1 3,244,592 United States Patent 0 Fatented Apr. 5, 1966

1 2 Starch .-—Elevated growth. Vegetative mycelium, 3,244,592 ASCOMYCIN AND PRQCESS FQR 1T§ cream colored. Aerial mycelium, mouse-gray, powdery. PRGDUCTEGN The surface of the growth, mosaic of gray and black. Tadashi Arai, 1-71 Nogata-machi, Nalranaku, No soluble pigment. Tokyo-to, Japan Yeast extract .-—Growth, yellowish-brown, N0 Drawing. Filed May 1, 1963, Ser. No. 277,111 wrinkled, with cracks on its surface. Aerial mycelium, Claims priority, application Japan, .l‘une 9, 1962, poor, white, grows on the surroundings of colony. No 37/215,253 soluble pigment.‘ 4 Claims. (Cl. 167-65) Potato plug.—Growth, ?at, spread. Aerial mycelium, This invention relates to a new and useful substance white, cottony. The color of the plug is not changed. called ascomycin, and to its production. More particu Carrot plug.—Growth, cream-colored, spotted, with larly it relates to. processes for its production by fermenta~ subsided center. Aerial mycelium, abundant, white, cot tion and methods for its recovery and puri?cation. The tony. No color change of the plug. invention embraces this compound in dilute solutions, Litmus milk.-Thin surface growth, pale-cream colored. as crude concentrates and as puri?ed solids. Ascomycin Aerial mycelium, white. No soluble pigment. Coagu is an effective inhibitor of. ?lamentous fungi, e.g. Penicil lated from seventh day, digested gradually. lium chlysogenum; at very low concentrations, e.g. about Gelatin.—No pigment formation. strong lysis. one part per million in a nutrient agar, and does not Blood' again-Colony, grayish-brown, round shaped, inhibit various Gram-positive and Gram-negative bac with subsided center. No aerial mycelium. No soluble teria even at much. higher concentrations. pigment. Strong hemolysis. There is now‘provided, according to the present inven Cellulose medium.—Grows in synthetic culture solu tion, a new antifungal substance identi?ed as ascomycin tion‘ containing cellulose as a sole carbon source. which is produced by a process which comprises cultivat Starch hydr0lysis.-—Strong. ing a strain of Streptomyces hygroscop‘icus var. arcomy Nitrate reduction.—-Negative. ceticus, and preferably A.T.C.C. 14891 or an isolate, Sugar utilization using Pridham’s synthetic media con mutant, descendant or variant thereof, in an aqueous taining various sugars is as follows: carbohydrate solution containing an organic nitrogenous Glucose ++, Starch ++, Xylose +, Dextrin —|—+, nutrient under submerged aerobic conditions, e.g. at about Arabinose +, Mannitol +»—|-, Rhamnose ++, Sorbitol 26° C., for. at least twelve hours until substantial activity i, Fructose ++, Dulcitol —, Galactose ++, lnositol ——, against ?lamentous fungi, e.g. Aspergillus niger, is im 30 Saccharose ++, Adonitol -|—, Maltose ++, Salicin —, parted to said. solution and then, if desired, recovering Lactose +, Sodium acetate -|—, Ra?inose +, Sodium said ascomycin. citrate -—, lnulin —, Sodium succinate +. There is further provided, according to the present in In several media, aerial mycelia have a tendency to be vention, a process of- producing an antifungal agent, wetted. identi?ed as. ascomycin, which comprises cultivating The above-described morphological characters and Streptomyces hygroscopicus var. ascomyceticus under sub ?ndings on the identi?cation media are compared with merged aerobic conditions in an aqueous carbohydrate those described in Bergey’s Manual (Williams and Wil solution containing a nitrogenous nutrient for at least kins, 7th edition, 1957), Pridham’s Classi?cation Stan twelve hours and then, if desired, recovering the so-pro dard (Applied , volume 6, pages 52-79, duced. ascomycin. from. the fermentation broth. 1958) and Waksman’s book (Actinomycetes, 2 volumes, ‘ Theworganisrn producing the ascomycin of the present 1961), and the classi?cation of this strain is investigated invention was isolated- from a sample of soil obtained in as follows: SannomiyaCity, Japan, and is a new variety, designated It is characteristic of this strain‘that its aerial mycelia ascomyceticus, of the-species Streptomyces hygroscopic-us. form spirals and it produces no speci?c soluble pigment A culture of the living organism, given the laboratory on any culture media including synthetic ones. designationKK 317, has. been deposited inthe American When only the colors of aerial mycelia and vegetative Type Culture Collection, Washington, D. C., and added mycelia on-synthetic'media are taken as important points, to its permanent collection or" micro-organisms as the resembling species, which have gray or black aerial A.T.C.C. 14891. mycelia and yellow or greenish yellow-brown vegetative With the color tones as de?ned by Maerz and Paulis ' mycelia, are found in the above-described classi?cation Dictionary of. Color, McGraw-Hill (1950 edition), the books. Streptomyces fungicidicus, Streptomyces albutr, representative strain of S. hygroscopicus var. ascomy Streptomyces niveus, Streptomyces platensis, Strepto‘myces ceticus has the following, characteristics: humz'dus, Streptomyces cacaoi, Streptomyces pseudogrise ]‘t/I0rphol0gy.