ANTIBIOTIC 10381A1

Abstract: Antibiotic 10381a1, wherein R is hydrogen, is a cytosine-containing antibiotic producible by culturing Streptomyces arginensis in an aqueous medium and isolation thereof. Derivatives wherein R is a C1 to C3 alkyl ester of antibiotic 10381a1, are also disclosed along with the pharmaceutically acceptable salts of both the native and esterified forms. The compounds inhibit the growth of selected species of yeast, fungi and bacteria.

Description: ANTIBIOTIC 10381 AT BACKGROUND OF THE INVENTION FIELD OF THE INVENTION This invention concerns a cytosine-containing antibiotic and isolation of this antibiotic from the fermentation beer of a new species of Streptomyces, S. arginensis. 5 INFORMATION DISCLOSURE Cytosine-containing antibiotics are known in the art. Congerotin, Fox, J.J., et al., Tett. Let., p. 3765, (1968); Plicacetin, Stevens, C.L., et al., J. Org. Chem., 31:2822 (1966); and blasticidin, Yonehara, H. and Otake, N., Tett. Let., p. 3785, (1966). Q None of the prior art antibiotics known to applicant teaches or suggests the specific invention disclosed herein. In addition, there are accordingly unexpected and advantageous properties associated with this new antibiotic, e.g., greatly reduced toxicity. SUMMARY OF THE INVENTION . This invention concerns cytosine-containing antibiotics of formula I wherein R is hydrogen or an alkyl of 1 to 1 carbon atoms, inclusive. The compounds are active in vitro against Gram-> positive bacteria, yeasts, and fungi. The compounds are particularly active against Candida albicans (UC R 1392), Penicillium oxalicum (UC 1268), and Sac 5 * 0 charorayces cerevisiae (UC 1342) and have a low toxicity in mammals. (UC is a registered trademark of The Upjohn Company). Specifically this invention concerns both a pure form of a naturally occurring cytosine«containing antibiotic named 103δ1a- | and synthetically derived alkyl esters. 5 The antibiotic of the present invention is produced by a naturally occuring microorganism^ As such, the antibiotic may be produced by this organism as it exists in its natural state. The present invention as it is directed to this antibiotic does not encompass any composition thereof as might have or does exist or occur in nature. Rather, the 0 present invention provides for the production and isolation of this antibiotic in a manner rendering it practically useful, e.g., for pharmacological and other antimicrobial purposes. In addition, the invention includes a process for culturing and purifying 1038lai« Accordingly, the antibiotics described herein can be used alone or 5 in combination with other antimicrobial agents to prevent the growth or reduce the numbers of microorganisms in various environments . For example , the compounds could be used to disinfect surfaces or as an additive to paint to prevent excess growth of microorganisms . DETAILED DESCRIPTION 5 Antibiotics of formula I where R is hydrogen are obtainable from the cultivation of S. arginensis . This antibiotic is designated 1038l a-j . The taxonomy of the organism is given below. The extraction , purification and physi co chemical analysis of the antibiotic are also described below . • JO Compound 1038l a- | has broad spectrum antibiotic properties having an especially strong growth inhibitory effect upon yeasts and fungi . In addition, 1038la- | has less toxicity than blasticidin, a known antibiotic of similar structure . Microorganism 15 The microorganism used for the production of 1038l a- j is a new species of streptomyces , Streptomyces arginensis bietz sp. , N RRL U *159 1 *1 . ' The organism was isolated from soil screening conducted by The Upjohn Company and was taxonomically characterized by Alma Dietz of The Upjohn Company. A subculture of the organism is deposited under the provisions 20 of the Budapest Treaty in the permanent collection of the Northern Region Research Center , ARS; U .S. Dept . of Agriculture; Peoria, Illinois , USA. Its accession number is NRRL->159 1 11 . Color Characteristics Aeri al mycelium is predominantly blue; Melanin- » positive . The 25 color pattern on Ektachrome is given in Table 1 . Reference color characteristics are given in Table 2. The culture may be placed in the Blue (B) color series of Tresner and Backus . (Appl . Microbiology , 11 :335-338, 1963) . Microscopic Characteristics 30 Spores are in short chains that are slightly curved or that have a simple coil near or at the tip . The spores are adorned with short spines and occasionally with fine hair-^llke structures . The spores are elliptical and measure 1 .2 x .8 ym. Growth on Carbon Compounds 5 The synthetic medi um of Shirling and Gottlieb (Int . J. Syst. Bac* teriol . , 16: 313-340, 1966) , was used for this determination . Growth was good on the positive control (D-glucose) , L-arabinose , sucrose, D»- xylose , D.