JPH0428278B2 - - Google Patents

Description

[Detailed description of the invention]

B. Industrial application field The present invention is based on the formula; (In the formula, R is

【formula】

[Formula] or H; R ₁ represents CH ₃ or H. However, R

When [Formula], R ₁ is not H. ) antibiotic PA−
The present invention relates to 45052, its pharmaceutically acceptable salts, its production method, its producing bacteria, and an animal growth promoter containing PA-45052 as an active ingredient. B. Prior Art With the recent widespread use of antibiotics, the emergence of multidrug-resistant bacteria, particularly methicillin-resistant bacteria, has become a problem. Methicillin-resistant bacteria are not only resistant to methicillin, but also to many other antibiotics, including aminoglycoside antibiotics, tetracycline antibiotics, cefem antibiotics, penicillin antibiotics, carbapenem antibiotics, and macrolide antibiotics. It is resistant to many antibiotics. It has been noticed that glycopeptide antibiotics, especially vancomycin antibiotics, are active against methicillin-resistant bacteria (ANTIMICROBIAL AGENT AND CHEMOTHERAPY).
CHEMOTHERAPY) 28 , 660−662 (1985)). Vancomycin is an antibiotic that has been known for a long time (Special Publication No. 33-8450), and new vancomycin analogs have also been discovered (ANTIMICROBIAL AGENT, AND CHEMOTHERAPY).
CHEMOTHERAPY) 28 , 660−662 (1985),
THE JOURNAL OF ANTIBIOTICS 37 ,
446-453 (1984), 38 , 1-8 (1985), 38 , 51-57
(1985), JP-A-60-139623, 60-199397, 60-
231698, 60−237099, etc.). In addition, the present inventors previously discovered that a strain belonging to the genus Nocardia is a vancomycin antibiotic PA-42867-, which has strong antibacterial activity.
A and PA-42867-B were found to be produced (Japanese Patent Application No. 61-14389 (Japanese Unexamined Patent Publication No. 62-174099)), and derivatives thereof were also created (Japanese Patent Application No. 61-188865 (Japanese Unexamined Patent Publication No. 63-44598)). )). However, the antibiotic PA-
45052 is a vancomidin antibiotic with a completely new structure that does not match any of the above compounds. Many vancomycins and their derivatives have been known for a long time, and it is also well known that by administering them to animals, their growth can be promoted (for example, in Japanese Patent Application Laid-Open No. 1983-1989).
129693, JP 59-213394, JP 59-213395,
Special publication 1986-502335, 1982-122300, 1982
-126970, JP-A-1993-199397, JP-A-1993-
237099, JP-A-60-231698, JP-A-61-251699,
US Patent 4558036, US Patent 4537770, European Published Patent 119575, etc.). Additionally, thiopeptin and the like are currently commercially available as growth promoters for animals. C. Problems to be Solved by the Invention Currently, infections caused by methicillin-resistant bacteria are becoming a serious clinical problem, and glycopeptide antibiotics, particularly vancomycin antibiotics, are considered to be effective. The purpose of the present invention is to provide a novel vancomycin antibiotic having such activity, and is expected to greatly contribute to the enrichment of medicine. Furthermore, the compounds of the present invention have a high growth-promoting effect and are useful as veterinary drugs. D. Means for Solving the Problems The present inventors have discovered that a bacterial strain belonging to the genus Nocardia has the following formula: (In the formula, R is