—Aerial mycelia form 3-5 tight spirals on us, Streptomyces halstedii, Streptomyces rutgersensis and synthetic medium and on glucose-.asparagine agar. Spores 55 Sr'reptomyces hygroscopicus have resembling points. are ellipsoidal or small cylindrical, 1.6-2.0 x 1.0-1.2,u in Among these species, Streptomyces albus forms white size, and have smooth surface under electron-microscope. aerial mycelium on glucose asparagine agar, S. macro The growth on the identi?cation media for streptomyces spol'eus does not form aerial mycelium on the same species is. as follows: medium, and Streptomyces niveus forms cream-pink Glucose-Czapek medium.-—Growth, pale-lemon color aerial mycelia. Thus they are clearly different from to brownish, with rugged ‘surface (13L6). Aerial myceli the strain of the present invention. um, at ?rst‘white, later mouse-gray (7C7). Surface of the Streptomyces fungicidicus is different in that it pro colony turns to- black when touched with loop. Very duces yellow soluble pigment in certain media, and pro small amount of greenish-brown soluble pigment is pro duces dark brown pigment in potato plug. Streptomyces duced. pseudogriseus forms grayish brown aerial mycelia on Glucose - asparagine agar.-—Colony, pale yellowish green, round. Aerial mycelium, abundant, gray. Soluble synthetic media and differs from the present strain in its pigment, yellowish, scarce. color. Streptomyces halstedii and Streptomyces rutger Nutrient agar.—-Growth, pale greenish-brown, flat. The sensz‘s show different growth on potato plug but their surface iscovered with grayish aerial mycelia, like velvet. dark black color is characteristic. In this point they No soluble pigment. are differentiated from the strain of the present invention. 3,244,592 3 4 Thus, this strain has much resemblance to Streptomyces tion. Iron-hydroxamate reaction is positive-like. Nin platensl's, Streptomyces humidus, Streptomyces cacaoi hydrin, biuret, Molisch, Fehling, iron-perchlorate, 2,4 and Streptomyces hygroscopicus. Among them, Strep dinitrophenol and hydrazine reactions are all negative. It tomyces platensis and Streptomyces humidus belong to is stable when it is heated at 100° C. for one hour. The the Streptomyces hygroscopicus group, according to the ultraviolet absorption in ethanol, indicates only end-ab idea of the species-group in the modern classi?cation of sorption. Streptomyces. This is discussed in the paper of Tresner Ascomycin forms a single antibacterial spot of Rf 1.0 and Baccus (Applied Microbiology, vol. 4, pages 243— when it is paper-chromatographed using solvent systems 250, 1956) and also described in Waksman’s book. such as water-saturated butanol; 80% Phenol; butanol Thus, this strain is considered most properly to be 10 methanol-water (2:111); benzene-methanol (3:1); buta long to Streptomyces hygroscopicus when a hygroscopic nol-ethanol-water (221:1); 50% acetone, pyridine-buta tendency of aerial mycelium is also taken in considera nol-water (4:6:3). ‘ ' ' tion. The comparison of various characteristics of this The in vitro antibiotic activity of ascomycin is as strain and the standard strain of Slreptomyces hygro follows: scopicus also showed good resemblance. A (fermentation broth containing ascomycin is pre Gone. for Gone. for Test organism complete incomplete Culture, - pared by inoculating spores or mycelia of the ascomycin inhibition inhibition hours producing organism into a suitable medium and then (meg/ml.) (meg/ml.) cultivating under aerobic conditions. For the production of ascomycin, cultivation on a solid medium is possible, 20 Staphylococcus aureus 2091:‘ ______24 but for production in large quantity cultivation in a Bacillus subtr'lz's (PC1219) 24 Escherichia coli (F1) ____ _- 24 liquid medium is preferable. The temperature of the Pseudomonas aelruginosa. 