WO2004081036A1 - Peptidic antibiotic - Google Patents

Abstract

It is intended to provide a compound which has an excellent efficacy against methicillin-resistant Staphylococcus aureus (MRSA) and penicillin-resistant Streptococcus pneumoniae (PRSP) and is useful as the active ingredient of drugs. A compound having an excellent efficacy against MRSA and PRSP which is produced by a microorganism belonging to the genus Kineosporia

Description

 Peptide antibiotics

 [Technical field to which the invention belongs]

 The present invention relates to novel antibiotics and their production and use.

[Prior art]

 Many antibiotics produced by microorganisms have been discovered so far, and they have already been put into practical use in the fields of pharmaceuticals, veterinary drugs, and agricultural chemicals.

 In the use of antibiotics, the development of resistant bacteria has become a problem. In particular, Staphylococcus aureus (S taphylococcus aureus) is known as a causative agent of purulent diseases, but its multidrug-resistant bacterium (Methicillinresistant staphylococcusaureus: hereinafter sometimes referred to as MRSA) is clinically large. This is a problem (Oguri et al., Clinical and Microbiological Sciences, Vol. 15, Vol. 7-15, 1988). Vancomycin, ticobranine, and bebekacin are known as anti-MRSA antibiotics, but even for these drugs, noncomycin low-sensitive bacteria (Vanco mycin intermediatestaphy lococcusaureus: sometimes abbreviated as VISA) ) And other resistant bacteria have become a problem (Hanagi, Nihon clinical, Vol. 55, No. 5, pp. 269-274, 1997). Streptococcus pneumoniae (S treptocoecus pneum oiae) is known to be the causative agent of pneumonia, otitis media, and purulent meningitis, but is resistant to the penicillin and cefem antibiotics conventionally used. In recent years, the number of penicillin-resistant pneumococci (Penicillinresistant streptococcus pneumon iae: hereafter sometimes abbreviated as PRSP) has been increasing and has become a major clinical problem (Iwata, Chemotherapy, Volume 16, Volume 5) , Pp. 29-37, 2000).

 <Title>

Non-Patent Document 1: Oguri et al., Clinical and Microorganisms, Vol. 15, pp. 7-15, 1988 Non-Patent Document 2: Eur. J. Biochem. 246, 193-199 (1997) [Disclosure of the Invention]

 There is a strong demand for new antibiotics that can solve problems such as the expression of resistant bacteria. The present inventors have extensively searched for microbial metabolites to find new antibiotics having antibacterial activity. As a result, from the culture of the actinomycetes AP17444 belonging to the genus Kineosporia isolated from the deciduous leaves of Oigawa Town, Shizuoka Prefecture, it was able to grow against various pathogenic microorganisms including MRSA and PRSP. The present inventors have found that a novel antibiotic exhibiting inhibitory activity (this compound is sometimes abbreviated as AP17444 compound) was produced, and the present invention was completed.

 That is, the present invention is as follows.

 (1) A peptide having the following physicochemical properties.

 [1] Amino acid analysis detects Asp, Thr, Gly, Val, He, Leu, Phe, Pro, Lantnionine, and Methyllanthionine.

 [2] Ultraviolet absorption spectrum: The main absorption peaks (nm) in methanol are as follows.

 End absorption, 2 6 7-2 7 3

C 3] Infrared absorption spectrum: KBr The main wave numbers (cm- ¹ ) in tablets are as follows.

 3 2 7 0, 3 0 5 4, 2 9 6 6, 1 6 6 0, 1 5 2 7, 1 4 2 7, 1 2 8 6, 1 24 7, 1 2 0 1, 1 1 3 3

 [4] R f value: about 0.5

 The conditions for thin layer chromatography are as follows.

 Fixed layer: Silica gel TLC

 Developing solvent: Mouth form-methanol (6: 4)

 (2) The peptide according to (1), which has the following physicochemical properties:

 [5] The N-terminus of the amino acid sequence is lie.

 [6] Color test: Positive for reaction with iodine vapor and decolorization reaction of permanganate power.

