DISCLOSURE OF INVENTION The present invention relates to a new bioactive compound, WF217 substance or its salt.

More particularly, it relates to a novel compound, WF217 substance or its salt which has an antimicrobial activity against pathogenic microorganisms, especially pathogenic fungi, to a process for preparation thereof, to a phannaceutical composition comprising the same, which is useful as an antimicrobial agent, and to a use thereof as a medicament.

Accordingly, one object of this invention is to providethe novel compound, WF217 substance or its salt which is of use for treating and preventing infectious diseases caused by pathogenic microorganisms.

Another object of this invention is to provide a process for production of the WF217 substance or its salt by fermentation of the WF217 substance- producing microorganism in a nutrient medium.

A further object of this invention is to provide a pharmaceutical composition containing, as an active ingredient, the WF217 substance or its salt.

Still further object of this invention is to provide a use of the WF217 substance or its salt for treating and preventing infectious diseases caused by pathogenic microorganisms.

The WF217 substance can be produced by fermentation of the WF217 substance-producing microorganism, especially, strain belonging to the genus Chaetomium sp. No. 217 in a nutrient medium.

It is to be understood that the production of the WF217 substance is not limited to the use of the particular organism described herein, which is given for the illustrative purpose only. This invention also includes the use of any mutants which are capable of producing the WF217 substance including natural mutants as well as artificial mutants which can be produced from the described organism by conventional means such as irradiation of X-ray, ultra- violet radiation, genetic engineering, treatment with N-methyl-N'-nitro-N- nitrosoguanidine, 2-aminopurine, and the like.

Characteristics of producing strain No. 217 The fungus strain No. 217 was originally isolated from a soil sample, collected in Ajigasawa-machi, Nishitsugaru-gun, Aomori-ken, Japan. This organism grew restrictedly on various culture media, and formed grayish colonies. On some agar media, strain No. 217 formed ascomata: perithecia with long terminal hairs. Its mycological characteristics were as follows.

Cultural characteristics on various agar media are summarized in Table 1. Culture on potato dextrose agar grew restrictedly, attaining 1. 5-2.0 cm in diameter two weeks later at 25°C. This colony surface was raised, sulcate to crusty, olive, and yellowish white at the margin. The reverse was olive.

Ascomata were not observed. Colonies on Emerson Yp Ss agar grew fairly rapidly, attaining 3.5-4.5 cm in diameter under the same conditions. The surface was plane, felty, sectoring, and showed several colors; white at the center, yellowish white at the margin, and grayish yellow to olive brown at the middle. Ascomata were observed abundantly as brown dots on the medium.

The reverse was light brown at the center, yellowish white at the margin, olive brown at the middle.

The morphological characteristics were determined from the cultures on Emerson Yp Ss agar. Ascomata of strain No. 217 were perithecial, discrete, superficial or immersed, sometimes attached by rhizoids, dark brown to nearly

black, oval to ovoid, with lateral and terminal hairs, ostiolate, and 120-210 x 180-350 mm in size. Their peridium was dark brown, with textura epidermoidea to textura angularis. Ascomatal hairs were numerus, unbranched, septate, brown, thick-walled, gradually tapering and paling to the tip. Terminal hairs were curved, with mostly 3-7 coils in the upper part, verrucose, 500-650 (-800) mm long and 5-6.5 mm thick near the base; lateral hairs were straight or undulate, verruculose, and 160-200 (-290) mm long. The ascomata matured within 28 days and produced asci and ascospores in them.

Asci were numerous, thin-walled, without apical structures, 8-spored, clavate, with a short stalk, 45-55 x 15-23 mm, and evanescent at an early age.

Ascospores were one-celled, smooth, thick-walled, hyaline when young and dark brown at maturity, broadly ellipsoidal to ovoid, sometimes lemoniform to biapiculate, with apical or slightly subapical germ pores, and 12-16 x 10-11.5 mm. The vegetative hyphae were smooth, septate, hyaline and branched. The hyphal cells were cylindrical and 1.5-8 mm thick. Mitosporic structures conidia and chlamydospores, were not observed.

Strain No. 217 was able to grow at the temperature range from 2 to 32°C, with the growth optimum at 22 to 26°C. These temperature data were detennined on potato dextrose agar (made by NISSUI).

