JP5144167B2 - Novel KB-3346-5 substance and production method thereof - Google Patents

Description

The present invention relates to a novel KB-3346-5 substance and its production method, Streptomyces sp. KB-3346, which enhances its effect when used in combination with β-lactam antibiotics used as antibacterial agents . -5 shares, related to antibacterial agents .

  In recent years, methicillin-resistant Staphylococcus aureus (MRSA) has become a social problem as the main cause of nosocomial infections. This pathogen has resistance to various drugs such as β-lactam antibiotics, and generally has not been reported to be resistant to MRSA infections at present. Glycopeptide vancomycin and aminoglycoside arbekacin Antibiotics are used. In addition, β-lactam antibiotics are combined with each other or β-lactam antibiotics and other antibiotics with different action points (Kesuke Sunagawa, Junichi Totsuka: Antibiotics Science, pp .264-273, Pharmaceutical Journal, 1998).

For vancomycin and arbekacin, resistant bacteria to these drugs have already appeared. Further, these drugs are known to have side effects such as hearing loss due to the eighth cranial nerve disorder, which is a problem. To date, substances that have the effect of restoring the efficacy of β-lactam antibiotics have been reported to address this situation. For example, this corresponds to a tea extract or an active fraction thereof that exhibits a synergistic effect when used in combination with an antibacterial agent containing β-lactam antibiotics.
JP 9-509677 A

  The new KB-3346-5 substance enhances the antibacterial activity of imipenem belonging to the carbapenem series of β-lactam antibiotics, and also enhances the antibacterial activity of cloxacillin, which is a penum series, and cefazoline, which is a cephem series. Therefore, it can be applied to combination therapy with antibacterial agents including β-lactam antibiotics. The novel KB-3346-5 substance is clearly distinguished in molecular formula and chemical structure from the polyphenol compound which is an active ingredient of tea extract or its active fraction.

  A drug that enhances the activity of β-lactam antibiotics is expected to reduce the frequency of appearance of resistant bacteria by reducing the dose of β-lactam antibiotics and shortening the administration period. At the same time, it is expected to overcome resistance to β-lactam antibiotics by using two drugs having different actions.

  In such a situation, providing a substance having an action of enhancing the activity of β-lactam antibiotics against MRSA is a new therapeutic agent for MRSA infections and infections caused by multidrug-resistant bacteria including β-lactam antibiotic resistance. It is something that provides and is useful.

  As a result of searching for imipenem activity-enhancing substances that are carbapenem β-lactam antibiotics for metabolites produced by microorganisms, the present inventors have found that in the culture solution of actinomycete KB3346-5 newly isolated from soil It was found that a substance having an imipenem activity enhancing action was produced.

Next, as a result of purifying and isolating the culture, KB3346-5-A ₁ substance, KB3346-5-A ₂ substance, KB3346-5-A ₃ substance, KB3346-5-A ₄ substance having imipenem activity enhancing action, KB3346-5-A ₅ substances, KB3346-5-A ₆ substances, KB3346-5-A ₇ substances, KB3346-5-A ₈ substances, KB3346-5-A ₉ substances, KB3346-5-A ₁₀ substances, KB3346- 5-A ₁₁ substances, KB3346-5-B ₁ substance, KB3346-5-B ₂ substances, KB3346-5-B ₃ substances, KB3346-5-B ₄ substances, KB3346-5-C ₁ substance and KB3346-5- 17 compounds of C ₂ substances (hereinafter collectively referred to as KB-3346-5 substances) were obtained. A substance having a chemical structure such as KB-3346-5 has never been known.

The present invention has been completed based on such findings, and is represented by the KB-3346-5-A substance group, which is a compound represented by the following general formula [A] , and the following general formula [B]. And a KB-3346-5-B substance group which is a compound represented by the following general formula [C].

（式中、R₁およびR₃は水素または塩素、R₂およびR₄は水素またはメチル基である。但し、次の（１）〜（５）の組合せは除く。（１）R ₁ ＝水素，R ₂ ＝水素，R ₃ ＝水素，R ₄ ＝メチル基、（２）R ₁ ＝塩素，R ₂ ＝水素，R ₃ ＝水素，R ₄ ＝水素、（３）R ₁ ＝塩素，R ₂ ＝水素，R ₃ ＝水素，R ₄ ＝メチル基、（４）R ₁ ＝塩素，R ₂ ＝水素，R ₃ ＝塩素，R ₄ ＝水素、（５）R ₁ ＝塩素，R ₂ ＝水素，R ₃ ＝塩素，R ₄ ＝メチル基）

(Wherein R ₁ and R ₃ are hydrogen or chlorine, and R ₂ and R ₄ are hydrogen or a methyl group, except for the following combinations (1) to (5). (1) R ₁ = hydrogen , R ₂ = hydrogen, R ₃ = hydrogen, R ₄ = methyl group, (2) R ₁ = chlorine, R ₂ = hydrogen, R ₃ = hydrogen, R ₄ = hydrogen, (3) R ₁ = chlorine, R ₂ = Hydrogen, R ₃ = hydrogen, R ₄ = methyl group, (4) R ₁ = chlorine, R ₂ = hydrogen, R ₃ = chlorine, R ₄ = hydrogen, (5) R ₁ = chlorine, R ₂ = hydrogen, R ₃ = chlorine, R ₄ = methyl group)

（式中、R₁およびR₃は水素または塩素、R₂は水素またはメチル基である。但し、次の（１）〜（４）の組合せは除く。（１）R ₁ ＝水素，R ₂ ＝水素，R ₃ ＝塩素、（２）R ₁ ＝塩素，R ₂ ＝水素，R ₃ ＝水素、（３）R ₁ ＝塩素，R ₂ ＝水素，R ₃ ＝塩素、（４）R ₁ ＝塩素，R ₂ ＝メチル基，R ₃ ＝塩素）

(In the formula, R ₁ and R ₃ are hydrogen or chlorine, and R ₂ is hydrogen or a methyl group, except for the following combinations (1) to (4). (1) R ₁ = hydrogen, R ₂ = Hydrogen, R ₃ = chlorine, (2) R ₁ = chlorine, R ₂ = hydrogen, R ₃ = hydrogen, (3) R ₁ = chlorine, R ₂ = hydrogen, R ₃ = chlorine, (4) R ₁ = Chlorine, R ₂ = methyl group, R ₃ = chlorine)

（式中、R ₁ 〜R ₃ は次の（１）、（２）の組合せである。（１）R ₁ ＝塩素，R ₂ ＝水素，R ₃ ＝水素、（２）R ₁ ＝水素，R ₂ ＝塩素，R ₃ ＝メチル基）

( Where R _{1 to} R ₃ are combinations of the following (1) and (2). (1) R ₁ = chlorine, R ₂ = hydrogen, R ₃ = hydrogen, (2) R ₁ = hydrogen, R ₂ = chlorine, R ₃ = methyl group)

The present invention also belongs to Streptomyces, claim 1, claim 2, and請 Motomeko 3 was cultured in a medium a microorganism having the ability to produce new KB-3346-5 material according, KB in culture -3346-5 A method for producing a new KB-3346-5 substance that accumulates the substance and collects the KB-3346-5 substance from the culture. In the present invention, the microorganism is Streptomyces sp. KB-3346-5 (FERM BP-10834) strain. The present invention further relates to an antibacterial agent comprising a combination of the KB-3346-5 substance according to claim 1, claim 2 or claim 3 and a β-lactam antibiotic.

As an example of the strain used to produce the novel KB-3346-5 substance represented by the general formulas [A], [B], and [C] of the present invention, Streptomyces sp. KB-3346-5 (FERM BP-10834) newly isolated from the soil of the main island. The culture properties of this strain are as follows.

1. Morphological properties No separation of vegetative mycelium is observed. Aerial hyphae are abundantly grown in yeast / malt extract agar or oatmeal agar. Under microscopic observation, more than 20 spore chains were found on the aerial hyphae, the morphology was spiral, and the size of the spores was about 0.5-0.6 x 0.7-0.8μm. The surface of the spore is smooth. Thick membrane spores, convergent hyphae, spore capsules and zoospores are not found.

2. Properties on various media
The KB-3346-5 strain exhibited normal or vigorous growth on commonly used synthetic and natural media, and the basic mycelium was beige or olive to brown. The medium produced soluble pigments of brown color. The characteristics of growth and color when cultured for 14 days at 28 ° C. on various media are shown in Table 1 below. In addition, the display of the color followed the color classification by the color arrangement card 129a (Japan Color Research Institute).

3. Physiological properties
The physiological properties of KB-3346-5 strain are shown below. For the growth temperature range, describe the results after 14 days of culture, and for the others after 28 ° C for 2 weeks.
(1) Formation of melanin pigment
(B) Tyrosine agar Negative
(B) Peptone / East / Iron Agar Negative
(2) Gelatin liquefaction (glucose, peptone, gelatin medium) Positive
(3) Starch hydrolysis positive
(4) Coagulation of skim milk negative
(5) Peptonization of skim milk negative

(6) Growth temperature range 15-42 ℃
(7) Utilization of carbon source (Prideham Gotrib Agar)
Use: D-fructose, rhamnose, D-mannitol Slight use: D-glucose, L-arabinose, D-xylose, raffinose Not used: sucrose, inositol

4). Chemical taxonomic properties Diaminopimelic acid in the cell wall is of type LL. The main cell menaquinone is MK-9 (H ₆ ) and MK-9 (H ₈ ), and the main fatty acids are iso-C16: 0, anteiso-C15: 0, and iso-C15: 0. The GC content is 74 mol%.

