NL1001012C2 - Antibiotic mixt. WAP-8294A prepd. by culturing a Lysobacter WAP-8294 - for use against pathogenic microorganisms, partic. methicillin-resistant Staphylococcus aureus - Google Patents

Abstract

Antibiotic WAP-8294A or its salt, with the following physico-chemical properties is new: (i) white powder (ii) m.pt. 213-220[deg]C (decomposed); (iii) soluble in water, MeOH, n-BuOH, DMF, and DMSO, insol. in acetone, EtOAc and CHCl3; (iv) gives positive ninhydrin, Ehrlich, Rydon-Smith, KMnO4, H2SO4, and iodine vapour colour reactions, negative in Molisch, AgNO3, and Dragendorff reactions, with UV spot from 254 nm radiation; (v) HPLC peaks at 4.0-6.1; 8.0; 11.1; 12.5; 16.5; 17.9 and 18.8 min; (vi) UV lambda max. 275, 280, 287 nm; (vii) NMR in D2O complex; (ix) mol. wt. 1400-1700; (x) elements contained C, H, O and N; (xi) acid hydrolysis gives Asp, Glu, Gly, Leu, Ser, Trp, Orn, N-Me-Val, beta -hydroxyaspartic acid, and N-Me-Phe; (xii) alpha -D/20[deg]C +42[deg]C (c = 0.5, water); and (xiii) amphoteric properties. Also new is a microoganism belonging to the genus Lysobacter and having the ability to produce WAP-8294A, more specifically the sp. WAP-8294 (FERM-BP 4990).

Description

0 "ANTIBIOTIC WAP-8294A, METHOD FOR THE PREPARATION THEREOF AND ANTIBACTERIAL COMPOSITION"

The present invention relates to a new antibiotic, WAP-8294A, which is useful as a means of treating diseases resulting from infection with pathogenic microorganisms, as well as a method for its preparation and use.

Since the history of chemotherapeutic agents for bacterial infectious diseases began with the synthesis of quinine and salvarsan and the discovery of penicillin, humans have been favored with these chemotherapeutic agents. Not long ago, however, it has emerged that the existence of these chemotherapeutic agents leads to the formation of resistant bacteria, as illustrated by the infectious disease with MRSA (methicillin resistant Staphylococcus aureus), against which disease chemotherapeutic agents other than vancomycin and habekacin are not effective. This leads to confusion in the field of medical treatment and poses a serious social problem.

Vancomycin as an antibiotic, was developed by Eli 20 Lilly & Co. in the United States, and has been used to treat MRSA infectious diseases, but such treatment is often accompanied by nephrotoxicity, hepatotoxicity, and scythe disorders (autotoxicity) of 1001012 2 cranial nerve VIII (octavus nerve). This treatment also requires a long time to perform pasteurization and it has been suspected that bacteria resistant to this antibiotic are reoccurring.

On the other hand, habekacin is a chemically prepared antibiotic that has the skeleton of kanamycin as one of the aminoglycoside antibiotics, and has an excellent antibiotic effect as well as an effect on various types of resistance bacteria. Accordingly, it is used as one of a limited number of chemotherapeutic agents for treating MRSA infectious diseases, but its use can sometimes be accompanied by severe nephrotoxicity and cranial nerve VIII (autotoxicity) disorders as side effects typical of the aminoglycoside. antibiotics. Therefore, from the medical point of view, it is not necessarily a safe antibiotic.

Under such circumstances, there has always been a desire for the discovery and development of novel low toxicity chemotherapeutic agents which exhibit antibiotic activity within a short period of time and show virtually no side effects. Many investigations have therefore been carried out to solve this problem.

The object of the present invention is therefore to provide a new antibiotic with antibacterial effect, which satisfies the aforementioned requirements. Another object of the present invention is to provide a method of preparing this antibiotic. Yet another object of the present invention is to provide a clinically suitable chemotherapeutic agent that exhibits low toxicity or very selective toxicity.

The inventors of the present invention have conducted several studies to develop a new and suitable antibiotic, with the result that they have succeeded in producing a strain belonging to the genus Lvsobacter. isolate it from the earth as a new microorganism, and they have found that this strain produces an antibiotic not yet described in the literature. In this way, they have accomplished the present invention.

The present invention therefore relates to the antibiotic WAP-8294A, or pharmaceutically acceptable salts thereof, with the physicochemical properties which will be explained in more detail below. The present invention also provides a method for the preparation of said antibiotic WAP-8294A, which method comprises the steps of cultivation, in a culture medium, of a microorganism belonging to the genus Lvsobacter and which has the ability to producing the antibiotic WAP-8294A, producing the antibiotic, and collecting this antibiotic in the culture medium; and then recovering the antibiotic. The invention further relates to the micro-organism belonging to the genus Lvsobacter, which is capable of producing the antibiotic WAP-8294A. And finally, the invention relates to an antibacterial composition comprising at least one member selected from the group consisting of the antibiotic WAP-8294A and pharmaceutically acceptable salts thereof.

Description of the drawings

Fig. 1 is a chromatogram that becomes visible when the antibiotic WAP-8294A is separated into a series of fractions by high performance liquid chromatography (HPLC).

Fig. 2 is a representation of a UV absorption spectrum (in water) of the antibiotic WAP-8294A (hydrochloride).

FIG. 3 depicts the IR absorption spectrum (FT-IR, KBr) of the antibiotic WAP-8294A (hydrochloride).

Fig. 4 depicts the 1 H-NMR spectrum (270 MHz, D20) of the antibiotic WAP-8294A (hydrochloride).

Fig. 5 depicts a two-dimensional TLC-35 chromatogram of the acid-complete hydrolyzate of the antibiotic WAP-8294A.

1001012 4 4

Fig. 6 is a chromatogram seen when the antibiotic WAP-8294AX is separated by high performance liquid chromatography (HPLC).

Fig. 7 depicts the UV absorption spectrum (in 5 water) of the antibiotic WAP-8294AX-8 (hydrochloride).

Fig. 8 depicts the IR absorption spectrum (FT-IR, KBr) of the antibiotic WAP-8294AX-8 (hydrochloride).

Fig. 9 is a 1 H NMR spectrum (270 MHz, D20) of the antibiotic WAP-8294AX-8 (hydrochloride).

Description of the preferred output formats

The present invention relates to a new antibiotic called WAP-8294A, which is found in the culture medium of a new strain belonging to the genus Lysobacter. This antibiotic WAP-8294A can be further divided into at least 7 components called AX, A1 (A2, A3, A4, A5 and A6 and the component AX is further fractionated into at least 13 components, named AX-1, AX-2, ax-3.20 AX-4, AX-5, AX-6, AX-7, AX-8, AX-9, AX-10, AX-11, AX-12, AX- 13. The term "antibiotic WAP -8294A ", used herein, refers to components AX, A1 (A2, A3, A4. A5, A6, AX-1, AX-2, AX-3. AX-4, AX-5, AX-6, AX-7, AX-8, AX-9, AX-10, AX-11, AX-12, AX-13 or a mixture thereof.

The present invention, in addition to the antibiotic WAP-8294A in the free form, also includes pharmaceutically acceptable salts thereof such as hydrochlorides, sulfates and methanesulfonates.

These compounds exhibit strong antibacterial activity against gram-positive bacteria, in particular, against methicillin-resistant Staphylococcus aureus (MRSA).

The antibiotic WAP-8294A is separated into fractions with retention times of 4.0 to 6.1 minutes, 8.0 minutes, 11.1 minutes, 12.5 minutes, 16.5 minutes, 17.9 minutes and 18.8 minutes, measured under eluting conditions for the C18 reverse phase silica gel high performance liquid chromatography (C18-RP silica gel HPLC) [column: YMC A-312 (6 x 150 mm): mobile phase: 0.05% triflu-1001012 oracetic acid-containing acetonitrile: water (45:55); detection wavelength: UV 214 nm; fluid rate: 1 ml / min], that is, the components AX, A1 (A2, A3, A4, As and A6 (Fig.

1).

The fraction AX is, as shown in Fig. 6, further separated into fractions with retention times of 5.3 minutes, 5.9 minutes, 6.2 minutes, 6.5 minutes, 6.9 minutes, 7.3 minutes, 8.1 minutes, 9.3 minutes, 9 , 8 minutes, 11.3 minutes, 12.1 minutes, 13.7 minutes and 15.0 minutes, measured under eluting conditions in the Cie-RP silica gel HPLC [column: YMC A-312 (6 x 150 mm); mobile phase: 0.05% trifluoroacetic acid containing acetonitrile: water (37:63); detection wavelength: UV 214 nm; liquid velocity: 1 ml per min., ie components AX-1, AX-2, AX-3, AX-4, AX-5, AX-6, 15 AX-7, AX-8, AX-9 , AX-10, AX-11, AX-12 and AX-13.

The antibiotics WAP-8294A, A1 (A2 and A1) have ultraviolet absorption spectra (in water) at Xmax 275 nm, 280 nm and 287 nm. The ultraviolet absorption spectrum of the antibiotic WAP-8294A2 is shown in Fig. 2. It is also clear that the antibiotics include WAP-8294A, A1 (A2 and A4) tryptophan as chromophor in the molecules, as confirmed by the analysis of the acid hydrolysates thereof, as will be described later.

The infrared absorption spectroscopic analysis of the antibiotics WAP-8294A, A1, A2 and A4 shows absorption at 3300 cm "1, attributed to OH and NH groups; 1720 to 1715 cm" 1, attributed to carboxyl or ester carbonyl groups ; 1636 and 1541 cm -1 attributed to amido bonds; and 1207 and 1137 cm -1 attributed to the C-O stretch 30 vibrations, but they do not exhibit characteristic absorption. The IR absorption spectrum of the antibiotic WAP-8294A2 is shown in Fig. 3.

In the 1 H-NMR spectroscopic measurement of the antibiotics WAP-8294A, A1 (A2 and A4), a number of complicated proton signals, which come from methyl, methylene, methine, as well as from heterocyclic or aromatic rings, are seen. spectrum of the antibiotic WAP-8294A2 is shown in Fig. 4.

1001012 6

The FAB mass spectrometric measurement of WAP-8294Alf A2 and A4, indicates that the (M + H) + ion of the component A3 has an m / z value of 1548.9, that the (M + H) + ion of the component A2 has an m / z value of 1562.9 and that the 5 (M + H) + ion of the component A4 has an m / z value of 1576.9. The results of a sodium melt test of the components Alf A2 and A4 make it clear that these components are each a compound comprising carbon, hydrogen, oxygen and nitrogen elements. The following 10 molecular formulas, shown in Table 1, can be derived from these facts, while also taking into account the structure estimation and molar ratios of all the amino acids and fatty acids that make up these components, as will be explained later, as well the 15 results of the high resolution FAB mass spectrometric measurements.

TABLE 1 2 0 Ai A2 A4 FAB mass (Μ + ΗΓ 1548.9 1562.9 1576.9 FAB mass (MH) '1546.7 1561.2 1575.4 Δ mass unit 14 mass 14 mass units units 25 A2 ------> A2 A3 ----> A4

Molecular Weight 1547.9 1561.9 1575.9 HR-FAB Mass 1458.8088 1562.8224 1576.8363

Molecular Formula O72H109021N17 C73H113021N17 C74H113021Nl7 30 The antibiotics WAP-8294A, A1 (A2 and A4) are positive in reactions of ninhydrin, Ehrlich, Rydon-Smith, iodine vapor, aqueous solution of potassium permanganate and sulfuric acid; ) by irradiation with light rays with a wavelength of 254 35 nm, emitted by a UV lamp, and are negative in the reactions of Molisch, silver nitrate, ferric chloride and Dradendorff Furthermore, the antibiotics WAP-8294A, Ax, A2 1001012 7 and A4, fully hydrolysed with an acid and then subjected to two-dimensional TLC and amino acid analysis, with the aim of investigating the amino acid compositions of these antibiotics The results obtained in this way indicate that each of these compounds is aspartic acid (Asp), glutamic acid (Glu), glycine (Gly), leucine (Leu), serine (Ser), tryptophan (Trp) and ornithine (Orn), as well as 3 unknown amino acids. Figure of the two-dimensional TLC analysis of the antibiotic WAP-8294A 10 is shown in FIG. 5.

