SE456588C - Biologically pure culture of Actinomadura yumaense NRRL 12515 and method for producing antibiotic LL-C23024 alpha, beta and iota - Google Patents

Description

456 588 and antibiotic LL-C23024 jota, which later proved to have the following structural formula: OM: in recoverable amounts during fermentation in a liquid nutrient medium containing assimilable sources of carbon, nitrogen and inorganic salts. The microorganism may be a spontaneous natural mutant, which is genetically modified, but has essentially the same properties as Actinomadura yumaense NRRL 12515 and still retains the “ability to synthesize antibiotic LL-C23024 alpha, beta and iodine. The microorganism may also have been mutated with mutagenic agents known to those skilled in the art.

The invention further relates to a process for the preparation of antibiotic LL-C23024 alpha, beta and iodine.

Antibiotic LL-C23024 beta according to the invention differs from previously known compounds due to its physical and chemical characteristics, including but not limited to its microanalysis, optical rotation, IR spectrum, 13 C nuclear magnetic resonance spectrum and 1 H nuclear magnetic resonance spectrum.

The above structure for LL-C23024 beta is based on elemental analysis, optical rotation, field desorption, 456 588 mass spectroscopic molecular weight, melting point, IR spectrum, 13 C nuclear magnetic resonance spectrum (13 C-NMR) and proton magnetic resonance spectrum (1H NMR). as detailed in the examples and shown in the accompanying figures: Fig. I: IR spectrum of LL-C23024 beta in KBr; 13 Pig. II: C-NMR spectrum of LL-C23024 beta. 20 MHz in CDCl 3, internal TMS reference equivalent; Fig. III: 1 H-NMR spectrum of LL-C23024 beta. 80 MHz in CDCl 3, internal TMS reference equivalent. 456 588 Table I Boman spectrum for LL-czaouß (ypm in ratio: in Tus) Chemical shift, (gym) Number of carbon atoms 10.5 1 11.0 1 12.2 1 17.0 l 17.7 1 17.9 1 22.3 1 26.1 1 26.8 1 27.6 1 30.2 1 32.0 1 33.3 1 33.7 2 33.8 2 36.5 1 36.8 l 39.0 1 39 .9 1 45.5 2 57.1 1 59.1 1 60.7 1 66.9 1 67.6 1 70.2 1 71.3 1 72.9 1 74.9 1 75.1 1 79.9 1 80.8 1 82.1 2 84.5 1 84.7 1 85.6 1 86.9 1 95.8 1 96.9 1 97.7 1 107.5 l 179.2 1 Carbon atoms, total 46 " './ 456 588 LL-C23024 jota is a new polyether antibiotic with very potent anticoccidial activity. It is at least ten times more potent than most other known polyethers. Field desorption mass spectral data show that it has a molecular weight of 930.

The only other reported polyether antibiotic with a molecular weight of 930 is antibiotic A28695B (hydroxyseptamycin) (J. Antibiotics 33 (2): 252 (l980) |, which differs significantly in structure and biological properties. The above observations are based on elemental analysis, optical rotation, calculated molecular weight by field desorption mass spectroscopy, IR spectrum, 13 C-NMR spectrum and proton nuclear magnetic resonance spectrum (1 H-NMR), as detailed in the examples and shown in the accompanying drawing figures: Fig. IV: IR- spectrum for LL-C23024 iodine in KBr; rig. v; ln NMR spectrum for LL-c23oz4 jaaa 80 MH: in CDCl3, internal TMS reference equivalent; 13 Fig. VI: C-NMR spectrum for LL-C23024 iota 20 MHz in CDCl 3, internal TMS reference equivalent; Fig. VII A: 13 C-NMR spectrum of LL-C23024 iodine (extended scale: 0-50 ppm). 20 MHz in CDCl 3, internal TMS: Fig. VII B: 13 C-NMR spectrum for LL-C23024 iodine (extended scale: 50-110 ppm) 20 MHz in CDCl internal TMS reference equivalent. C23024 iodine was obtained in deuterated chloroform (CDCl3) at a field strength of 20 MHz. The peaks with their respective chemical shifts in parts per million in relation to tetramethylsilane (TMS) and the estimated number of carbon atoms per peak are given in Table II. Peaks were observed for chloroform at 75.4, 77.0 and 78.6. 456 588 6 Table II Chemical shift Number of carbon atoms chemical shiftLntal carbon atoms 10.6 10.9 11.7 l6.U 17.6 18.0 21.7 22.9 2U.l 28.2 31.2 32.2 33- 33. 3U. 36. 37. 39.

H0.

UN. 12.1: ° ~ IU1 O \ U1U1LIJJ = N ~ 1NCD- = UI O \ @ F 'FJF- * UJI-'bJD-'D-'l- * WÅIO-'I-'MFJFJI- * I-'l- * b- * P-'l-'k fl 59.9 60.8 68.5 68.8 71-3 72.2 76.1 76.9 78.5 81.0 81.3 81.8 88.8 85.3 85.6 86.8 88.5 95.9 97.9 99.6 107.2 173.0 Total. = oolw- w- F- v- v- w- v - vf F- F- P- h- w- v- v- h- r- r- F- have new pa o Antibiotic LL-C23024 alpha, beta and iodine are organic carboxylic acids and can thus form salts with non-toxic pharmaceutically acceptable cations. The salts formed by mixing the antibiotic free acid with stoichiometric amounts of cations, suitably in a neutral solvent, can be formed with cations such as sodium, potassium, calcium, magnesium and ammonium as well as with organic amine cations such as' lv a »456 588 tri (lower alkyl) amine (eg triethylamine, triethanolamine), procaine and the like. The cationic salts of antibiotic LL-C23024 beta are generally crystalline solids, relatively insoluble in water and soluble in most common organic solvents such as methanol, ethyl acetate, acetone, chloroform, heptane, ether and benzene. For the purposes of the invention, the antibiotic free acid is equivalent to its pharmaceutically acceptable non-toxic salts.

Antibiotic LL-C23024 beta and iota are active in vitro against gram-positive bacteria when tested according to the standard agar dilution procedure. The results are given as the minimum inhibitory concentration (MIC) in pg / ml in Tables III and IV. Antibiotic LL-C23024 beta and iota are not active against gram-negative organisms at concentrations of 256 pg / ml or less. 456 588 Table III In vitro antibacterial activity of LL-czaozmó ORGANISM Minimum inhibitory concentration (pg / ml) Enterococcus SSC-80-62 "S $ C-80-53 Staphylococcus aureus Smith" "SSC 80-ll" "SSC 80 -32 HH ssc ao-38 "" LL-lä uu LL _) _ | 5 nn LL_27 "" ATCC 25923 Streptococcus pyogenes C203 "B-hemblytic Keller T623" pneumoniae 78-l en eu sd 6ä su en 128 en Öü 128 Table IV In vitro antibacterial activity of LL-C23024 jota Organism minimum inhibitory concentration island (n / ml) LL-C23024 Jota Enterococcus OSU 75-1 1 Enterococcus SM 77-15 1 Staphylococcus aureus SSC 79-18 1 Staphylococcus aureus FU 79-19-2 2 Mícrococcus Igšgg PCI 1001 1 Stanhylococcus aureus Smith 0.5 Staphylococcus aureus ATCC 25923 0.5 vi 456 588 As can be seen from the data above, the antibiotic substances LL-C23024 inhibit alpha, beta and jota growth of certain gram-positive bacteria and are thus antiseptic or antiseptic useful as a surface antiseptic in washing solutions for skin, equipment, walls, floors etc.