5). Conclusion As described above, spore chains are formed in the aerial hyphae in terms of morphology, and the cell wall is type I (
LL-diaminopimelic acid) and the main quinone is trisaturated MK-9 (H ₆ ) of menaquinone 9 and tetrasaturated MK-9 (H ₈ ) of menaquinone 9. Among fungi, it was classified as Streptomyces. Therefore, this strain was named Streptomyces sp. KB-3346-5.

This strain is Streptomyces sp. KB-3346-5, based on the Budapest Treaty concerning the international approval of the deposit of microorganisms in patent procedures, 1 1-1 Higashi 1-chome, Tsukuba, Ibaraki, Japan Deposited on June 6, 2007 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, located in the 6th (Postal Code 305-8566). The accession number is FERM BP-10834.

Next, the physicochemical properties of the KB-3346-5 substance of the present invention will be described.
1. KB3346-5-A ₁ substance
(1) Property: Yellow powder
(2) Molecular formula: C ₂₈ H ₂₂ O ₈ HR-FAB-MS (m / z) [M + H] ⁺ calculated value 487.1393, measured value 487.1407
(3) Molecular weight: Observe [M + H] ⁺ 487 with 486 FAB-MS (m / z)
(4) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in methanol solution is as shown in Fig. 1. λ _max (e): 279 nm (26924), 304 nm (17885), 380 nm (6853) The absorption maximum of is shown.

(5) Red external absorption spectrum: The infrared absorption spectrum measured by the potassium bromide tablet method is as shown in FIG. 2, and ν _max 3361, 2929, 2852, 1676, 1608, 1462, 1331, 1255, 1159, 1089 , 1020, 984, 935 cm ^-1 etc.
(6) Solubility in solvents: Soluble in methanol, acetone and chloroform. Insoluble in water
(7) Proton and carbon nuclear magnetic resonance spectra: Hydrogen chemical shift (ppm) and carbon chemical shift (ppm) measured with Varian 300 MHz nuclear magnetic resonance spectrometer in heavy acetone are as shown below. .

d _H : 1.83 (3H), 1.87 (3H), 1.99 (3H), 3.61 (3H), 6.30 (1H), 6.41 (1H), 6.41 (1H), 6.74 (1H), 6.95 (1H), 7.46 ( 1H), 12.96 (1H)
d _C : 20.5, 29.8, 30.1, 39.8, 55.8, 97.7, 101.7, 106.3, 109.7, 110.4, 112.1, 121.0, 125.8, 126.5, 137.0, 137.3, 137.6, 141.0, 155.4, 156.0, 158.0, 158.4, 161.5,165.9 , 165.9, 182.2, 186.1, 186.6

Thus it, KB3346-5-A ₁ materials various physicochemical properties and spectral data result of studying in detail the, KB3346-5-A ₁ substance is a chemical structure represented by the following formula [I] Was decided.

2. KB3346-5-A ₂ substances
(1) Property: Yellow powder
(2) Molecular formula: C ₂₈ H ₂₁ ClO ₈ HR-FAB-MS (m / z) [M + H] ⁺ calculated value 521.1003, measured value 521.102
(3) Molecular weight: [M + H] ⁺ 521 observed with 520 FAB-MS (m / z)
(4) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in a methanol solution is as shown in FIG. 5. λ _max (e): 280 nm (25584), 304 nm (17316), 378 nm (8840) The absorption maximum of is shown.

(5) Red external absorption spectrum: Infrared absorption spectrum measured by the potassium bromide tablet method is as shown in FIG. 6, and ν _max 3359, 2927, 2856, 1674, 1604, 1458, 1406, 1323, 1257, 1161 , 1076, 1022, 935 cm ^-1 etc.
(6) Solubility in solvents: Soluble in methanol, acetone and chloroform. Insoluble in water
(7) Proton and carbon nuclear magnetic resonance spectra: Hydrogen chemical shift (ppm) and carbon chemical shift (ppm) measured with Varian 300 MHz nuclear magnetic resonance spectrometer in heavy acetone are as shown below. .

d _H : 1.84 (3H), 1.86 (3H), 1.94 (3H), 3.50 (3H), 6.31 (1H), 6.75 (1H), 6.77 (1H), 7.00 (1H), 7.56 (1H), 12.88 ( 1H)
d _C : 20.1, 29.9, 30.0, 39.9, 60.5, 101.7, 106.3, 110.5, 112.7, 113.0, 114.1,125.4, 125.6, 127.7, 136.3, 136.9, 137.2, 140.2, 153.9, 154.5, 155.7, 155.9, 161.8,165.8 , 165.9, 182.4, 186.1, 186.4

Thus it, KB3346-5-A ₂ substance various physicochemical properties and spectral data result of studying in detail the, KB3346-5-A ₂ substance is a chemical structure represented by the following formula [II] Was decided.

3. KB3346-5-A ₃ substances
(1) Property: Yellow powder
(2) Molecular formula: C ₂₉ H ₂₄ O ₈ HR-FAB-MS (m / z) [M + H] ⁺ calculated value 501.1549, actual value 501.1540
(3) Molecular weight: [M + H] ⁺ 501 observed with 500 FAB-MS (m / z)
(4) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in methanol solution is as shown in FIG. 9, and λ _max (e): 276 nm (21000), 306 nm (12800), 368 nm (5100) The absorption maximum of.

(5) Red external absorption spectrum: The infrared absorption spectrum measured by the potassium bromide tablet method is as shown in FIG. 10, and ν _max 3354, 3205, 2929, 2850, 1670, 1618, 1525, 1464, 1392, 1313 , 1159, 1092, 1026, 957 cm ^-1 etc.
(6) Solubility in solvents: Soluble in methanol, acetone and chloroform. Insoluble in water
(7) Proton and carbon nuclear magnetic resonance spectra: Hydrogen chemical shift (ppm) and carbon chemical shift (ppm) measured by Varian 300 MHz nuclear magnetic resonance spectrometer in deuterated chloroform are as shown below. .

d _H : 1.79 (3H), 1.85 (3H), 2.02 (3H), 3.64 (3H), 3.94 (3H), 6.28 (1H), 6.39 (1H), 6.40 (1H), 6.56 (1H), 7.00 ( 1H), 7.52 (1H), 12.90 (1H)
d _C : 20.4, 29.6, 30.1, 39.2, 55.7, 55.8, 97.1, 101.5, 105.3, 109.4, 109.5, 110.2, 120.4, 124.7, 125.9, 135.6, 135.8, 137.1, 140.2, 154.8, 155.3, 156.4, 156.8, 162.6 , 163.6, 165.0, 182.5, 185.1, 185.8

As described above, KB3346-5-A ₃ substance various physicochemical properties and spectral results data was investigated in detail in, that KB3346-5-A ₃ substance is a chemical structure represented by the following formula [III] It has been determined.

4). KB3346-5-A ₄ substances
(1) Property: Yellow powder
(2) Molecular formula: C ₂₉ H ₂₃ ClO ₈ HR-FAB-MS (m / z) [M + H] ⁺ calculated value 535.1160, actual value 535.1147
(3) Molecular weight: Observe [M + H] ⁺ 535 with 534 FAB-MS (m / z)
(4) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in a methanol solution is as shown in FIG. 13, and λ _max (e): 275 nm (25098), 304 nm (17302), 362 nm (7423) The absorption maximum of.

(5) Red external absorption spectrum: The infrared absorption spectrum measured by the potassium bromide tablet method is as shown in FIG. 14, and ν _max 3379, 3184, 2931, 2852, 1687, 1628, 1464, 1331, 1290, 1188 , 1161, 1095, 993 cm ^-1 etc.
(6) Solubility in solvents: Soluble in methanol, acetone and chloroform. Insoluble in water
(7) Proton and carbon nuclear magnetic resonance spectra: Hydrogen chemical shift (ppm) and carbon chemical shift (ppm) measured by Varian 300 MHz nuclear magnetic resonance spectrometer in deuterated dimethyl sulfoxide are as shown below. is there.

d _H : 1.67 (3H), 1.75 (3H), 1.95 (3H), 3.46 (3H), 3.92 (3H), 6.19 (1H), 6.26 (1H), 6.31 (1H), 6.59 (1H), 7.12 ( 1H), 9.41 (1H), 12.95 (1H)
d _C : 20.3, 28.2, 30.2, 38.9, 55.1, 57.2, 96.7, 100.8, 105.6, 108.2, 108.9, 118.3, 118.6, 120.0, 127.1, 132.4, 134.5, 136.5, 137.1, 154.3, 156.2, 157.3, 157.7, 157.8 , 164.5, 165.3, 180.5, 183.5, 185.9

Thus it, KB3346-5-A ₄ substances various physicochemical properties and spectral data result of studying in detail the, KB3346-5-A ₄ substance is a chemical structure represented by the following formula [IV] Was decided.