In this way, unknown amino acid 1, unknown amino acid 2 and unknown amino acid 3 were isolated by re-hydrolyzing the WAP 8294A with an acid, with the aim of identifying these unknown amino acids. The unknown 15 amino acids 1, 2 and 3 were found to be β-hydroxy aspartic acid, N-methylvaline and N-methylphenylalanine, respectively, based on the results of the different instrumental analyzes.

On the other hand, 3-hydroxyoctanoic acid, 3-hydroxy-7-methyloctanoic acid or 3-hydroxy-8-methylnonanoic acid were isolated from the ether extract of each solution obtained by acid hydrolysis of the individual component WAP-8294A1 (A2 or A".

Furthermore, the antibiotic WAP-8294AX was separated into at least 13 fractions, ie AX-1: retention time 5.3 min .; AX-2: retention time 5.9 min .; AX-3: retention time 6.2 min .; AX-4: retention time 6.5 min .; AX-5: retention time 6.9 min .; AX-6: retention time 7.3 min .; AX-7: retention time 8.1 min .; AX-8: retention time 9.3 min .; AX-9: retention time 9.8 30 min .; AX-10: retention time 11.3 min .; AX-11: retention time 12.1 min .; AX-12: retention time 13.7 min .; and AX-13: retention time 15.0 min., as is evident from the chromatogram of Cie reverse phase silica gel "high performance liquid chromatography" shown in FIG. 6.

The antibiotic WAP-8294AX, AX-8, AX-9 and AX-13, have ultraviolet absorption spectra (in water) at λΜΧ 273 nm, 280 nm and 289 nm. It also becomes clear that the WAP-8294AX, AX-8, AX-9 and AX-13 comprise one mole of tryptophan 1001012 8 as chromophor in the molecule, as confirmed by the analysis of its acid hydrolysates, as detailed later will be explained. The ultraviolet absorption spectrum of the antibiotic WAP-8294AX-8 is shown in FIG. 7.

The infrared absorption spectroscopic analysis of the antibiotics WAP-8294AX, AX-8, AX-9 and AX-13 show absorption at 3300 cm'1, attributed to OH and NH groups, 1720 to 1715 cm'1, attributed to carboxyl or ester carbonyl-10 groups; 1636 and 1541 cm -1, attributed to amido bonds; and 1207 and 1137 cm -1, attributed to C-O stretch fibers, but they do not exhibit other characteristic absorbances. The IR absorption spectrum of the antibiotic WAP-8294AX-8 is shown in Fig. 8.

In the ^ -NMR spectroscopic measurements of the antibiotics WAP-8294AX, AX-8, AX-9 and AX-13, a number of complicated proton signals are seen, which come from methyl, methylene, methine, and heterocyclic or aromatic rings. The -NMR spectrum of the antibiotic WAP-20 8294AX-8 is shown in Fig. 9.

The FAB mass spectrometric measurements of the WAP-8294AX-8, AX-9 and AX-13 show that the (M + H) * ion of the component AX-8 has an m / z value of 1549, that it (M + H) * - ion of component AX-9 has an m / z value of 15 1549 and that the (M + H) 'ion of component AX-13 has an m / z value of 1577 The results of the sodium melt test of components AX-8, AX-9 and AX-13 make it clear that these components are each individually compounds containing carbon, hydrogen, oxygen and nitrogen elements. The following molecular formulas, shown in Table 2, can be derived from these facts, while also taking into account the structure estimate and molar ratios of all the amino acids and fatty acids that make up these components, as well as the results of higher resolution 35 FAB mass spectrometric measurements, as will be explained later.

1001012 9 TABLE 2

Component AX-8 AX-9 AX-13 FAB mass (Μ + ΗΓ 1549 1549 1577 5 FAB mass (MH) '- 1547 Δ mass unit 14 mass 14 mass 14 mass units units units A2 ---> AX - 8 Ai ---> AX- 9 A3> A4

Molecular weight 1548 1548 1576 10 HR-FAB mass 1548.8079 1548.8065 1576.8324

Molecular Formula O72H109O21N17 C72H109O21N17 C74H113021N17

The antibiotics WAP-8294AX, AX-8, AX-9 and AX-13 are positive in reactions of ninhydrin, Ehrlich, Rydon Smith, iodine vapor, aqueous solution of potassium permanganate and sulfuric acid; they exhibit quenching spots by "irradiation with light rays having a wavelength of 254 nm, emitted by a UV lamp; and are negative in the reactions of Molic, silver nitrate, ferric chloride and Dragendorff.

To examine the amino acid compositions of the antibiotics WAP-8294AX, AX-8, AX-9 and AX-13, each of them was fully hydrolyzed with an acid and then subjected to two-dimensional TLC analysis [cellulose plate; Solvent 1: n-butanol: acetic acid: water (4: 1: 2); solvent 2: n-butanol: pyridine: acetic acid: water (15: 10: 3: 12); spray reagent: ninhydrin] and amino acid analysis. As a result, WAP-8294AX was found to include the following amino acids, which are positive in the ninhydrin reaction: aspartic acid (Asp), glutamic acid (Glu), glycine (Gly), 3-alanine (β-Ala), leucine (Leu), serine (Ser), tryptophan (Trp), ornithine (Orn), valine (Val), N-methylvaline (N-MeVal), S-hydroxyaspartic acid (β-ΟΗ-Asp), pheneylalanine (Phe) and N-methylphenylalanine (N -MePhe).

On the other hand, it was also confirmed that the ether extract of the acid-complete hydrolyzate of the antibiotic WAP-8294AX included the 3-hydroxy-4-methyloctanoic acid.

1001012 10

The antibiotics WAP-8294AX-8, ΑΧ-9 and AX-13 were treated by the same procedure used for the treatment of the antibiotic WAP-8294AX and the amino acids and fatty acids that make up these components could be identified based on from the two-dimensional TLC analysis, amino acid analysis, and GC mass analysis of the ether extract of the acid-complete hydrolysates. The results are shown in Table 3, together with the results noted for the antibiotic WAP-10 8294A2.

TABLE 3

Amino acids and fatty acids that make up each fraction (molar number is given in brackets) 15

Compo-AX-8 AX-9 AX-13 A, (main component)

Amino- Asp (l) Asp (l) Asp (l) Asp (l) acid Ser (2) Ser (2) Ser (2) Ser (2)

Glu (l) Glu (l) Glu (l) Glu (l)

Gly (l) Gly (l) Ê-Ala (l) Gly (l)

Leu (l) Leu (l) Leu (l) Leu (l)

Trp {1) Trp (1) Trp (1) Trp (1)

Orn (2) Orn (2) Orn (2) Orn (2) β-ΟΗ-Asp- β-ΟΗ-Asp- β-ΟΗ-Asp- β-ΟΗ-Asp- (1) (1) (1) ( 1)

Val (1) N-MeVal (1) N-MeVal (l) N-MeVal (l) N-MePhe (1) Phe (l) N-MePhe (l) N-MePhe (l)

20 Fat- 3H-7M-OA 3H-7M-OA 3H-7M-OA 3H-7M-0A

acid (1) (1) (1) (1)

Note: 3H-7M-OA stands for 3-hydroxy-7-methyloctanoic acid.

The inventors have searched for known compounds based on the physicochemical properties and knowledge of the acid-complete hydrolysates of the 1001012 11 antibiotics WAP-8294A, A1 (A2, A4, AX, AX-8, AX-9 and AX) described above. -13, and the amino acids and fatty acids that make up these antibiotics.

As a result, they have confirmed positive evidence that these are new compounds not previously described in the literature.

The antibiotics WAP-8294A, Au A2 (A1, AX, AX-8, AX-9 and AX-13, according to the present invention, can be prepared by the method comprising the steps of: growing in a culture medium of a bacterium-10, which produces the antibiotic WAP-8294A, which bacterium belongs to the genus Lysobacter · isolating and collecting the antibiotic WAP-8294A, and, if necessary, additionally separating and purifying it An example of a bacterium producing WAP-8294A belonging to the genus Lvsobacter is a Lysobacter sp. WAP-8294 strain isolated by the inventors of the present invention from the earth of Shimoda City, Shizuoka Prefecture , Japan The taxonomic bacteriological properties of the strain are as follows: 1. Morphology:

The strain was grown for 3 days at 25 ° C, on a broth agar plate, viewed under a microscope, and was found to be a rod-shaped cell with a diameter ranging from 0.4 to 0.6 μτη and a length varying from 3.2 to 4.2 μτη. The strain is a gram-negative bacterium, free from flagellae but shows a sliding movement, does not form spores and is free from acid fastness.

30 2. Growth characteristics in a variety of culture media

The strain was grown at 25 ° C in a variety of culture media and observed for 3 to 14 days.

(1) Broth agar plate culture 35 The formed colony has a translucent pale yellow circular shape, a convex circular surface and a full to undulating periphery. The trunk does not form a diffusing pigment.

1001012 12 (2) Broth agar-slant culture

The trunk grows in the form of a scattered tissue and has a translucent weak yellow color.

(3) Liquid culture broth 5 The medium becomes slightly cloudy, the stem forms a smooth ring and deposits of bacterial cells on the bottom of the culture vessel become visible.

(4) Broth-gelatin perforated culture

The trunk grows within an area extending from the surface to the center of the medium, while the gelatin is liquefied.

(5) Litmus milk culture

The strain appears to be unable to reduce the litmus and is free from coagulation, but capable of peptone.

(6) Culture skim milk-acetate agar

The strain shows strong proteolytic activity and grows on the agar plate to form highly viscous gel-like masses.

20 3. Physiological properties:

The physiological properties of the WAP-8294 strain are as follows: 25 (1) Nitrate reduction (2) Denitrification reaction (3) MR test (4) VP test (5) Indole formation 30 (6) Hydrogen sulfide ( 7) Hydrolysis of starch (8) Application of citric acid 1. Koser culture medium 2. Simmonds culture medium 35 3. Christensen culture medium + (9) Application of inorganic nitrogen source 1. Sodium nitrate + 2. Ammonium sulfate + 1001012 13 3. Sodium glutamate + (10) Formation of pigment 1. King A culture medium 2. King B culture medium 5 (11) Urease (12) Oxidase + (13) Catalase +

(14) Growth temperature 15 to 37 ° C

(15) Growth pH 5 to 8 10 (16) Behavior towards oxygen aerobic (17) OR test non-decomposing type (18) Deoxyribonuclease + (19) Phosphatase + (20) Hemolysis β 15 (21) Cellulose degradation (22) Degradation of Tween 1. Tween 20 + 2. Tween 40 + 3. Tween 60 + 20 4. Tween 80 + (23) Degradation of colloidal chitin + (24) Degradation of polysaccharide 1. CMC + 2. Alginic acid 25 3 Pectic acid (25) Bacteriolytic activity 1. Saccharomyces cerevisiae + 2. Bacillus subtilis + 3. Escherichia coli + 30 (26) GC content 68.3% (HPLC method) (27) Acid and gas production 10 010 12 14

Sugar Formation Formation Application of sugar gas acid (Pepton (Bouillon (Davis water) agar) culture medium) L-Arabinose - D-Xylose - - - 5 D-Glucose + - D-Mannose + D-Fructose + D-Galactose +

Maltose + - + 10 Sucrose + -

Lactose +

Trehalose + - D-Sorbitol D-Mannitol - 15 Inositol -

Glycerin -

Starch + - +

Cellobiose + - + 20 If the foregoing results regarding WAP-8294 strain are compared to the details of bacterial species, which are described in "Bergey's Manual of Systematic Bacteriology", vol. 3 (1989), it can be concluded that the strain is a gram-negative aerobic rod, flagella-free and has motility by sliding and therefore the strain belongs to the sliding bacteria.