It has also been found that the antibacterial agents LL-C23024 alpha, beta and iota are active in vivo as an anticoagulant for poultry, as demonstrated by the following tests.

Two days before inoculation, prepared feed with different levels of drug was given to different groups of one-day-old test chickens. The poultry feed used in the test was prepared as follows: Vitamin-amino acid premix 0.5% Trace mineral 0.1% Sodium chloride 0.3% Dicalcium phosphate 1.2% Ground limestone 0.5% Stabilized fat 4.0% Dehydrated alfalfa, 17 % protein 2.0% Corn gluten meal, 41% protein 5.0% Menhaden fish meal, 60% protein 5.0% Soybean oil meal, 44% protein 30.0% Ground yellow corn, to 100% Vitamin-amino acid premix in the above feed the composition was prepared according to the following composition. Quantitative terms refer to units per kg of the finished feed composition.

Butylated hydroxytoluene 125.0 mg DL-methionine 5000, o mg Vitamin A 3300.0 I.U.

Vitamin D ll00.0 I.C.U. 3 Riboflavin 4.4 mg 456 588 Vitamin E 2.2 1.0, Niacin 27.5 mg Pantothenic acid 8.3 mg Choline chloride a 500.0 mg Folic acid, 1.43 mg Menadione sodium hydrogen sulphate 1.1 mg Vitamin B12 11.0 pg Ground yellow maize, to 5.0 g The test chickens were then inoculated by direct inoculation into the inoculum of each chicken with a mixed inoculum of 5000 sporulated oocysts of Eimeria acervulina and a sufficient number of oocysts of Eimeria tenella to produce 85-100 % mortality in untreated controls. The chickens were given free access to feed and water throughout the test period. 7 days after inoculation, the tests were completed and the birds were weighed and killed and their gastrointestinal tract examined for damage. The results are set forth in Tables V and VI below and show that improved survival in infected chickens is obtained when 10 ppm or less of the antibiotic LL-C23024 beta or iota is added to the diet. The results also show a significant inhibition of intestinal damage caused by Eimeria tenella and Eimeria acervulina. u: 456 588 ll HÜHHOHUCOX UÜUHUUMÜMGH ÜMUH ~ UÜN fi UCNÃU fl ÜXUH H UZZ ¥ || || oo fi ma ozz 0 0 ßm ma o O ß ßv m fl m O O om ma o fi oo fi oo oo fl m fl mä ooH ä oo fi 3 oN ïàoåolqq nu || oo fi md ozz ooo ~ m fi o oo fi oo fl oo fi mo om oo fi oo fi oo fi ma om oo fi oo fi oo fi m fl ow fi «\« ~ om ~ o | qq oo o_o «m fi o« uzz oo fl o om mm Q «~ oo | Aq oo fl oo ooH m oH Kåomwuaqq oo fl oo fi oo fi m mä @ \ <~ om ~ u | Au m: flfi:> uwum .W m fifl mcmu .m cmnußn: www Emm .cwuww fl mc fl cmunh HOÜNMW UÜÜMWEME ÜÜÉ HMHmW% W Ü m UU WHU A COHU fl HUCOUGO ¥ ummc fifi xuwx hmm Hwwwš v fi m fiflfl xuox fl v fl m Mum Eomwm Qèmcmmuxq fl Edx fl uo fl nwucm> ø mc fl Hw®uw>> Haw fl m fi 12 Oo fi O OOH OH OO OOH. oo fl m md mc fl H:> um0m .N ma fl wcwu .m cmnumn: www Hoømxm mwmxmc fl š øwë Hm fl mwu w wmG> wHum> © w um fi mwm fi mucd Åëmm. uw fl m fl co fl umuucwocøx ummc flfi xuæx www fi wuws u fl m fi w fi xuox fl ucm »um sowwm ~» owÅ «~ om ~ o | 4A snx fl uo fl n fi ucw> ~ m = Huwøpm> H> H t tfi westermälande 45.

Example A Evaluation of test compositions for goat nematocidal activity using the free-living hermaphroditic microbivora nematode Caenorhabditis elegans II In the following tests, C. elegans was used to determine the nematocidal activity of the fermentation broth and the crop mass produced by the process of the invention.

This organism was also used to evaluate LL-C230240C and LL-C2302¶ß to determine their nematocidal activities.

In these tests, the fermentation broth constitutes the fermentation broth and mixed solids are taken up before the solids, ie. the harvest, is separated from the liquid. The harvest mass is the solids that remain after separation.

To evaluate the crop mass, the solids are dispersed therein 1: 1 in distilled water. The fermentation broth is used as such and contains both liquid and solids.

At the values given below, C. elegans was maintained in a C. briggsae maintenance medium. This medium is commercially available from Grant Island Biological Company, Grant Island, New York, and has the following composition: "C. briggsae MAINTENANCE MEDIUM" (l) Component mg / l Inorganic salts CaCl2. 2H2O 220.50 CuCl2. 2H2O 6.50 Fe (NH4) 2 (so4) 2. 62120 58.80 KHZPQ4 1225.5O Con (a) MnCl 2. 4H2O 22,20 Znclz 10:20 456 588 14 Other components N-acetylglucosamine Adenosine-3 '- (2') - phosphoric acid.H2O Cytidine-3 '- (2') - phosphoric acid _ D-glucose Glutathione, reduced Guanosin-3 '- (2') * PO4Na2.H2O Magnesium citrate.5H20 (dibasic) Potassium citrate.H2O DL-thioctic acid Thymine Uridine-3 '- (2') - phosphoric acid Amino acids L-alanine L-arginine L-aspartic acid L-cysteine HCl.H2O L-glutamate (Na) .H2O L-glutamine Glycine L-histidine L-isoleucine L-leucine L-lysine HCl L-methionine L-phenylalanine L-proline L-serine L-threonine L-tryptophan L-tyrosine L-valine Vitamins p-aminobenzoic acid Biotin Choline dihydrogen citrate 15.00 365.00 323.00 1315, o0 204.00 363.00 915.00 486.00 3.75 126.00 324.00 1395.00 975.00 1s20.00 28.00 550.00 1463.00 722.00 283.00 361.00 1439.00 1283.00 389.00 803.00 653.00 788.00 717.00 184.00 272.00 J020_00 7.50 3175 88.50 w 456 588 Vitamins (cont.) Cyanocobalamin (B12) 3.75 Folinate (Ca) 3:75 Myo-inositol 64.50 Niacin 7.50 'Niacinamide 7.50 Pantetin 3.75 Pantothenate (Ca) 7.50 Pterolyglutamic acid 7.50 Pyridoxal phosphate 3 , 75 Pyridoxamine 2HCl 3.75 Pyridoxine HCl 7.50 Riboflavin-5'-PO4 (Na) .2H2O 7.50 Thiamine HCl 7.50 References: (1) Hansen, E.L., Proc. Soc. Exp. Cinema. & Med. 121: 30-393 (1966).

