CA1334180C - Antibiotics r106 - Google Patents

Abstract

Novel antibiotics R106 represented by general formula (I):

... (I) wherein:
R is methyl or ethyl;
X1 is MePhe, .beta.-HOMePhe or Phe;
X2 is allo-Ile, Val or Leu;
X3 is MeVal or Val;
X4 is .beta.-HOMeVal, .gamma.-HOMeVal, MeVal, Val, N,.beta.-MeAsp, .beta.-HOMePhe, MePhe, MeDH2,3Val or MeDH3,4Val are useful as therapeutic agents for fungal infection.

Description

The present invention relates to novel antibiotics R106 which are useful as therapeutic agents for fungal infection, and a process for producing the same as well as use thereof.
As therapeutic agents for fungal infection, there are known approximately 20 antibiotics including amphotericin B, nystatin, trichomycin, griseofulvin, pyrrolnitrin, clotrimazole, miconazole nitrate, etc. However, these antibiotics are questionable in activity and toxicity.
An object of the present invention is to provide novel antibiotics which have a high activity but low toxicity as therapeutic agents for fungal infection.
The present inventors isolated a number of microorganisms from the surface of plant leaves or from the soil, for purposes to discover novel antibiotics, purified antibiotics produced by these microorganisms and examined the biological properties of these antibiotics. As a result, it has been found that a series of antibiotics showing an antimicrobial activity against pathogenic fungi such as Candida albicans, Cryptococcus neoformans, etc. can be produced in fermentation broth of microorganisms belonging to the genus Aureobasidium.
These antibiotics have been isolated from the fermentation broth; and as the result of examination of physicochemical pro-perties of the antibiotics, it has been confirmed that these antibiotics are novel compounds that are not found in litera-tures. These antibiotics have been named R106. Thus, the present invention provides antibiotics R106 having the following structural formula and a process for production thereof as well as application thereof. ~

RCHCHCO > MeVal ~ Phe > Xl 1 --(I) 4 Leu ~ X3 C X2~ Pro wherein:
R is methyl or ethyl;
Xl is MePhe, ~-HOMePhe or Phe;
X2 is allo-Ile, Val or Leu;
X3 is MeVal or Val;
X4 is ~-HOMeVal, ~-HOMeVal, MeVal, Val, N,~-MeAsp, ~-HOMePhe, MePhe, MeDH2 3Val or 3,4 Abbreviations for amino acids used in the above formula (I) are given in Table 9 later shown.
First, microorganisms used for the present invention can be a strain of the genus Aureobasidium which is capable of producing antibiotics R106 of the present invention represented by the general formula (I). An example of the microorganisms is Aureobasidium No. R106 (hereinafter this strain is referred to as strain No. R106), which the present inventors newly isolated from a soil sample collected at Kamitushima-cho, Kamiagata-gun, Nagasaki-ken, Japan. This strain is a new strain that has the characteristics described above and can produce advantageously novel antibiotics R106 of the present invention. Thus, this strain is one of the microorganisms which can be effectively used for the method of the present invention. Further, not only mutants of strain No. R106 which are prepared by natural or artificial mutation, but all micro-t 33 4 1 8~

organisms of the genus Aureobasidium which are capable of pro-ducing the antibiotics R106 of the present invention can be used for the method of the present invention.
Strain No. R106 has the following mycological charac-teristics.
(1) Growth on various nutrient media.
The following table shows cultural characteristics of strain No. R106 on various nutrient agar media after incubation for 4, 7, and 14 days at 25C.
Strain No. R106 shows good growth on potato-dextrose agar, Czapek agar, and malt extract agar media. Colonies of the strain are usually mucoid, pasty, or rarely velvety, and then become leathery as time goes by. The colonies are white to creamy or light pink in color, then changing olive green to light brown or brown, sometimes finally to black with production of dark brown pigments, which is insoluble, as time goes by. Rhizoid-like structures are often formed around the colonies. Hyphae with a size of 2 to 15 ~m in diameter elongate into the agar medium without forming aerial mycelia. Blastic conidia with a size of 1-5 x 2-10 ~m, are often formed intercalary or terminal on hyphae like finger tips and sometimes form ball-like clusters.
Vegetative cells in early stage of growth are yeast-like, and ellipsoidal or lemon-like in shape with a size of 3-5 x 8-15 ~m and multiply by polyblastic budding. The strain forms arthrospores with a size of 4-10 x 8-20 lum and chlamydospores with a size of 5-25 x 10-25 /um. No ascospores are observed.

