KR100309804B1 - Chaetoatrosin A, a Novel Chitin Synthase II Inhibitor, and Antifungal Composition Containing Same - Google Patents

Abstract

The present invention is a novel compound ketoatrosin A (chaetoatrosin A), which inhibits chitin synthase II, a new strain Ketoium atrobrunne F449 which produces the same , ketoart from the culture of this strain. A method for purifying Rosin A and an antifungal composition containing KetoArosine A. Since the novel compound ketoatrosine A of the present invention is excellent in selective inhibitory activity against chitin synthase II and antibacterial activity against pathogenic fungi, pharmaceutical compositions containing the same are useful as antifungal agents.

Description

Novel Chitosynsin A Inhibiting Chitin Synthetase 2 and Antifungal Composition Containing the Same {Chaetoatrosin A, a Novel Chitin Synthase II Inhibitor, and Antifungal Composition Containing Same}

The present invention relates to a novel compound ketoatrosine A which inhibits chitin synthase II, a new strain Keetomium atrobrunneum F449, which produces the same, a method for purifying ketoatrosine A from a culture of this strain, and An antifungal composition containing ketoatrosine A is disclosed.

Although research on fungal cell wall and membrane synthase inhibitors has been under way for a long time, the rate of development is slower than that of antibacterial antibiotics, and the overall knowledge of fungi is insufficient. Therefore, the number of enzyme inhibitors that have good efficacy against fungi is very limited. It is. Moreover, most of the enzyme inhibitors found are highly toxic and have many side effects. Therefore, the necessity for new enzyme inhibitors has been emerging for a long time. Recently, opportunistic infections caused by long-term use of AIDS, immunosuppressants or anticancer agents in organ transplantation, etc. Visceral mycosis is increasing more and more (TJ Walsh, et al., Science, 264 , 371-373, 1994).

Thus, as a countermeasure against such infections urgent early development of the establishment of early diagnostic and selection toxicity excellent chemotherapeutic agents, and In addition, Candida development of a chemotherapy for various fungi, such as (Candida) and Aspergillus (Aspergillus) This is urgently needed.

Antifungal agents currently used in clinical practice are polyene and azole derivatives and allylamine-thiocarbamates, all of which interact with ergosterol, a fungal cell membrane component. Or inhibits the synthesis of ergosterol. Among these, polyene increases cell membrane permeability, causes cell contents to leak, kills cells, and has many side effects, but amphotericin B is still the only polyene antibiotic used for systemic fungal infection. . The azole compounds are mostly synthetic products, and many new compounds are used and inhibit the ergosterol synthesis by inhibiting C-14 demethylase in the synthesis pathway of ergosterol, but some imidazole compounds (clotrimazole, miconazole, etc.) Because of its direct toxicity and damage to cell membranes, it is highly toxic and used only locally (DP Hanger, et al., Antimicrob. Agents Chemother., 32 , 646, 1988). Recently developed azole compounds (ketoconazole, itraconazole, etc.) have been reported to have side effects of inhibiting enzymes that depend on cytochrome P-450 and inhibiting endocrine steroid hormone synthesis while being less toxic. In addition, fungicides such as allylamine compound naftifine, terbinafine, and thiocarbamate compound tolnaftate, such as squalene epoxidase and oxidose squalene cycle, It is known to inhibit ergosterol synthesis by inhibiting oxidosqualene cyclase, but these compounds are widely used in skin diseases because they have broader antifungal activity in vitro and are less toxic than azole compounds, but have weaker pharmacokinetics. have.