5). KB3346-5-A ₅ substances
(1) Property: Yellow powder
(2) Molecular formula: C ₂₉ H ₂₃ ClO ₈ HR-FAB-MS (m / z) [M + H] ⁺ calculated value 535.1160, actual value 535.1168
(3) Molecular weight: Observe [M + H] ⁺ 535 with 534 FAB-MS (m / z)
(4) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in a methanol solution is as shown in FIG. 17, and λ _max (e): 280 nm (29210), 304 nm (19705), 408 nm (11641) The absorption maximum of is shown.

(5) Red external absorption spectrum: The infrared absorption spectrum measured by the potassium bromide tablet method is as shown in FIG. 18, and ν _max 3400, 2931, 2854, 1674, 1626, 1593, 1456, 1392, 1329, 1259 , 1196, 1161, 1101, 980 cm ^-1 etc.
(6) Solubility in solvents: Soluble in methanol, acetone and chloroform. Insoluble in water
(7) Proton and carbon nuclear magnetic resonance spectra: Hydrogen chemical shift (ppm) and carbon chemical shift (ppm) measured with Varian 300 MHz nuclear magnetic resonance spectrometer in heavy acetone are as shown below. .

d _H : 1.83 (3H), 1.85 (3H), 2.01 (3H), 3.50 (3H), 3.95 (3H), 6.30 (1H), 6.73 (1H), 6.85 (1H), 6.98 (1H), 7.56 ( 1H), 12.89 (1H)
d _C : 20.4, 29.9, 30.0, 39.9, 56.5, 60.5, 101.7, 106.3, 110.0, 110.4, 113.2, 114.0, 125.3, 125.4, 128.6, 136.3, 136.8, 137.2, 140.1, 154.6, 155.8, 155.9, 155.9,162.1 , 165.8, 166.1, 182.4, 186.0, 186.4

Thus it, KB3346-5-A ₅ substances various physicochemical properties and spectral data result of studying in detail the, KB3346-5-A ₅ substances is a chemical structure represented by the following formula [V] Was decided.

6). KB3346-5-A ₆ substances
(1) Property: Yellow powder
(2) Molecular formula: C ₂₉ H ₂₃ ClO ₈ HR-FAB-MS (m / z) [M + H] ⁺ calculated value 535.1160, actual value 535.1173
(3) Molecular weight: Observe [M + H] ⁺ 535 with 534 FAB-MS (m / z)
(4) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in a methanol solution is as shown in FIG. 21, and λ _max (e): 276 nm (25525), 305 nm (14845), 392 nm (6568) The absorption maximum of.

(5) Red external absorption spectrum: Infrared absorption spectrum measured by the potassium bromide tablet method is as shown in FIG. 22, and ν _max 3379, 2931, 2850, 1676, 1630, 1577, 1462, 1404, 1308, 1244 , 1159, 1076, 955 cm ^-1 etc.
(6) Solubility in solvents: Soluble in methanol, acetone and chloroform. Insoluble in water
(7) Proton and carbon nuclear magnetic resonance spectra: Hydrogen chemical shift (ppm) and carbon chemical shift (ppm) measured with Varian 300 MHz nuclear magnetic resonance spectrometer in heavy acetone are as shown below. .

d _H : 1.85 (3H), 1.86 (3H), 1.93 (3H), 3.49 (3H), 4.04 (3H), 6.30 (1H), 6.74 (1H), 6.77 (1H), 7.09 (1H), 7.61 ( 1H), 12.88 (1H)
d _C : 20.1, 30.0, 30.0, 39.9, 56.5, 60.5, 101.8, 106.4, 110.4, 110.9, 112.7, 114.1, 124.5, 126.5, 127.5, 136.4, 136.9, 137.0, 140.0, 154.0, 154.5, 155.8, 155.9,163.5 , 165.9, 166.2, 182.5, 185.9, 186.3

Thus it, KB3346-5-A ₆ substances various physicochemical properties and spectral data result of studying in detail the, KB3346-5-A ₆ material is a chemical structure represented by the following formula [VI] Was decided.

7). KB3346-5-A ₇ substances
(1) Property: Yellow powder
(2) Molecular formula: C ₂₉ H ₂₂ Cl ₂ O ₈ HR-FAB-MS (m / z) [M + H] ⁺ calculated value 569.0770, actual value 569.0757
(3) Molecular weight: 568 [M + H] ⁺ 569 observed with FAB-MS (m / z)
(4) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in a methanol solution is as shown in FIG. 25. λ _max (e): 276 nm (25446), 304 nm (17778), 396 nm (10110) The absorption maximum of is shown.

(5) Red external absorption spectrum: The infrared absorption spectrum measured by the potassium bromide tablet method is as shown in FIG. 26, and ν _max 3388, 2929, 2854, 1685, 1631, 1576, 1460, 1406, 1327, 1286 , 1230, 1163, 1078, 999, 947 cm ^-1 etc.
(6) Solubility in solvents: Soluble in methanol, acetone and chloroform. Insoluble in water
(7) Proton and carbon nuclear magnetic resonance spectra: Hydrogen chemical shift (ppm) and carbon chemical shift (ppm) measured with Varian 300 MHz nuclear magnetic resonance spectrometer in heavy acetone are as shown below. .

d _H : 1.84 (3H), 1.86 (3H), 1.93 (3H), 3.51 (3H), 4.08 (3H), 6.30 (1H), 6.74 (1H), 6.78 (1H), 7.22 (1H), 12.98 ( 1H)
d _C : 20.1, 29.8, 29.9, 40.1, 57.6, 60.5, 101.9, 106.4, 109.7, 112.7, 114.1, 120.2, 121.1, 127.0, 127.4, 134.1, 135.6, 136.5, 137.8, 154.2, 154.5, 155.4, 158.6,159.4 , 166.3, 166.6, 181.6, 184.9, 187.0

As described above, KB3346-5-A ₇ various physicochemical properties and spectral results data was investigated in detail the materials, it KB3346-5-A ₇ substance is a chemical structure represented by the following formula [VII] It has been determined.

8). KB3346-5-A ₈ substances
(1) Property: Yellow powder
(2) Molecular formula: C ₃₀ H ₂₅ ClO ₈ HR-FAB-MS (m / z) [M + H] ⁺ calculated value 549.1316, actual value 549.1302
(3) Molecular weight: 548 [M + H] ⁺ 549 observed with FAB-MS (m / z)
(4) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in a methanol solution is as shown in FIG. 29, and λ _max (e): 276 nm (27510), 302 nm (19344), 373 nm (8110) The absorption maximum of is shown.

(5) Red external absorption spectrum: The infrared absorption spectrum measured by the potassium bromide tablet method is as shown in FIG. 30, and ν _max 3402, 2927, 2850, 1685, 1630, 1458, 1331, 1281, 1198, 1163 , 1093, 1057, 999, 951 cm ^-1 etc.
(6) Solubility in solvents: Soluble in methanol, acetone and chloroform. Insoluble in water
(7) Proton and carbon nuclear magnetic resonance spectra: Hydrogen chemical shift (ppm) and carbon chemical shift (ppm) measured by Varian 300 MHz nuclear magnetic resonance spectrometer in deuterated chloroform are as shown below. .

d _H : 1.76 (3H), 1.85 (3H), 2.08 (3H), 3.62 (3H), 3.83 (3H), 3.95 (3H), 6.27 (1H), 6.39 (1H), 6.46 (1H), 6.54 ( 1H), 6.93 (1H), 13.02 (1H)
d _C : 20.7, 28.5, 30.8, 39.4, 55.2, 55.7, 56.8, 96.3, 101.3, 105.4, 106.9, 109.2, 119.2, 120.3, 120.4, 127.1, 133.0, 134.6, 137.1, 137.4, 154.3, 156.4, 158.1, 158.4 , 160.2, 164.3, 165.0, 181.4, 183.8, 186.5

As described above, KB3346-5-A ₈ material various physicochemical properties and spectral results data was investigated in detail in, that KB3346-5-A ₈ material is a chemical structure represented by the following formula [VIII] It has been determined.

9. KB3346-5-A ₉ substances
(1) Property: Yellow powder
(2) Molecular formula: C ₃₀ H ₂₆ O ₈ HR-FAB-MS (m / z) [M + H] ⁺ calculated value 515.1706, actual value 515.1718
(3) Molecular weight: Observe [M + H] ⁺ 515 with 514 FAB-MS (m / z)
(4) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in methanol solution is as shown in FIG. 33. λ _max (e): 277 nm (24364), 303 nm (15266), 374 nm (5962) The absorption maximum of is shown.