Sliding bacteria are classified into non-fruiting sliding bacteria, which do not form fruiting bodies and fruiting sliding bacteria, which do form fruiting bodies. The trunk does not form a fruiting body and therefore belongs to the non-fruiting sliding bacteria. The sliding bacteria are further divided into three 1001012 15 orders, namely, Cvtophagales. Beggiotales and Lysobacter ales. which can be clearly distinguished from each other based on the GC content of each DNA, which content is one of the important key properties. To this end, the DNA from the strain was extracted by the conventional method and as a result, its GC content was found to be high, on the order of 68.3%, as determined by the HPLC method is described in FEMS Microbiol. Letters, 1984, 25, p. 125. Accordingly, it can be concluded that the strain belongs to the genus Lvsob acter of the order Lysobacterales which have a high GC content, ranging from 65 to 71%.

When the properties of the strain are compared with the properties of the species belonging to the same genus, the properties thereof appear to correspond to those of Lysobacter enzymogenes. but do not correspond in detail to the latter. For example, a difference is the ability to assimilate citric acid. Accordingly, the strain Lvsobacter sp. WAP-8294 2 0.

The WAP-8294 strain was deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science & Technology, Japan, on January 31, 1994, under the accession number FERM BP-4990 (FERM P-14093).

Bacteria, including Lysobacter. fairly easily undergo changes in their taxonomic properties, as is their general nature, and the WAP-8294 strain is no exception. All microorganisms capable of producing the antibiotic WAP-8294A can be used in the method of the present invention. These microorganisms include variants that are naturally mutated or artificially mutated with a variety of mutagens.

35 (Breeding and production)

The antibiotic WAP-8294A of the present invention is produced by inoculating a culture medium containing a nutrient source with said WAP-8294A-10 010 12 16 producing bacteria, and culturing the bacteria under aerobic conditions. For example, if the antibiotic WAP-8294A-producing bacteria are grown, the bacteria use assimilable organic carbon containing compounds such as glucose, fructose, starch, dextrin, glycerine, molasses, starch syrup, fats and oils, and organic acids as carbon sources, and organic and inorganic nitrogen-containing compounds such as soybean flour, cottonseed flour, grain extract, casein, peptone, yeast extract, meat extract, seed germs, urea, amino acids and ammonium salts as nitrogen sources. In addition, examples of such salts are inorganic salts such as sodium, potassium, calcium and magnesium salts and phosphates which can be used either singly or in combination. In addition, it is desirable to optionally add heavy metal salts such as iron salts, copper salts, zinc salts and cobalt salts to the culture medium; vitamins necessary for the growth of microorganisms, such as biotin and vitamin Bx; as well as other organic and / or inorganic compounds capable of enhancing the growth of the bacteria producing the antibiotic WAP-8294A, as well as improving the yield of the WAP-8294A. The culture medium may likewise comprise an anti-foaming agent, such as silicone oil or a polyalkylene glycol ether, as well as a surfactant.

The strain is cultivated by any method commonly employed for antibiotic production, but culture techniques using submerged aeration agitation are preferred to maintain aerobic conditions and the usual protective culture method in a bottle or flask is suitable for cultivation on a laboratory scale. Culturing can generally be carried out at a temperature ranging from 20 to 40 ° C and preferably from 25 to 30 ° C. Culturing is carried out at a pH ranging from about 6 to 8 and preferably about 7. The culture time ranges from about 2 to 6 days and is sufficient to ensure a desired yield of the WAP-8294A, which has accumulated in the culture medium.

(Isolation and Purification) The recovery of the antibiotic WAP-8294A accumulated in the culture medium in this manner can be advantageously carried out by using the physicochemical properties of the antibiotic compounds of the present invention, such as 10 will be explained later. More specifically, the antibiotic WAP-8294A is present in the filtrate of the culture medium. To this end, the filtrate is collected from the culture medium by first adding a filter aid, such as Celite or Radiolite, and then removing the bacterial cells by filtration under reduced pressure or by centrifugation.

The antibiotics WAP-8294A are considered to be water-soluble polypeptide antibiotics, in view of their physicochemical properties, which are described in detail below. The antibiotics are therefore made up of relatively large compounds, each having a molecular weight ranging from 1400 to 1700. When collecting the WAP-8294A from the filtrate of the culture medium, if these properties are taken into account, adsorbents are resins such as Diaion HP-20, Sepabeads SP207, CHP-20 (available from Mitsubishi Chemical Industries Ltd.), Amberlite XAD-2 (available from Rohm & Haas Co.), Duolite S-30 (available from Diamond Shamrock Chemical Co.) useful. In particular, the antibiotic is efficiently purified by activated carbon column chromatography.

For example, the filtrate of the culture medium, having a pH of 6.9, is passed through a column filled with Amberlite IR-120B (H + type), the resulting acidic liquid passed through the column is then passed through a column filled with activated carbon without any treatment, in order to adsorb the filtrate in this way, followed by washing with water and eluting with a mixture of 80% 10 010 12 18 acetone-water or an alkaline mixture of 80% acetone -water as an eluate to recover the antibiotic WAP-8294A in this way. After distilling off the acetone from the eluate under reduced pressure, the resulting residue is extracted with ethyl acetate under acidic conditions to remove the oil-soluble impurities. Then, the aqueous phase is re-extracted with a high-polarity organic solvent, such as, for example, N-butanol, to recover the WAP-8294A. After distilling the N-butanol from the extract at a low temperature, the residue is per se concentrated to dryness or a poor solvent is added to the concentrate of the residue to precipitate the WAP-8294A in this way, followed by centrifugation or filtration under reduced pressure to give a crude compound.

The crude compound recovered in this way can be further purified by countercurrent partitioning methods, including centrifugal liquid-liquid partition chromatography, adsorption chromatography, such as silica gel column chromatography, and partition chromatography, such as cellulose column chromatography, used individually or in combination can be used to obtain a highly purified compound, because the WAP-8294A can be extracted with polar organic solvents such as n-25 butanol under acidic conditions.

Alternatively, it is also possible to purify the compound using the molecular sieve effect of Sephadex G-10, G-15, LH-20 (available from Pharmacia Company) or Biogel P-4 gel (available from Bio-Rad 30 laboratories) and then developing and eluting the compound with water, water-containing lower alcohols, such as water-containing methanol, dilute alkaline or acidic aqueous solutions, and aqueous solution comprising suitable salts, which can be used individually or in combination.

The individual 7 components AX, Alt A2, A3, A4, As and A6 are obtained by further purifying and separating the WAP-8294A using, for example, the countercurrent distribution techniques described above and / or "high performance liquid". chromatography "(" HPLC ") techniques. The 13 types of components AX-l, AX-2, AX-3, AX-4, AX-5, AX-6, AX-7, AX-8, AX-9, AX-10, AX-ll, AX- 12 and AX-13 can be obtained in the same manner by purifying and separating the AX component in the manner described above.

The fillers used in the HPLC chromatography may be silica gel, commercially available protruding supports in which alkyl groups such as octadecyl and octyl-10 groups are chemically bonded to the silanol groups of silica gel, or general purpose supports such as polystyrene type porous polymer gels. The WAP-8294A and the AX component can be separated into separate components, through the use of these filler materials, with high effectiveness. The mobile phase usable in the HPLC chromatography can be aqueous acetonitrile acidified by, for example, trifluoroacetic acid, or aqueous lower alcohols such as aqueous methanol, as well as buffer solutions.

20 (Physicochemical properties)

The physicochemical properties of the WAP-8294A, Alf A2, A4, AX, AX-8, AX-9 and AX-13 obtained by the above-described various chromatographic techniques, the compounds obtained by the complete acid hydrolysis thereof and which are positive in the ninhydrin reactions, as well as ether extracts of the acid hydrolysates, will be described below.

30 Antibiotic WAP-8294A

(1) Appearance: white powder; (2) Melting point: 213-220 ° C (decomposition); (3) Solubility: soluble in water, methanol, n-butanol, dimethylformamide and dimethyl sulfoxide and insoluble in acetone, ethyl acetate and chloroform, (4) Color reaction: positive in reactions of ninhydrin,

Ehrlich, Rydon-Smith, aqueous solution of potassium permanganate, sulfuric acid and iodine vapor, and exhibits fading spots upon irradiation with light beams of a wavelength of 254 nm emitted by a UV lamp; negative in reaction of Molic, silver nitrate, ferric chloride and Dragendorff; 5 (5) Retention time in HPLC (Fig. 1): 4.0 to 5.1 minutes, 8.0 minutes, 11.1 minutes, 12.5 minutes, 16.5 minutes, 17.9 minutes, 18.8 minutes; (6) Ultraviolet absorption spectrum {in water):

Xmax; 275 nm, 280 nm, 287 nm; (7) Infrared absorption spectrum (FT-IR, KBr). Characteristic absorption spectrum: 3300 cm'1, 1720-1715 cm'1, 1636 cm'1, 1541 cm'1, 1404 cm'1, 1207 cm'1, 1137 cm -1; (8) Molecular weight: 1400-1700, - (9) Qualitative test for organic compound (sodium melting method): the antibiotic is a compound comprising carbon, hydrogen and oxygen and nitrogen elements; (10) Acid complete hydrolysis provides, as ninhydrin positive compounds, Asp, Glu, Gly, Leu, Ser, Trp, 20 Orn, N-methylvaline, β-hydroxyaspartic acid and N-methylphenylalanine; (11) Specific rotation: [a] d20 = + 42 ° (c = 0.5, H20), (12) Classification in base, acid and neutral: amphotheer.

25 Antibiotic WAP-8294A, (1) Appearance: white powder, (2) Melting point: 215-225 ° C (decomposition), - (3) Solubility: soluble in water, methanol, n-butanol, dimethylformamide and dimethyl sulfoxide and insoluble 30 in acetone, ethyl acetate and chloroform, (4) Color reaction: positive in reactions of ninhydrin,

Ehrlich, Rydon-Smith, aqueous solution of potassium permanganate, sulfuric acid and iodine vapor, and exhibits fading spots upon irradiation with 254 nm wavelength light rays emitted by a UV lamp; negative in reaction of Molic, silver nitrate, ferric chloride and Dragendorff, - (5) Retention time in HPLC: 8.0 minutes; 1001012 21 (6) Ultraviolet absorption spectrum (in water):

Xmax: 275 nm, 280 nm, 287 nm; (7) Infrared absorption spectrum (FT-IR, KBr): characteristic absorption spectrum: 3300 cm'1, 1720-1715 cm'1, 1636 5 cm'1, 1541 cm'1, 1404 cm'1, 1207 cm'1, 1133 cm'1; (8) Molecular weight: 1547.9 (FABS-MS m / z 1548.9 (M + H) +, m / z 1546.7 (M-H) +); (9) Molecular formula: C-, 2H109021N17 (measured m / z value of (M + H) + ion as determined by high resolution 10 FAB mass: 1548,8088; calculated m / z value: 1548 , 8063]; (10) Acid complete hydrolysis provides: Asp (1 mol), Glu (1 mol), Gly (1 mol), Leu (1 mol), Ser (2 mol), Trp (1 mol), Orn (2 mol), N-methylvaline (1 mol), β-hydroxyaspartic acid (1 mol), N-methylphenylalanine (1 mol) and 15 3-hydroxyoctanoic acid (1 mol); (11) Specific rotation: [a] / c = + 41 ° (c = 0.5, H 2 O), - (12) Classification in base, acid and neutral: amphoteric;

Antibiotic WAP-8294A-, 20 (1) Appearance: white powder, (2) Melting point: 215-225 ° C (decomposition); (3) Solubility: soluble in water, methanol, n-butanol, dimethylformamide and dimethyl sulfoxide and insoluble in acetone, ethyl acetate and chloroform; 25 (4) Color reaction: positive in reactions of ninhydrin,

Ehrlich, Rydon-Smith, aqueous solution of potassium per manganate, sulfuric acid and iodine vapor, and exhibits fading spots when irradiated with light beams of a wavelength of 254 nm emitted by a UV lamp, · negative in reaction of Molic, silver nitrate, ferric chloride and Dragendorff; (5) Retention time in HPLC: 11.1 minutes; (6) Ultraviolet absorption spectrum (in water) (Fig. 2): Xmax: 275 nm (Ej cm1% 31.8), 280 nm (E2 OT1% 33.4), 287 nm (Ei 35 ", 1 * 29, 2); (7) Infrared absorption spectrum (FT-IR, KBr) (Fig. 3): Characteristic absorption spectrum. 3300 cm'1, 1720-1715 1001012 22 cm'1, 1636 cm'1, 1541 cm'1, 1404 cm'1, 1207 cm'1, 1137 cm '1.

