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HiROSH KAWAGUCH MASANOR OKANSH TAKEO MYAK INVENTORS

BY CURTS W. CARLSON RCHARD H. BRINK HERBERT W., TAYLOR JR. ATTORNEYS Aug. 17, 1965 HiROSH KAWAGUCH ETAL 3,201,386 COUMERMYCIN AND SALTS THEREOF Filed Aug. 25, 1964 6 Sheets-Shee, 2 d O w | |

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BY CURTS W. CARLSON RICHARD H. BRINK HERBERT W., TAYLOR JR. ATTORNEYS Aug. 17, 1965 HiROSH KAWAGUCH ETAL 3,201,386 COUMERMYCIN AND SALTS THEREOF Filled Aug. 25, 1964 6 Sheets-Sheet 3

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HiROSH KAWAGUCH MASANOR OKAMSH TAKEO MYAK INVENTORS

BY CURTS W. CARLSO N RCHARD H. BRINK HERBERT W. TAYLOR JR. ATTORMEYS Aug. 17, 1965 HiROSH KAWAGUCH ETAL 3,201,386 COUMERMYCIN AND SALTS THEREOF Filed Aug. 25, l964 6 Sheets-Sheet 4

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HiROSH KAWAGUCH MASANOR OKANISH TAKEO MYAK INVENTORS

BY CURTS W. CARLSON RCHARD H. BRMK HERSERT W., TAYLOR JR. ATTORNEYS Aug. 17, 1965 HiROSH KAwAGUCH: ETAL 3,201,386 COUMERMYCIN AND SALTS THEREOF Filed Aug. 25, 1964 6 Sheets-Sheet 5

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HiROSH KAWAGUCH MASANOR OKANSH TAKEO MYAK INVENTORS BY CURTS W. CARLSON RCHARD H. BRINK HERBERT W., TAYLOR JR. ATTORNEYS Aug. 17, 1965 HiROS - KAWAG c. A 3,20386 COUAERMYCIN AND SALTS THEREO Filed Aug. 25, 1964 W 6 Sheets-Sheet 6

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ROS- KAAAGUC NASANOR O CANS AKEO NYAK INVENTORS

BY CRS W. CAR SON Ric-ARD H, SR NK HERBERT WAYOR R. AORNEYS 3,20,386 Patent Office Patented Aug. 17, 1965

and in N/10 NaOH having a maximum at 3,201,386 280 mu (E=766) HiroshiCOUMERMYCIN Kawaguchi, Masanori AND Okaaishi,SALTS THEREOF and Takeo Mii has a neutral equivalent of 548, has the following average yaki, Tokyo, Japan, assignors to Bristol-Banyu Re elemental analysis: C, 59.1%; H, 5.35%; N, 5.90% and O search institute, Ltd., Tokyo, Japan, a Japanese 5 (by difference), 29.65%; and when pelleted in potassium corporation bromide exhibits characteristic absorption in the infrared Filed Aug. 25, 1964, Ser. No. 392,003 region of the spectrum as shown in FIGURE 1; and salts 14 Clains. (Cl. 260-210) of said acidic substance. This invention relates to a new and useful substance Referring to the drawings: called coumermycin and to processes for its production. O FIGURE 1 is a curve of the infrared absorption spec More particularly, it relates to processes for its production trum of coumermycin free acid when pelleted in potas by fermentation and methods for its recovery and puri sium bromide. fication. The invention embraces this acidic antibacterial FIGURE 2 is a curve of the ultraviolet absorption agent and its alkaline salts in dilute solutions, as crude 5 spectrum of counermycin in solution in ethanol, in 0.1 concentrates, as purified solids and in pure crystalline N HCl and in 0.1 N NaOH. forms. Coumermycin is effective in inhibiting the growth FIGURES 3 and 4 taken together are the infrared ab of Gram-positive bacteria. Coumermycin is nontoxic and sorption spectrum of the monosodium salt of coumermy exhibits a therapeutic effect on mice infected with Gram cin A1 when pelleted in potassium bromide. postive bacteria. Coumermycin has also been called FIGURES 5 and 6 taken together are the infrared ab Bu-620 and "Notomycin.” Coumermycin itself sorption spectrum of the monosodium salt of coumermy is a complex or mixture of five compounds which have cin A2. been given the individual names of coumermycin A1, There is further provided, according to the present in coumer mycin A2, coumermycin B, coumermycin C and vention, the process for the production of an antibiotic coumermycin D. substance, designated coumermycin, which comprises cul This application is a continuation-in-part of our prior tivating a strain of Streptomyces rishiriensis in an aqueous cope:Inding applications Serial No. 247,435, filed December carbohydrate solution containing a nitrogenous nutrient 17, 1962 and Serial No. 366,461, filed May 11, 1964 and under submerged aerobic conditions until substantial ac both now abandoned. tivity versus Gram-positive bacteria is imparted to said The most important members of the coumermycin 30 solution and then recovering said coumermycin from said complex are coumermyin A and coumermycin A which solution. have the formula The organism producing the antibiotic of the present C3 CH 3C O. O. O O O O CH itsc? -O IN O 4CH,