(5) Red external absorption spectrum: The infrared absorption spectrum measured by the potassium bromide tablet method is as shown in FIG. 34, and ν _max 3357, 3082, 2931, 2843, 1678, 1630, 1583, 1462, 1317, 1236 , 1196, 1157, 1093, 1030, 955 cm ^-1 etc.
(6) Solubility in solvents: Soluble in methanol, acetone and chloroform. Insoluble in water
(7) Proton and carbon nuclear magnetic resonance spectra: Hydrogen chemical shift (ppm) and carbon chemical shift (ppm) measured by Varian 300 MHz nuclear magnetic resonance spectrometer in deuterated chloroform are as shown below. .

d _H : 1.77 (3H), 1.83 (3H), 2.06 (3H), 3.65 (3H), 3.80 (3H), 3.94 (3H), 6.25 (1H), 6.39 (1H), 6.44 (1H), 6.55 ( 1H), 6.99 (1H), 7.52 (1H), 12.86 (1H)
d _C : 20.6, 29.6, 30.0, 39.2, 55.2, 55.7, 55.8, 96.4, 101.6, 105.5, 106.9, 109.5, 110.0, 120.9, 124.6, 125.8, 135.5, 135.8, 137.0, 140.1, 154.7, 155.4, 156.7, 159.9 , 162.6, 163.9, 165.0, 182.6, 185.0, 185.7

Thus it, KB3346-5-A ₉ material various physicochemical properties and spectral results data was investigated in detail in, KB3346-5-A ₉ material is a chemical structure represented by the following formula [IX] Was decided.

10. KB3346-5-A ₁₀ substances
(1) Property: Yellow powder
(2) Molecular formula: C ₃₀ H ₂₄ Cl ₂ O ₈ HR-FAB-MS (m / z) [M + H] ⁺ calculated value 583.0927, actual value 583.0920
(3) Molecular weight: 582 [M + H] ⁺ 583 observed with FAB-MS (m / z)
(4) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in a methanol solution is as shown in FIG. 37. λ _max (e): 274 nm (27529), 303 nm (18740), 394 nm (7450) The absorption maximum of.

(5) Red external absorption spectrum: The infrared absorption spectrum measured by the potassium bromide tablet method is as shown in FIG. 38, and ν _max 3438, 2925, 2854, 1685, 1630, 1583, 1462, 1394, 1331, 1288 , 1234, 1201, 1169, 1103, 1053, 1003, 958 cm ^-1 etc.
(6) Solubility in solvents: Soluble in methanol, acetone and chloroform. Insoluble in water
(7) Proton and carbon nuclear magnetic resonance spectra: Hydrogen chemical shift (ppm) and carbon chemical shift (ppm) measured by Varian 300 MHz nuclear magnetic resonance spectrometer in deuterated chloroform are as shown below. .

d _H : 1.82 (3H), 1.85 (3H), 1.98 (3H), 3.55 (3H), 3.93 (3H), 3.97 (3H), 6.27 (1H), 6.55 (1H), 6.67 (1H), 6.95 ( 1H), 12.89 (1H)
d _C : 20.2, 29.3, 30.1, 39.6, 56.3, 57.0, 60.5, 101.7, 105.2, 109.4, 109.6, 113.9, 119.1, 121.5, 126.0, 126.6, 133.0, 134.1, 134.9, 137.3, 153.5, 154.4, 155.4, 158.5 , 158.8, 163.6, 165.4, 181.4, 184.0, 186.0

Thus it, KB3346-5-A ₁₀ material various physicochemical properties and spectral results data was investigated in detail in, KB3346-5-A ₁₀ material is a chemical structure represented by the following formula [X] Was decided.

11. KB3346-5-A ₁₁ substances
(1) Property: Yellow powder
(2) Molecular formula: C ₃₀ H ₂₅ ClO ₈ HR-FAB-MS (m / z) [M + H] ⁺ calculated value 549.1316, actual value 549.1325
(3) Molecular weight: 548 [M + H] ⁺ 549 observed with FAB-MS (m / z)
(4) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in a methanol solution is as shown in FIG. 41, and λ _max (e): 278 nm (33866), 354 nm (20495), 376 nm (8165) The absorption maximum of.

(5) Red external absorption spectrum: The infrared absorption spectrum measured by the potassium bromide tablet method is as shown in FIG. 42, and ν _max 3411, 2929, 2854, 1678, 1630, 1587, 1460, 1394, 1311, 1246 , 1198, 1163, 1103, 1034, 958 cm ^-1 etc.
(6) Solubility in solvents: Soluble in methanol, acetone and chloroform. Insoluble in water
(7) Proton and carbon nuclear magnetic resonance spectra: Hydrogen chemical shift (ppm) and carbon chemical shift (ppm) measured by Varian 300 MHz nuclear magnetic resonance spectrometer in deuterated chloroform are as shown below. .

d _H : 1.80 (3H), 1.82 (3H), 2.00 (3H), 3.53 (3H), 3.92 (3H), 3.95 (3H), 6.24 (1H), 6.54 (1H), 6.66 (1H), 7.00 ( 1H), 7.59 (1H), 12.80 (1H)
d _C : 20.3, 29.8, 30.0, 39.3, 55.9, 56.2, 60.5, 101.5, 105.4, 109.5, 110.1, 110.5, 113.8, 124.2, 125.0, 127.1, 134.9, 135.0, 135.8, 139.6, 153.4, 154.6, 155.2, 155.7 , 162.8, 163.9, 165.0, 182.3, 184.9, 185.4

Thus it, KB3346-5-A ₁₁ material various physicochemical properties and spectral results data was investigated in detail in, KB3346-5-A ₁₁ material is a chemical structure represented by the following formula [XI] Was decided.

12 KB3346-5-B ₁ substance
(1) Property: Yellow powder
(2) Molecular formula: C ₂₇ H ₂₂ O ₇ HR-FAB-MS (m / z) [M + H] ⁺ calculated value 459.1444, actual value 459.1446
(3) Molecular weight: [M + H] ⁺ 459 observed with 458 FAB-MS (m / z)
(4) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in a methanol solution is as shown in FIG. 45. λ _max (e): 247 nm (21618), 287 nm (15389), 353 nm (11861) , 417 nm (15343).

(5) Red external absorption spectrum: The infrared absorption spectrum measured by the potassium bromide tablet method is as shown in FIG. 46, and ν _max 3400, 2970, 2925, 2856, 1604, 1441, 1373, 1333, 1275, 1174 , 1155, 1034, 980 cm ^-1 etc.
(6) Solubility in solvents: Soluble in methanol, acetone and chloroform. Insoluble in water
(7) Proton and carbon nuclear magnetic resonance spectra: Hydrogen chemical shift (ppm) and carbon chemical shift (ppm) measured by Varian 300 MHz nuclear magnetic resonance spectrometer in deuterated methanol are as shown below. .

d _H : 1.70 (3H), 1.71 (3H), 1.88 (3H), 6.20 (1H), 6.23 (1H), 6.26 (1H), 6.65 (1H), 6.72 (1H), 7.05 (1H), 7.36 ( 1H)
d _C : 20.7, 34.5, 34.7, 39.7, 100.8, 102.1, 107.1, 107.6, 108.6, 108.8, 110.1, 116.1, 118.2, 122.5, 124.5, 138.3, 140.8, 142.9, 146.5, 155.4, 155.8, 157.5, 160.1, 166.6 , 166.7, 166.9, 191.6

As described above, it KB3346-5-B ₁ substances various physicochemical properties and spectral results data was investigated in detail in, KB3346-5-B ₁ substance is a chemical structure represented by the following formula [XII] It has been determined.

13. KB3346-5-B ₂ substances
(1) Property: Yellow powder
(2) Molecular formula: C ₂₈ H ₂₄ O ₇ HR-FAB-MS (m / z) [M + H] ⁺ calculated value 473.1601, actual value 473.1586
(3) Molecular weight: [M + H] ⁺ 473 observed with 472 FAB-MS (m / z)
(4) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in a methanol solution is as shown in FIG. 49. λ _max (e): 246 nm (28745), 287 nm (19777), 354 nm (15906) , 416 nm (20674).

(5) Red external absorption spectrum: The infrared absorption spectrum measured by the potassium bromide tablet method is as shown in FIG. 50, and ν _max 3361, 2972, 2927, 1604, 1464, 1439, 1373, 1335, 1277, 1180 , 1155, 1103, 1024, 937 cm ^-1 etc.
(6) Solubility in solvents: Soluble in methanol, acetone and chloroform. Insoluble in water
(7) Proton and carbon nuclear magnetic resonance spectra: Hydrogen chemical shift (ppm) and carbon chemical shift (ppm) measured by Varian 300 MHz nuclear magnetic resonance spectrometer in deuterated methanol are as shown below. .

d _H : 1.49 (3H), 1.57 (3H), 1.91 (3H), 3.49 (3H), 6.33 (1H), 6.50 (1H), 6.51 (1H), 6.68 (1H), 6.88 (1H), 7.18 ( 1H), 7.34 (1H)
d _C : 20.9, 34.5, 34.7, 39.5, 55.9, 97.6, 102.2, 107.3, 107.6, 108.4, 109.4, 110.1, 116.0, 118.0, 122.6, 125.6, 137.9, 141.2, 142.8, 146.3, 155.6, 158.4, 158.9,160.4 , 166.6, 166.7, 166.9, 191.2

As described above, it KB3346-5-B ₂ substances various physicochemical properties and spectral results data was investigated in detail in, KB3346-5-B ₂ substances is a chemical structure represented by the following formula [XIII] It has been determined.

