture. Nitrogen sources include, for example, yeast extract, peptone, meat extract, soy flour, corn steep liquor,
Urea, ammonium salt or the like is used alone or as a mixture. As the inorganic salt, for example, monopotassium phosphate, magnesium sulfate, sodium chloride, calcium carbonate and the like are used alone or as a mixture. Adecanol as an antifoaming agent,
A silicon compound or the like can be used.

Aerobic culture such as shaking culture and aeration and stirring culture is suitable for the culture method.
0 ° C. for 2-4 days, desirably pH 6-7, 26-28
Incubate at 4 ° C for 4 days. AEA produced by this culture
0060, AEA0061, AEA0062, AEA0
In order to isolate 063, AEA0064 and AEA0065, a general method for collecting fermentation products may be used. That is, after culturing, AEA0060, AEA0061,
AEA0062, AEA0063, AEA0064 and AEA0065 are extracted with an organic solvent such as lower alcohol and acetone, and the extract is concentrated under reduced pressure to remove the organic solvent, and then transferred to a water-insoluble organic solvent such as ethyl acetate, benzene and chloroform. Then, this is concentrated under reduced pressure to form a syrup. Add the syrup again to ethyl acetate, benzene,
Dissolved in an organic solvent such as chloroform, acetone, and methanol, and subjected to column chromatography using silica gel, high-performance liquid chromatography packed with silica gel ODS for reverse phase distribution, and gel filtration column chromatography to obtain AEA0060, AEA0061,
AEA0062, AEA0063, AEA0064 and AEA0065 can be purified and isolated.

The target substances of the present invention, AEA0060, AEA0061, and AEA0, obtained by the above purification
062, AEA0063, AEA0064 and AEA0
No. 065 has the following physicochemical properties. No antibacterial substance with the same physicochemical properties as this substance is known.

[1] Physicochemical properties of AEA0060 (1) Appearance: white powder (2) Melting point: 209-211 ° C. (3) High resolution mass spectrum: measured value; m / z 893.2843 (M + K) ⁺ theory Value; m / z 893.2949 (C ₄₅ H ₅₆ Cl ₂ N ₂
(Calculated as O ₁₀ K) (4) Mass spectrum: FABMS (+) m / z 855 (M + H) ⁺ FABMS (+) m / z 893 (M + K) ⁺ (5) Molecular weight: 855.85 (6) Molecular formula: C ₄₅ H ₅₆ Cl ₂ N ₂ O ₁₀ (7) Specific rotation: [α] _D ²⁶ = −28.0 ° (c = 0.1, methanol) (8) Ultraviolet absorption spectrum: λ _max 270 nm (ε = 22000) (Measured in methanol solution) (9) Infrared absorption spectrum: FIG. 1 shows the result of measurement by the KBr method.

(10) ¹ H-NMR spectrum: CD ₃ O
The result measured at 500 MHz in D is shown in FIG.

(11) ¹³ C-NMR spectrum: CD ₃
FIG. 3 shows the result of measurement at 125 MHz during OD.

(12) Solubility in solvent: insoluble in water,
Chloroform, methanol, ethanol, ethyl acetate,
Soluble in chloroform and benzene (13) Color reaction; Positive: iodine, sulfuric acid Negative: ferric chloride [2] Physicochemical properties of AEA0061 (1) Appearance: white powder (2) Melting point: 224-226 ° C ( 3) high resolution mass spectrum: Found; m / z 819.3642 (M- H) - _{theory; m / z 819.3663 (C 45} H 56 ClN 2 O
(Calculated as _10. ) (4) Mass spectrum: FABMS (+) m / z 821 (M + H) ⁺ FABMS (+) m / z 859 (M + K) ⁺ (5) Molecular weight: 821.41 (6) Molecular formula: C ₄₅ H ₅₇ ClN ₂ O ₁₀ (7) Specific rotation: [α] _D ²⁶ = −26.0 ° (c = 0.1, methanol) (8) Ultraviolet absorption spectrum: λ _max 271 nm (ε = 22000) (methanol solution (9) Infrared absorption spectrum: FIG. 4 shows the result of measurement by the KBr method.

(10) ¹ H-NMR spectrum: CD ₃ O
The result measured at 500 MHz in D is shown in FIG.

(11) ¹³ C-NMR spectrum: CD ₃
FIG. 6 shows the result of measurement at 125 MHz during OD.

