WO2007007399A1

WO2007007399A1 - Thiazole compound

Info

Publication number: WO2007007399A1
Application number: PCT/JP2005/012894
Authority: WO
Inventors: Yuriko Nozawa; Yi-Wen Chu; Jun-Ying Li
Original assignee: Taisho Pharmaceutical Co., Ltd.; Sichuan Industrial Institute Of Antibiotics Of China National Pharmaceutical Group Corporation
Priority date: 2005-07-13
Filing date: 2005-07-13
Publication date: 2007-01-18
Also published as: CN101400684B; CN101400684A

Abstract

Compounds having an anti-MRSA activity and having a skeleton and action mechanism different from those of existing drugs, which are useful as a novel antimicrobial agent. Conventionally, vancomycin is used as a drug for MRSA. However, recently, as, for example, vancomycin-resistant enterococcus (VRE) is reported and resistant bacteria occur, there is a demand for other drugs. Thiazole compounds of the formula: [in the formula, R is the group of the formula: or an amino] have an anti-MRSA activity and an anti-VRE activity, being useful as drugs.

Description

Specification

Thiazole compounds

Technical field

[0001] The present invention relates to a novel compound having anti-methicillin-resistant Staphylococcus aureus (hereinafter referred to as MRSA) activity and anti-vancomycin-resistant enterococci (VRE) activity.

Background art

[0002] MRSA has acquired resistance to β-ratata antibacterial agent, which is a treatment for Staphylococcus aureus, and was reported in the 1980s in Japan. In recent years, multi-drug resistant MRSA has become mainstream, and nosocomial infections have become a major problem in many hospitals.

[0003] A compound having anti-MRSA activity and having a skeleton or action mechanism different from that of existing drugs is useful as a new antibacterial drug.

[0004] Conventionally, vancomycin has been used as a drug for MRSA. Other resistant drugs have been desired due to the occurrence of vancomycin-resistant enterococci (VRE). .

[0005] Conventionally, nosiheptide (Nosih tide) is known as a thiazole compound having an antibacterial action (Non-patent Document 1).

[0006] Non-Patent Document 1: K. Umemura et al. Bull. Chem. Soc. Jpn., 71, 1391-1396 (1998).

Disclosure of the invention

Problems to be solved by the invention

[0007] An object of the present invention is to provide a novel physiologically active substance having inhibitory activity against MRSA and VRE.

Means for solving the problem

[0008] The present inventors isolated a large number of strains from soil and plants in order to achieve the above object, and as a result of various studies on the metabolites of the strains, As a result, the present invention was completed.

That is, the present invention relates to a formula [0010] [Chemical 1]

[0011] [where R is the formula

[0012] [Chemical 2]

[0013] or an amino group. In the following, R is a compound represented by the formula:

[0015] [Chemical 3]

[0016] The compound of the group represented by is WSS2258, and the compound of R is an amino group is WSS2260.

[0017] The strains producing WSS2258 and WSS2260 were obtained by the present inventors from soil in Yunnan, China. It is an isolated actinomycete, and this strain was entrusted to the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology on April 12, 2004, under the accession number FERM BP-10354.

[0018] The following are mycological properties.

A. Morphological properties

The basic mycelium grows and branches well, but no fragmentation is seen. No aerial hyphae are formed, but shorter hyphae than the basic hyphae rise and form sporangia at the tip. The sporangia are spherical and subspherical, the surface is wrinkled, and the size is about 4 10 x m. Also, the size of the spores released from the mature spore sac is about:! ~ 1.5 x m.

[0019] B. Culture properties

The following table 1 shows the results of macroscopic observation when cultured at 28 ° C for 3 weeks on various media. For the color display, the system color names from the Japanese Standards Association and JIS Color Name Book (1985) are used.

[0020] [Table 1]

[0021] C. Physiological properties

(1) Growth temperature range

'Growth temperature range: 18 32 ° C

• Optimal growth temperature range: 25 28 ° C

(2) Melanin-like pigment production: None (3) Carbon source availability

Table 2 shows the results of macroscopic observation when cultured at 30 ° C for 2 weeks on Prideham Gotleyve medium. In the table, “+” indicates growth and “w” indicates weak growth.

[Table 2]

[0023] D. Chemical taxonomic properties

Meso-diaminopimelic acid and glycine are detected from the cell wall of the bacterial cell component, and the cell wall type is type II. All microbial sugar components have arabinose detected and the sugar pattern is type D. It has MK9 (H) as the main menaquinone. The phospholipid pattern is saddle-shaped with only phosphatidylethanolamine (PE).

[0024] E. Phylogenetic analysis based on 16S rDNA gene

Determine the partial base sequence of the 16S rDNA region of the bacterium and search the database (DDBJ; DN

As a result of A Database bank of Japan, only the genus Actinoplanes was confirmed to have a homology of 98% or more.

[0025] Based on the results described above, according to the isolation and identification of actinomycetes (edited by the Japanese Society for Actinomycetes, 2001), it is appropriate to classify this strain as an actinomycete belonging to the genus Actinoplanes. This strain was named Actinoplanes sp. TA0455 (Actinoplanes sp. TA0455).