[7] Enzyme stability: S ubutilisin, T hermoly When each enzyme was digested with sin, proline-specific endopeptidase, or Chymotrypsin, the degradation did not progress.

 (3) The peptide according to claim 1 or 2, which has the following physicochemical properties.

 [8] Molecular weight: 2 1 0 7

[9] Composition: C ₈₉ H ₁₂₉ N ₂₅ O ₂₅ S ₅

 (4) A method comprising culturing a microorganism belonging to the genus Kineosporia having the ability to produce the compound according to any one of (1) to (3) above, and collecting the compound from the culture. And a method for producing the compound or a salt thereof.

 (5) A medicament comprising the compound according to any one of (1) to (3) above as an active ingredient.

 (6) A medicament for preventing and / or treating infectious diseases, comprising as an active ingredient the compound described in any of the above (1) to (3).

[Brief description of drawings]

FIG. 1 shows the infrared absorption spectrum of the API 7444 compound in the KBr tablet. Figure 2 were measured in DM SO- d ₆ of AP I 7444 Compound ¹ H- NM R (4

00 MHz) Indicates the spectrum.

Figure 3 shows a ^{13 C- NMR (1 00 MH z} ) spectrum was measured in DMS O-d ₆ of AP 1 7444 compound.

[Best Mode for Carrying Out the Invention]

 BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be specifically described below, but is not particularly limited thereto.

The compound of the present invention is represented by a combination of any of the above-mentioned physicochemical properties [1] to [9], and further a combination to which other properties described in the present specification are added. Or a compound represented by any combination of two or more of these. For example, as described above, [1]-[4] A compound having properties, or a compound having the physicochemical properties of [8] or [9] in addition to the physicochemical properties of [1]-[4], Preferred examples include compounds having the physicochemical properties of [1] and [8] and [1] and [9].

The confirmation items in the research process of the compound of the present invention will be further described. As a result of infrared absorption spectrum measurement of the compound of the present invention, strong absorption was observed at 1660 and 1527 cm- ¹ It was suggested to be a compound of structure. After complete hydrolysis using 6 N hydrochloric acid (24 hours, 110 ° C), quantitative analysis of amino acids by ion exchange chromatography (JEOL JLC

-500 / V type device), at least aspartic acid (which may have been changed from asparagine by hydrolysis), threonine, glycine, valinine, isocyanate, leucine, fenylalanine and proline. It was detected and found to be a compound with a peptide structure. In addition, it was determined that at least 4 molecules of glycine and 2 molecules of proline. Furthermore, D and L analyzes of these amino acids were performed by the Marfey method (P. Marfey, Carlsberg Res. Commun. 49, 591-596, u984), K. rujn et al, Anal. Chem. 69, 5146-5151 (1997) ). The complete hydrolyzate was used as a derivatization reagent for 1-fluoro-2, 4-dinitrophenyl-5-alanine amide (FDAA) .After derivatizing each amino acid with FDAA, LC / MS analysis showed that It was supposed to be a body. In the above amino acid analysis, peaks of two kinds of amino acids other than the above amino acids were detected. As a result of LC-MS and LC-NMR measurements, these amino acids showed La 11 thionine and Methyl 1 ant li ionine (Hans-George SAHL et al., European Journa 1 of Biochemistry ^ 230 vol. , Pp. 827-853, 1995). Furthermore, amino acid sequence measurement by the Edman degradation reaction (Shimazu Seisakusho's fully automatic protein primary structure analyzer PPSQ-23A type) was performed, but only I 1 e was detected in the first place, and after the second, the amino acid sequence was measured. No peak was detected. It is presumed that the second amino acid is not dehydrogenated by dehydroa 1 anine or dehydrobutyrine. Subutilisin (treated in an lmM Tris (pH 8) buffer at 30 ° C for 4 days), T her mo lysin (treated in an ammonium bicarbonate buffer at 37 ° C for 3 days), Proline-specific Enzymes with donpeptidase (treated in 0.1M phosphate buffer at 30 ° C for 1 day) or Chymotrypsin (treated in lOOmMTris-HC1 at 25 ° C for 2 days) Decomposition showed that decomposition hardly proceeded. The amounts of the compound of the present invention and each of the above enzymes were approximately 20: 1 by weight. This result indicated that the compound of the present invention was very stable to the enzyme. Therefore, a reaction to cleave the Lanthionine structure and a reaction to convert Dehydroalanine or Dehydrobutyrine to a thioatenole derivative (reaction in ethanol / aqueous solution, 5 M sodium hydroxide aqueous solution, ethanethiol at 50 ° C for 1 hour ( H elmut E. Meyer et al., Analytical 1 Biochemistry, Vol. 23, Vol. 23, pp. 185-1900, 1994))) and amino acid sequence measurements up to the second As a result, the following sequence was obtained. _{IX 1 X 2 VX 3 LX,} X 5 in _{P GX 6 TX 7 P GGGX S} NX 9 X 10 F ( wherein and X ₅ represents an NH (CH) C0-CH ( CH 3) SCH 2 CH 2 0H , X _2, X had _{X 6, X 7, X 3} , X 9, and X ₁₀ are each independently, NH (CH) C0-CH 2 SCH 2 CH 2 0H, or NH (CH) C0_CH ₂ SH indicates one of, X ₃ in NH (CH) C0_CH shows a ₂ SCH ₂ CH ₂ 0H. above SL, alpha Beck preparative other than X "X _1Q is one letter code Amino acid.) the present invention the molecular weight of the compound, the 2 1 0 is determined to be 7, high resolution Masusu Bae-vector (ABI Co. QSTAR Pulsari) was estimated composition formula _{C 89 H 129 N 25 0 25} S 5 from. present compound Analysis of the NMR spectrum (Varian's Unity IN0VA600) suggested the presence of a single NH—CH = CH— structure at the C-terminal site, which was presumed to be bound to Lanthionine.