In addition, many compounds have been reported as inhibitors of ergosterol synthesis, but the low toxicity and excellent ergosterol synthesis inhibitors are very small, so the need for new ergosterol inhibitors has been steadily increasing. As a new target, research on squalene synthase, which is involved in the squalene synthesis of ergosterol synthase, and the search for a substance that inhibits its activity are being actively conducted. It is an enzyme that plays an important role in the formation of intermediate metabolites in the synthesis of ergosterol and cholesterol, and has already been used as an inhibitor of the enzyme, squalestatin (MJ Dawson, et al ., J. Antibiot., 45 , 639-647). , 1992) and zaragozic acid (JD Bergstrom, et al., Proc. Natl. Acad. Sci., 90 , 80-84, 1993) have been published, which not only have antifungal activity but also cholesterol lowering effects. In addition, it becomes the target of the search for a new enzyme inhibitor.

On the other hand, since fungi have a cell wall unlike mammalian cells, fungal cell walls have long received a lot of attention as selective targets. The main components of the fungal cell wall are mainly composed of the polysaccharides chitin and glucan (DJ Frost, et al., J. Antibiot., 48 , 306-310, 1995), and chitin is β--1 in N-acetyl-D-glucosamine. It is synthesized by chitin synthase I, II, III as a homopolymer consisting of a 4-bond (W, J. Choi, et al., Proc. Natl. Acad. Sci., 90 , 80-84, 1994). As the characteristics and functions of these chitin enzymes are known, developed countries have been involved in the formation of chitin synthase II and chitin ring, which are involved in the formation of septa by developing strains capable of selectively producing each enzyme using molecular biology techniques. An inhibitor of chitin synthase III is searched for. Inhibitors of chitin synthesis were polyoxin (J. Nagatsu, et al., Agric. Biol. Chem., 29 , 848-854, 1965) and nikkomycin (HP Fiedler, et al., J. Chem. Tech.Biotechnol ., 32 , 271-280, 1982), and glucan synthesis inhibitors include aculeacin (J. Mizoguchi, et al., J. Antibiotics, 30 , 308-313, 1977). ) And echinocandin (F. Benz, et al., Helv. Chim. Acta, 57 , 2459-2477, 1974) have been reported but are not widely used due to various problems.

Therefore, there is a need for a new antifungal agent that inhibits chitin synthase II and exhibits effective antifungal activity while having low toxicity.

It is an object of the present invention to provide novel compounds that inhibit chitin synthase II, which is essential for cell wall synthesis of fungi.

Another object of the present invention is to provide a novel microorganism producing the novel compound.

Still another object of the present invention is to provide a method for purifying the novel compound from the culture medium of the novel microorganism.

Another object of the present invention is to provide an antifungal composition containing the novel compound.

1A to 1D show optical micrographs of Ketomium atrobrunium F449, FIG. 1A is an ascospore, FIG. 1B is an ascus, FIG. 1C is an ascomata, Figure 1d shows the peridium (子 殼, peridium) in the form of thickened capsules, respectively.

Figure 2 shows the hydrogen nuclear magnetic resonance spectrum of ketoatrosine A.

Figure 3 shows the carbon nuclear magnetic resonance spectrum of ketoatrosine A.

Figure 4 shows the hydrogen-carbon nuclear magnetic resonance spectrum of ketoatrosine A.

In accordance with the above object, the present invention provides a novel compound ketoatrosine A having the structure of formula (1) and exhibiting inhibitory activity against chitin synthase II.

Formula 1

The compound ketoatrosine A can be represented by the molecular formula C ₁₄ H ₁₄ O ₅ as a yellow powder and has a molecular weight of 262, and is soluble in methanol, dimethyl sulfoxide (DMSO) and water, but acetone, ethanol, CH ₃ CN, hexane And insoluble in chloroform. Ketoatrosine A exhibits an excellent antimicrobial activity against various pathogenic fungi, with an IC ₅₀ of about 104 μg / ml having an inhibitory activity against chitin synthase II.

In order to effectively inhibit pathogenic fungi, anti-fungal compositions can be prepared by mixing ketoatrosine A as an active ingredient with a pharmaceutically acceptable carrier. The antifungal composition may further include conventionally used excipients, disintegrants, sweeteners, lubricants, flavoring agents, etc., and tablets, capsules, powders, granules, suspensions, emulsifiers, syrups, liquids by conventional methods Or in dosage unit forms or as multi-dose pharmaceutical preparations, such as preparations for parenteral administration.