Note: (a) As required for adjustment to pH 5.9 + 0.1.

A test plate having a series of small depressions was used for the evaluations. 25 ml fermentation broth, 1: 1 water / crop suspension or a water / acetone solution of LL-C23024d. or LL-C23024ß containing from 31.25 to 500 ppm of the test compound was aseptically deposited in separate wells. To each well was then added 25 ml of C. elegans maintenance medium containing 10-20 adult worms and larvae of various ages as well as eggs. The plates were then placed under a guard and examined 48 hours after inoculation to assess the rate and degree of nematicidal activity of the test compositions. The data obtained are given below. Where activity was observed in the fermentation broth, it was further diluted to obtain a 1: 4 ratio of broth to C. elegans. 456 588 1.6 Table VII Composition Concentration ppm Nematocidal activity or dilution at 48 hours Fermentation D = 1: 1 8 (no motility) broth D = 1: 4 Harvest mass LL-230240 <500 ppm 7 250 ppm 7 125 ppm 7 62.5 ppm 6 31.25 ppm O LL-2302% ß 500 ppm 7 250 ppm 0 The activity scale used in these evaluations was as follows: Activity scale 8 = no motility (obviously dead) = markedly reduced motility 7 6 = reduced motility O = normal motility for the species .Example B Evaluation of LL-C23024u as a nematocidal agent against infectious larvae ** of ruminant nematodes.

Evaluation of LL-C23024 ° as a nematocidal agent against the infectious larvae of ruminant nematodes: Haemonhmus contortus, Ostertagia circumcincta, Trichostrongylus axei, T. Colubriformis; and against vinegar worm, Turbatrix acetí, was determined using the above-mentioned nematode species instead of C. elegans.

The data obtained are given in the table below.

** Det third täckta larvstadíet 456 588 17 umšëwu wm Eon fl cmuum www uou fififl uoë HmEH0: n umu fl a fi uoë omnwo fl wwu u umuw fifi uoš wmumuz fi wn ucmxnmš uw v- \ o Ow fi um mvß fl mwß v ms fi øåw than mw fi ßw fifl u. äñdšwoumaw: ä 2 fi o Näää? xåoßotoq ä 3 vmnw flfi wummo fi wnns fl fi m flfifl mumñmum .dwmæv fl x .ummcwcmsnunwu mm Umë uouum fl mn fl uHøxmøm:> m> M fl hmwëøcmø ": wuum> N ooooo fl wß ww mßw. nw no w oom Huwom .a .nø fi oo .a Üxm .a .Eøuu fi o .o .fi oucøu .m Kvwommoaqq É COHQMHHCUUCOM Åumäš fl u mv ®fi> y uoë uwuwb fl uxm m 0uu fl>: Hfl W588 H5 V5. antibiotic LL-C230240 <against Nematospiroides dubius and Aspicularis tetraptera on fflöåâ In the following tests, Swiss-Webster female white mice were infected with Nematospiroides dubius and Aspicularis tetraptera and kept for three weeks for the infections to mature.

After the rest period, the mice were divided into groups of four and the groups were placed in separate cages. Medicated feeds containing from approximately 31.25 ppm to 4,000 ppm test compound were then offered ad libitum to the mice for one week. Water was also added ad libitum throughout the test period. During the treatment period, the mice's feces were examined to determine whether worms passed. All treatment groups were found to pass worms, thus indicating nematocidal activity at all concentrations against both nematodes. At the end of the one-week treatment with medicated feed, the mice were sacrificed and the contents of the gastrointestinal tract thereof were examined for the presence of worms. The data obtained are given below as a percentage reduction of worms in comparison with infected and non-medicated controls.

In these tests, raw fermentation broths obtained before harvesting the pulp and containing from 1000 ppm to 4000 ppm of the nematocidal antibiotic were evaluated.

Similarly, mouse feedstocks treated with from 31.25 ppm to 500 ppm of LL-C23024p (dissolved in acetone / water (Izl) were evaluated.

Activity Test composition Dietens conc. (% reduction) *** against ppm N. dubius A. tetraptera * Fermentation- 4.000 73 - broth with LL-C230240ç 2'OOO 44 53 1.000 65 33 500 70 SA ** 250 63 SA 125 61 SA 62.5 29 SA 31.25 32 SA 456 588 19 All the amount of LL-C23024 @ ¿indeterminate ** light activity: increased number of tapeworms was recovered in faecal examination ti * compared with infected non-medicated controls The new antibacterial and anticockidal agents according to the invention are formed during culture under the regulated conditions of Actinomadura yumaense sp. nov.

A representative strain of this microorganism was isolated from a soil sample taken in Yuma County, Arizona, and kept in the culture collection of the Lederle Laboratories Division, American Cyanamid Company, Pearl River, New York, as culture no.

LL-C23024. A viable culture of this representative strain has been deposited with the Culture Collection Laboratory, Northern Regional Center, U.S.A. Department of Agriculture, Peoria, Illinois 61604, and has been included in its permanent collection under the accessibility number NRRL 12515.

Taxonomic characterization of NRRL 12515 The strain NRRL 12515 has been taxonomically characterized and identified as the type strain of a new species of Actinomadura known as Actinomadura yumaense sp. nov. observations have been made of the cultural, physiological and morphological characteristics of the representative strain NRRL 12515 using the methods set forth by E.B. Shirling and D. Gottlieb. "Methods for characterization of Streptomyces species", Internat. J. Syst. Bacteriol. 16: 31-33 (1966), and R.E. Gordon et al., "Nocardia coeliaca, Nocardia autotrophica, and the nocardin strain", Internat. J. Syst. Bacteriol. 24: 54-63 (1974). The media used for this study were selected from those recommended by T.G. Pridham et al., "A selec- tion of media for maintenance and taxonomic study of Strepto- mycetes", Antibiotics Ann., Pp. 947-953 (1956/1957); GF Gauze et al., "Problems in the classification of antagonistic actinomycetes ", State Publishing House for Medical Literature, Medgiz, Moscow (1957); and RE Gordon et al., opra for taxonomic 456 588 20 study of actinomycetes and soil bacteria. Chemical composition of the cell walls of the microorganism was determined using - of the method specified by HA Lechevalier et al., "Chemical composition as a criterion in the classification of actinomycetes", Adv. Appl. Microbiol. 14: 47-72 (1971). The phospholipid patterns were determined using the method set forth by M.P. Lechevalier et al., "Chemotaxonomy of aerobic actinomycetes; phospholipid composition", Biochem. System Ecol. 5: 249-260 (1977). Details are given in Tables IV-IX and a general description of the culture is given below. Underlined descriptive colors have been taken from K.L. Kelly and D.B. Judd, "Color. Universal Language and Dictionary of Names," U.S. Pat. Nat Bur. Stand., Spec. Publ. 440. Washington D.C. (1976) and the accompanying Inter- Society Color Council, Natl. Cage. Stand. Centroid Color Charts.