 Therefore, based on amino acid analysis, amino acid sequence and molecular weight, the number of constituent amino acids is considered to be 22 or 23 (lanthionine and methyllanthionine structures are counted as 2 amino acids), and the results of NMR analysis are considered. It was presumed that there was a high possibility that the number would be two or three.

In addition, the ultraviolet absorption spectrum of the compound of the present invention was measured with a methanol solution, and as a result, niaxnm (E 1 cm ^1% ) was found to have a terminal absorption of 267-127 (45). As a result of measurement with an alkaline methanol solution, the maxnm (Elcm ^1% ) was found to be terminal absorption, 251-25 (195). There was no compound having each of the above physicochemical properties, and the compound of the present invention was considered to be a novel compound.

 According to the present invention, there is also provided a compound of the present invention (including a salt thereof), wherein an actinomycete belonging to the genus Kinesporia having the ability to produce the compound of the present invention is cultured, and the compound of the present invention is collected from the culture. Is provided.

 Microorganisms belonging to the genus Kineosporia capable of producing the compound of the present invention are not particularly limited as long as they have the ability to produce the compound of the present invention. For example, the following Kineosporiasp. AP The I 7444 strain is exemplified.

 The mycological properties of the above-mentioned A P 17444 strain are as follows.

 1. Morphological properties

 The cells were cultured on a yeast extract / malt extract agar medium at 28 ° C for 20 days, and observed with an optical microscope and a stereoscopic microscope. The fungus forms a filament and develops a well-branched basal hypha. Usually, no hyphal rupture is observed. The diameter of the mature basal hypha is about 0.7-1.2 X m. A septum is formed at the tip of a portion of the basal hypha and forms a spore or spore-like morphology. The spores are spherical, subspherical, oval, and ovoid, and are about 0.8-1.6 At mX 0.8-1.6 μπι. Spores are motile. The spore surface is observed to be smooth under a light microscope (XI500). Aerial hyphae cannot be confirmed.

 2. Cultural properties

 The cells were cultured in various media at 28 ° C for 20 days and observed. The color description is in accordance with the Color Harmony Manual, 4th ed. 1958, from the Container Corporation of America.

 (1) East extract 'malt extract agar medium'

 Very good growth. The color of the colony is Light Apricot (4ea) to Apricot (4ga). The condition of the colony surface is wet. No aerial mycelium forms. No pigment production is observed.

