JPH01240196A - Novel glycopeptide-based antibiotic pa-45052 - Google Patents

Abstract

PURPOSE:To produce the title antibiotic in high efficiency by making a culture of PA-45052 producing bacteria classified as Nocardia in a culture medium and separating PA-45052 from the resultant culture product. CONSTITUTION:Nocardia orientalis PA-45052 classified as Nocardia (FERM P-1320) is put to culture in a nutritive culture medium under aerobic conditions, and after completion of the culture, PA-45052 is separated and collected from the resultant culture product. This glycopeptide compound PA-45052 is represented by formula I (R is H, of formula II or III; R1 is H or CH3). The PA-45052 thus obtained and its salts permitted pharmaceutically can orally or parenterally administered to humans or animals as an active ingredient of antifungus agent.

Description

DETAILED DESCRIPTION OF THE INVENTION A. Industrial Application Field The present invention relates to the antibiotic PA-45052 represented by the formula; Salt, its manufacturing method, its producing bacteria, and PA-
The present invention relates to an animal growth promoter containing 45052 as an active ingredient.

BACKGROUND OF THE INVENTION 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 aminoglycoside antibiotics, tetracycline antibiotics, cephem antibiotics, penicillin antibiotics, carbapenem antibiotics, and macrolide antibiotics. Resistant to many antibiotics.

It has been noticed that glycopeptide antibiotics, especially vancomycin antibiotics, are active against methicillin-resistant bacteria (Antimicrobial Agents and Chemotherapy (AN'fIMICROBIA)).
LAGENI AND CHEMOIHERAPY)
660-662 (1985)). Vancomycin is an antibiotic that has been known for a long time.
50), and new vancomycin analogs have also been discovered (antimicrobial agents and chemotherapy).
AGENT AND CHEMOTHERAPY)
28, 660-662 (1985), The Journal of Antibiotics (rHEJOURNAL
OF ANTIBIOTIC5) 37.446-4
53 (1984), Kuni, 1-8 (1985), Kuma, 51-57 (1985), JP-A-60-1396
23.60-199397.6o-231698,60
-237099, etc.), and the present inventors previously discovered vancomycin antibiotics PA-42867-A and PA-4, in which strains belonging to the genus Nocardia have strong antibacterial activity.
Patent application Sho 6l-1 discovered that 2867-B could be produced.
4389), and also created its derivatives (patent application Sho 6l).
-188865).

However, the antibiotic PA-45052 of the present invention is a vancomycin antibiotic having a completely new structure that does not match the above compounds.

A large number of vancomycin and its derivatives have been known for a long time, and it was also well known that administration of these to animals can promote their growth (
For example, JP-A-57-129693, JP-A-59-21
3394, JP 59-213395, JP 61-5
02335, JP-A-61-122300, JP-A-62-
126970, Japanese Patent Publication No. 1983-199397, Japanese Patent Publication No. 1987-199397
-237099, JP-A-60-231698, JP-A-6
1-251699, US Pat. No. 4,558,036, US Pat. No. 4,537,770, European Published Patent No. 119575, etc.). Additionally, thiopebutin and the like are currently commercially available as growth promoters for animals.

Problems to be Solved by the Invention At present, infections caused by methinlin-resistant bacteria are becoming a serious clinical problem, and glycopeptide antibiotics, especially 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.

Means for Solving the Problems The present inventors discovered that a bacterial strain belonging to the genus Nocardia has the formula; or H:R, is 01. Or represents H. ) was found to produce a compound with strong antibacterial activity, and a compound in which R is H is PA-45052-A, a compound in which R1 is H.
is 0)I The compound in which R7 is H is PA-45052-B, in which R
) I, R.

is H, PA-45052-C, where R is R
The compound in which 2 is CH is PA-45052-D, the compound in which R is HR and CH, is PA-45052-E, and the compound in which R is H5R1 is CHs is PA-4505.
It was named 2-F.

The present invention encompasses not only these compounds, but also their pharmaceutically acceptable salts. In addition, PA-45 referred to in this specification
052 mainly refers to any one of the above six 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 (HCI), PA-45052-
B (HCI), PA-45052-C (HCI), PA
-45052-D (HCI), PA-45052-E (
HCI) and PA-45052-F (HCI) each indicate a hydrochloride.