The data observed for this new species represented by the strain NRRL 12515 were compared with data published for the known species of the genus Actinomadura [M. Goodfellow et al., "Numerical Taxonomy of Actinomadura and related actinomycetes", J. Gen. Microbiol. L22: 95-lll (l979); L.H. Huang "Actinomadura macra sp. nov., the producer of antibiotic CP-47,433 and CP- 47,434 ", Internat. J. Syst. Bacteriol. 30: 565 ~ 568 (l980); J. Meyer," New species of the genus Actinomadura ", Z. Allgem.

Microbiol. 19: 37-44 (1979); H. Nomura and Y. Ohara, "Distribution of actinomycetes in soil. XI. Some new species of the genus Actinomadura, Lechevalier et al.," J. Ferment. Technol. 49: 9o4-912 (l97l); and TP Preobrazhenskaya et al., "Key for identification of the species of the genus Actinomadura" The Biology of Actinomycetes and Related Organism 12: 30-38 (1977L7. The culture NRRL 12515 shows a certain slight resemblance to Actinomadura pelletieri, but does not resemble any other described species and differs from A. pelletieri in a number of characteristics, thus the strain NRRL 12515 has been designated as the type strain of a new species known as Actinomadura yumaense, sp. nov., named on the basis of the place 456 588 21 Micromorphology The spores are formed in short spiral chains (maximum length about ~ 20 spores per chain) on branched, almost verticillate air sporopores. The spores are ovoid, 0.6-0.8 micron times. 1.0-1.4 microns, with a smooth surface.

Cell wall composition Whole cell hydrolyzate of this culture contains madurose (3-O-methyl-D-galactose) and the mesoisomer of diaminopimelic acid (DAP). The culture also has a phospholipid pattern of type P-1 and no other diagnostic phospholipids than any phosphatidylglycerol. These characteristics are all very typical of members of the genus Actinomadura.

Degree of growth Good growth was observed on Bennett's agar, Gauze No. 2-agar, Nz-amine starch glucose agar (ATCC Medium 172), tomato paste / oatmeal agar and yeast extract / malt extract agar; moderate growth was observed on Benedict's agar, Czapek's sucrose agar, glycerol / asparagine agar, Hickey-Tresner agar and oat agar; poor growth was observed on calcium malate agar, Gauze No. l-agar and inorganic salts / starch agar.

Vegetative mycelium In media where good growth took place, it was observed that the vegetative mycelium was raised and spiral and generally yellowish gray in color.

Aerial mycelium and trace color Aerial mycelium and / or trace masses were white to 264. light gray in color. Aerial mycelium production was weak in most media.

Soluble pigments Absent in many media; yellow pigment on Benedicts and glycerol / asparagine agar; yellow-green pigment on calcium malate agar; greenish brown pigment on NZ-amine / starch / glucose agar; orange pigment on Bennett's and yeast extract / malt extract agar. 456 588 22 Physiological reactions No melanin pigments on peptone / iron agar and tyrosine agar (ISO-7); strong peptonization of litmus milk; strong proteolysis of nutrient gelatin; moderate reduction of nitrate; no hydrolysis of adenine or guanine; strong hydrolysis of hypoxanthine, tyrosine and xanthine; weak hydrolysis of starch; hydrolysis of esculin; variable hydrolysis of urea. No growth at 4 ° C, 10 ° C or 50 ° C; moderate growth at 2s ° c and 4s ° c; good growth at 32 ° C and 37 ° C. Carbohydrate utilization according to the method set forth by T.G. Pridham and D. Gottlieb, "The utilization of carbon compounds by some actinomycetales as an aid for species determination", J. Bacteriol. 56: 10? -114 (1948): good utilization of glucose, glycerol and trehalose; moderate utilization of maltose and sucrose; poor utilization of fructose, galactose, inositol, mannose and melezitose; no use of adonitol, arabinose, dulcitol, lactose, mannitol, melibiosis, raffinose, ramnose, salicin, sorbitol and xylose.