14 KB3346-5-B ₃ substances
(1) Property: Yellow powder
(2) Molecular formula: C ₂₈ H ₂₃ ClO ₇ HR-FAB-MS (m / z) [M + H] ⁺ calculated value 507.11211, actual value 507.1194
(3) Molecular weight: [M + H] ⁺ 507 observed with 506 FAB-MS (m / z)
(4) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in a methanol solution is as shown in FIG. 53, and λ _max (e): 247 nm (22821), 287 nm (15838), 354 nm (13055) , 414 nm (17508) absorption maximum.

(5) Red external absorption spectrum: Infrared absorption spectrum measured by the potassium bromide tablet method is as shown in FIG. 54, and ν _max 3400, 2970, 2927, 2856, 1604, 1462, 1442, 1373, 1325, 1277 , 1169, 1076, 1016, 850 cm ^-1 etc.
(6) Solubility in solvents: Soluble in methanol, acetone and chloroform. Insoluble in water
(7) Proton and carbon nuclear magnetic resonance spectra: Hydrogen chemical shift (ppm) and carbon chemical shift (ppm) measured with Varian 300 MHz nuclear magnetic resonance spectrometer in heavy acetone are as shown below. .

d _H : 1.75 (3H), 1.75 (3H), 1.92 (3H), 3.48 (3H), 6.31 (1H), 6.76 (1H), 6.79 (1H), 6.86 (1H), 7.26 (1H), 7.55 ( 1H), 12.80 (1H), 14.58 (1H)
d _C : 20.2, 34.3, 34.5, 39.5, 60.2, 102.0, 107.0, 107.5, 108.3, 110.4, 112.3, 113.4, 116.1, 117.3, 121.9, 131.2, 136.3, 139.3, 142.3, 146.4, 153.4, 154.2, 155.6, 159.4 , 166.0, 166.1, 166.6, 191.2

As described above, it KB3346-5-B ₃ substances various physicochemical properties and spectral results data was investigated in detail in, KB3346-5-B ₃ substances is a chemical structure represented by the following formula [XIV] It has been determined.

１５．KB3346-5-B₄ 物質
(1) 性状：黄色粉末
(2) 分子式：C₂₈H₂₃ClO₇ HR-FAB-MS (m/z) [M+H] ⁺ 計算値507.1211, 実測値507.1188
(3) 分子量：506 FAB-MS (m/z)で[M+H] ⁺ 507 を観測
(4) 紫外部吸収スペクトル：メタノール溶液中で測定した紫外部吸収スペクトルは図５７に示すとおりであり、λ_max (e) : 249 nm (26211), 288 nm (17204), 352 nm (12448), 416nm (17912) の吸収極大を示す。

15. KB3346-5-B ₄ substances
(1) Property: Yellow powder
(2) Molecular formula: C ₂₈ H ₂₃ ClO ₇ HR-FAB-MS (m / z) [M + H] ⁺ calculated value 507.11211, actual value 507.1188
(3) Molecular weight: [M + H] ⁺ 507 observed with 506 FAB-MS (m / z)
(4) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in a methanol solution is as shown in FIG. 57. λ _max (e): 249 nm (26211), 288 nm (17204), 352 nm (12448) , 416 nm (17912).

(5) Red external absorption spectrum: Infrared absorption spectrum measured by the potassium bromide tablet method is as shown in FIG. 58, and λ _max 3400, 2968, 2927, 2852, 1603, 1466, 1441, 1369, 1335, 1281 , 1153, 1090, 1018, 982 cm ^-1 etc.
(6) Solubility in solvents: Soluble in methanol, acetone and chloroform. Insoluble in water
(7) Proton and carbon nuclear magnetic resonance spectra: Hydrogen chemical shift (ppm) and carbon chemical shift (ppm) measured with Varian 300 MHz nuclear magnetic resonance spectrometer in heavy acetone are as shown below. .

d _H : 1.79 (3H), 1.80 (3H), 1.91 (3H), 3.58 (3H), 6.32 (1H), 6.40 (2H), 6.81 (1H), 6.94 (1H), 7.92 (1H), 12.74 ( 1H), 14.51 (1H)
d _C : 20.5, 34.5, 34.9, 39.8, 55.7, 97.2, 102.1, 107.2, 107.6, 108.2, 109.0, 111.7, 113.3, 118.6, 121.5, 124.4, 137.2, 138.1, 139.3, 147.8, 155.0, 155.5, 158.0,158.3 , 166.2, 166.5, 166.6, 191.2

Thus it, KB3346-5-B ₄ material various physicochemical properties and spectral data result of studying in detail the, KB3346-5-B ₄ material is a chemical structure represented by the following formula [XV] Was decided.

１６．KB3346-5-C₁ 物質
(1) 性状：黄色粉末
(2) 分子式：C₃₂H₂₉ClO₉ HR-FAB-MS (m/z) [M+Na] ⁺ 計算値615.1397, 実測値615.1376
(3) 分子量：592 FAB-MS (m/z)で[M+H] ⁺ 593 を観測
(4) 紫外部吸収スペクトル：メタノール溶液中で測定した紫外部吸収スペクトルは図６１に示すとおりであり、λ_max (e) : 248 nm (19095), 410 nm (18561)の吸収極大を示す。

16. KB3346-5-C ₁ substance
(1) Property: Yellow powder
(2) Molecular formula: C ₃₂ H ₂₉ ClO ₉ HR-FAB-MS (m / z) [M + Na] ⁺ calculated value 615.11397, actual value 615.1376
(3) Molecular weight: 592 Observation of [M + H] ⁺ 593 with FAB-MS (m / z)
(4) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in a methanol solution is as shown in FIG. 61, and shows the absorption maximum at λ _max (e): 248 nm (19095), 410 nm (18561).

(5) Red external absorption spectrum: The infrared absorption spectrum measured by the potassium bromide tablet method is as shown in FIG. 62, and ν _max 3186, 2931, 1716, 1685, 1599, 1562, 1462, 1327, 1271, 1161 , 1016, 997 cm ^-1 etc.
(6) Specific rotation: [α] _D ²⁴ + 86.4 ° (c 0.1, methanol)
(7) Solubility in solvents: Soluble in methanol, acetone and chloroform. Insoluble in water
(8) Proton and carbon nuclear magnetic resonance spectra: Hydrogen chemical shift (ppm) and carbon chemical shift (ppm) measured by Varian 300 MHz nuclear magnetic resonance spectrometer in heavy acetone are as shown below. .

d _H : 1.13 (3H), 1.53 (3H), 1.92 (3H), 2.01 (3H), 2.83 (1H), 3.54 (1H), 3.64 (3H), 4.03 (3H), 6.24 (1H), 6.37 ( 1H), 6.39 (1H), 6.53 (1H), 7.09 (1H), 12.23 (1H) d _C : 20.7, 21.4, 27.4, 29.9, 43.1, 49.8, 54.9, 55.9, 57.3, 97.4, 102.3, 106.3, 108.0 , 108.2, 109.4, 120.8, 120.8, 123.7, 126.7, 135.4, 137.0, 139.3, 153.6, 158.1, 158.4, 158.6, 166.3, 166.4, 169.6, 189.1, 196.5, 205.4

As described above, it KB3346-5-C ₁ materials various physicochemical properties and spectral results data was investigated in detail in, KB3346-5-C ₁ substance is a chemical structure represented by the following formula [XVI] It has been determined.

17. KB3346-5-C ₂ substances
(1) Property: Yellow powder
(2) Molecular formula: C ₃₃ H ₃₁ ClO ₉ HR-FAB-MS (m / z) [M + Na] ⁺ calculated value 629.1554, actual value 629.1549
(3) Molecular weight: [M + H] ⁺ 607 observed with 606 FAB-MS (m / z)
(4) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in a methanol solution is as shown in FIG. 65, and shows the absorption maximum at λ _max (e): 249 nm (19513), 410 nm (19271).

(5) Red external absorption spectrum: The infrared absorption spectrum measured by the potassium bromide tablet method is as shown in FIG. 66, and ν _max 3431, 2935, 2848, 1714, 1674, 1599, 1464, 1387, 1329, 1261 , 1200, 1161, 1103, 1024 cm ^-1 etc.
(6) Specific rotation: [α] _D ²² + 32.6 ° (c 0.1, methanol)
(7) Solubility in solvents: Soluble in methanol, acetone and chloroform. Insoluble in water
(8) Proton and carbon nuclear magnetic resonance spectra: Hydrogen chemical shift (ppm) and carbon chemical shift (ppm) measured by Varian 300 MHz nuclear magnetic resonance spectrometer in heavy acetone are as shown below. .

d _H : 1.01 (3H), 1.59 (3H), 1.89 (3H), 1.96 (3H), 2.89 (1H), 3.52 (1H), 3.63 (3H), 3.94 (6H), 6.26 (1H), 6.46 ( 1H), 6.61 (1H), 6.98 (1H), 7.73 (1H), 12.52 (1H), 14.74 (1H)
d _C : 20.3, 21.0, 26.5, 29.6, 43.3, 52.4, 53.2, 55.8, 56.3, 60.5, 102.1, 105.4, 107.6, 108.6, 108.6, 110.1, 113.5, 123.8, 124.6, 130.2, 134.9, 137.3, 139.3, 152.9 , 153.1, 154.7, 161.9, 162.6, 165.4, 168.8, 188.5, 197.8, 205.4

As described above, it KB3346-5-C ₂ material various physicochemical properties and spectral results data was investigated in detail in, KB3346-5-C ₂ material is a chemical structure represented by the following formula [XVII] It has been determined.