 (2) Oatmeal agar medium

Good growth. The color of the colony is Pearl Pink (4ca) to Shell Pink (5ba). Colony table Surface condition is wet, but not as high as yeast extract / malt extract agar medium. No aerial mycelium forms. No pigment production is observed.

 (3) StarchInorganic salt agar medium

 Weak growth. The color of the colony is from Shell Tint (2ba;) to Shell Tint (3ba). The colony surface is not wet. No aerial mycelium forms. No pigment production is observed.

 (4) Glycerol.asparagine agar medium

 The growth is weak, and it grows like a thin trace of the trace applied with Ese. The color of the colony is white (. The colony surface is not wet. No aerial mycelium forms. No pigment production is observed.

3. Physiological properties

 (1) Temperature (cultured on yeast extract / malt extract agar medium for 14 days)

 5 ° C No growth

 7 1 1 ° C Slight growth confirmation

 1 7 ° C growth

 20, 23, 26, 29 ° C vigorous growth

 Weak growth

 3 7 ° C Slight growth confirmation

 3 9. C, 42 ° C No growth

 (2) Melanin pigment productivity

 Peptone 'East · Iron agar: No pigment production (weak growth)

Tyrosine agar medium: no pigment production (weak biological)

 (3) Utilization of carbon source (Pridham-Gottlieb agar medium as basal medium)

Use L-arabinose, D-xylose, D-gunoresose, D-fructose, inositol and L-rhamnose. Raffinose is not used. Based on the above morphological, cultivation and physiological characteristics, the strain was identified as an actinomycete belonging to the genus Kineosporia, and Kineosporia sp. AP 1 744 was identified. Named 4.

In addition, this strain was transferred to Tsukuba East 1-chome, Ibaraki Prefecture, Japan on February 13, 2003. Land 1 Deposited at the National Institute of Advanced Industrial Science and Technology (AIST), the 6th independent administrative institute, Patent Organism Depositary (FERM P—192 2 12; FERM B P — 085 75).

 A typical and preferred method for producing the compound of the present invention according to the present invention is a method for culturing the compound-producing bacterium belonging to the genus Kineosporia (for example, Kineosporiasp. API 7444) in a suitable medium, and isolating from the culture. Illustrated. The medium used for producing the substance of the present invention is most preferably, but not limited to, shaking culture or aeration-agitation culture in a liquid medium. The medium is not particularly limited as long as the compound-producing bacterium of the present invention grows and accumulates the compound in the medium. Examples of the carbon source include glucose, sucrose, dextrin, starch, glycerin, molasses, and the like. Organic acids and the like can be used. As the nitrogen source, for example, yeast extract, peptone, meat extract, soy flour, gluten meal, wheat germ, corn steep liquor, amino acids, ammonium salt, nitrate, and various other organic or inorganic nitrogen compounds are used. . As inorganic salts, sodium chloride, potassium chloride, calcium carbonate, various phosphates, magnesium salts, copper salts, cobalt salts, and the like may be added. Vitamins, coenzymes, and the like may be added to promote the growth of bacteria and the production of the compound of the present invention. In particular, if the medium foams strongly, liquid paraffin, animal oil, mineral oil, silicon, etc. may be added when necessary.

 Conditions such as culturing temperature, culturing time, stirring speed, aeration rate, and pH of the culture solution in culturing the bacterium of the present invention are appropriately selected and adjusted so that the accumulated amount of the present compound is maximized. For example, in the case of ordinary aeration and stirring culture, the culture temperature is usually 20 to 35 ° C, preferably 25 to 30 ° C. The cultivation time is usually 2 to 10 days, preferably 3 to 8 days. The pH of the culture solution is generally adjusted to pH 5.0 to 9.0, preferably 6.0 to 8.0.

 The time-dependent change in the amount of the compound of the present invention accumulated in the culture solution over the course of culture should be measured by liquid chromatography or thin-layer chromatography which can be confirmed during the separation and purification described below. Can be.

For separation and purification of the compound of the present invention from the culture solution, various methods based on its physicochemical properties can be used. In that case, for example, liquid chromatography under the following conditions It can be traced by confirmation by chromatography or thin layer chromatography.