t (8) -NMR spectrum: (270 MHz, D20) (Fig. 3): complicated proton signals are seen, which come from methyl, methylene, methine and heterocyclic or aromatic rings; (9) Molecular weight: 1561.9 [FAB mass m / z 1562.9 (Μ + ΗΓ, m / z 1561.2 (MH) 'J; (10) Molecular formula: C73H111021N17 [measured m / z value of 10 (M + H) + - ion as determined by high resolution FAB mass: 1562.8224, - calculated m / z value: 1562.8219], - (11) Acid complete hydrolysis, as hydrolysates, provides Asp (1 mol), Glu (1 mol, Gly (1 mol), Leu (1 mol), Ser (2 mol), Trp (1 mol), Orn (2 mol), N-methylvaline (1 15 mol), β-hydroxyaspartic acid (1 mol), N-methylphenylalanine (1 mol), and 3-hydroxy-7-methyloctanoic acid (1 mol), - (12) Specific rotation: [0-) 020 = + 42 ° (c = 0.5, - H 2 O), - (13) Classification in base, acid and neutral: amphotheer, 20

Antibiotic WAP8294A, (1) Appearance: white powder; (2) Melting point: 215-225 ° C (decomposition); (3) Solubility: soluble in water, methanol, n-butanol, dimethylformamide and dimethyl sulfoxide and insoluble in acetone, ethyl acetate and chloroform, (4) Color reaction. · Positive in reactions of ninhydrine, Ehrlich, Rydon-Smith, aqueous solution of potassium per manganate, sulfuric acid and iodine vapor, and exhibits effective stains upon irradiation with light beams of a wavelength of 254 nm emitted by a UV lamp, negative in reaction of Molic, silver nitrate, ferric chloride and Dragendorff; (5) Retention time in HPLC: 16.5 minutes, · 35 (6) Ultraviolet absorption spectrum (in water): Xmax: 275 nm, 280 nm, 289 nm; 1001012 23 (7) Infrared absorption spectrum (FT-IR, KBr): Characteristic absorption spectrum: 3300 cm'1, 1720-1715 cm'1, 1636 cm'1, 1541 cm'1, 1404 cm'1, 1207 cm'1, 1137 cm1, (8) Molecular weight: 1400-1700; 5 (9) Molecular formula: C73H113021N17 [measured m / z value of (M + H) + ion as determined by high resolution FAB mass: 1576.8363, calculated m / z value: 1576.8375 ]; (10) Acid hydrolysis, as hydrolysates, provides Asp (1 mol), Glu (1 mol, Gly (1 mol), Leu (1 mol), Ser (2 10 mol), Trp (1 mol), Orn (2 mol), N-methylvaline (1 mol), β-hydroxyaspartic acid (1 mol), N-methylphenylalanine (1 mol), and 3-hydroxy-8-methylnonanoic acid (1 mol); (11) Specific rotation: [o '] D20 = + 43 ° (c = 0.5; H20); (12) Classification in base, acid and neutral: amphotheer; 15

The elution conditions for the Cle-RP silica gel HPLC

of the antibiotics WAP-8294A, Aj, A2 and A4 are as follows:

Column: YMC A-312 (6 x 150 mm)

Mobile phase: 0.05% trifluoroacetic acid containing acetonitrile: water (45:55)

Wavelength detection: UV 214 nm

Liquid speed: 1 ml / min.

Antibiotic WAP-8294AX

25 (l) Appearance: white powder; (2) Melting point: 196 ~ 200 ° C (decomposition); (3) Solubility: soluble in water, methanol, n-butanol, dimethylformamide and dimethyl sulfoxide and insoluble in acetone, ethyl acetate and chloroform; 30 (4) Color reaction: positive in reactions of ninhydrin,

Ehrlich, Rydon-Smith, aqueous solution of potassium permanganate, sulfuric acid and iodine vapor, showing fading spots when irradiated with light beams of a wavelength of 254 nm emitted by a UV lamp; Negative in reaction of Molic, silver nitrate, ferric chloride and Dragendorff; (5) Retention time in HPLC (Fig. 6): 5.3 minutes, 5.9 minutes, 6.2 minutes, 6.5 minutes, 6.9 minutes, 7.3 minutes 1001012 24 hours, 8.1 minutes 9.3 minutes, 9.8 minutes, 11.3 minutes, 12.1 minutes, 13.7 minutes and 15.0 minutes; (6) Ultraviolet absorption spectrum (in water):: 273 nm, 280 nm, 28 "nm, 5 (7) Infrared absorption spectrum (FT-IR, KBr): Characteristic absorption spectrum: 3300 cm'1, 1720 ~ 1715 cm ' 1.1636 cm -1, 1541 cm -1, 1404 cm -1, 1207 cm -1, 1137 cm -1; (8) Molecular weight: 1575.9 (FAB mass m / z 1576.9 (M + H) *, m / z 1575.4 (MH) ']; 10 (9) Qualitative test for organic compounds (sodium melting method): the antibiotic is a compound comprising carbon-hydrogen-oxygen and nitrogen elements; (10) Acid complete hydrolysis, as ninhydrin positive compounds and fatty acids, provides: Asp, Glu, Gly, β-

Ala, Leu, Ser, Trp, Orn, Val, N-methylvaline, β-hydroxyaspartic acid, Phe, N-methylphenylalanine and 3-hydroxy-7-methyloctanoic acid; (11) Specific rotation: [a] d20 = +24 ° (c = 0.5, H20), 20 (12) Classification in base, acid and neutral: amphotheer, ·

Antibiotic WAP-8294AX-8 (1) Appearance: white powder; (2) Melting point: 196 ~ 200 ° C (decomposition); (3) Solubility: soluble in water, methanol, n-butanol, dimethylformamide and dimethyl sulfoxide and insoluble in acetone, ethyl acetate and chloroform; (4) Color reaction: positive in reactions of ninhydrin,

Ehrlich, Rydon-Smith, aqueous solution of potassium permanganate, sulfuric acid and iodine vapor, and exhibits adequate stains upon irradiation with 254 nm wavelength light emitted by a UV lamp; negative in reaction of Molic, silver nitrate, ferric chloride and Dragendorff; (5) Retention time in HPLC: 9.3 minutes (6) Ultraviolet absorption spectrum (in water) (Fig. 7): Xmax: 273 nm, 280 nm, 289 nm; 1001012 25 (7) Infrared absorption spectrum (FT-IR, KBr) (Fig. 8).

Characteristic absorption spectrum: 3300 cm1, 1720-1715 cm'1, 1636 cm1, 1541 cm 1, 1404 cm'1, 1207 cm1, 1133 cm '1 # t 5 (8) ^ -NMR spectrum: (270 MHz, DMS0-D «) (Fig. 9): Complicated prototypes are measured from methyl, methylene, methine and heterocyclic or aromatic rings, · (9) Molecular weight: 1548 (FAB mass m / z 1549 (M + H) + ); 10 (10) Molecular formula · C72H109O21N17 (measured m / z value of (M + H) * ion as determined by high resolution FAB mass: 1548.8079; calculated m / z value. (11) Acid complete hydrolysis provides, as hydrolysates, Asp (1 mol), Glu (1 mol), Gly (1 mol), Leu (1 mol), Ser 15 (2 mol), Trp (1 mol) , Orn (2 mol), Val (1 mol),,-hydroxyaspartic acid (1 mol), N-methylphenylalanine (1 mol) and 3-hydroxy-7-methyloctanoic acid (1 mol); (12) Specific rotation: [οΊβ20 = + 25 °) c = 0.5, H 2 O); (13) Classification in base, acid and neutral: amphotheer; 20

Antibiotic WAP-8294AX-9 (1) Appearance: white powder; (2) Melting point: 216 ~ 220 ° C (decomposition); (3) Solubility: soluble in water, methanol, n-butanol, dimethylformamide and dimethyl sulfoxide and insoluble in acetone, ethyl acetate and chloroform; (4) Color reaction: positive in reactions of ninhydrin, Ehrlich, Rydon-Smith, aqueous solution of potassium permanganate, sulfuric acid and iodine vapor, and exhibits effective staining upon irradiation with light beams of a wavelength of 254 nm emitted by a UV lamp. ; negative in reaction of Molic, silver nitrate, ferric chloride and Dragendorff, · (5) Retention time in HPLC: 9.8 minutes 35 (6) Ultraviolet absorption spectrum (in water): Xmax: 273 nm, 280 nm, 289 nm; 1001012 26 (7) Infrared absorption spectrum (FT-IR, KBr): Characteristic absorption spectrum: 3300 cm'1, 1720-1715 cm "1, 1636 cm" 1, 1541 cm'1, 1404 cm'1, 1207 cm'1, 1137 cm -1; (8) Molecular weight: 1548 (FAB mass m / z 1549 (M + H) *; 5 m / z 1547 (M-H) "); (9) Molecular formula: C72H109O21N17 [measured m / z value of (M + H) + ion as determined by high resolution FAB mass: 1548,8065, calculated m / z value: 1548,8063], (10) Acid complete hydrolysis, as hydrolysates, provides 10 Asp (1 mol), Glu (1 mol), Gly (1 mol), Leu (1 mol), Ser (2 mol), Trp (1 mol) Orn (2 mol), Val (1 mol), β-hydroxyaspartic acid (1 mol), Phe (1 mol) and 3-hydroxy-7-methyloctanoic acid (1 mol); (11) Specific rotation: [a] c20 = + 28 ° (c = 0.5, H20); (12) Classification in base, acid and neutral: amphotheer;

Antibiotic WAP-8294AX-13 (1) Appearance: white powder, - (2) Melting point: 205 ~ 210 ° C (decomposition); (3) Solubility: soluble in water, methanol, n-butanol, dimethylformamide and dimethyl sulfoxide and insoluble in acetone, ethyl acetate and chloroform; (4) Color reaction: positive in reactions of ninhydrin,

Ehrlich, Rydon-Smith, aqueous solution of potassium permanganate, sulfuric acid and iodine vapor, and exhibits adequate stains upon irradiation with 254 nm wavelength light emitted by a UV lamp; negative in reaction of Molic, silver nitrate, ferric chloride and Dragendorff; (5) Retention time in HPLC: 15.0 minutes (6) Ultraviolet absorption spectrum (in water): Xmax: 273 nm, 280 nm, 289 nm; (7) Infrared absorption spectrum (FT-IR, KBr): Characteristic absorption spectrum: 3300 cm'1, 1720-1715 cm'1, 1636 35 cm'1, 1541 cm'1, 1404 cm'1, 1207 cm'1, 1137 cm1; (8) Molecular weight: 1548 (FAB mass m / z 1577 (M + H) *]; 1001012 27 (9) Molecular formula: C74H113021N17 [measured m / z value of (M + H) + ion as determined by high resolution FAB mass: 1576.8324; calculated m / z value: 1576.8375]; (10) Acid complete hydrolysis, as hydrolysates, provides: 5 Asp (1 mol), Glu (1 mol) fi-Ala (1 mol), Leu (1 mol),

Ser (2 mol), Trp (1 mol), Orn (2 mol), N-methylvaline), β-hydroxyaspartic acid (1 mol), N-methylphenylalanine (1 mol) and 3-hydroxy-7-methyloctanoic acid (1 mol) ; (11) Specific rotation: [α] D20 = + 27 ° (c = 0.5, H20), (10 (12) Classification in base, acid and neutral: amphotheer;

The elution conditions for the C18 reverse phase silica gel HPLC of the antibiotics WAP-8294AX, AX-8, AX-9 and AX-13 are as follows: 15 Column: YMC A-312 (6 x 150 mm)

Mobile phase: 0.05% trifluoroacetic acid containing acetonitrile: water (37: 663)

Detection wavelength: UV 214 nm Liquid speed: 1 ml / min.

20 (Biological properties) (1) Antibacterial activity

Table 4 shows the results of the inspection of WAP 8294A, A1, A, and A with respect to their antibacterial activity. Determination of the minimum inhibitory concentration (MIC) was performed by the broth dilution method using a sensitive broth medium (available from Eiken Chemical Co., Ltd.).