The antifungal composition of the present invention containing ketoatrosine A as an active ingredient may be parenterally or orally administered as desired, and may be 1 to 50 mg / kg body weight, preferably 10 to 1 kg per day of body weight per day. It may be administered at least once a day to be an active ingredient of 15 mg / kg body weight, more preferably 12 to 13 mg / kg body weight. However, the dosage level for a particular patient may vary depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion, severity of disease, and the like.

Also provided in the present invention is a new strain, Keetomium atrobrunneum F449 , which produces ketoatrosine A. In the present invention, the strain Ketomium atrobrunium F449 is most vigorous at 25 to 30 ° C., but does not grow above 42 ° C., but generally has a very slow growth rate and does not show a significant difference depending on the type of medium. The morphology of this strain is as shown in Figures 1a to 1d, Figure 1a is an ascospore, Figure 1b is an ascus, Figure 1c is an ascomata and Figure 1d Each thickened form of perception (子 殼, peridium) represents. Comparison of the color of this strain based on the ISCC-NBC chromaticity results showed that Czapek medium (0.2NaNO ₃ , 0.1K ₂ HPO ₄ , 0.05MgSO ₄ · 7H ₂ O, 0.05KCl, 0.001FeSO ₄ · 7H ₂ O, 3.0 Sucrose, 1.5 agar), MEA medium (2.5 malt extract, 1.5 agar), MEA1C medium (2.5 malt extract, 1.0 cellulose, 1.5 agar), CYA medium (0.5 yeast extract, 0.3 NaNO ₃ , 0.1K ₂ HPO ₄ , 0.05 MgSO ₄ · 7H ₂ O, 0.05KCl, 0.001FeSO ₄ · 7H ₂ O, 0.0005CuSO ₄ · 5H ₂ O, 0.001ZnCl ₂ · 7H ₂ O, 3.0 sucrose, 1.5 agar), CYA 20S medium (0.5 yeast extract, 0.3NaNO ₃ , 0.1K ₂ HPO ₄ , 0.05MgSO ₄ · 7H ₂ O, 0.05KCl, 0.001FeSO ₄ · 7H ₂ O, 0.0005CuSO ₄ · 5H ₂ O, 0.001ZnCl ₂ · 7H ₂ O, 20.0 sucrose, 1.5 Agar) and PDA medium (Difco. Co., product number: 0013-17-6) showed yellowish color, whereas YMA medium (0.3 yeast extract, 0.3 malt extract, 0.5 tryptone, 1.5 agar) showed white color. The back side of the medium was all dark brown in color. It is estimated to produce. Also, as a morphological feature, the vesicles have eight spherical spherical spores, and the vesicles are gray, fusiform and long pyriform. In addition, a smooth form, and long as the septum is attached to the long scapula and ascomatal setae is observed, the round is 40 to 70 ㎛ diameter yellow-yellow and black where the bristles are produced There is a form of pericardium. Ketomeum Acrobrunium F449 has been deposited with Accession No. KCTC 0612BP dated May 17, 1999 to the Institute of Biotechnology Gene Bank (KCTC).

In order to produce ketoatrosine A by culturing ketodium atrobrunium F449, the seed medium (0.5 glucose, 1.5 soluble starch, 0.2 yeast extract, 0.5 polypeptone, 0.1 KH ₂ PO ₄ and 0.05 MgSO ₄ · 7H ₂ O) was inoculated with ketium atrobrunium F449 and shaken for 3 to 4 days at 25 to 30 ° C., followed by production of cultured spawn, for example, YM broth medium (0.3 yeast extract, 0.3 malt extract). , 0.5 tryptone) and incubated for 3 to 5 days at 25-30 ℃.