-mannitol , D-«fructose , and rhamnose; and fair on inositol and raff inose. There was no growth on cellulose or on the negative control (synthetic medium ISP-9) without added carbon compound. Whole Cell Analysis L^diaminopimelic acid was detected in the whole cell hydrolysate. Culture Characteristics General culture characteristics are given in Table 3. Temperature Growth on Bennett' s , Czapek' s Sucrose, and Maltose a Tryptone Agars was good at 24°C to 32°C. Optimum growth (good blue aerial growth) was f ound on Bennett' s and Maltose-Tryptone Agars . Growth was fair at 18°C , and moderate at 37°C. There was vegetative growth on Bennett's and Czapek' s Sucrose Agars and aerial growth on Maltose-Tryptone Agar at M5°C There was no growth at 55°C . Streptomyces arginensis was compared to S. cineroviridis ATCC 29776 and S. viridochromogenes ss . sulfomycini ATCC 14920. A summary of the taxonomic comparison is presented in Table* 4. Fermentation and Recovery of Antibiotic 1038l a-ι Antibiotic 103β1a-j (Formula I) is produced when S. arginensis is grown in an aqueous nutrient medium under submerged aerobic conditions . Typically the microorganism is grown in a nutrient medium containing a carbon source and an assimilable nitrogen compound or proteinaceous material. Preferred carbon sources incl ude glucose , brown sugar , sucrose, glycerol , starch , cornstarch, lactose , dextrin, molasses , and the like. Preferred nitrogen sources include corn steep liquor , yeast , autolyzed brewer' s yeast with milk solids , soybean meal , cottonseed meal , cornmeal , milk solids , pancreatic digest of casein , distillers' solids , animal peptone liquors , meat and bone scraps , and the like . Combinations of these carbon and nitrogen sources can be used advanta- geously. Trace metals , for example , zinc, magnesium, manganese , cobalt, iron and the like need not be added to the fermentation medium since tap water and unpurified ingredients are used as medium components . Production of Antibiotic 1038la-j can be induced at any temperature conducive to satisfactory growth of the microorganism preferably between about 20° and 32° C . Ordinarily, optimum production of the compound is obtained in about 2 to 10 days. The medium normally remains weakly basic (pH 7.4 .- 9.0) during the fermentation. The f inal pH is dependent , __l|«- in part, on the buffers present, if any, and in part, on the initial pH of the culture medium which is advantageously adjusted to about pH 7.2 prior to sterilization. When growth is carried out in large vessels and tanks, it is preferable to use the vegetative form, rather than the spore form, of the microorganism for inoculation to avoid a pronounced lag in the production of the new compound and the attendant inefficient utili- zation of the equipment. Accordingly, it is desirable to produce a vegetative inoculum in a nutrient broth culture by inoculating this broth culture with an aliquot from a soil or a slant culture. When a young, active vegetative inoculum has thus been secured, it is trans¬ ferred aseptically to large vessels or tanks. The medium in which the vegetative inoculum is produced can be the same as, or different from, that utilized for the production of the new compound, as long as it is such that adequate growth of the microorganism is obtained. A variety of procedures can be employed to isolate and purify 1038-1a-j, for example, chromatographic absorbtion procedures followed by elution with a suitable solvent, column chromatography, partition chromatography, and crystallization from solvents and combinations thereof. In the preferred recovery process the clear filtrate is used to extract 1038la-|. Column chromatography techniques, preferably IRC- 50(H + ), and Amberlite XAD-4, (Rohm and Haas; Phil, PA, USA) are