Next, the biological properties of the KB-3346-5 substance of the present invention will be described in detail.
1. Evaluation method of imipenem (IPM) activity enhancing action by paper disc method Clinically isolated methicillin resistant Staphylococcus aureus (MRSA) K24 strain was used as a test bacterium. S. aureus K24 strain was cultured in Mueller-Hinton broth (2.1% w / v, Difco) at 37 ° C for 20 hours, then suspended in the same medium to 0.5 McFarland (approximately 10 ⁸ CFU / mL) Medium (Mueller-Hinton broth 2.1% w / v, Agar 1.5%) and medium with the same composition were treated with imipenem (Ahiyari Pharmaceutical Co., Ltd., Thienam intramuscular titer 0.5). It was smeared on the medium added to a concentration of 10 μg / mL. The smearing was performed using a sterile swab (Kawamoto Sangyo) according to the US National Committee for Laboratory Standard (NCCLS) method.

  The antibacterial activity on each medium against the test bacteria was determined by measuring the diameter of the inhibition circle diameter after culturing at 37 ° C. for 20 hours by the paper disk method (diameter 6 mm, thin: Advantec). The measured inhibition circle diameter was expressed in units of mm and is shown in Table 2 below. As a result, all KB-3346-5 substances showed no inhibition circles in the absence of imipenem under the condition of 0.3 μg / disk, whereas in the presence of imipenem, all of the diameters ranged from 10 mm to 21 mm. The inhibition circle was observed, and the imipenem activity enhancing action was confirmed.

2. Evaluation method of imipenem activity enhancement by micro liquid dilution method
Regarding KB3346-5 substances, activity enhancement against imipenem was evaluated according to the micro liquid dilution method. The evaluation was performed by partially modifying the standard method of the Japanese Society of Chemotherapy (Chemotherapy 38, 103-105, 1990). Add 85 μL of Mueller-Hinton broth (2.1% w / v) to each well of a 96 well plate (Corning, USA), and then add imipenem serially diluted with sterile water in advance to a final concentration of 4.8 × 10 ⁻⁴ From this, 5 μL was added to each well so as to be 256 μg / mL.

Furthermore, 5 μL of KB-3346-5 substance methanol solution was added to each of these wells so that the final concentration was 0.5 μg / mL without affecting growth. After mixing well, the MRSA K24 strain was suspended in an amount equivalent to 0.5 McFarland (about 10 ⁸ CFU / mL), and 5 μL of each well was inoculated with an inoculum solution diluted 60-fold with the same medium. After culturing at 37 ° C. for 20 hours, among the wells in which the growth of bacteria was not visually observed, the minimum concentration of imipenem was defined as the minimum growth inhibitory concentration (MIC).

  The MIC when imipenem alone and the MIC when imipenem and KB-3346-5 are used in combination are shown in Table 3 below. Table 3 shows the results of evaluating the imipenem activity enhancing action of the KB-3346-5 substance according to the present invention by the micro liquid dilution method. As a result, when imipenem alone was used, the MIC was 32 μg / mL, but the combined use of the KB-3346-5 substance decreased the MIC to 0.06 μg / mL, confirming a 100-500-fold increase in activity.

3. Antibacterial action against various test bacteria
For KB3346-5-A ₄ and KB3346-5-B _2, 6 kinds of test bacteria Bacillus subtilis PCI 219, Micrococcus luteus PCI 1001, Escherichia coli NIHJ, against Xanthomonas campestris pv. Oryzae KB88, Mucor racemosus IFO 4581 and Candida albicans KF1 Antibacterial activity was measured by the paper disk method (diameter 6 mm, thin). The culture temperature was 27 ° C. for X.campestris pv. Oryzae , M. racemosus and C. albicans , and 37 ° C. for the other test bacteria. The culture time was 24 hours. The inhibition circle diameter (mm) for each test bacterium is as shown in Table 4 below.

EXAMPLES The present invention will be specifically described below with reference to examples of the present invention, but the present invention is not limited to these examples.
Agar slope medium (starch 1.0% (Kanto Chemical), N-Zet Amin 0.3% (Wako Pure Chemical Industries), yeast extract 0.1% (Oriental yeast), meat extract 0.1% (Kyokuto Pharmaceutical), calcium carbonate 0.3% (Kanto) Chemical) and KB-3346-5 strain cultured in 1.2% agar (prepared to Shimizu Foods pH 7.0), seed medium (starch 2.4% (Kanto Chemical), glucose 0.1% (Wako Pure Chemicals), peptone 0.3% ( Far East Pharmaceutical Industry), Meat Extract 0.3% (Farto Pharmaceutical Industry), Yeast Extract 0.5% (Oriental Yeast), CaCO ₃ 0.4% (Kanto Chemical) adjusted to pH 7.0) After inoculating platinum ears and incubating on a shaker (300 rpm) for 3 days at 27 ° C, inoculate 10% each into 6 500 mL Erlenmeyer flasks containing 100 mL of the same type medium, and 3 days at 27 ° C. The cells were cultured on a rotary shaker (210 rpm).

After that, production medium (glucose 0.5% (Wako Pure Chemicals), corn steep powder 0.5% (Marco), oatmeal 1.0% (Japanese food manufacturing), Pharmamedia 1.0% (Iwaki), K ₂ HPO ₄ 0.5% (Kanto Chemical) , MgSO ₄ · 7H ₂ O 0.5% (Wako Pure Chemicals), metal salt solution 0.1% (FeSO ₄ · 7H ₂ O 0.1% (Kanto Chemical), MnCl ₂ · 4H ₂ O 0.1% (Kanto Chemical), ZnSO ₄ · 7H ₂ O 0.1% (Kanto Chemical), CuSO ₄ · 5H ₂ O 0.1% (Kanto Chemical), CoCl ₂ · 6H ₂ O 0.1% (Wako Pure Chemical Industries, Ltd.), pH 7.0) adjusted to 100 mL) 500 mL 50% Erlenmeyer flasks were inoculated 10% each, and cultured at 28 ° C. for 8 days on a rotary shaker.

  After completion of the culture, acetone (5 L) was added to the culture solution (5 L), and after stirring for 30 minutes, the cells were separated by filtration to obtain an acetone extract. From the water residue obtained by evaporating acetone from the acetone extract under reduced pressure, the active ingredient was extracted with ethyl acetate (10 L), and the ethyl acetate layer was concentrated to dryness to obtain the active crude substance (6.73 g). Got. The crude material was roughly purified using a silica gel column (silica gel 60 (mesh 60-230), Merck, 220 g).

  After washing the silica gel column with chloroform, chromatography is performed using each mixed solvent of chloroform-methanol (100: 0, 100: 1, 50: 1, 10: 1, 1: 1, 0: 100) as a developing solvent. The eluate was fractionated for each elution condition. The active fraction (100: 1) was concentrated to dryness to obtain 4370 mg of a brown oily substance, and the active fraction (10: 1) was concentrated to dryness to obtain 830 mg of a brown oily substance.

  Subsequently, 1000 mg of the active fraction (100: 1) was purified with a silica gel column (silica gel 60 (mesh 230-400), 30 g). Chromatography was performed using each mixed solvent of chloroform-methanol (100: 0, 100: 1, 50: 1, 0: 100) as a developing solvent, and the eluate was fractionated into 20 10 mL portions under each condition. Active fraction 1 (100: 0 fraction numbers 1 to 100: 2 fraction number 16) was concentrated to dryness to give 900.6 mg of a brown oily substance, and active fraction 2 (100: 2 fraction numbers 17 to 100: 2 Fraction number 20) was concentrated to dryness to obtain 69.2 mg of a brown oily substance.

137.0 mg of the active fraction 1 was dissolved in a small amount of methanol and purified by preparative HPLC (column: Pegasil ODS, 20φx 250 mm, Senshu Science). Absorption at UV 210 nm was monitored at a flow rate of 8 mL / min using a 60% acetonitrile aqueous solution as a mobile phase. Peaks showing activity were observed at retention times of 43 min, 49 min, 51 min, and 55 min, and these peaks were collected. By concentrating and drying the preparative solution under reduced pressure, a mixture of KB3346-5-A ₈ substances and KB3346-5-A ₉ substances, KB3346-5-A ₁₀ substances, KB3346-5-A ₁₁ substances, KB3346-5 -C ₂ substances were isolated in yields 60.8 mg, 17.3 mg, 27.8 mg and 2.0 mg, respectively.

Furthermore, a mixture of KB3346-5-A ₈ substance and KB3346-5-A ₉ substance was dissolved in a small amount of methanol, and final purification was performed by preparative HPLC (column: Develosil, 20φ × 250 mm, Nomura Science). Absorption at UV 210 nm was monitored at a flow rate of 8 mL / min using 70% aqueous acetonitrile as a mobile phase. Peaks showing activity were observed at retention times of 23 min and 24 min, and these peaks were collected. The fractions were concentrated to dryness under reduced pressure to isolate KB3346-5-A ₈ substance and KB3346-5-A ₉ substance in a yield of 27.4 mg and 21.8 mg, respectively.