・Physicochemical properties Ultraviolet absorption spectrum PA-45052-A (HCI>λ0°01NHc1nm (I14H-: 281.0
(36,4)max A'``NNa0''8qnm(E'): 301
．． 8 (40,4>max 1cm PA-45052-B(HCI>λ' 01NHc1nm(E jHl,l)' 281
．． 2 (41,1)max (margin below) PA-45052-E (HCI) [α]25°: -59,9±1.9° (c・o
, 52, Water) PA-45052-D (HCI) [α]25°: -86,1±2.5” (c・o, 5
03, Wednesday) PA-45052-E (HCl [α 25°: -71,2±2.1@ (c=0.
52, Water) Infrared absorption spectrum IR (KBr) am
-' (see Figure 1) PA-45052-A()IcI) 3412, 1658. t5g9(sbn,
1506. 1424. 1397°1336
, 1232.1133.1065.1019.902.
843PA-45052-B (HCI) 3392, 1665.1589 (sh), 1505.
1423.1400°1332, 1231.1132.
1064.1015.892.848PA-45052
-C(HCl> 3392, 1665.1624(sh), 1603(
sh), 1515°1501(sh), 1430.
1399.1233.1133.1060°1002,
889.848 PA-45052-D (HCI) 3412, 1655. 158g, 1504.
1420. 1396. 1227°1130, 10
62. 1025. 1014. 998. 900P
A-45052-E (HCl) 3400, 1654. 1587. 1507. 1
489(sb), 1420°1395, 1228
．． 1130. 1062. 1026. 1014.
1000PA-45052-F (HCI) 3400, 1687. 1622 (sh), 15
95 (sh), 1518°1491 (sh),
1431. 139g, 1233. 11
33. 1061°1 (115,999 Mass spectrometry (5IM5) <See Figure 2) PA-4505
2-A (Free) 1591 (W+H)”PA-
45052-B (Free) 1448 (M+H)
"PA-45052-C (HCI) 1286 (M
+H)”PA-45052-D(HCl) 160
5 (M+H)”PA-45052-E(HCI)
1462 (M+H)”PA-45052-F(HC
I) 1300 (M+H)” Elemental analysis PA-45052-A (HCI) Cy+HssO*sN+oC1*・3/2HC1・8H
*Theoretical value as O: C; 48.95. H;5.
94. N;7.82. C1; 6.93 actual arrogance value (″)
:):C;49.01. H;5.82. N; 7.9
8. C1 Near, 29PA~45052-B (HCI) Ca*HtiO+aNsC1*'HC1'5H*O theory (am:C;50.21.u;s, as, N
;7.98. C1; 6.74 experimental value (X>:C:50
．． 38. H:5.60. N1.13. C1;6.
75PA-45052-C (HCI) C6゜HaiO+ 5Nsc1t・2HC1・6H90
Theoretical value (X): C; 49.09. u;s, 4z
, N1.59. C1; 9.66 Experimental value m: c; 49
．． 14. H:5.49. N1.76, C1;9.
88PA-45052-D (HCI> Cy=H-ao+sN+-C1a・2HC1・10H,
Theoretical value for O<X'): C: 47.80. H:6.
07. N;7.53. C1; 7.63 experimental value (X)
:C;47.62. Hl, OO, N; 7.50. C
1;7.53PA-45052-E (HCI) Cg yHt? O! aNsc112HC1・8H8
Theoretical value m: c; 47.89.1 (; 5.70.
N; 7.50. C1; 8.44 experimental value <%'): C
:47', 79. H;5.46. N;7.80.
C11,47 nuclear magnetic resonance spectrum 'H-NMR [400MHz, ds-DM50 (i
internal TMS)] (ppm) (See Figure 3 WA) PA-45052-A (free) [temp,
60°C] 8.45 (br-d, -5), 8.
29 (d, ~6.2>, 8.07 (d.

7.5>, 7.89 (s-1ike), 7.
78 (br), 7.49 (br-d.

-8,5>, 7.36 (s-1ike), 7
．． 36 (br-d, ~8), 7.26 (b
r-d, ~8), 7.25 (d, 8.5
), 7.14 (s-1ike>, 6.77
(br-d, ~8.5>, 6.70 (d,
8.5), 6.59(d, ~7), 6.
40 (s-1ike), 6.34 (s-1ik
e), 6.13 (br), 5.69 (b
r-d, ~7.5), 5.69 (s-1ik
e).

5.65 (d, 7.5>, 5.34 (br
), 5.19 (s-1ike>.

5.19 (s-1ike), 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-1ike, 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.

~L3.5), 1.74 (m), 1.66
(br), 1.65 (br), 1°49(m
), 1.42 (ffl), 1.21 (!
l), 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-1ike>, 7.34 (br-d,
8.4), 7.27 (d, 84),
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-1ike), 5.71 (br-
d, ~8.5>, 5.36 (d, 7.4),
5.23 (s-1ike), 5.20 (
s-1ike), 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-1ike, 11.5), 3.62 (
q-d, 6.0. 9.7>, 3.55
(dd, 11. Milk 45).

3.44 (t-1ike), 3.13 (t-1
ike), 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(H
CI) [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-1ike), 6.84
(br-d-1ike, ~8.5).

6.57 (d, ~8.5>, 6.43 (d
, 2.3>, 6.33 (d.

2.3>, 5.62 (s-1ike), 5.
57 (br), 5.23 (s-1ike),
5.21 (br), 5.18 (s-1ik
e), 4.87 (br), 4.76 (d
, 4.0>, 4.57 (d, 5.
8>, 4.50 (d, 5.8), 4.2
7 (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), 151
(m).

1.49 (m), 1.48 (br),
1.23 (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), 7.88
4 (br-d, 9.0), 7.874 (d,
1.5), 7.508 (br-d.