The present invention also provides a method for isolating ketoatrosine A from a culture of Ketomium atrobrunium F449. The method of the present invention specifically includes the following steps. First, ethyl acetate is added to a fermentation broth of Ketodium Atrobrunium F449 and extracted, followed by concentration under reduced pressure to obtain a yellow crude extract. This crude extract is separated by normal phase silica gel column chromatography using chloroform-methanol mixture as eluent, wherein the mixing ratio of chloroform and methanol in the eluate can be in the range of 98: 2 to 95: 5, and 97: 3 ( v / v) is preferred. The fractions showing inhibitory activity were concentrated under reduced pressure and separated by reverse phase silica gel column chromatography using a mixture of methanol and water as eluent, wherein the mixing ratio of methanol and water in the eluate could be in the range of 50:50 to 60:40. , 50:50 (v / v) is preferable. The active fractions obtained here are adsorbed on a Sephadex LH-20 column and then eluted with methanol to give a yellow partially purified active material. The pure ketoatrosine A is then separated by partially purified purification of the active substance using preparative TLC. In the preparative TLC, a mixed solvent of hexane, ethyl acetate and isopropyl alcohol can be used as the developing solvent, and the mixing ratio thereof can be in the range of 60 to 70:30 to 40: 0 to 10, and these are 60 A solvent mixed at a ratio of: 30: 10 (v / v / v) is preferred. According to the method of the present invention, pure ketoatrosine A can be obtained in a yield of 400 µg per liter of fermentation broth of Ketodium atrobrunium F449.

Hereinafter, the specific configuration and operation of the present invention will be described with reference to Examples, but the scope of the present invention is not limited only to the following Examples.

Example 1 Selection of Production Strains

1 g of soil collected from Odaesan, Gangwon-do, Korea was added a pre-treatment solution (composition: 0.02Tween 80, 6 yeast extract, 0.01MgSO ₄ · 7H ₂ O) and diluted 10 ³ times, and 0.1 ml of the diluted solution was added to YMA medium (0.3 yeast extract, Single malt (Haykawa et al. , J. Ferment. Technol., 65 (5), 501-509 (1987)) was cultured in 0.3 malt extract, 0.5 tryptone and 1.5 agar) for 7 days. The strain named F449 was isolated and bacteriologically studied according to the method of De Hong and Guano (Atlas of Clinical Fungi, Universitat Kovira: Nirgili, Reus, Spain , pp 282-283 (1995)). Was performed. To identify isolated strains, Czapek medium (0.2NaNO ₃ , 0.1K ₂ HPO ₄ , 0.05MgSO ₄ · 7H ₂ O, 0.05KCl, 0.001FeSO ₄ · 7H ₂ O, 3.0 sucrose, 1.5 agar), MEA medium (2.5 malt extract, 1.5 agar), MEA1C medium (2.5 malt extract, 1.0 cellulose, 1.5 agar), CYA medium (0.5 yeast extract, 0.3 NaNO ₃ , 0.1K ₂ HPO ₄ , 0.05MgSO ₄ · 7H ₂ O, 0.05KCl , 0.001 FeSO ₄ · 7H ₂ O, 0.0005CuSO ₄ · 5H ₂ O, 0.001 ZnCl ₂ · 7H ₂ O, 3.0 sucrose, 1.5 agar), CYA 20S medium (0.5 yeast extract, 0.3NaNO ₃ , 0.1K ₂ HPO ₄ , 0.05MgSO ₄ · 7H ₂ O, 0.05KCl, 0.001FeSO ₄ · 7H ₂ O, 0.0005CuSO ₄ · 5H ₂ O, 0.001ZnCl ₂ · 7H ₂ O, 20.0 sucrose, 1.5 agar), PDA medium (Difco.Co , Product number: 0013-17-6) and inoculated in YMA medium and observed for 14 days at 25-42 ℃.