Acid production from carbohydrates according to the method given by Gordon et al., Supra: good acid production of glucose, glycerol, maltose, sucrose and trehalose; weak acid production from galactose, inositol and mannose. Utilization of organic acids according to the method set forth by Gordon et al., Supra: utilization of acetate, malate, propionate, pyruvate, succinate and tartrate; no use of benzoate, citrate, lactate, mucate and oxalate. »F> uwwuxm Fi if .Nm .aø flfi wuæapms al 456,588 Ham umummoë uwum microns al UXO Fi if .mm Ed flfl woæñ u>« u Hmm uwmcmuø uxnwä .Nß UMMC fl | mummm> ABU fi nøaøx 0Umnwu flfi o>: 0x wwmummum wow IN .OZ wusmw E flflfl woæäuun al Hmm MSS fi muww ummnw NUH> vëmmummm «uxw > H ~ «u uum fifl mm al muww et al AWO na .OZ musmw Hmm Ham microns al uxmwum .If uxw> HH fl u> al umummm> umuwøoš nmoumxumw u fi ømxwan .mm UMMC al Ed flfl wohñumø fi umuwwoñ" UXM> HH f u uumam fiflfl u et al aw al mxwmwnv H fl m ummmlumamë umwuxmcmum .If Gwum al on e flflfi wuæšum flfl uu «> uëwmummw ~ UXM> HH fl u uum fi etc. flfl mw nëswu fi MX B: Small microns al UXM c fl un fi nm um al uxmwum l f sm microns al UXM .If H fifl u Wum microns al uxmasm .mm UXM> flfifl u> fl u hmm uwum uxumš .Hm wmcmuo Imummm> wmuwu: H0> G0x NEN flfi muæäuws al uwmø fi vom muuwncmm Wummn fifi .www H fifi u m » FL> um flfi m flfi WØ H fifl u hmm Hsmxw f n .mm microns al uxmasm lmuæëuwø fi ABU f co f ox mm al Eu0uum> H fl m .uum f m umumwoå muuwwmcmm ucmñm fl m mmnm mHwwmum> mm mm fifl MMQ uwuomm uwAHm \: O0 E flflfl woæëuwøq | uxw> HH "B E flfifl mz UOæN Musumuømëwa ummmw VA nmcwuwnsxc fi mother Ammz wmcmmä al ä musømñoc al UOM Hmm uwmmxmcmmmmmc fifi WO HH > H fi mnmä m: nun E flfifl wumëuud fi ummc fl umcsun um al UXM f ow um fi UXM Hmm »al UXM f sw ..: www .if HHH" www pm fi UXM f am .mm .uw al a luxßnuxwu al ms uxumë MSS wmcmno | 0H0x et al mumußa0>: 0x .wømuHßXm> ~ 0 © mumwum vom \ uxmuuXwummn Hmm Ed al awuæåuwøa uu fl>> m Mmmm "H fl m um al UXM | mHmwEwH> m: uwmc fi | wHm uxumå .Hm .uxm> HHwu wmuumXm> uum fi m vom \ mummmumEoH Ham uu fl> .E flfl Hwo> Euw fl H umum fl oä “Ham Hmmm um fl uxmmum .Om um um m>. mm mumws fifl. «w ~ Hmmm: sun H flfl u uu fl> .ë fiflfl woæëawd fi umuw fl o fi“ uum> m xmoxø fl m um fl uxm: sun um fl ugmcsun .mm H fifi u ucnun »Ham um fl uxm \ wwHwx» m »m» m »m» m. > -fl u © muwusH0> c0x m fl uwøux wow \ c fl Em | Nz 4 ummmlwm fi wx 2 E fl «HwU> Euw fl H uu fi>« nw fi: 0fl Ox tHw @ m \ uwu fi mm mwamuwm uwmc fl mm fl mumm u mm «. FL> Hmm H50 ~ E9 «Huo> Euw fl H uämmummm uaøw microns al UXM lumcmwuh microns al uxmwum .If uwmc fi: mmm .If ABU fi coaox wumuumxm> muum f m vmuwooë -æmxu fl m ummm Es fi amoaävm flfl umnwwoä uc al mmummmm Hsmxw f n .mm NANM uu fl> ABU f co f ox mm al Eucuuw> Hsm .muum fl m flflfl u m flfi mo \ Houwu> Hw ucmäm fl m nanm mHmw mHw> wm mm flfl mmq umuomm umHHm \: o0 Ed flfi wuæäuwøé | uxm> HH «B E flfifl mz 456 588 A.w fl HOuV HH> flfl mnmß 456 588 25 cøux fl å vH | O. fi uwuomw mcmoë> u u w m m ux m ux : moumxumm: 0HX fl E w.0 | mO mu fi O> O Mumma .wømcwum fi uwuømøuommum flfl mxwmmuu Hwuøuxduum muæuomm Mw al uøuwuomm Euøuuomw muow fi uømm uwHHm \ nu0 Ed fifl wüæšwm flfl Ed fifl wz m fi mm fi Aamz mmcwmëdæ mn al ømšoc al uu al UWM al mo al owuoäoux fl z HHH> HHNQMB 456 588 26 Mæ al ou al Æs CMMC al wOM Hmm al am Mmmm MH fi ouøæs CMMC fi vom Hmmmw w fl |: «CMO Fi Go fl ux flfl mu umuwvoä vom Hnmmw mm UCON fifl n co al uxsømu mm> m uwxuæå UOM ummmw v fl numuu fl z _m> HomuoHm al mvøu nom ummmw mm fi WWM% m% Mæ al owucum ABU fi mum ummm al va lmmn fi NMZ .m c al umm fi coumwm xumvm vom uømmw mm xamwë mc al UWM flfi oumwm wow FL about ummm al va Imsäxomâ wcmim f m um al uzm fi WOW ummmo w fi ummw; ucwEvHm uwmz al umnwwoä ummmw> lc fi mouæa mc al cmumwcønn Wuma subsection ummm al va mm% w mc al mmumucøun uum al vom ummmu ß \ n0ummw uo al UWM co fi uxmwu xw al mo f O fl MAEM @mumuxm> HH f B | m O fl umn: x: H E flfl WMZ mother QMMZ mmZw <2D % XH Ham 45 mß 456 588 27 fl wnm fl um> mc fl n fl wwuw fl nw m vom ummmw æw mnø fifi ønmwub uoßm: oo u Nm _ Awß fi .oz ø fl> una »m _ _ o vw: ousmv _. H flfl u w0 .Uømv: OO Uoæm Uømm Hmmmtmoxb fi m ø fl> uxw> H ~ H »umnwøos No mm zoo o o fi: uo wm fi wx ~ U w © fl> uxm> a m 0 0 | umum m flfi wm fl 0. : HH. ~ o ~ @> n Hmßou wow »Mmmm v fi»: fi »nmxøm> = mw fl ou fl æs cmm fifl wow ummmw HN ummm m> fi OuUæ: cwmc fl Uom Hmmm fl va lc fi cm fl w C Uo fl umm O fl uxmmu m um> x H H fi wnmæ 456 588 28 Mæ al ouøæn CMMC al nom Hmm al OH name Mæ fi ouwæs cmmcw vom ummmw m LMM fi mxnmuw maHonm>: ¶ vom nmmmø AEW MCON al ON Mæ al onøæz nom HOMM al wa |: fifi øxmm WO al UWM cowuxmmu microns al Mo f O fl MAEM Umumuxm> HH fl B | Wc0 al umn5x f H Es al uwz ~ .mßH0uv KH flfi w f m f 456 588 29 Table X Carbon source utilization for Actinomadura yumaense NRRL 12515 in ISP-9 carbohydrate release medium Incubation: 28 days Temperature: 28 ° C Carbon source Utilization Adonitol - 1-arabinose - Dulcitol - Fructose bad d-Galactose bad d-Glucose good Llycer Good Glycer Good Glycer - Maltose moderate d-Mannitol ~ d-Mannos bad d-Melezitos bad d-Melibious - d-Raffinose - 1-Ramnos - Salicin - Sorbitol - Sucrose moderate d-Trehalos gOd d-Xylose - Negative control 456 588 30 Table XI Acid production ur various carbohydrates of Actinomadura yumaense NRRL 12515 on Gordon's basal inorganic nitrogen Incubation: 28 days Temperature: 28 ° C Carbon source Acid production * 7 days 28 days Adonitol - - 1-arabinos - - Dulcitol 7 - - Fructose - - d-Galactose - + d- Glucose +++ +++ Glycerol ++ +++ i-Inositol - + Lactose - - Maltos - +++ d-Maanitol - '- d-Mannos - + d-Melezitos - - a-Melibios - d - d-Raffinos - - l -Ramnos - - Salicin - - Sorbitol - - Sucrose - +++ d-Trehalos - +++ d-Xylos - - Negative control - - *) +++ = strongly positive rgspons ++ = moderate positive response + = somewhat positive response - = negative response 456 583 31 Table XII Utilization of organic acids by Actinomadura yumaense NRRL 12515 on Gordon's modification of Koser's basal agar (Koser's citrate agar) Incubation: 28 days Temperature: 28 ° C Carbon source Utilization * Acetate + Benzoate - Citrate - Lactate Malate + Mucic acid - Oxalate - Propionate + Pyruvate + Succinate _ + Tartrate + *) + = positive response - = negative response It should be understood that the expression Actinomadura yumaense is not limited to the strain Actinomadura yumaense NRRL 12515 or to strains that fully correspond to the above the growth and microscopic characteristics, which are given by way of illustration only. The Actinomadura yumaense described in the present context includes all strains of Actinomadura yumaense, which may form antibiotic LL-C23024 alpha, beta and and iodine and which cannot be definitively differentiated from Actinomadura yumaense NRRL 12515 and its subcultures, including mutants and variants thereof. The term "mutants" includes the natural (spontaneous) mutants of this organism having substantially the same properties as well as induced 456 588 32 mutants produced by this organism by various mutagenic agents known to those skilled in the art, such as exposure to X-rays, ultraviolet radiation. , mustard gas, actinophages, nitrosamines and the like. It is also desirable and intended to include inter- and intraspecific genetic recombinants produced by genetic engineering known to those skilled in the art. for example, conjugation, transduction and genetic engineering procedures.

Cultivation of Actinomadura yumaense can be performed with a wide variety of solid or liquid culture media. Media useful for the production of antibiotics LL-C234024 alpha, beta and iodine include an assimilable nitrogen source, such as protein, protein hydrolyzate, polypeptides, amino acids, corn steep liquor, etc., and inorganic anions and cations, such as potassium, sodium, ammonium, calcium, sulfate , carbonate, phosphate, chloride, etc. Trace elements such as boron, molybdenum, copper, etc. are added as impurities of other constituents in the media. Aeration in tanks and bottles is accomplished by causing sterile air to flow through or toward the surface of the fermentation medium. Furthermore, stirring in containers is effected by means of a mechanical stirrer. A defoamer such as ice oil or silicone type may be added as needed.