used to perform the initial purification. Final purification of 10381a- j is achieved by counter current distribution. Bioassays on separate, or combined fractions can be conducted on Micrococcus luteus or Streptococcus pyogenes. Bioassays are conducted by applying 80 μl of test solution to a 1/2 inch paper disc and applying the disc to a growing plate of either test organism. A biounit of activity (BU) is defined as the quantity of antibiotic necessary to achieve a 20 mm zone of growth inhibition around the 1/2 inch paper disc. Antibiotic 1038la-j is an amphoteric compound forming salts with acids, alkalinemetals (includingammonia) alkaline earthmetals (including magnesium and aluminum, and amines). Metal salts can be formed by dissolving 10381a! in water and adding a dilute metal base until the pH is between 7 and 8. Metal salts include sodium, potassium and calcium salts. Amine salts, including those with organic bases such as primary, tertiary, mono-, di>-', and polyamines can also be formed using the above-described or other commonly used procedures. Further, ammonium salts can be made by well-known procedures. Acid salts can be made by neutralizing 10381 a- | with the appropriate acid to below pH 7.0, and preferably to between pH 2 and 6. Suitable acids for this purpose include hydrochloric, sulfuric, phosphoric, sulfamic, hydrobromic, and the like. Acid and base salts of 10381 a can be used for the same biological purposes as the parent compound. Antibiotic 10381 a^ can be esterified under standard esterif ication conditions. Most preferably compounds of formula I can be treated with alcohol, such as methanol, ethanol, n-propanol, or .isopropanol and at reflux temperatures in the presence of hydrogen chloride to yield the (C1HC3) alkyl esters of Formula I. Both the free base and pharmaceutically acceptable salts as defined above can be used for the same biological purposes as antibiotic 10381a- ) . It will be apparent to those "skilled in the art that compounds of formula I and II contain several assymetric carbons. All of the enant iomorphic and stereoisomeric forms of the compounds of formulas I and II are included within the scope of the invention. Antibiotic 10381a! is active against Candida albicans (UC 1392), Penicillium oxalicum (UC 1268), Saccaromyces cerevisiae (UC 1342), Streptococcus pyogenes (UC 152), and Streptococcus faecalis (UC 694). The compounds of formula I are effective for treating bacterial infections in mammals, including humans. Various compositions of the present invention are presented for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, eye drops, oral solutions or suspensions, and water- in-oil emulsions containing suitable quantities of compounds of Formula I. For oral administration, either solid or fluid unit dosage forms can be prepared. For preparing solid compositions such as tablets, the compounds of formula I are mixed with conventional ingredients such as talc, magnesium stearate, dicalcium phosphate, magnesium aluminum silicate, calcium sulfate, starch, lactose, acacia, methyl cellulose, and functionally similar materials as pharmaceutical diluents or carriers. Capsules are prepared by mixing the compound with an inert pharmaceutical diluent and filling the mixture into a hard gelatin capsule of appropriate size. Soft gelatin capsules are prepared by machine encapsulation of a slurry of the compound wi th an acceptable vegetable oil , light liquid petrolatum or other inert oil . Fluid unit dosage forms for oral administration such as syrups , elixirs , and suspensions can be prepared. The water-soluble forms can be dissolved in an aqueous vehicle together with sugar , aromatic flavoring agents and preservatives to form a syrup. An elixir is prepared by using a hydroalcoholic (ethanol ) vehicle with suitable sweeteners such 10 as sugar and saccharin , together with an aromatic flavoring agent . Suspensions can be prepared with an aqueous vehicle with the aid of a suspending agent such as acacia , tragacanth , methylcellullose and the like . , - For parenteral administration, fluid unit dosage forms are prepared utilizing the compounds and a sterile vehicle , water being preferred . The compounds , depending on the vehicle and concentration used, can be either suspended or dissolved in the vehicle . In preparing solutions the compound can be dissolved in water for inj ection and filtered sterilized before filling into a suitable vial or ampoule and sealing . 