NL8204069A - ANTIBACTERIALS LL-C23024BETA AND JOTA AND THE MICRO-ORGANISM ACTINOMADURA YUMAENSE NRRL 12515. - Google Patents

Description

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Antibacterial agents LL-C2302 ^ $ and iota and the microorganism Actino-madura yumaense NRRL 12515.

The invention relates to antibacterial and anticoccidinal agents designated 1.1-02302, and iota, as well as to a new microorganism Actinomadura yumaense sp. nov. or mutant and so on.

The invention further relates to a new additive for producing the antibiotics 11-02302, β and iota by fermentation under controlled conditions of the new microorganisms Actinomadura yumaense sp. nov.

The antibiotic LL-C23Q2ha {has the structure according to formula 1 of the formula sheet.

The antibiotic 11-02302 of the present invention differs from known compounds in its physical and chemical properties, including, but not limited to, its microanalysis, optical rotation, infrared spectrum, C nuclear magnetic resonance spectrum, and H ceramic resonance spectrum.

Antibiotic LL-C2302U / 3 has the postulated structure of formula 2 of the formula sheet, wherein R1 and Rg are different and each is selected from the group of hydrogen and methyl.

The above postulated structure is based on the elemental analysis, optical rotation, field desorption mass spectroscopy molecular weight, melting point, infrared spectrum (IR), ^ C nuclear magnetic resonance spectrum (C-HMR), and proton magnetic resonance spectrum (1H-NMR), as detailed described in the examples and shown in the accompanying figures: Fig. 1: IR spectrum of 11-02302 ^ / 3 in KBr; 13

Fig. 2: C-NMR spectrum of LL-C2302U 3; 20 MHz in CDCl 2 internal TMS reference equivalent;

Fig. 3: 1 H-NMR spectrum of LL-C2302U / &; 80 MHz in CDCl ^, internal TMS reference equivalent.

8204069 »* - 2 -

Table A

1? JC NMR spectrum of LL-C2302 ^ / 3 (ppm relative to IMS)

Chemical shift (ppm) Number of carbon atoms 5 10.5 1 11.0 1 12.2 1 17.0 1 17.7 1 10 17.9 1 22.3 1 26.1 1 26.8 1 27.6 1 15 30.2 1 32.0 1 33.3 1 33.7 2 33.8 2 20 36.5 1 36.8 1 39.0 1 39.9 1 ^ 5.5 2 25 57.1 1 59.1 1 60.7 1. 66.9 1 6 7.6 1 30 70.2 1 71.3 1 72.9 1 7 ^, 9 1 75.1 1 35 79.9 1 8204069% 4 - 3 -

Table A (continued) 80.8 1 82.1 2 · 8 ^, 5 1 5 8 ^, 7 1 85.6 1 86.9 1 95.8 1 96.9 1 10 97.7 1 107.5 1

179.2 JL

total number of carbon atoms U6 LL-C2302k iota is a new polyether antibiotic with very strong anticoccidial activity. It is at least 10 times stronger than most other known polyethers. Data from the field desorption mass spectrum show that it has a molecular view of 930, which, together with its carbon-13 nuclear magnetic resonance spectrum 13 (JC-NMR), allows the proposal of a preliminary molecular formula CU8H82 ° 17. It appears that LL-2302U iota has four methoxy groups, the same sugar seen in the polyether antibiotic X-11 + 868A (U.S. Patent 1 + .278,663), and has a carboxyl group. The only other known polyether antibiotic with a molecular weight of 930 is the antibiotic A28695B (hydroxyseptamycin), see J. Antibiotics 25 33 (2): 252 (1980), which is significantly different in structure and biological properties. The above observations are based on the elemental analysis, optical rotation, the calculated molecular weight by field desorption mass spectroscopy, the infrared spectrum (CER), the CC-NMR spectrum, and the proton nuclear magnetic resonance spectrum (1H-NMR), as described in detail in the examples and shown in the accompanying figures:

Fig. 1+: IR spectrum of LL-C23021 + iota in KBr;

Fig. 5: H-UMR spectrum of LL-C23021 + iota; 80 MHz in CDCl 3, internal TMS reference equivalent; Fig. 6: C-NMR spectrum of LL-C23024 iota; 20 MHz in CDCl 3, in internal TMS reference equivalent; 8204069 13 »ί - k -

Fig. 7A: C-NMR spectrum of LL-C2302U iota (extended scale; 0-50 ppm); 20 MHz in CDCl 4, internal TMS reference equivalent; 13

Fig. 8B: C-NMR spectrum of LL-C2302U iota (extended scale: 50-110 ppm); 20 MHz in CDCl 3, internal TMS reference equivalent.