Color of colonies after Growth Media 4-7-14 days cultivation characteristics Malt extract agar Creamy - olive green - Good growth light brown Chlamydospores are formed.
Potato dextrose Creamy - olive green - Good growth agar light brown Blastic conidia are abundant and form ball-like clusters.
Czapek agar Creamy - brown - black Good growth Hyphae are abundant, and often thick-walled.
Sabouraud agar Creamy - olive green - Good growth dark green Chlamydospores are observed.
Oatmeal agar Creamy gray - Moderate growth light brown - dark brown Hyphae are abundant.
YpSs agar Creamy - light olive Good growth green - olive green Hyphae are thin.
Blastic conidia are abundant and form ball-like clusters.

(2) Physiological characteristics.
1) Temperature range permitting growth Temperature permitting growth : 12.5-29.0C
Optimum temperature for growth : 23.0-29.0C
2) Vitamin requirement Growth in vitamin-free medium : Good growth.

3) Pigment formation Insoluble dark brown pigments are produced. From the foregoing mycological characteristics, strain No. R106 is considered to belong to the genus Aureobasidium. Among the known species of Aureobasidium listed in "W.B. Cooke: Mycopathologia et Mycologia Applicata, 17 , 1-43 (1962)", "J.A. von Arx: The Genera of Fungi Sporulating in Pure Culture, J. Cramer, Lehre (1970)", "G.S. de Hoog & E.J. Hermanides-Nijhoff: Studies in Mycology, No. 15, p. 141-166, CBS, Baarn (1977)" and other references, strain No. R106 is considered to be a member of A. pullulans from its characteristics. But known A. pullulans did not produce an antibiotic R106. So we designated the strain as Aureobasidium pullulans No. R106, and the strain was deposited on July 8, 1988 at the Fermentation Research Institute, Agency of Industrial Science and Technology, Japan, under deposit number FERM BP-1938.
The antibiotics R106 of the present invention can be produced by inoculating and culturing the strain described above in a nutrient medium.
In culturing the R106-producing organisms, there may be appropriately used as carbon sources, for example, glucose, fructose, saccharose, starch, dextrin, glycerin, molasses, thick malt syrup, oils and fats, organic acids, etc.; as nitrogen sources, organic nitrogen compounds or inorganic nitrogen compounds such as soybean powder, cotton seed powder, corn steep liquor, casein, peptone, Casamino acids, yeast extract, meat extract, germ, urea, amino acids, ammonium salts, etc.; as salts, inorganic salts, for example, sodium salts, potassium salts, calcium salts, magnesium salts, phosphates, etc.; singly or in appropriate combination. If necessary and desired, heavy metals salts, e.g., iron salts, copper salts, zinc salts, cobalt salts, etc.; vitamins such as biotin, vitamin Bl, etc.; other organic or inorganic compounds which can assist growth of the producing organisms and accelerate production of R106, may be suitably added. Furthermore, defoaming agents or surface active agents such as silicone oil, polyalkylene glycol ethers, etc. may also added to the medium.
For culture, conventional techniques used for production of antibiotics by fermentation of microorganisms may be adopted, and liquid culture methods especially by shaking or tank fermentation with aeration and agitation are most suitable. Further by appropriately supplementing carbon sources, nitrogen sources, trace salts, etc. during the course of incubation, the amount of antibiotics R106 production can be increased. A preferred temperature for the culture is generally in a range of 15 to 30C. A pH value for the culture is preferably in a range of 2 to 8. Days for fermentation may vary depending upon culture conditions but are generally 1 to 14 days.
The R106 thus accumulated in the fermentation broth can be advantageously collected therefrom by utilizing the physico-chemical properties of the antibiotics.
That is, antibiotics R106 are contained in the fermentation broth and mycelial cake and can thus be obtained by extracting the whole fermentation broth with a hydrophobic organic solvent, for example, an organic solvent such as ethyl acetate, butyl acetate, chloroform, butanol, methyl isobutyl ketone, etc.
Furthermore, antibiotics R106 can also be obtained after separa-tion of the fermentation broth into the broth filtrate and t 334 t 8~