The active fraction 2 was dissolved in a small amount of methanol and purified by preparative HPLC (column: Pegasil ODS, 20φ × 250 mm). Absorption at UV 210 nm was monitored at a flow rate of 8 mL / min using a 55% aqueous acetonitrile solution as a mobile phase. Peaks showing activity were observed at retention times of 25 min, 36 min, and 38 min, and these peaks were collected. Concentrate and dry the fractionated solution under reduced pressure to isolate KB3346-5-A ₅ substances, KB3346-5-A ₆ substances, KB3346-5-A ₇ substances in yields 26.4 mg, 11.5 mg, and 14.3 mg, respectively. did.

  Moreover, the active fraction (10: 1) of the silica gel chromatography performed first was also purified on a silica gel column (silica gel 60 (mesh 230-400), 25 g). Chromatography was performed using each mixed solvent of chloroform-methanol (50: 1, 10: 1, 0: 100) as a developing solvent, and the eluate was fractionated into 20 10 mL portions under each condition. Active fraction 3 (50: 1 fraction number 13 to 10: 1 fraction number 4) was concentrated to dryness to obtain 170.8 mg of a brown oily substance, and active fraction 4 (10: 1 fraction number 7 to 10: 1 fraction number 11) was concentrated to dryness to obtain 251.3 mg of a brown oily substance.

The active fraction 3 was dissolved in a small amount of methanol and purified by preparative HPLC (column: Pegasil ODS, 20φ × 250 mm). The absorption at UV 220 nm was monitored using a 75% aqueous methanol solution as a mobile phase at a flow rate of 8 mL / min. Peaks showing activity were observed at retention times of 17 min, 19 min, 22 min, 27 min, and 36 min, and these peaks were collected. By concentrating and drying the preparative solution under reduced pressure, KB3346-5-A ₂ substances, KB3346-5-B ₃ substances, KB3346-5-C ₁ substance mixture, KB3346-5-B ₄ substances, KB3346-5 A mixture of ₃ substances -A3 and ₄ substances KB3346-5-A was obtained in a yield of 13.9 mg, 10.0 mg, 20.2 mg, 30.5 mg and 61.2 mg, respectively.

Furthermore, a mixture of KB3346-5-C ₁ substance was dissolved in a small amount of methanol, and purified by preparative HPLC (column: Pegasil ODS, 20φ × 250 mm). Absorption at UV 210 nm was monitored at a flow rate of 8 mL / min using a 55% aqueous acetonitrile solution as a mobile phase. A peak showing activity at a retention time of 18 min was observed, and this peak was collected. The fractionated solution was concentrated to dryness under reduced pressure to isolate KB3346-5-C ₁ substance in a yield of 14.2 mg.

Further, a mixture of KB3346-5-A ₃ substance and KB3346-5-A ₄ substance was dissolved in a small amount of methanol and purified by preparative HPLC (column: Pegasil ODS, 20φ × 250 mm). Absorption at UV 210 nm was monitored at a flow rate of 8 mL / min using a 55% aqueous acetonitrile solution as a mobile phase. Peaks showing activity were observed at retention times of 21 min and 24 min, and these peaks were collected. The fractionated solution was concentrated to dryness under reduced pressure to isolate KB3346-5-A ₃ substance and KB3346-5-A ₄ substance in a yield of 21.5 mg and 32.8 mg, respectively.

The active fraction 4 was dissolved in a small amount of methanol and purified by preparative HPLC (column: Pegasil ODS, 20φ × 250 mm). Absorption at UV 210 nm was monitored at a flow rate of 8 mL / min using 50% aqueous acetonitrile as a mobile phase. Peaks showing activity were observed at retention times of 12 min and 19 min, and these peaks were collected. The fractionated solution was concentrated to dryness under reduced pressure to obtain a mixture of KB3346-5-B ₁ substance, KB3346-5-A ₁ substance and KB3346-5-B ₂ substance in a yield of 29.9 mg and 89.6 mg, respectively.

Furthermore, a mixture of KB3346-5-B ₁ substance and KB3346-5-A ₁ substance was dissolved in a small amount of methanol and purified by preparative HPLC (column: Pegasil ODS, 20φ × 250 mm). UV absorption at 220 nm was monitored at a flow rate of 7 mL / min using a 65% aqueous methanol solution as a mobile phase. Peaks showing activity were observed at retention times of 36 min and 44 min, and these peaks were collected. The fractions were concentrated to dryness under reduced pressure to isolate KB3346-5-B ₁ substance and KB3346-5-A ₁ substance in a yield of 10.0 mg and 14.2 mg, respectively.

  As described above, a microorganism represented by the strain KB-3346-5 belonging to the genus Streptomyces having the ability to produce a new KB-3346-5 substance was cultured in a medium, and KB-3346 collected from the culture solution. -5 Since the substance has the effect of enhancing its effect when used in combination with β-lactam antibiotics that are used as antibacterial agents, methicillin-resistant Staphylococcus aureus (MRSA) infections and β-lactam antibiotic resistance It is expected to be useful as a therapeutic agent for infectious diseases caused by multidrug-resistant bacteria including