Ds ^ C-NMR spectra of LL-C2302U iota were obtained in dechlorinated chloroform (CDCl2) at a field strength of 20 MHz. The peaks with their associated chemical shifts in parts per million to tetramethylsilane (TMS) and the estimated number of carbon atoms per peak are summarized in Table B. Peaks were plotted for chloroform at 75.77.0 and 78 , 6.

Table B

Chemical Number of carbon— Chemical Number of carbon shift atoms shift atoms 10.6 1 59.9 1 10.9 1 60.8 2 n, 7 1 68.5 1 l6, U 1 68.8 1 20 17.6 1 71.3 1 18 .0 1 72.2 1 21.7 1 76.1 1 22.9 1 76.9 1 2U, 1 1 78.5 1 25 28.2 1 81.0 1 31.2 2 81.3 1 32.2 1 81.8 1 33, h 1 8U, 8 1 33.8 2 85.3 1 30 3 ^, 2 1 85.6 1 36.7 1 86.8 1 37.2 1 88.5 1 39 Λ 1 95.9 1 10.3 1 97.9 1 35 11.5 1 99.6 1 8204069 -5-.

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Table B (continued) 1 * 5.5 1 107.2 1

1 * 7, 6 1 173.0 __L

56.8 1 Total 1 + 8 5 The antibiotics LL-C23Q2cat, / 3 and iota are organic carboxylic acids and therefore capable of forming salts with non-toxic pharmaceutically acceptable cations. Therefore, salts formed by mixing the antibiotic free acid with nitrogen biometric amounts of cations, preferably in a neutral solvent, can be formed with cations such as sodium, potassium, calcium, magnesium and ammonium, as well as organic amine cations such as tri (lower alkyl) amine (eg triethylamine, triethanolamine), procaine and the like. The cat ion salts of the antibiotic LL-C2302k / 3 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 this invention, the antibiotic free acid is equivalent to its pharmaceutically acceptable non-toxic salts.

The antibiotics LL-C2302U / 3 and iota are active in vitro against 20 gram positive bacteria and they are tested by the standard agar dilution procedure. The results are reported as minimum inhalative concentrations (MIC) in micrograms / cm in Tables C and D.

The antibiotics LL-C23021 * / 3 and iota are not active against gram-negative organisms in concentrations of 256 micrograms / cm or less.

Table C

In vitro antibacterial activity of LL-C2302U

Organism minimal inhibitory concentration (mcg / cm ^)

Staphylococcus aureus Smith 6h 30 "" SSC 80-11 6h "" SSC 80-32 61+ "" SSC 80-38 oh "" LL-11 + 61+ "" LL-1 + 5 61+ 35 "" LL-27 128 "" ATCC 25923 61+ 8204069 • * - 6 -

Table C (continued)

Streptococcus pyogenes C203 1 "/ 3-hemolytic Keller T623 8" pneumoniae 78-1 8 5

Table D

In vitro antibacterial activity of LL-C23021 * iota

Organism minimal inhibitory concentration (mcg / cm ^) LL-C23024 iota 10 _ ___

Enterococcus OSU 75-1 1

Enterococcus SM 77-15 1

Staphylococcus aureus SSC 79-18 1

Staphylococcus aureus FU 79-19-2 2 15. Micrococcus lutea PCI 1001 .1

Staphylococcus aureus Smith 0.5

Staphylococcus aureus AICC 25923 0.5

As shown by the above data, the antibiotics LL-C2302Uoc, and iota inhibit the growth of certain gram-positive baeterins and are therefore useful as topical or surface antiseptic in skin washes, equipment, walls, floors, etc.