20 Advantageously, adjuvants such as a local anesthetic, preservative and buffering agents can be dissolved in the vehicle. To enhance the stability, the composition can be frozen after filling into the vial and the water removed under vacuum. The dry lyophilized powder Is then 25 sealed in the vial and an accompanying vial of water for inj ection is supplied to reconstitute the liquid prior to use. Parenteral sus- 1 pensions can be prepared in substantially the same manner except that the compounds are suspended in the vehicle instead of being dissolved and sterilization cannot be accomplished by filtration. The compound 30 can be sterilized by exposure to ethylene oxide before suspending in the sterile vehicle. Advantageously, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the compound. Additionally, a rectal suppository can be employed to deliver the active compound. This dosage form is of particular interest where the 5 mammal cannot be treated conveniently by means of other dosage forms, such as orally or by insufflation, as in the case of young children or debilitated persons. The active compound can be incorporated into any of the known suppository bases by methods known in the art. Examples of such bases include cocoa butter, polyethylene glycols (carbowaxes) , polyethylene sorbitan monostearate, and mixtures of these with other compatible materials to modify the melting point or dissolution rate. These rectal suppositories can weigh from.about 1 to 2.5 gm. The term "unit dosage form", as used in the specification, refers to physically discrete units suitable as unitary dosages for human subjects and animals, each unit containing a predetermined quantity of active material calculated to produce the desired pharmaceutical effect in association with the required pharmaceutical diluent, carrier or vehicle. The specifications for the novel unit dosage forms of this invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular effect to be achieved and (b) the limitations inherent in the art of compounding such an' active material for use in humans and animals, as disclosed in detail in this specification, these being features of the present- invention. Examples of suitable unit dosage forms in accord with this invention are tablets, capsules, pills, suppositories, powder packets, wafers, granules, cachets, teaspoonfuls, tablespoonfuls, dropperfuls, ampoules, vials, aerosols with metered discharges, segregated multiples of any of the foregoing, and other forms as herein described. An effective quantity of antibiotic is employed in treatment. The dosage of the specific compound for treatment depends on many factors that are well known to those skilled in the art. They include for example, the route of administration and the potency of the particular compound. A dosage schedule for humans of from about 1 to 2 grams of compound in a single dose, administered parenterally or in the compositions of this invention, are effective for treating bacterial infections. More specifically, the single dose is about 2 grams of compound. The oral and rectal dose is from about 1 to 2 grams in a single dose. More speci ically, the single dose is about 2 grams of compound. Without further elaboration, it is believed that one skilled in the art can, using the preceding description, practice the present invention to its fullest extent. The following detailed examples describe how to prepare the various compounds and/or perform the various processes of the invention and are to be construed as merely illustrative, and not limitations of the preceding disclosure in any way whatsoever . Thos skilled in the art will promptly recognize appropriate variations fro the procedures both as to reactants and as to reaction conditions an techniques . Example 1 A. Fermentation A soil stock of Streptomyces arginensis , NRRL"15941 is cultured o HickneyTresner Agar and stored at 4°C. Samples are homogenized i water and replated on Hickney-Tresner Agar . After 7 days of incubatio a 28° C , the growth is sufficient for a seed culture inoculum . Seed cultures are grown in seed medi um (SSM..1 ) containing pe liter of tap water: blackstrap molasses 5.8 g , Difco peptone 10 g Difco yeast extract 4 g, dextrin 4 g, Liasparagine 0.2 g , COCI2.6H2O mg; the medium is adj usted to pH 7.2 with KOH before sterilization The SSM.1 is dispensed into plain 500 ml wide-mouth Erlenmeyer flask (capped with 2 milk filters) at 100 ml/ flask and autoclaved for 3 minutes at 121 °C , 15 psi . Flasks' are inoculated with homogenized aga plugs at a rate of .5-1 plug/ flask and shaken (250 rpra, 2.5" stroke for 3 days at 28°C . The harvest pH is 8.3. The seed cultures are fermented in media containing in amounts pe liter of tap water: soybean meal 20 g , brewer' s yeast 2 g , Cerelose 2 g (added as 50?. solution after autoclaving) ; the medium is adj usted t pH 7.2 before sterilization . The medium is dispensed, sterilized an shaken as described above. The inoculation rate is 5 ml/1 00 ml of see culture . Fermentation conditions are for 3 days at 28°C . B. Purification for a 10 liter fermentation beer . The beer is filtered using a f ilter aid as required and the soli cake is washed in water in an amount one-tenth the volume of the filtrat filtered and added to the beer-filtrate which is saved. A one liter • column of IRC-50 (H + ) is prepared and the filtrat mixture is adj usted to pH 8.5 using ammonium hydroxide . The filtrat is passed through the column at a rate of 50H50 ml/minute and collecte as a single fraction . The antibiotic fraction is eluted off the IRO50 column using 2 aqueous ammonium chloride collecting 2 liter-fractions and combinin active fractions . An 800 ml column of Amberlite XAD-4 is prepared. The precedin ammonium chloride eluate is adj usted to a pH of 8.0 using ammoniu hydroxide and passed over the column at a rate of 50*-60 ml/ minute . Th column is washed with 3 liters of water . The active fraction is elute off by 5 elutions with met hanol- water 70:30 w/v. The 5 elutions o met hanol* 1 water are concentrated to an aqueous solution , freeze r -drie and tested for activity. The various filtrate or eluant solutions are bioassayed as previousl described. A typical assay is presented in Table 5. The fraction from the IRC-50 column are being tested against M . luteus . Final purification of 10381 a! is achieved by countercurrent distri bution (CCD) . Active fractions from an Amberlite XAD ^ «4 column ar dissolved in both phases of the solvent system consisting of 1-butanol/p 7.0 phosphate buffer and distributed in an all-glass countercurren distribution apparatus . After approximately 500 transfers , the bioactivit of the CCD-fractions is determined and the active fractions concen trated to an aqueous solution which is' freeze^dried. The residue i dissolved in water and pH adj usted to 8. The aqueous solution is passed through a one liter column o Amberlite XAD-4 , and washed three times with three one-liter portion of water (wash 1 , 2 and 3) . The column is then eluted with methanol water ( 70 :30 v/v) . The aqueous-methanolic fractions are assayed fo antimicrobial activity (Table 6) . Biologically 11 active fractions ar combined , concentrated to an aqueous solution and freeze-'dried to yiel pure antibiotic 10381 a! . The physicochemical properties of 1038l aι ar presented in Table 7. The spectrum of 10381 a ' s antimicrobial activit is presented in Table 10. Example 2. Antibiotic 10381 a! Hydrochloride Antibiotic 10381 a! , 300 mg is dissolved in 10 ml of 1N methanoli hydrogen chloride . The solution is clarified by f iltration over sintered glass filter and mixed with 100 ml of acetone and 50 ml o ether . The precipitated colorless material is isolated by filtratio and dried. Physicochemical data for 10381 a! hydrochloride are presente in Table 8. Example 3 Antibiotic 10381 a Methyl Ester Hydrochloride Antibiotic 10381 a! , 500 mg is dissolved in 20 ml of 0.8 N_ methanoli hydrogen chloride . The solution is kept at reflux for 3 hours and the cooled and clarified by filtration over a sintered glass filter . Th clear solution is mixed with 200 ml of acetone and 50 ml of ether. The precipitated colorless material is isolated by filtration and dried. Physicochemical data for 10381a! methyl ester hydrochloride is presented in Table 9. Utilizing a procedure similar to that described in Example 3 but substituting ethanolic or propanolic hydrogen chloride, there is obtained the corresponding ethyl or propyl ester. __!!