mycelial cake by filtration or centrifugation. For isolation of R106 from the broth filtrate, the broth filtrate may be ext-racted with the aforesaid hydrophobic organic solvents.
Alternatively, the broth filtrate may be brought into contact with an appropriate supporting resins to adsorb R106 in the filtrate thereto followed by elution with an appropriate solvent.
For the purpose, supporting resins such as, for example, acti-vated charcoal, cellulose powder, adsorptive resin, etc., which separate compounds according to their differences in the adsorb-ability to the resin, can be advantageously used. In order to elute antibiotics R106 from these resins aqueous solution of hydrophilic organic solvents, for example, aqueous acetone, aqueous alcohol, etc. can be used in appropriate combination, though the combination varies depending upon kind and property of the resin. Antibiotics R106can be obtained from mycelial cake by extracting with a hydrophilic organic solvent such as acetone or the like.
Crude R106 thus obtained can be further purified by con-ventional purification methods used for lipophilic antibiotics.
A example of the methods is a column chromatography with a sup-porting resin such as silica gel, activated aluminum, activated charcoal and adsorbtive resin.
In the silica-gel column chromatography antibiotics R106 can be eluted with chloroform, ethylacetated, methanol, acetone, water, etc. are singly or in appropriate combination.
Isolation and purification by high performance liquid chromatography can also be advantageously utilized. Examples of the supporting resins which can be used include silica gel, silica gel with chemical bonds such as octadecyl, octyl or amino groups, or polystyrene type porous polymer gel, etc.
As a mobile phase, there may be used a mixed solvent of hexane, isopropyl alcohol and chloroform, etc., aqueous methanol or aqueous acetonitrile, etc.
Counter current chromatography which is a purification method based on differences of compounds in partition between two liquid phases can also be advantageously utilized. As the partition solvent system, there may be used a solvent mixture of hexane-ethyl acetate-acetonitrile, chloroform-methanol-water or the like.
The physicochemical and biological properties of these new antibiotics R106 are as explained below by referring partly to the accompanying drawings wherein:
Fig. 1 is a graph showing the ultraviolet absorption spectrum of the compound 1, and Fig. 2 is a graph showing the infrared absorption spectrum of the same compound.
(1) Physicochemical properties Structures of antibiotics R106 obtained in accordance with the present invention are shown in Table 1.

Table 1 RCHCHCO ~ MeVal - ~ Phe > X
0~ 1 x4 ~ Leu < X3 X2 ~ ro Compound No. R Xl _2 ~ _4 1 C2H5 MePheallo-Ile MeVal B-HOMeVal 2 CH3 MePheallo-Ile MeVal B-HOMeVal 2 5 MePheVal MeVal ~-HOMeVal 2 5 MePheallo-Ile MeVal ~-HOMeVal C2H5 B-HoMePhe allo-Ile MeVal B-HOMeVal 6 C2H5 MePheallo-Ile Val B-HOMeVal 2 5 MePheallo-Ile MeVal MeVal 2 5 MePheallo-Ile MeVal Val 9 C2H5 MePheLeu MeVal B-HOMeVal C2H5 MePheallo-Ile MeVal N,B-MeAsp 11 CH3 MePheallo-Ile MeVal MeVal 12 C2H5 MePhe Val MeVal MeVal 13 C2H5 Phe allo-Ile MeVal MeVal 14 C2H5 MePhe allo-Ile MeVal e 3,4V
C2H5 MePhe allo-Ile MeVal B-HOMePhe 16 C2H5 MePhe allo-Ile Val MeVal 7 C2H5 MePhe allo-Ile MeVal MePhe 18 C2H5 MePhe allo-Ile MeVal 2,3 _ g _ Abbreviations for the amino acids in Table l are indicated in Table 9 later presented.