3CO OHI --NE-O Ö -NH HO d I E3 =0 NIH

R wherein both R groups represent methyl in the case of invention was isolated from a sample of soil collected coumermycin A1 and represent hydrogen in coumermycin 45 in Rishiri Island, Hokkaido, Japan and is a new species, A2. The coumermycin complex is referred to herein designated Streptomyces rishiriensis of the genus Strep simply as coumermycin and is referred to in applicants' tomyces. A culture of the living organism, given the parent application Serial No. 247,435 simply as noto laboratory designations 404Y3 and A9795, has been de mycin. posited in the American Type Culture Collection, Wash There is now provided, according to the present inven- 50 ington, D. C. and added to its permanent collection of tion, an antibiotic substance effective in inhibiting the microorganisms as A.T.C.C. 14812. growth of Gram-positive bacteria which is selected from , the group consisting of an acidic substance, coumermycin, The representative strain, No. 404Y3 of S. rishiriensis which is readily soluble in acetone, dioxane and alkaline has the following characteristics: water, moderately soluble in ethanol, butanol, ethyl ace 5 5 CULTURAL CHARACTERISTICS tate, butyl acetate and methyl isobutyl ketone, less soluble G-growth; A-aerial mycelium; S-c soluble pigment; B-bio in benzene, methanoi and chloroform and insoluble in chemical property carbon tetrachloride, petroleum ether and acidic water, 1. Czapek's agar: which gives positive Fehling and Molisch reactions, de G: moderate, yellowish gray to light brownish gray colorizes bromine and gives negative ninhydrin, Tollens 60 or ivory yellow and anthrone reactions, and which in purified form melts A: poor, powdery, white at 222-224° C., exhibits (ox) D20 of -134 (c. = 1.0. ace S: none tone), exhibits an ultraviolet absorption spectra in ethanol 2. Glycerol Czapek's agar: having maxima at G: moderate, yellowish gray or light brownish gray 275 mu (E=595) 65 to pale yellow with faint brown and at - A: poor, powdery, white S: none or pale yellow with faint brown 335 mu (E4,-498) 3. Glycerol ammonium salt agar: in NA 10 HCl having maxima at G: moderate, light yellowish gray to yellowish white 275 mu (E=287) 70 A: scant, powdery, light yellowish gray to light gray and at 345 mp. (E=277) S: none 3,201,386 3 4. Glucose asparagine agar: 4. G: poor, thin, glossy, light yellowish gray branched hyphae, occasionally tuft, and sporophores A: none which produced sinistrorse spirals. On electron micro S: none Scopic observation, the shape of spores was found to be 5. Starch agar: elliptical to oval and the surface was smooth. G: good, light yellowish gray to ivory yellow 5 Streptomyces 404Y3 has a brownish gray aerial my A: moderate, powdery, spreading, faint brown to celium and produces brown or dark brown pigment on pale brown with gray organic media. Gelatin liquefaction is negative both in S: none gelatin stab and tyrosine yeast gelatin stab and milk is not B: hydrolysis is moderately strong digested. Nitrite is produced from nitrate and starch 6. Nutrient agar: 0 is hydrolyzed. Strain 404Y3 resenbles S. diastatochromo G: moderate, burnt umber to brown genes, S. griseoruber, S. olivochromogenes, S. aureus, S. A: poor, powdery, white griseochromogenes, S. haivaiensis and S. naganishi in S: dark brown some respects such as spiral formation, color of aerial 7. Bennett's agar: mycelium and nelanoid pigment, but differs in certain G: moderate, grayish olive brown to grayish brown cultural and physiological properties as shown below: A: moderate, white to pale brown to brownish gray S. diastatochromogenes.-According to Waksman and S: yellow brown Curtis, sporophores arc straight, and according to Jensen, 8. Oatmeal soyton agar: sinisrotrose spirals are produced. The color of aerial my G: good, pale yellow with faint brown celium is reported to be white to ash gray on Czapek's A: moderate, white to gray with faint brown 20 and gray on glucose asparagine agar. Liquefaction on S: pale yellow with faint brown gciatin stab is fairly rapid. 9. Potato plug: S. griseoruher.-The shape of spore is reported to be G: good, glossy, wrinkled surface cylindrical. The color of growth is reddish orange on A: none Czapek's and light reddish orange to reddish purple on S: plug changed brownish black 25 starch agar. Differences are also found on utilization of 10. Gelatin Stab: carbon sources such as sucrose, raffinose, citrate and suc G: brownish black colony on the surface cinate. A: none S. olivochromogenes.--Differences are found in growth S: dark brown color, soluble pigment on glucose asparaginc agar and B: negative liquefaction 30 digestion of gelatin and milk. 11. Tyrosine yeast gelatin stab: S. aureus-The color of growth is dark brown on G: brownish black colony on the surface Czapek's and light orange on glucose asparagine agar. A: none Gelatin liquefaction is rapid later slowing down. S: dark brown S. griseochromogenes.--The color of aerial mycelium B: negative liquefaction is white to light neutral gray. Gelatin liquefaction is 12. Milk: moderate. Milk is peptionized without coagulation. Dif G: brownish ring formation ferences are also found in utilization of carbon sources A: none such as rhamnose, inulin, Salicin, citrate and succinate. S: faint brown S. hawaiiensis.--The growth color on glucose aspara B: not digested 46) gine agar is light brown and aerial mycelium is white to gray. Differences are also found in utilization of carbon 13. Nitrate solution: sources such as Sorbitol and succinate. G: white pellet mass on the surface S. naganishi.-The growth color on starch agar is B. positive reduction to nitrite cream with reddish purple and soluble pigment on starch 14. Melanin formation media: agar is faint pink. Gelatin is moderately liqucfied. Milk G: poor, thin, grayish black is coagulated and peptionized. Differences are observed A: scant, white in utilization of carbon sources such as Sucrose and S: brownish gray to grayish brown sorbitol. B: nelanin positive In order to select strains of high productivity, ultra 15. Potato dextrose agar: violet irradiation was done on the original strain and G: good, glossy, brownish black mutant colonies were selected. Somc of the higher pro A: moderate, white to pale pink to pale brown with ducers among these strains were investigated mycologic faint gray ally, but notable differences were not observed except for S: gray or warm gray obtaining a few strains of white aerial mycelium. 55 In view of the above characteristics of the strain, UTILIZATION OF CARBON SOURCES Streptomyces 404Y3 was determined to be a new species Xylose ------Inulin ------and designated Streptomyces rishiriensis nov. Sp. Arabinose ------Salicin ------The species Streptomyces rishiriensis described herein Glucose ------. Mannitol ------includes all strains of streptomyces which form coumer Galactose ------Sorbitol ------60 mycin and which cannot be definitely differentiated from Fructose ------Cellobiose ------the strain No. 404Y3 and its subcultures including mul Sorbose ------Rhamnose ------tants and variants. The properties of councrmycin are Sucrose ------Sodium citrate ------described herein and, after these propertics are known, it Maltose ------Sodium succinate -- -- is easy to differentiate the strains producing coumermycin Lactose ------Inositol ------65 from others. Raflinose ------Control ------au Streptomyces rishiricnsis when grown undcr suitable -----good growth. conditions, produces collmermycin. A fermentation ---fair growth. broth containing coumermycin is prepared by inoculating doubtful growth. -cino growth. spores or mycelia of the councimycin-producing organ 70 isms into a suitable medium and then cultivating under MYCOLOGICAL CHARACTERISTICS acrobic conditions. For the production of cott mermycin, cultivation on a solid nedium is possible, but for pro The morphological properties of the strain were ob duction in a large quantity cultivation in a liquid medium served on starch agar and Bennett's agar. Microscopic is preferabic. The temperature of thc cultivation may be examination of the aerial mycelium revealed tangled and y - varied over a wide range, 20-35° C., within which the 3,201,386 5. 6 organism may grow but a tenperature of 26-30° C. and Example 5 a Substantially neutral pH is preferred. In the submerged CONSTITUENTS OF MEDIUM aerobic fermentation of the organism for the production Soybean Meal ------percent-- 2.0 of counternhycin, the medium contains as the source of Glycerine ------do---- 1.5 carbon, a commercially available glyceride oil or a car Yeast extract ------do---- 0.2 bohydrate such as glycerol, glucose, maltose, sucrose, Potassium phosphate, dibasic ------do---- 0.1 lactose, dextrin, starch, etc. in pure or crude states, and Sodium chloride ------do---- 0.1 as the Source of initrogen an organic material such as Calcium chloride ------do...-- 0.05 Soybean meal, distillers' solubles, peanut meal, cotton Magnesium sulfate ------do---- 0.05 Seed meal, meat extract, peptone, fish meal, yeast ex IO tract, corn steep liquor, etc., and, when desired, inorganic Zinc sulfate ------do---- 0.00 Sotirces of nitrogen such as nitrates and ammonium salts, Pl ------7.0 and miner:l salts such as sodium chloridc, potassium A culture medium (100 m.) containing the above chloride and magnesium sulfate, and buffering agents components was sterilized in a flask of 500 ml. volume, such as calcium carbonate or phosphates and trace inoculated with a seed culture of Streptomyces rishiriensis amounts of heavy metal salts; such medium ingredients strain 404Y3 and cultivated at 27-h1 C. for six days include those listed in Canadian Patent 513,324 and in with shaking, whereupon the production of coumermycin British Patents 730,341 and 736,325 and in Uniter States in tha fermentation broth reached 150 mcg../mi. Paints 2,691,618; 2,658,018; 2,653,899; 2,586,762; 2,516 080; 2,483,892; 2,609,329 and 2,709,672. In aerated sub 20 Example 6 Incrged cuttire an antifoam Such as liquid para?lin, fatty CONSTITUTENTS OF MEUM oils or silicone is used. More than one kind of carbon Cottonseed endosperm flour source, nitrogen source or antifoam may be used for the ("Pharman edia") ------percent.-- 2.0 production of coumermycin. Generally, the cultivation Soluble starch ------do---- 1.5 is continued until at least several hundred mcg../mi. of 2 5 Yeast extract ------do---- 0.2 coumermycin has accumulated in the medium. In some Potassium phosphate, dibasic ------do---- 0.1 cases the broth pH decreased at the beginning and then Sodium chloride ------do---- 0. gradually rose. Calcium chloride ------do. --- 0.05 The following exampies will serve to illustrate this Magnesium sulfate ------do--- 0.05 invention without limiting it thcreto. 30 Zinc sulfate ------do---- 0.001 Evample 1 pH ------7.0 Fermentation conditions suitable for production of the antibiotic concrimycin were studied and the following A culture medium (100 ml) containing the above composition of medium was found to be useful in shake 3. 5 components was sterilized in a flask of 500 ml. volume, culture: 1.5% soluble starch, 2.0% cottonseed endosperm inoculated with a seed culture of Streptomyces rishiriensis flour ("Pharmamedia'), 0.1% KHPO, 0.1% NaCl, strain 404Y3-30 (which is a mutant strain of 404Y3 ob 0.05% MgSO.7HO, 0.05% CaC 0.001% tained by irradiation of ultraviolet ray) and cultivated ZnS).7H2O and 0.2% yeast extracts. at 27-h1° C. for nine days with shaking, whereupon Coumermycin is produced in three to five days' fer the production of coumermycin in the fermentation broth nientation under adequate aeration and agitation and the reached 400 mcg../ml. activity is found in both the broth filtrate and the inny Example 7 celium. Counernycin is assayed against Staphylococcus ature its FDA 209P by paper-disc or cylinder plate method. A culture medium (30 i.) containing the same compo nents as in Example 6 was sterilized in a stainless steel Example 2 tank of 50 liter volume, inoculated with a seed culture of Submerged aerobic fermentation of S. rishiiensis Streptomyces rishiriensis, and cultivated at 28--1 C. (strain 404Y3) in shake flasks in a medium containing for 90 hours, whereupon the production of coumermycin 6% starch (Staclipse J"), 3% debittered brewers' yeast in the fermentation broth reached 120 mcg../ml. and 1% CaCO3 produced 300-400 meg/ml. counermy The fermentation broth (5 l.) was filtered at pH 8.0 and Cll. the filtrate was adjusted to pH 6.0 and extracted with 2 Example 3 liters methyl isobutyl ketone. The mycelial cake was extracted with 500 ml. of acetone with vigorous stirring Submerged aerobic fernmentation of S. rishiriciisis and the extract was evaporated in vacuo to remove the (strain 404Y3) in shake flasks in a medium containing 5 5 solvent. The resultant aqueous concentrate was extracted 6% starch (“Staciipse J"), 3% cottonseed endorsperm with 200 ml. of methyl isobutyl ketone at pH 6.0. The two ketone extracts were combined and washed with 500 flour ("Pharmamedia”), 2.5% distillers' solubles and 1% ml. of water and then the activity was transferred into CaCO produced 150-200 mcg/ml. coumermycin. 1000 ml. of cold watcr adjusted to ph 10.0 by sodium Example 4 hydroxide. The aqueous extract was adjusted to pH 6.0 60 and again extracted by 300 ml. of ethyl acetate which Submerged aerobic fermentation of S. rishiriensis was concentrated in vacuo to 30 ml. Upon the addition (strain 404Y3) in shake flasks in a medium containing of 150 ml. of petroleum ether to the concentrate a light 1.0% soybean neal, 2.0% cottonseed endosperm flour brown annorphous precipitate appeared which was col (“Pharmamcdia”), 2.0% soluble starch, 0.5% glycerine, lected, washed with small amounts of methanol to remove 0.2% yeast extracts, 0.2% KHPO, 0.1% MgSO4, 1.0% 65 colored contaminant and dried to give 750 mg. of cou CaCO and 0.05% silicone antifoam at pH 7.0 gave mermycin as a powder. coumermycin as follows: The coumermycin powder (400 mg.) thus obtained was