It has also been found that the antibacterial agents LL-C2302hot, yS and iota are active in vivo as anticoccidial agents for poultry as shown by the following tests.

Two days before inoculation, a medicated feed of different drug levels was fed to several groups of one day old test chicks. The poultry feed used throughout the test was prepared as follows: premix of vitamins and amino acids 0.5% 30 trace minerals 0.1% sodium chloride 0.3% dicalcium phosphate 1.2% ground limestone 0.5% stabilized fat. 0% 35 dehydrated alfalfa, 17% protein 2.0% 8204069 - τ - corn gluten meal, kl% eggit 5.0 # menhaden fish tit, 60% eggit 5.0 # soybean oil meal, bk% eggit 30.0 # ground yellow corn, fine to 100 # 5 The premix of vitaznines and amino acids in the above nutrient composition prepared from the following composition. The quantity indications refer to units per kg of the finished food composition.

Butylated Hydroxytoluene 125.0 mg 10 dl-Methionine 500.0 mg Vitamin A 3300.0 I.U.

vitamin 1100.0 I.C.E.

riboflavin b, k mg of vitamin E 2.2 I.E.

15 niacin 27.5 mg pantothenic acid 8.8 mg choline chloride 500.0 mg folinic acid 1.3 mg menadione sodium bisulfate 1.1 mg 20 vitamin 11.0 meg ground yellow corn, fine to 5.0 g

The test chicks then were inoculated by direct inoculation into the head of each chick with a mixed inoculum of 5,000 spore cysts of Eimeria acervulina and a sufficient number of oocytes from Eimeria tenella to a mortality rate of 85-100 # in the untreated obtain control animals. The chick is given free access to food and fed throughout the test period. Seven days after the inoculation, the tests ended and the birds were found to have been subjected to body shells and their intestines examined for damage. The results are shown in Tables E and F and show that better survival of infected chickens is obtained when 10 ppm or less of the antibiotics LL-C2302b / 3 or iota are administered in the diet. The results also show a significant suppression of intestinal lesions to be infested with Eimeria tenella and Eimeria acervulina.

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The following tests demonstrate nematocidal activity. Example A

Evaluation of Test Compositions for Nematocidal Activity Using Free-Living Hermaphroditic Microbe-Eating Nematodes 5 Caenorhabditis elegans II.

In the following tests, C. elegans was used to determine the nematocidal activity of the fermentation broth and the harvest kernel prepared by the procedure of the invention. This organism was also used to evaluate LL-C2302b ^ and LL-C2302 ^ to determine its nematodde activity.

In these tests, the fermentation broth was the mixture of fermentation broth and solid taken prior to separating the solid, i.e. the harvest, of the liquid. The crop was the solid material remaining after the separation.

For evaluation of the harvesting pulp, the solid harvesting pulp was dispersed in a 1: 1 ratio in distilled water. The fermentation broth was used as such and contained both liquid and solid material.

In the evaluations now described, C. elegans was collected in a C. briggsae storage medium. The storage medium is commercially available from Grant Island Biological Comapny, Grant Island, New York, and trembles the following compositions: C. briggsae Storage Medium ^ * 3

Component mg / dm 25 INORGANIC SALTS:

CaCl2.2H20 220.50

CuC12.2H20 6.50

Fe (lffll +) 2 (S01;) 2.6H20 58.80 KH2P0 ^ 1225.50 30 Κ0Η (a)

