WO2000055186A1

WO2000055186A1 - Antibiotic wap-2607b, process for producing the same and antibacterial agents

Info

Publication number: WO2000055186A1
Application number: PCT/JP2000/001428
Authority: WO
Inventors: Azusa Katoh; Seigo Nakatani; Naomi Kokubo; Haruhisa Hirata
Original assignee: Wakamoto Pharmaceutical Co.,Ltd.
Priority date: 1999-03-16
Filing date: 2000-03-09
Publication date: 2000-09-21
Also published as: AU2939900A; JP2003113192A

Abstract

An antibiotic showing an excellent antibacterial effect on gram-positive sphericals (in particular, methicillin resistant Staphylococcus aureus) and being produced by a strain belonging to the genus Flavobacterium isolated from soil is provided to thereby give chemotherapeutics having a low toxicity, being capable of achieving an antibacterial effect within a short time and having little side effects.

Description

Specification

Antibiotic WAP-2607B, its manufacturing method and antibacterial agent

The present invention relates to a novel antibiotic WAP-2607B useful as a therapeutic agent for infectious diseases caused by pathogenic microorganisms, a method for producing the same, and uses thereof. Background art

The history of chemotherapeutic agents for bacterial infections began with the discovery of synthetic penicillin in quinine and salvarsan, and has greatly benefited humankind until now. Recently, MRSA (methicillin-resistant Staphylococcus aureus) infections As typified, resistant bacteria that do not respond to chemotherapeutic agents other than vancomycin and eightbekacin have emerged, causing confusion at medical sites and causing serious social problems. Vancomycin is currently used in the treatment of MRSA infections as an antibiotic developed by Eli Lilly of America in 1958, but is often associated with renal and hepatic disorders, and even with eighth neuropathy, However, it also has the disadvantage that the sterilization rate is low. There is also concern about the emergence of more resistant bacteria.

Habekacin, on the other hand, is chemically derived from the aminoglycoside antibiotic kanamycin as a skeleton and is used as a chemotherapeutic agent for MRSA infections, which is one of the few that has excellent antibacterial activity and is effective against various resistant bacteria. However, it is not necessarily an antibiotic that can be safely used from a medical point of view, since it may cause serious renal damage and the risk of complications of the eighth cranial nerve disorder, which are inherent side effects of aminoglycoside antibiotics.

In view of these circumstances, there has always been a demand for the discovery or creation of a new chemotherapeutic agent that has low toxicity, can exhibit a bactericidal action in a short time, and has few side effects. For that purpose, much research has been conducted. Disclosure of the invention

An object of the present invention is to provide a novel antibacterial substance having an antibacterial activity capable of meeting the above demand. Another object of the present invention is to provide a clinically useful chemotherapeutic agent having lower toxicity, ie, superior selectivity.

The present inventors have conducted intensive research to discover useful new antibiotics, and as a result, succeeded in isolating a strain belonging to the genus Flavobacterium as a novel microorganism from soil, and this strain has become a new antibiotic. They found that they produced substances, and completed the present invention.

The present invention is a novel antibiotic WAP-2607B found in a culture solution of a strain belonging to the novel genus Flavobacterium. This antibiotic WAP-2607B is further fractionated into at least five components Bl, B2, B3, B4 and B5. In this specification, the term “antibiotic WAP-2607B” shall mean the above B1, B2, B3, B4, B5 or a mixture of two or more thereof. The present invention also includes, in addition to the free form of the antibiotic WAP-2607B, pharmaceutically acceptable salts thereof, for example, hydrochloride, sulfate, methanesulfonate, trifluoroacetate and the like. These substances have strong antibacterial activity against gram-positive bacteria, especially methicillin-resistant Staphylococcus aureus (MRSA).

The antibiotic WAP-2607B was used for elution conditions of _C18 reverse phase silica gel high performance liquid chromatography [column: YMC A-312 (6 (id) xl50mm), mobile phase: acetonitrile containing 0.05% trifluoroacetic acid: water ( 38: 62), detection wavelength: UV280 nm, flow rate: 1 ml / min], and the retention times obtained at 5.8, 8.0, 11.7, 12.8, and 19.5 minutes, respectively, are: Bl, B2, B3, B4, It is fractionated into B5 (Fig. 1).

The antibiotics WAP-2607B1, B2, B3, B4, and B5 give a UV absorption spectrum (in MeOH) of / Lmax 281 nm, 290 jobs. FIG. 2 shows the ultraviolet absorption spectrum of the antibiotic WAP-2607B2. The antibiotics WAP-2607B1, B2, B3, B4, and B5 incorporate tributophan as a chromophore in the molecule from the confirmation of the acid hydrolyzate described below. The infrared absorption spectrum of the antibiotic WAP-2607B1 B2 B3 B4 B5 shows 0H NHs around 3300 cm " ¹ and CH stretching at 2960 cm- ¹ and amide bonds around 1660 cm- ¹ and 1540 cm- ^1. The absorption spectrum of the antibiotic WAP-2607B2 is shown in Fig. 3.

¹ H-NMR of the antibiotic WAP-2607B1 B2 B3 B4 B5 shows complex proton signals from numerous methyl, methylene, methine, and heterocyclic or aromatic rings. FIG. 4 shows the ¹ H-NMR spectrum of the antibiotic WAP-2607B2.

From the FAB-mass spectrum measurement of the antibiotic WAP-2607B1 B2 B3 B4 B5, -260781 ^ + 11) ⁺ ion is 1603.9, \ ^ -260782 (+11) ⁺ ion is 1617.9 WAP-2607B3 (+11). M + H) + ion gives 1631.8, ¾ ^ -260784 (> 1 + 11) ⁺ ion gives 1631.9 WAP-2607B5 +11) ⁺ ion gives 1645.8, and all the constituent amino acids and Considering the structural determination of the constituent fatty acids and their molar ratios, as well as the measurement results of the high-resolution FAB-mass vector, the molecular formula shown in Table 1 is submitted for the antibiotic WAP-2607B1 B2 B3 B4 B5 .

Table 1 Molecular formula of each component, constituent amino acids and constituent fatty acids

Antibiotic WAP-2607B1 B2 B3 B4 B5 is ninhydrin, Sakaguchi, Ehrlich, replacement paper (Rule 26) Rydon Smith, potassium permanganate aqueous solution, sulfuric acid, positive reaction in sodium hydroxide, appearance of quenching spots due to irradiation with UV lamp 254 nm, Mauritsch, Dragendorf, silver nitrate, salt Negative for diiron reaction.

In addition, in order to know the constituent amino acids of the antibiotics WAP-2607B1, B2, B3, B4, and B5, each fraction was subjected to complete acid hydrolysis, and two-dimensional TLC and amino acid analysis were performed. As a result, all fractions contained glycine (Gly), leucine (Leu), tryptophan (Trp), glucumate (Glu), arginine (Arg), serine (Ser), threonine (Thr) and one unknown amino acid. In addition, Bl and B4 were found to contain palin (Val), and B2, B3 and B5 contained iso-isocyanate (lie). Antibiotic The results of the two-dimensional TLC of the antibiotic WAP-2607B are shown in FIG.

To further determine the unknown amino acids, the antibiotic WAP-2607B was acid hydrolyzed again and the unknown amino acids were isolated. The results of various instrumental analyzes revealed that the unknown amino acid was N-methylphenylalanine.

On the other hand, from ether extracts of the acid hydrolysates of the antibiotics WAP-2607B1, B2, B3, B4, and B5, 3-hydroxy-5-methylhexanoic acid was obtained from Bl and B2, and 3- 3-Hydroxy-7-methyloctanoic acid was obtained from hydroxy-6-methylheptanoic acid, B4 and B5.