On the basis of comparing the morphological characteristics with fungal taxonomic criteria by von Arx (J. A. von Arx: The Genera of Fungi- Sporulating in Pure Culture. 3rd ed., pp. 145-163, J. Cramer, Vaduz, 1974) and by Gams et al. strain No. 217 was considered to belong to the ascomycete genus Chaetomium Kunze 1817 (Sphaeriales). Thus, the strain was signed Chaetomium sp. No. 217. This strain has been deposited to the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan, as FERM BP-6917 (deposited date: October 15,1999) under the Budapest Treaty.

Table 1. Cultural characteristics of strain No. 217.

Media Cultural characteristics Malt extract agar* G: Very restrictedly, 0.5-1.0 cm S: Circular to irregular, scanty, plane to sulcate, orange gray (5B2), not formed ascomata R: Grayish yellow (4B3) Potato dextrose agar G: Restrictedly, 1.5-2.0 cm (Difco 0013) S: Circular to irregular, raised, sulcate to crusty, olive (3D3), and yellowish white (3A2) at the margin, not formed ascomata R: Olive (3D3) Czapek's solution agar* G: Very restrictedly, 1.0-1.5 cm S: Irregular, thin, plane, yellowish white (3A2) to dull yellow (3B3), producing immature ascomata R: Yellowish white (3A2) to dull yellow (3B3) Sabouraud dextrose agar G: Very restrictedly, 0.5-1.0 cm (Difco 0190) S: Irregular, scanty, convex, crusty, grayish yellow (4B3-4C3), not formed ascomata R: Grayish yellow (4B3-4C3) Emerson Yp Ss agar G: Fairly rapidly, 3.5-4.5 cm (Difco 0739) S: Circular, plane, felty, sectoring, white (IAI) at the center, yellowish white (4A2) at the margin, grayish yellow (4C3) to olive brown (4D3) at the middle, producing abundant ascomata as brown (6D7) dots on the medium R: Light brown (6D6) at the center, yellowish white (4A2) at the margin, olive brown (4E3) at the middle Corn meal agar G: Rather rapidly, 2.5-3.5 cm (Difco 0386) S: Circular, thin, submerged, plane, white (IAI) to yellowish white (4A2), producing abundant ascomata as light brown (6D6) to dark brown (6F6) dots on or in the medium R: White (ici) to yellowish white (4A2)

MY20 agar* G: Very restrictedly, 1. 0-1.5 cm S: Circular, convex, felty, sulcate to crusty, orange white (5A2), and white (lAl) at the center, not formed ascomata, R: Light yellow (4A4-4A5) Abbreviation G: growth, measuring colony size in diameter, S: colony surface, R: reverse.

* : The compositions of malt extract agar, Czapek's solution agar and MY20 agar were based on JCM Catalogue of Strains (Nakase, T., 5th ed., 503p., Japan Collection of Microorganisms and Life Science Research Information Section of the Institute of Physical and Chemical Research, Saitama, 1992).

These characteristics were observed after 14 days of incubation at 25°C.

The color descriptions were based on Methuen Handbook of Colour (Kornerup, A. and J. H. Wanscher, 3rd ed., 525p., Methuen, London, 1978).

Production of the WF217 substance The WF217 substance is produced when the WF217 substance-producing strain belonging to the genus Chaetomium is grown in a nutrient medium containing sources of assimilable carbon and nitrogen under aerobic conditions (e. g. shaking culture, submerged culture, etc.).

The preferred sources of carbon in the nutrient medium are carbohydrates such as glucose, sucrose, starch, fructose or glycerin, or the like.

The preferred sources of nitrogen are peanut powder, yeast extract, peptone, gluten meal, cotton seed flour, soybean powder, soybean meal, corn steep liquor, dried yeast, wheat germ, etc., as well as inorganic and organic nitrogen compounds such as ammonium salts (e. g. ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.), urea or amino acid, or the like.

The carbon and nitrogen sources, though advantageously employed in combination, need not to be used in their pure form because less pure materials, which contain traces of growth factors and considerable quantities of mineral nutrients, are also suitable for use.

When desired, there may be added to the medium mineral salts such as sodium or calcium carbonate, sodium or potassium phosphate, sodium or potassium chloride, sodium or potassium iodide, magnesium salts, copper salts, zinc salts, iron salts, or cobalt salts, or the like.

If necessary, especially when the culture medium foams seriously, a defoaming agent such as liquid paraffin, fatty oil, plant oil, mineral oil or silicone, or the like may be added.

Agitation and aeration of the culture mixture may be accomplished in a variety of ways, such as agitation by a propeller or similar mechanical agitation equipment, by revolving or shaking the fermenter, and the like.

The fermentation is usually conducted at a temperature between about 10°C and 40°C, preferably 20°C to 35°C, for a period of about 24 hours to 120 hours, which may be varied according to fermentation conditions and scales.