The data, shown in Table 4, clearly indicate that WAP-8294A, A3, A2 and A4 exhibit strong antibacterial activity against gram-positive bacteria, including MRSA (methicillin-resistant Staphylococcus aureus). The activity is characterized in that it is not less than 8 times greater than that seen in the previous culture medium when 10% calf serum is added to the culture medium.

10 010 12 28

On the other hand, these compounds show no activity against gram-negative bacteria, fungi and yeasts.

TABLE 4-1 5

Bacteria MIC ^ g / ml) A A,

Addition of serum None 10% None 10%

Staphylococcus aureus Nr. 0.78 <0.10 0.39 <0.10 I (MRSA clinically isolated strain)

Staphylococcus aureus Nr. 0.78 <0.10 0.39 <0.10 II (MRSA clinically isolated strain)

Staphylococcus aureus Nr. 0.78 <0 10 0.39 <0.10 15 371 R (MRSA clinically isolated strain)

Staphylococcus aureus 0.78 <0.10 0.39 <0.10 (ATCC 25923)

Staphylococcus epidermis 0.78 <0.10 0.39 <0.10 20 (ATCC 12228)

Streptococcus pvrogenes 6.25 25 6.25 25 (ATCC 19615)

Bacillus subtilis (ATCC 0.78 <0.10 0.39 <0.10 6633) 25 Escherichia coli (ATCC> 100> 100> 100> 100 25 922)

Pseudomonas aeruginosa> 100> 100> 100> 100 (ATCC 9027)

Candida albicans (TIMM> 100> 100> 100> 100 30 0239)

Aspergillus fumiqatus> 100> 100> 100> 100 (IAM 2004) 1001012 29 TABLE 4-2

Bacteria MIC {μς / χύΐ) A Aj

Addition of serum None 10% None 10% 5 Staphylococcus aureus No. 0.78 <0.10 0.78 <0.10 I (MRSA clinically isolated strain)

Staphylococcus aureus Nr. 0.78 <0.10 0.78 <0.10 II (MRSA clinically isolated strain)

Staphylococcus aureus Nr. 0.78 <0 10 0.78 <0.10 371 R (MRSA clinically isolated strain)

Staphylococcus aureus 0.78 <0.10 0.78 <0.10 15 (ATCC 25923)

Staphylococcus epidermis 0.78 <0.10 0.78 <0.10 (ATCC 12228)

Streptococcus pvrogenes 6.25 25 6.25 25 (ATCC 19615) 20 Bacillus subtilis (ATCC 0.78 <0.10 0.78 <0.10 6633)

Escherichia coli (ATCC> 100> 100> 100> 100 25922)

Pseudomonas aeruginosa> 100> 100> 100> 100 25 (ATCC 9027) |

Candida albicans (TIMM> 100> 100> 100> 100 0239)

Aspergillus fumigatus> 100> 100> 100> 100 (IAM 2004) 30

The antibacterial activities of the antibiotic WAP-8249AX, AX-8, AX-9 and AX-13 are determined in the same way. The results are shown in Table 5. The 1001012 determination of their minimum inhibitory concentration (MIC) was performed by the broth dilution method using Muller-Hinton broth (available from DIFCO Company) supplemented with 2% sodium chloride for MRSA 5 and Muller-Hinton broth as such, for other bacteria.

The data presented in Table 5 indicate that the antibiotics WAP-8294AX, AX-8, AX-9 and AX-13 show strong antibacterial activity against gram-positive bacteria including methicillin-resistant Staphylococcus aureus and have been found to the activity increases about twice, compared to the value from the previously described culture medium, when 10% calf serum is added to the culture medium.

On the other hand, these compounds show no activity against gram-negative bacteria, fungi and yeasts.

1001012 TABLE 5-1 31

Bacteria MIC ^ g / ml) A Ax

Addition of serum

None 10% None 10% 5 Staphylococcus aureus Nr. 1.56 0.78 3.13 3.13 1 (MRSA clinically isolated strain)

Staphylococcus aureus 1.56 0.78 3.13 1.56 (ATCC 25923) 10 Staphylococcus epidermis 1.56 0.78 1.56 0.78 (ATCC 12228)

Streptococcus pyrogenes> 100> 100> 100> 100 (ATCC 19615)

Bacillus subtilis (ATCC 1.56 0.78 1.56 0.78 15 6633)

Escherichia coli (ATCC> 100> 100> 100> 100 25922)

Pseudomonas aeruginosa> 100> 100> 100> 100 (ATCC 9027) 20 Candida albicans (TIMM> 100> 100> 100> 100 0239)

Aspergillus fumiqatus> 100> 100> 100> 100

(IAM 2004) I

25 1001012 TABLE 5-2 32

Bacteria MIC (μg / ml) tested A Ax

Addition of serum

None 10% None 10% 5 Staphylococcus aureus Nr. 3.13 1.56 3.13 3.13 1 (MRSA clinically isolated strain)

Staphylococcus aureus 3.13 1.56 3.13 3.13 (ATCC 25923) 10 Staphylococcus epidermis 1.56 0.78 1.56 0.78 (ATCC 12228)

Streptococcus pyrooenes> 100> 100> 100> 100 (ATCC 19615)

Bacillus subtilis (ATCC 3.13 1.56 3.13 1.56 15 6633)

Escherichia coli (ATCC> 100> 100> 100> 100 25922)

Pseudomonas aeruginosa> 100> 100> 100> 100 (ATCC 9027) 20 Candida albicans (TIMM> 100> 100> 100> 100 0239)

Aspergillus fumiaatus> 100> 100> 100> 100 (IAM 2004) 25 (2) Protective effect of WAP-8294A against experimental MRSA infection in mice

Since the antibiotic WAP-8294A exhibits a strong antibacterial activity against gram-positive bacteria, including MRSA, as evidenced by its in vitro antibacterial activity as shown in Table 4, a test was performed protecting mice against infection with Staphylococcus aureus, No. 1 (a clinically isolated strain of MRSA).

1001012 33

The experiments were performed by intraperitoneally administering 0.5 mg per 0.5 ml per mouse cyclophosphamide to ICR-MCS mice [4 week old male mice (available from Nippon Clea Co., Ltd.), 8 animals per group] . Administration was done 3 days before infection of the animals by intraperitoneal administration of 0.5 ml each of a liquid containing 2 x 106 / roll Staphylococcus aureus No. 1 strain supplemented with 5% mucin. 1 hour after infection, once, subcutaneously, 10 mg / kg vancomycin HCl (available from Shionogi & Co., Ltd.) and 0.2 ml / mouse of the antibiotic WAP-8294A (containing 10 mg / kg, 5 mg / kg, 2.5 mg / kg, 1.0 mg / kg, 0.5 mg / kg, 0.25 mg / kg, 0.1 mg / kg or 0.05 mg / kg of the antibiotic), injected into the mice. To the control groups, 0.2 ml of physiological saline was administered subcutaneously to each mouse.

As can be seen from the results of these experiments, which are presented in Table 6 below, all control animals not receiving treatment died within 3 days of infection. On the other hand, in the antibiotic-treated group, 5 out of 8 animals administered 10 mg / kg vancomycin remained alive up to 5 days after infection. In contrast, WAP-8294A showed excellent therapeutic activity at low dose levels, as evidenced by the results set forth in Table 6. Its EDS0 value was determined by the "probit" method and found to be 0.155 mg / kg.

1001012 34 TABLE 6

Results of WAP-8294A treatment of experimental systemic MRSA infection in mice 5 Test group Dose (mg / kg) Survival / # test animals

Control (physiological 0/8 logical saline)

Vancomycin 10 5/8 10 Antibiotic WAP- 10 8/8 8294A 5 8/8 2.5 8/8 1.0 8/8 0.5 7/8 0.25 5/8 0.10 3/8 0, 05 1/8

Because the antibiotic WAP-8294AX showed strong antibacterial activity against gram-positive bacteria, including MRSA, as evidenced by its in vitro antibacterial activity, as shown in Table 5, a test for protecting mice against infection with Staphylococcus aureus JCM8702 (a clinically isolated strain of MRSA).

The experiments were performed by intraperitoneally administering 0.5 mg / 0.5 ml / mouse cyclophosphamide to iCR-MCH mice (4 week old male mice (available from Nippon Clea Co., Ltd.), 8 animals per group]. The administration was performed 3 days before the 25 animals by intraperitional administration of 0.5 ml each of a liquid containing 2 x 104 / ml of a Staphylococcus aureus JCM98702 strain, supplemented with 5% mucin, were infected.

1001012 35 One hour after infection, 10 mg / kg vancomycin HCl (available from Shionogi & Co., Ltd.) and 0.2 ml / mouse of an antibiotic WAP-8294AX (containing 10 mg / kg, 2.5 mg / kg, 1.0 mg / kg or 0.5 mg / kg of the antibiotic 5) injected into the mice. Mice from the control groups were each administered 0.2 ml of physiological saline subcutaneously.

As shown in the results of these experiments, shown in Table 7 below, 6 of the 8 10 untreated control animals died within 3 days of infection. On the other hand, out of the antibiotic-treated group, 5 out of 8 animals administered 10 mg / kg vancomycin remained alive up to 5 days after infection. In contrast, the WAP-8294AX showed excellent therapeutic activity at a lower dosage level, as evidenced by the results shown in Table 7. Its EDS0 value was determined by the probit method and found to be 1.25 mg / kg.

20 1001012 TABLE 7 36

Results of WAP-8294AX treatment of experimental systemic MRSA infection in mice 5 Test group Dose (mg / kg) Survival / # test animals

Control (physiological 2/8 saline)

Vancomycin 10 5/8 10 Antibiotic WAP- 10 7/8 8294A 2.5 5/8 1.0 4/8 0.5 2/8 (3) Acute toxicity test in mice

The antibiotic WAP-8294A was administered intravenously to mice (4 week old ICR-MCH male mice; 5 animals per group) at a dose of 25 mg / kg, 50 mg / kg, 100 mg / kg or 250 mg / kg, no acute toxicity was seen.

The antibiotic WAP-8294AX was administered intraperitoneally to mice (4 week old ICR-MCH male mice; 5 animals per group) at a dose of 25 mg / kg or 100 mg / kg. However, no acute toxicity was seen.

From the above it appears that the antibiotics WAP-8294A, A1 (A2, A3, A ,, As, A6, AX, AX-1, AX-2, AX-3, AX-4, AX-5, AX-6, AX-7, AX-8, AX-9, AX-10, AX-11, AX-12 and AX-13 are effective as a therapeutic agent for treating bacterial infectious diseases, in particular, infectious diseases resulting from infection with methicillin-resistant Staphylococcus aureus, in humans and animals.

30 (Applications)

When the new antibiotics of the present invention are administered as medicaments, they can be administered in various dosage forms, in T 0 01 6 T 2 37 according to the conventional manner. For example, they can be used in orally administrable forms, such as powder, granules, tablets, capsules and syrups. They can be safely administered in parenterally administrable forms, such as (intravenous, intramuscular, subcutaneous) injections, drops, suppositories, spreads and ointments. Furthermore, the new antibiotics of the present invention can be safely used in the field of ophthalmic medicine in the same manner as parenterally administrable dosage forms such as eye drops and eye ointments. It is hereby taken into account that their bactericidal action is faster than that of vancomycin and that their effectiveness is manifested at low concentrations.

These different pharmaceutical preparations can be prepared by the usual methods, ie, by including in the main ingredient pharmaceutically acceptable and currently known known auxiliaries, such as excipients, binders, disintegrants, coatings, lubricants, stabilizers , Corrective or flavoring agents, solubilizing agents, suspending agents and diluents, and then bringing the mixture into the desired dosage form.

The dosage thereof may vary depending on, for example, the diseases to be treated, the routes of administration and the dosage time, but they are preferably administered in a dosage range ranging from 5 mg to 2000 mg / day, for adults, which doses at one time or can be administered in portions at different times.

The present invention will be described in more detail below, referring to the following examples, but the present invention is not intended to be limited to these examples.