24 cultivation may be varied over a wide range, 20-35“ C., enteritidis on nutrient agar at; 27° C_____ 24 within which the organism may grow but a temperature Candida krusel ______-_ 24 of about 27° C. and a neutral pH, i.e. pH 6.0—8.0, are Candida paralcrusei. _ 2A Candida nlbz'cans ______24 preferred. In the submerged aerobic fermentation of Cryptococcus neoformans_.__ 24 the organism for the production of ascomycin, the medi Trichophyton interdz'gz'tale__-_ 96 Trichophyton mentagrl» um contains as the source of carbon a commercially phytes ______1 72 available glyceride oil or a carbohydrate such as glycerol, Trichophyton rubr um ______7 5 50 144‘ 30 Trichophyton tonsulans 830 glucose, maltose, sucrose, lactose, dextrin, starch, etc. in on at 37° pure or crude states and as the source of nitrogen an C ______. 50 240 Zygosaccharomyces salsus organic material such as soybean meal, distillers’ solubles, lagao ______48 Saccharomyces sake 48 peanut meal, cottonseed meal, meat extract, .peptone, ?sh Wrllz‘a anomala____ 48‘ meal, yeast extract, corn steep liquor, etc., and, when Mycoderma ______48 Mucor macaw...“ 7. 5 72 desired, inorganic sources of nitrogen such as nitrates Rlrizopus m'grz'cans ...... -. 1 0.5 72 and ammonium salts, and mineral salts such as sodium Aspergillus m'ger ATCC 62-75 ______0.75 72 chloride, potassium chloride and magnesium sulfate, and Aspergillus oryzae. . 0.5 72 buffering agents such as calcium carbonate or phosphates glaucum ______25 48 ‘and trace amounts of heavy metal salts; such medium Penicillimn chrysogenum 40 76 ______1 ______. 48 ingredients include those listed in Canadian Patent Neurospora crassa ATGC 9277 ______._ 10 72 513,324 and in British Patents 730,341 and 736,325 and Mycobacterium tuberculosis in U.S. Patents 2,691,618, 2,658,018, 2,653,899, 2,586,762, 607 on 1% sugar-nutrient 2,516,080, 2,483,892, 2,609,329, and 2,709,672. In agar at; 27° C ______.. 50 ______. 72 aerated submerged culture an antifoam such as liquid para?in, fatty oils or silicone is used. More than one ’ This antibiotic substance is, as indicated in the above-dc‘ kind of carbon source, nitrogen. source or antifoam may scribed antibiotic spectrum, effective against fungi, espe be used for the production of ascomycin. Generally, cially against ?lamentous fungi. but ineffective against com» the cultivation is continued for about 12-72 hours, e.g. mon . It is exceptionally active against acid-fast until at least several hundred meg/ml. of ascomycin is bacteria, and incompletely inhibits Trichophyton at lower accumulated in the medium. 50 concentrations. The active substance is contained mainly in mycelia, The acute toxicity of ascomycin was tested by intra but ascomycin exists also in fermentation liquor. The peritoneal injection in mice. All the mice survived 100 potency of ascomycin is represented by units such that mg./kg. injection. 1 unit/ml. equals the minimum concentration to inhibit The above-described characteristics are compared with the spore~formation of Aspergillus niger. “ those of known antibiotics abtained from Streptomyces, The mycelia is separated from fermentation liquor, especially with those of colorless antibiotics. Ascomycin was clearly differentiated from various polyene antifungal washed with water and then extracted by Water-soluble antibiotics by its ultraviolet absorption. solvents such as acetone, methanol, ethanol, and Ascomycin differs from endomycin and helixin in that other low alcohols or by water-immiscible solvents the former is completely inactive against bacteria and the such as ether, chloroform and others. The solvent-extract 60 is concentrated and the active substance is extracted by latter is active against both fungi and bacteria. Asco water-insoluble solvents from the water-containing con mycin also differs from acti-dione (cycloheximide) and centrate. The extract is dried, chromatographed using its related substances, streptobiocin or naramycin, because ascomycin is ineffective against yeasts such as Saccharo active carbon, cellite, alumina, silica gel and others and de myces. veloped with ether, chloroform and others. By repeating Similarly, ascomycin is clearly ineffective on Candida fractional precipitation by solvents, after active fractions and thus differs from amidomycin, querulomycin, date are combined and concentrated, puri?ed ascomycin is ob mycin, eumycetin, ?avofungin and toyokamycin. tained as a colorless syrup. Ascomycin is obtained as In addition, ascomycin has only end-absorption in its white solid by drying this syrup. ultraviolet