When the fermentation is completed, the culture broth is then subjected for recovery of the WF217 substance to various procedures conventionally used for recovery and purification of biological active substance, for instance, solvent extraction with an appropriate solvent or a mixture of some solvents, chromatography or recrystallization from an appropriate solvent or a mixture thereof.

The WF217 substance obtained can be converted to its salt in a conventional manner. The salt of the WF217 substance may include a salt with an organic or inorganic base such as alkaline metal salt (e. g. sodium or potassium salt), alkaline earth metal salt (e. g. calcium or magnesium salt), organic amine salt (e. g. ethanolamine salt, etc.) amino acid salt (e. g. arginine salt, lysine salt, histidine salt, etc.) and the like.

The WF217 substance has the following physico-chemical properties: (1) Appearance: white needle crystal (2) Molecular : C38H62 I 0 (3) Elementary Analysis: Calcd for C38H62Olo 1/2H20

C 66.35, H 9.23 Found: C 66. 59, H 9. 15 (4) Molecular weight: ESI-MS (positive): m/z 679 (M+H) (5) Melting point: 250-255 °C (dec.) (6) Optical rotation: [a] D (23°C) =-22 ° (c=0. 5, pyridine) (7) Ultraviolet absorption spectrum: Smax (methanol): end absorption (8) Solubility: Soluble: dimethylsulfoxide, tetrahydrofuran Slightly soluble: methanol Insoluble: water (9) Color reaction: Positive: cerium sulfate reaction, iodine vapor reaction, Negative: ninhydrin reaction, Dragendorff reaction, Ehrlich's reaction, ferric chloride reaction (10) Thin layer chromatography (TLC): Stationary phase Developing solvent Rf value Silica Gel 60 chloroform : methanol = 5 : 1 0.44 F254* * made by E. Merck (11) High performance liquid chromatography (HPLC) : Condition: Column: YMC-Pack ODS-AS-302 ** (4.6 mm x 150mmL) Mobile phase: 70% aqueous acetonitrile containing 0.1% trifluoroacetic acid Flow rate: 1 ml/min.

Detection: UV at 210 nm Retention time: 6.3 min.

** : made by YMC Co., Ltd.

(12) Infrared absorption spectrum: vmax (KBr): 3400,2960,1730,1720,1650,1460,1380,1370,1250, 1080,1040,1020 cm-' (13)'H Nuclear magnetic resonance spectrum: (500 MHz, DMSO-d6) 8 (ppm): 4.98 (1H, m), 4.19 (1H, d, J=8 Hz), 3.65 (1H, m), 3.48 (1H, m), 3.21 (1H, d, J=10 Hz), 3.11 (1 H, m), 3.07-3.02 (2H, m), 2.91 (1 H, m), 2.60 (1H, s), 2.18-2.12 (2H, m), 2.00 (3H, s), 2.00-1.80 (4H, m), 1.71-1.53 (4H, m), 1.40-1.29 (2H, m), 1.22-1.10 (SH, m), 1.13 (3H, s), 1.08 (3H, s), 1.03 (3H, s), 0.99 (3H, s), 0.95 (3H, s), 0.86 (3H, d, J=7 Hz), 0.85 (3H, s) (14)'3C Nuclear magnetic resonance spectrum: (125 MHz, DMSO-d6) 8 (ppm): 175.2 (s), 170.0 (s), 133.6 (s), 133.4 (s), 104.8 (d), 87.8 (d), 77.0 (d), 76.5 (d), 74.2 (d), 70.0 (d), 69.3 (d), 61.1 (t), 52.3 (d), 49.4 (d), 47.7 (d), 41.0 (t), 40.4 (s), 40.0 (s), 39.7 (s), 37.7 (s), 37.1 (s), 30.8 (t), 28.9 (t), 27.6 (q), 26. 3 (t), 25.9 (d), 25.2 (t), 24.7 (q), 21.7 (q), 21. 0 (q), 20.8 (q), 20.6 (q), 18.9 (t), 18.3 (t), 18.2 (q), 17.4 (q), 16.5 (q), 8.0 (q) Biological properties of WF217 substance Antimicrobial activity Antimicrobial activities of WF217 substance were determined by a serial broth dilution method using 96-well microtiter plate in 100 u. l of MEM (Eagle's minimum essential medium) for Candida albicans and in 100 p1 of yeast nitrogen base dextrose medium for both Aspergillus fumigatus and Cryptococcus neoformans. The inoculum was adjusted to 1 x 105 colony forming units/ml. Candida albicans and Aspergillusfumigatus were cultured at 37°C for 24 hours and Cryptococcus neoformanswas cultured at 37°C for 48 hours in 5% CO2 incubator. After incubation, the growth inhibition of microorganism in each well was determined by microscopic observation. The results were shown as MEC (minimum effective concentration: ptg/ml) value (Table 2).