__ Table 1. Color characteristics 1 on Ektachrome2 of Streptomyces arginensis NRRL-15941. Agar Medium Chip Color Bennett's S 190 light blue gray R 77 moderate yellow brown Czapek's sucrose S 263 white R 70 light olive Maltose-tryptone S 190 light blue gray R 81 dark grayish yellowish brown Peptone-iron S 81 dark grayish yellowish brown R 81 dark grayish yellowish brown • 0.1% Tyrosine S 191 bluish gray R 76 light yellowish brown Casein starch S 191 bluish gray R 77 moderate yellowish brown S = surface, R « reverse. 1 Growth on media in tubes was photographed after seven days incubation at 28°C. Color was determined by comparison with NBS color chips. (Kelly, K.L., and D.B. Judd. 1976. Color. Universal Language and Dictionary of Names. BS Spec. Publ. 440. Superintendent of Documents , U.S. Government Printing Office, Washington, D.C. 20942; and SRM 2106. 1958, ISCC-NBS Centroid Color Charts. Office of Standard Reference Material, Room B311, Chem. Building, National Bureau of Standards, Washington, D.C. 20234.) Dietz, A. and Thayer, D.W. (ed.) 1980. SIM Special Publ. No. 6. Soc. for Ind. Microbiol., Arlington, VA. Table 2. Reference color characteristics! Streptomyces arginensis NRRL-15941. Agar Medium Chip Color - Bennett' s S 184 very pale blue R 87 moderate yellow P «*«* Czapek's sucrose S 7 pale pink 9 pink white R 33 brownish pink P r— Maltose" tryptone S 10 pinkish gray (edge) . 60 light grayish brown (center) R -π moderate yellowiέh brown P 90 grayish yellow Yeast extract- S 190 light bluish gray malt extract R 88 dark yellow ( ISP- 2) P 90 grayish yellow Oatmeal S 189 bluish white (ISP-3) R 105 grayish greenish yellow P 93 yellowish gray Inorganic salts S : 184 very pale blue starch R 105 grayish greenish yellow (ISP-4) P fltth Glycerol** S 190 light bluish gray asparagine R 105 grayish greenish yellow (ISP-»5) S - surface, R - reverse, P ■ pigment. 1 Color determination was made on growth on plates incubated 14 days at 28°C. Color was determined by comparison with NBS color chips. (Kelly, K.L., and D.B. Judd. 1976. Color. Universal Language and Dictionary of Names. NBS Spec. Publ. 440. Superintendent of Documents, U.S. Govern¬ ment Printing Office, Washington, D.C. 20942; and SRM 2106. 1958. ISCC^NBS Centroid Color Charts. Office of Standard Reference Material, Room B311 . Chem. Building, National Bureau of Standards, Washington, D.C. 20234.) « 4r- Table 3. Culture characteristics! - a general Streptomyces arginensis NRRLΓ 5941 . Medium≥ Culture Characteristics Agar Peptone* iron S brown vegetative growth R brown P brown 0 melanin 8 * positive Calcium malate S pale gray R pale gray P « * 0 malate solubilized Glucose as par'agine S pale gray R pale cream Skim milk s pale tan vegetative R o ange÷' an P orange-tan 0 casein not solubilized Tyrosine s trace gray R brown P pink- brown 0 tyrosine not solubilized Xanthine s very slight trace gray R pale yellow^tan 0 xanthine solubilized Nutrient starch S very slight trace gray R pale yellow P pale yellow -15H 0 starch solubilized Yeast extract-* S pale blue-white malt extract R yellow tan P yellow tan Peptone- S tan vegetative yeast extract R tan brown iron P tan brown ( ISP a 6) 0 melanin- doubtful Tyrosine S pale blue ( ISP -7) R gray brown P trace tan 0 melanin^negative * Gelatin Plain s tan surface ring P tan 0 trace liquefaction Nutrient s tan surface ring P tan 0 no liquefaction Broth Synthetic nitrate wide surface ring with trace pink aerial growth P pale yellow 0 flocculent throughout and at base no reduction Nutrient nitrate S no growth P brown ( 1 tube ) , tan (2 tubes ) 0 flocculent at base no reduction Litmus milk S brown ring P 0 no change in litmus pH 6.3 (control, 6.5) S * surface, R = re-verse, P = pigment, 0 = other characteristics 1 Dietz, A. and Thayer, D.W. (ed.) 1980. SIM Special Publ. No. 6. Soc. for Ind. Microbiol., Arlington, VA. 2 Growth on media in tubes was photographed after seven days incubation at 28°C. Color was determined by comparison with NBS color chips. (Kelly, K.L., and D.B. Judd. 1976. Color. Universal Language and Dictionary of Names. NBS Spec. Publ. 440. Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20942; and SRM 2106. 