8.8), 7.466 (br-d, 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 (b
rd, 7.0>, 6.375 (s-1ike)
, 5.950 (br-4, ~10), 5.7
0 (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°66
7 (d, 4.2>, 4.522 (d,
6.4>, 4.506 (d, 60), 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 (b
r-d, 96), 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), 1.86
3 (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 6
0F254 Developing solvent chloroform: methanol: concentrated ammonia water: secondary butanol: water (5: 10: 5: 5: 2')
PA-45052-A (HCI) Rf=0.22P
A-45052-B (HCI) Rf=0.22PA
-45052-C (HCI) Rf=0.26 High performance liquid chromatography column: Cosmosil 5Ph dr 4.6 X L5Q
mm (Hakai Chemical Co., Ltd.) Detection: UV 220 nm Flow rate: 1 ml/
min.

Motion phase: 10z acetonitrile-0.05M phosphate buffer (pH 3.5) PA-45052-A (HCI) Retention time: 4.1 min.

PA-45052-B (HCI) Holding time: 9°2 min.

PA-45052-D (HCI) Retention time: 6.22 min.

PA-45052-E (HCI) Retention time: 13.67 min: 17z Acetonitrile-0.05M phosphate buffer (pH 3,5) PA-45052-C (HCI) Retention time: 8.8 min.

PA-45052-F (HCI> Retention time: 7.93 min.

Column: Nucleosil 5C8-4,6x 150 mm (Chemco) Detection: UV
220 nm Flow rate: 1 ml/min Motion phase:
10z acetonitrile-0.05M phosphate buffer (
pH 3,5> PA-45052-A (HCI) Retention time: 5.6 min.

PA-45052-B (HCI) Retention time: 8.8 m1n = 17z acetonitrile-0.05M phosphate buffer (pH 3,5) PA-45052-C (HCI > Retention time: 7.6 min.

Color reaction: Ninhydrin positive Solubility: Soluble in water, dimethyl sulfoxide, slightly soluble in methanol, ethanol, insoluble in ether, benzene, chloroform, ethyl acetate Diamphoteric substance, physicochemical properties of white amorphous powder or higher , the compound of the invention PA-45
052 was estimated to have the following three-dimensional structure.

(The following is a blank space. R represents α or H.) As described above, PA-45052 has properties that are different from known glycopeptide antibiotics, and it is determined that it is a new glycopeptide antibiotic. be done.

Examples of PA-45052-producing bacteria include - PA-45052 strain isolated from a soil sample. This strain was identified as Nocardia orientalis (N.
Nocardia orientalis PA-45052 (Nocardia orientalis) was identified as the same species as Nocardia orientalis PA-45052 (Nocardia orientalis).
aorientalis PA-45052>, has been deposited as FERM BP-1320. In addition, PA-450 referred to in this specification
52-producing bacteria are PA-45052-A, -B, -C1
A strain that produces at least one compound among -D, -E, and -F.

The mycological properties of this strain are as shown below.

(1) Morphological properties This strain shows good growth on yeast malt agar, tyrosine agar, Pennet agar medium, etc., and has good adhesion of aerial mycelium and abundant sporulation. No whorl technique is seen on the 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.
The surface structure is smooth (Smooth type).
), sporangia, flagellated spores, and sclerotia are not observed.

(Margins below) (2) Various culturing properties (cultivated at 28°C for 14 days) Color names are displayed according to one color standard J (Japanese Color Research New Edition).

Growth temperature (Pennet agar medium, cultured at each temperature for 14 days) 10°C: No growth 28°C: Good growth, aerial mycelium formation, and spore settlement 37°C: Good growth, slightly aerobic Formation of hyphae at 45℃:
No growth (3) Physiological properties (cultured at 28°C for 114 days) Melanoid pigment production ability Negative tyrosinase reaction Negative milk coagulation Negative milk peptonization Positive gelatin liquefying ability Positive starch ice-melting ability Negative (4) Carbon source Sugars that can be used well for growth: L-arabinose, D-xylose, D-glucose,
D-fructose, sucrose, inositol, rhamnose, D-7 nitol Sugars not utilized for growth Raffinose (5) Composition of cell wall The type of diaminopimelic acid is meso-type.

Based on the above properties, this strain is judged to belong to the genus Nocardia. Waxman, The Actinomycetes, No. 2
Volume (1961), Scherling and Gottlieb's I, S, P, (Internationala
l StreptomycesProject) report [International Journal of Systematics Bacteriology (International
al Journal of SystematicB
acteriology) Volume 18 (1968),
Volume 19 (1969), Volume 22 (1972)
] and Bergey's Manual of Terminal Batteriology.
null of Determina-tive'
Bacteriology) 8th edition (1974) and other literature related to actinomycetes for related species of this strain, Nocardia orientalis (N.
ocardia orientalis, Stre-ptomyces orientalis in the following literature
is written as ea) [International
al Journal of SystematicB
Acteriology Vol. 18, pp. 154-157 (
1968), rhe Actinomycetes,
Vol. 2, pp. 254-255 (1961)] is listed as the most closely related species. As a result of comparing the properties of Nocardia orientalis and strain PA-45052, a difference was 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, and Nocardia orientalis PA-45052 (N
ocardia orienta-Lis PA-45
052).