[0026] Production of WSS2258 and WSS2260 is generally carried out by culturing the TA0455 strain under aerobic conditions in a medium containing various nutrients, in the same manner as when producing general fermentation products.

[0027] A liquid medium is mainly used as a medium, which is composed of a carbon source, a nitrogen source, and an inorganic salt. Vitamins, precursors and antifoaming agents can be added as necessary, and the pH is adjusted to around 7. As the carbon source, for example, glucose, sucrose, dextrin, glycerin, starch or the like is used alone or in combination. Examples of nitrogen sources include meat extract, oatmeal, yeast Extract, soy flour, polypeptone, corn steep liquor, urea, ammonium salt, etc. are used alone or in combination. As the inorganic salt, for example, monopotassium phosphate, magnesium sulfate, sodium chloride, calcium carbonate and the like are used alone or in combination. As the antifoaming agent, Ade force knoll, silicon compound and the like can be used.

[0028] The culture method is suitable for aerobic culture such as shaking culture, aeration and agitation culture, pH 4-10, 25-35 ° C for 2-5 days, preferably _PH 6-7, 25- Incubate at 28 ° C for 4 days.

[0029] The compound of the present invention can be obtained by purifying a fermentation product by a general method. That is, after culturing, a culture filtrate is obtained by centrifugation or filtration, adsorbed on a polystyrene resin such as Diamond HP-20 (trade name, manufactured by Mitsubishi Chemical Corporation), and then adsorbed with an organic solvent such as lower alcohol or acetone. Elute. The cells are extracted with an organic solvent such as lower alcohol or acetone. Next, the bacterial cell extract and the eluate from the adsorption resin were combined and concentrated under reduced pressure to remove the organic solvent, and then transferred to a water-insoluble organic solvent such as ethyl acetate, black mouth form, and n-butanol. This is concentrated to form a syrup. This syrup is again dissolved in an organic solvent such as benzene, ethyl acetate, acetone, methanol, chloroform, etc., and silica gel column chromatography, gel filtration column chromatography, and column chromatography packed with ODS for reverse layer distribution. In addition, the compound of the present invention can be purified and isolated by being exposed to high performance liquid chromatography.

[0030] The structures of WSS2258 and WSS2260 obtained by the above method were determined by analysis of their molecular weight, ultraviolet absorption spectrum, ^ -NMR spectrum, ¹³ C_NMR spectrum, and the like.

[0031] The physicochemical properties of WSS2258 are as follows.

(a) Appearance: Yellow powder

(b) Molecular weight: 1206

(c). Molecular formula: C H N 0 S

51 43 13 11 6

(d). HR- TOF Mass Spectrum

Actual value: 1206.1625

Theoretical value: 1206.1608 (calculated as C H N 0 S)

51 43 13 11 6

(e). H-NMR spectrum: Figure 1 shows the results measured at 500 MHz in deuterated dimethyl sulfoxide.

(f). ¹³ C_NMR spectrum:

Figure 2 shows the results measured at 125 MHz in deuterated dimethyl sulfoxide.

(g) Solubility in solvents:

Dimethyl sulfoxide, soluble in black mouth form

Insoluble in water, hexane, methanol, ether, acetone, ethyl acetate

(h) Basic, acidic or neutral distinction: neutral

[0032] The physicochemical properties of WSS2260 are as follows.

(a) Appearance: Yellow powder

(b) Molecular weight: 1137

(c). Molecular formula: C H N 0 S

48 40 12 10 6

(d). HR-TOF Mass Spectrum

Actual value: 1137.1375

Theoretical value: 1137.1393 (calculated as C H N 0 S)

48 41 12 10 6

(e) Specific rotation: [α] ⁵ : 80.91 (c 0.26, CHC1)

D 3

(f). ^ -NMR spectrum:

Figure 1 shows the results measured at 500 MHz in deuterated dimethyl sulfoxide.

(g). ¹³ C_NMR spectrum:

(h) Solubility in solvents:

Dimethyl sulfoxide, soluble in black mouth form

Insoluble in water, hexane, methanol, ether, acetone, ethyl acetate G). Basic, acidic, neutral: neutral

The invention's effect

[0033] The compound of the present invention was found to have inhibitory activity against MRSA and VRE.

Brief Description of Drawings

[0034] FIG. 1 shows a ¹ H-NMR spectrum of WSS2258 measured at 500 MHz in deuterated dimethyl sulfoxide. FIG. 2 shows the C-NMR spectrum of WSS2258 measured at 125 MHz in deuterated dimethyl sulfoxide.

FIG. 3 shows the ¹ H-NMR spectrum of WSS2260 measured at 500 MHz in deuterated dimethyl sulfoxide.

FIG. 4 shows the ¹³ C-NMR spectrum of WSS2260 measured at 125 MHz in deuterated dimethyl sulfoxide.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be specifically described with reference to Examples and Test Examples.