【formula】

[Formula] or H; R ₁ represents CH ₃ or H. ) was found to produce a compound with strong antibacterial activity, where R is The compound in which R ₁ is H is PA-45052-A, in which R
but The compound in which R ₁ is H is PA-45052-B, in which R
is H and R ₁ is H, PA-45052-C,
In the formula, R is The compound in which R ₁ is CH ₃ is referred to as PA-45052-D, where R is The compound in which R ₁ is CH ₃ is PA-45052-E, and the compound in which R is H and R ₁ is CH ₃ is PA-45052-E.
It was named E. The present invention encompasses not only these compounds, but also their pharmaceutically acceptable salts. In addition,
In this specification, PA-45052 mainly refers to the above 6
Refers to any one of the compounds, but in some cases refers to all the compounds. The physicochemical properties of the compound of the present invention are shown below. In addition, PA-45052-A (HCl), PA-45052-
B (HCl), PA-45052-C (HCl), PA-45052-
D (HCl), PA-45052-E (HCl), PA-45052-
F(HCl) each indicates a hydrochloride. ●Physicochemical properties Ultraviolet absorption spectrum PA-45052-A (HCl) λ0.01NHClmaxnm (E1% 1cm): 281.0 (36.4) λ0.01NNaOH・aqmaxnm (E1% 1cm): 301.8
(40.4) PA-45052-B (HCl) λ0.01NHClmaxnm (E1% 1cm): 281.2 (41.1) λ0.01NNaOH・aqmaxnm (E1% 1cm): 302.0
(46.8) PA-45052-C (HCl) λ0.01NHClmaxnm (E1% 1cm): 279.6 (46.7) λ0.01NNaOH・aqmaxnm (E1% 1cm): 296.4
(87.0) PA-45052-D (HCl) λ0.01NHClmaxnm (E1% 1cm): 280.7 (34.5) λ0.01NNaOH・aqmaxnm (E1% 1cm): 301.8
(39.4) PA-45052-E (HCl) λ0.01NHClmaxnm (E1% 1cm): 280.9 (39.0) λ0.01NNaOH・aqmaxnm (E1% 1cm): 302.5
(47.1) Specific optical rotation PA-45052-A (HCl) [a] ²⁵ ° _D : -87.2 ± 2.5 ° (c = 0.52, water) PA-45052-B (HCl) [a] ²⁵ ° _D : -67.3 ±2.1° (c = 0.51, water) PA-45052-C (HCl) [a] ²⁵ ° _D : -59.9 ± 1.9° (c = 0.52, water) PA-45052-D (HCl) [a] ²⁵ ° _D : -86.1±2.5° (c=0.503, water) PA-45052-E (HCl) [a] ²⁵ ° _D : -71.2±2.1° (c=0.52, water) Infrared absorption spectrum IR (KBr) cm ^-1 (See Figure 1) PA-45052-A (HCl) 3412, 1658, 1589 (sh), 1506, 1424, 1397,
1336, 1232, 1133, 1065, 1019, 902, 843 PA-45052-B (HCl) 3392, 1665, 1589 (sh), 1505, 1423, 1400,
1332, 1231, 1132, 1064, 1015, 892, 848 PA-45052-C (HCl) 3392, 1665, 1624 (sh), 1603 (sh), 1515, 1501
(sh), 1430, 1399, 1233, 1133, 1060, 1002,
889, 848 PA-45052-D (HCl) 3412, 1655, 1588, 1504, 1420, 1396, 1227,
1130, 1062, 1025, 1014, 998, 900 PA-45052-E (HCl) 3400, 1654, 1587, 1507, 1489 (sh), 1420,
1395, 1228, 1130, 1062, 1026, 1014, 1000 PA-45052-F (HCl) 3400, 1667, 1622 (sh), 1595 (sh), 1518, 1491
(sh), 1431, 1398, 1233, 1133, 1061, 1015,
999 Mass spectrometry (SIMS) (see Figure 2) PA-45052-A (Free) 1591 (M+H) ⁺ PA-45052-B (Free) 1448 (M+H) ⁺ PA-45052-C (HCl) 1286 (M+H) ⁺ PA-45052-D (HCl) 1605 (M+H) ⁺ PA-45052-E (HCl) 1462 (M+H) ⁺ PA-45052-F (HCl) 1300 (M+H) ⁺ Elemental analysis PA-45052-A (HCl) C ₇₃ H ₈₈ O ₂₆ N ₁₀ Cl ₂・3/2HCl・8H ₂ O Theoretical value (%): C; 48.95, H; 5.94, N; 7.82,
Cl; 6.93 Experimental value (%): C; 49.01, H; 5.82, N; 7.98,
Cl; 7.29 PA-45052-B (HCl) C ₆₆ H ₇₅ O ₂₄ N ₉ Cl ₂・HCl・5H ₂ O Theoretical value (%): C; 50.21, H; 5.68, N; 7.98,
Cl; 6.74 Experimental value (%): C; 50.38, H; 5.60, N; 8.13,
Cl; 6.75 PA-45052-C (HCl) C ₆₀ H ₆₅ O ₁₉ N ₉ Cl ₂・2HCl・6H ₂ O Theoretical value (%): C; 49.09, H; 5.42, N; 8.59,
Cl; 9.66 Experimental value (%): C; 49.14, H; 5.49, N; 8.76,
Cl; 9.88 PA-45052-D (HCl) C ₇₄ H ₉₀ O ₂₆ N ₁₀ Cl ₂・2HCl・10H ₂ O Theoretical value (%): C; 47.80, H; 6.07, N; 7.53,
Cl; 7.63 Experimental value (%): C; 47.62, H; 6.00, N; 7.50,
Cl; 7.53 PA-45052-E (HCl) C ₆₇ H ₇₇ O ₂₄ N ₉ Cl ₂・2HCl・8H ₂ O Theoretical value (%): C; 47.89, H; 5.70, N; 7.50,
Cl; 8.44 Experimental value (%): C; 47.79, H; 5.46, N; 7.80,
Cl; 8.47 Nuclear magnetic resonance spectrum ¹ H-NMR [4000MHz, d ₆ -DMSO (internal
TMS)] {ppm} (see Figure 3) PA-45052-A (free) [temp.60℃] 8.45 (br-d, ~5), 8.29 (d, ~6.2), 8.07
(d, 7.5), 7.89 (s-like), 7.78 (br), 7.49 (b
r
-d, ~8.5), 7.36 (s-like), 7.36 (br-d,
~8), 7.26 (br-d, ~8), 7.25 (d, 8.5),
7.14 (s-like), 6.77 (br-d, ~8.5), 6.70
(d, 8.5), 6.59 (d, ~7), 6.40 (s-like),
6.34 (s-like), 6.13 (br), 5.69 (br-d, ~
7.5), 5.69 (s-like), 5.65 (d, 7.5), 5.34