Physicochemical properties of compound 1 are as follows.
(1) Molecular formula: C60H92N8Oll (2) Elemental analysis: C 65.0%, H 8.5%, N 9.9% (found) C 65.45%, H 8.36%, N 10.18% (calcd.) (3) Melting point: 138-140C

(4) Specific rotary power: [a] D -254.3 (C 1.0, methanol) (5) Molecular weight: FAB-MS m/z 1101 (M+H), 1123 (M+Na) (6) Ultraviolet absorption spectrum (in methanol): as shown in Fig. 1 (7) Infrared absorption spectrum (KBr method): as shown in Fig.

8) Amino acid analysis: Proline, alloisoleucine, leucine and phenylalanine are detected (device: JCL-300 manufactured by JEOL Co., Ltd.; Detection: ninhydrin reaction) 9) Color forming reaction: positive in 50% sulfuric acid and potassium permanganate and negative in ninhydrin and ferric chloride 10) Solubility in solvent: soluble in chloroform, methanol, ethanol, N,N-dimethylformamide and dimethylsulfoxide;
sparingly soluble in water 11) Acidic, neutral or basic: neutral substance (12) Color: white substance Physicochemical properties of compounds 2 through 18 are shown in Table 2.

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t 3341 80 (2) Biological properties The antibiotics R106 of the present invention are active against various fungi including pathogenic fungi. The minimum inhibitory concentration (MIC) of each antibiotic against various fungi was determined by agar dilution method using casitone agar medium (2.0% glucose, 0.9%
bacto-casitone, 1.0% yeast extract, 0.1% KH2PO4, 0.1% Na2HPO4, 1.0% sodium citrate, 2.0% agar: concentrations are all by w/v).
The results determined with compound 1 are shown in Tables 3 and 4. The results determined with compounds 2 through 18 are shown in Table 5.

t 334 1 80 Table 3 Minimum Inhibitory Strain TIMM No. Concentration (~g/ml) Candida albicans 0144 0.04 Candida albicans 1529 0.04>
Candida albicans 1623 0.04>
Candida albicans 1308 0.04 var. stellatoidia Candida tropicalis 0312 0.08 Candida tropicalis 0315 0.08 Candida kefyr 0298 0.16 Candida parapsilosis 0287 0.16 Candida krusei 0270 0.04 Candida guilliermondii 0257 0.08 Candida glabrata 1062 0.04 Candida glabrata 1064 0.08 Cryptococcus neoformans 0354 0.63 Cryptococcus neoformans 0355 0.31 Cryptococcus neoformans 0363 1.25 Cryptococcus laurentii 0352 0.31 Cryptococcus terreus 0424 0.31 Rhodotorula rubra 0923 0.63 Each minimum growth inhibitory concentration was determined after culturing at 27C for 4 days.

Table 4 Minimum Inhibitory Strain TIMM No. Concentration (~g/ml) Aspergillus clavatus 0056 0.16 Aspergillus flaw s 0058 ?80 Aspergillus nidulans 0112 0.16 Aspergillus niger 0113 ~80 Aspergillus terreus 0120 5 Aspergillus citrinum 1330 ~80 Aspergillus commune 1331 1.25 Aspergillus crustorum 1332 ~80 Trichophyton mentagrophytes 1177 >80 Trichophyton rubrum 1216 ~80 Microsporum canis 0760 ~80 Epidermophyton floccosum 0431 2.5 Fonsecaea pedrosoi 0482 0.31 Phialophora verrucosa 0903 >80 Exophiala werneckii 1334 1.25 Cladosporium bantianum 0343 0.63 Cladosporium carrionii 0337 1.25 Sporothrix schenckii 0959 ~ 80 Histoplasma capsulatum 0713 0.16 Histoplasma capsulatum 0714 0.08 Paracoccidioides brasiliensis 0878 80 Paracoccidioides brasiliensis 0880 0.31 Geotrichum candidum 0694 0.63 Trichosporon cutanum 1318 >80 Blastomyces dermatitidis 1690 0.04>
Blastomyces dermatitidis 0126 0.31 After culturing at 27C for 7 days, each minimum inhlbitory concentration was determined.