 One condition of liquid chromatography

 Separation column: Showa Denko Shode X C I 84.6 X 150 mm Solvent: acetonitrile—0.05% TFA aqueous solution (3: 7)

 Flow velocity: 1.0 ml z min

 Detection: U V 220 nm

 Retention time: 13.3 minutes

 One condition of thin layer mouth chromatography

 Fixed layer: Tokyo Kasei Silikki Gel fS2 0 1

 Developing solvent: chloroform-methanol (6: 4)

 Detection: U V 2 54 n m

 R f value: about 0.5

 As a method for separation and purification, for example, the compound of the present invention present in a culture solution can be extracted and purified using an organic solvent immiscible with water, for example, butanol. Alternatively, for example, Diaion HP-20 (manufactured by Mitsubishi Kasei) or the like is used as an adsorbent. Either the fraction containing the compound of the present invention is passed through the above-mentioned layer of the adsorbent to adsorb and remove impurities, or after adsorbing the compound of the present invention, elution is carried out using methanol water, acetone water or the like. The compound can be obtained. Furthermore, adsorption column chromatography using a carrier such as silica gel, alumina, and florisil, Sephadex LH-20 (manufactured by Pharmacia), and distribution column chromatography using Toyopearl HW-40 (manufactured by Tosoh Corporation). The compound of the present invention can be separated and purified by chromatography, high-performance liquid chromatography using a normal phase, reverse phase column, or the like.

 The compound of the present invention may be free or formed as any salt, and these forms are included in the compound of the present invention. Of course, when used as a medicament, a pharmaceutically acceptable salt is preferable.

The antibacterial activity of the compounds of the present invention was measured against various pathogenic microorganisms, and their minimum inhibitory concentrations (MIC) were determined. The results are shown in Table 1. As shown in Table 1, The compound of the present invention is useful for various pathogenic microorganisms including MRSA (KU16 strain), vancomycin-insensitive MRSA (MU550 strain), penicillin-resistant pneumococcus (ASP965 strain), and pathogenic anaerobic bacteria. Shows antibacterial activity against

 In addition, an MRS A infection treatment test was performed with the compound of the present invention. The results are shown in Table 2. As shown in Table 2, the compound of the present invention shows an excellent therapeutic effect as compared with vancomycin, which has been conventionally regarded as most effective in the treatment of MRSA infection.

 A therapeutic test was performed in a leukopenia mouse thigh infection model using the S. aureus MU50 strain, which is a vancomycin-insensitive MRSA, as an infectious bacterium. The AP17444 compound shows a superior therapeutic effect on vancomycin-resistant bacteria as compared to vancomycin.

 In addition, a treatment test for the treatment of PRSP infection was performed with the compound of the present invention. The results are shown in Table 3. As shown in Table 3, the compound of the present invention shows a therapeutic effect superior to vancomycin, which has been effective in the treatment test for PRPS infection.

 Therefore, the compound of the present invention is useful as an agent for treating or preventing infectious diseases caused by these pathogenic microorganisms.

 From the above results, the compound of the present invention is useful as a therapeutic and / or prophylactic agent for the above-mentioned infectious diseases caused by pathogenic microorganisms. The microorganism used for the assay is not limited to the microorganism used in the present application, and standard strains of various depository institutions ゃ clinical isolates in each facility can be used, and the effect of the antimicrobial activity of the present application can be easily obtained. Will be confirmed.

 When the compound of the present invention of the present invention is used as a medicine, it can be composed of a pharmaceutically acceptable carrier in addition to the compound of the present invention. Known carriers can be used, and depending on their properties, they can be classified as excipients, binders, disintegrants, lubricants, flavoring agents, dissolution aids, suspending agents, coating agents, etc. There are also, for example, lactose, corn starch, magnesium stearate and the like. A typical example of the drug is a 5% glucose aqueous solution of the compound of the present invention.

The medicament of the present invention is, for example, an antibiotic, and more specifically, an anti-MRSA agent and an anti-PRSP agent. Although the medicament of the present invention can be administered in various forms, for example, oral administration or injection by tablets, capsules, granules, syrups and the like (Intravenous, intramuscular, subcutaneous), parenteral administration by eye drops, suppositories, ointments, sprays, lotions and the like.