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, PA-45052-A, -B, -C, -
Any strain that produces 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 medium composition, medium conditions, etc., those generally used for the production of antibiotics may be used. The medium, in principle, contains a carbon source, a nitrogen source, an inorganic salt, etc. If necessary, vitamins, precursors, etc. may be added. As the carbon source, for example, gluten, starch, dextrin, glycerin, molasses, organic acids, etc. are used alone or in a mixture. As the nitrogen source, for example, soybean flour, corn steep liquor, meat extract, yeast extract, cottonseed flour, peptone, wheat germ, ammonium sulfate, ammonium nitrate, etc. are used alone or in a mixture. Examples of inorganic salts include calcium carbonate, sodium chloride, potassium chloride, magnesium sulfate, steel sulfate, manganese chloride, zinc sulfate, cobalt chloride, and various phosphates.
Add to the medium as necessary.

Cultivation may be carried out according to methods generally used for the production of antibiotics, preferably liquid culture, but for mass production, deep aeration culture is preferred.

If the pH of the medium is variable, a buffer such as calcium carbonate may be added to the medium. Culture at approximately 20-40℃
The temperature is preferably 25 to 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.

After culturing, PA-45052 can be separated and collected from the culture using appropriate methods for separating and collecting ordinary fermentation products. For example, filtration, centrifugation, adsorption and desorption using various ion exchange resins or other active adsorbents. and chromatograph i-1
Extraction using various organic solvents may be appropriately combined.

It may be desirable to convert PA-45052 into a salt for convenience of separation operations and 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 antimicrobial 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 semi-open formulations. It can also be administered to 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.

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 effects, and have added these compounds to animal feed to raise animals. They found that their body weight increased significantly.

When administering the growth promoter of the present invention to animals, the glycopeptides related to the present invention may be administered orally directly; (e.g., defatted rice bran, defatted soybean flour, bran, kaolin, talc, calcium carbonate, lactose, water, etc.), or such mixtures or glycopeptides alone can be administered in animal feed or It is preferable to administer the glycopeptides by mixing them with water. The glycopeptides used here do not necessarily have to be pure products. In addition, pharmaceutically acceptable salts of the glycopeptide may be used, which may be partially purified, such as alkali metals such as potassium, sodium, alkaline earth metals such as aluminum, magnesium, hydrochloric acid, Examples include salts with inorganic acids such as sulfuric acid and nitric acid, and organic acids such as acetic acid and fumaric acid.

The animal feed containing one or more of the glycopeptides related to the present invention may be any feed that can be generally used as animal feed.

Examples are as follows. Namely, corn, bran, rice, wheat, cottonseed meal, milo, soybean meal, fishmeal, defatted rice bran, oil and fat, alfalfa, calcium carbonate, calcium phosphate, sodium chloride, choline chloride, vitamin A1, vitamin D, vitamin E1, vitamin Bl.
s Vitamin B8, Vitamin B6, Vitamin B12,
Vitamins such as calcium pantothenate, nicotinamide, and folic acid; inorganic salts such as magnesium sulfate, iron sulfate, copper sulfate, zinc sulfate, potassium iodide, and cobalt sulfate;
It is used by mixing some or all of the following.

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 where a crude extract containing glycopeptides is used, the appropriate amount of glycopeptides to be used is in the range of 0.5 to 1100 pp.

The growth promoter of the present invention can be used for animals in general, such as poultry and livestock such as chickens, turkeys, ducks, quail, cows, horses, pigs, sheep, goats, mink, and rabbits.

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. Kirani has low toxicity to animals and has the characteristic that it does not remain in the animal body at all.
Extremely excellent. Below, LD in mice intravenously injected with the compound according to the present invention. Show value.

LD (m/K) PA-45052-A >1.000PA-450
52-81,439 28 Effects The compound PA-45052 of the present invention exhibits strong antibacterial activity against Durham-positive bacteria, particularly against methicillin-resistant bacteria, and is useful as a medicine for humans or animals. In addition, since it can be collected with high purity, it can be used not only as an oral preparation, but also as a
Suitable for use 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 with reference to Examples below, 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 0.25
Nocardia orientalis PA-45052 (
Inoculate the seed culture slant of Kaikoken Joyori No. 1320),
Shaking culture is performed at 28° C. for 48 hours at 180 revolutions per minute. Add 800ml each of this culture solution to 3.6% dextrin.
, 2.4% molasses, 0.84% Batatopebutone, 0.12% L-tyrosine, 0.05% antifoaming agent P-2000 (manufactured by Dainippon Ink), and tap water (pH 7.0, before sterilization). Inoculate into a 5 ON jar fermenter containing medium 301, aeration rate 3o1/min, internal pressure 0, 35 kg/cm"
G, Fil stirring rotation speed: 55 Orp (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 3312 is obtained by centrifugation. After adjusting this supernatant to pH'7.5,
3.32 HP-20 (Mitsubishi Kasei Co., Ltd.) column, washed with water 232, and then 15% methanol-0.005N
Wash with hydrochloric acid 102, 50% methanol-0.005N
Dissolve with hydrochloric acid. Fraction 9 shows activity using the valve disk diffusion method using Bacillus subtilis! Collect yA! to pH 7!
I do. When this is concentrated under reduced pressure and freeze-dried, PA-45
46.3 g of coarse powder of 052 is obtained.