2 shows an ultraviolet absorption spectrum (in a methanol solution) of KB3346-5-A ₁ substance according to the present invention. 2 shows an infrared absorption spectrum (potassium bromide method) of KB3346-5-A ₁ substance according to the present invention. 2 shows a proton nuclear magnetic resonance spectrum (in heavy acetone) of KB3346-5-A ₁ substance according to the present invention. 2 shows a carbon nuclear magnetic resonance spectrum (in heavy acetone) of KB3346-5-A ₁ substance according to the present invention. ₂ shows an ultraviolet absorption spectrum (in a methanol solution) of KB3346-5-A ₂ substance according to the present invention. ₂ shows an infrared absorption spectrum (potassium bromide method) of KB3346-5-A ₂ substance according to the present invention. ₂ shows proton nuclear magnetic resonance spectra (in heavy acetone) of _two KB3346-5-A substances according to the present invention. ₂ shows a carbon nuclear magnetic resonance spectrum (in heavy acetone) of _two KB3346-5-A substances according to the present invention. ₃ shows an ultraviolet absorption spectrum (in a methanol solution) of KB3346-5-A ₃ substance according to the present invention. ₃ shows an infrared absorption spectrum (potassium bromide method) of KB3346-5-A ₃ substance according to the present invention. FIG. 2 shows a proton nuclear magnetic resonance spectrum (in deuterated chloroform) of KB3346-5-A ₃ substance according to the present invention. FIG. 2 shows a carbon nuclear magnetic resonance spectrum (in deuterated chloroform) of KB3346-5-A ₃ substance according to the present invention. 2 shows an ultraviolet absorption spectrum (in a methanol solution) of the KB3346-5-A ₄ substance according to the present invention. 2 shows an infrared absorption spectrum (potassium bromide method) of KB3346-5-A ₄ substance according to the present invention. FIG. 3 shows a proton nuclear magnetic resonance spectrum (in deuterated dimethyl sulfoxide) of KB3346-5-A ₄ substance according to the present invention. FIG. FIG. 5 shows a carbon nuclear magnetic resonance spectrum (in deuterated dimethyl sulfoxide) of KB3346-5-A ₄ substance according to the present invention. FIG. It shows the ultraviolet absorption spectrum of KB3346-5-A ₅ substances according to the invention (methanol solution). 2 shows an infrared absorption spectrum (potassium bromide method) of KB3346-5-A ₅ substance according to the present invention. FIG. ₅ shows a proton nuclear magnetic resonance spectrum (in heavy acetone) of KB3346-5-A ₅ substance according to the present invention. FIG. 2 shows a carbon nuclear magnetic resonance spectrum (in heavy acetone) of KB3346-5-A ₅ substance according to the present invention. 2 shows an ultraviolet absorption spectrum (in a methanol solution) of KB3346-5-A ₆ substance according to the present invention. 2 shows an infrared absorption spectrum (potassium bromide method) of KB3346-5-A ₆ substance according to the present invention. FIG. 5 shows a proton nuclear magnetic resonance spectrum (in heavy acetone) of KB3346-5-A ₆ substance according to the present invention. FIG. 3 shows a carbon nuclear magnetic resonance spectrum (in heavy acetone) of KB3346-5-A ₆ substance according to the present invention. FIG. 5 shows an ultraviolet absorption spectrum (in a methanol solution) of KB3346-5-A ₇ substance according to the present invention. FIG. 2 shows an infrared absorption spectrum (potassium bromide method) of KB3346-5-A ₇ substance according to the present invention. FIG. 5 shows a proton nuclear magnetic resonance spectrum (in heavy acetone) of KB3346-5-A ₇ substance according to the present invention. FIG. FIG. 5 shows a carbon nuclear magnetic resonance spectrum (in heavy acetone) of KB3346-5-A ₇ substance according to the present invention. FIG. 2 shows an ultraviolet absorption spectrum (in a methanol solution) of KB3346-5-A ₈ substance according to the present invention. 2 shows an infrared absorption spectrum (potassium bromide method) of KB3346-5-A ₈ substance according to the present invention. FIG. 5 shows a proton nuclear magnetic resonance spectrum (in deuterated chloroform) of KB3346-5-A ₈ substance according to the present invention. FIG. 2 shows a carbon nuclear magnetic resonance spectrum (in deuterated chloroform) of KB3346-5-A ₈ substance according to the present invention. 2 shows an ultraviolet absorption spectrum (in a methanol solution) of KB3346-5-A ₉ substance according to the present invention. 3 shows an infrared absorption spectrum (potassium bromide method) of KB3346-5-A ₉ substance according to the present invention. FIG. 5 shows a proton nuclear magnetic resonance spectrum (in deuterated chloroform) of KB3346-5-A ₉ substance according to the present invention. FIG. 2 shows a carbon nuclear magnetic resonance spectrum (in deuterated chloroform) of KB3346-5-A ₉ substance according to the present invention. FIG. 5 shows an ultraviolet absorption spectrum (in a methanol solution) of KB3346-5-A ₁₀ substance according to the present invention. FIG. 2 shows an infrared absorption spectrum (potassium bromide method) of KB3346-5-A ₁₀ substance according to the present invention. FIG. 2 shows a proton nuclear magnetic resonance spectrum (in deuterated chloroform) of KB3346-5-A ₁₀ substance according to the present invention. FIG. 2 shows a carbon nuclear magnetic resonance spectrum (in deuterated chloroform) of KB3346-5-A ₁₀ substance according to the present invention. It shows the ultraviolet absorption spectrum of KB3346-5-A ₁₁ material according to the present invention (methanol solution). It shows the infrared absorption spectrum of KB3346-5-A ₁₁ material according to the present invention (potassium bromide method). It shows the proton nuclear magnetic resonance spectrum of KB3346-5-A ₁₁ material according to the present invention (in deuterated chloroform). Shows a carbon nuclear magnetic resonance spectrum of KB3346-5-A ₁₁ material according to the present invention (in deuterated chloroform). 2 shows an ultraviolet absorption spectrum (in a methanol solution) of KB3346-5-B ₁ substance according to the present invention. 2 shows an infrared absorption spectrum (potassium bromide method) of KB3346-5-B ₁ substance according to the present invention. 2 shows a proton nuclear magnetic resonance spectrum (in deuterated methanol) of KB3346-5-B ₁ substance according to the present invention. 2 shows a carbon nuclear magnetic resonance spectrum (in deuterated methanol) of KB3346-5-B ₁ substance according to the present invention. ₂ shows an ultraviolet absorption spectrum (in a methanol solution) of KB3346-5-B ₂ substance according to the present invention. ₂ shows an infrared absorption spectrum (potassium bromide method) of KB3346-5-B ₂ substance according to the present invention. FIG. 2 shows a proton nuclear magnetic resonance spectrum (in deuterated methanol) of KB3346-5-B ₂ according to the present invention. FIG. ₂ shows a carbon nuclear magnetic resonance spectrum (in deuterated methanol) of KB3346-5-B ₂ material according to the present invention. 2 shows an ultraviolet absorption spectrum (in a methanol solution) of KB3346-5-B ₃ substance according to the present invention. ₃ shows an infrared absorption spectrum (potassium bromide method) of KB3346-5-B ₃ substance according to the present invention. FIG. ₃ shows a proton nuclear magnetic resonance spectrum (in heavy acetone) of KB3346-5-B ₃ according to the present invention. FIG. 2 shows a carbon nuclear magnetic resonance spectrum (in heavy acetone) of KB3346-5-B ₃ substance according to the present invention. 2 shows an ultraviolet absorption spectrum (in a methanol solution) of KB3346-5-B ₄ substance according to the present invention. 2 shows an infrared absorption spectrum (potassium bromide method) of KB3346-5-B ₄ substance according to the present invention. FIG. 5 shows a proton nuclear magnetic resonance spectrum (in heavy acetone) of KB3346-5-B ₄ according to the present invention. FIG. FIG. 5 shows a carbon nuclear magnetic resonance spectrum (in heavy acetone) of KB3346-5-B ₄ substance according to the present invention. FIG. 3 shows an ultraviolet absorption spectrum (in a methanol solution) of KB3346-5-C ₁ substance according to the present invention. 3 shows an infrared absorption spectrum (potassium bromide method) of KB3346-5-C ₁ substance according to the present invention. FIG. 2 shows a proton nuclear magnetic resonance spectrum (in heavy acetone) of KB3346-5-C ₁ substance according to the present invention. FIG. 3 shows a carbon nuclear magnetic resonance spectrum (in heavy acetone) of KB3346-5-C ₁ substance according to the present invention. ₂ shows an ultraviolet absorption spectrum (in a methanol solution) of KB3346-5-C ₂ substance according to the present invention. 3 shows an infrared absorption spectrum (potassium bromide method) of KB3346-5-C ₂ substance according to the present invention. FIG. 3 shows a proton nuclear magnetic resonance spectrum (in deuterated chloroform) of KB3346-5-C ₂ substance according to the present invention. FIG. ₂ shows a carbon nuclear magnetic resonance spectrum (in deuterated chloroform) of KB3346-5-C ₂ substance according to the present invention.

Claims (6)

The following general formula [A]

(Wherein R ₁ and R ₃ are hydrogen or chlorine, and R ₂ and R ₄ are hydrogen or a methyl group, except for the following combinations (1) to (5). (1) R ₁ = hydrogen , R ₂ = hydrogen, R ₃ = hydrogen, R ₄ = methyl group, (2) R ₁ = chlorine, R ₂ = hydrogen, R ₃ = hydrogen, R ₄ = hydrogen, (3) R ₁ = chlorine, R ₂ = Hydrogen, R ₃ = hydrogen, R ₄ = methyl group, (4) R ₁ = chlorine, R ₂ = hydrogen, R ₃ = chlorine, R ₄ = hydrogen, (5) R ₁ = chlorine, R ₂ = hydrogen, KB-3346-5-A substance group, which is a compound represented by R ₃ = chlorine, R ₄ = methyl group ). The following general formula [B]

(In the formula, R ₁ and R ₃ are hydrogen or chlorine, and R ₂ is hydrogen or a methyl group, except for the following combinations (1) to (4). (1) R ₁ = hydrogen, R ₂ = Hydrogen, R ₃ = chlorine, (2) R ₁ = chlorine, R ₂ = hydrogen, R ₃ = hydrogen, (3) R ₁ = chlorine, R ₂ = hydrogen, R ₃ = chlorine, (4) R ₁ = KB-3346-5-B substance group, which is a compound represented by chlorine, R ₂ = methyl group, R ₃ = chlorine ). The following general formula [C]

( Where R _{1 to} R ₃ are combinations of the following (1) and (2). (1) R ₁ = chlorine, R ₂ = hydrogen, R ₃ = hydrogen, (2) R ₁ = hydrogen, KB-3346-5-C substance group, which is a compound represented by R ₂ = chlorine, R ₃ = methyl group ). Belong to actinomycetes, the following general formula [A]

(Wherein R ₁ and R ₃ are hydrogen or chlorine, and R ₂ and R ₄ are hydrogen or a methyl group, except for the following combinations (1) to (5). (1) R ₁ = hydrogen , R ₂ = hydrogen, R ₃ = hydrogen, R ₄ = methyl group, (2) R ₁ = chlorine, R ₂ = hydrogen, R ₃ = hydrogen, R ₄ = hydrogen, (3) R ₁ = chlorine, R ₂ = Hydrogen, R ₃ = hydrogen, R ₄ = methyl group, (4) R ₁ = chlorine, R ₂ = hydrogen, R ₃ = chlorine, R ₄ = hydrogen, (5) R ₁ = chlorine, R ₂ = hydrogen, KB-3346-5-A substance group represented by R ₃ = chlorine, R ₄ = methyl group ), the following general formula [B]

(In the formula, R ₁ and R ₃ are hydrogen or chlorine, and R ₂ is hydrogen or a methyl group, except for the following combinations (1) to (4). (1) R ₁ = hydrogen, R ₂ = Hydrogen, R ₃ = chlorine, (2) R ₁ = chlorine, R ₂ = hydrogen, R ₃ = hydrogen, (3) R ₁ = chlorine, R ₂ = hydrogen, R ₃ = chlorine, (4) R ₁ = Chlorine, R ₂ = methyl group, R ₃ = chlorine ) KB-3346-5-B substance group, and the following general formula [C]

( Where R _{1 to} R ₃ are combinations of the following (1) and (2). (1) R ₁ = chlorine, R ₂ = hydrogen, R ₃ = hydrogen, (2) R ₁ = hydrogen, A microorganism having the ability to produce the KB-3346-5-C substance group (hereinafter collectively referred to as KB-3346-5 substance) represented by R ₂ = chlorine, R ₃ = methyl group ) cultured, allowed accumulate KB-3346-5- substance in the culture, a method of manufacturing a K B-3346-5 material collecting KB-3346-5 substance from the culture,
The micro organism, preparation of KB-3346-5 material, which is a Streptomyces sp. (Streptomyces sp.) KB-3346-5 (FERM BP-10834). Streptomyces sp. KB-3346-5 (FERM BP-10834) strain.   Any of the KB-3346-5-A substance group according to claim 1, the KB-3346-5-B substance group according to claim 2, and the KB-3346-5-C substance group according to claim 3. An antibacterial agent characterized by combining a substance group of the above and a β-lactam antibiotic.

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