As a result of searching and comparing known compounds based on the above physicochemical properties, complete acid hydrolyzate, constituent amino acids, constituent fatty acids, etc. of the antibiotics WAP-2607B1, B2, B3, B4, B5, the compounds are new substances Was concluded.

The antibiotic WAP-2607B1, B2, B3, B4, B5 of the present invention is obtained by culturing an antibiotic WAP-2607B producing bacterium belonging to the genus Flavobacterium, and isolating the antibiotic WAP-2607B from the culture. It can be produced by collecting and, if necessary, further separating and purifying. One example of the WAP-2607B-producing bacterium belonging to the genus Flavopacterium used in the method of the present invention is as follows: Flavobacterium sp. WAP-2607 strain that has been isolated is listed. The bacteriological properties of this strain are as follows.

1.Form

After culturing on a broth agar plate at 24 ° C for 3 days, when observed under a microscope, the cell size is 0.5 to 0.7〃111 in diameter and 1.4 to length: L-shaped 8〃m rod-shaped, negative for Gram staining No flagella, no spores, no acid resistance.

2. Growth status in various media

(1) Gravy agar plate culture

The colonies are translucent light yellow, circular, and the surface is convex, peripheral to wavy. No diffuse pixels are generated.

(2) Gravy agar slope culture

It grows in a good spread and has an opaque light yellow color.

(3) Liquid broth culture

It grows in a light turbid state, forms a bacterial ring, and can be found on the bottom of the culture vessel.

(4) Juice gelatin puncture culture

It grows from the surface to the mid-depth while squeezing gelatin.

(5) Litmus. Milk culture

Litmus has no reducing ability, but has peptone formation and coagulation effects.

3.physiological properties

The physiological properties of the WAP-2607 strain are as follows.

(1) Reduction of nitrate

(2) Denitrification reaction

(3) MR test +

(4) VP test + Generation of indol

(6) Generation of hydrogen sulfide

(7) Hydrolysis of starch-

(8) Use of cunic acid

①Kozel medium +

② Simmons medium +

③ Christensen medium +

(9) Use of inorganic nitrogen source

Acid ammonium + ③ Sodium glutamate +

(10) Dye formation

① King A medium

② King B medium ―

11) Ureze I

12) Oxy Yuichi +

13) Rikyuu Rase +

14) Growth temperature 15-37 ° C

15) Growth pH 5-9

16) Attitude to oxygen Aerobic

17) 0-F test Non-disassembly type

18) Phosphatase +

19) Hemolytic β

20) Decomposition of cellulose

21) Decomposition of Tween ®Tween20 +

② Tween40 +

③ Tween60 +

④ Tween80 +

(21) Decomposition of colloidal chitin +

(22) Decomposition of polysaccharide

① CMC +

② Alginate +

③ ぺ Cutic acid +

(23) Generation and utilization of acids and gases from sugars (Table 2)

Table 2

Acid generation—gas generation

: ぺ (ぺ: (Davis's medium) Realabinose

D-xylose

D-glucose +

D-Mannose +

D-fructose +

D-galactose +

+ Sucrose + sucrose + trueno, mouth +

D-Sorbit

D-Mannit

Wild boar

Glycerin

+ Cellobiose +

Replacement form (Rule 26) Purification of the WAP-2607 strain having the above mycological properties was carried out by purging the manual, which was described in [Bergey, s Manual of Systematic Bacteriology ¥ ₀ 1.1 (1984)]. Is a yellow gram-negative bacillus with no flagella and no motility.It is aerobic, does not form microcysts, and produces oxidase and ribonuclease. Therefore, it is considered to belong to Flavobacteriumg.

When compared with known species of the same genus, their properties are similar to those of Flavobacterium breve. sp. WAP-2607).

WAP-2607 strain was deposited with the Research Institute of Biotechnology, Industrial Technology Institute on March 8, 1999 and was deposited under accession number FEPP-17284. As a general property of the bacterium of the genus Flavobacterium, its mycological properties are extremely mutated and the strain WAP-2607 is no exception. However, even if those mutants are naturally mutated or artificial mutants by various mutagenic agents, etc., all of the present invention is applicable as long as it has the ability to produce the antibiotic WAP-2607B. Can be used. BEST MODE FOR CARRYING OUT THE INVENTION

(Culture and production)

The antibiotic WAP-2607B of the present invention is produced by inoculating the above-mentioned production bacterium into a nutrient source-containing medium and culturing aerobically. In culturing bacteria producing the antibiotic WAP-2607B, organic carbon compounds that can be used as carbon sources such as glucose, fructose, starch, dextrin, glycerin, molasses, starch syrup, oils and fats, and organic acids On the other hand, nitrogen sources include, for example, soybean flour, cottonseed flour, cornstarch, primate, casein, peptone, yeast extract, meat extract, germ, urea, amino Organic nitrogen compounds and inorganic nitrogen compounds such as acids and ammonium salts can be used. Further, as the salts, for example, inorganic salts such as sodium salt, potassium salt, calcium salt, magnesium salt and phosphate can be used alone or in an appropriate combination. If necessary, promote the growth of heavy metals such as iron, copper, zinc, and cobalt salts, vitamins such as biotin and vitamin B, which are necessary for the growth of microorganisms, and promote the growth of other producing bacteria. -It is desirable to add an organic or inorganic substance that can produce a significant amount of 2607B. An antifoaming agent such as silicone oil or polyalkylene glycol ether or a surfactant may be added to the medium.

As the culture method, a method generally used for the production of antibiotics is employed.In the liquid culture method, deep aeration and stirring culture is preferable, particularly in order to maintain aerobicity. Shaking culture is suitable. The cultivation temperature can be usually 20 to 40 ° C, but it is preferable to keep it at 25 to 30 ° C. The pH of the culture can be around 6-8, but it is preferable to keep it around 7. Production of WAP-2607B in the culture is sufficiently achieved in about 2 to 6 days.

(Isolation and purification)

As described above, WAP-2607B accumulated in the culture can be advantageously collected from the culture by utilizing the physicochemical properties of the antibiotic of the present invention described later. That is, since the antibiotic WAP-2607B is contained in the culture filtrate and the cultured cells, the culture is filtrated under reduced pressure or centrifuged to obtain the cultured cells and the culture filtrate. The cultured cells are further extracted with aqueous alcohol or aqueous acetone, filtered under reduced pressure, and acetone is distilled off to obtain an extract.

Antibiotic WAP-2607B is considered to be a water-soluble polypeptide antibiotic based on the physicochemical properties described below, and is composed of components consisting of relatively large compounds with molecular weights of about 1,600 to 1,700. ing. Considering these properties, when collecting WAP-2607B from the culture filtrate and the cell extract, an adsorption resin such as Diaion HP20, Sepabeads SP207, CHP20 (Mitsubishi Kasei), Amberlite XAD-2 (Kichiichi Muandhas), Duolite S-30 (Diamond Shark Rock Chemical), activated carbon, etc. can be used advantageously. However, purification by DIA-ON HP-20 column chromatography is particularly efficient.