MnClg.UHgO 22.20

ZnCl2 10.20 OTHER COMPONENTS: N-acetylglucosamine 15.00 35 adenosine-3 '- (2') - phosphoric acid.HgO 365.00 8204069 - n - cytidine-S '- ^') - phosphoric acid 323.00 D-glucose 1315.00 glut athion, reduced 201.00 guanosine-3 (2'J-PO ^ Na ^. HgO 363.00 5 magnesium citrate. 55 ^ 0 (dibasic) 915.00 potassium citrate. H ^ O 136.00 DL -thioctic acid 3.75 thymine 126.00 uridine-3 '- (2') - phosphoric acid 321.00 10 AMRU ACIDS: L-alanine 139 5.00 L-arginine 9T5.00 L-aspartic acid 1620.00 L-cystelne HCl. H ^ O 28.00 15 L-glutamate (Na). HgO 550.00 L-glutamine 1163.00 glycine 722.00 L-histidine 283.00 L-isoleucine 861.00 20 L-leucine 1139.00 L-lysine HCl 1283.00 L-methionine 389.00 - L-phenylalanine 803.00 L-proline 653.00 25 L-serine 788.00 L-threonine 717.00 L-tryptophan 181.00 L-tyrosine 272.00 L- valine 1020.00 30 VTTAiMIITES: p-aminobenzoic acid 7.50 biotin 3.75 choline dihydric citrate 88.50 cyanooob alanine (B) 3.75 35 folinate (Ca) 3.75 8204069 - 12 - myo-inositol 6U, 50 niacin 7, 50 niacinamide 7.50 pantethine 3.75 5 pantothen ate (Ca) 7.50 pterolyglutamic acid 7.50 pyridoxal phosphate 3.75 pyridoxamine 2HC1 3.75 pyridoxine HCl 7.50 10 riboflavin ^ - PO ^ iNa) .2Hg0 7.50 thiamine HCl 7.50

References: (1) Hansen, E.L., Proc. Soc. Exp. Bio. & Med. 12130-393 (1966).

Notes: (a) as needed to adjust the pH to 5.9 + 0.1.

A well plate with a series of small wells was used for the evalua. 3 ties used. Aseptically 25 cm fermentation broth, 1: 1 water / harvesting suspension, or an aqueous / acetone solution of LL-C2302hoc or LL-C2302U / 3 containing 31.25 to 500 ppm of the test compound was introduced. 3 separate wells. To each well was then added 25 carg of the C. elegans storage medium containing 10-20 itol wax worms plus larvae of various ages plus eggs. The plates were then placed in a fume hood and examined bQ hours after the inoculation to determine the rate and degree of nematocidal activity of the test compositions. The data obtained is below. When activity was observed for the fermentation broth, the broth was further diluted to a 1: k broth to C. elegans ratio.

Table G

Composition Concentration, ppm, Nematocidal activity or dilution after U8 hours of fermentation broth D = 1: 1 8 (no motility) D = 1: b 8204069 - 13 -

Table G (cont'd) Harvest Prut LL-C2302U * 500ppm 7 250ppm 7 5 125ppm 7 62.5ppm 6 31.25ppm 0 LL-C2302ly3 500ppm 7 250ppm 0 10 The activity designation used in these evaluations was as follows: 8 * no motility (apparent death) 7 = greatly reduced motility 6 = decreased motility 15 0 = normal motility for the species.

Example B

Evaluation of LL-C2302Uc <, as a nematocidal agent against infective larvae of ruminant nematodes.

Evaluation of LL-C2302 Onion as a nematocidal agent against the infective larvae of ruminant nematodes: Haemonhmus contortus, Ostertagia circumcincta, Trichostrongylus axei, T. colubriformis; and against the vinegar worm, Turbatrix aceti, was determined using the above nematode species in place of C. elegans.

The data obtained are shown in the table below.

25 Third stage sheathed larvae.

Table H

In vitro activity against (after 8 hours) concentration of H. contort. 0. circum. T. axei T.colub. T.aceti LL-C2302lw <, ppm 30 500 8 31 ° 813 8 6 250 7 8b 8b 7 6 125 7 7 7 6 6 62.50 7 6 7 66 31.25 6 6 066 35 0 (control) 0 0 000 a Performed in 96-well tissue culture plates, X1U868A prepared in 8204069-ll * - ο double-distilled water: 25 µm of LL-C2302l * cf solution and 25 µm nematode culture were added per well.

The activity indications sail: 8 = no motility (apparent death) 5 T = strongly impaired motility 6 = impaired motility, 0 = normals motility for the species within 2 hours.

Example C

Evaluation of nematocidal activity of the antibiotic LL-C2302UOC against Nematospiroides dubius and Aspicularis tetraptera in mice.