 These medicaments vary depending on symptoms, age, body weight, administration method, dosage form, etc., but the dose is usually preferably 0.1 mg or more, more preferably 1 mg or more, per day for an adult, or 1 0 mg or more is preferred. The upper limit is preferably 3000 mg or less, more preferably 300 mg or less, and particularly preferably lOOmg or less. These may be administered once or in several divided doses.

 Next, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

 [Example 1]

 (1) Fermentative production of AP 1 7444 compounds

Medium with glucose 1 o / ₀ , dextrin 1 o / ₀ , yeast extract 0.5%, casein hydrolyzate 0.5%, CaCO ₃ 0.1% (pH 7. 0) was dispensed 100 ml each into four 500 ml Erlenmeyer flasks, and 1 15. Sterilized with C for 15 minutes. A platinum loop of Kineosporiasp.AP 1 7444 (FE RM BP—08 5 7 5) was inoculated onto each of these, and the seeds were cultured at 29 ° C for 72 hours on a rotary shaker (200 rpm) for 72 hours. A culture solution was used. 2% glucose, starch 2%, defatted soybean meal 2%, Ys Toekisu 0. 5%, N a C 1 0. 2 5%, C a CO 30. 3 5%, Z n SO 4 · 7 tetrahydrate 0. 0 0 5%, M n C 1 2 · 7 hydrate 0. 000 5%, C u SO 4 * 5 tetrahydrate 0.000 5% chemically defined medium (before sterilization p H 7. 0) to 500 m 1 volume Dispense 100 ml each into one Erlenmeyer flask, inoculate 4 ml of the above seed culture solution into 100 medium sterilized at 115 ° C for 15 minutes, and incubate at 29 ° C. The culture was carried out on a mouthpiece for one hour (200 revolutions per minute).

 (2) Purification of AP1 7444 compound

 10 L of broth obtained by the above culture method was separated into bacterial cells (800 g) and filtrate (8 L) by a centrifuge (3,000 rpm, 10 minutes).

The obtained filtrate was applied to a styrene-divinylbenzene-based polymer resin HP-20 (manufactured by Mitsubishi Diaion) column (800 ml) which had been washed in advance with water to adsorb the AP174444 substance. Rinse the column with 4 L of water and use 4 1 of 30% acetone water After washing with water, elution was carried out with 50% acetone water, and fractionation was carried out in increments of 400 ml. As a result, the AP17444 compound was eluted in fractions Nos. 2 to 5. The brush (No. 2 to 5) containing the above-mentioned AP 17444 was collected and concentrated under reduced pressure. The obtained concentrated and dried product was mixed with 250 ml of a mixed solvent (chloroform: methanol = 3: 2). , And centrifuged (30000 rpm, 10 minutes) to obtain a supernatant. The obtained supernatant is concentrated under reduced pressure, dissolved in a small amount of a mixed solvent (chloroform: methanol = 7: 3), and silica gel previously prepared with chromate-form-methanol (7: 3) The column was subjected to column chromatography (300 ml), eluted with the above solvent, and fractionated by 20 ml. The AP17444 compound was eluted in fraction No. 42-95.

The fractions (No. 42-95) containing the above-mentioned AP 17444 were collected and concentrated under reduced pressure to obtain 310 mg of a coarse powder. Next, this coarse powder was dissolved in 6 ml of dimethyl sulfoxide, and a 1/2 part thereof was subjected to reverse phase fractionation prepared in advance with acetonitrile_0.05% TFA (30:70). The mixture was applied to a column (Showa Denko, Shodex C 18 M 20 ιηπιΦ X 250 mm), developed with the same mixed solvent, and fractionated by 5 ml. A fraction of No. 46 to 54 was obtained as a component of AP1444. The same procedure was repeated once or twice, and the fractions containing the AP17444 compound were collected, concentrated in vacuo, and lyophilized to give a white powder of a single AP17444 compound (164 mg). Obtained. When the various properties of the obtained AP17444 compound were tested, the following results were shown.