For example, the culture filtrate of PH2.5 is adsorbed on a Diaion HP20 column, washed with water, and eluted with 80% acetone water to recover the antibiotic WAP-2607B. After acetone was distilled off under reduced pressure, ethyl acetate was extracted under acidic conditions to remove fat-soluble impurities, and the aqueous layer was again subjected to WAP with a highly polar organic solvent such as n-butanol. -Can extract 2607B. After evaporating n-butanol at low temperature, freeze-dry as it is, or add a solvent insoluble in WAP-2607B, such as acetone, to the concentrate to form a precipitate, and then centrifuge or vacuum filtration to obtain a crude substance. Can be collected. In order to further purify the crude material collected in this way, it is extracted by a polar organic solvent such as n-butanol under S denaturing conditions, so it is typified by centrifugal liquid separation chromatography. A purified product of high purity can be obtained by a single or appropriate combination of distribution chromatography such as counter-current distribution method, adsorption chromatography using silica gel column chromatography, and cellulose column chromatography. In addition, by using molecular sieve effects such as Sephadex G-10, G-15, LH-20 (manufactured by Pharmacia) and Biogel P-4 gel (manufactured by Bio-Rad), it is possible to use low-grade water or hydrated methanol. It is also possible to purify by developing and eluting a suitable combination of a water-containing solvent such as an alcohol, a dilute alkaline or dilute acidic aqueous solution, or an aqueous solution containing an appropriate salt.

The WAP-2607B thus obtained was further separated and purified by the above-described countercurrent distribution method or high performance liquid chromatography, and the five components WAP-2607B1, B2, B3, B4, and B5 were separated. Obtainable. Silica gel and alkyl groups such as octyl decyl and octyl groups are used as packing materials for high performance liquid chromatography. Excellent carriers chemically bonded to silanol groups of lycagel are commercially available, and carriers such as polystyrene-based porous polymer gels are also widely used, and can be efficiently separated using these fillers. . As the mobile phase, under an acidic condition, for example, a mixed alcohol acetonitrile containing trifluoroacetic acid or the like, a lower alcohol such as hydrated methanol, a buffer solution or the like can be used.

(Physicochemical properties)

Physicochemical properties of WAP-2607B1, B2, B3, B4, and B5 (all of which are hydrochlorides) obtained using the above-mentioned various chromatography, and ether extraction of ninhydrin-positive substances and acid hydrolysates by complete acid hydrolysis Things are described below.

• Antibiotic WAP-2607B

(1) Appearance: White powder

(2) Melting point: 215-225 ° C (decomposition)

(3) Solubility: Soluble in water, methanol, n-butanol, dimethylformamide, dimethylsulfoxide, insoluble in acetone, ethyl acetate, and chloroform.

(4) Color reaction: Positive for ninhydrin, Sakaguchi, Ehrlich, Leydon Smith, aqueous potassium permanganate, sulfuric acid, and Rhodoboper reaction, showing a quenching spot by irradiation with UV lamp 254.

Maurish, silver nitrate, ferric chloride, negative for Dragendorf reaction.

(5) Retention time in high performance liquid chromatography (Figure 1): 5.8 minutes, 8.0 minutes, 11.7 minutes, 12.8 minutes, 19.5 minutes

(6) UV absorption spectrum (in MeOH): Amax 281 nm, 290 nm

(7) Infrared absorption spectrum (FT-IR ヽ KBr method): characteristic absorption 3342 cm ¹ , 2964 cm- ¹ , 1655 cm " ¹ , 1539 cm— ¹

(8) Molecular weight: 1,600-1,700

(9) Ninhydrin positive substance by acid complete hydrolysis: Gly, Leu, Val, lie, Trp, Glu, Gives Arg, Ser, Thr, and N-methylphenylalanine.

(10) Specific rotation: [H] _D ²⁰ = + 30 ° (c = 0.5, MeOH)

(11) Basic, acidic and neutral: amphoteric

• Antibiotic WAP-2607B1

(1) Appearance: White powder

(2) Melting point: 215-225 ° C (decomposition)

(3) Solubility: Soluble in water, methanol, n-butanol, dimethylformamide, dimethyl sulfoxide, insoluble in acetone, ethyl acetate, and chloroform.

(4) Color reaction: Positive for ninhydrin, Sakaguchi, Ehrlich, Leydon-Smith, aqueous potassium permanganate, sulfuric acid, and oxidative reaction, showing a quenching spot by irradiation with UV lamp 254ηι.

Maurish, silver nitrate, ferric chloride, negative for Dragendorf reaction.

(5) Retention time in high performance liquid chromatography: 5.8 minutes

(6) UV absorption spectrum (in MeOH): person max 282 ran, 290 run

(7) Infrared absorption spectrum (FT-1 KBr method): characteristic absorption 3328 cm-960 cm " ¹ , 1655 cm" ¹ , 1541 cm ¹

(8) Molecular weight: 1602.9 [FAB-MS m / z 1603.9 (M + H) +]

(9) Molecular Formula: C ₇₄ H ₁₁₄ N _2. 0 ₂ . [Measured value of (M + H) ⁺ ion from high resolution FAB-Mass: m / z 1603.8547, calculated value: 1603.8597]

(10) Complete acid hydrolyzate: Gly (1 mol), Leu (1 mol), Val (1 mol), Trp (1 mol), Glu (2 mol), Arg (2 mol), Ser (1 mol) And Thr (2 mol) N-methylphenylalanine (1 mol) and 3-hydroxy-5-methylhexanoic acid (1 mol).

(11) Specific rotation: [H] _D ²⁰ = + 32 ° (c = 0.5, MeOH)

(12) Basic, acidic and neutral: amphoteric • Antibiotic WAP-2607B2

(1) Appearance: White powder

(2) Melting point: 220-225 ° C (decomposition)

(4) Color reaction: Positive for ninhydrin, Sakaguchi, Ehrlich, Leydon Smith, potassium permanganate solution, sulfuric acid, and greenhouse reaction, quenching spot by irradiation with UV lamp 254 Is shown.

Maurish, silver nitrate, ferric chloride, negative for Dragendorf reaction.

(5) High-performance liquid chromatography retention time: 8.0 minutes

(6) UV absorption spectrum (in MeOH) (Figure 2): Amax 281 nm, 290 ran

(7) Infrared absorption spectrum (FT-IR, KBr method) (Figure 3): Characteristic absorption 3320 cm— ¹ , 2964 cm ¹ , 1655 cm ¹ , 1539 cm ” ¹

(8) Ή nuclear magnetic resonance (NMR) scan Bae spectrum (Figure 4): (270MHz, DMS0- d 6), are observations methyl, methylene, complex proton signals originated from methine and heterocyclic or aromatic ring .

(9) Molecular weight: 1616.9 [FAB-MS m / z 1617. 9 (M + H) ⁺ ]

(10) Molecular _{_{_{formula: C 75 H 116 N 2fl 0}}} 2. [Measured value of (M + H) + ion from FAB-Mass with high resolution: m / z 1617.8782, calculated value: 1617.8753]

(11) Complete acid hydrolyzate: Gly (1 mol), Leu (1 mol), lie (1 mol), Trp (1 mol), Glu (2 mol), Arg (2 mol), Ser (1 mol) ), To give Thr (2 mol) N-methylphenylalanine (1 mol) and 3-hydroxy-5-methylhexanoic acid (1 mol).

(12) Specific rotation: [HI] _D ² . = +31. (c = 0.5, MeOH)

(13) Basic, S-denatured, neutral: amphoteric • Antibiotic WAP-2607B3

(1) Appearance: White powder

(2) Melting point: 220-230 ° C (decomposition)

(4) Color reaction: Positive for ninhydrin, Sakaguchi, Ehrlich, Rydsmith, aqueous solution of potassium permanganate, sulfuric acid, and oxidative vapor, showing a quenching spot by irradiation with a UV lamp at 254 nm.

Maurish, silver nitrate, ferric chloride, negative for Dragendorf reaction.