(c) Purification step: Purification of PA-45052-A, -B, -C 10 g of the above crude powder was suspended in 100 ml of water, adjusted to pH 2.0 with IN hydrochloric acid, dissolved, and MCI GEL 0IF.
-20P (manufactured by Mitsubishi Chemical Corporation) 100ml, HPLC
While checking the content in the fraction with
, 0IN hydrochloric acid, 10% methanol-0.0IN hydrochloric acid, 2
Elute with 5% methanol-0.0IN hydrochloric acid, PA-45
It is divided into a fraction containing 052-A%B and a fraction containing PA-45052-C.

The fraction containing PA-45052-A and -B was adjusted to pH 6.
After adjusting to 0 and concentrating, add to 100ml of CHP-20P,
Water and then 30% while checking the content using PLC.
The PA-45052-A containing fraction and PA-45052 were sequentially eluted with methanol-water and 50% methanol-water.
-Separate into B-containing fractions. The PA-45052-A-containing fraction was adjusted to pH 7.0 and concentrated using back column RQ-2.
(Fujigel Sales Co., Ltd.), and while checking the purity by HPLC, 13% acetonitrile-0.05M phosphate buffer (hereinafter abbreviated as PBS) (pH 7.0) was added.
5% acetonitrile-0.

PA-4505 was eluted with 05MPBS (pH 7.0).
2-A (HPLC purity 95% or more) fractions were combined, concentrated, applied to 12 ml of CHP-20P, washed with water, and eluted with 50% methanol-water to pH 4. After adjusting to Q, concentrated and freeze-dried, PA-45052-A (HCI) 198 m
g Get it.

The PA-45052-B-containing fraction was adjusted to pH 7.0, concentrated, and then applied to backed column RQ-2 and washed with 8% acetonitrile-O. while checking the purity by HPLC. .

5MPBS (pH 3,5) and then eluted with 10% acetonitrile-0.05MPBS (pH 3.5).
Combine the A-45052-B (HPLC purity 95% or higher) fractions, adjust to pH 7.0, and concentrate before adding CHP-20P12m.
After washing with water, elute with 50% methanol and 100% methanol.
Because it is colored, adjust the pH again to 7.0, and then adjust the pH after concentration.
Adjust to 4.0, apply to 2ml of CHP-20P, and add to 0.0.
Elute with 01N hydrochloric acid and decolorize, PA-45052-
B(7) Collect fractions/) 1) Adjust H7.04m, concentrate, adsorb to 12 ml of CHP-20F, wash with water, elute with 50% methanol-water, concentrate, adjust to pH 4, 0, freeze-dry, PA-45052 -B (HCI) 290 mg is obtained.

Adjust the PA-45052-C-containing fraction to pH 8.5 CH
It was adsorbed onto 100 ml of P-20F, washed with water and then with 50% methanol-water, and then eluted with 50% methanol-0.01N hydrochloric acid. After combining and concentrating the PA-45052-C containing fractions, they were applied to backed column RQ-2 and purified with 15% acetonitrile-0.05MP while checking the purity by HPLC.
BS (pH 3,5) then 18% acetonitrile-0,
Elute with 05MPBS (pH 3,5). PA-45
The fractions of 052-C (HPLC purity 90% or more) were combined and adjusted to pH 8.0, and after concentration, CHP-20P12m
18% acetonitrile-0.05 MPBS (pH 7.0) and then 2.0 ml of 18% acetonitrile-0.05 MPBS (pH 7.0) while checking purity by HPLC.
1% acetonitrile-0.05MPBS (pH 7).

0) and PA-45052-C (HPLC purity 9
After combining and concentrating the fractions (more than 5X), CHP-2
Added to 12ml of 0P, washed with water, desalted, and then added with 50% methanol
Elute with water, then 50% methanol-0.01N hydrochloric acid, concentrate, adjust to pH 2.5, freeze-dry, and PA-4505
365 mg of 2-C(HCI) are obtained.

- Purification of PA-45052-D After suspending 20 g of the above crude powder in 140 ml of water, the suspension was adjusted to pH 2.0 with IN hydrochloric acid and dissolved. Add this solution to MCI
Apply to 100 ml of GEL CHP20P (Mitsubishi Kasei) and elute with 0.0 IN hydrochloric acid. Check the fraction by HPLC and obtain PA-45052-B.