In the following tests, white Swiss-Webster female mice were infected with Nematospiroides dubius and Aspicularis tetraptera and held for 3 weeks to allow the infections to mature.

After this period, the mice were randomly divided into groups of four, and the groups were placed in separate cages. Medicinal feeds containing about 31.25 ppm to 1000 ppm test compound were then offered to the mice ad libitum for 1 week. Water was also given ad libitum during the test period. During the treatment period, the excrements of the mice were examined to determine if they contained worms. Worms were found in the excrements in all treatment groups, indicating nematocidal activity at all concentrations of the compound against both nematodes. At the end of the 1 week medicated treatment, the mice were autopsied, and the contents of their intestines were examined for worms.

The data obtained are presented below as a percentage reduction in worms compared to infected controls that had not undergone medical treatment.

In these tests, crude fermentation broths obtained from the prut harvest containing 1000 to 600 ppm of the nematocidal antibiotic were evaluated. Mice whose feed was treated with 31.25 to 500 ppm of I * L-C2302koc dissolved in a 1: 1 mixture of acetone and water were also evaluated.

- 15 -

Test composition Diet Activity (% reduction) cone. 4® "teg" N. dubius A. tetraptera * fermentation broth k,000 73 5 with LL-C2302UQC 2,000 U4 53 LL-C2302MX 1,000 65 33 500 70 SA **

250 63 SA

125 6l SA

10 62.5 29 SA

31.25 32 SA

* a quantity of LL-C2302Uo £ not determined O. = light activity: Increased number of pin shapes found in faeces study.

15 dost * Compared to infected controls who had not been on medication.

The novel antibacterial and anticoccidial agents of the invention are formed during cultivation under controlled conditions of Actinomadura yumaense sp. nov.

A representative strain of this microorganism was isolated from a soil sample collected in Yuma County, Arizona and stored in the culture collection of the Lerle Laboratories Division, American Cyanamid Company, Pearl River, Rev York, under culture number LL-C2302k. A vigorous culture of this representative strain has been deposited with the Culture Collection Laboratory, Northern Regional Center, U.S.

Department of Agriculture, Peoria, Illinois 6l60b, and was added to its permanent collection under the accession number NRRL 12515-Taxonomic characterization of NRRL 12515

The strain NRRL 12515 has been taxonomically characterized and identified as the strain type of a new species of the genus Actinomadura known as Actinomadura yumaense sp. nov.

The culture features, physiological features * and morphological features of the representative strain NRRL 12515 were observed using detailed methods by E.B. Shirling and D. Gottlieb, "Methods for characterization of

Streptomyces species ", Internat. J. Syst. Bacteriol. 16: 313-3-0-16 - (1966), and R.E. Gordon et al.," Nocardia coeliaca, Nocardia autotrophica. and the nocardin strain ", Internat. J. Syst. Bacteriol. 2b: 5 ^ - 63 (197 * 0. Media used in this study were selected from the media recommended by TGPridham et al.," A selection of media 5 for maintenance and taxonomic study of StreptomycetesAntibiotics Ann., biz. 9 * + 7 - 953 (1956/1957); GFGauze et al., "Problems in the classification of antagonistic actinomycetes," State Publishing House for Medical Literature, Medgiz, Mowcos (1957); and REGordon et al., Supra, for the taxonomic study of actinomycetes and soil bacteria 10. The chemical composition of the cell walls of the microorganism was determined using the method of HALechevalier et al, , "Chemical composition as a criterion in the classification of actinomycetes," Adv. Appl. Microbiol. IU: U7 - 72 (1971) Phospholipid patterns were determined using the method of 15 MPLechevalier et al., "Chemotaxonomy of aerobic actinomycetes: phopholipid composition, Biochem. Syst. Ecol. 5: 2b9-260 (1977). Details are listed in Tables I - N, and a general description of the culture is provided below. The underlined descriptive colors are taken from K.L. Kelly and D.B. Judd, "Color. Universal 20 Language and Dictionary of Names," U.S. Pat. Wet. Bur. Stand., Spec. Publ.

UlQ, Washington, D.C. (1976) and the accompanying Inter-Society Color Council, Natl. Bur. Stand. Centroid Color Charts.