(5) Retention time in high performance liquid chromatography: 11.7 minutes

(6) UV absorption spectrum (in MeOH): human max 281 nm, 290 ran

(7) Infrared absorption spectrum (FT-IR, KBr method): characteristic absorption 3412 cm ¹ , 2932 cm " ¹ , 1663 cm— ¹ , 1541 cm" ¹

(8) Molecular weight: 1630.8 [FAB-MS m / z 1631.8 (M + H) +]

(9) Molecular Formula: C ₇₆ H ₁₁₈ N _2. 0 _2fl [Measured value of (M + H) + ion from high resolution FAB-Mass: m / z 1631.8888, calculated value: 1631.8910]

(10) Complete acid hydrolyzate: Gly (1 mol), Leu (1 mol), lie (1 mol), Trp (1 mol), Glu (2 mol), Arg (2 mol), Ser (1 mol) ), Thr (2 mol) N-methylphenylalanine (1 mol) and 3-hydroxy-6-methylheptanoic acid (1 mol).

(11) Specific rotation: [H]. ² . = + 5.8 ° (c = 0.5, MeOH)

(12) Basic, acidic and neutral: amphoteric

• Antibiotic WAP-2607B4

(1) Appearance: White powder

(2) Melting point: 220-225 ° C (decomposition) Solubility: Soluble in water, methanol, n-butanol, dimethylformamide, dimethyl sulfoxide, insoluble in acetone, ethyl acetate, and chloroform.

(4) Color reaction: Positive for ninhydrin, Sakaguchi, Ehrlich, Leydon Smith, aqueous potassium permanganate, sulfuric acid, and Rhodoboper reaction, showing quenching spots by irradiation with a UV lamp at 254 nm.

Maurish, silver nitrate, ferric chloride, negative for Dragendorf reaction.

(5) Retention time in high performance liquid chromatography: 12.8 minutes

(6) UV absorption spectrum (in MeOH): human max 282 nm, 290 nm

(7) Infrared absorption spectrum (FT-IR, KBr method): characteristic absorption 3340 cm " ¹ , 2956 cm" ¹ , 1657 cm " ¹ , 154 cm" ¹

(8) Molecular weight: 1630.9 [FAB-MS m / z 1631.9 (M + H) ⁺ ]

(9) Molecular Formula: C ₇₆ H ₁₁₈ N _2. 0 ₂ . [Measured value of (M + H) ⁺ ion from FAB-Mass with high resolution: m / z 1631.8888, calculated value: 1631.8910]

(10) Complete acid hydrolyzate: Gly (1 mol), Leu (1 mol), Val (1 mol), Trp (1 mol), Glu (2 mol), Arg (2 mol), Ser (1 mol) , Thr (2 mol) N-methylphenylalanine (1 mol) and 3-hydroxy-7-methyloctanoic acid (1 mol).

(11) Specific rotation: [HI] _D ² . = + 29 ° (c = 0.5, MeOH)

(12) Basic, S-denatured, neutral: amphoteric

• Antibiotic WAP-2607B5

(1) Appearance: White powder

(2) Melting point: 220-230 ° C (decomposition)

(4) Color reaction: ninhydrin, Sakaguchi, Ehrlich, Ryd Smith, overman Positive for potassium gunate aqueous solution, sulfuric acid, and rhodobapper reaction, showing quenching spots by irradiation with a UV lamp at 254 nm.

Maurish, silver nitrate, ferric chloride, negative for Dragendorff reaction.

(5) Retention time during high performance liquid chromatography: 19.5 minutes

(6) Ultraviolet absorption spectrum (in MeOH): human max 281 nm, 290 nm

(7) Infrared absorption spectrum (FT-m ヽ KBr method): characteristic absorption 3288 cm- ¹ , 2955 cm-1655 cm- ', 1541 cm- ¹

(8) Molecular weight: 1644.8 [FAB-MS m / z 1645.8 (M + H) ⁺ ]

(9) Molecular _{_{Formula: C 77 H 12 () N}} 2Q 0 2Q [ high resolution FAB-Mass than (M + H) + Found ions: m / z 1645. 9022, Calculated: 1645.9066]

(10) Complete acid hydrolyzate: Gly (1 mol), Leu (1 mol), lie (1 mol), Trp (1 mol), Glu (2 mol), Arg (2 mol), Ser (1 mol) ), To give Thr (2 mol) N-methylphenylalanine (1 mol) and 3-hydroxy-7-methyloctanoic acid (1 mol).

(11) Specific rotation: [H] _D ²⁰ = + 30 ° (c = 0.5, MeOH)

(12) Basic, acidic and neutral: amphoteric

The elution conditions for the C18 reversed-phase silica gel high-performance liquid chromatography of the antibiotics WAP-2607B1, B2, B3, B4, and B5 are as follows.

Column: YMC A-312 (6 (i.d.) xl50 customer)

Mobile phase: acetonitrile containing 0.05% trifluoroacetic acid: water (38:62) Detection wavelength: 280 nm

Flow rate: 1ml / min

(Biological properties)

(1) Antibacterial activity

Table 3 shows the results of the antibacterial activity test for the antibiotics WAP-2607B1, B2, B3, B4, and B5. minimum The growth inhibitory concentration (MIC) was measured using a medium in which 2% sodium chloride was added to Mueller-Hinton Broth (DIFC0) for MRSA, and a Mulla-Hinton medium for other bacteria. This was performed using only the liquid medium dilution method.

As shown in Table 3, WAP-2607B1, B2, B3, B4, and B5 show strong activity against methicillin-resistant Staphylococcus aureus, and the activity is more than 8-fold enhanced in the same medium supplemented with 10% bovine serum. Is the feature. In addition, gram-negative bacilli, mold, and yeast have almost no activity.

Further, by a method according to the MIC measuring method, scan evening Fuirokokkasu 'Aureusu JCM 8 7 0 2 (MRSA, viable cell number 1 0 ⁶ / ml) for WAP- 2 6 0 7 B1, B2 , B3, B4, B5 As a result of examining the sterilization rate, it was found that all the antibiotics could be sterilized to a viable cell count of less than 10 cells / ml after 3 hours at the MIC concentration. On the other hand, vancomycin in after 3 hours, the number of viable cells is remained 1 0 ⁵ / ml, was able to be sterilized up to 2 4 hours after last viable cell number 1 0 fewer than / m 1.

Table 3

Antibacterial activity of WAP-2607B

MIC (g ml)

S «Ji¾ bacteria Β Β1 Β2 Β3 Β4 Β5 Suefu Kobucus' Aureus JCM8702 1.56 1.56 1.56 3.13 1.56 1.56

(MRSA) 1 (1% serum added (1.20 0.20 0.20 0.39 0.20 0.20 Star 7 cas * Aureus JCM8703 0.78 0.78 0.78 1.56 0.78 0.7Η

(MRSA) 1 〖)% serum added 0.10 0.10 0.10 0.20 0.10 0.10 Sphere α 7 ds * Aureus JCM8704 1.56 1.56 1.56 3.13 1.56 1.56

(MRSA) 10% serum added 0.20 0.20 0.20 0.39 0.20 0.20 staphy Π] ₇ dregs << Ryoreus ATCC25923 1.56 1.56 1.56 3.13 1.56 1.56

(MSSA) 10% serum added 0.20 0.2 () 0.20 0.39 0.20 () .20 Staff: Kas * Ebite Lumitice ATCC12228 0.78 0.78 0.78 1,56 0.39 0.39

1% serum added 0.10 0.10 Ο.ΐϋ 0.20 0.10 0.10 Hachi-Sachil AT ATCC6633 1.56 1.56 1.56 3.13 1.56 1.56