Actinomadura yumaense is grown and maintained on sloping days, for example Bennett's agar, yeast malt or ATCC Medium no. 172.

ATCC Medium no. 172 is preferred. The oblique culture is inoculated with a culture of Actinomadura yumaense and incubated at 28-37 ° C, preferably at about 32 ° C, for about 7 days. These stock cultures can be maintained by serial transfers to fresh slant cultivations or ah plug of agar containing mycelium from well-grown slant days can be used for inoculation of liquid media.

Shake bottle inoculations of Actinomadura yumaense are prepared by inoculating 100 ml of sterile liquid medium into 500 ml flasks with scrapes or washed spores from an oblique agar culture. Examples of suitable inoculum media are as follows: Medium A Meat extract 0.3% Basta trypton10.5 Glucose 1.0% Yeast extract 0.5% Water qs 100% 1. ... .

[A peptone, registered trademark of Difco Laboratories, Detroit, Michigan]. The pH is adjusted to 6.8-7.2 with diluted base, e.g. sodium hydroxide.

Medium B Glucose 1% Starch 2% Yeast extract 0.5%. ® 2 N-Z-amine A 0.5% Calcium carbonate 0.1% Water qs 100% ['2 Casein digestion, registered trademark of Sheffield Chemical Co., Div. Nat'l. Dairy Products Corp., Norwich, N.y.].

Medium B is preferred.

The flasks are incubated at a temperature of 25-35 ° C, preferably at 32 ° C, and stirred vigorously on a rotary shaker for 1-4 days. This inoculum is then used to inoculate the fermentation culture or this culture can be frozen and stored to provide inoculum for subsequent inoculum cultures. The following media are examples of those suitable for the fermentation of Actinomadura yumaense for the production of antibiotic LL-C23024 alpha, beta and iodine.

Medium C Glucose 1.5% Glycerol 1.5% sqæ@m.3 Lsæ Calcium carbonate 0.1% Sodium chloride 0.3% Water qs 100% [3 Can be replaced with cotton seed flour or soluble meat substances with equally good effect.

Medium D Glucose 3% Soybean meal 1.5% Calcium carbonate 0.1% Sodium chloride 0.3% Water qs 100% Medium E Starch l% Syrup 2% Soybean. L5% Calcium carbonate 0.1% water qs 1000% Medium F Glucose 3% Soluble meat substances 2.5% Sodium chloride 0.2% Calcium carbonate 0.1% Water qs 100% m 456 588 35 Medium G Glucose 3% Soybean meal 0.5 g Ammonium sulphate 0,3 g Sodium chloride 0,2 s Calcium carbonate 0,1% Water qs 100% Medium H Glucose 3% Ammonium sulphate 0,3% Diabasic potassium phosphate 0,1% Calcium carbonate 0,2% Sodium chloride 0,1% Water qs 100 % Medium D is preferred.

The fermentation can be carried out in 100 ml of medium in a 500 ml flask inoculated with 3-10% (v / v) of a seed culture prepared as described above and incubated at 25-35 ° C, preferably at about 3200, for 24 hours. 72 hours during aeration.

Samples of the fermentation culture can be frozen and stored for later use as inoculum for seed cultures.

Alternatively, the fermentation can be carried out in larger fermentation vessels provided with means for aeration and stirring. Each container is inoculated with 3-10% (v / v) inoculum prepared as described above. Aeration is performed at a rate of 0.5-2.0 liters of sterile air per liter of culture fluid per minute and the medium is stirred with a stirrer operated at 200-400 rpm. The temperature is maintained at 25-3500, preferably at 320 ° C. The fermentation is continued until antibiotic substance accumulates in the fermentation medium, usually after 100-150 hours, at which time antibiotic substance is harvested. The antibiotic substance can be harvested and purified according to the methods described in U.S. Patent No. 4,278,663 or according to the following procedure: The crude fermentation broth containing whole cells, prepared as described above, is mixed with an equal volume of one with water. immiscible non-hydrocarbon organic solvent. Methylene chloride or ethyl acetate is preferred.

The organic phase is separated and concentrated in vacuo to an oily syrup.

The oily syrup is dissolved in methylene chloride and added to a column of silica gel, alumina, Sephadex LH-20 (Pharmacy Fine Chemicals, a subsidiary of Pharmacia, Inc. Piscataway, N.J.) or magnesium aluminum silicate. Examples of suitable solvents for developing the column are diethyl ether, ethyl acetate, a 1: 1 to 1: 7 (v / v) mixture of methylene chloride and ethyl ether, 10-40% acetone in methylene chloride, 2-10% lower alcohol (methanol is preferred) in methylene chloride, 2-15% acetonitrile in methylene chloride, or 2-15% dioxane in methylene chloride. Methylene chloride / ethyl acetate (1: 1, v / v) is preferred.

Fractions are collected and examined for the presence of antibacterial activity by bioassay against a susceptible organism, e.g. Bacillus subtilis. Active fractions are combined and concentrated in vacuo to a residue.

This residue is dissolved in an organic solvent, e.g. t-butanol (preferred), benzene or p-dioxane, and lyophilized.

The lyophilized solid is dissolved in a suitable organic solvent, e.g. methylene chloride, hexane, methylene chloride / ethyl acetate, diethyl ether, hexane / ethyl acetate, hexane / chloroform or hexane / ether. Diethylene: preferred. This solution is shaken with water and the pH is adjusted to 1.5-4.0, preferably about 2.5, with some dilute mineral acid. The organic phase is separated, washed with water to remove any excess acid, dried over a suitable desiccant, filtered and concentrated to a residue in vacuo. 456 588 37 This residue is dissolved in a suitable solvent and the solution is allowed to rise slowly, preferably at about 4 ° C. Examples of suitable solvents are methylene chloride, hexane, methylene chloride / ethyl acetate, diethyl ether, hexane / ethyl acetate, hexane / chloroform or hexane / ether. Hexane / ether (5: 2, v / v) is preferred. The resulting crystals are collected and washed, preferably at about 40 ° C with some moderate boiling hydrocarbon, for example hexane or heptane; and air dried to obtain the final product antibiotic X-1468A in the form of the free acid.

If it is desired to obtain the product in the form of a salt, the free acid can be converted by treatment with the corresponding cation, preferably in the form of a dilute mineral base, according to methods well known to those skilled in the art.

The following examples illustrate the invention. Media A, B, C, etc. are those listed above. Unless otherwise indicated, all procedures were performed at room temperature (approximately 22 ° C) and at 1 atm. print.

Example 1 Washed spores from an oblique day of Actinomadura yumaense NRRL 12515 were used to inoculate a 500 ml flask containing 100 ml of sterile medium B. The flask was incubated in a rotating oak caper at 2 ° C for 2 days.

A 5% inoculum of this culture was then transferred to 100 ml of sterile medium C in a 500 ml flask and incubated at 28 ° C for 5 days in a rotary shaker.