(br), 5.19 (s-like), 5.19 (s-like), 5.14
(d, ~3.5), 4.81 (br), 4.69 (br), 4.48 (br)
,
4.45 (d, 6.2), 4.36 (br-m), 4.22 (br), 4.14
(qd, ~6, 9.5), 3.74 (br-d), 3.67 (t-
like, dd, 7.5, 9), 3.67 (br-m), 3.29 (m),
2.99 (br-d, 9.5), 2.96 (br-d, ~9.5), 2.47
(br), 2.31 (s), 2.16 (dd, 16, ~7), 1.95 (br
-d, 13.5), 1.88 (br-d, ~13.5), 1.74
(m), 1.66 (br), 1.65 (br), 1.49 (m), 1.42
(m), 1.21 (s), 1.18 (d, ~6), 1.17 (s),
1.09 (d, 6), 0.91 (d, 6.7), 0.87 (d, 6.7) PA-45052-B (free) [temp.100℃] 8.40 (br), 8.29 (d, 6.5), 8.04 (d, 8.5), 7.88
(d, 1.8), 7.52 (dd, 8.4, ~2), 7.40 (br-s
-like), 7.34 (br-d, 8.4), 7.27 (d, 8.4),
7.23 (br), 7.15 (d, 2.2), 6.80 (dd, 8.4, 2.2)
,
6.71 (d, 8.4), 6.45 (br), 6.39 (d, 2.2), 6.38
(d, 2.2), 5.96 (br-d, ~11), 5.73 (s-
like), 5.71 (br-d, ~8.5), 5.36 (d, 7.4),
5.23 (s-like), 5.20 (s-like), 5.14 (d,
4.0), 4.76 (d, 4.0), 4.67 (d, 4.2), 4.52 (d
,
6.5), 4.50 (d, ~6.5), 4.35 (br-m), 4.23 (br
-d, ~11), 3.71 (d-like, 11.5), 3.62 (q
-d, 6.0, 9.7), 3.55 (dd, 11.5, 4.5), 3.44
(t-like), 3.13 (t-like), 3.1 (m), 2.86
(d, 9.7), 2.55 (dd, 16, 7.2), 2.32 (s), 2.17
(dd, 16, 7.2), 1.90 (br-d, 13.5), 1.77
(m), 1.61 (dd, 13.5, 4.2), 1.52 (m), 1.42
(m), 1.17 (d, 6.0), 1.13 (s), 0.92 (d,
6.5), 0.89 (d, 6.5) PA-45052-C (HCl) [temp.100℃] 8.63 (br), 8.37 (d, 5.8), 7.96 (br-d, ~
8.5), 7.80 (br), 7.59 (br-d, ~8.5), 7.36
(br), 7.33(br), 7.30(br), 7.19(br), 7.18(
br
-s-like), 6.84 (br-d-like, ~8.5), 6.57
(d, ~8.5), 6.43 (d, 2.3), 6.33 (d, 2.3),
5.62 (s-like), 5.57 (br), 5.23 (s-like),
5.21 (br), 5.18 (s-like), 4.87 (br), 4.76 (d
,
4.0), 4.57 (d, 5.8), 4.50 (d, 5.8), 4.27 (d
,
12.0), 3.63 (q-d, 6.1, 9.5), 3.28 (br-d,
~9.7), 2.51 (unknown), 2.39 (s), 2.25 (unknown),
1.77 (m), 1.61 (m), 1.49 (m), 1.48 (br), 1.
twenty three
(d, 6.1), 0.92 (d, 6.5), 0.90 (d, 6.5) PA-45052-D (HCl) [temp.100℃] 8.312 (br-d, 4.3), 8.283 (d, 6.4), 7.884 (br
-d, 9.0), 7.874 (d, 1.5), 7.508 (br-d,
8.8), 7.466 (br-b, 8.8), 7.338 (d, 8.4),
7.327 (br-s), 7.23 (br-d, 8.2), 7.225 (d,
8.4), 7.153 (d, 2.0), 6.795 (d, d, 2.0,
8.4), 6.711 (d, 8.4), 6.646 (br-d, 7.0),
6.375 (s-like), 5.950 (br-d, ~10), 5.70
(br-s), 5.667 (d, 7.5), 5.642 (br-d,
8.0), 5.328 (d, 4.5), 5.218 (br-s), 5.193
(br-s), 5.172 (d, 4.0), 4.831 (d, d,
4.0, 9.0), 4.667 (d, 4.2), 4.522 (d, 6.4),
4.506 (d, 6.0), 4.411 (br-m), 4.236 (br-
d, 8.2), 4.131 (q, d, 6.8, 9.5), 3.732 (br
-d, 10.2), 3.708 (br-t, 8.0), 3.607 (m),
2.913 (br-d, 9.6), 2.865 (br-d, 9.6),
2.419 (d, d, 6.2, 15.9), 2.315 (s), 2.173
(d, d, 6.5, 15.9), 1.886 (br-d, ~13.5),
1.863 (br-d, ~13.5), 1.38 (m), 1.185 (s),
1.165 (d, 6.5), 1.140 (s), 1.094 (d, 6.0),
0.878 (d, 6.7) Thin layer chromatography Merck Precoated TLC plate Silica gel 60F254 Developing solvent Chloroform: Methanol: Concentrated aqueous ammonia:
Secondary butanol: water (5:10:5:5:2) PA-45052-A (HCl) Rf = 0.22 PA-45052-B (HCl) Rf = 0.22 PA-45052-C (HCl) Rf = 0.26 High speed Liquid chromatography column: Cosmosil 5Phφ4.6×150mm (Hakai Chemical Co., Ltd.) Detection: UV220nm Flow rate: 1ml/min. Motion phase: 10% acetonitrile-0.05M phosphoric acid buffer (PH3.5) PA-45052-A (HCl) Retention time: 4.1min. PA-45052-B (HCl) Retention time: 9.2min. PA-45052-D (HCl) Retention time: 6.22min. PA-45052-E (HCl) Retention time: 13.67min :17% acetonitrile-0.05M phosphate buffer (PH3.5) PA-45052-C (HCl) Retention time: 8.8min. PA-45052-F (HCl) Retention time: 7.93min. Column: Nucleosil 5C8φ4.6 ×150mm (Chemco) Detection: UV220nm Flow rate: 1ml/min. Motion phase: 10% acetonitrile-0.05M phosphoric acid buffer (PH3.5) PA-45052-A (HCl) Retention time: 5.6min. PA-45052- B (HCl) Retention time: 8.8 min.: 17% acetonitrile in 0.05M phosphate buffer (PH3.5) PA-45052-C (HCl) Retention time: 7.6 min. Color reaction: Ninhydrin positive Solubility: Water, Soluble in dimethyl sulfoxide, sparingly soluble in methanol and ethanol, insoluble in ether, benzene, chloroform, and ethyl acetate Characteristics: Amphoteric substance, white amorphous powder Based on the above physicochemical properties, the compound of the present invention
PA-45052 was estimated to have the following three-dimensional structure. (In the formula, R is