(12) Solubility in solvent: insoluble in water,
Chloroform, methanol, ethanol, ethyl acetate,
Soluble in chloroform and benzene (13) Color reaction; Positive: iodine, sulfuric acid Negative: ferric chloride [3] Physicochemical properties of AEA0062 (1) Appearance: white powder (2) Melting point: 220-221 ° C ( 3) high resolution mass spectrum: Found; m / z 839.3068 (M- H) - _{theory; m / z 839.3064 (C 44} H 53 Cl 2 N 2
Calculated as O ₁₀₎ (4) Mass ^{Spectrum: FABMS (+) m / z} 841 (M + H) + FABMS (+) m / z 879 (M + K) + (5) Molecular weight: 841.83 (6) Molecular formula: C _{44 H 54 Cl 2 N 2 O 10} (7) specific ^{_{rotation: [α] D 26 = -36.0}} ° (c = 0.1, methanol) (8) ultraviolet absorption _{spectrum: λ max 268nm (ε = 23000} ) (Measured in methanol solution) (9) Infrared absorption spectrum: FIG. 7 shows the result of measurement by the KBr method.

(10) ¹ H-NMR spectrum: CD ₃ O
FIG. 8 shows the results measured at 500 MHz in D.

(11) ¹³ C-NMR spectrum: CD ₃
The result measured at 125 MHz during OD is shown in FIG.

(12) Solubility in solvent: insoluble in water,
Chloroform, methanol, ethanol, ethyl acetate,
Soluble in chloroform and benzene (13) Color reaction; Positive: iodine, sulfuric acid Negative: ferric chloride [4] Physicochemical properties of AEA0063 (1) Appearance: white powder (2) Melting point: 214-215 ° C ( 3) High-resolution mass spectrum: measured value; m / z 887.2829 (M−H) ⁺ theoretical value; m / z 887.2880 (C ₄₅ H ₅₄ Cl ₃ N ₂₎
(Calculated as O ₁₀ ) (4) Mass spectrum: FABMS (−) m / z 889 (M−H) ⁻ FABMS (+) m / z 929 (M + K) ⁺ (5) Molecular weight: 890.30 (6) Molecular formula: _{C 45 H 55 Cl 3 N 2} O 10 (7) specific ^{_{rotation: [α] D 26 = -2.0}} ° (c = 0.1, methanol) (8) ultraviolet absorption spectrum: λ _max 273nm (ε = (19000) (measured in methanol solution) (9) Infrared absorption spectrum: FIG. 10 shows the result of measurement by the KBr method.

(10) ¹ H-NMR spectrum: CD ₃ O
FIG. 11 shows the results measured at 500 MHz in D.

(11) ¹³ C-NMR spectrum: CD ₃
FIG. 12 shows the result of measurement at 125 MHz during OD.

(12) Solubility in solvent: insoluble in water,
Chloroform, methanol, ethanol, ethyl acetate,
Soluble in chloroform and benzene (13) Color reaction; Positive: iodine, sulfuric acid Negative: ferric chloride [5] Physicochemical properties of AEA0064 (1) Appearance: white powder (2) Melting point: 204-205 ° C ( 3) high resolution mass spectrum: Found; m / z 867.3398 (M- H) - _{theory; m / z 867.3392 (C 46} H 57 Cl 2 N 2
(Calculated as O ₁₀ ) (4) Mass spectrum: FABMS (−) m / z 867 (M−H) ⁻ FABMS (+) m / z 907 (M + K) ⁺ (5) Molecular weight: 869.88 (6) Molecular formula: _{C 46 H 58 Cl 2 N 2} O 10 (7) specific ^{_{rotation: [α] D 26 = -14.0}} ° (c = 0.1, methanol) (8) ultraviolet absorption spectrum: λ _max 268nm (ε = (19000) (measured in methanol solution) (9) Infrared absorption spectrum: FIG. 13 shows the result of measurement by the KBr method.

(10) ¹ H-NMR spectrum: CD ₃ O
FIG. 14 shows the results measured at 500 MHz in D.

(11) ¹³ C-NMR spectrum: CD ₃
FIG. 15 shows the result of measurement at 125 MHz during OD.

(12) Solubility in solvent: insoluble in water,
Chloroform, methanol, ethanol, ethyl acetate,
Soluble in chloroform and benzene (13) Color reaction; Positive: iodine, sulfuric acid Negative: ferric chloride [6] Physicochemical properties of AEA0065 (1) Appearance: white powder (2) Melting point: 213-215 ° C ( 3) high resolution mass spectrum: Found; m / z 901.2988 (M- H) - _{theory; m / z 901.3001 (C 46} H 56 Cl 3 N 2
(Calculated as O ₁₀ ) (4) Mass spectrum: FABMS (−) m / z 903 (M−H) ⁻ FABMS (+) m / z 943 (M + K) ⁺ (5) Molecular weight: 904.32 (6) Molecular formula: _{C 46 H 57 Cl 3 N 2} O 10 (7) specific ^{_{rotation: [α] D 26 = -18.0}} ° (c = 0.1, methanol) (8) ultraviolet absorption spectrum: λ _max 272nm (ε = (17000) (measured in methanol solution) (9) Infrared absorption spectrum: FIG. 16 shows the result of the measurement by the KBr method.