As a result, this strain was the most vigorous growth at 25 ~ 30 ℃ and did not grow above 42 ℃ but in general the growth rate is very slow and did not show a big difference depending on the type of medium. The morphology of the strain was observed using an optical microscope with a magnification of 800 times, and the results are as shown in FIGS. 1A to 1D, where FIG. 1A is an ascospore, FIG. 1B is an ascus, and FIG. Fruits (子囊 果, ascomata) and Figure 1d shows the peridium (子 殼, peridium) in the form of a thickened capsule. The color comparison of this strain was based on the ISCC-NBC chromaticity. All six media except YMA medium showed similar yellow color, whereas YMA medium showed white color and the back side of the medium was dark brown. It was found that color produced soluble pigment. Also, as a morphological feature, the scapula has eight spherical spherical spores, and the scapula are gray and fusiform and long pyriform. In addition, smooth and long septum and bristles (ascomatal setae) with diaphragm were observed, and rounded pericardium with a diameter of 40-70 μm, yellowish green and black, were observed. . In addition, a similar form of texture augularis was observed, and thus, the F449 strain was identified as Chaetomium atrobrunneum .

The inventors deposited Ketoum Astrobrunium F449 with the Accession No. KCTC 0612BP dated May 17, 1999 at the Biotechnology Research Institute Gene Bank.

Example 2 Isolation and Purification of Chitin Synthase II Inhibitors Produced by Ketomium Atrobrunium F449

(Step 1) Cultivation of Strains

Frozen F449 strain (10 glycerol, -80 ° C) was placed in a 500 ml Erlenmeyer flask with a baffle in a 50 ml seedling medium (glucose 2, yeast extract 0.2, polypeptone 0.5, potassium phosphate 0.1, magnesium sulfate heptahydrate). 0.05, adjusted to pH 5.9 and then sterilized) and incubated for 4 days at 26 ℃. This culture was inoculated at a concentration of 2 (v / v) in 1 L of YM medium (yeast extract 0.3, tryptone 0.5, glucose 1), and then cultured at 26 ° C. at 130 rpm for 5 days.

(Step 2) Isolation and Purification of Chitin Synthase II Inhibitors

Ketomium atrobrunium obtained in (Step 1) After extracting twice with the same amount of ethyl acetate, 12 ml of F449 was concentrated under reduced pressure. The ethyl acetate extract was adsorbed onto a normal chromatography column (Merck, Kieselgel 60, 230-400 mesh), and then a mixture of chloroform and methanol (97 : Eluted with 3 (v / v)) to obtain an active fraction, which was concentrated and again adsorbed onto a reversed phase column (Merck, Lichroprep RP-18, 40-63 μm). The active ingredient adsorbed on the reverse phase column was eluted with a mixture of methanol and water (50:50 (v / v)) to obtain an active fraction, which was separated by Sephadex LH-20 chromatography using methanol as eluent. A partially purified active material was obtained. Finally, chitin synthesis was carried out using partially purified active fraction using preparative TLC using a mixed solvent of hexane, ethyl acetate and isopropyl alcohol (60:30:10 (v / v / v)) as a developing solvent. Pure compounds that inhibit the activity of enzyme II were isolated and termed ketoatrosine A. At this time, the yield of pure ketoatrosine A finally obtained was 400 μg per 1 liter of culture.

The physicochemical properties of ketoatrosine A are shown in Table 1.

division Physicochemical Properties Appearance Yellow powder Molecular formula C ₁₄ H ₁₄ O ₅ Molecular Weight 262 Availability MeOH, DMSO, Water Insoluble Acetone, EtOH, CH ₃ CN, Hexane, Chloroform

Example 3: Instrument Analysis and Structure Determination

In order to determine the structure of ketoatrosine A, various instrumental analyzes were performed as follows.

(1) UV-Vis absorbance analysis

Ketoatrosine A was dissolved in 100 methanol, and the absorption wavelength was analyzed using an ultraviolet-visible spectrometer (Shimazu, UV-265). As a result, maximum absorption was obtained at UV 225, 272, and 408 nm.