【formula】

[Formula] or H; R ₁ represents CH ₃ or H; ) As described above, PA-45052 has properties different from those of known glycopeptide antibiotics, and is considered to be a novel glycopeptide antibiotic. Examples of PA-45052 producing bacteria include the PA-45052 strain isolated from a soil sample. This strain was identified as being the same species as Nocardia orientalis based on the results of taxonomic examination, and since March 26, 1988, Nocardia・Orientalis PA-45052 (Nocardia
orientalis PA-45052) and has been deposited as FERM BP-1320. In addition, the PA-45052-producing bacteria referred to in this specification refers to PA-45052-producing bacteria.
-45052-A strain that produces at least one compound among -A, -B, -C, -D, -E, and -F. The mycological properties of this strain are as shown below. (1) Morphological properties This strain was tested on yeast malt agar, tyrosine agar,
It shows good growth on Bennet's agar medium, etc., has good aerial mycelium, and has abundant spore formation.
Branches are simple, with no whorled branches. Aerial hyphae are relatively long and have straight or wavy tips. As a result of observation using an electron microscope, the spores have an elongated cylindrical shape with a width of 0.3 to 0.5 μm and a length of 1.2 to 1.8 μm, and the surface structure is smooth. No sporangia, flagellated spores, or sclerotia are observed. (2) Culture properties (cultured at 28℃ for 14 days)