Table 5 TIMM Compound No.
Strain No. 2 3 4 S 6 7 8 9 Candida albicans0136 0.10 0.10 0.200.05~ 0.78 3.12 1.56 0.05 Candida albicans0171 0.10 0.10 0.100.05> 0.39 1.56 1.56 0.05 Candida albicans1768 0.78 1.56 0.39 0.05> 0.393.12 12.5 0.20 Candida kefyr 0301 0.20 0.20 0.78 0.20 0.39 2525< 0.20 Candida glabrata1062 0.20 0.20 1.56 0.20 1.56 25< 25< 0.39 Cryptococcus neoformans 03541.56 1.56 25 3.12 25 25< 25< 25 TIMM Compound No.
Strain No. 10 11 12 13 14 15 16 17 18 Candida al~icans 0136 0.78 1.56 6.25 6.25 1.56 12.5 3.12 12.5 6.25 Candida albicans 0171 0.78 1.56 6.25 12.5 1.56 25 1.56 25 3.12 Candida albicans 1768 12.5 1.56 25 12.5 12.5 25 25 25< 25 Candida kefyr 0301 3.12 6.25 12.5 25 6.25 25< 3.12 25C 25 Candida glabrata 1062 3.12 25< 25< 25< 12.5 25< 25< 25< 25<

Cryptococcus neoformans 0354 25 25< 25C 25~ 25( 25 25< 25~ 25<

Antibiotics R106 exhibit a potent therapeutic effect on systemic candidiasis model of mice prepared by intravenous injection of Candida albicans. Candida albicans TIMM 1768 was cultured in Sabouraud-dextrose broth at 37C overnight, and the cells were collected and suspended in physiological saline.
A cell suspension containing 1 x 106 cells was intravenously injected to ICR strain mice (5 week age, female). After 3 hours, a solution of an anti~iotic R106 in Tween 80 -ethanol-physlological saline (1 : 9 : 90, v/v) was subcutaneously, intravenously or orally given to the mice in various concentra-tions, and then once a day for 4 days the R106 drug solution was given. Mortality was assessed for 30 days after infection to determine the therapeutic effect. The results obtained by measurement with compound 1 are shown in Table 6.

trade-mark ", Table 6 Number of Animal Survived Dose Mean /Number of Route (mg/kg) Survival Day T/C (%) Animal Tested p.o. 7 14.8 + 4.8 121 0/5 14 23.2 + 6.2 189 0/5 28 30.0 + 0 245 5/5 s.c. 1.75 14.2 + 2.9 116 0/5 3.5 18.8 + 3.5 153 0/5 7 22.4 + 7.0 183 2/5 14 27.2 + 6.3 222 4/5 i.v. 0.875 14.6 + 1.9 119 0/5 1.75 18.2 + 5.0 149 0/5 3.5 19.2 + 6.3 157 0/5 7 29.0 + 2.2 237 4/5 14 26.6 + 5.0 217 3/5 Control 0 12.3 + 3.2 100 0/12 T/C is a value which expresses the mean survival day of the treated group to the mean survival day of the not treated control group by %.

All of the ant~biotics R106 show low toxicity. The results of 50%
lethal dose (LD50) obtained when each of representative compounds of the present invention was given to mice intravenously, intraperitoneally and orally are shown in Table 7.