- Adjust the C-containing fraction to pH 6.4 and concentrate it using CHP20P
100 ml and again eluted with 0.0 IN hydrochloric acid.
45052-B, -C-containing fractions are obtained. Next, this PA
-45052-B, -C-containing fractions were adjusted to pH 7.0, concentrated, and applied to back column RQ-2 (Fujigel Sales Co., Ltd.) in 0.05MPBS (pH 7.0), 13%
CH+CN-0, 05MP BS (pH 7, 0), 15
%CH, CN-0, 05MPBS (pH 7, 0), 18
%CH,CN-0.05MPBS (pH 7.0), 30
Elute sequentially with %CH, C and 0.05MPBS (pH 7,0). 30% CH, CN-0, 05MPB 5 (pH
7,0) Obtain a PA-45052-C containing fraction from the eluate. After concentrating the fraction containing PA-45052-C, MCI
After adsorbing on 10ml of GEL CHP20P and washing with water, 25% CH, 0H-H, 0150% CH$0H-H,
Elute sequentially with O. After collecting and concentrating the desalted fractions,
The pH was adjusted to 4.0 with hydrochloric acid and lyophilized to obtain PA-45052-D (665 mg, hydrochloride) as a white amorphous powder.

- Purification of PA-45052-H After suspending 20 g of the above crude powder in 140 ml of water, the suspension was adjusted to pH 2.0 with IN hydrochloric acid and dissolved. Add this solution to MCI
The mixture was applied to 100 ml of GEL CHP20P (Mitsubishi Kasei) and sequentially eluted with 0.0 IN hydrochloric acid, 10% methanol-0.0 IN hydrochloric acid, and 25% methanol-0.0 IN hydrochloric acid.

Check the fraction by HPLC and find PA-45052
-A, -B, -D, -E containing fractions and PA-450
A fraction containing 52-C, -F is obtained. PA-45052-
Adjust the A, -B, -〇, -E containing fractions to pH 6.4, concentrate, apply to 100 ml of CHP20F, and elute again with 0.01N hydrochloric acid to obtain A-containing fractions and B, D%E-containing fractions. 0
Next, the B, D, and E-containing fractions were adjusted to pH 7.0, concentrated, applied to back column RQ-2 (Fujigel Sales Co., Ltd.), and washed with 0.05 MPBS (pH 7.0), 13
%CH, CN-0, 05MPBS (pH 7,0),
15% CH, CN-0.05MPBS (pH 7.0)
, 18% CH,CN-0.05MPBS (pH 7.0
), 30% CH, CN-0.05MPBS (pH 7.0
). 30%C1, CN-0, 05MPB
A C-containing fraction and an E-containing fraction are obtained from the S(p'H7,o) elution part. After concentrating the fraction containing PA-45052-E, MC
I GEL CHP20F 10ml adsorbed, washed with water, 25% CHsOH-H*0.50% CH,0H
-H and 0 are sequentially eluted. After collecting and concentrating the E-containing fractions, the pH was adjusted to 4.0 with hydrochloric acid and lyophilized to obtain PA-45052-E (245 m
g.

hydrochloride) was obtained.

・Purification of PA-45052-F PA-45052-C, -F-containing fraction was adjusted to pH 8.0, adsorbed on 100 ml of CHP20F, water, 50% methanol water fis, and then eluted with so% methanol-0.0IN hydrochloric acid. let After concentrating the fraction containing PA-45052-C, -F, it was applied to backed column RQ-2, and 15% CH
, CN-0,05MPBS (pH 3,5), 18% CH
, eluted with CN-0,05MPBS (pH 3,5), P
A fraction containing A-45052-C1-F is obtained. This C,F
The containing fraction was adjusted to pH 8.0, concentrated, and CHP20P1
0 ml and 18% CH,CN-0.05MPBS (
pH 7.0), 21% CH, CN-0.05MPBS (
pH 7,0), 30% CH, CN-0, 05MPBS (
PH7,0), eluted sequentially with 50% CH, 0H-0, and 0I N hydrochloric acid to separate the PA-45052-C-containing fraction and PA-4.
A fraction containing 5052-F is obtained. After concentrating the Pa-45052-F containing fraction, it was adsorbed on 10 ml of CHP20F, washed with water and desalted, and then mixed with 50% methanol water, 50% methanol-0,
Elute with 0IN hydrochloric acid, concentrate, adjust to pH 2.5, and lyophilize to obtain 30 mg of PA-45052-F (hydrochloride).

Example 2 (a) Fermentation process: soluble starch 0.5%, glucose 0.5%, polypeptone 0.5%, beef extract 0.5%, yeast extract 0.25
Nocardia orienglis PA-45052 (
Inoculate the seed culture slant of Kaikoken Joyori No. 1320),
Shaking culture is carried out at 28°C for 148 hours at 180 revolutions per minute. 800 ml of this culture solution was mixed with 18! of a medium consisting of the same components as above! 3 (inoculated into B volume fermenter, aeration rate 18j2/min, internal pressure 0, 35 kg/c
Culture solution 812 was cultured at 32°C for 23 hours at a stirring speed of 20 rpm, 2.0% bote starp, 1.0% glucose, 3.0% molasses, 1.1% bactopebutone, and 25 containing medium 165P consisting of foaming agent P-2000 (manufactured by Dainippon Ink) 0.05% and tap water (pH 7.0, before sterilization) (inoculated into a B-capacity fermentation tank,
Air flow rate 165P/min, internal pressure 5psi, stirring rotation speed 35
Culture in Orpm at 32°C for 164 hours.