The presence of antibiotic activity was examined daily by bioassay against Staphylococcus aureus ATCC 6538 P, Bacillus subtilis and Streptococcus faecalis and the anthelmintic activity was monitored by bioassay against the free-living nematode Caenorhabditis elegans. 456 588 38 Example gel 2 Seven 500 ml flasks were prepared, each containing 100 ml of the following sterile media: Flask 1: 100 ml medium C Flask 2: 100 ml medium C with 1.5% cotton seed flour instead of soy flour Flask 3: 100 ml medium C with 1.5% soluble meat substances instead of soy flour Flask 4: 100 ml medium D Flask 5: 100 ml medium E Flask 6: 100 ml medium F Flask 7: 100 ml medium G These flasks were each inoculated with a 5% inoculum of Actinomadura yumaense NRRL 12515 grown in medium B. The flasks were then incubated on a rotary shaker at 28 ° C for 4-6 days.

Each of the above cultures was found to be active in the presence of antibiotic activity as mentioned in Example 1 and in the study of anticoccidial activity against Eimeria tenella in chicken kidney-based tissue cultures.

Example Gel 3 Frozen fermentation culture cells of Actinomadura yumaense NRRL 12515 were used to inoculate an SOO ml flask containing 100 ml of sterile medium B. The flask was then incubated at 32 ° C for 4 days in a rotary shaker.

A 5% inoculum of this culture was then transferred to 100 ml of sterile medium H in a 500 ml flask and incubated at 28 ° C for 5 days in a rotary shaker.

Antibacterial activity was confirmed as in Example 1 and anticockidial activity as in Example 2. The presence of antibiotic activity was also analyzed by thin layer chromatography on silica gel plates developed in ethyl acetate / chloroform (70:30, v / v).

Example 4 100 ml of sterile medium A in a 500 ml flask was inoculated with washed spores from a slant of Actinomadura yumaense NRRL 12515. The flask was incubated at 32 ° C for 2 days in a rotary shaker.

A 5% inoculum of this culture was then transferred to 100 ml of sterile medium H in a 500 ml flask and incubated in a rotary shaker at 32 ° C for 2 days.

A 5% inoculum of this culture was then transferred to a 500 ml flask containing 100 ml of fresh sterile medium H and incubated at 28 ° C for 6 days in a rotary shaker.

Antibacterial activity was confirmed as in Example 1.

Example 5 Two 500 ml flasks each containing 100 ml of sterile medium A were inoculated with a frozen seed culture of Actinomadura yumaense NRRL 12515 and incubated at 32 ° C for 2 days in a rotary shaker.

The contents of the two flasks were then combined and added to 12 liters of fresh sterile medium A in a 20 liter bottle.

This culture was then incubated for two days at 2800 under aeration.

The contents of this bottle were then transferred to a 300 liter inoculum container containing 288 liters of sterile medium A and this culture was aerated and stirred for 25 hours at 32 ° C.

At the end of 25 hours of incubation, the 300 liters of inoculum were transferred to a 1500 liter fermentor containing 1200 liters of sterile medium C. This culture was incubated under aeration and stirring for 115 hours at 28 ° C.

The fermentation broth was analyzed for antibiotic activity by thin layer chromatography on silica gel plates developed in ethyl acetate / chloroform (70:30, v / v).

Alternatively, a number of the developed plates were subjected to bioanalysis against Bacillus subtilis which showed the presence of antibiotic activity.

Example 6 A typical medium used to grow the primary inoculum was prepared according to the following formula: Meat extract 0.3% Bacto ® tryptone 0.5% Glucose 1.0% Yeast extract. 0.5% Bacto ® aqar 0, 15% Water qs 100% Washed or scraped spores and mycelia from an oblique actinomadura yumaense NRRL 12515 were used to inoculate a 500 ml flask containing 100 ml of the above sterilized medium. The flask was placed in a rotary shaker and stirred vigorously for 48 hours at 32 ° C. The resulting flask inoculum (100 ml) was used to inoculate 1 liter of the same sterile medium into a 2-liter flask. This inoculum was aerated with sterile air while culturing for 48 hours at 28 ° C. This 1 liter culture was then used to inoculate a 30 liter fermentor container containing the same sterile medium and this tank was aerated with sterile air and incubated at 28 ° C for 42 hours.

Bxemgel 7 A fermentation medium was prepared according to the following formula: 456 588 41 Dextrose 1.5% Glycerol 1.5% Soybean meal 1.5% Calcium carbonate 0.1% Sodium chloride 0.3% Water qs 100% pH was adjusted to 7.0 with 6N sodium hydroxide and the medium was sterilized. A 30 liter portion of inoculum prepared as described in Example 1 was used to inoculate 250 liters of the above medium into a 300 liter fermentor. Sterile aeration was applied and the mass was stirred by an impeller operated at 220 rpm. The fermentation was carried out at 32 ° C for 97 hours after which the pulp was harvested.

Example Gel 8 A fermentation broth was prepared according to the following formula: Dextrose 3.0% Soybean Flour 1.5% Calcium Carbonate 0.1% Sodium Chloride 0.3% Water at 100% pH was adjusted to 7.0 with 6N sodium hydroxide and the medium was sterilized. 300 liters of inoculum prepared according to Example 1 was used to inoculate 3000 liters of the above medium into a fermenter. Sterile aeration was applied. The mass was stirred with an impeller driven at 100 rpm. Fermentation was performed at 3200 for 115 hours at which time the pulp was harvested.

Example 9 1. The fermentation broth (100 liters) was adjusted to pH 4.0 using 6N HCl. The acidified mass was then stirred for 1-2 hours with an equal volume of methylene chloride. The mixture of pulp and methylene chloride was then filtered using diatomaceous earth as a filter aid. The methylene chloride extract was evaporated and concentrated in vacuo to about 2 liters of partially purified antibiotic substances. 2. Chromatography of the partially purified antibiotics on silica gel A glass column 7 cm in diameter was packed to a height of 91 cm with Woelm Silica Gel TSC. The crude concentrate (see 1 above) with a volume of about 2 liters was allowed to seep into the silica gel column. The column was then first developed with 3 liters of methylene chloride followed by methylene chloride / ethyl acetate (1: 1 by volume) to obtain a total of 126 fractions each having a volume of 80 ml. It was then further developed using ethyl acetate / ethanol (7: 3) to give an additional 87 fractions.

Fractions 58-87, rich in C23024X, were combined and concentrated in vacuo to a residue weighing 90.4 g. This residue was dissolved in a mixture of 300 ml of diethyl ether and 600 ml of hexane. The resulting suspension was filtered to give a clear filtrate. The clear filtrate was concentrated in vacuo until crystals began to form.

This suspension was allowed to age overnight. The crystalline substance C23024K was collected in a funnel, washed with cold ether and air dried to obtain 33.1 g of crystalline C23024K. An additional 12.4 g was obtained upon continued volume reduction of the combined filtrate and washings.

Fractions 177-203 from the elution with ethyl acetate / ethanol and enriched for C23024 / 3 were combined and concentrated in vacuo to give 6.7 g of partially purified C23024 / 6 and treated as above.