(10) ¹ H-NMR spectrum: CD ₃ O
FIG. 17 shows the results measured at 500 MHz in D.

(11) ¹³ C-NMR spectrum: CD ₃
FIG. 18 shows the result of measurement at 125 MHz during OD.

(12) Solubility in solvent: insoluble in water,
Chloroform, methanol, ethanol, ethyl acetate,
Soluble in chloroform and benzene (13) Color reaction; Positive: iodine, sulfuric acid Negative: ferric chloride

[0037]

The compounds of the present invention have antibacterial activity and are useful as pharmaceuticals.

[0038]

The present invention will be described in more detail with reference to the following Examples and Test Examples.

Example 1 [AEA0060, AEA00
61, Preparation of AEA0062, AEA0063] (1) pH7 consisting of 2 g of soluble starch, 0.5 g of glucose, 0.2 g of yeast extract, 0.3 g of NZ Keith, 0.5 g of meat extract, and 0.3 g of calcium carbonate per 100 ml.
Of sterile liquid medium 50 ml Ac in 500ml Erlenmeyer flask containing tinomadura sp. Were inoculated with TA-0327 strain, 28 ° C., 1
Rotary culture was performed for 92 hours. Then 100ml per glucose 2.5g, Konsute _Lee Purika powder 0.75
500m containing g, Famamede _Lee A 0.5g, de _Lee stearyl _Lee Razusoruburu 1.0 g, cobalt chloride 0.001 g, polyethylene glycol (400) 0.25 g, a sterile liquid medium 100ml of pH7 consisting of calcium carbonate 0.3g
A 1-L Erlenmeyer flask was inoculated with the cells in the seed culture,
Rotary culture was performed at 8 ° C for 216 hours. Next, using one 200-L culture tank, 1.5 L of the seed culture was inoculated into 120 L of a sterile medium having the same composition as that of the seed culture.
The culture was stirred and aerated for 96 hours.

(2) After completion of the culture, the supernatant was separated from the cells by a centrifuge. The supernatant was adsorbed on 6 L of HP-20,
After washing with L of purified water, elution was performed with 12 L of acetone, followed by concentration under reduced pressure. After removing the acetone, the residue was extracted twice with 5 L of ethyl acetate. The obtained cells were extracted with 24 L of acetone and concentrated under reduced pressure. After removing the acetone, the residue was extracted twice with 5 L of ethyl acetate. These ethyl acetate extraction sections were combined, dehydrated with anhydrous sodium sulfate, and concentrated to dryness under reduced pressure to obtain 32.522 g of a brown substance.

(3) The brown substance was treated with chloroform 200
of silica gel [Kieselgel-60 (trade name, manufactured by Merck)] dissolved in chloroform and dissolved in chloroform.
It was adsorbed on a ml column. After washing with 2000 ml of chloroform, chloroform-methanol (97: 3 to 50:
The mixture was eluted sequentially with the mixed solvent of 50). The active fractions eluted at a mixed solvent ratio (90:10 to 80:20) were combined, concentrated under reduced pressure to dryness, and 2.75 g of a brown syrup was obtained.

(4) The brown substance described in the preceding paragraph was dissolved in a small amount of methanol, and the mixture was mixed using a 580 ml column packed with Sephadex LH20 (trade name, manufactured by Pharmacia) prepared with chloroform-methanol (1: 1). Gel filtration was performed with the solvent. Collect the active fractions and brown matter 1.15
g was obtained.

(5) 950 mg of the brown substance described in the preceding paragraph was dissolved in methanol, and this solution was subjected to preparative high performance liquid chromatography using an acetonitrile-phosphate buffer, pH 3.5 (75:25) as a mobile phase. Waters; Column: Kanto Chemical MightysilRP-18 (2
0φ × 200 mm)] and monitoring at UV absorption 210 nm at a flow rate of 9.45 ml / min.
Min, peaks eluting at 15 to 17.5 minutes and 17.5 to 25.0 minutes were collected. The three fractions obtained by fractionation were combined, concentrated under reduced pressure to remove acetonitrile, and then extracted twice with a half amount of ethyl acetate. The ethyl acetate extracted sections were combined, dehydrated with anhydrous sodium sulfate, concentrated under reduced pressure to dryness, and dried.
78 mg of Fr1, 69.0 mg of AEA0060, 7
4.5 mg of Fr2 were obtained.