Example 1 To each of four Erlenmeyer flasks with a volume of 500 ml was added 100 ml of a culture medium (pH 7.2) containing 2.5% glucose, 2.0% defatted soybean flour, 0.4% soybean oil, 0.25% sodium chloride and 0.5% calcium carbonate, 1001012 38 administered. This was followed by sterilization, inoculating each culture medium with one platinum eye of Lvsobacter sp. WAP 8294 strain, which was grown in a slant agar medium. Cultivation was carried out at 30 ° C for 2 days while the 5 bottles were shaken in a circulating shaker at a rate of 180 movements per minute. Erlenmeyer flasks with a volume of 500 ml (150 pieces), to which each 100 ml of the culture medium described above were added and which were then sterilized, were then prepared, followed by seeding each with 2 ml of the culture medium and cultivation for 4 days at 30 ° C while moving the flasks in a rotary shaker at 180 movements per minute. The antibacterial activity was determined by the agar plate method using Staphylococcus aureus No. 1.

Example 2

The culture media obtained in Example 1 were subjected to continuous centrifugation (9000 rpm) to obtain 15 l of supernatant. The supernatant (pH 6.9) was loaded onto a column packed with Amberlite IR-120B (H + type, 1.5 L, available from Rohm & Haas Co.), to collect 15 L of effluent and 3 L of an aqueous wash. After the effluent and washings were collected and their pH adjusted to 3, the combined fluid was chromatographed by loading it onto a column packed with activated carbon (700ml, available from Wako Pure Chemical Co., Ltd. ). After washing with 4 L of water, the active compounds were eluted with 4 L of an 80% acetone-30 water mixture and 4 L of an 80% acetone-water mixture with a pH of 12. The resulting eluate was concentrated to 800 ml, followed by adjusting the pH to 3 with hydrochloric acid, and extraction with ethyl acetate (3 x 400 ml) and then with n-butanol (3 x 400 ml). Concentration of the butanol phase and lyophylization thereof gave 6.4 g of a crude product (hydrochloride).

1001012 39

Example 3

The crude compound (6.4 g) obtained in Example 2 was chromatographed by loading onto a column packed with cellulose (450 ml, available from E.

5 Merck Company), then eluting and fractionating it with a solvent system: acetonitrile acetic acid water (75: 1: 25). The active fractions were collected, concentrated and then lyophilized to yield 880 mg of hot WAP-8294A (hydrochloride) as a white powder.

10

Example 4

The WAP-8294A (700 mg) obtained in Example 3 was loaded onto a reverse phase high performance liquid chromatography (RP-HPLC) column packed with 15 octadecyl silica gel [column: YMC-GEL SH-343- 5.20 x 250 mm, available from Yamamura Chemical Research Laboratory; mobile phase: 0.05% trifluoroacetic acid containing acetonitrile water (45:55)], in 10 portions, to collect the fractions containing WAP 8294A1, A3 and A4. This was followed by concentrating each fraction to obtain WAP-8294AX (50mg), A2 (210mg) and A4 (16mg) as a white powder (hydrochloride).

Example 5

Amino acid analysis of WAP-8294A

The WAP 8294A (5 mg) obtained in Example 3 was dissolved in 0.5 ml 4N methanesulfonic acid solution, to which 0.2% 3- (2-aminomethyl) indole was added to completely hydrolyze the antibiotic with the acidic for 24 hours at 110 ° C. The resulting hydrolyzate was concentrated to dryness, dissolved in a small amount of water, followed by loading the solution onto a column packed with Dowex 50WX-8 (H + type, 1ml, available from Dow Chemical Co.), wash with 20 ml of water and then elute with 20 ml of a 0.5 N NH4OH solution. The eluate was concentrated to dryness, dissolved in a small amount of water, then subjected to two-dimensional thin layer chromatography [cellulose plate, solvent 1: 10 01 0 12 40 n-butanol: acetic acid: water (4: 1: 2); solution 2: n-butanol: pyridine: acetic acid: water (15: 10: 3: 12)] and amino acid analysis (Hitachi Amino Acid Analyzer L-8500). In this way the eluate was found Asp (1 mol), Glu (1 mol), Gly 5 (1 mol), Ser (2 mol), Leu (1 mol), Trp (1 mol) and Orn (2 mol) , included, as well as three kinds of unknown amino acids. The results of this two-dimensional TLC analysis of the antibiotic WAP-8294A are shown in Fig. 5.

10 Example 6

Identification of the unknown amino acids

To 100 mg of the WAP-8294A, obtained in Example 3, was added 10 ml of a 6N HCl solution containing 4% thioglycolic acid. This was followed by hydrolysis of the antibiotic at 110 ° C for 20 hours. After concentrating the hydrolyzate, its pH was adjusted to 7.0 and the hydrolyzate was subjected to column chromatography using Sepabeads SP207 (available from Mitsubishi Chemical Industries, Ltd.) while developing with water. The fraction eluting between 18 ml and 27 ml from the column, and comprising the unknown amino acid -1, was concentrated and loaded onto a column packed with Dowex 50WX-8 (H + type, 9 ml). After eluting with 0.1N HCl solution, the resulting eluate was concentrated to dryness, yielding 4 mg of the unknown amino acid 1-hydrochloride, as a white powder. The amino acid was found to have a molecular weight of 149 [FAB-MS m / z: 150 (M + H) *] and -NMR spectrum [D20, 270 MHz; chemical shift: ((ppm)] at 4.53 (1H, d, J = 2.9Hz) and 5.00 (1H, d, 30J = 2.9Hz). In this way it supported the assumption that it was β-hydroxyaspartic acid.

When compared with an authentic sample of β-hydroxyaspartic hydrochloride (available from Sigma Company), the--NMR spectra (chemical shifts) and Rf value (0.20) measured on the cellulose TLC [ solvent: n-butanol: pyridine: acetic acid: water (15: 10: 3: 12)] of the unknown amino acid 1-hydrochloride to be completely identical to those measured 1001012 41 for the authentic sample. Accordingly, the unknown amino acid-1 was identified as β-hydroxyaspartic acid.

In addition, the fraction eluting between 30 ml and 45 ml from the Sepabeads SP207 column containing the unknown amino acid-2 was concentrated and loaded onto a column packed with 10 ml of activated carbon. The fraction eluting between 24 and 34 ml was collected, loaded onto a Dowex 50WX-8 column (1 ml), eluted with 0.5 N NH 4 OH solution, followed by concentration to dryness. Thereby 2.1 mg of the unknown amino acid-2 was obtained as a white powder. This amino acid was found to have a molecular weight of 131 [FAB-MS m / z: 132 (M + H) *] and 1 H-NMR spectrum [D20, 27 MHz; chemical shift: δ (ppm)] at 1.18 (6H, dd, J = 7.0Hz), 2.38 (1H, dq, J = 4.8, 7.0 Hz), 2.87 (3H , s) and 3.55 (1H, d, J = 4.8Hz). In this way it seemed to be N-methylvaline. This amino acid was therefore compared with an authentic sample of N-methyl-DL-valine (available from Sigma Company) and it was found that the 1 H-NMR spectrum (chemical shift-20 gene) and the Rf value (0.71), measured on cellulose TLC (solvent: n-butanol: pyridine: acetic acid: water (15: 10: 3: 12)] of the unknown amino acid-2 were completely identical to the values measured for the authentic sample. the unknown amino-25 acid-2 identified as N-methylvaline.

Furthermore, the fraction eluting with 80% acetone from the Sepabeads SP207 column, containing the unknown amino acid-3, was concentrated and loaded onto a Dowex 50WX-8 column (H + type, 1 ml). After eluting with 1N HCl solution, the resulting eluate was concentrated to dryness to yield 3.5 mg of the unknown amino acid-3 hydrochloride, as a white powder. This amino acid was found to have a molecular weight of 179 [FAB-MS m / z. 180 (M + H) +] and 1 H-NMR spectrum [D20, 270 MHz, chemical shift: δ (ppm)] at 2.78 (1H, s), 3.32 (2H, d, J = 6.2Hz), 3.95 (1H, t, J = 6.2Hz), 7.4 ~ 7.5 (5H, m). It therefore seemed to be N-methylphenylalanine. Therefore, this amino acid was compared with an authentic sample of N-methyl-L-phenylalanine 1001012 42 (available from Sigma Company) and it was found that the 1 H-NMR spectrum (chemical shifts) and the Rf value (0.82) measured on cellulose TLC (solvent: n-butanol: pyridine: acetic acid: water (15: 10: 3: 12)] of the unknown 5 amino acid-3 were completely identical to the values measured for the authentic sample. Accordingly, the unknown amino acid-3 is identified as N-methylphenylalanine.

10 Example 7

Amino acid analysis of WAP-8294A !, k2 and A4

About 30 μg of WAP-8294A1 (A2 or A4, obtained in Example 4, was completely hydrolysed with a 6N HCl solution at 110 ° C for 24 hours, then concentrated and subjected to amino acid analysis. of them Asp (1 mol), Glu (1 mol), Gly (1 mol), Ser (2 mol), Leu (1 mol), Orn (2 mol), Trp (1 mol), β-hydroxyaspartic acid ( 1 mole), N-methylvaline (1 mole) and N-methylphenylalanine (1 mole).

20

Example 8

Isolation of the fatty acids that make up WAP-8294A !, A2 and A4 and their structure

The WAP 8294Aj (10mg) obtained in Example 25 4 was hydrolyzed with 6N HCl for 2 hours at 110 ° C. The resulting hydrolyzate was extracted with ether, followed by concentration of the ether phase to dryness to yield 0.8 mg of white powder. This compound was found to have a molecular weight of 160 [FAB-MS m / z: 161 (M + H) *] and 1 H-NMR spectrum [CDCl 3, 270 MHz; chemical shift: ö (pprn) J at 0.89 (3H, t, J-7.0Hz), 1.3-1.55 (8H, m), 2.46 (1H, dd, J = 16.5 , 8.8Hz), 2.58 (1H, dd, J = 16.5, 3.3Hz) and 4.03 (1H, m). It therefore appeared to be 3-hydroxyoctanoic acid. This compound (100 μg) was dissolved in 0.1 ml of benzene-methanol (8: 2) and converted into its methyl ester at room temperature for 10 min by adding a drop of trimethylsilyldiazomethane (available from Tokyo Chemical Industry Co ., Ltd.). When the 1001012 43 obtained methyl ester was subjected to EI-MS measurements, an ion peak was seen at m / z = 173 (M + -1), fragment ion peaks at 156 (M * -18), 143 (M + -31) and 125 (M * -31-18) and a characteristic base fragment ion peak at m / z 5 103, derived from the cleavage of C3-C4 from the 3-hydroxy fatty acid methyl ester. This compound was accordingly identified as 3-hydroxyoctanoic acid.

The WAP-8294A2 (10mg) was hydrolyzed with an acid in the same manner as described above, followed by extraction with ether and concentration of the ether phase to dryness, yielding 0.9mg of white powder. This compound was found to have a molecular weight of 174 [FAB-MS m / z: 175 (M + H) *] and 1 H-NMR spectrum [CDCl 3, 270 mHz; chemical shift: δ (ppm)] at 0.87 (6H, d, J = 6.6Hz), 1.15-1.56 (7H, m), 2.50 (1H, dd, J = 16, 5, 8.8Hz), 2.60 (1H, dd, J = 16.5, 3.3Hz) and 4.03 (1H, m), and the methyl ester derivative thereof showed EI-MS peaks at m / z 187 (NT-1), 170 (M * -18), 157 (M + -31), 139 (M + -31) 018) and 103 (base fragment ion peak from C3-CJ cleavage. This compound was accordingly identified as 3-hydroxy-7-methyloctanoic acid.

The WAP-8294A, (10 mg) was hydrolyzed with an acid in the same manner as described above, followed by extraction with ether and concentration of the ether phase to dryness, whereby 0.8 mg of white powder was obtained. This compound was found to have a molecular weight of 188 [FAB-MS rn / z: 189 (M + H) *] and -NMR spectrum [CDCl1, 270 MHz; chemical shift: δ (ppm)] at 0.80 (6H, d, J = 6.8,

Hz), 1.15-1.6 (9H, m), 2.43 (1H, dd, J-16.5, 8.8Hz), 2.50 (1H, dd, J = 16.5, 3.3Hz) and 3.95 (1H, m) and the methyl ester derivative thereof showed EI-MS peaks at m / z 2 01 (Μ * -1), 184 (M + -18), 171 (M + -31) , 153 (M + -31 -18) and 103 (base fragment ion peak from C3-C4 cleavage).