The data observed for this new species, as represented by the strain NRRL 12515, were compared with the data published for the known species of the genus Actinomadura (M. Goodfellow et al., "Numerical Taxonomy of Actinomadura and related actinomycetes , "J. Gen. Microbiol. 122: 95-111 (1979); LH Huang," Actinomadura macra sp. Nov., the producer of antibiotic CP-17, * + 33 and CP-17, ^ 3 ^, "Internat J, Syst. Bacteriol. 30: 565-568 (1980); 30 J.Meyer, "New species of the genus Actinomadura / Z. Allgem. Mikrobiol. 19: 37-1 (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. 90l - 912 (1971); and TPPreobrazhenskaya et al., "Key for identification of the species of 35 the genus Actinomadura, "The 3iology of Actinomycetes and Related - IT -

Organisms 12: 30-38 (1977)). The NRRL 12515 cattle breed has little resemblance to Actinomadura pelletieri, but is unlike any other species described and differs in some properties from A. pelletieri. Therefore, voice NRRL 12515 has been designated as the Stan type of a new species called Actinomadura yumaense, sp. Nov., named after the collection site of the soil sample from which the strain type was isolated.

Spores are formed in short spiral chains (maximum length about 20 spores per chain) on branched, almost wreath-like air sporophores. The spores are ovoid, 0.6 - 0.8 µm by 1.0 to 1.1 µm, with a smooth surface.

Cell wall arsenal

Whole cell hydrolysates of this culture contain madurose 15 (3-0-methyl-D-galactose) and the meso isomer of diaminopimelic acid (DAP). The culture also has a type P-1 phospholipid pattern and no diagnostic phospholipid other than a little phosphatidylglycerol. These properties are all very typical of members of the genus Actinomadura.

20 Hayness growth

Good growth is observed on Bennett's agar, Gauze No, 2 agar, NZ amine starch glucose agar (ATCC medium 172), tomato pie 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 oatmeal agar; poor growth is observed on calcium malate agar, Gauze No.1 agar, and inorganic salt starch agar.

Vegetative mycelium

Good growth was observed on media over which the vegetative mycelium rose and curled up, and generally had yellowish-gray hues.

Aerial mycelium and spore color

The aerial mycelia and / or the spore masses had a pale to 261 light gray color. The aerial mycelia production is light on most media.

- 18 -

Soluble pigments

Absent on many media; yellow pigment on Benedicts and glycerol asparagine agars; yellow-green pigment on calcium malate agar; greenish-brown pigment on NZ amine starch glucose agar; orange pigment on 5 Bennettt's and yeast extract malt extract agars.

Physiological reactions

No melanin pigments on peptone iron agar and tyrosine agar (ISO-7); strong peptonization of litmus milk; strong proteolysis of food gelatin; moderate nitrate reduction; no hydrolysis of ade-10 nine or guanine; strong hydrolysis of hypoxanthine, tyrosine and xanthine; such hydrolysis of starch; hydrolysis of esculin; variable hydrolysis of urea. No growth at U ° C, 10 ° C, or 55 ° C; moderate growth at 25 ° C and U5 ° C; good growth at 32 ° C and 37 ° C. Carbohydrate consumption, as determined by the method of TGPridham and D. Gottlieb, "The 15 utilization of carbon compounds by some actinomycetales as an aid for species determination, 1 'J. Bacteriol. 56: 107 - ll1 * (19 ^ 8): good use of glucose, glycerol and trehalose; moderate use of maltose and sucrose; poor use of fructose, galactose, inositol, mannose, and melezitose; no use of adonitol, arabinose, dulcitol, lactose, mannitol, melibiosis, raffinose, rhamnose, salicin, sorbitol and xylose. Acid production from carbohydrates according to the method of Gordon, et al., supra: good acid production from glucose, glycerol, maltose, sucrose and trehalose; weak acid product from galactose, inositol and mannose. Use of organic acids according to the method of Gordon et al., supra: use of acetate, malate, propionate, pyruvate, succinate and tartrate, no use of benzoate, citrate, lactate, nucleate and oxalate.