[Table] Growth temperature (Bennet's agar medium, cultured for 14 days at each temperature) 10℃: No growth 28℃: Good growth, aerial mycelium formation, and spore settlement 37℃: Good growth, slightly aerial mycelium Formed at 45℃: No growth (3) Physiological properties (cultured at 28℃ for 14 days) Melanoid pigment production ability Negative tyrosinase reaction Negative milk coagulation Negative milk peptonization Positive gelatin liquefaction ability Positive starch water decomposition ability Negative (4) Carbon source availability Sugars commonly used for growth L-arabinose, D-xylose, D-glucose, D-fructose, sucrose, inositol, rhamnose, D-manitol Sugars that are not used for growth Ruffinose (5) Cell wall The composition of diaminopyrimenoic acid is meso (meso).
−) type. Based on the above properties, this strain is judged to belong to the genus Nocardia. Written by Waczman,
The Actinomycetes
Volume 2 (1961), Shearling and Gottlieb's ISP (International Streptomyces)
Project) report [International Journal of Systematic Bacteriology]
Bacteriology, Vol. 18 (1968), Vol. 19 (1969), Vol. 22 (1972)] and Versey's Manual of Determinative
Bacteriology (Bergey's Manual of
Determinative Bacteriology) 8th edition (1974
Searching for related species of this strain from the literature related to actinomycetes), Nocardia
Nocardia orientalis (described as Stre-ptomyces orientalis in the following literature) [International Journal of
Systematic Bacteriology Vol. 18, pp. 154-157 (1968), The Actinomycetes, Vol. 2, pp. 254-
255 (1961)] is listed as the most closely related species. Nocardia orientalis and PA−45052
As a result of comparing the properties of the strains, differences were observed in the ability to utilize sucrose, but other major properties were in good agreement. Therefore, strain PA-45052 was identified as being homologous to Nocardia orientalis;
Nocardia orientalis PA−45052
(Nocardia orienta-lis PA-45052). In the present invention, not only the above-mentioned strain PA-45052 and its natural and artificial mutant strains, but also those belonging to the genus Nocardia, such as PA-45052-A, -B, -C,
Any strain producing -D, -E and/or -F can be used and is within the scope of the present invention. Production of PA-45052 is carried out by culturing a PA-45052 producing strain in a nutrient medium under aerobic conditions, and after completion of the culture, PA-45052 is separated and collected from the culture.
A general method for producing PA-45052 is described below. As for the culture medium composition, culture conditions, etc., those generally used in the production of antibiotics may be used. In principle, the medium contains a carbon source, a nitrogen source, inorganic salts, etc. Vitamins, precursors, etc. may be added as necessary. Examples of carbon sources include glucose, starch, dextrin, glycerin, molasses,
Organic acids and the like can be used alone or in mixtures. Examples of nitrogen sources include soybean flour, corn steep liquor, meat extract, yeast extract, string flour,
Peptone, wheat germ, ammonium sulfate, ammonium nitrate, etc. can be used alone or as a mixture. Examples of inorganic salts include calcium carbonate, sodium chloride, potassium chloride, magnesium sulfate, copper sulfate, manganese chloride, zinc sulfate, cobalt chloride, and various phosphates, which are added to the medium as necessary. Cultivation may be carried out according to methods generally used for the production of antibiotics, and preferably liquid culture is used.
For mass production, deep aeration culture is recommended.
If the pH of the medium is variable, a buffer such as calcium carbonate may be added to the medium. Culture is approximately 20
It is preferable to carry out the reaction at a temperature of -40°C, particularly preferably 25-32°C. Although the culture time largely depends on the scale of fermentation, about 5 to 7 days is the culture time required for mass production. If severe foaming occurs during culturing, an antifoaming agent such as vegetable oil, lard, or polypropylene glycol may be appropriately added before or during culturing. To separate and collect PA-45052 from the culture after completion of the culture, a conventional method for separating and collecting fermentation products is appropriately used. For example, filtration, centrifugation, adsorption/desorption using various ion exchange resins or other active adsorbents, chromatography, extraction using various organic solvents, etc. may be appropriately combined. It may be desirable to form PA-45052 into a salt for separation operations and for convenience of use as a medicine or veterinary drug. Bases that can form salts with PA-45052 include alkali metals such as potassium and sodium, alkaline earth metals such as aluminum and magnesium, and acids include inorganic acids such as hydrochloric acid, sulfuric acid, and nitric acid, and acetic acid and fumaric acid. Examples include organic acids. PA-45052 and its pharmaceutically acceptable salts can be administered to humans or animals orally or parenterally as the active ingredient of an antibacterial agent. Using commonly used excipients, stabilizers, preservatives, wetting agents, surfactants, etc., it can be administered orally in the form of tablets, capsules, and powders, and parenterally as injections, liniments, and suppositories. It can also be administered directly. The dosage varies depending on the therapeutic purpose, age of the patient, symptoms, etc., but when administered intravenously, it is approximately 0.1 to 10 g per day for adults. Furthermore, the present inventors have discovered that the above-mentioned compounds have strong antibacterial activity against strains belonging to the genus Clostridium, which is an indicator of animal growth promoting activity. , found that their body weight increased significantly. When administering the growth promoting agent of the present invention to an animal, the glycopeptides related to the present invention may be directly orally administered, but in general, a commonly used carrier ( (e.g., defatted rice sugar, defatted soybean flour, bran, kaolin, talc, calcium carbonate, lactose, water, etc.), or administer such mixtures or glycopeptides alone to animal feed or water. A method in which the drug is administered by mixing with the drug is preferred. The glycopeptides used here do not necessarily have to be pure products; for example, they may be partially purified from a culture obtained by culturing the glycopeptide-producing bacteria related to the present invention in a medium. . Additionally, pharmaceutically acceptable salts of the glycopeptides may be used, including alkali metals such as potassium, sodium,
Examples include salts with alkaline earth metals such as aluminum and magnesium, inorganic acids such as hydrochloric acid, sulfuric acid, and nitric acid, and organic acids such as acetic acid and fumaric acid. 1 or 2 of glycopeptides related to the present invention
The animal feed containing the above may be any feed commonly used as animal feed.
The columns are as follows. Namely, corn, bran, rice, wheat, cottonseed meal, milo,
Soybean meal, fish meal, defatted rice bran, oil, alpha alpha, calcium carbonate, calcium phosphate, sodium chloride, choline chloride, vitamin A, vitamin D, vitamin E, vitamin B ₁ , vitamin B ₂ , vitamin B ₆ , vitamin B ₁₂ , pantothene Vitamin supplements such as calcium acid, nicotinamide, and folic acid,
Magnesium sulfate, iron sulfate, copper sulfate, zinc sulfate,
It is used in combination with some or all of inorganic salts such as potassium iodide and cobalt sulfate. In addition, other antibiotics, bactericides, anticoccidial agents,