dissolved in 100 ml. of ethyl acetate and adsorbed on a column of 20 gm. alumina which was previously treated Time, days pH Potencymcg.fml. in 70 with sulfuric acid. The column was washed with 200 ml. of ethyl acetate and eluted with methanol. The eluate 5 7.8 O was taken fractionally in 10 ml. portions and the active 6 S. 1 SO fractions were combined and concentrated in vacuo to 7 8.3 120 dryness. The pale yellow solid counermycin thus ob 3,201,386 7 tained was recrystallized twice from methanol to give 70 8 mg. of white crystalline countermycin which melted at Coumermycin is readily soluble in acetone, dioxane 28-220° C. with decomposition. and alkaline water, moderately soluble in ethanol, buta Counermycin was further purified by Craig's technique nol, ethyl acetate, butyl acctate and methyl isobutyl ke of counter-current distribution using a solvent system five tone, less soluble in benzene, methanol and chloroform parts (by volume) carbon tetrachloride, onc part chloro 5 and insoluble in carbon tetrachloridc, petroleum cther form, five parts methanol and onc part water. After 50 and acidic water. transfers, the peak of concentration was found in tube As shown in FIGURE 2, coumermycin cxhibits an number 26 by bioassay curve, by ultraviolet assay curve ultraviolet absorption spectrum containing the following and by weight curve; these curves agreed with the theoreti maxima: cal curve. Lyophilization of ten tubes around this peak 10 gave pure coumermycin. Maxima in ethanol, 275 m.p. (E-595), 335 mp (1:...F498 Coumermycin has also been purified by recrystalliza Maxima N/10 IICI, 275mu (IE =287), 345 inpi (14,5-277) tion from aqueous acetone and then from absolute meth anol to give material melting at 220 C. with decomposi Maxima N/10 NaOH, 280 mu (E = 766) tion. Coumermycin has also been purified by alumina chro As shown in FIGURE 1, coumermycin when pelleted matography, in which 39.7 mgm. (potency 500 mcg. / in potassium bromide exhibits characteristic infrared ab mgm.) was dissolved in ethyl acetate and adsorbed on 2 sorption maxima at the following wave lengths in mi g. AlO (pH 4.7, treated with H2SO4). After washing crons: 2.99; 3.36 (shoulder); 3.45; 5.88 (shoulder); 5.92; with ethyl acetate, the column was eluted successively 6.10; 6.24; 6.45; 6.55; 6.7: 7.2; 7.65; 7.85; 8.9; 9.2; 9.6; with methanol and acidic methanol. The active fractions 10.05; 10.3; 10.6; 11.3; 12.25; 12.65; 13.1; 13.35. were combined, neutralized, concentrated in vacuo and Thus coumermycin and are readily differen then lyophilized to give 12.4 mgm. as pale yellowish pow tiated, e.g. by melting point, elemental analysis, specific der (potency 700 mcg../ngm.). : rotation, ultraviolet and infrared absorption spectra, an Coumermycin (480 mgm.) was treated with a small 2 5 throne reaction and behavior in paper strip chromatog amount of active carbon in acetone solution and then Iaphy. recrystallized from hot ethanol to give 230 mgm. of crys Coumermycin is an acidic organic compound which is tailine coumermycin which was dried in vacuo for three easily soluble in alkaline solutions such as aqueous solu hours at 50° C. Melting point 222-224 C. (dec.), pKa tions of alkali metal and alkaline earth metal hydroxides. 6.32 in 75% acetone. Titration equivalent: 526.5. 30 Thus solution of coumermycin in aqueous sodium hy Analysis.--Calc'd. for C2H8N2O10: C, 59.35; H, 4.98; droxide or calcium hydroxide forms the sodium or cal N, 5.32. Found: C, 59.1; H, 5.35; N, 5.90. cium salt, respectively, which can be isolated if desired, A molecular weight determination by the cryoscopic as by lyophilization. In like manner are formed salts method in dioxane gave a value of 500-20. The specific with organic bases; addition of streptomycin or dihydro rotation c. 20 was -134.4 (c. = 1%, acetone) as com streptomycin base to coumcrmycin forms the strepto pared to -69.6 for novobiocin under the same condi mycin and dihydrostreptonycin salts of councrmycin, re tions. Coumermycin gave a negative (brown) anthrone spectively; for this reaction it is convenient to use an or test whereas novobiocin gave a positive (green) anthrone ganic solvent such as ethyl acetate. Other amine salts test. 40 which can be used include those used in therapy as Salts A solution of 100 mgm. coumermycin in 120 ml. ab of benzylpenicillin, e.g. procaine, N-benzyl-6-phenethyl solute ethanol was stirred with 0.25 gm. of Adams' cata amine, hydrabamine, ephenamine, N,N'-dibenzylethyl lyst under atmospheric pressure of hydrogen for 30 min enediamine, dehydroabietylamine and dicyclohexylamine. utes at room temperature. Almost no hydrogen uptake Acidification of an aqueous solution of sodium coumer was observed. After removal of the catalyst, 35 mi. of mycin serves to precipitate purified coumermycin as the water was added to the filtrate with vigorous stirring. Ad free acid. dition of 0.5 ml. of one normal hydrochloric acid to the One preferred method of isolating coumcrimycin from resulting emulsion precipitated a white solid which was a fermentation broth comprises extracting whole broth at collected, dried and found to weigh 88.7 mgm. and to be pH 6.0 with one-half volume of methyl isobutyl ketone, original starting material, i.e., coumermycin as confirmed back extracting at pH 10.0 into water (one-quarter the by paper chromatography. Under the same conditions volume of the methyl isobutyl ketone phase) and re-cX 1000 mgm. novobiocin gave 946 mgm. dihydronovobiocin tracting at pH 6.0 into cthyl acetate or methyl isobuty as confirmed by paper chromatographic analysis accord ketone (one-third the volume of the last aqueous phase). ing to the literature (F. J. Wolf et al., Annual The final concentrated solution of coumermycin in the 1956-1957, page 1035). organic solvent is then concentrated to onc-tenth or One Coumermycin gives positive Fehling and Molisch re 5 5 twentieth its original volume by distillation in vacuo. actions and decolorizes bromine. Ninhydrin, Tollens and Coumermycin is then precipitated, if it does not precipi anthrone reactions are negative. tate spontaneously, by the addition of mixed lower al Paper strip chromatography of coumermycin gave the kanes, e.g. by the addition of ten volumes of “Skellysolve following results: B.' . 60 Councrmycin is also easily recovered from filtered Solvent system: Rf value broths by adsorption on activated carbon ("Darco KB') Wet butanol ------0.15 and Subsequent clution therefrom but this proccss is not 3% Aqueous ammonium chloride ------0.03 superior to solvent extraction of whole broth as in many 75% Phenol------0.95 broths nost of the counnermycin is in the mycelium. 50% Acetone ------0.75 65 Microbiological Studies on Counterniycin in Vitro Anti Butanol-methanol-water (4:1:2)--2% methyl bacterial Activity.-The minimum inhibitory concentra Orange ------0.45 tions (MIC) of counermycin against a variety of micro Butanol-methanol-water (4:1:2) ------0.40 organisms were determined by serial agar dilution meth Benzene-methanol (4:1) ------0.05 od, using horse blood agar for hemolytic streptococci and Distilled Water ------0.05 pneumococci, glucose-yeast extract-peptione agar for lac Butanol-water (4:1)--0.25% p-toluenesulfonic tic bacilli, nutrient agar for other bacteria, 2% glucose Sabouraud agar for fungi and Kirchner's hiroth for tuber acid ------0.30 cle bacilli. The results are tabulated below along with Butanol-water-acetic acid (2:1:1) ------0.50 those obtained with novobiocin which was used as a refer Butanol-water (4:1)+2% piperidine ------0.25 CCC. 3,201,386 O ANTI3ACTERIAL SPECTRUM OF COUMERMYCIN however, reduced activity against the staphylococci which (AGAR IDILUTION AIETFIOD) were made resistant to novobiocin in the laboratory. Effect of inoculum size on the minimum inhibitory MIC (mcg...fcc.) Test Organisin concentration.-Using Staph. aureus Smith strain and 5 Staph. aureus strain 193 as test organisms in nutrient Cottermyci Novoliocin broth, tube dilutions were run with various inchculum sizes. The results are tabulated below and it will be tril- (give: f'self 'ichiti coli. Ni------>50 3, 13 seen that the effect of heavy inocula is similar to that l:(litriclict cyli () 45... ---- >50 50 obtained with novobiocin. -- Fl's ille pirat anoniae Tyl)cy All----- 3, 13 2.5 almm lla typhi >50 (5.25 O Effect of pH of medium on the niinimum inhibitory Sligola fle Thri------...------>50 6.25 concentration.-The MIC of counermycin was treated Shiti lit clusc interine A.------>50 25 Neisserift sp. (CP-R)------>50 2.5 by broth dilution method, the pH of the media being Bordetella bronchiseptica ATCC 4617- >50 >50 adjusted to several levels. Nutrient broth and Difico's F's pition on a 3 arrattinosa.------>50 >50 libri) in Citchinikozi------0.025 0.1 brain-heart infusion broth were used as test media, and rail-positive: a 104 dilution of an overnight culture of Staph. aureus Staphylococcus aureus FI) A 209 P---- O, O(3 0.046) 5 S. (ture its FI) A 2011 (SM, ST-R)--- 0.03 0.049 Smith (105 cell/ml.) was inoculated into each tube. As S. (ture its FA 209 (N3-R). 1,56 6.25 shown below, the activity of coumermycin increases in S. aurents 52-34 (TC, EM, CM, 'C-l).------. (), (c)3 0.1 acidic pH and decreases in alkaline pH. The compara S. aire 18 Slit st': i. (). (? 0.049 S. nitri, its 123 ('C. IC-R)------(), (?.3 0.04) tive data with novobiocin indicate that coumer mycin is S. aureus 93 (PC, 'J'?, EM, ('M-R). 0.3 0.1 20 affected by media pH to a greater extent than novobiocin. S. atre 8 193 (C, C, EM-R)----- (), ()(3 (), (4) S. ?tatre its Terajina------. (). 003 0.012 EFFECT OF MEDIA pI ON MIC S. (all 18-----...------(). (Of 0.049 Micrococci's flat its -- - - (), ()() 0.049 N N M.I.C. in mcg.fml. Sarcinct litte?t P(: 1001------. 0.025 0. 13. Sultilis PC? 219.------50 0, Media B, 8plerict 822. ------().78 (). Coumermycin Novobiocin 13. Ricoides "O'. ------().78 0.39 2 5 13. cit feats ATCC 10702------3.13 0.78 18, it ill racis : 115------().8 (). NB 1 B2 NB B Lacto acilis casei ATCC 746.) >50 0. l, acidophilus B-4 (3-1-. >50 0. Streptococcus faecalis B- 3.13 0.39 3 0.0001 0.00005 0.024 0.24 S. ngog"ifs Type 3------3.13 0.78 0. 0.003 0.006 0.098 0.098 Diplococcits pneutnoniae Type II... 1.56 0.78 30 6 0.049 0.098 0.195 0.39 tycolacterium 07------() 50 'ycolacterial in uhlei. -- 50 2.5 2 0.39 ().78 0.78 0.78 Fug.: 5 1.56 3, 13 56 1.56 Al's pergillais miger van Tieghem------. >50 >50 niciliatin chrysogen it in------>50 >50 • Canfilt illicans- >50 >50 1 NB = nutrict broth, Saccharomyces certisiac ATCC 9763. >50 >50 23IForail-heart infusion broth. Trichophyton mentagrophytes------>50 >50 Effect of media on the minimum inhibitory concentra tion.--The effect was examined using four kinds of Abbreviations: - R=resistint; SM-list reptomycin; ST=streptothricin NB = lovoliocin; TC= tetracycline; PCsipenicillin; EM=erythromycin media; namely, nutrient broth, brain-heart infusion broth CMI = carbonycin. (Difco), trypticase-soy broth and 1% yeast extract broth. The minimum inhibitory concentrations of couner 40 The MIC was not affected by the media used for the mycin were also tested by two-fold tube dilution method determination as long as the pH of the media was con using nutrient broth except for hemolytic streptococci troled. Effect of serum on the minimum inhibitory concentra and pneumococci which were tested by brain-heart in tion-Increasing concentrations of human serum were fustion broth. The results are tabulated below along added to nutrient broth containing phosphate buffer in with the activity ratio of countermycin and novobiocin. a final concentration of 1/0 M to maintain the media ANTIBACTERIAL SPECT RUM OF COUMERMYCIN (TUBE pH at 7.2. Staphylococcus aureus Smith was used as a DiUTION METHOD) test organism, inoculum size being 10 cell/ml. As tabu Coumer- Novobio lated below, the MIC was markedly affected by serum. Test Organism mycin, cin, Ratio mcg...fill. mcg../nl. 50 EFFECT OF SERUM ON MIC