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- 21 -Table K

jysiological reactions of Actinomadura yumaense NRRL 12515

Medium Incubation- How much- Physiological reaction period growth peptone iron- 7 days good light browning a®ar lb days good light browning tyrosine agar 7 days - moderate no pigment lU days good yellowish pigment 10 litmus cream lb days good good peptonization 28 days good strong peptonization nutritional gelatin lk days good licorice proteolysis 28 days good total proteolysis nitrate broth lU days good very weak reduction 15 28 days good moderate reduction adenine agar lU days good no hydrolysis 21 days good no hydrolysis guanine agar lk days good no hydrolysis 21 days good no hydrolysis 20 hypoxanthine- 1¼ days good total hydrolysis 21 days good total hydrolysis tyrosine agar 1 ^ days good strong hydrolysis 21 days good strong hydrolysis xanthine agar lb days good moderate hydrolysis 25 21 days good strong hydrolysis NZ amine with at - 5 days of poor or no growth at 4 ° C, detachable starch 10 ° C and 55 ° C; moderate growth.

and glucose agar 25 ° C; 28 ° C and i + 5 ° C; good growth

(ATCC Med. No. at 32 ° C and 37 ° C

172) urea stock 28 days good decomposition variable esculin stock lU days good hydrolysis 28 days good hydrolysis starch agar 5 days good no hydrolysis 10 days good no hydrolysis 35 8204069 - 22 -

Table L

Carbon source utilization of Actinomadura yumaense NRRL 12515 on ISP-9 carbohydrate utilization

Ineubation: 28 days Temperature: 28 ° C

5

Carbon source Use adonitol 1-arabinose dulcitol 10 fructose only d-galactose only d-glucose good glycerol good i-inositol only 15 lactose matose reasonable d-mannitol mann-mannose only d-melezitose only 20 d-melibiose d-raffnose 1-raffinose sorbitol 25 sucrose reasonable d-trehalose good d-xylose negative control 820 4 0 6 9 - 23 -Table Μ

Acid production from various carbohydrates by Actinomadura yumaense NEEL 12515 on Gordon's basic inorganic nitrogen medium

5 Incubation: 28 days. Temperature: 28 ° C

Carbon source Acid production _ 7 days 28 days adonitol 1-arabinose 10 dulcitol fructose d-galactose - + d-glucose +++ +++ glycerol ++ +++ 15 i-inositol - + lactose maltose - +++ d-mannitol d -mannose - + 20 d-melezitose d-melibioe d-raffinose 1-rhamnose salicin 25 sorbitol sucrose - +++ d-trehalose - +++ d-xylose negative control 30 +++ = strong positive response ++ = moderate positive response + = slight positive response - = negative response 8204069

- 2k -Table N

Use of Organic Acids by Actinomadura yumaense NEEL 12515 on Gordon's Modification of Koser's Basal Agar (Koser's Citrate Agar)

5 Incubation: 28 days. Temperature: 28 ° C

Carbon source Use acetate + benzoate, citrate 10 lactate malate + mucous acid oxalate propionate + 15 pyruvate + succinate + tartrate + + = positive response - = negative response

It is noted that the term Actinomadura yumaense is not limited to the stain Actinomadura yumaense NEEL 12515 or to strains that fully correspond to the above growth and microscopic characteristics, which are given for illustrative purposes only. The Actinomadura yumaense described herein encompasses all strains of Actinomadura yumaense which produce the antibiotics 11-02302 ^ and iota and which cannot be sharply distinguished from Actinomadura yumaense NEEL 12515 and its subcultures, including mutants and variants thereof. The term "mutants" includes the natural (spontaneous) mutants of this organism as well as induced mutants obtained from this organism by various mutagens known to those skilled in the art, including exposure to X-rays, ultraviolet rays, nitrogen mustard, actinophages , nitrosamines, etc. It is also desired and contemplated to include inter- and intra-specific genetic recombinants obtained by genetic techniques known to those skilled in the art, eg, by conjugation, transduction and genetic engineering techniques.