1958. ISCC-NBS Centroid Color Charts. Office of Standard Reference Material, Room B311, Chem. Building, National Bureau of Standards, Washington, D.C. 20234.) * « Table 4. Comparison of S. arginensis NRRL-15941 with S. viridochromogenes ss . sulfomycini ATCC 29776 and S . viridochromogenes ATCC 14920! . Characteristic NRRLH15941 ATCC 29776 ATCC 1 920 Aerial mass color blue + + + , a Colony reverse color Peptone- iron agar brown brown ' - Melanoid pigments Peptone^ yeast- 1 iron agar a ή Tyrosine agar « + > * + , •* ■ ' Utilization of # sucrose ++ 4-+ doubtful Spore chain morphology short moderate long loose coil compact open spiral spiral (spores/ chain) 10*15 <50 >50 Spore surface spiny spiny spiny Production of 10381 a not tested !Dietz, A. and Thayer, D.W. (ed.) 1980. SIM Special Publ. No. 6. Soc. for Ind. Microbiol., Arlington, VA. Table 5. Bioassay ZONES (mm) Diluti on FS 1/2 1/4 1/8 1/16 Filtrate 26 23 21 18 Trace Spent 0 0 0 0 I 0 Wash 0 0 0 0 0 NHι,Cl (9L) 25 22 19 15 Trace Table 6. Bioassay Full 1/10 1/100 Amount Strength (zone of inhibition vs. M.luteus) SPENT 0 0 0 WASH→I 0 0 0 WASH- 2 16(1) 0 0 WASHn3 0 0 0 Methanol-1 500 ml 19 0 0 Methanol-2 500 ml 44 35 25 Methanol-3 500 ml 35 25 0 Methanol-4 500 ml 27 traces 0 Methanol 5 1,000 ml 23 Table 7. CHARACTERIZATION OF ANTIBIOTIC 10381a 1. Appearance: Colorless amorphous material. 2. Solubility: Soluble in water and lower alcohols. Insoluble in acetone, ethyl acetate, ether and chlorinated or saturated hydrocarbon solvents . 3. Molecular Composition: C!8H28N8θ5 Calcd molecular weight: 436.2183 Found (HR-FAB/MS): 436.21 76. 4. [o] 25 D: +44° (C, 0.55, water). 5. Melting point: Decomposition at ca. 218° C. 6. UV Spectrum: λmax(nm) α Solvent 268 1 .02 " " 6100 water 268 14.25 6200 0.1N NaOH 276 19.55 8500 0.1N HC1 7. The PMR spectrum of antibiotic 10381a! is presented in Figure 1. 8. The Rf vaiues of. Antibiotic 103δ1aι using paper chromatography and various solvent systems are listed below: Table 7 (Continued) PAPER CHROMATOGRAPHIC MOBILITIES OF ANTIBIOTIC 10381 A! No.(#) Solvent System! if_ 1 1 -Butanol: water , 21:4 0 2 #1 with 0.25? p^toluene sulfonic acid 0.2 3 1-Butanol: acetic acid: water, 2:1:1 0.2 4 #1 with 2% piperidine NZ 0 5 1*Butanol: water, 1:24 0.7 6 #5 with 0.25J p-*toluene sulfonic acid 0.8 7 0.1 M_ potassium phosphate, pH 7-0 0.9 : 8 0.075 _ ammonium hydroxide saturated 0.9 with methyl isobutyl ketone 5 9 Benzene: methanol: water, 1:1:2 0 (upper phase) • 10 #1 with 2% p 4 toluene sulfonic acid • 0.2 1 ' 1 • Methanol :15% NaCl, 4:1 0.5 0 ! Liquid ■ Liquid ratios are v/v; solid percentages are w/v. NZ = No zone. 9. IR Band Tabulation: IR BAND TABULATION OF ANTIBIOTIC 10381A! , METHYL ESTER 5 Band Band Frequency Intensity Type Frequency Intensity Type 3197.0 13 BRD 1605.7 6 BRD 2953.9 0 BRD M 1522.7 17 AVG 2916.3 0 BRD M 1490.9 !4 AVG 0 2868.1 3 SH M 1464.9 9 AVG M 2854.6 2 AVG M 1411.8 32 SH 2735.0 59 SH M 1391.6 23 SH 2681.0 64 SH M 1378 * 1 13 AVG M. 1649.1 4 BRD 1354.9 37 SH 5 Band Band Frequency Intensity Type Frequency Intensity Type 1296.1 39 SH 944.1 62 AVG 1280.7 36 AVG 896.8 65 SH 1229.6 38 AVG 868.9 57 AVG 1203.5 45 AVG 826.4 47 AVG 1184.2 53 SH 788.8 37 AVG 1 1 17.7 44 AVG 778.2 41 SH 1070.4 33 AVG 721 .3 33 AVG M 983.6 59 AVG Band Frequency: Band frequencies in wavenumbers (CM^l) Intensity: Intensity in percent transmittance ($T) Data Type in Local Peak Region: BRD = Broad; AVG - Average; SHP Sharp; SH = Shoulder. . M: Possible interference from mineral oil. Table 8. CHARACTERIZATION OF ANTIBIOTIC 10381a! HYDROCHLORIDE 1. Appearance: Colorless amorphous material. 2. Meltini 5 poiπ it: ca 120°C wi ,h decomposit ;ion. 3. [ct]25 D ,+40° (C, 0. 897, water). 4. UV: λmax=271 ; α=!4 .64; i ε=7950. 5. IR Band Tabulation : Band Band Frequency Intensity Type Frequency Intensity Type 3343.5 18 BRD 2019.4 86 BRD 3200.8 18 BRD 1727.2 25 AVG 3066.8 24 BRD 1655.8 5 BRD 2948.1 0 BRD M 1632.7 9 SH 2909.6 0 BRD M 1537.2 26 BRD 2868.1 1 SH M 1493.8 33 AVG 2853.6 0 AVG M 1462.0 12 AVG M 2733.1 • 52 SH M 1398.3 42 BRD 2674.2 57 SH M 1378.1 .21 SHP M Band Band Frequency Intensity Type Frequency Intensity Type 1340.5 52 SH 897.8 72 BRD 1296.1 49 SH 865.0 67 AVG 1271.0 42 AVG 826.4 59 AVG 1242.1 40 AVG 804.3 60 SH 1197.7 52 AVG 779.2 51 AVG 1120.6 48 AVG 763-8 56 SH 1074.3 39 AVG 721.3 45 AVG M 965.3 68 BRD 649.0 44 SH Band Frequency: Band frequencies in wavenumbers (CM"!) Intensity: Intensity in percent transmittance (JT) Data Type in Local Peak Region: BRD » Broad; AVG = Average; SHP Sharp; SH = Shoulder. M: Possible interference from mineral oil. Table 9. CHARACTERIZATION OF ANTIBIOTIC 10381a METHYL ESTER 1. Appearance: Colorless amorphous material. 