leliscilla pneumonicle.------3.13 6.25 2 MIC in Incg.fm. Staphylococcus aureaus FDA. 209 P 0.0015 0.049 32 Serum Concen Stiphylococci is a virtuits erjilla- - 0.00(5 0.012 16 tration, iercent Stuphylococcits attré as Smith-- -- 0.0015 0.024 6 Councrmycin Novobiocin Sarcina litect PCI 1001------0.005 0.049 8 Corynebitcteriatin cerosis 53-K- -- 0.0005 0.024 32 Without scrum 0.003 0.098 Bacilits certs ATCC 1002 - 0.78 0.78 2.5 0.024 (). 195 Hacilitats anthracis l15------0.39 0.95 -3 5 0.098 .95 Streptococcus pyogcles Type 3-- 0.049 0.39 8 i) 0.098 0.39 Diplococcus pictionicle Type II------0.093 0.098 20 0.08 0.18 50 0.195 : 3.13 Counermycin inhibits the growth of various Gram positive bacteria except for Bacillus subtilis and lactic bacilli which are sensitive to novobiocin. Counermycin Activity of councerniycin against clinically isolated is especially active against staphylococci, the activity staphylococci.-A variety of Staph. aureus cultures (126 being 10 to 30 times greater than novobiocin. It shows, strains) isolated from clinical sources were tested in vitro EFFECT OF INO CULUM SIZE ON MIC - Inoculum Size (cellfull.) Test Organism Antibiotic 107 100 105 04 103 02 (MIC in Inc.g.fml. S. aureus Smith.... Coumormycin.-- 0.012 : 0.012 0.003 (). 003 0, 003 0.003 S. aureus Smith--- Novoliotii -- 0.39 (), 39 (). 195 (). (398 0.098 0.098 S. aureus strait 193- Counernycin (). 024 0.003 (), ()()15 (), ()()15 0.0015 0.0015 S. aureus strain 193 Novolioci.... 0.78 (). 1950,098 0.098 (,098 0.098 3,201,386 2 against counermycin and six commercial antibiotics; Counermycin is a useful agent for the treatment of Thaiinely, benzylpenicillin, dihydrostreptomycin, tetracy mastitis in cattle or calf scours; for this purpose use is cline, crythromycin, kanamycin and novobiocin. The made, for example, of suspensions in vegetable oil for in MiC values were obtained by ten-fold serial agar dilu stillation in the teats to treat mastitis, containing 1 to 1000 tion method as follows: 5 mgm./ml., and preferably about 50 mgm., of the anti SENSITIVITY OF CLINICAL SOLATES (STAPLIYIOCoCCI) TO COUMER MYCIN AND COMMERCIAL ANTIBIOICS