Anthelmintics and the like may also be added. In addition, the content of one or more glycopeptides according to the present invention in the feed, when each of them is used alone, when any mixture thereof is used, the bacterial cells containing them, In any case when using a crude extract containing
A range of 100ppm is appropriate. The growth promoter of this invention can be used for animals in general,
Examples include poultry and farm animals such as chickens, turkeys, ducks, quail, cows, horses, pigs, sheep, goats, mink, rabbits, etc. In addition, as for the breeding method of the animal, a commonly used method may be used as is. The growth promoter of the present invention not only promotes the growth of animals, but also improves the animal's feed conversion rate. It is also effective against bacterial diseases. Furthermore, it is extremely superior because it has low toxicity to animals and does not remain in the animal body at all. The LD ₅₀ values of the compounds according to the present invention obtained by intravenous injection into mice are shown below. LD ₅₀ (mg/Kg) PA-45052-A ＞1000 PA-45052-B 1439 D Effect The compound PA-45052 of the present invention exhibits strong antibacterial activity against Gram-positive bacteria, especially against methicillin-resistant bacteria.
Useful as a medicine for humans or animals.
Furthermore, since it can be collected with high purity, it is suitable for use not only as an oral preparation but also as an injection. Furthermore, it has a growth-promoting effect on animals and is an active ingredient in growth-promoting agents for animals. E. Examples The present invention will be explained in detail below with reference to Examples, but the present invention is not limited in any way. Example 1 (a) Fermentation process: soluble starch 0.5%, glucose 0.5%, polypeptone 0.5%, beef extract 0.5%, yeast extract
Nocardia orientalis PA- in a 2-volume Erlenmeyer flask containing 800 ml of a medium consisting of 0.25% NaCl, 0.25% NaCl, and deionized water (PH7.0, before sterilization).
45052 (Feikoken Jokyo No. 1320) was inoculated with a seed culture slant, and cultured with shaking at 180 revolutions per minute at 28°C for 48 hours. Add 800ml each of this culture solution to 3.6% dextrin, 2.4% molasses, and bactopeptone.
0.84%, L-tyrosine 0.12%, antifoaming agent P-2000
(manufactured by Dainippon Ink) 0.05%, tap water (PH7.0, before sterilization) was inoculated into a 50-volume jar fermenter containing a medium of 30%, aeration rate 30/min, and internal pressure.
0.35Kg/cm ² G, stirring speed 550rpm, 32℃,
Incubate for 137 hours. (b) Separation step: The culture solution obtained in the above step is adjusted to pH 10.5 with 10% sodium hydroxide, and a supernatant liquid 33 is obtained by centrifugation. After adjusting this supernatant to pH 7.5, it was poured into a 3.3 HP-20 (manufactured by Mitsubishi Chemical Corporation) column, washed with water 23, and then 15% methanol-
Wash with 0.005N hydrochloric acid 10-50% methanol.
Elute with 0.005N hydrochloric acid. Fraction 9 shows activity using pulp disk diffusion method using Bacillus subtilis
Collect and adjust the pH to 7. After concentrating this under reduced pressure and freeze-drying, 46.3g of coarse powder of PA-45052 was obtained.
get. (c) Purification process: ●Purification of PA-45052-A, -B, -C Suspend 10g of the above crude powder in 100ml of water, adjust the pH to 2.0 with 1N hydrochloric acid, dissolve, and MCI GEL CHP-
Add to 100ml of 20P (manufactured by Mitsubishi Kasei Corporation) and add 0.01N hydrochloric acid, 10% methanol - 0.01N hydrochloric acid, and
Elute with 25% methanol-0.01N hydrochloric acid, PA-
Separate into fractions containing 45052-A and B and fractions containing PA-45052-C. The fraction containing PA-45052-A and -B was PH
Adjust to 6.0, concentrate and add to 100ml of CHP-20P.
While checking the degree of content by HPLC, elute with water, then 30% methanol-water, and 50% methanol-water to obtain a fraction containing PA-45052-A and PA-45052-A.
-45052-B-containing fraction. PA−45052−
The A-containing fraction was adjusted to pH 7.0, concentrated, and applied to packed column RQ-2 (Fujigel Sales Co., Ltd.).
While checking the purity by HPLC, add 13% acetonitrile to 0.05M phosphate buffer (hereinafter abbreviated as PBS) (PH7.0) and then 15% acetonitrile to
Elute with 0.05MPBS (PH7.0) PA-45052-A
(HPLC purity 95% or higher) Combine and concentrate the fractions.
CHP-20P 50% methanol after washing with water (12ml)
Elute with water, adjust to pH 4.0, concentrate and lyophilize.
198 mg of PA-45052-A (HC1) is obtained. The PA-45052-B-containing fraction was adjusted to pH 7.0, and after concentration, it was applied to back column RQ-2 and purified with 8% acetonitrile while checking the purity by HPLC.
PA-45052-0.05MPBS (PH3.5) then eluted with 10% acetonitrile-0.05MPBS (PH3.5)
Combine the B (HPLC purity 95% or higher) fractions, adjust to pH 7.0, concentrate, apply to 12 ml of CHP-20P, and wash with water.
Elute with % methanol-water. Since it is colored, the pH was adjusted to 7.0 again, and after concentration, the pH was adjusted to 4.0, applied to 2 ml of CHP-20P, eluted with 0.001N hydrochloric acid, decolorized, and collected the PA-45052-B fraction.
Adjusted to 7.0, concentrated, adsorbed on 12ml of CHP-20P, washed with water, eluted with 50% methanol-water, concentrated, and PH
Adjusted to 4.0, freeze-dried, PA-45052-B
(HC1) Obtain 290 mg. PA-45052-C-containing fraction adjusted to PH8.5 CHP
Adsorb onto 100ml of -20P, wash with water, then 50% methanol-water, and elute with 50% methanol-0.01N hydrochloric acid. The PA-45052-C containing fractions were combined and concentrated, then subjected to packed column RQ-2 and HPLC
Elute with 15% acetonitrile-0.05MPBS (PH3.5) and then with 18% acetonitrile-0.05MPBS (PH3.5) while checking the purity. PA-
The fractions of 45052-C (HPLC purity 90% or higher) were combined and adjusted to pH 8.0, and after concentration, they were added to 12 ml of CHP-20P, and while checking the purity by HPLC, they were mixed with 18% acetonitrile - 0.05 MPBS (PH 7.0) and then 21 Elute with % acetonitrile-0.05MPBS (PH7.0),
The fractions of PA-45052-C (HPLC purity 95% or higher) were combined and concentrated, then added to 12 ml of CHP-20P, washed with water, desalted, then 50% methanol-water, then 50%
% methanol - PH after concentration by elution with 0.01N hydrochloric acid
Adjust to 2.5, freeze dry, PA-45052-C
(HC1) Obtain 365 mg. ●Purification of PA-45052-D After suspending 20 g of the above crude powder in 140 ml of water, adjust the pH to 2.0 with 1N hydrochloric acid and dissolve. MCI this solution
Attached to 100ml of GEL CHP20P (Mitsubishi Kasei),
Elute with 0.01N hydrochloric acid. Check the fraction by HPLC and find the fraction containing PA-45052-B and -D.
Adjust to PH6.4, concentrate and add to 100ml of CHP20P again.
Elute with 0.01N hydrochloric acid to obtain fractions containing PA-45052-B and -D. Next, this PA-45052-B, -
The D-containing fraction was adjusted to pH 7.0, concentrated, and applied to packed column RQ-2 (Fuji Gel Sales Co., Ltd.) with 0.05 MPBS (PH 7.0), 13% CH ₃ CN-
0.05MPBS (PH7.0), 15% _CH3CN −0.05MPBS
(PH7.0), 18% _CH3CN −0.05MPBS (PH7.0), 30
Elute sequentially with % _CH3CN -0.05MPBS (PH7.0). A PA-45052-D containing fraction is obtained from the 30% CH ₃ CN-0.05 MPBS (PH 7.0) eluate. PA-
After concentrating the 45052-D containing fraction, MCI GEL
Adsorbed on 10ml of CHP20P, after washing with water, 25%