10% serum added 0.20 0.20 0.20 0.39 0.20 0.20 Gent 7L Charis J MSS03> 1 (Χ)> 100> 100> 100> 100> 100

10% serum addition> 10> 101)> 10ϋ> 1 (Μ)> 100> 1 (Κ) Gent 7 Q7 77ι Shim JCM580> 1 (Κ)> 1 () 0> 1 (Κ)> 1 ( Κ)> 10 ()> 1 (Κ)

10% serum addition> 1 ()> 10ϋ> 1 (Κ)> 1 (Χ)> 10 ()> 1 <Μ)

I series I ATTR25922> 1 (Κ)> 100> 1 (Χ)> 1 (Κ)> 10 ()> 1 () 0

10% serum addition> 100> 1 (Κ)> 100> 1 (Κ)> 1 () 0> 100 Pseudomonas 1 llucinoser * ATCC 027> 1 (Κ)> 100> 100> 1 ( Κ)> 1 () 0> ιοο

1 () ¾Serum addition> 1 () 0> 10ϋ> 1 (Κ)> 1 (Κ)> ΚΜ)> 10 () Kansi 'Yu' 'Albicans ΉΜΜ0239> 10 ()> 100> 1 (Μ)> 100 > 1 (Μ)> 100

10% serum addition> 100> 1 (Κ)> 1 (Μ)> 1 (Κ)>! () ()> 1 () ϋ

(2) Acute mouse toxicity test

The antibiotics WAP- 2607Β (25 mg / kg, 50 mg / kg, 100 mg, 250 mg / kg) and WAP- 2607B2 (25 mg / kg ヽ 50 mg / kg, 100 mg, 250 mg / kg) were administered to mice (ICR, 4 weeks old, male) , 5 per group) Intraperitoneally, no acute toxicity was observed.

From the above, it can be seen that the antibiotic WAP-2607B1, B2, B3, B4, B5 of the present invention is useful as a therapeutic agent for bacterial infections in humans and animals, and sometimes for methicillin-resistant Staphylococcus aureus infections.

(3) Infection protection test in mice

Since the antibiotic WAP-2607B shows strong antibacterial activity against Gram-positive cocci including MRSA based on the in vitro antibacterial activity shown in Table 2, the infection protection test in mice using S. phylococcus apereus JCM8702 (MRSA) Was done. Experimental method: ICR-MCH mice, 4-week-old, male (CLEA Japan) 8 mice per group, 3 days before infection, intraperitoneal injection of cyclophosphamide 0.5 mg / 0.5 ml per mouse 3 days before infection After that, infection was carried out by intraperitoneal administration of 0.5 ml / mouse of a bacterial solution of 2 × 10 ⁴ cfu / ml of S. aureococcus apereus JCM8702 strain supplemented with 5% mucin. One hour after infection, 10 mg / kg of vancoma isin hydrochloride (manufactured by Shionogi & Co., Ltd.) and 2.5 mg / kg, l.Omg / kg and 0.5 mg / kg of WAP-2607B1 and WAP-2607B2 were administered in a single dose of 0.2 ml per mouse. Subcutaneous administration was performed in one time. The untreated control group was subcutaneously administered with 0.2 ml of physiological saline.

The results of the treatment are shown in Table 4. In the untreated control group, 7/8 died 7 days after infection. On the other hand, on the 7th day after infection in the antibiotic group, 5/8 survived in the vancomycin 10 mg / kg group. In contrast, as shown in Table 4, WAP-2607B1 and WAP-2607B2 showed superior treatment results at lower doses than vancomycin.

Table 4

WAP-2607B infection protection test

Test fresh cast (, me / kv no ^ surviving number

1 / Η

, Z ~ J in

2.5 8/8

AP-2607B1 1.0 7/8

0.5 5/8

2.5 8/8

WAP-2607B2 1.0 8/8

0.5 6/8

(Application)

When the novel antibiotic of the present invention is administered as a medicine, it is administered in various forms according to a conventional method. Examples of the administration form include powders, granules, tablets, capsules, syrups, etc., orally, or injections (intravenous, intramuscular, subcutaneous), infusions, suppositories, liniments, ointments. It can be safely administered parenterally in the form of an agent or the like. In view of the fact that the novel antibiotic of the present invention has a higher bactericidal rate than noncomycin and is effective at a low concentration, it can be safely administered parenterally to eye drops and ointments in the ophthalmic field.

These various preparations can be used in the usual manner in the form of active ingredients such as excipients, binders, disintegrants, coating agents, lubricants, stabilizers, flavoring agents, solubilizing agents, suspending agents, diluents, etc. It can be formulated using known adjuvants which can be usually used in the field of formulation technology. The dose varies depending on the target disease, the route of administration, the number of administrations, and the like. For example, it is preferable to administer 5 mg to 2000 m / day to adults once or several times depending on the symptoms. Example

Next, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.

Example 1

After pouring 100 ml of a medium (pH 7.2) consisting of 2.5% glucose, 2.0% defatted soy flour, 0.4% soybean oil, 0.25% sodium chloride, and 0.5% calcium carbonate into a 500ml Erlenmeyer flask, prepare eight sterilized mediums. Then, after inoculating one platinum loop from a slant agar medium of Flavobacterium sp. WAP-2607 strain, the cells were cultured in a mouth shaker at 180 ° C / min, 30 ° C for 2 days. After injecting 100 ml of the above-mentioned medium, prepare 300 sterilized 500 ml triangular flasks, inoculate 2 ml of the culture wave of the Erlenmeyer flask that had been previously cultured, and reciprocate at 180 ° / min, 30 ° with a mouth-to-mouth shaker. C. Culture was performed for 4 days. The antibacterial activity was measured by the agar plate method of S. aureus J. aureus JCM8702.

Example 2

The culture solution obtained in Example 1 was continuously centrifuged (10,000 rpm) to obtain 30 liters of the culture filtrate. The culture filtrate (PH2.5) was subjected to column chromatography using Diaion # 20 (manufactured by Mitsubishi Kasei Corporation, 6 liter). After washing with 15 liters of water, the active substance was eluted with 15 liters of 80% acetone water containing 0.05% trifluoroacetic acid. The eluate was concentrated to 1 liter and extracted three times with 1 liter of ethyl acetate. Subsequently, extraction was performed three times with 1 liter of η-butanol, and the η-butanol layer was concentrated and lyophilized to obtain 14.7 g of a crude filtrate. The cultured cells were extracted twice with 3 liters of 80 acetone water, and acetone was distilled off by concentration under reduced pressure to obtain 500 ml of a crude extract. This extract was extracted three times with 500 ml of ethyl acetate. Subsequently, extraction was performed three times with 500 ml of n-butanol, and the n-butanol layer was concentrated and lyophilized to obtain 16.8 g of crude bacterial cells. Defeat 3

14.7 g of the crude filtrate obtained in Example 2 was subjected to column chromatography with Chromatorex 0DS (manufactured by Fuji Silicon Chemical Co., Ltd., 600 ml), and eluted and fractionated with 40% acetonitrile water containing 0.05% trifluoroacetic acid. . The active fractions were collected, concentrated and freeze-dried to obtain 6.6 g of WAP-2607B.

Example 4

6.6 g of WAP-2607B obtained in Example 3 was subjected to column chromatography using Chromatorex 0DS (manufactured by Fuji Silicon Chemicals, Inc., 600 ml), and eluted and fractionated with 38% acetonitrile water containing 0.05% trifluoroacetic acid. The active fractions were collected, concentrated and freeze-dried to obtain WAP-2607Bl (800 mg), B2 (1490 mg), a mixture of B3 and B4 (480 mg), and B5 (810 mg).