(2) Infrared (IR) absorbance analysis

2 mg of powdery ketoatrosine A was put into a KBr window and analyzed by an infrared spectrometer (Bruker EQuinox 55). Spectroscopy shows the presence of hydroxyl groups at 3,400 cm ^-1 and CH stretching vibrations due to methine and methyl groups at 2,921 and 2,851 cm ^-1 , carbonyl and aromatic C at 1,500-1,750 cm ^-1 An absorption peak attributable to C was observed.

(3) molecular weight analysis

As a result of high resolution mass spectrometry using a mass spectrometer (Hewlett packard 5989A), the molecular weight of the purified purified ketoatrosine A was 262, and the molecular formula was C ₁₄ H ₁₄ O ₅ . It turned out to be.

(4) nuclear magnetic resonance (NMR) analysis

10 mg of ketoatrosine A was dissolved in CD ₃ OD, placed in a 5 mm NMR tube, and subjected to NMR analysis using a Brucker AMX 500. ¹ H-NMR was measured at 500 MHz and ¹³ C-NMR at 125 MHz. 2 and 3). The structure was determined by measuring the hydrogen-carbon-linked nuclear magnetic resonance spectra and the results are shown in FIG. 4.

Example 4: Inhibitory Activity Assay of Chitin Synthetase II

Inhibitory activity of ketoatrosine A on chitin synthase II was based on UDP- [ ¹⁴ C] GlcNAc (N-acetyl-D-glucosamine) (400,000 cpm / μmol) as a substrate (Won-Ja). Choi, et al., Anal. Biochem., 219 , 368-372, 1994). As test group, 32 mM Tris-HCl (pH 8.0), 1.6 mM cobalt acetate, 1.1 mM UDP- [ ¹⁴ C] GlcNAc, 20 μl of chitin synthase II coenzyme solution, 14 μl of ketoatrosine A, 2 μl Trypsin (2 mg / ml) was added to the reaction for 30 minutes at 30 ℃.

2 μl of trypsin inhibitor (4 mg / ml) was added to the reaction solution, which was left on ice for 10 minutes, and 32 mM N-acetylglucosamine was added so that the total volume was 50 μl, followed by 90 minutes at 30 ° C. Reacted for a while. After the reaction was stopped by adding 1 ml of 10 trichloroacetic acid to the reaction solution, the reaction solution was filtered using GF / C filter paper (Whatman Co.). After drying the filter paper, 3 ml of cocktail (LUMAC LSC B.V.) was added and radioactivity was measured using a liquid scintillation counter.

At this time, the control group did not add ketoatrosine A to the reaction solution, and the blank test group (Blank) repeated the same experiment without adding ketoatrosine A and chitin synthase II to measure radioactivity. Inhibitory activity of ketoatrosine A against chitin synthase II was calculated according to Equation 1.

As a result, the inhibitory activity of the chitin synthase II of ketoatrosine A was IC ₅₀ of 104 g / ml.

Example 5: Activity Screening Using Pathogenic Strains

Yeasts among the pathogenic strains were cultured overnight using Sabouraud's broth, and the fungi were adjusted to 10 ⁷ CFU / mL and the fungus was used as inoculation fungi. Incubated in Sabouraud's agar medium at 25 ° C. for 7-14 days and used as inoculum. At this time, the fungi forming spores were used as the inoculated fungi solution by adjusting the final concentration to 10 ⁷ CFU / mL. The test organisms without forming spores were tested using agar bloc. Was inoculated. Ketoatrosine A was prepared in 2-fold dilution series using sterilized distilled water, and then mixed with potato dextrose agar medium or saburo agar medium at a ratio of 9: 1 by sterilization and maintained at 50 ° C. Aliquots were plated onto 5 cm sized plates to -1.95 μg / ml. When the medium is solidified, the inoculated bacteria or agar blocks of the above-described test bacteria are inoculated in a predetermined amount for each plate, incubated for 1-5 days at 25-30 ° C, and visually observed to minimize the concentration at which the growth of bacteria is suppressed. Low concentration (MIC) was determined.