(6) Fr1 of the preceding paragraph was dissolved in a small amount of methanol, and this solution was subjected to preparative high performance liquid chromatography using acetonitrile-phosphate buffer pH 3.5 (70:30) as a mobile phase [apparatus used: Waters Company;
Column: Kanto Chemical MightysilRP-18 (20φ × 20
0)), and the peak eluted at 14.9 minutes and 21.2 minutes at a flow rate of 9.45 ml / min was monitored while monitoring the UV absorption at 210 nm. The two fractions obtained by fractionation were combined, concentrated under reduced pressure to remove acetonitrile, and then extracted twice with a half amount of ethyl acetate. The ethyl acetate extractions were combined, dehydrated with anhydrous sodium sulfate, concentrated to dryness under reduced pressure, and dried with 28.1 mg of AEA0061 and 8.0 mg of A.
EA0062 was obtained.

(7) Fr2 of the preceding paragraph was dissolved in a small amount of methanol, and this solution was subjected to preparative high performance liquid chromatography using acetonitrile-phosphate buffer pH 3.5 (80:20) as a mobile phase [apparatus used: Waters Company;
Column: Kanto Chemical MightysilRP-18 (20φ × 20
0 mm)], and a peak eluted at 16.5 minutes at a flow rate of 9.45 ml / min was collected while monitoring the UV absorption at 210 nm. The peak obtained by fractionation was concentrated under reduced pressure to remove acetonitrile, and then extracted twice with a half amount of ethyl acetate. The ethyl acetate extracted sections were combined, dehydrated with anhydrous sodium sulfate, concentrated under reduced pressure to dryness, and 9.4 mg of A
EA0063 was obtained.

[0046] Example 2 Preparation of AEA0064,0065 (1) 100ml per soluble starch 2g, glucose 0.5g, yeast extract 0.2 g, NZ Keith 0.3 g, Famamede _Lee A 0.5g, fish meal 0.5g, Sterile liquid medium of pH 7 consisting of 0.2 g of calcium carbonate 1
Actinomadu in 500ml Erlenmeyer flask containing 00ml
The ra sp. TA-0327 strain was inoculated and rotary-cultured at 28 ° C. for 144 hours. Then, 2.5 ml of glucose per 100 ml
g, Konsute _Lee Purika powder 0.75 g, Famamede _Lee A 0.5g, de _Lee stearyl _Lee Razusoruburu 1.0 g, cobalt chloride 0.001 g, polyethylene glycol (40
0) pH7 consisting of 0.25 g and calcium carbonate 0.3 g
2% of the seed culture was inoculated into 100 500 ml Erlenmeyer flasks containing 100 ml of a sterile liquid medium at
Rotary culture was performed for 92 hours.

(2) After completion of the culture, the supernatant was separated from the cells by a centrifuge. The supernatant was adsorbed on 0.5 L of HP-20,
After washing with 1 L of purified water, the mixture was eluted with 2 L of acetone and concentrated under reduced pressure. The obtained cells were extracted with 6 L of acetone,
It was concentrated under reduced pressure. Both residues were extracted twice with 1 L of ethyl acetate. These ethyl acetate extracted sections were combined, dehydrated with anhydrous sodium sulfate, concentrated to dryness under reduced pressure, and dried to give a brown syrup.
57 g were obtained.

(3) This brown substance was treated with chloroform 20m
l, and silica gel [Kieselgel-60 (trade name, manufactured by Merck)] prepared with chloroform was 1200 m
1 column. After washing with 2000 ml of chloroform, chloroform-methanol (97: 3 to 50: 5
It eluted in order with the mixed solvent of 0). Among these, the active fractions eluted at a mixed solvent ratio (95: 5) were combined, concentrated under reduced pressure and dried to give 1.50 g of a brown syrup.

(4) The brown substance described in the preceding paragraph was dissolved in a small amount of methanol, and Sephadex LH2 prepared with methanol was used.
1200ml filled with 0 (trade name, manufactured by Pharmacia)
Gel filtration was performed with the same solvent using the column described above. The active fraction was collected to obtain 1.20 g of a brown substance.

(5) The brown substance described in the preceding paragraph was dissolved in methanol, and this solution was prepared in ODS prepared with 75% acetonitrile.
Medium-pressure liquid chromatography [Prepared by Kusano Kagaku Kikai Seisakusho, Ltd.]
Flow rate 3 while monitoring with UV absorption 270 nm.
Peaks eluted at 82.5 minutes and 115.0 minutes at 0.0 ml / min were collected. The two fractions obtained by fractionation were combined, concentrated under reduced pressure to remove acetonitrile, and then extracted twice with a half amount of ethyl acetate. The ethyl acetate extractions were combined, dehydrated with anhydrous sodium sulfate, concentrated to dryness under reduced pressure, and dried to obtain 88.0 mg of AEA0064 and 60.0 mg of AEA0.
065 was obtained.