Example 5

16.8 g of the crude cell material obtained in Example 2 was subjected to column chromatography with Chromatorex 0DS (manufactured by Fuji Silicon Chemical Co., Ltd., 500 ml), and eluted and fractionated with 38% acetonitrile water containing 0.05% trifluoroacetic acid. did. The active fractions were collected, concentrated, and freeze-dried to obtain WAP-2607Bl (214 mg), B2 (580 mg), a mixture of B3 and B4 (230 mg), and B5 (490 mg).

Example 6

The respective fractions obtained in Example 4 were subjected to high-performance liquid chromatography using a reverse-phase octane decyl silica gel column [column: YMC-GEL SH-343-5, 20 (id) X 250 mm, YMC Corp. And mobile phase: acetonitrile containing 0.05% trifluoroacetic acid: water (40-45: 60-55)], injected into dozens of batches, and fractions containing WAP-2607BK B2, B3, B4, B5 collected. The obtained fraction was passed through an activated carbon column (5-22 ml, manufactured by Wako Pure Chemical Industries, Ltd.) for decolorization, and then acetonitrile was distilled off under reduced pressure, passed through Dyaion SA11A, and lyophilized to WAP. -2607Bl (50mg), A white powder (hydrochloride) of B2 (830 mg), B3 (10 mg), B4 (240 mg), and B5 (340 g) was obtained.

Example 7 Amino acid analysis of WAP-2607B

5 mg of WAP-2607B 5m obtained in Example 3 and 5 mg of WAP-2607B1, B2, B3, B4, B5 obtained in Example 6 were dissolved in 1 ml of 6N hydrochloric acid containing 4 thioglycolic acid, and the solution was dissolved at 110 ° ( The hydrolyzed solution was concentrated to dryness, dissolved in a small amount of water, and then subjected to two-dimensional thin-layer chromatography on a cell source. n-butanol: pyridine: succinic acid: water (15: 10: 3: 12), second dimension, n-butanol: acetic acid: water (4: 1: 2)]. As a result, each component contains glycine (Gly), leucine (Leu), tribubutan (Trp), glutamic acid (Glu), arginine (Arg), serine (Ser), threonine (Thr) and one unknown amino acid, In addition, Bl and B4 were found to contain palin (Val) and B2, B3, and B5 contained isoleucine (lie). Figure 5 shows the results of TLC.

Example 8 Identification of unknown amino acid

To 100 mg of WAP-2607B4 obtained in Example 6, 4 ml of 6 HC1 containing 4% thioglycolic acid was added, and the mixture was hydrolyzed at 110 ° (for 20 hours. After the hydrolyzate was concentrated, the pH was adjusted to 7.0, Loaded on Dowex 50WX-8 (H ⁺ type, 10ml) and eluted with 0.1N, 0.2N, 0.4N, 1.0N, 2. ON HCl Unknown amino acids were present in the fraction eluted with 2.ON HC1 This fraction was concentrated and evaporated to dryness The concentrate was subjected to column chromatography on Sepabeads SP207 (Mitsubishi Kasei Co., Ltd., 30 ml), and a fraction containing unknown amino acids eluted with 80% acetone Was concentrated to give 2.3 mg of a powder of an unknown amino acid by scraping with cellulose TLC [developing solvent: n-butanol: pyridine: acetic acid: water (15: 10: 3: 12)]. The amino acid of this amino acid had a molecular weight of 179. CFAB-MSm / z: 180 ( M + H) + ] and ¹ H- NMR scan Bae spectrum (D ₂ 0, 270MHz) chemical Shi full h (5 (ppm): 2.78 (3H, s), 3.32 (2H , d, J = 6.2Hz), 3.95 ( 1H, t, J = 6.2Hz), 7.4-7.5 (5H, m), which was presumed to be N-methylphenylalanine. Compared to the standard N-methyl-L-phenylalanine (Sigma). where was, of preparation ¹ H- NMR The chemical shift of the spectrum, cellulose TLC value: 0.82 [Developing solvent: n-butanol: pyridine: acetic acid: water (15: 10: 3: 12)] It was identified as N-methylpheniralanine.

Example 9 Amino acid analysis of WAP-2607B1, B2, B5

About 30 μg of each of WAP-2607B1, B2, and B5 obtained in Example 6 was dissolved in 1 ml of 6N hydrochloric acid containing 4% thioglycolic acid, acid-completely hydrolyzed at 110 ° C. for 24 hours, and concentrated. Amino acid analysis was performed. As a result, Gly (1 mol), Leu (1 mol), Trp (1 mol), Glu (2 mol), Arg (2 mol), Ser (1 mol), Thr (2 mol), N-methylphenyl B1 contained Val (1 mol), B2 contained B2, and B5 contained ILe (1 mol).

Example 10 Isolation and Constitution of Constituent Fatty Acids of WAP-2607BU B2 B3, B4, and B5 10 mg of WAP-2607B1 obtained in Example 6 was hydrolyzed with 6NHC1 at 110 ° C. for 2 hours. The hydrolyzed solution was extracted with ether, and the ether layer was concentrated and dried to obtain 0.7 mg of a white powder. The substance is the molecular weight: 146 CFAB-MS m / z 145 (M - H) - ], E- Momosupe vector (CDC1 _3, 270MHz) of chemical shift δ (ppm): 0.93 (6H , d, J = 6.6Hz), 1. 17~1.57 ( 2H, m), 1.75~1.85 (1H, m), 2.46 (1H, dd, J = 16.7 3 8.8Hz), 2.56 (1H, dd, J = 16.7, 3.6Hz), 4.07 to 4.17 (1H, m), it was estimated to be 3-hydroxy-5-methylhexanoic acid. 100 / g of this substance was dissolved in 0.1 ml of benzene: methanol (8: 2), and one drop of trimethylsilyldiazomethane (manufactured by Tokyo Chemical Industry Co., Ltd.) was added, followed by methyl esterification at room temperature for 10 minutes. As a result of EI-MS measurement of the methyl ester, m / zl59 (M + -1) ion peak and each fragment ion of 142 (M + -18), 129 (M ⁺ -31), 111 (M + -3 tri 18) The peak and the characteristic reference fragment ion peak m / z 103 derived from C ₃ -C ₄ cleavage of ₃ -hydroxy fatty acid methyl ester were observed, indicating that the substance was 3-hydroxy-5-methylhexanoic acid. Was determined.

WAP-2607B2 lOm is acid-hydrolyzed in the same manner as above, and extracted with ether. The tellurium layer was concentrated and dried to obtain 0.8 mg of white powder. This substance has a molecular weight of 146 [FAB-MSm / z 145 (Μ-Η) _], a chemical shift of -ΝΜΕ spectrum (CDCl ₃ , 270 MHz) d (ppm): 0.93 (6H, d, J = 6.6Hz) ), 117-17.57 (2H, m), 1.75-1.85 (1H, m), 2.46 (1H, dd, J = 16.7, 8.8Hz), 2.56 (1H, dd, J = 16.7, 3.6Hz), 4.07 to 4.17 (1H, m) and EI-MS m / zl59 (M ⁺ -1), 142 (M ⁺ -18), 129 (M ⁺ -31), 111 (M +-31-18) of methyl ester form ), 103 (C ₃ -C ₄ cleavage, standard fragment ion peak), it was determined to be 3-hydroxy-5-methylhexanoic acid.