Elute sequentially with _CH3OH - _H2O and 50% _CH3OH - _H2O . The desalted fractions are collected and concentrated, adjusted to pH 4.0 with hydrochloric acid, and lyophilized to obtain PA-45052-D (665 mg, hydrochloride) as a white amorphous powder. ●Purification of PA-45052-E After suspending 20 g of the above crude powder in 140 ml of water, adjust the pH to 2.0 with 1N hydrochloric acid and dissolve. MCI this solution
Attached to 100ml of GEL CHP20P (Mitsubishi Kasei),
0.01N hydrochloric acid, 10% methanol - 0.01N hydrochloric acid, 25
Elute sequentially with % methanol and 0.01N hydrochloric acid.
Check the fraction by HPLC and PA-45052
-A, -B, -D, -E containing fractions and PA-45052-
A fraction containing C and -F is obtained. PA-45052-A, -
After adjusting the pH of the B, -D, and -E containing fractions to 6.4 and concentrating them.
Apply to 100ml of CHP20P and elute again with 0.01N hydrochloric acid.
A fraction containing A and a fraction containing B, D, and E are obtained. Next, this B, D, and E-containing fraction was adjusted to pH 7.0, concentrated, and applied to a packed column RQ-2 (Fujigel Sales Co., Ltd.) to 0.05 MPBS (PH 7.0), 13
% _CH3CN −0.05MPBS(PH7.0), 15% _CH3CN
−0.05MPBS (PH7.0), 18% _CH3CN−
0.05MPBS (PH7.0), 30% _CH3CN −0.05MPBS
(PH7.0) and sequentially elute. 30% _CH3CN−
D-containing fraction and E from 0.05MPBS (PH7.0) elution part
Obtain the containing fraction. After concentrating the PA-45052-E-containing fraction, it was adsorbed on 10 ml of MCI GEL CHP20P, washed with water, and then dissolved in 25% CH ₃ OH-H ₂ O, 50% CH ₃ OH
Elute sequentially with −H ₂ O. After collecting and concentrating the E-containing fractions, the pH was adjusted to 4.0 with hydrochloric acid and lyophilized to produce PA-45052-E as a white amorphous powder.
(245 mg, hydrochloride) was obtained. ●Purification of PA-45052-F The fraction containing PA-45052-C and -F was adjusted to pH 8.0, adsorbed on 100ml of CHP20P, washed with water and 50% methanol, and eluted with 50% methanol and 0.01N hydrochloric acid. . After concentrating the PA-45052-C, -F-containing fraction, it was applied to packed column RQ-2 and 15%
_CH3CN −0.05MPBS(PH3.5), 18% _CH3CN−
Elute with 0.05MPBS (PH3.5), PA−45052−
A fraction containing C and -F is obtained. This C and F-containing fraction was adjusted to pH 8.0, concentrated, and applied to 10 ml of CHP20P.
18% _CH3CN −0.05MPBS(PH7.0), 21%
_CH3CN −0.05MPBS(PH7.0), 30% _CH3CN−
Sequentially elute with 0.05MPBS (PH7.0) and 50% CH ₃ OH-0.01N hydrochloric acid to separate the PA-45052-C-containing fraction and PA.
A fraction containing -45052-F is obtained. PA-45052-F
After concentrating the containing fraction, it was adsorbed on 10 ml of CHP20P, washed with water and desalted, eluted with 50% methanol water, 50% methanol-0.01N hydrochloric acid, adjusted to pH 2.5 after concentration, and lyophilized to obtain PA-45052-F. (hydrochloride) to obtain 30 mg. Example 2 (a) Fermentation process: soluble starch 0.5%, glucose 0.5%, polypeptone 0.5%, beef extract 0.5%, yeast extract
Nocardia orientalis PA− in a 2-volume Erlenmeyer flask containing 800 ml of a medium consisting of 0.25% NaCl, 0.25% NaCl, and deionized water (PH7.0, before sterilization).
45052 (Feikoken Jokyo No. 1320) was inoculated with a seed culture slant, and cultured with shaking at 180 revolutions per minute at 28°C for 48 hours. 800 ml of this culture solution was inoculated into a 30-volume jar fermenter containing medium 18 consisting of the same components as above, and was incubated at 32°C at an aeration rate of 18/min, an internal pressure of 0.35 Kg/cm ² G, and a stirring speed of 200 rpm.
Incubate for 23 hours. Next, this culture solution 8 was mixed with 2.0% potato starch, 1.0% glucose, and 3.0% molasses.
%, bactopeptone 1.1%, antifoaming agent P-2000 (manufactured by Dainippon Ink) 0.05%, and tap water (PH 7.0, before sterilization). Incubate at 32°C for 164 hours at 350 rpm, internal pressure 5 psi, and stirring speed 350 rpm. (b) Separation step: The culture solution obtained in the above step is adjusted to pH 10.5 with 10% sodium hydroxide, and a supernatant liquid 140 is obtained by centrifugation. After adjusting the pH of this supernatant to 7.5, it was poured into a 15 HP-20 (Mitsubishi Kasei Co., Ltd.) column, washed with water 23, and then 15% methanol-
Wash with 30% 0.005N hydrochloric acid and 50% methanol.
Elute with 0.005N hydrochloric acid. Fraction 53 showing activity using pulp disk diffusion method using Bacillus subtilis
Collect and adjust the pH to 7. When this is concentrated under reduced pressure and freeze-dried, the crude powder of PA-45052 becomes 150.8
get g. Example 3 Examples of adding each compound to feed as a growth promoter are shown below. (1) Corn 46.45% Milo 15.00% Soybean meal 5.00% Fish meal 3.00% Defatted rice bran 25.00% Alpha alpha 3.00% Calcium carbonate 1.00% Calcium phosphate 0.70% Sodium chloride 0.40% Vitamin A, D, E mixture 0.05% ^* Inorganic salt mixture 0 .1 % ^** Vitamin B group mixture 0.1% PA-45052-A 10ppm Mix the above well. ^* Inorganic salt mixture: manganese sulfate, zinc sulfate, copper sulfate, cobalt sulfate, potassium iodide ^** Vitamin B group mixture: vitamin B ₁ , vitamin B ₂ , vitamin B ₆ , vitamin B ₁₂ , biotin,
Folic acid, calcium pantothenate (2) Corn 41.00% Milo 25.00% Soybean meal 19.10% Fish meal 8.00% Oil 4.00% Calcium carbonate 1.40% Calcium phosphate 0.85% Vitamin inorganic salt mixture 0.26% Methionine 0.10% Sodium chloride 0.29% PA− 45052 -B 20ppm Mix the above well. ^* Vitamin inorganic salt mixture: vitamin A, vitamin _D3 , vitamin E, vitamin _B1 , vitamin _B2 ,
Vitamin _B6 , vitamin _B12 , calcium pantothenate, nicotinamide, vitamin _K4 , choline chloride, magnesium sulfate, iron sulfate, copper sulfate, zinc sulfate, cobalt sulfate, potassium iodide (3) Corn 78% Soybean oil meal 9 % Fish meal 10% Fat 3.9% Crude fiber 2.4% Crude ash 5.1% Calcium 1.07% Phosphoric acid 0.73% Mixture of alpha-alphamil, salt, calcium carbonate 3% PA-45052-B 20ppm Mix the above well. (4) PA-45052-C, -D, -E, and -F are also mixed in the same manner as in (1), (2), and (3) above to prepare drug-added feed. F Effect of the invention PA-45052-A, -B, -C, -D, -E and -
The in vitro antibacterial activity of F was measured according to the method prescribed by the Japanese Society of Chemotherapy under the following conditions. Preparation of bacterial cell suspension A loopful of the test bacterial strain 1 on a slant medium is inoculated into 1 ml of a growth medium (trypto soy broth, Eiken Chemical Co., Ltd.) and cultured at 37°C for 18 to 20 hours. of culture
Use the 100-fold dilution as the cell suspension for inoculation. Sample solution Weigh accurately 9-10 mg of the sample and dissolve it in distilled water to a concentration of 2 mg/ml. Agar plate The sample solution is serially diluted with sterile water (2000-0.25 μg/ml). Transfer 0.51 ml of each diluted sample solution to a sterile plastic petri dish (9 cm in diameter), pour 9.5 ml of agar medium (susceptibility test medium, Eiken Chemical Co., Ltd.) there, stir gently, and allow to solidify. . Measurement of MIC value 1 platinum loop of inoculation suspension (0.5-
Transfer 1 μl) onto the surface of an agar plate containing various concentrations of samples prepared as described above. 18− at 37℃
After culturing for 20 hours, observe the growth of bacteria on the agar surface. The lowest concentration at which bacterial growth is observed to be completely inhibited is defined as the MIC and is expressed in μg/ml. Addition of horse serum in special medium Agar medium for determining MIC against Streptococcus 0.5% (V/V) before injection
Add horse serum. The results are shown in Table 1.