(b) Separation step: The culture solution obtained in the above step was adjusted to pH 10.5 with 10% sodium hydroxide, and the supernatant liquid was separated by centrifugation.
0! get. After adjusting this supernatant to pH 7.5, 151
of HP-20 (manufactured by Mitsubishi Kasei Co., Ltd.) and water 23
After washing with j2, 15% methanol-0,005N hydrochloric acid 3
Wash with 01 and elute with 50% methanol-0,005N hydrochloric acid. Fraction 53j showing activity by valve disc diffusion method using Bacillus subtilis! Collect and adjust to pH 7. When this is concentrated under reduced pressure and lyophilized, PA-45052
150.8 g of coarse powder was obtained.

Example 3 Examples of adding each compound to feed as a growth promoter are shown below.

(1) Corn 46.45'X
Milo L5.00% soybean meal
5.00% fishmeal
3.0OX defatted rice bran 25. OOX
Alfalfa 3.0OX Calcium Carbonate T,OOX Calcium Phosphate
0.70% sodium chloride 0.40% vitamin A, D, E mixture 0.05% 0 inorganic salt mixture 0.1xO vitamin B group mixture
0.1xPA-45052-A 1
0 ppm Mix the above well.

・Inorganic salt mixture: manganese sulfate, zinc sulfate, steel sulfate, cobalt sulfate, potassium iodide 1 vitamin B group mixture: vitamin Bls vitamin B
1. Vitamin L, vitamin B12, biotin, folic acid,
Calcium pantothenate ■ Corn 41.00 X Milo 25. OOX soybean meal
19.10χ fishmeal s,
oox oil 4.00% calcium carbonate 1.40% calcium phosphate 0.85%0 vitamin inorganic salt mixture
0.26% methionine 0. LOX Sodium Chloride 0.29XPA-45052-
B 20 ppffi Mix the above well.

8 vitamin inorganic salt mixture: vitamin A, vitamin D1,
Vitamin E1 Vitamin B1, Vitamin B3, Vitamin B, Vitamin B11, Calcium pantothenate, Nicotinic acid amide, Vitamin 4% Choline chloride, Magnesium sulfate, Iron sulfate, Copper sulfate, Zinc sulfate, Cobalt sulfate,
Potassium iodide (3) Corn 78% Soybean oil meal 9x Fishmeal 10 x fat
3.9x coarse fiber fi
2.4% crude ash 5.1x calcium 1.07% phosphoric acid
Mixture of 0.73% Alfalfa Meal - Salt 1 Calcium Carbonate 3z PA-45052-820ppm Mix the above well.

(4) PA-45052-C, -D, -E, and -F are also mixed in the same manner as in (1), (R, 0) above to prepare drug-added feed.

Effects of the Invention The in vitro antibacterial activity of PA-45052-A, -B, -C, -D, -E and -F was measured under the following conditions according to the method prescribed by the Japanese Society of Chemotherapy. did.

(2) Preparation of bacterial cell suspension A platinum loop of test bacterial strain 1 on a slant medium is inoculated into 1 ml of a growth medium (Tryptosoypros, Eiken Chemical Co., Ltd.) and cultured at 37°C for 18 to 20 hours. A 100-fold dilution of the culture is used as a cell suspension for inoculation.

■ Accurately weigh 9-10 mg of the sample solution sample and add it to distilled water for 2 m.
Dissolve to give a concentration of g/ml.

■ Stepwise dilute the agar plate sample solution with sterile water (2000 ~
0.25 μs/ml), 0.5 ml of each diluted sample solution was placed in a sterile plastic Petri dish (9 cm diameter).
), and 9.5 ml of agar medium (susceptibility test medium, Eiken Chemical Co., Ltd.) is poured therein, stirred gently, and solidified.

■ Measurement of MIC value 1 platinum loop of inoculation suspension (0°5-1
μm) onto the surface of an agar plate containing various concentrations of samples prepared by the method described above. After incubation at 37° C. for 18-20 hours, the growth of bacteria on the agar surface is observed. The lowest concentration at which bacterial growth is completely inhibited is defined as MIC.
Expressed in g/ml.

■ Addition of horse serum in special medium The agar medium for measuring the MIC against Streptofuccus is supplemented with 0.5% (V/V) horse serum before injection.

The results are shown in Table 1.

(Left below) Next, in vivo PA-45052-A, -B
, and () are shown in Table 2.

Test method: Test bacteria were intraperitoneally inoculated into 5lc-ICR female mouse groups (8 mice), and PA-45052-A, -B, or -C was inoculated twice, 1 hour and 5 hours later.
(multiple dilution) is administered subcutaneously.

Results From the survival rate of mice after near 8, 50% effective dose (ED
s*).

(Left below) Clostridium perfringens of PA-45052
An in vitro test on s) and a growth promoting effect test on broiler chicks were conducted.