spectrum and if its solubility in various sol Puri?ed ascomycin is easily soluble in carbon tetra vents color reactions and othercharacteristics are also chloride, benzene, chloroform, ether, acetone, esters and considered this substance is easily determined to be a new lower alcohols, but hardly soluble or insoluble in Water, antibiotic substance. petroleum ether and n-hexane. The following examples are for purposes of illustration Ascomycin decolorizes potassium permangenate solu only and not of limitation and illustrate the recovery 3,244,592 5 6 of ascomycin from fermentation broth in both crude and I claim: puri?ed form. 1. The process of producing an antifungal agent, iden Example 1 ti?ed as ascomycin, which comprises cultivating a strain of Streptomyces lzygroscopicus var. ascomyceticus under Glucose 5 g., starch 5 g., beef extract 5 g., polypeptone submerged aerobic conditions in an aqueous carbohy 10 g., sodium chloride 5 g., tap water 1000 ml, pH 7.0. drate solution containing a nitrogenous nutrient for at Culture medium which has the above-described com position is autoclaved at 120° C. for 20 minutes. To least twelve hours. this medium, Streptomyces hygroscopica's No. KK 317 is 2. The process of producing an antifungal agent, iden inoculated and aerobically fermented in submerged cul ti?ed as ascomycin, which comprises cultivating a strain ture at 25-27° C. The growth of mycelium reaches the 10 of Streptomyces hygroscopicus var. ascomyceticus under maximum after three days fermentation. Then the myce submerged aerobic conditions in an aqueous carbohydrate solution containing a nitrogenous nutrient for at least lium is collected, washed with water and extracted with twelve hours and then recovering from the broth the acetone (?ve volumes) to obtain an extract containing 320 units per ml. This extract is concentrated under ascomycin thus produced. atmospheric pressure to 1,4,5 volume, and the turbid yel 3. The process of claim 1 in which the microorganism iowish-brown concentrate is extracted with two volumes is A.T.C.C. 14891. of ether, divided into two parts. The ether-extract is 4. A new antifungal agent identi?ed as ascomycin washed with small quantities of water, dried and con which is prepared by the process which comprises cultivat centrated under atmospheric pressure to evaporate off the ing a strain of Streptomyces hygroscopicus var. ascomyce acetone and ether completely. The residue obtained is ticus under submerged aerobic conditions in an aqueous dissolved in ether, and passed through a 2 x 30 cm. glass carbohydrate solution containing a nitrogenous nutrient ?lled with a mixture of active carbon and diatomaceous for at least twelve hours and then recovering from the earth (1:1), developed with ether, and highly potent broth the ascomycin thus produced. fractions are collected to obtain ascomycin as a pale References Cited by the Examiner yellow syrup. This is passed through a similar glass tube 25 ?lled with silica gel and developed with ether. Next, UNITED STATES PATENTS highly-potent fractions are combined and concentrated to 3,032,470 5/1962 Ziffer et al ______195-80 X obtain ascomycin as a colorless syrup. Further, this 3,094,460 6/1963 Boer et al. ______19S—80 X syrup is dissolved in ether and n-hexane is added to obtain a white ?ocullant precipitate. By drying this precipitate OTHER REFERENCES about 200 mg. of puri?ed ascomycin is obtained as a Ascomycin, An Antifungal Antibiotic, T. Arai et al., J. powder. Antibiotics (Japan), Series A, 15, 231-232 (1962). Example 2 Nakazawa et al.: I. Agr. Chem. Soc. (Japan) 28 (4), Soybean ?our 20 g., “ebios” 0.5 g., starch 2.5 g., mag 296—299, April 1954. nesium chloride 0.007 g., copper sulfate 0.007 g., zinc Nakazawa et al.: J. Agr. Chem. Soc. (Japan) 28 (9), sulfate 0.03 g., calcium carbonate 3.5 g., tap water 1000 7l5—716, September 1950. ml, pH 7.0. Ten ml. of a liquid medium with the above Tresner and Baccus: Applied Microbiology, 4, 243-250‘ described composition is autoclaved at 120° C. for 20 (1956). minutes, inoculated with Slreplomyces hygroscopicus var. 40 Wakaki et al.: I. Antibiotics, Ser. B 5(1), 24-29, ascomyceticus and shake-cultured aerobically at 27° C. January 1952. The growth reaches the maximum after three days and the mycelial extract by ?ve volumes of acetone has a potency A. LOUIS MONACELL, Primary Examiner. of 160 units per ml. By the similar treatment as in Ex ample 1, about 100 mg. of ascomycin is obtained. DAREN M. STEPHENS, Assistant Examiner.