Table 7 LD50 (mg/kg) Compound No. i.v. i.p. p.o.
1 >200 >400 >1000 2 >100 ~200 >200 >100 ~200 >200 7 >100 ~200 ~200 9 >100 ~200 ~200 From the foregoing biological properties, it is evident that the antibiotics R106 are useful as therapeutic agents for various fungal infections such as candidiasis, histoplasmosis, blastmycosis, etc.
When the compound of the present invention is administered as drugs, the compound can be administered to animals including human as it is, or as pharmaceutical compositions comprising, for example, 0.1 to 99.5~, preferably 0.5 to 90% of the compound in pharmaceutically acceptable non-toxic inert carrier.
As the carrier, solid, semisolid or liquid diluents, fillers and one or more of other aids for formulation can be used. The pharmaceutical composition is desirably administered in a single dose unit form. The pharmaceutical composition of the present invention can be administered orally, intratissularly, topically ~ 3341 80 (percutaneously) or per rectU~ but, oral compositions and injections are preferred. Needless to say, the compound can be administered in preparations suited for these routes for administration.
It is desired that dose as the antifungal agent be determined, taking into account conditions of the patient such as age, body weight, etc., route for administration, condition and degree of disease, etc. In general, it is general to administer the compound of the present invention calculated as an effective ingredient in a range of 10 to 2000 mg per day. If the occasion demands, the dose may be smaller or larger than the range. When a large dose is required, the dose may be desirably portionwise given several times a day.
Next, the present invention is described in more detail by referring to the examples below.
Example 1 One platinum loop from a slant culture of strain No. R106 ~deposited at the Fermentation Research Institute or the Agency of Industrial Science & Technology under deposition No. 1938 (FERM BP-1938)~ was inoculated in 100 ml of liquid medium (0.67% (w/v) Difco yeast nitrogen base, 2 ~ (w/v) glucose) in a 500 ml Erlenmeyer flask and shaken at 27C for 2 days to give seed culture. The seed culture, 1400 ml, was transferred into a 200-liter fermentor of containing 140 liters of the liquid medium described above followed by fermentation at 25C for 63 hours with aeration (100 liters/min.) and agitation (150 rpm). The fermentation broth was cent-rifuged to separate into the supernatant and the mycelial cake. To the mycelial cake was added 8 liters of acetone.

After thoroughly mixing, acetone extract of mycelial cakewas obtained. The acetone extract was concentrated under reduced pressure to give 81.5 g of the residue. Methanol was added to the residue to give an active extract. The methanol extract was condensed under reduced pressure to give 65.4 g of the residue. The resulting residue was subjected to a silica gel (made by Merck) column (9 cm x 35 cm).
The column was eluted with 7 liters of chloroform-methanol (49 : 1) to give the active fraction. The fraction was con-densed under reduced pressure to give 10.6 g of the residue.
The resulting residue was subjected to preparative high per-formance liquid chromatography ¦column : PrepPak-500/C18 (5.7 cm x 30 cm) (manufactured by Waters), mobile phase :
70% (v/v) acetonitrile-wate~, to give the active fraction.
The fraction was condensed under reduced pressure to give 1.5 g of crude R106. The crude substance, 475 mg, was again subjected to preparative high performance liquid chro-matography ~column : Capcell Pak-500/C18 (1 cm x 25 cm) (manufactured by Shiseido Co., Ltd.), moving phase : 70 %
(v/v) acetonitrile-water~ to obtain the active fraction that shows the largest peak in active fractions. The fraction was condensed under reduced pressure to give 370 mg of compound 1 as white powders. The activity was determined by measuring an antifungal activity against Candida albicans TIMM 0136 according to the paper disk diffusion method using Casitone agar plate. Further the peak in high performance liquid chromatography was detected by measuring ultraviolet absorbance at 230 nm.

* trade-mark 1 334t 80 Example 2 The seed culture, lO00 ml, of strain No. R106 prepared in a manner similar to Example 1, was inoculated in lO0 liters of liquid medium (2% glucose, 0.5% ammonium sulfate, 0.15% KH2PO4, 0.05% MgSO4 7H2O, 0.01% CaCl2, 0.01% NaCl (concentrations are all by w/v), 0.5 ~ug/ml FeCl2, 0.5 lug/ml ZnSC-4) in a 200-liter fermentor followed by fermentation at 25C for 72 hours with aeration (lO0 liter/min) and agitation (lO0 rpm). To the culture was supplemented 20 liters of liquid medium (10% glucose, 2.5% ammonium sulfate, 5% poly-peptone, 0.75% KH2PO4, 0.25% MgSO4 7H2O, 0.05% CaC12, 0.05%
NaCl (concentrations are all by w/v), 2.5 lug/ml FeC12, 2.5 jug/ml ZnSO4), and fermentation was further carried out at 25C for 65 hours with aeration (120 liters/min) and agita-tion (100 rpm).
The thus obtained fermentation broth was centrifuged to separate into the supernatant and the mycelial cake.
To the obtained mycelial cake was added lO liters of ethanol to extract Rl06. The ethanol extract was concentrated under reduced pressure to remove ethanol and the residue was extracted twice with l liter of ethyl acetate. The ethyl acetate extract was concentrated under reduced pressure to dryness, and the residue was dissolved in chloroform. The chloro-form solution was subjected onto 1.5 liter of a silica gel column which had been previously saturated with hexane.
After washing with 3 liters of hexane, the column was developed and eluted with 6 liters of hexane-isopropanol (7 : 3).