5 mg of WAP-2607B3 was subjected to acid hydrolysis in the same manner as described above, and after ether extraction, the ether layer was concentrated and dried to obtain 0.4 mg of a white powder. This material has a molecular weight: 160 CFAB-MSm / z 159 (M- !!广], ¹ !! - NMR scan Bae spectrum (CDC1 _3, 270MHz) chemical shift d (ppm): 0.89 (6H , d, J = 1.62 (5H, m), 2.47 (1H, dd, J = 16.2, 8.8Hz), 2.58 (1H, dd, J = 16.2, 3.4Hz), 3.99 to 4.06 (1H, m) EI-MS m / z 173 (M ⁺ -1), 156 (M ⁺ -18), 143 (M ⁺ -31), 125 (M ⁺ -31-18), 103 (C ₃ -C ₄ Cleavage, standard fragment ion peak) was determined to be 3-hydroxy-6-methylheptanoic acid.

10 mg of WAP-2607B4 was subjected to acid hydrolysis in the same manner as described above, and after ether extraction, the ether layer was concentrated and dried to obtain 0.9 m of a white powder. This material has a molecular weight: 174CFAB- MSm / z 173 (M- H) one], the chemical shift 5 (ppm) of ¹ H-NMR scan Bae spectrum _{(CDC1 3, 270MHz): 0.87} (6H, d, J = 6.6Hz ), 1-16 to 1.58 (7Η, m), 2.46 (1Η, dd, J 26.6, 8.8Hz), 2.58 (1H, dd, J = 16.6, 3.4Hz), 4.03 to 4.05 (1H, m) and EI-MS m / z 187 methyl ester (M + - 1), 170 (M + - 18), 157 (M + - 31), 139 (M + - 31- 18), 103 (C 3 -C 4 cleavage Determined from the standard fragment ion peak) to be 3-hydroxy-7-methyloctanoic acid c

10m of WAP-2607B5 was acid-hydrolyzed in the same manner as described above, and after ether extraction, the ether layer was concentrated and dried to obtain 0.8 mg of a white powder. The substance Molecular weight: 174 [FAB-MSm / z 173 (M- H) - ], the chemical shift d (ppm) of the E- NMR scan Bae spectrum _{(CDC1 3, 270MHz): 0.87} (6H, d, J = 6.6 Hz), 1-16 to 1.58 (7H, m), 2.46 (1H, dd, J = 16.6, 8.8Hz), 2.58 (1H,

Z5 dd, J = 16.6, 3.4Hz), 4.03 to 405 (1H, m) and EI-MS m / zl87 (M + -l), 170 (M + -18), 157 (M ⁺ -31) ), 139 (M + -31 - 18), 103 (C 3 - C 4 cleavage, reference fragment ion peak) from 3-hydroxy - was determined to be 7-Mechiruoku evening phosphate. Example 11 Formulation of WAP-2607B

A representative example of preparing a pharmaceutical composition is shown below.

Injection: In accordance with the rules of the 12th revised edition of the Japanese Pharmacopoeia, General Rules for Preparations for Injections, dissolve 40m of WAP-2607B hydrochloride in 3ml of sterile physiological saline (JP), and aseptic in a 3ml injection ampoule After filling, it was sealed to produce an injection.

Tablets: In compliance with the provisions of “Tablets”, lactose (JP) 65mg, starch (JP) 14.2mg, excipient using WAP-2607B hydrochloride lOOmg, polyvinylpyrrolidone K25 (JP) 20 mg, 0.8 mg of magnesium stearate (JP) as a lubricant were added, mixed uniformly, and compression-molded with a tableting machine to produce 200 mg plain tablets per tablet.

Ointment: WAP-2607B hydrochloride 40m in 1m of polyethylene glycol 4000 (JP), 600mg of polyethylene glycol 400 (JP) 156mg of hydrophilic petrolatum (JP) ) And 0.8 mg of propyl parabenzoate (JP) and 3.2 mg of ethyl ethyl paraoxybenzoate (JP) were melted by heating and uniformly kneaded to produce 4 g of an ointment per tube.

Eye drops: In accordance with the same general rules as “Eye drops”, dissolve 25 mg of WAP-2607B hydrochloride in 5 ml of 0.9 sterile saline and add 0.5 mg of benzalkonium chloride (JP) as a preservative to obtain a uniform solution. After a sterile filtration week, the mixture was aseptically filled in a 5 ml ophthalmic container to produce eye drops. Industrial applicability

As described above, the novel antibiotic WAP-2607B of the present invention has excellent antibacterial activity against gram-positive cocci, especially methicillin-resistant Staphylococcus aureus, which is currently becoming increasingly confused in medical practice. Therefore, it is useful as a therapeutic agent for diseases in which gram-positive bacteria combined with MRSA infection cause causative bacteria. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a drawing showing a chromatogram obtained by separating the antibiotic WAP-2607B by high performance liquid chromatography.

Figure 2 is a drawing showing the ultraviolet absorption spectrum (in MeOH) of the antibiotic WAP-2607B2 (hydrochloride).

Figure 3 is a drawing showing the infrared absorption spectrum (FT-IR, KBr method) of the antibiotic WAP-2607B2 (hydrochloride).

Figure 4 is a view showing an antibiotic WAP- 2607B2 ¹ HN R scan Bae spectrum of _{(hydrochloride) (270MHz, DMS0-d 6} ).

FIG. 5 is a drawing showing a two-dimensional TLC chromatogram of the complete acid hydrolysis product of the antibiotic WAP-2607B.

Claims

The scope of the claims

1. An antibiotic WAP-2607B having the following physicochemical properties or a pharmaceutically acceptable salt thereof.

(1) Appearance: White powder

(2) Melting point: 215-225 ° C (decomposition)

(4) Color reaction: Positive for ninhydrin, Sakaguchi, Ehrlich, Leidensmith, aqueous potassium permanganate, sulfuric acid, and Rhodoboper reaction.

Maurish, silver nitrate, ferric chloride, negative for Dragendorff reaction.

(5) Retention time in high performance liquid chromatography: 5.8 minutes, 8.0 minutes, 11.7 minutes, 12.8 minutes, 19.5 minutes

(6) UV absorption spectrum (in MeOH): ληιαχ 281 mn, 290 nm

(7) Infrared absorption spectrum (FT-m, r method): characteristic absorption 3342 cm " ¹ , 2964 cm" ¹ , 1655 cm— ¹ , 1539 cm— ¹

(8) Molecular weight: 1,600-1,700

(9) Ninhydrin-positive substance by acid complete hydrolysis: Gly, Leu, Val, lie, Trp, Glu, Arg, Ser, Thr, and N-methylphenylalanine are provided.

(10) Specific rotation: [h] _D ² ° = + 30 ° (c = 0.5, MeOH)

(11) Basic, acidic and neutral: amphoteric

2. Antibiotic WAP-2607B1 having the following embedded chemical properties, or its pharmaceutically acceptable ¾mo

(1) Appearance: White powder (2) Melting point: 215-225 ° C (decomposition)

(4) Color reaction: Positive for ninhydrin, Sakaguchi, Ellrich, Rydsmith, aqueous potassium permanganate, sulfuric acid, and cold vapor, and shows a quenching spot by irradiation with UV lamp 254 歷.

Maurish, silver nitrate, ferric chloride, negative for Dragendorf reaction.