This compound was accordingly identified as 3-hydroxy-8-methanonanoic acid.

1001012 44

Example 9

A crude compound (9.0 g), obtained in the same manner as used in Example 2, was chromatographed by loading it onto a column packed with octade-5 cylsilicagel (chromatolex ODS-DM1020T from Fuji Silicia Chemical Co ., Ltd., 450 ml). It was then eluted and fractionated with a 0.05% trifluoroacetic acid containing acetonitrile: water (4: 6) solvent system. The active fractions were collected, concentrated, and then lyophylized to yield 450 mg of WAP-8294AX (hydrochloride) as a white powder.

Example 10

The WAP-8294AX (120mg) obtained in Example 15 9 was loaded, in 10 portions, onto a column packed with octadecyl silica gel for high performance liquid chromatography (HPLC) [column: YMC-GEL SH-343 -5.20 x 250 mm, available from Yamamura Chemical Research Laboratory, mobile phase: 0.05% trifluoroacetic acid-containing acetoni-20 vibratory water (37:63)], in ten sections, with fractions containing each WAP-8294AX -8, AX-9 or AX-13, were collected, followed by concentration of each fraction with WAPO-8294AX-8 (14 mg), AX-9 (10 mg) or AX-13 (12 mg) when white powder (hydrochloride) was obtained.

25

Example 11

Amino acid analysis of WAP-8294AX-8, AX-9, AX-13

The WAP-8294AX-8 (5 mg), which was obtained in Example 10, was dissolved in 0.5 ml of 4N methanesulfonic acid solution supplemented with 0.2% 3- (2-aminomethyl) indole to obtain acid complete hydrolysis run for 24 hours at 110 ° C. Then, the resulting hydrolyzate was concentrated to dryness, dissolved in a small amount of water, followed by loading onto a column packed with Dowex 50WX-8 (H + type, available from Dow Chemical Co., 1 ml). This was followed by washing with 20 ml of water and eluting with 20 ml of 0.5 N NH4 OH. The resulting eluate was concentrated to dryness, dissolved in a small amount of 100101245 water and then subjected to two-dimensional thin layer chromatography (developing solvent for first dimension: n-butanol: acetic acid: water (4: 1: 2); developing liquids for the second dimension: n-butanol, pyridine-5 ne: acetic acid: water (15: 10: 3: 12)) and amino acid analysis (Hitachi Amino Acid Analyzer L-8500). As a result, it was found that the WAP-8294AX-8 Asp (1 mol), Glu (1 mol), Gly (1 mol), Ser (2 mol), Leu (1 mol), Trp (1 mol), Orn (2 mol), Val (1 mol), N-methylphenylalanine (1 mol) and β-hydroxyaspa-10 raginic acid (1 mol).

In addition, WAP-8294AX-9 and AX-13, each 5 mg, were subjected to acid complete hydrolysis and then the resulting hydrolysates were subjected to amino acid analysis in the same manner as described above. As a result, it was found that the WAP-8294AX-9 Asp (1 mol), Glu (1 mol), Gly (1 mol), Ser (2 mol), Leu (1 mol), Trp (1 mol),

Orn (2 moles), Phe (1 mole), N-methylvaline (1 mole) and β-hydroxyaspartic acid (1 mole), included, and that the WAP-8294AX-13 Asp (1 mole), Glu (1 mol), β-Ala (1 mol), Ser (2 mol), Leu 20 (1 mol), Trp (1 mol), Orn (2 mol), N-methylphenylalanine (1 mol), N-methylvaline (1 mol ) and β-hydroxyasapartic acid (1 mol).

Example 12 Fatty acid analysis of WAP-8294AX-8, AX-9, AX-13

The WAP-8294AX-8, AX-9 and AX-13 (1 mg each), obtained in Example 10, were hydrolyzed for 2 hours at 110 ° C, with 6N HCl. Each hydrolyzate was extracted with ether, followed by concentration of the ether phase to dryness, dissolving the residue in 0.1 ml of a mixture of benzene and methanol (8: 2) and converting to its methyl ester for 10 min at room temperature , by adding a drop of trimethylsilyldiazomethane (available from Tokyo Chemical Industry Co., Ltd.). Each methyl ester derivative 35 was subjected to GC-MS analysis (column: DB-5 (inner diameter: 0.25 mm, length: 30 m; film thickness: 0.25 μπί; available from J&W Company); temperature: 100 up to 240 ° C (heating rate: 10 ° C / min.), gas phase: helium, liquid 1001012 46 t dust rate: 0.993 ml / min.] In all cases, the retention time (9.7 min.) was the GC analysis and m / z, as determined by EI-MS, of 187 (M + -1), 170 (M + -18), 157 (M + -31), 139 (M * -31 -18) and 103 (from splitting 5 C3-C4; a characteristic base fragment ion peak) in accordance with the measurements seen for the methyl ester derivative of 3-hydroxy-7-methyloctanoic acid (a fatty acid constituting the WAP-8294A2). that both WAP-8294AX-8, AX-9 and AX-13 comprise 3-hydroxy-7-methyloctanoic acid as a fatty acid component.

Example 13

Pharmaceutical preparations of WAP-8294A

Typical examples of pharmaceutical preparations of the WAP-8294A will be described below.

Injection:

According to the rules laid down in the general rules for preparations, "injections", in the Pharmacopoeia 20 of Japan, 12th edition, 40 mg of WAP-8294A hydrochloride dissolved in 3 ml of sterilized physiological saline (Japanese Pharmacapoeia) was sterile infused. in a 3 ml ampoule and sealed by fusion to be ready for injection.

25

Tablet:

According to the rules laid down in the general preparation rules, "tablets", 100 mg of WAP-8294A2 hydrochloride, 65 mg of lactose (Japanese Pharmacopoeia) and 14.2 mg of starch (Japanese Pharmacopoeia) were added as excipients; 20 mg of polyvinylpyrrolidone K25 (Japanese Pharmacopoeia) added as a binder; and 0.8 mg of magnesium stearate (Japanese Pharmacopoeia) added as a lubricant followed by uniform mixing, pressurizing with a tabletting machine to form uncoated tablets each weighing 200 mg.

10 0 10 12 47

Ointments:

According to the rules laid down in the General Preparation Rules, "ointments", 40 mg of WAP-8294A2 hydrochloride, 1.2 g of polyethylene glycol 4000 (Japanese Pharmacopoeia), 600 mg polyethylene glycol 400 (Japanese

Pharmacopoeia), 156 mg hydrophilic Vaseline (Japanese Pharmacopoeia), and 0.8 mg propyl p-oxybenzoate (Japanese Pharmacopoeia) and 3.2 mg ethyl p-oxybenzoate (Japanese Pharmacopoeia) added as antifungals, followed by melting and uniform kneading heating, whereby an ointment is obtained (4 g / squeezable tube).

Eye drops:

According to the rules laid down in the general rules of preparation, "eye drops", 25 mg of WAP-8294A2 hydrochloride was dissolved in 5 ml of a 0.9% sterilized sodium chloride solution. Then 0.5 mg of benzalkonium chloride (Japsne Pharmacopoeia) as a preservative was added thereto, whereby a uniform solution was obtained, followed by sterile filtration and sterile packaging in an eye dropper with a volume of 5 ml, yielding eye drops.

As described above, the use of a small animal infection model demonstrates that the novel antibiotic WAP-8294A of the present invention exhibits excellent therapeutic effects on infectious diseases resulting from infection with gram-positive bacteria, in particular, with MRSA (methicillin-30 resistant Staphylococcus aureus), which has become a serious problem in medical medicine today. As a result, the antibiotic can be effective in treating diseases, including MRSA infectious diseases, that result from infection with gram-positive bacteria as infectious bacteria.

1001012

Claims (12)