1, in for C1, perfringens
C1 perfringens in vitro test
A total of 11 strains derived from cattle and chicken were used. The susceptibility measurement method used CAM agar medium (Japan) as a measurement medium and performed anaerobic culture (gas back method) by agar plate dilution method. The MIC value of the drug was determined as the lowest concentration (MIC) at which growth was clearly inhibited with the naked eye after culturing at 37'C for 24 hours.

In vitro screening of antibiotics for the purpose of growth promotion, the activity against Durham-positive bacteria, especially C1
, perfringens is considered to be one indicator (Poultry 5science 62.
1633-1638.1983; Poultry 5
science 63.2036-2042.1984)
.

The results are shown in Table 3.

(Left below) Table 3 In for C1, perfringens
Vitro performance NCfC-10239 cow 0.2
G, 2 0.1FC2208/75
Cow 0.39 0.39 0. l5-
79 Cow 0.39 0.39
0.1CM-1970 Cow 0.39
0.39 0.26to10 Cow
0.39 0.39 0.2NαC-8239
Cow 0.2 0.2 Q, 2NC
rC-8798 cow 0.39 0.39
0.25PO7 chicken 0.2 0.2
0.19C-02 chicken 0.39 0.3
9 0.23C-01 chicken 0.78 0
．． 78 0.202441 Chicken
0.39 0.39 0.2 Compound 1 :P
A-45052-A Compound 2: PA-45052-B Compound 3: PA-50527-C Growth effect test on 2,'M The growth-promoting effect of thiobebden, a known antibiotic, was investigated as a control drug.
Antibacterial additive-free feed matshu (crude protein concentration (CP)
) : 18X) with 20ppm addition aljJ degree 14
A study was conducted using a daily battery breeding method. The experimental method was to raise day-old chicks free-range on feed with a CPa degree of 23X until they were 8 days old, and then conduct the experiment in a total of 5 groups of 24 birds per group (12 males and 12 females) so that the weight of each group was equal. It was divided into groups. Each experimental group consisted of 20p of PA-45052-A, -B and -E groups.
pmx 14 days, Thiobeptin 20ppm x 14 days,
Additive-free control group CP18% feed for 14 days. Each of these experimental groups was housed in a cage (435x600x4
10a+m) and then reared for 14 days (222 days old) at an environmental temperature of 26±2"C.Efficacy was evaluated from body weight gain and feed conversion rate during the test period (14 days). did.

The test results are shown in Table 4.

[Brief explanation of the drawing]

Figure 1 shows PA-45052-A, PA-45052-
B%PA-45052-C%PA-45052-D,
PA-45052-E and PA-45052-F (7
) Infrared absorption spectrum, Figure 2 is PA-45052
-A. PA-45052-B, PA-45052-C, PA
-45052-D, PA-45052-E and PA
Mass spectrometry spectrum of -45052-F (however, the peaks marked with # are the peaks of the standard material for marker correction)
, Figure 3 shows PA-45052-A%PA-45052-
B, pA-asosz-c Oyohi PA-45052-
D'H-NMRt hectorum is shown. Patent applicant: Shionogi & Co., Ltd. Figure 1 (Part 1) PA-45052-APA-45
052-B WAVENUM BER (cm 2nd figure (Part 1) PA-45052-A 2nd figure (Ino 2) PA-45052-B 2nd figure (Part 13) PA-45052-C

Claims (14)

[Claims] (1) Formula; ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R is ▲There are mathematical formulas, chemical formulas, tables, etc.▼, ▲There are mathematical formulas, chemical formulas, tables, etc.▼ or H; R_1 is CH_3 or H. Compound PA-45052 and its pharmaceutically acceptable salts. (2) Compound PA-450 according to claim 1, in which R is ▲ a mathematical formula, a chemical formula, a table, etc. ▼ R_1 is H
52-A. (3) Compound PA-450 according to claim 1, in which R is ▲ a mathematical formula, a chemical formula, a table, etc. ▼ R_1 is H
52-B. (4) The compound PA-45052-C according to claim 1, wherein R is H and R_1 is H. (5) The compound PA- according to claim 1, in which R is ▲a mathematical formula, a chemical formula, a table, etc.▼R_1 is CH_3
45052-D. (6) The compound PA- according to claim 1, in which R is ▲a mathematical formula, a chemical formula, a table, etc.▼R_1 is CH_3
45052-E. (7) The compound PA-45052-F according to claim 1, wherein R is H and R_1 is CH_3. (8) A method for producing PA-45052, which comprises culturing PA-45052-producing bacteria belonging to the genus Nocardia in a medium, and separating and collecting PA-45052 from the culture. (9) The production method according to claim 8, wherein the producing bacterium is a bacterium belonging to the species Nocardia orientalis. (10) The producing bacterium is Nocardia orientalis PA
-45052.The manufacturing method according to claim 8. (11) PA-45052-producing bacteria belonging to the genus Nocardia. (12) The producing bacterium according to claim 11, which belongs to the species Nocardia orientalis. (13) Nocardia orientalis PA-4505
12. The producing bacterium according to claim 11, which is No. 2. (14) An animal growth promoter containing as an active ingredient one or more compounds selected from the group consisting of the compounds and salts thereof according to claims 1 to 7.