(5) Retention time in high performance liquid chromatography: 5.8 minutes

(6) Ultraviolet absorption spectrum (in MeOH): max. 282 dishes per person, 290 nm

(7) Infrared absorption spectrum (FT-IR, KBr method): characteristic absorption 3328 cm- ¹ , 2960 cm ¹ , 1655 cm " ¹ , 1541 cm— ¹

(8) Molecular weight: 1602.9 [FAB-MS m / z 1603.9 (M + H) ⁺ ]

(9) Molecular formula: C ₇₄ H _U4 N ₂₀ 0 ₂₀

(10) Complete acid hydrolyzate: Gly (1 mol), Leu (1 mol), Val (1 mol), Trp (1 mol), Glu (2 mol), Arg (2 mol), Ser (1 mol) , Thr (2 mol), N-methylpheniralanine (1 mol) and 3-hydroxy-5-methylhexanoic acid (1 mol).

(11) Specific rotation: [ _H ] _B ²⁰ = + 32 ° (c = 0.5, MeOH)

(12) Basic, acidic and neutral: amphoteric

3. Antibiotic WAP-2607B2 having the following in-built chemical properties, or a pharmaceutically acceptable salt thereof.

(1) Appearance: White powder

(2) Melting point: 220-225 ° C (decomposition)

(3) Solubility: Soluble in water, methanol, n-butanol, dimethylformamide, dimethyl sulfoxide, insoluble in acetone, ethyl acetate, and chloroform. (4) Color reaction: Positive for ninhydrin, Sakaguchi, Ehrlich, Leydon Smith, aqueous potassium permanganate, sulfuric acid, and greenhouse reaction, showing a quenching spot by irradiation with a UV lamp at 254 nm.

Maurish, silver nitrate, ferric chloride, negative for Dragendorf reaction.

(5) Retention time in high performance liquid chromatography: 8.0 minutes

(6) UV absorption spectrum (in MeOH): max 281 people employed, 290 meetings

(7) Infrared absorption spectrum (FT-IR ヽ KBr method): characteristic absorption 3320 cm " ¹ , 2964 cm— ¹ , 1655 cm" ¹ , 1539 cm " ¹

(8) Ή nuclear magnetic resonance (NMR) scan Bae _{spectrum: (270MHz, DMS0-d 6} ), methyl; methylene emissions, complicated proton signals originated from methine and heterocycles or aromatic rings Ru observed.

(9) Molecular weight: 1616.9 [FAB-MS m / z 1617. 9 (read) ⁺ ]

(10) Molecular formula: C ₇₅ H ₁₁₆ N ₂₀ 0 ₂₀

(12) Specific rotation: [HI] _D ² . = + 31 ° (c = 0.5, MeOH)

(13) Basic, acidic, and neutral: amphoteric

4. An antibiotic WAP-2607B3 having the following in-built chemical properties, or a pharmaceutically acceptable salt thereof.

(1) Appearance: White powder

(2) Melting point: 220-230. C (decomposition)

(3) Solubility: Soluble in water, methanol, n-butanol, dimethylformamide, dimethyl sulfoxide, insoluble in acetone, ethyl acetate, and chloroform. (4) Color reaction: Positive for ninhydrin, Sakaguchi, Ehrlich, Raidsmith, aqueous potassium permanganate, sulfuric acid, and redox reaction, showing quenching spots by irradiation with a UV lamp at 254 nm.

Negative for Moritzsch, silver nitrate, ferric chloride, and Dragendorf reactions.

(5) Retention time in high performance liquid chromatography: 11.7 minutes

(6) Ultraviolet absorption spectrum (in MeOH): max. 281 humans, 290 nm

(7) Infrared absorption spectrum (FT-IR, KBr method): characteristic absorption 3412 cm- ¹ , 2932 cm- ¹ , 1663 cm " ¹ , 1541 cm" ¹

(8) Molecular weight: 1630.8 [FAB-MS m / z 1631.8 (M + H) ⁺ ]

(9) Molecular formula: C ₇₆ H ₁₁₈ N ₂₀ 0 ₂₀

(10) Complete acid hydrolyzate: Gly (1 mol), Leu (1 mol), He (1 mol), Trp (1 mol), Glu (2 mol), Arg (2 mol), Ser (1 mol) And Thr (2 mol) N-methylphenylalanine (1 mol) and 3-hydroxy-6-methylheptanoic acid (1 mol).

(11) Specific rotation: [ ^H ] _D ^2fl = + 5.8 ° (c = 0.5, MeOH)

(12) Basic, acidic and neutral: amphoteric

5. An antibiotic WAP-2607B4 having the following in-built chemical properties, or a pharmaceutically acceptable salt thereof.

(1) Appearance: White powder

(2) Melting point: 220-225. C (decomposition)

(4) Color reaction: Positive for ninhydrin, Sakaguchi, Ehrlich, Rydsmith, aqueous potassium permanganate, sulfuric acid, and odobepa reaction, showing quenching spots by irradiation with a UV lamp at 254 nm. Maurish, silver nitrate, ferric chloride, negative for Dragendorf reaction.

(5) Retention time in high performance liquid chromatography: 12.8 minutes

(6) Ultraviolet absorption spectrum (in MeOH): max. 28 dishes per person, 290 nm

(7) Infrared absorption spectrum (FT-IR, KBr method): characteristic absorption 3340 cm ¹ , 2956 cm " ¹ , 1657 cm" ¹ , 1545 cm— ¹

(8) Molecular weight: 1630.9 [FAB-MS m / z 1631.9 (M + H) +]

(9) Molecular formula: C ₇₆ H ₁₁₈ N ₂₀ 0 ₂₀

(11) Specific rotation: [H] _D ² ° = + 29 ° (c = 0.5, MeOH)

(12) Basic, acidic and neutral: amphoteric

6. An antibiotic WAP-2607B5 having the following in-built chemical properties, or a pharmaceutically acceptable salt thereof.

(1) Appearance: White powder

(2) Melting point: 220-230. C (decomposition)

(4) Color reaction: Positive for ninhydrin, Sakaguchi, Ehrlich, Rydsmith, aqueous potassium permanganate, sulfuric acid, and oxidative vapor reaction.

Maurish, silver nitrate, ferric chloride, negative for Dragendorf reaction.

(5) Retention time in high performance liquid chromatography: 19.5 minutes

(6) UV absorption spectrum (in MeOH): Am x 281 dish, 290 nm (7) Infrared absorption spectrum (FT-IR, KBr method): characteristic absorption 3288 cm— ¹ , 2955 cm ” ¹ , 1655 cm” ¹ , 1541 cm ” ¹

(8) Molecular weight: 1644.8 [FAB-MS m / z 1645.8 (M + H) ⁺ ]

(9) Molecular formula: C ₇₇ H ₁₂₀ N ₂₀ 0 ₂₀

(10) Complete acid hydrolyzate: Gly (1 mol), Leu (1 mol), He (1 mol), Trp (1 mol), Glu (2 mol), Arg (2 mol), Ser (1 mol) , Thr (2 mol) N-methylphenylalanine (1 mol) and 3-hydroxy-7-methyloctanoic acid (1 mol).

(11) Specific rotation: [HI] _D ² . = +30. (C = 0.5, MeOH)

(12) Basic, acidic and neutral: amphoteric

7. A microorganism belonging to the genus Flavobacterium sp. And capable of producing the antibiotic WAP-2607B according to any one of claims 1 to 6 is cultured in a medium, and the microorganism is cultured during the culture. The method for producing the antibiotic WAP-2607B according to any one of claims 1 to 6, wherein the antibiotic is produced and accumulated, and then collected.

8. WAP-2607B-producing bacterium belonging to the genus Flavopacterium.

9. The production bacterium according to claim 8, which is Flavobacterium sp. WAP-2607.

10.The antibiotic WAP-2607B according to any one of claims 1 to 6, and

An antibacterial agent containing at least one pharmaceutically acceptable salt thereof.