Table 2. Antimicrobial activities of WF217 substance.

Microorganisms MEC (pLg/ml) FP633 1.56 Candida albicans FP 1305 0.04 Aspergillus fumigatus YC203 15.6 Cryptococcus neoformans The phannaceutical composition of this invention can be used in the form of pharmaceutical preparation, for example, in solid, semisolid or liquid form, which contains the WF217 substance or its pharmaceutically acceptable salt, as an active ingredient in admixture with an organic or inorganic carrier or excipient suitable for external, enteral or parenteral administrations. The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, injections, ointments, liniments, eye drops, lotion, gel, cream, and any other form suitable for use.

The carriers which can be used are water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea and other carriers suitable for use in manufacturing preparations, in solid, semisolid, or liquid form, and in addition auxiliary, stabilizing, thickening, solubilizing and coloring agents and perfumes.

For applying the above pharmaceutical composition to patients including human beings and animals, it is preferable to apply it by intravenous, intramuscular, topical percutaneous, pulumonary, insufflation or oral administration. While the dosage oftherapeutically effective amount of the WF217 substance or a pharmaceutically acceptable salt thereof varies depending upon the age and conditions of each individual patient to be treated, the optimal dosage for the treatment of the patient may be selected from the range of 0.01-50 mg of the WF217 substance per kg weight of the patient.

The following examples are given for the purpose of illustrating the present invention, but not limited thereto.

(1) Fermentation of the strain No. 217 for the production of the WF217 substance An aqueous seed medium (100 ml) containing sucrose 4%, glucose 1 %, soluble starch 2%, pharmamedia 3%, soybean powder 1.5%, KH2PO4 1 %, CaC03 0.2%, Adekanol LG-109 0.05%, and Silicone KM-70 0.05% was placed in each of ten 500-ml Erlenmeyer flasks and was sterilized at 120°C for 30 minutes. A loopful of the slant culture of the strain No. 217 was inoculated in each of the seed flasks. The inoculated flasks were shaken on a rotary shaker (220 rpm, 5.1 cm throw) at 25°C for 8 days, and 20 ml of the seed culture was transferred to each of forty 500-ml Erlenmeyer flasks containing 130 ml of the above sterile seed medium. The flasks were shaken on a rotary shaker (220 rpm, 5.1 cm throw) at 25°C for 4 days, and 3 L (20 flasks) of the second seed culture was inoculated to each of two 30-liter jar fermentors containing 17 liters of the above sterile seed medium.

Fermentation was carried out at 25°C for 3 days under aeration of 20 liters/minute and agitation of 200 rpm, and 40 L (two jars) of the third seed culture was inoculated to 160 liters of sterile production medium consisting of glucose 1 %, pinedex 14%, wheat germ 2%, Adekanol LG-109 0.05%, and Silicone KM-70 0.05% in a 300-liter jar fermentor. Fermentation was carried out at 25°C for 8 days under aeration of 20 liters/minute and agitation of 200 rpm.

The production of the WF217 substance in the fermentation broth was monitored by HPLC analysis indicated below.

# Analytical HPLC condition Column: YMC Pack ODS-AS 302, S-5 120A (150 x 4.6 mm I. D., YMC Co., Ltd.) Mobile phase: 70% aqueous acetonitrile containing 0.1 % TFA Flow rate : 1 ml/min.

Detection : UV at 210 nm

Retention time: WF217 6.3 min.

(2) Isolation and purification of the WF217 substance The culture broth (160 liters) was extracted with the mixture of 80 liters of acetone and 80 liters of methanol by stirring for 2 hours at room temperature.

The resultant mixture was filtered with an aid of diatomaceous earth. The filtrate was passed through a column (6 L) of SEPABEADS SP-207 (Mitsubishi Chemical Co., Ltd). The column was washed with 50% aqueous methanol (18 L) and then eluted with methanol (30 L). The active fraction (5-25 L) was concentrated under reduced pressure to give crude crystals.

The crude crystals were filtered, dried up, dissolved in a small amount of tetrahydrofuran, and recrystallized from methanol. The crystals were filtered and dried up to give 10.28 g of WF217 substance as white needle.