Example 1

[0036] Manufacture of WSS2258 and WSS2260

(1) Solvent starch 2.5%, glucose 1%, fish meal 0.5%, Pharmamedia 0 · 3%, NZ case 0.3%, yeast extract 0.2%, calcium carbonate 0.2% 60 ml liquid medium 300 ml Erlenmeyer flask And sterilized at 120 ° C and 2 atm for 20 minutes. Next, this sterile medium was inoculated with Actino planes sp. TA0455 strain, and cultured with shaking at 28 ° C, 200 mm for 3 days.

[0037] Next, 100 ml of a sterile liquid medium consisting of 2% oatmeal, 1% glucose, 2.5% dextrin, 1% pharmamedia, 0.5% food schmeal, 0.5% sugarcane is placed in a 500 ml Erlenmeyer flask. Add 2 ml of the seed culture solution above and incubate with shaking at 28 ° C and 200 mm for 3 days.

[0038] Next, using two 50 L culture tanks, 600 ml of the preculture solution was added to 30 L of each sterile medium having the same composition as that of the preculture, and cultured with shaking at 28 ° C and 200 mm for 5 days. .

[0039] After completion of the culture, 30 L of n-butanol was added to 60 L of the obtained culture broth and stirred, and the cells were separated into cells and n-butanol extracted fractions by centrifugation. The n-butanol fraction was concentrated under reduced pressure to obtain 120.27 g of a brown oily substance.

[0040] (2) Dissolve 120.27 g of the oily substance in the previous paragraph in 750 ml of a water-methanol (10:90) mixed solution, stir 750 ml of hexane, centrifuge, and concentrate the water-methanol layer under reduced pressure. Solidified. 65.35 g of the obtained brown substance was dissolved in a mixed solvent of formaldehyde-methanol, 200 ml of silica gel [Silica Gel 60 (manufactured by Merck)] was added and concentrated under reduced pressure to be adsorbed. This is black It was placed on an upper layer of 800 ml of silica gel prepared by mouth form, washed with 1000 ml of mouth form form, and then eluted sequentially with a mixed solvent of form mouth form methanol (99: 1 to 80:20). Of these, fractions eluted with a mixed solvent ratio (96: 4) were combined and concentrated to dryness under reduced pressure to obtain 2.014 g of a brown substance. Add 20 ml of methanol to this brown substance, dissolve 323. lmg of the precipitate obtained by filtration in Kuroguchi Form, and prepare a preparative TLC with a mixed solvent of Kuroguchi Form 1 Methanol (8: 1) as the developing solvent. Thin layer plate silica gel Merck F254 (manufactured by Merck)] was used for fractionation. The portions of Rf.0.62 to Rf.0.56 were scraped off, and the resulting silica gel was extracted with a mixed solvent of black mouth form monomethanol (1: 1) and concentrated to dryness under reduced pressure to obtain 69.3 mg of WSS2258. Similarly, remove the portion of Rf.0.06-0.37, extract the resulting silica gel with a mixed solvent of black mouth methanol (1: 1), concentrate under reduced pressure and dry to obtain 36.3 mg of WSS2260. It was.

[0041] Test Example 1 MIC measurement for MRSA

(Sample)

As WSS2258, WSS2260 and positive control, vancomycin was dissolved in DMSO to a concentration of 10 mg / ml, and diluted with sterilized water to a target concentration.

(Test species)

Staphylococcus aureus (MRSA) SA_9 (clinical isolate)

(Test method)

The MIC measurement was performed by the following micro liquid dilution method.

[0042] The strength of the heart-infusion agar medium cultured in sputum The colonies were picked and the turbidity was adjusted to McFarl and O.5. The final inoculum was taken up in the drug-containing medium so that the amount of inoculum was 10 X 10 ⁵ CFU / ml. After culturing at 35 ° C for 17 hours, the determination is shown in Table 2 as MIC (ag / ml) with the lowest concentration at which no bacterial growth was observed.

[0043] [Table 3] Specimen name lC C ^ g / ml)

WSS2258 0. 06

WSS2260 0. 03

Hydrochloric acid / Ncomycin 2 [0044] Test example 2 MIC measurement for VRE

(Sample)

As WSS2258, WSS2260, and positive control, linezolid was dissolved in DMSO to a concentration of 10 mg / m 1, and diluted with sterilized water to a target concentration was used. (Test species)

VRE (owned by van (A), clinical isolate)

(Test method)

[0045]-The colonies from the heart infusion agar medium cultured in spear were picked and the turbidity was adjusted to McFarl and 0.5. And ingestion into the drug-containing medium to a final inoculum volume mosquitoes S5 X 10 ⁵ CFU / ml. After culturing at 35 ° C for 17 hours, the determination is shown in Table 3 as the MIC (μg / ml) with the lowest concentration at which no bacterial growth was observed.

[0046] [Table 4]

Industrial applicability

[0047] Since the compound of the present invention has strong antibacterial activity against MRSA and VRE,

Can be used.

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