【table】

[Table] * indicates methicillin-resistant bacteria, ** indicates penicillin-resistant bacteria Next, Table 2 shows the antibacterial activities of PA-45052-A, -B, and -C in vivo. Test method: Slc-ICR female mice (8 mice per group) were intraperitoneally inoculated with the test bacteria, and inoculated twice 1 hour and 5 hours later with PA-45052-A, -B, or -C (multiple dilutions). ) is administered subcutaneously. Results: The 50% effective dose ( _ED50 ) is calculated from the survival rate of mice after 7 days.

[Table] *Methicillin-resistant bacteria
PA-45052 against Clostridium perfringens
Vitro tests and growth promoting effects tests on broiler chicks were conducted. 1 In vitro test on Cl.perfringens A total of 11 strains of Cl.perfringens from cows and chickens were used as test bacteria. The sensitivity measurement method uses GAM agar medium (Nissui) as the measurement medium.
Anaerobic culture (gaspack method) was performed using the agar plate dilution method. Judgment was made by determining the lowest concentration (MIC) at which growth was clearly inhibited with the naked eye after culturing at 37°C for 24 hours. In in vitro screening of antibiotics for the purpose of growth promotion, activity against Gram-positive bacteria, particularly against Cl.perfringens, is considered as one guideline (Poultry Science 62, 1633).
~1638, 1983; Poultry Science63, 2036~
2042, 1984). The results are shown in Table 3.

[Table] 2 Growth effect test on chicks The growth promoting effect of PA-45052 on broiler chicks (8 days old, Averacre breed) was investigated using the known antibiotic thiopeptin as a control drug. The additive concentration was 20ppm for each antibacterial-free feed mash (crude protein concentration (CP): 18%).
The study was carried out using a battery rearing method in which the animals were fed for 14 days at an additive concentration of . The experimental method involved raising first-born chicks until they were 8 days old.
Group 1 after being raised free-range on feed with a CP concentration of 23%.
24 birds (12 males and 12 males) were divided into a total of 5 experimental groups so that each group had an equal weight. Each experimental group is PA-45052-A, -B and -E groups.
20ppm x 14 days, thiopeptin 2ppm x 14 days, non-additive control group CP18% feed for 14 days. Each of these experimental groups contained 6 to 7 birds in cages (435
600×410mm) for 14 days (22 days old)
The animals were reared at an environmental temperature of 26±2°C. Efficacy was evaluated based on body weight gain and feed conversion rate during the test period (14 days). The test results are shown in Table 4.

【table】 [Brief explanation of drawings]

Figure 1 shows PA-45052-A, PA-45052-B,
PA-45052-C, PA-45052-D, PA-45052-
Infrared absorption spectra of E and PA-45052-F, Figure 2 shows PA-45052-A, PA-45052-B,
PA-45052-C, PA-45052-D, PA-45052-
Mass spectrometry spectra of E and PA-45052-F (however, the peaks marked with # are the peaks of the standard material for marker correction), Figure 3 shows the mass spectra of PA-45052-A,
PA-45052-B, PA-45052-C and PA-
The ¹ H-NMR spectrum of 45052-D is shown.

Claims (1)

[Claims] 1 formula; (In the formula, R represents [formula] [formula] or H; R ₁ represents CH ₃ or H. However, when R is [formula], R ₁ is not H.) Compounds PA-45052 and its pharmaceutically acceptable salts. 2 In the formula, R is Compound PA- according to claim 1, wherein R ₁ is H
45052-B. 3. The compound PA-45052-C according to claim 1, wherein R is H and _R1 is H. 4 In the formula, R is Compound PA- according to claim 1, wherein R ₁ is CH ₃
45052-D. 5 In the formula, R is Compound PA- according to claim 1, wherein R ₁ is CH ₃
45052-E. 6. The compound PA-45052-F according to claim 1, wherein R is H and _R1 is _CH3 . 7. The plant according to claim 1 belonging to the genus Nocardia.
A method for producing PA-45052, which comprises culturing a bacterium that produces PA-45052 in a medium, and separating and collecting the PA-45052 from the culture. 8. The production method according to claim 7, wherein the producing bacterium is a bacterium belonging to the species Nocardia orientalis. 9 The producing bacterium is Nocardia orientalis
The manufacturing method according to claim 7, which is PA-45052 (Feikoken Joyori No. 1320). 10. The bacterium producing PA-45052 according to claim 1, which belongs to the species Nocardia orientalis. 11 Nocardia orientalis PA-45052
(Feikoken Jokyo No. 1320).The producing bacterium according to claim 10. 12. An animal growth promoter containing as an active ingredient one or more compounds selected from the group consisting of the compounds according to any one of claims 1 to 6 and salts thereof.