MIC in Antibiotics mcg...final. Coumcrnycin PC IDSM TC EM

>100 O O 3 0 O Ot) O 16 2 1. 0 O 7 03 O 3 i O 89 2 8 117 0.1 1 2 0. 4. 3. (. ) 52 7 2 2 O (). (01 48 () 0 O 2 (. )) 21 () () () (). OOOO. 4. 0. O O O

Sane strain. C = benzylpenicillin. TSM = dihydrostreptomycin. TCastetracycline. thromycin. KAI = kailannycill. NB =inovobiocin. EM-sery. The results indicate that coumermycin is highly active biotic, or enough capsules to provide a total dosage of against staphylococci and it is not cross-resistant with 0.25 to 2.0 grams by oral administration as for calf other antibiotics. Only one strain out of 126 strains which SCOS, was resistant to novobiocin was found to be less sensitive When desired for specific purposes and rendercd phar to countermycin, but the MIC value of coumermycin to 30 maceutically compatible, there may be admixed with the that strain was 0.1 mcg../ml. compounds of the present invention other medicaments N VIVO EXPERIMENTS ON COUMERMYCIN such as antihistamines, sulfa drugs, e.g. , sulfabenzamide, , , sulfapyri Toxicity-Coumermycin is an antibiotic of low toxic dine, , sulfapyrazine, sulfaguanidine, sulfa ity. The intramuscular LD50 was found to be 500 mg/kg. thalidine, sulfasuxidine, sulfisoxazole, sulfamylon, phthal in mice. ylsulfacetamide, N' - 3,4-dimethylbenzoylsulfanilamide, Chemotherapeutic effect.-The in vivo activity of cou benzylsulfanilamide and N'-2-(2-quinoxalyl)sulfanila inhermycin was tested on mice against an experimental in mide J, lipotropic agents (particularly methionine, choline, fection of Staphylococcus aureus Smith strain. Mice were inositol and betasitosterol and mixtures thereof), stimu W. infected intraperitoneally with 100XLD50 of the pathogen 40 lants of the central nervous system (e.g. caffeine, amphet and the antibiotic was administered intramuscularly or amines), local anesthetics, analgesics (e.g. aspirin, Sal orally just after the bacterial challenge. A single intra icylamide, sodium gentisate, p-acetylaminophenol, phen muscular CDso value (50% curvative dose) of 0.3 mg. / acetin, codeine), sedatives, (e.g. barbiturates, bromides), kg, and a single oral CD50 of 4.5 mg/kg, were obtained. salts of benzylpenicillin (e.g. potassium penicillin G, pro In a comparative test, intramuscular and oral CDso values caine penicillin G, 1-ephenahine penicillin G, dibenzyl of novobiocin were found to be 6.0 mg./kg. and 10.0 mg/ amine penicillin G, other saits disclosed by U.S. Patent kg., respectively. The data are presented below. 2,627,491; these combinations are particularly useful to CENOT II ERAPETIC, EFFECT OF COUMERMYCIN enable variation of the pattern of blood levels obtained), (INT RAAU SCULAR T1 ERAPY) phenoxymethylpenicillin, phenethicillin, methicillin, ox

acillin, cloxacillin, nafcillin, cepholathin anti other syn thetic penicillins and salts thereof, other antibiotic agents Those ill Coutheriycin Novolbiocin (e.g. streptomycin, dihydrostreptomycin, bacit racin, po lymixin, tyrothricin, erythromycin, chlorict racycline, 50 15.5 676 coxytetracycline, tetracycline, oleandomycin, chloram 25 55 6/6 55 phenicol, magnamycin, novobiocin, cycloserine, neomy 2. 515 4f6 1111 4f6 cin, kanamycin; in some cases such combinations attack . 646 lf. a wider range of organisms or show synergistic efficacy or ... 5 6.6 (iG S 1:24 12 provide decreased toxicity with equal efficacy), vitamins (). 39 4iii 0.19 16 (e.g. A, A, B1, B2, B6, B12 and members of that family, (). 10 f(5 00 folic acid and members of that family, vitamins C, D, Da 0.05 0/6 and E), hormones (e.g. cortisone, hydrocortisone, 9-a- Control 0/12 fluorocortisone, 9-cy-florohydrocortisone, prednisone and CIEMOTERA. IPF, UTIC, EFFECT OF prednisolone), anabolic agents, (c.g. 1 1,17-dihydroxy-9- C() ill, RMY IN or - fluoro - 17 - O - methyl - 4 - androsten - 3 - one; 17 - a - (ORAL TIERAIPY) ethyl-19-nortestosteronic) and antifungal agents (e.g. ny costatin). 10) 515 () lfli The antibiotic of the present invention is a useful agent 25 (3/6 for the detection of contamination by Gram-negative bac 12.5 (f 6.25 1(). 12 teria, fungi, yeasts and the like in the course of the corn 3.2 113 70 mercial production of the enzymes streptokinase and strep ... 6 ()/ (), S Oli todornase by the growth of streptococci and the produc C(trol 0112 tion of amylase by fermentation of B. subtilis or B. cereits. Thus, the addition of 1 to 1000 meg/ml. and preferably Nutler of lice survived flyer of licy about () mcg../ml., of the antibiotic to an aliquot of in infected. oculated medium followed by incubation, permits tile 3,201,386 3. 4. growth of undesirable contaminants and their visual de 250 F. containing the following ingredients: 7.0% tection. starch ("Staclipse J"), 2.0% cottonseed endosperm The data given above were obtained, as previously flour ("Pharmamedia'), 1.0% debittered yeast, 0.4% stated, or the countermycin complex, called simply cou (NH)2SO4, 1.0% raw linseed oil (containing cobalt mermycin. Paper chromatography serves to separate the 5 salts), 0.5% CaCO3, 0.25% KHPO, 2.0% lard oil and mixtures of counermycins A1 and A. from zones of anti 0.05% of a water-soluble condensation product, having bacterial components which have been named coumerny a total molecular weight of about 1900-2000, of ethylene cin B, coumer mycin C and coumermycin D. oxide with a water-insoluble, hydrophobic polyoxypropyl Mixtures of coumermycins A1 and A2, free of other ene chain formed by condensing propylene oxide with components, are easily obtained by the procedures set O propylene glycol, said hydrophobic chain having a molecu forth below. The identity and amount of each of cou lar weight of about 1750 and constituting about 90% by mermycin A1 and coumermycin A in such mixtures is weight of said water-soluble condensation product determined by the use of gas chromatography. In this (“Pluronic L61). The pH was initially 6.8, fell to 5.3 procedure a mixture of collmer mycins A and A (10 at 40 hours and then rose gradually to a final value of 6.4. mgm.) is suspended in dry reagent grade methanol (200 From this fermentation 2650 l. whole broth was ad microliters) in a 5 mm. glass tube having one end sealed. justed to pH 6 with sulfuric acid and extracted with an To this suspension is added 2.5 M sodium methoxide in equal volume of methyl isobutyl ketone (MIBK) by two methanol (17 microliters). The Solids dissolve. The stage counter-current extraction. The MIBK was con tube is chiled in a solid CO2-acctone bath, sealed, placed centrated to one-tenth volume by vacuum distillation to in a 60 C. bath for 15 minutes, removed and opened. precipitate the crude antibiotic. As it was contaminated Of the reaction mixture therein, 10 microliters are with linseed oil, it was slurried several times in MIBK and chromatographed on an 8 foot by 4 inch copper column in methanol. The product (367.3 g.) was then recrystal containing 13.5 grams of five percent by weight of (Craig) lized in the acid form by slurrying it in a mixture of two polyester succinate (supplied by Wilkins instrument and parts water and eight parts acetone in which it was then Research Inc.) on dichlorodimethylsilane-treated diatoma 2 5 dissolved by the addition of NHOH to pH 10. After ceous earth ("Gas-Crom Z' supplied by Applied Science carbon decolorization the pH was lowered to 6 to pre Laboratories Inc., State College, Pa.). The instrument cipitate the purified product, coumermycin A1, as the is a gas chromatograph supplied by F & M Scientific Corp., acid (303.3 g.). Avondale, Pa. The bridge power is 200 milliamperes, the The acid was then converted to the monosodium salt. attenuation is 2X, the starting column is 100° C., the 30 Thus, 300 g. acid was dissolved in 1500 ml. dimethyl heating rate is 4 C. per minute, the detector block tem formamide and filtered. To the filtrate there was added perature is 260' C., the injection port is 280° C. and the 100 ml. of a 42% by weight solution of sodium 2-ethyl gas (helium) flow from the column is 60 ml/minute and hexanoate in dry n-butanol and then 8500 ml. dry n from the detector is 20 ml./min. Under these conditions butanol. Counermycin A1 spontaneously crystallized as the methyl esters of pyrrole-2-carboxylic acid (from cou 35 the monosodium salt and was collected by filtration and nermycin A2) and 5-methyl-pyrrole-2-carboxylic acid found to weigh 272.5 g. and to be over 99% pure as from Coumermycin A1 have retention times of 12 and 15 determined by vapor phase chromatography. This mate minutes respectively. The detector measures thermal rial was then converted to the free acid, recrystallized conductivity versus helium alone and when recorded is from aqueous methyl ethyl ketone and from methanol used to make a quantitative determination of the amounts 40 methylene chloride and reconverted by the above pro present of the two methyl esters and thus of the amounts cedure to the monosodium salt which was recrystallized of counermycins A1 and A2 in the original mixture. from ethanol-tetrahydrofuran to give 187 g. pure couEner Of these components of the courinernycin complex, the mycin A. When pelleted in potassium bromide this one of greatest interest as an antibacterial agent is sample of coumertnycin A1, as shown in FIGURES3 and coumermycin A1. Unless special fermentation techni 4, exhibited infrared absorption maxima at the following ques are used, it is accompanied by a roughly equal wave numbers in reciprocal centimeters: 3380, 2990, amount of coumermycin A2 from which it is extremely 2940, 1685, 1640, 1605, 1530, 1495, 1430, 1400-1380, difficult to separate by any process of a commercial 1320, 1265, 1225, 1140 (shoulder), 1120, 1090, 1045, ature. 1000, 975, 890, 820, 795 and 770. The maxima at 1495 and 1225 cm. l are exhibited by coumermycin A1 but not COUMERMYCIN A AND COUMERMYCIN A by coumermycin A2. A highly efficient method of causing this fermentation The antibacterial spectrum of the sample of coumer to produce coumermycin A1 to the virtual exclusion of mycin A prepared above was tested by the tube dilution counermycin A2, that is, less than one or two percent of technique to determine the minimum concentrations the latter, is a method which is not a part of the present 5 5 (MIC) of the antibiotic completely inhibiting growth of invention but which comprises adding a salt, preferably a bacteria for 25 hours. The following results were ob water-soluble salt, of cobalt to the medium at or near the tained: beginning of the fermentation. A typically useful Salt is CoCl2. Amounts of 0.0001, 0.0004, 0.0008 and 0.006% have been found to be highly effective in the 60 MIC in mcgiml. usual media. Typical broths contain 500-1000mcg../ml. Modium i of coumermycin A1 and contains zero, less than one or 1st, run 2nd run less than two percent coumermycin A by analysis. A Bacilits 8tbtilis------IIB------7.3 2.5 typical medium is that set forth in Example 8 with the Escherichic coli------FIB------2.5 2.5 omission of the linseed oil. fielsiella pneumonite. IIB ------6.3 6.3 Oels m0rgani------HIB.----- 6.2 6.3 Protcil 8 tillaris------IIIB.----- 3, 3. Example 8 Pscation. Oncts aeruginosa, 8602, A HI8------12.5 25 Pseudomonas (teratinosu (Yale) IIIB ------25 25 'COUMERMYCIN A Saintinella entritidis.------IB------2.5 25 Stilnionicla typhosa.-- Elits------2.5 2.5 O Staph focaccus (urcats (Smith) -- IIIB ------.04 , 002 Streptomyces rishiriensis (six gallons of inoculum) was Staphylococcat.8 aureus (Smith) ------HIB-S- 6 6.3 fermented with agitation beginning at the twentieth hour Streptococcits pyogeti?s------HI8------0.02 0.062 (two sets of impellers at 55 r.p.m.) and with aeration Proteas vulgaris No. 329------HIB------2.5 at 75 cubic feet per minute at 81 F. for 90 hours in HB-Difco Heart Infusion broth, IIIB --S-Same mixed 1:1 with 800 gallons of a medium sterilized thirty minutes at 5 poolcd lunal Scrun. 3,201,386 5 6 Coumermycin A1 has the structural formula:

CI CH IIC O O O O O O CH HCN, =O IN O-e N Ca

E33CO OH ill-t HO OO H Hs d-o C-O - Sir.

Production of coumermycin Al-Streptomyces risliiri 20 methylformamide, moderately soluble in acetone, meth chisis grows luxuriantly at 26-30 C. in agitated or yl isobutyl ketone, methyl ethyl kctone and ethyl acetate, aerated submerged culture. A typical fermentation less soluble in butanol, ethanol, methanol, chloroform medium suitable for the production of coumermycin A1 and benzene, and insoluble in carbon tetrachloride, pe contains the following ingredients: 4.0% hydrolyzed corn troleum ether and acidic water. Mono- and disodium starch, 3.5% "Pharmamedia,' 0.4% glycerin, 0.2% yeast 25 Saits of the antibiotic are more soluble in ethanol than extract, 0.8% CaCO3, 1.0% KHPO, 0.2% KCl. the free acid. Counermycin A is produced in six to eight days' fer The free acid of coumermycin A turns brown at 240 mentation under adequate aeration and agitation, the 245 C. and decomposes at 258-260° C. It analyzes well activity being found mainly in the mycelium. Coumer for Cs5H5NSOo. Calculated: C, 59.51; H, 5.36; N, 6.31. mycin is assayed against Staphylococcus aureus FDA 209P 30 Found: C, 60.5, 59.98; H, 5.59, 5.49; N, 6.10, 6.22. The by cylinder plate method, the inhibition Zone being 20 molecular weight was determined osmometrically to be 21 mm. with a solution of 0.5 mcg../ml. of coumermycin approximately 1 100 compared to the theoretical value of A1. 11 10.1. Titration equivalent was found to be 566 in 75% Extraction and purification of coumermycin. A 1 aqueous dioxane solution with a pKa' of 7.76. Councrmycin A was isolated from the fermentation The crystalline monosodium salt of coumcrmycin A1 broth by a solvent extraction procedure. The broth was deconposes at 245 C. with gas evolution. filtered at pH 5 with filter aid, more than 95% of the Analysis.--Calculated for C5H5NO2Na: C, 58.35; activity being found in the mycelial cake which was ex H, 5.16; N, 6.19; Na, 2.03. Found: C, 58.03; H, 5.26; tracted twice with a solvent mixture of acetone and di N, 6.18. The sodium content was found to be 1.99% by oxane (10:1). The combined extracts were distilled in 40 vacuo to remove the solvents, and the aqueous concen flame spectrophotometric assay. The monosodium salt trate was extracted with ethyl acetate or methyl isobutyl was titrated both by 0.1 N NaOH and 0.1 NHCl with an ketone at pH 6.0. The solvent extract was washed with equivalent weight of 1121 by NaOH and 1182 by HC1. water and then transferred to half volume of water at pH Coumernycin A1 is levorotatory: or D2--141.1 (c. 1, 9.0, extracted again into ethyl acetate at pH 6.0 and then 75% aqueous acetone). concentrated in vacuo to a small volume. Upon the addi In the ultraviolet absorption spectra of monosodium tion of seven volumes of petroleum ether to the concen coumermycin A1 absorption maxima are observed at the trate, the activity separated as a light brown flocculent following wave lengths: precipitate which was collected by filtration, washed with In ethanol: methanol to remove colored impurities, and then dried in vacuo. The crude antibiotic was dissolved in a small 50 214(E=362) mu (E = 670), 273 mu (E=485), 330 mp. amount of dioxane, filtered and precipitated with the addition of five to seven volumes of methanol. The light In acidic ethanol: 1 yellow solid thus obtained was reprecipitated from hot 274 mu (E = 432), 344 mu (E=500) ethanol to give white solid with a potency of 800-900 mcg../mg. 5 5 In alkaline ethanol: 2 Further purification of the antibiotic could be achieved 213 mit (E =861), 237 mu (E = 510), 283 mp. by partition chromatography or counter-current distribu (E=576) tion, but recrystallization of the monosodium salt of Coumermycin A gave a positive reaction with Fehling, coumermycin A1 was found to be more efficient. Molisch and Ehrlich reagents and decolorized bromine in A semi-pure preparation of the antibiotic was dis 60 acetic acid. Tollens reagent gave a silver mirror after solved in tetrahydrofuran (10% w./v.) then 0.1 N aque about eight hours, and anthrone gave a brown color. ous sodium hydroxide solution was added dropwise to give The ninhydrin reaction was negative. The following Rf a pH of 6.5. The solution was diluted with five volumes values were obtained with monosodium coumermycin A1 of ethanol then concentrated in vacuo to a small volume. by ascending paper strip chromatography: butanoi, 0.30; Needle crystals were formed when the concentrate was aqueous 3% ammonium chloride, 0.03; 50% acetone, kept standing overnight at room temperature. These 0.69; butanol-methanol-water (4:1:2), 0.25; benzene were recrystallized from boiling ethanol using a small methanol (4:1), 0.16; water, 0.06. One solvent system amount of active carbon. The colorless needles thus was used to separate other closely related antibiotic frac obtaincd were analytically pure monosodium salt of tions formed during the fermentation and isolation coumermycin A. Pure free acid of counermycin A1 70 studies. This descending system consists of two parts was prepared from the monosodium salt. of acetone and three parts of 0.1 molar triethanolamine Physico-chemical properties of councrimycin Al adjusted to pH 7 with glacial acetic acid. Countermycin A is a white acidic antibiotic, readily solu 195% ethanol containing N/100 HC). ble in alkaline watcr, dioxane, tetrahydrofuran and di 95% ethanol containing N/100 NaOI. 3,20,386 17 8 N VITRO MICROBIOLOGICAL STUDIES 5X10, 5X 103, 5X102, and 50 cell/ml. were used and Antibacterial Spectrum.-The minimum inhibitory con the MIC was determined by two-fold serial tube dilution centration (MIC) of coumermycin A1 against a variety of method, adjusting the initial pH of the test medium to microorganisms was determined by the serial two-fold 7.0. It was found that heavy inocula of more than 108 dilution method. Crystalline monosidium salt of coum 5 cell/ml. increase the MIC value of coumermycin A and ermycin A1 was sterilized by dissolving it in 80% aqueous a similar effect was observed with novobiocin. ethanol at a concentration of 2000 mcg/ml. and serial Effect of media pH on the minimum inhibitory con dilutions were made by sterile water in a set of sterile centration. The MIC of coumermycin A1 was tested by test tubes. From each tube 0.5 ml. was taken and mixed broth dilution method, the pH of the media being con with 9.5 ml. of the respective test medium, the pH of the O trolled by M/10 phosphate buffer in heart infusion broth. media being adjusted to 7.0. Mostly, nutrient agar was A 10 dilution of an overnight culture of Staphylococcus employed as the test medium, however 10% horse blood aureus 209P (5X 10 cell/ml.) was inoculated into each agar was used for hemolytic streptococci and pneumo tube. It was found that the activity of coumermycin in cocci, glucose-yeast-peptone agar for lactobacilli, 2% creases in acidic pH and decreases in alkaline pH. The glucose-Sabouraud agar for fungi and Kirchner's broth comparative data with novobiocin indicate that cou for tubercle bacilli. The results are tabulated along mermycin A1 is affected my media pH to a greater ex with those obtained with novobiocin which was used as tent than novobiocin. a reference: Effect of serum on the minimum inhibitory concentra ANTIBACTERIAL SPECTRUM OF COUMERMYCIN A MIC mcg.fm. Test Ratio Test Organism Media 1 NBINM mycin.Coumer- A Novobiocin Gram-negative:

E. coli NIII------A. 6.25 12.5 E. coli PO 1495------A. 12.5 00 E. coli PO 1495 (CP, TC-R) --- A. 6.25 >100 IE. coli ACC 9637 ------A 6.25 50.0 K.pneumoniae Type A. ---- A 0,78 3.12 Salmonella typhi------A 6.25 6.25 Salmonella paratyphi Al- ---- A 1.56 56 Shigella dysenteriae A- ---- A. 6.25 25.0 Shigellafletneri------A 6.25 3.2 Shigella 80m nei------A. 1.56 50. B. brochiseptica ATCC 4617 ---- A 12, 5 >100 Neisseria sp. (CP-R)------A 12.5 12.5 Pseudomonas (terugin.08d.------A. 2.5 100 Gram-positive: Staph. aureus FDA 209 P------A. 0.0012 0.039 Staph. aureus Fl). A 209-P (ST, SM-R). A 0.002 0.019 Staph. aureus FDA 209-P (NB-R).--- A 0.56 5.0 Staph. aureus 52-34 (PC, TC, EM, CM-R)------A. 0.002 0.078 Staph. aureus 193 (PC, TC, EM, CM R)------A. 0.0006 0.039 Staph. aureus 193 (PC, TC-R) A. 0.0012 0.039 Staph. aureus Smith strain A. 0.0012 0.039 Staph. alb8---- A. 0.0025 0.09 Micrococcu8 flavus ---. A. 0.0025 0.039 Sarcina latted PCI 1001 A. 0.005 0.078 Corynebacterium aerosis A. 0.0006 0.002 Bacilits subtilis PCI219 A. 0.312 0.312 Bacilitats 8phericts 122------A. 0.156 0.156 Bacilius mycoides strain 'O' A. 0.078 0.312 Bacitats cerets ATCC 10702. A. 0.56 0.625 Bacilius anthracis 115------A 0.156 0.56 Streptococcus pyogenes Type 3.-- - B 0.78 0.78 Diplococcus pneumoniae Type ------B 0.78 0.78 Diplococcus pneumoniae DP-3-5A.----- B 0.78 6.25 L. casei ATCC 7469------C >10 0.039 I. acidophilus B-406-1------C >10 0.078 Acid-fast: Mycobacterium tuberculosis war hominis 37 RV------D 0.78 2.5 Mycobacterium. 607- -- D 12.5 25 Mycobacterium phlei------D 1.56 6.25 Fungi: 1spergilius niger war Tieghell.------E >100 >00 Penicilium chrysogenium. -- E >100 >100 Candida albicans------E. >100 >00 Saccharomyces cercviciae ATCC 9763--- E >100 >100 1 Asia nutrient agar. B = nutrient agar containing 10% lorse blood. Ceglucose 1.0%, dry yeast extract 0.5%, polypeptone 0.5%, sodium acetate i.0%, agar 1.5%. pH 7.4. ID is Kirchner's broth (MIC after 10 days). E=Sabouraud agar containing 2% glucose. Abbreviations: -R=resistant; CM=carbomycin; Cl’= chloranphericol; romycin; NB=novobiocin; PC = penicillin; SM=streptomycill; ST=streptothricin; TC= tetracycline. Coumermycin A inhibits the growth of Gram-positive, tion-Increasing concentrations of human serum were Gram-negative and acid-fast bacteria. It is remarkably 65 added to heart infusion broth containing phosphate buf active against staphylococci, the activity being about 30 fer in a final concentration of M/10 to maintain the times greater than novobiocin. It shows, however, re medium pH at 7.0. Staphylococcus aureus 209P was duced activity against a laboratory strain of staphylococ used as a test organism, the inoculum size being 5x iO4 cus which was made resistant to novobiocin. Two strains 70 cell/ml. It was found that the MIC of coumermycin of lactobacilli tested, which are sensitive to novobiocin, A and novobiocin was markedly affected by serum. were found to be resistant to coumermycin A1. Activity of coumermycin against clinically isolated Effect of inoculum size on the minimum inhibitory con staphylococci.-A variety of Staphylococcus aureus cul centration.-The test was done using Staphyloccus tures (126 strains) isolated from clinical sources were aureus 209P as the test organism and heart infusion broth tested in vitro against coumermycin along with six com as the medium. Six levels of inocula, 5x10, 5X105, 75 mercial antibiotics: namely, benzylpenicillin, dihydro 3,201,386 20 streptomycin, tetracycline, erythromycin, kanamycin, and The product was then purified by Craig counter-cur novobiocin. The MIC values were obtained by ten-fold rent distribution using 40 ml. of aqueous phase and 20 SSrial agar dilution method and the material used was a ml. of solvent upper phase in a 200-tube apparatus. The coumermycin complex containing approximately 70% of solvent system used was 15 liters of 0.25 molar triethyl coumermycin A1. The data are presented above. amine in water mixed with 30 liters methyl ethyl ketone The results indicate that coumermycin is highly active and adjusted to pH 8.0 with glacial acetic acid. Twenty against staphylococci and is not cross-resistant with other grams of product were put into 16 tubes and 700 transfers antibiotics. Only one strain out of 126 strains was were made. In two runs the coumermycin A found in found to be resistant to novobiocin. This strain was tubes 105-129 was combined (9.0 g.) put into the first also found to be more resistant to coumermycin. eight tubes of the Craig apparatus using the same solvent N VIVO EXPERIMENTS ON COUMERMYCIN A, system and, after 700 transfers, collected from tubes 115 Toxicologic studies.-Columermycin A is an antibiotic 134 (2.5 g.), converted to the free acid and reconverted of low toxicity. The acute subcutaneous LD50 in mice to 1.145 g. crystalline sodium salt of coumermycin A2 was 380 mg/kg. No death occurred by oral admini for which the following analytical results were obtained: stration of 2000 mg/kg. in the same species. Chronic toxicity was examined in a group of ten rats which re Theory, Found, percent Average, ceived intramuscularly 25 mg./kg./day or orally 100 pcrcent percent mg/kg/day of the antibiotic daily for 60 days (6 day/ 57.6 57.75 57. A() 57.58 4.94 4.54 4.72 4.63 week basic). In this experiment, novobiocin was used as 6,34 5, 62 6.8 5,90 a reference and saline was given to control groups. The 5.82 cor.) (6.41 cor.) (6.13) weight gain was normal and no adverse effect was ob Ash------(Na 2.08) 3.5 3.00 3, 25 served during the treatment period. Percent Volatile------4.15 3, 9 4.0 Chemotherapeutic effect on mice.-The in vivo ac tivity of coumermycin A was tested on mice against an When pelleted in potassium bromide this sample of experimental infection of Staphylococcus aureus, Smith coumermycin A2 as shown in FIGURES 5 and 6, exhib strain. The material used was the free acid form of the ited infrared absorption maxima at the following wave antibiotic, dissolved in dioxane and diluted to the appro numbers in reciprocal centimeters: 3380, 2980, 2940, priate concentrations with sterile water. Mice were in 1695, 1638, 1604, 1535, 1413, 1315, 1265, 1160, 1120, fected intraperitoneally with 100XLDs of the pathogen 1090, 1030,995,973, 950, 885, 820, 765, 750. The bands and the antibiotic was administered subcutaneously or at 1413 and 950 cm. are present in coumermycin A2 but orally just after the bacterial challenge. The log-probit not in coumermycin A1. method of Litchfield and Wilcoxon in J. Pharmacol. Exp. Coumermycin A2 has the structural formula:

CEI (H, H C o O O O O O CH HC sO HN O CHs HCO NH.OC. CONH OCH V O HO E. Ea O l Cs-O O

Dr. H{-- Therap. 96(2):99-113, (1949) was used for the calcu The antibacterial spectrum in vitro of the sample of lation of median curative dose (CD50). A subcutaneous coumermycin A2 prepared above was tested by the tube CD50 of 0.13 mg/kg. and an oral CDso of 4.3 mg/kg. dilution technique to determine the minimum concentra were obtained. In a comparative test, subcutaneous and tions (MIC) of the antibiotic completely inhibiting oral CDso values of novobiocin were found to be 3.0 mg/ growth of bacteria for 24 hours. The following results kg, and 7.6 mg/kg, respectively. were obtained: Example 9 MIC in mcg...fm. COUMERMYCIN Aa Medium Streptomyces rishiniensis was fermented 171 hours with agitation and aeration at 83° F. in a medium containing 1st run 2nd run the following ingredients: 7.0% starch ("Staclipse J'); Bacius subtilis. HIB------12.5 12.5 Escherichia coli.--- HIB------>100 >100 3.0% cottonseed endosperm flour ("Pharmamedia'); Klebsiella pneumoniae- - HIB------12.5 12.5 1.5% debittered yeast; 0.5% CaCO; 0.4% KHPO and Proteus morgani------HII3------25 25 Proteus vulgaris------IIIB ------>00 >00 0.01% "Pluronic L61.' The coumermycin complex so Pseudomonas aeruginosa, 8602/A HIB------> 100 >00 produced contained a large proportion of coumermycin Pseudomonas aeruginosa (Yale IIIB------>100 >100 Salmonella enteritidis.------lils------50 >100 A2 and was isolated as a solid by solvent extraction Salimonella typhosa.------IIIB ------>100 >100 (MIBK). Staphylococcats auretta (Smith).------. HIB------0.25 >1 Staphylococcus aureu8 (Suith)-- IIB --S--- 50 60 Three hundred grams of the above material were con verted to Sodium coumermycin. A by the procedure of Streptococcus pyogenes.----- lt IB------0.5 0.5 the preceding example after removing the 17% of start Proteus vulgaris No. 329. IIB------>100 ing material which was insoluble in tetrahydrofuran. pooledIIIB-Di?co human serum. Heart, Infusion broth, HIB--S Same mixed 1:1 with The product was recrystallized by dissolving in 15 ml. 70 (per gram of Solid) of a solution of equal parts of meth We claim: ylene chloride and methanol by warming to boiling, stir 1. The process for the production of an antibiotic sub ring vigorously, filtering, boiling off some methylene chlo stance, designated coumermycin, which comprises culti ride, adding methanol to a haze point and cooling slowly vating a strain of Streptomyces rishiriensis in an aqueous to allow crystals to form. 75 carbohydrate Solution containing a nitrogenous nutrient 3,201,386 22 under Submerged aerobic conditions until substantial ac 5. A member selected from the group consisting of an tivity versus Gram-positive bacteria is imparted to said acidic compound of the formula CH (I, I O O O O O. O. CE H3C N -O IN O N 4-CH 3CO NI-C C-NH OCEI. O s t HO H H (=o 2H3 d=0

NII

R solution and then recovering said coumermycin from said wherein R is a member selected from the group consisting Solution. of hydrogen and methyl and both R groups are alike; and 2. The process of claim 1 in which the ogranism is nontoxic, pharmaceutically acceptable cationic salts Streptomyces rishiriensis, A.T.C.C. 14812. thereof. 3. A process according to claim 1 wherein the antibi 20 6. The compound of the formula CH3 (H, O O o, o CI3 N -O IN -o/N Y 4.-CH3

-NH-C C-NH- OCEI3 O I y s HO (bit H 3H3 r p=0 NII HX =/ N= otic coumermycin is separated from the fermentation 7. The compound of the formula C CH3 IIC, o O o, o, o, CH, H3C-N N scO IN O 4 -CH3 NH-C C-NH. OCH3 3CO O Y . HO E. - - H3

NH H3 ". 8. A nontoxic, pharmacetulically acceptable cationic broth by extraction at an acidic pH in the range about salt of the compound of claim 6. 50 9. A nontoxic, pharmaceutically acceptable cationic salt 5 to about 6 of said antibiotic with a water-immiscible of the compound of claim 7. solvent in which said antibiotic is soluble. 10. The sodium salt of the compound of claim 7. 4. A process according to claim 1 wherein the anti 11. The calcium salt of the compound of claim 7. biotic coumermycin is separated from the fermentation 12. The streptomycin salt of the compound of claim 7. broth by extraction at an acidic pH in the range about 55 13. The dihydrostreptomycin salt of the compound of 5 to about 6 of said antibiotic into a water-immiscible claim 7. solvent in which said antibiotic is soluble followed by con 14. The compound of claim 7 in substantially pure, centration of said coumermycin-containing solvent. solid form. References Cited by the Examiner UNITED STATES PATENTS 2,123,530 3/64 Rhodes et al. ------195-80 3,126,317 3/64 Heinemann et al. ------195-80 References Cited by the Applicant - UNITED STATES PATENTS 3,049,534 8/62 Wallick. OTHER REFERENCES Hinman et al.: J. Amer. Chem. Soc. 79, 3789-3800 (1957). The Merck Index, Seventh Edition, Merck and Co. Inc., Rahway, N.J. (1960), page 738. LEWIS GOTTS, Primary Examiner. Disclaimer 3,201,386. -Hiroshi Kawaguchi, Masanori Okanishi, and Takeo Miyaki, Tokyo, Japan. COUMERMYCIN AND SALTS THEREOF. Patent dated Aug. 17, 1965. Disclaimer filed Oct. 24, 1966, by the assignee, Bristol-Banyu Research Institute, Ltd. . . Hereby enters this disclaimer to claims 6 and 8 of said patent. Official Gazette January 24, 1967.