The active fraction was condensed under reduced pressure to give 15 g of the residue. The residue was dissolved in 100 ml of acetonitrile and the solution was divided into 30 and subjected to preparative high performance liquid chromatography [column : Soken Pak/C18 (5 cm x 50 cm) (manufactured by Soken Chemical Co., Ltd.), mobile phase :
70% (v/v) acetonitrile-water~. The 18 active fractions eluted at the retention time shown in Table 8 were respectively collected and condensed under reduced pressure to give compounds 1 through 18 as white powders in amounts shown in Table 8. The activity detection and the peak detection of high performance liquid chromatography was carried out in the same manner as shown in Example 1.

* trade-mark Table 8 CompoundNo. Retention Time (min) Yield (mg) 167.0 3500 2 58.8 55 3 60.0 54 4 54.3 96 50.6 81 6 42.7 72 7 131.6 1380 8 72.5 32 9 62.2 60 44.5 12 11 111.4 76 12 112.6 60 13 109.8 24 14 91.6 107 90.4 42 16 75.0 78 17 146.7 18 18 102.2 15 Table 9 Val : valine MeVal: N-methylvaline B-HOMeVal: ~-hydroxy-N-methylvaline -N-CH-CO-y-HOMeVal: y-hydroxy-N-methylvaline -N-CH-CO-CH

MeDH2 3Val: N-methyl-2,3-didehydrovaline -N-C-CO-C

MeDH3 4Val: N-methyl-3,4-didehydrovaline -N-CH-CO-~ 334 1 80 Phe: phenylalanine MePhe: N-methylphenylalanine B-HOMePhe: B-hydroxy-N-methylphenylalanine -N-CH-CO-CHOH

allo-Ile: alloisoleucine Leu: leucine Pro: proline N,B-MeAsp: N,B-dimethylaspartic acid -N-CH-CO-CH

The antibiotics R106 of the present invention are novel antibiotics produced by the strains belonging to the genus Aureobasidium, have low toxicity and high antifungal activity against pathogenic fungi such as Candida albicans, Cryptococcus neoformans, etc. Therefore, the antibiotics are useful as clinical drugs, for example, therapeutic agents for fungal infection.

Claims (4)

1. A strain of the genus Aureobasidium designated Aureobasidium pullulans No. R106, having deposit number FERM
BP-1938 at the Fermentation Research Institute, Agency of Industrial Science and Technology, Japan.

2. A process for producing an antibiotic R106 represented by the general formula (1) described below, comprising culturing Aureobasidium pullulans No. R106 and collecting the said antibiotic from the fermentation broth:

... (I) wherein:
R is methyl or ethyl;
X1 is MePhe, .beta.-HOMePhe or Phe;
X2 is allo-Ile, Val or Leu;
X3 is MeVal or Val;
X4 is .beta.-HOMeVal, .gamma.-HOMeVal, MeVal, Val, N,.beta.-MeAsp, .beta.-HOMePhe, MePhe, MeDH2,3Val or MeDH3,4Val

3. An antibiotic R106 represented by general formula (I):

... (I) wherein:
R is methyl or ethyl;
X1 is MePhe, .beta.-HOMePhe or Phe;
X2 is allo-Ile, Val or Leu;
X3 is MeVal or Val;
X4 is .beta.-HOMeVal, .gamma.-HOMeVal, MeVal, Val, N,.beta.-MeAsp, .beta.-HOMePhe, MePhe, MeDH2,3Val or MeDH3,4Val, whenever produced by the process of claim 2.

4. An antifungal agent containing the antibiotic R106 defined in claim 3.