A P 17 44 Properties of compound

 1) Overview: white powder

2) Specific rotation: [a] _D ²² = +4 0.2 ± 5 (c = 1.0, DMS O)

3) Molecular weight: 2 1 0 7

 4) Ultraviolet absorption spectrum: The main absorption peaks (nm) in methanol are as follows.

λ max nm (E 1 cm ^1% ) end absorption, 2 6 7 2 7 3 (4 5)

The main absorption peaks (nm) in the alkaline methanol solution are as follows. λ max nm (E 1 cm ^1% ) end absorption, 25 1— 25 5 (1 ⁹⁵ ) 5) Infrared absorption spectrum: KBr The main wave number (cm- ¹ ) in the tablet is as follows.

 3 2 7 0, 3 0 5 4, 2 9 6 6, 1 6 6 0, 1 5 2 7, 1 4 2 7, 1 2 8 6, 1 24 7, 1 20 1, 1 1 3 3

6) Contains at least the following amino acids. Asn, Thr, Gly, Val, lie, Leu, Phe, Pro, Lanthionine, Methyllanthionine _{0 At} least four Gly and two Pro.

7) — NMR spectrum (400 MHz, DMS O-d ₆ ): as shown in FIG.

8) ¹³ C-NMR spectrum (100 MHz, DMS O-d ₆ ): As shown in FIG.

 9) Solubility: Soluble in dimethyl sulfoxide (soluble to about 50 mg / m1), relatively soluble in methanol, relatively insoluble in ethyl acetate o

 10) Color test: Positive for reaction with iodine vapor and decolorization reaction of permanganate power.

 [Example 2]

 Antibacterial activity

After dissolving the AP17444 compound obtained in Example 2 in DMSO, it was diluted with water to prepare aqueous solutions of various concentrations, and diluted with agar for antibacterial activity against various pathogenic microorganisms shown in Table 1. The test was performed according to the plate method (standard method of the Japanese Society of Chemotherapy). The results are shown in Table 1.

[table 1]

表 1に示したごとく、 AP 1 7444化合物は各種の病原性微生物に対して 抗菌活性を示す。 従って AP 1 7444化合物はこれら病原菌に起因する感染 症に対する抗菌剤として有用である。

As shown in Table 1, the AP17444 compound exhibits antibacterial activity against various pathogenic microorganisms. Therefore, the AP17444 compound is useful as an antibacterial agent against infectious diseases caused by these pathogenic bacteria.

 [Example 3]

 Therapeutic effect of MR S A animal infection

The I CR mice (4 weeks old male), the S. aureus KU 1 6 (MRSA), were infected in 4 X 1 0 ⁷ CFU intraperitoneally per mouse. One hour after infection, the AP 1744 compound and vancomycin were administered intraperitoneally. From the number of surviving mice up to 3 days after infection, the ED50 value was determined by the Vander Waerden method. The results are shown in Table 2.

[Table 2] Dose EDso

 Number used Number survived

 (mg / kg) (mg / kg)

 5 5

 1.25 5 0.36

 API 7444

 0,31 5

 0.08 5

 2.5 5

 1.25 4 0.67

 / Ncomycin

 0.63 3

 0.31 0

As shown in Table 2, the AP 17444 compound shows a superior infection therapeutic effect on MRSA as compared to vancimacycin.

 [Example 4]

Vancomycin low sensitivity MR S A Animal infection treatment effect

S. aureus MU50 strain was added to the thigh of leukopenic mice to which cyclophosphamide (cyclophosphamide) was administered to ICR mice (male, 4 weeks old) with 3.22 × 10 ⁶ CFU Inoculated and infected. Two hours after infection, the AP17444 compound and vancomycin were administered intraperitoneally. Twenty-four hours after infection, the thighs of the mice were cut out, homogenized with physiological saline, diluted, and then applied to mannitol saline medium. After culturing at 37 ° C at −47 ° C., the number of colonies was counted and converted to the number of mouse thigh bacteria.