2. Melti: ng point: ca 110°C (d decomposition ). 3. C ]25 ; D.-H5 0 (C, 1. 03, water). 4. UV: λmax=268; a-13 .21; ( £=5950. 5. IR Band Tabulation IR BAND TABULATION OF ANTIBIOTIC 10381 A! , METHYL ESTER HYDROCHLORIDE Band Band Frequency Intensity Type Frequency Intensity Type 3333.9 21 BRD 1735.9 11 AVG 3194.1 18 BRD 1679.9 4 ! AVG 3047.5 20 BRD 1659.7 8 SH 2960.7 0 BRD M 1631.7 13 AVG 2853.6 0 AVG M 1539.1 29 AVG 2733.1 47 SH M 1461.0 11 AVG M 2671.4 55 * SH M 1377.1 23 ■ SHP M 2639.5 58 SH 1366.5 37 AVG 2007.8 91 BRD 1312.5 56 SH Band Band Frequency Intensi y Type Frequency Intens ty Type 1267.2 32 AVG 936.4 76 SH 1239.2 35 AVG 894.0 77 BRD 1215.1 46 AVG 866.0 70 AVG 1195.8 48 AVG 824.5 61 AVG 1122.5 47 AVG 779.2 57 AVG 1082.0 45 AVG 764.7 60 AVG 1013-5 66 AVG 721.3 49 AVG M 965.3 74 BRD 647.1 45 BRD Band Frequency: Band frequencies in wavenumbers (CM"1) Intensity: Intensity in percent transmittance (ΪT) Data Type in Local Peak Region: BRD « Broad; AVG - Average; SHP Sharp; SH » Shoulder. M: Possible interference from mineral oil. Table 10. ANTIBACTERIAL SPECTRUM OF ANTIBIOTIC 1038^! Organism Culture if Zone of Inhibition (mm) Bacillus subtilis UC 564 0 Staphyloeoccus aureus UC 80 1δh Staphylococcus aureus UC 3665 0 Micrococcus luteus UC 130 32 Micrococcus luteus UC 3383 32 Klebsiella pneumoniae UC 57 0 Escherichia coli UC 51 0 Salmonella schottmuelleri UC 126 0 Proteus vulga is UC 93 0 Mycobacteriαn avium UC 159 17 PeniciIlium oxalicum UC 1268 36 Pseudomonas aeruginosa UC 95 0 Rhodopseudomonas UC 3238 33 ' sphaeroides Streptococcus pyogenes UC 152 25 Clostridium perfrigens UC 6509 18 Bacteroideβ fragilis UC 6513 21 •Staphylococcus aureus UC 6685 0 ♦ Staphylococcus epidermidis UC 719 tr •Streptococcus faecalis UC 221 10 vl •Streptococcus pneumoniae UC 9207 tr •Hemophilus influenzae UC 6483 10 •Serratia marcescens UC 6888 • 0 •Neisseria gonorrhoeae UC 3065 tr •Candida albicans UC 1392 12 Saccharomyces cerevisiae UC 1342 31 Bordetella bronchiseptica UC 6481 0 Pasteurella hemoly ica UC 6531 0 !Assays marked * used 1/4 inch (6.35 mm) discs and 20 μl samples, All other assays used 1/2 inch (12.7 mm) discs and 80 μl samples. h = hazy; vl = very light; tr = trace. Assay procedures are described in Hanka, L.J., M.R. Burch and W.T. Sokolski. Psicofuranine. IV. Micro? 4 biological assay. Antibiot. and Chemoth. 9:432-435 (1959). FORMULA CHART H 2 N- C- NH 27 a International Application No: PCT/ (JS86 ' 00657

Claims: 28 β CLAIMS We claim: 1 . An antibiotic of formula I , except as existing or occuring in nature , represented by the following formula: JIJU H 2 N- C- NH wherein R is either hydrogen or an alkyl of 1 to 3 carbon atoms * inclusive , and pharmaceutically acceptable salts thereof . 2. The antibiotic described in claim 1 wherein R is hydrogen and pharmaceutically acceptable salts thereof . 3. The antibiotic described in claim 1 wherein R is methyl and pharman ceutically acceptable salts thereof . . The hydrochloride salt of the antibiotic described in claim 1 wherein R is hydrogen. 5. The hydrochloride salt of the antibiotic described in claim 1 wherein R is methyl . 6. An antibiotic according to claim 1 in an essentially pure form. 7. A process for producing an antibiotic of formula I which comprises cultivating Streptomyces arginensis in an aqueous nutrient medium containing a source of assimilable carbohydrate and assimilable nitrogen under aerobic conditions until substantial antibiotic activity is imparted to the medi um and isolating the antibiotic. « *29 8. A process according to claim 7 in which the isolation comprises filtering the fermentation medium on ion exchange resins, eluting the antibiotic from the resin and subjecting the eluates to purification procedures.

Inventors: ARGOUDELIS ALEXANDER D, LABORDE ALICE L, SEBEK OLDRICH K, TRUESDELL SCOTT E, ARGOUDELIS, ALEXANDER, D., LABORDE, ALICE, L., SEBEK, OLDRICH, K., TRUESDELL, SCOTT, E.
Applicants: UPJOHN CO, THE UPJOHN COMPANY
Application year: 1986
Main classification: C07D405
IPC codes: A23K1, C07D405, C07H19, C12P17