Test Example (Antibacterial Activity) Antibacterial activity (MIC; minimum growth inhibitory concentration) was measured by a microfluidic dilution method.

(Specimen) AEA obtained in Examples 1 and 2
0060, AEA0061, AEA0062, AEA0
Each of 0.63, AEA0064 and AEA0065 was dissolved in dimethyl sulfoxide (1.0 mg each) and serially diluted.

(Test Bacteria) Staphylococcus aureus ATCC 33591 (Methicillin-resistant Staphylococcus aureus) (Medium) Mueller Hinton liquid medium (Test method) Test strain Staphylococcus aureus ATCC 3359
1 (methicillin-resistant Staphylococcus aureus) 1.7 × 10 ⁴ C
FU / ml was prepared, each sample was added, and the mixture was cultured in an incubator at 37 ° C. for 24 hours, and then the minimum inhibitory concentration (MIC) was measured.

(Results) AEA0060, AEA006
1, AEA0062, AEA0063, AEA0064
And AEA0065 exhibited excellent antibacterial activity against methicillin-resistant Staphylococcus aureus (Table 2).

[0055]

[Table 2]

Also, AEA0060, AEA0061,
AEA0062, AEA0063, AEA0064 and AEA0065 are available from Coagulase (-) Staphylococcus , Ent.
It also showed antibacterial activity against erococcus faecium and Moraxella cata rralis .

[Brief description of the drawings]

FIG. 1 shows an infrared absorption spectrum of AEA0060 measured by a KBr method.

FIG. 2: AEA measured at 500 MHz in CD ₃ OD
¹ shows a ¹ H-NMR spectrum of 0060.

FIG. 3 AEA measured at 125 MHz in CD ₃ OD
1 shows a ¹³ C-NMR spectrum of 0060.

FIG. 4 shows an infrared absorption spectrum of AEA0061 measured by a KBr method.

FIG. 5: AEA measured at 500 MHz in CD ₃ OD
¹ shows a ¹ H-NMR spectrum of 0061.

FIG. 6: AEA measured at 125 MHz in CD ₃ OD
1 shows a ¹³ C-NMR spectrum of 0061.

FIG. 7 shows an infrared absorption spectrum of AEA0062 measured by a KBr method.

FIG. 8: AEA measured at 500 MHz in CD ₃ OD
¹ shows the ¹ H-NMR spectrum of 0062.

FIG. 9: AEA measured at 125 MHz in CD ₃ OD
¹³ shows the ¹³ C-NMR spectrum of 0062.

FIG. 10 shows an infrared absorption spectrum of AEA0063 measured by the KBr method.

FIG. 11: AE measured at 500 MHz in CD ₃ OD
¹ shows a ¹ H-NMR spectrum of A0063.

FIG. 12: AE measured at 125 MHz in CD ₃ OD
3 shows the ¹³ C-NMR spectrum of A0063.

FIG. 13 shows an infrared absorption spectrum of AEA0064 measured by the KBr method.

FIG. 14: AE measured at 500 MHz in CD ₃ OD
¹ shows a ¹ H-NMR spectrum of A0064.

FIG. 15: AE measured at 125 MHz in CD ₃ OD
1 shows the ¹³ C-NMR spectrum of A0064.

FIG. 16 shows an infrared absorption spectrum of AEA0065 measured by a KBr method.

FIG. 17: AE measured at 500 MHz in CD ₃ OD
¹ shows a ¹ H-NMR spectrum of A0065.

FIG. 18: AE measured at 125 MHz in CD ₃ OD
3 shows the ¹³ C-NMR spectrum of A0065.

[Procedure amendment]

[Submission date] June 10, 1997

[Procedure amendment 1]

[Document name to be amended] Drawing

[Correction target item name] All figures

[Correction method] Change

[Correction contents]

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Continuation of the front page (72) Inventor Satoshi Akashi 3-24-1, Takada, Toshima-ku, Tokyo Inside Taisho Pharmaceutical Co., Ltd.

Claims (1)

[Claims] (1) Formula (1) (Wherein, R ₁ is a methyl group or an ethyl group, and R ₂ and R
₃ is a hydrogen atom or a chlorine atom, respectively, and R ₄ is a hydrogen atom or a methyl group. Antibacterial agent represented by).