As a result of measuring the antimicrobial activity of pathogenic fungi derived from the flora and fauna of ketoatrosine A, microsporum canis that causes body whiteness and athlete's foot with human pathogensMicrosporum canis) And tricophyton mentagrophytes (Trichophyton mentagrophytes) And Cryptococcus neoformans causing meningitisCryptococcus neoformansMIC for) was 100 μg / ml each. In addition, Botrytis cineraria, a plant pathogen, causes gray fungal and mozalococcal diseases.Botrytis cinerea) And Pittium UltimumPythium ultimumThe MIC value for) was 100 μg / ml, and especially the Lysatonia solani that caused rice paddy disease.Rhizoctonia solaniMIC values for) were 50 μg / ml, and polyoxin D (E. Cabib,Antimicrob. Agents Chemother., 35, 170-173, 1991).

Example 6: Acute Toxicity Test of Active Inhibitors

Acute toxicity test was performed on 5 weeks old ddy mice (Korean Association of Experimental Animals) weighing about 25 g on ketoatrosine A. First, the test substance (ketoatrosine A) was mixed with sterile distilled water and prepared immediately before administration. As a control group, sterile distilled water was used, and the test substance was forcibly administered using the sonde for oral administration to the test system fasted for about 3 hours. On the day of administration, changes in general condition, symptoms of intoxication and the presence of dead animals were observed every hour from 1 hour to 6 hours after administration and once daily from the next day of administration to 14 days. After the end of the observation period, heat dissipation under CO ₂ anesthesia was performed to observe the visual abnormality of internal organs in detail. As a result, even when the compound was administered up to 100 mg once and continuously observed for 14 days, it was confirmed that there was no great toxicity.

Formulation example

The following formulation is an example for the preparation of an antifungal agent containing ketoatrosine A according to the present invention as an active ingredient. The antifungal agent according to the present invention is not limited by the following formulation examples.

Formulation Example 1 Preparation of Tablet for Oral Administration

After preparing the following composition with keto atosine as an effective drug, a tablet for oral administration was prepared by a conventional method.

ingredient content

Ketoatrosine A 100 mg

Hard silicic anhydride 10 mg

190 mg of microcrystalline cellulose

Magnesium Stearate 5mg

Sodium starch glycolate 60 mg

Calcium monohydrogen anhydride 135 mg

Formulation Example 2: Preparation of Injection

Injectables were prepared in a conventional manner after preparing the following composition using ketoatrosine A as an effective drug.

ingredient content

Ketoatrosine A 100 mg

Mannitol 180 mg

Na ₂ HPO ₄ 12H ₂ O 26 mg

Distilled water 2,974 mg

The novel compound ketoatrosine A of the present invention has excellent selective inhibitory activity against chitin synthase II, and thus excellent antimicrobial activity against pathogenic fungi, so that the pharmaceutical composition comprising the same may be usefully used as an antifungal agent.

Claims (4)

Ketoatrosine A of Formula 1 that inhibits chitin synthase II: Formula 1 Chaetomium atrobrunneum F449 strain (KCTC 0612BP) that produces ketoatrosine A. (a) adding ethyl acetate to the culture solution of Ketodium atrobrunium F449 and extracting to obtain a crude extract; (b) separating the crude extract by normal phase silica gel column chromatography using chloroform-methanol mixture as eluent to obtain an active fraction: (c) separating the active fraction obtained in step (b) by reverse phase silica gel column chromatography using a mixture of methanol and water as eluent to obtain an active fraction; (d) separating the active fraction obtained in step (c) by Sephadex LH-20 column chromatography using methanol as eluent to obtain partially purified material; And (e) separating pure ketoatrosine A by separating and purifying the partially purified material obtained in step (d) using preparative TLC. A method for purifying ketoatrosine A from a culture solution of Ketodium atrobrunium F449. An antifungal composition comprising, as an active ingredient, ketoatrosine A of Formula 1 and a pharmaceutically acceptable carrier: Formula 1