Fractions 204-213 from the ethyl acetate / ethanol elution enriched for LL-C23024 iota were combined and concentrated in vacuo to a paste and extracted repeatedly with methanol. The methanol extract was extracted with methylene chloride, which was loaded on a column containing Woelm silica gel. The column was washed with methylene chloride and then eluted with methylene chloride / ethyl acetate (2: 3). The fractionation was controlled by thin layer chromatography and the active fractions were combined, treated with charcoal, concentrated and extracted repeatedly with heptane. The final heptane extract was cooled at 0 ° C for 48 hours and the crystals were removed by filtration from the mother liquor.

This mother liquor was dissolved in methylene chloride and allowed to seep into a glass column packed with Woelm silica gel. The column was then eluted sequentially with 2 liters of methylene, 8 liters of methylene chloride / ethyl acetate (1: 1) and 4 liters of ethyl acetate / methanol (9: 1). The eluate was collected in fractions and examined for their antibiotic composition. The active fractions were combined and concentrated in vacuo to give a residue containing LL-C23024 iota.

Example 10 3. Further purification of C23024 / 3 from chromatography on silica gel Sephadex (â) LH2O was allowed to swell in a mixture of hexane / methylene chloride / methanol (10: 5: 1). A glass column with a diameter of 5 cm was packed to a height of 86 cm with the gel. The charge, 3 g of purified LL-C23024 / 3, was dissolved in 10 ml of methylene chloride, 1 ml of methanol and 10 ml of hexane, and allowed to seep into the gel column. Column 3 was then developed with solvent of the same composition as that used to add the antibiotic to the column. Fractions were collected using a collector. The first 23 fractions each had a volume of 17 ml. Fractions 17-26 contained C23024¿3 according to thin layer chromatography. These fractions were combined and concentrated in vacuo to give pure amorphous LL-C2302 at a weight of 1269 mg. 456 588 44 Example 11 Crystallization of LL-C2302§® as sodium salt Purified LL-C23024 / 3 (2.4 g) prepared as described in sections B and C was dissolved in a mixture of diethyl ether (500 ml), hexane (160 g). ml) and methylene chloride (25 ml). The resulting solution was transferred to a separatory funnel containing 300 ml of distilled water. Diluted HCl (1N) was added dropwise thereto until the pH became 2.0-2.5 after shaking and sedimentation.

The acidic aqueous phase was discarded and the acid-treated organic layer was washed three times successively with 400 ml volumes of water. The washed organic layer was stirred with a teaspoon of Darco G-60 powder for 15 minutes and filtered through celite. The decolorized organic layer was placed over 500 ml of distilled water and SN NaOH was added dropwise until the pH reached 11.0-11.5 after shaking and sedimentation.

The alkaline aqueous phase was discarded and the remaining organic layer was washed twice with 400 ml portions of water. The washed organic layer was dried over sodium sulfate and then concentrated in vacuo to a volume of 150 ml. This concentrate was kept under protection for several hours. The crystalline substance LL-C23024 / 3 was collected in a funnel, washed with cold hexane and air dried to obtain 402 mg of the crystalline salt of LL-C23024 / 9. This sample was used for microanalyses and selected spectral data.

Analysis: Calculated for C 46 H 77 O 17 Na: C 59.70 H 8.33 O 29.46 ash 2.49 Found: C 61.55 H 8.79 ash 3.31 N O Melting point (Fisher-John apparatus) = 174 ° C.

(FDMass Spec.) = 924; yÅï] š6 = + 3.2 ° (in methanol). 456 588 45 Example 12 Purification of LL-C23O24Ajota A high efficiency liquid chromatography instrument (Waters Prep. SOOA) was fitted with a silica gel cartridge under a pressure of 38.5 kp / cm 2. The charge, which consisted of 5.0 g of the crude material from Example 3, was dissolved in 50 ml of heptane / ethyl acetate (45:55) and injected onto the silica gel column.

The column was then developed with the same solvent mixture at a flow rate of 200 ml / minute, recovering forty 200 ml fractions. Fractions 21-32 were combined, concentrated to a residue, triturated with hexane and evaporated to give pure LL-C23024 iota. The above procedure was repeated four times to obtain a total of 280 mg of amorphous LL-C23024 iodine.

A 90 mg portion of amorphous LL-C23024 iodine was dissolved in 10 ml of diethyl ether, mixed with 10 ml of water and adjusted to pH 2.5 with 0.1N hydrochloric acid. The ether layer was washed with water, adjusted to about pH 11 with 0.1N sodium hydroxide, separated and washed again with water. The ether phase was dried over sodium sulfate, filtered and concentrated to a residue. A solution of this residue in ether / hexane was slowly evaporated to give an amorphous white solid.

This amorphous white solid had the following properties; Elemental analysis: C 61.79 H 8.84 ash 0.32; gxíšs = + z7 ° (c = o, 724, metano1> ..

Field desorption mass spectrometry: (M + Na) + = m / e 953, so the calculated molecular weight is 930.

Claims (6)

456 588 ”IG PATENT REQUIREMENTS 1. l. Biologically pure culture of Actinomadura yumaense NRRL 12515 or a mutant thereof having essentially the same properties as Actinomadura yumaense NRRL 12515, which culture can form antibiotic LL-C23024 alpha with the structural formula: OMe Meq, NMe 0 lm š 0 on HH Me M2 antibiotic LL-C23024 beta with the structural formula: OMe MeO Me I, \\\ and antibiotic LL-C23024 jota, which later turned out to have the following structural formula: Eel? 456 588 in recoverable amounts for use in controlling the coccidiosis of poultry during fermentation in a liquid nutrient medium containing assimilable sources of carbon, nitrogen and inorganic salts. Biologically pure culture of the microorganism Actinomadura yumaense NRRL 12515 according to claim 1, characterized in that the microorganism is a spontaneous (natural) mutant, which is genetically modified but has essentially the same properties as Actinomadura yumaense NRRL 12515 and still retains the ability to tetisera antibiotic LL-C23024, alpha, beta and jota. Biologically pure culture of the microorganism Actinomadura yumaense NRRL 12515 according to claim 1, characterized in that the microorganism is mutated with mutagens known to those skilled in the art, so that the microorganism is genetically modified but has essentially the same properties and still retains the ability to synthesize antibiotic LL-C23024 alpha, beta and jota. A process for the preparation of antibiotic LL-C23024 alpha, beta and iodine having the formulas: 456 588 W OMQ Me fl b “pm for use in controlling coccidiosis in poultry characterized by aerobically fermenting Actinomadura yumaense NRRL 12515 or a mutant thereof having substantially the same properties in a liquid medium containing assimilable sources of carbon, nitrogen and inorganic salts until substantial antibiotic activity has been imparted to the fermentation broth, and the antibiotic substance is subsequently recovered therefrom. 5. A process according to claim 4, characterized in that the temperature in the fermentation culture is maintained at 25-3S ° C for a period of 24-150 hours. Process according to Claim 5 for the preparation of antibiotic LL-C23024 alpha, characterized in that Actinomadura yumaense NRRL 12515 or a mutant thereof having substantially the same properties is aerobically grown in a liquid medium containing assimilable sources of carbon, nitrogen and inorganic salts. that significant antibiotic activity has been imparted to the fermentation broth, and that the antibiotic substance is subsequently recovered therefrom.