1. Antibiotic WAP-8294A or pharmaceutically acceptable salt thereof, having the following physicochemical properties: (1) Appearance: white powder; (2) Melting point: 213-220 ° C (decomposition); (3) Solubility: soluble in water, methanol, n-butanol, dimethylformamide and dimethyl sulfoxide and insoluble in acetone, ethyl acetate and chloroform, (4) Color reaction: positive in reactions of ninhydrine, Ehrlich, Rydon-Smith, aqueous solution of potassium per -manganate, sulfuric acid and iodine vapor, and exhibits fading spots upon irradiation with 254 nm wavelength light rays emitted by a UV lamp; negative in reaction of Molic, silver nitrate, ferric chloride and Dragendorf f, (5) Retention time in HPLC (Fig. 1): 4.0 to 5.1 minutes, 8.0 minutes, 11.1 minutes, 12.5 minutes, 16.5 minutes, 17.9 20 minutes, 18.8 minutes, - (6) Ultraviolet absorption spectrum (in water): Xmax: 275 nm, 280 nm, 287 nm, (7) Infrared absorption spectrum (FT-IR , KBr): characteristic absorption spectrum: 3300 cm -1, 1720-1715 cm -1, 1636 25 cm -1, 1541 cm -1, 1404 cm -1, 1207 cm -1, 1137 cm -1; (8) H- NMR spectrum: (270 MHz, D20): complicated proton signals are seen, which come from methyl, methylene, methine and heterocyclic or aromatic rings; 30 (9) Molecular weight: 1400-1700; (10) Qualitative test for organic compound (sodium melting method): the antibiotic is a compound which includes carbon, hydrogen and oxygen and nitrogen elements; 35 (11) provides acid complete hydrolysis, as ninhydrin positive compounds, in Asp, Glu, Gly, Leu, Ser, Trp, Orn, N-me thylvaline, β-hydroxyaspartic acid and N-methylphenylalanine; 0 010 12 (12) Specific rotation: [a] d 2 O = + 42 ° (c = 0.5, H 2 O); (13) Classification in base, acid and neutral: amphotheer. 2. Xmax: 275 nm, 280 nm, 287 nm; (7) Infrared absorption spectrum (FT-IR, KBr): characteristic absorption spectrum: 3300 cm'1, 1720-1715 cm'1, 1636 cm'1, 1541 cm'1, 1404 cm "1, 1207 cm" 1, 1133 cm 1; (8) Molecular weight: 1547.9 (FABS-MS m / z 1548.9 (M + H) *, 25 m / z 1546.7 (M-H)); (9) Molecular Formula: C72H109O2lN17 (10) Acid Complete Hydrolysis Provides: Asp (1 Mol), Glu (1 Mol), Gly (1 Mol), Leu (1 Mol), Ser (2 Mol), Trp (1 mol), Orn (2 mol), N-methylvaline (1 mol), β-hydroxyaspartic acid (1 mol), N-methylphenylalanine (1 mol) and 3-hydroxyoctanoic acid (1 mol); (11) Specific rotation: [or] d20 = + 41 ° (c = 0.5, H20), (12) Classification in base, acid and neutral: amphoteric, · 2. Antibiotic WAP-8294A1 (or pharmaceutically acceptable salt thereof, with the following physicochemical properties: (1) Appearance: white powder; (2) Melting point: 215-225 ° C (decomposition); (3) Solubility: soluble in water, methanol, n-butanol, dimethylformamide and dimethylsulfoxide and insoluble in acetone, ethyl acetate and chloroform; (4) Color reaction: positive in reactions of ninhydrin, Ehrlich, Rydon-Smith, aqueous solution of potassium permanganate, sulfuric acid and iodine vapor, and exhibits fading spots upon irradiation with light beams with a wavelength of 254 nm emitted by a UV lamp; negative in reaction of Molisch, silver nitrate, ferric chloride and Dragendorff; (5) Retention time in HPLC: 8.0 minutes; (6) Ultraviolet absorption spectrum (in water): 3. Antibiotic WAP-8294A2 or pharmaceutically acceptable salt thereof, having the following physicochemical properties: (1) Appearance: white powder; (2) Melting point: 215-225 ° C (decomposition); 1001012 (3) Solubility: soluble in water, methanol, n-butanol, dimethylformamide and dimethyl sulfoxide and insoluble in acetone, ethyl acetate and chloroform, (4) Color reaction: positive in reactions of ninhydrin, 4. Antibiotic WAP-8294A4 or pharmaceutically acceptable salt thereof, having the following physicochemical properties: (1) Appearance: white powder, (2) Melting point: 215-225 ° C (decomposition); (3) Solubility: soluble in water, methanol, n-butanol, dimethylformamide and dimethyl sulfoxide and insoluble in acetone, ethyl acetate and chloroform, (4) Color reaction: positive in reactions of ninhydrine, Ehrlich, Rydon-Smith, aqueous solution of potassium per -manganate, sulfuric acid and iodine vapor, and exhibits extinguishing spots upon irradiation with light rays of 254 nm wavelength emitted by a UV lamp, negative in reaction of Molic, silver nitrate, ferric chloride and Dragendorff; (5) Retention time in HPLC: 16.5 minutes. (6) Ultraviolet absorption spectrum (in water): Xm, x: 275 nm, 280 nm, 289 nm; (7) Infrared absorption spectrum (FT-IR, KBr): Characteristic absorption spectrum: 3300 cm'1, 1720-1715 cm'1, 1636 10 cm'1, 1541 cm'1, 1404 cm "1, 1207 cm'1, 1137 cm'1, (8) Molecular weight: 1400-1700, - (9) Molecular formula: C73H113021N17, (10) Acid hydrolysis, as hydrolysates, provides Asp (1 mol), Glu (1 mol, Gly (1 mol), Leu (1 mol), Ser (2 15 mol), Trp (1 mol), Orn (2 mol), N-methylvaline (1 mol), β-hydroxyaspartic acid (1 mol), N-methylphenylalanine (1 mol ), and 3-hydroxy-8-methylnonanoic acid (1 mol); (11) Specific rotation. [or] D20 = + 43 ° (c = 0.5; H, 0); (12) Classification in base, acid and neutral: amphotheer; 5. Antibiotic WAP-8294AX or pharmaceutically acceptable salt thereof having the following physicochemical properties: (1) Appearance: white powder; (2) Melting point: 196 ~ 200 ° C (decomposition); (3) Solubility: soluble in water, methanol, n-butanol, dimethylformamide and dimethyl sulfoxide and insoluble in acetone, ethyl acetate and chloroform, (4) Color reaction: positive in reactions of ninhydrine, Ehrlich, Rydon-Smith, aqueous solution of potassium per - manganate, sulfuric acid and iodine vapor, and exhibits adequate stains upon irradiation with light rays having a wavelength of 254 nm emitted by a UV lamp; negative in reaction of Molic, silver nitrate, ferric chloride and Dragendorff, · 35 (5) Retention time in HPLC: 5.3 minutes, 5.9 minutes, 6.2 minutes, 6.5 minutes, 6.9 minutes, 7, 3 minutes, 8.1 minutes, 9.3 minutes, 9.8 minutes, 11.3 minutes, 12.1 minutes, 13.7 minutes and 15.0 minutes; 1001012 (6) Ultraviolet absorption spectrum (in water): \ nax ·. 273 nm, 280 nm, 287 nm; (7) Infrared absorption spectrum (FT-IR, KBr): Characteristic absorption spectrum: 3300 cm'1, 1720-1715 cm1, 1636 5 cm'1, 1541 cm 1, 1404 cm'1, 1207 cm "1, 1137 cm'1 (8) 1 H NMR spectrum: (270 MHz, DMS0-d6): complicated proton signals are measured, which come from methyl, methylene, methine and heterocyclic or aromatic rings, · 10 (9) Molecular weight: 1400-1700 (10 ) Qualitative test for organic compounds (sodium melting method): The antibiotic is a compound that includes carbon-hydrogen-oxygen and nitrogen elements; 15 (11) Provides acid complete hydrolysis, as ninhydrin-positive compounds and fatty acids, in: Asp, Glu, Gly, β-Ala, Leu, Ser, Trp, Orn, Val, N-methylvaline, β-hydroxyaspartic acid, Phe, N-methylphenylalanine and 3-hydroxy-7-methyloctanoic acid; 20 (12) Specific rotation : [ar] d 2 O = + 24 ° (c = 0.5, H 2 O) (13) Classification in base, acid and neutral: amphother; 5 Ehrlich, Rydon-Smith, aqueous solution of potassium permanganate, sulfuric acid and iodine vapor, and exhibits fading stains upon irradiation with 254 nm wavelength light emitted by a UV lamp; negative in reaction of Molic, silver nitrate, ferric chloride and Dragendorff; (5) Retention time in HPLC: 11.1 minutes; (6) Ultraviolet absorption spectrum (in water): Xmax: 275 nm, 280 nm, 287 nm; (7) Infrared absorption spectrum (FT-IR, KBr): Characteristic absorption spectrum: 3300 cm'1, 1720-1715 cm'1, 1636 cm'1, 1541 cm'1, 1404 cm "1, 1207 cm'1 , 1137 cm -1; (8) Molecular weight: 1561.9 [FAB mass m / z 1562.9 (M + H) \ m / z 1561.2 (MH)]]; (9) Molecular formula · C73H11102lN17 20 (10) Acid complete hydrolysis provides, as hydrolysates, Asp (1 mol), Glu (1 mol, Gly (1 mol), Leu (1 mol), Ser (2 mol), Trp (1 mol), Orn ( 2 mol), N-methylvaline (1 mol), β-hydroxyaspartic acid (1 mol), N-methylphenylalanine (1 mol), and 3-hydroxy-7-methyloctanoic acid (1 25 mol); (11) Specific rotation: [or] D20 = + 42 ° (c = 0.5; H20); (12) Classification in base, acid and neutral: amphotheer, 6. Antibiotic WAP-8294AX-8 or pharmaceutically acceptable salt thereof, having the following physicochemical properties: (1) Appearance: white powder, (2) Melting point: 196 ~ 200 ° C (decomposition); (3) Solubility: soluble in water, methanol, n-butanol, dimethylformamide and dimethyl sulfoxide and insoluble in acetone, ethyl acetate and chloroform; (4) Color reaction: positive in reactions of ninhydrin, Ehrlich, Rydon-Smith, aqueous solution of potassium per manganate, sulfuric acid and iodine vapor, and exhibits fading spots when irradiated with light beams of a wavelength of 254 nm emitted by a UV lamp, · Negative in reaction of Molic, silver nitrate, ferric chloride and Dragendorff; (5) Retention time in HPLC: 9.3 minutes 1001012 t (6) Ultraviolet absorption spectrum (in water): \ max: 273 nm, 280 nm, 289 nm, (7) Infrared absorption spectrum (FT-IR, KBr): Characteristic absorption spectrum: 3300 cm'1, 1720-1715 cm'1, 1636 5 cm ", 1541 cm'1, 1404 cm'1, 1207 cm'1, 1133 cm'1; (9) Molecular weight: 1548 (FAB- mass m / z 1549 (M + H) +); (9) Molecular formula: C72Hl09O21N17; (10) Acid complete hydrolysis provides, as hydrolysates, Asp (1 mol), Glu (1 mol), Gly (1 mol ), Leu (1 mol), Ser 10 (2 mol), Trp (1 mol), Orn (2 mol), Val (1 mol), β-hydroxyaspartic acid (1 mol), N-methylphenylalanine (1 mol) and 3 -hydroxy-7-methyloctanoic acid (1 mol); (11) Specific rotation: [a] D 2 O = + 25 ° C = 0.5, H 2 O) (12) Classification in base, acid and neutral: amphother; Antibiotic WAP-8294AX-9 or pharmaceutically acceptable salt thereof, having the following physicochemical properties: (1) Appearance: white powder, (2) Melting point: 216 ~ 220 ° C (decomposition); (3) Solubility: soluble in water, methanol, n-butanol, dimethylformamide and dimethyl sulfoxide and insoluble in acetone, ethyl acetate and chloroform; (4) Color reaction: positive in reactions of ninhydrin, Ehrlich, Rydon-Smith, aqueous solution of potassium permanganate, sulfuric acid and iodine vapor, and exhibits effective staining when irradiated with light beams of a wavelength of 254 nm emitted by a UV lamp; negative in reaction of Molic, silver nitrate, ferric chloride and Dragendorf f (30) Retention time in HPLC: 9.8 minutes (6) Ultraviolet absorption spectrum (in water): XMX: 273 nm, 280 nm, 289 nm; (7) Infrared absorption spectrum (FT-IR, KBr): Characteristic absorption spectrum: 3300 cm'1, 1720-1715 cm'1, 1636 35 cm'1, 1541 cm "1, 1404 cm'1, 1207 cm'1, 1137 cm'1; (8) Molecular weight: 1548 (FAB mass m / z 1549 (M + H) +; m / z 1547 (MH) '); (9) Molecular formula: C72H109O21N17; 1001012 ♦ (10) Acid Complete hydrolysis, as hydrolysates, provides Asp (1 mol), Glu (1 mol), Gly (1 mol), Leu (1 mol), Ser (2 mol), Trp (1 mol), Orn (2 mol) Val (1 mol), β-hydroxyaspartic acid (1 mol), Phe (1 mol) and 3-hydroxy-7-methyloctanoic acid (1 mol); (11) Specific rotation: [alt, 20 = + 28 ° (c = 0.5, H 2 O); (12) Classification in base, acid and neutral: amphotheer, 8. Antibiotic WAP-8294AX-13 or pharmaceutically acceptable salt thereof, having the following physicochemical properties: (1) Appearance: white powder; (2) Melting point: 205 ~ 210 ° C (decomposition); (3) Solubility: soluble in water, methanol, n-butanol, dimethylformamide and dimethyl sulfoxide and insoluble in acetone, ethyl acetate and chloroform, (4) Color reaction: positive in reactions of ninhydrine, Ehrlich, Rydon-Smith, aqueous solution of potassium per manganese, sulfuric acid and iodine vapor, and exhibits fading spots upon irradiation with 254 nm wavelength light rays emitted by a UV lamp, negative in reaction of Molic, silver nitrate, ferric chloride and Dragendorff; (5) Retention time in HPLC: 15.0 minutes (6) Ultraviolet absorption spectrum (in water): Xmax: 273 nm, 280 nm, 289 nm; (7) Infrared absorption spectrum (FT-IR, KBr): Characteristic absorption spectrum: 3300 cm'1, 1720-1715 cm'1, 1636 cm ", 1541 cm'1, 1404 cm'1, 1207 cm'1, 1137 cm 1; (8) Molecular weight: 1548 (FAB mass m / z 1577 (M + H) *]; 30 (9) Molecular formula: C74Hu3021Nl7, (10) Acid complete hydrolysis provides, as hydrolysates,: Asp (1 mol), Glu (1 mol), β-Ala (1 mol), Leu (1 mol), Ser (2 mol), Trp (1 mol), Orn (2 mol), N-methylvaline), β -hydroxyaspartic acid (1 mol), N-methylphenylalanine 35 (1 mol) and 3-hydroxy-7-methyloctanoic acid (1 mol); (11) Specific rotation: [or] D20 = + 27 ° (c = 0.5, H20 ); (12) Classification in base, acid and neutral: amphotheer. 1001012 4 * A method of producing the antibiotic WAP-8294A according to any one of claims 1 to 8, which method comprises the steps of culturing, in a culture medium, a microorganism belonging to the genus Lysob-5 actor. having the ability of producing the antibiotic WAP-8294A, according to any one of claims 1 to 8, to produce the antibiotic and allow it to accumulate in the culture medium; and then win the antibiotic. 10. Micro-organism belonging to the genus Lvsobacter. having the ability to produce the antibiotic WAP-8294A according to any one of claims 1 to 8. Microorganism according to claim 10, wherein the microorganism Lysobacter sp. WAP-8294. An antibacterial composition comprising at least one member selected from the group consisting of the antibiotic WAP-8294A and pharmaceutically acceptable salts thereof, as set out in any one of claims 1 to 8. 1001012