As a result, the number of thigh bacteria in the untreated group was 6.89 × 10 ⁸ , whereas that of the AP 17444 compound 25 mg / kg intraperitoneal administration group was 9 × 06 × 10 ⁴ a number, equal administration group Pankomaishin is 5. was 1 X 1 0 ⁸ pieces. The API 7444 compound also showed excellent therapeutic effects against vancomycin-resistant bacteria.

 [Example 5]

 PR SP Animal infection treatment effect

S. pne umo niae AS P965 was injected into ICR mice (4 week-old male). The (P RS P), were infected per pet 1. 8 X 1 0 ⁷ in C FU vein. One hour after infection, the AP17444 compound and vancomycin were administered intraperitoneally. To determine the ED ₅ o value by V ander Wa erden method from the survival number of mice until after the infection 5 days. The results are shown in Table 3.

[Table 3]

 10 5 4

 2 5 4 0.89

 AP 17444

 0.4 5 1

 0.08 5 0

 10 5 2

 2 5 1 3.24

 Pancomycin

 0.4 5 0

 0.08 5 0

As shown in Table 3, the AP 17444 compound shows a better infection treatment effect on PRSP than vancomycin.

 [Example 6]

 Toxicity test

 The AP17444 compound obtained in Example 2 was dissolved in DMSO and diluted with water to prepare a 20% DMSO aqueous solution, and 100 mg / kg was intravenously administered to three ICR mice to determine the state of death. confirmed. As a result, none of the individuals died, and it was confirmed that the compound of the present invention was highly safe.

[Industrial applicability]

According to the present invention, it is possible to provide a novel antibiotic which is effective for infectious diseases caused by various pathogenic bacteria including MRSA and PRSP. [Reference to deposited biological material]

The name and address of the depositary institution that deposited the biological material

 National Institute of Advanced Industrial Science and Technology (AIST) Patent Organism Depositary

 1, Tsukuba, Higashi, Ibaraki, Japan 1 1 Chuo No. 6 (zip code 305-8566) Date of deposit of biological material at the depository institution

 February 13, 2003 (Deposit by Hara)

 February 16, 1995 (Transfer date to deposit based on the Budapest Treaty by original deposit)

The accession number assigned to the deposit by the high depositary institution

 F E RM B P— 08 5 7 5

Claims

1. A peptide having the following physicochemical properties:  [1] Amino acid analysis can detect Asp, Thr, Gly, Val, lie, Leu, Phe, Pro, Lanthionine, and Methyllanthionine.  [2] Ultraviolet absorption spectrum: The main absorption peaks (nm) in methanol are as follows.  End absorption, 2 6 7—2 7 3  Education [3] Infrared absorption spectrum: KBr The main wave numbers (cm- ¹ ) in tablets are as follows.  of  3 2 7 0, 3 0 5 4, 2 9 6 6, 1 6 6 0, 1 5 2 7, 1 4 2 7, 1 2 8 6, 1 24 7, 1 2 0 1, 1 1 3 3  [4] R f value: about 0.5  The conditions for thin layer chromatography are as follows.  Fixed layer: Shiri gel TLC  Developing solvent: Mouth form-methanol (6: 4)  2. The peptide according to claim 1, which has the following physicochemical properties.  [5] The N-terminus of the amino acid sequence is lie.  [6] Color test: Positive for reaction with iodine vapor and decolorization reaction of permanganate power.  [7] Stability to enzymes: Degradation did not progress when enzymatic digestion was performed with Subutilysin, Thermolisin, proline-specific endopeptidase, or Chymotrypsin.  3. The peptide according to claim 1, which has the following physicochemical properties.  [8] Molecular weight: 2 1 0 7 [9] Composition: C ₈₉ H ₁₂₉ N ₂₅ 0 ₂₅ S ₅ 4. A method for culturing a microorganism belonging to the genus Kineosporia capable of producing the compound according to any one of claims 13 to 13, and collecting the compound from the culture, wherein the compound or a salt thereof is obtained. Production method. 5. A medicament comprising the compound according to claim 13 as an active ingredient. 6. A medicament for preventing and / or treating infectious diseases, comprising the compound according to any one of claims 13 to 13 as an active ingredient.