8204069 - 25 -

Cultivation of Actinomadura yumaense can be performed with a variety of rast or liquid culture media. Media useful for the production of the antibiotics LL-C2302U ¢ £ and iota include an assimilable source of nitrogen such as protein, egg vitamin hydrolyzate, polypeptides, amino acids, corn broth, 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 supplied as impurities of other media constituents. Aeration in tanks and 10 bottles is accomplished by passing sterile air through or on the surface of the fermentation broth. A mechanical stirrer ensures further agitation in tanks. An anti-foaming agent such as pork lard oil or a silicone defoamer may be added as needed. Actinomadura yumaense is grown and delivered on agarhellin-15 gene, e.g. Bennett's agar, yeast malt agar, or ATCC medium 172. ATCC medium 172 is preferred. The slope is inoculated with a culture of Actinomadura yumaense and incubated at 28-37 ° C, preferably at about 32 ° C, for about 7 days. These starting cultures can be serially transferred onto fresh agar slopes, or a plug of the agar-containing mycelia of the well-grown agar slope can be used to seed liquid media.

Shake bottle inoculations of Actinomadura yumaense are prepared to inoculate 3 · 3 for 100 cm stem fluid medium with 500 cm bottles ir. With spores obtained from the agar slope of the culture by scraping or washing. Examples of suitable inoculation media are:

Medium A.

beef extract 0.3%

Bacto®trypton ^ 0.5¾ glucose 1.0% 30 yeast extract 0.5% water q.s. 100¾ (a peptone, branded product from Difco Laboratories, Detroit, Michigan.)

The pH is adjusted with a dilute base, e.g. sodium hydroxide set at 6.8-7.2.

35 8204069 - 26 -

Medium B

glucose 1 $ starch 2 $ yeast extract 0.5 $ 5 N-Z amine A®2 0.5 $ calcium carbonate 0.1 $ water 100 $

Casexne digest, branded product from Sheffield Chemical Co., Div. Nat'1 Dairy Products Corp., Norwich, N.Y.) Medium B is preferred.

The bottles are incubated at a temperature of 25 - 35 ° C, preferably at 32 ° C and agitated vigorously on a rotary shaker for 1 - U days. This inoculum is then used to inoculate a fermentation culture, or this culture can be frozen and stored to provide inoculum for later inoculation cultures.

The following media are examples of media suitable for the fermentation of Actinomadura yumaense for the production of antibiotics LL-C2302UdC, 3 and iota.

20 Medium C

glucose 1.5 $ glycerol 1.5 $ soybean meal-3 "1.5 $ calcium carbonate 0.1 $ 25 sodium chloride 0.3 $ water qs, 100 $ (J Can be replaced with cottonseed meal or soluble meat ingredients with similar effect. )

Medium D.

30 glucose 3 $ soybean meal 1.5 $ calcium carbonate 0.1 $ sodium chloride 0.3 $ water q.s. 100 $ 3204069 - 27 -

Medium E

starch 1 $ molasses 2 $ soybean meal 1.5 $ 5 calcium carbonate 0.1 $ water q.s. 100 $

Medium F

glucose 3 $ soluble meat ingredients 2.5 $ 10 sodium chloride 0.2 $ calcium carbonate 0.1 $ water q.s. 100 $

Medium G

glucose 3 $ 15 soybean meal 0.5 $ ammonium sulfate 0.3 $ sodium chloride 0.2 $ calcium carbonate 0.1 $ water q.s. 100 $

20 Medium H

glucose 3 $ ammonium sulfate 0.3 $ dibasic potassium phosphate 0.1 $ calcium carbonate 0.2 $ 25 sodium chloride 0.1 $ water q.s. 100 $

Medium D is preferred.

The fermentation can be carried out in 100 ml media in a 500 ml bottle inoculated with 3-10 $ (v / v) from a seed culture prepared as described above and for 2k-72 hours under aeration incubated at 25 - 35 ° C, preferably at about 32 ° C. Samples of the fermentation culture can be frozen and stored for later use as inoculations for inoculation cultures.

On the other hand, the fermentation can be carried out in larger fermentation tanks equipped with aeration and agitating agents.

3 2 3 4 0 6 9 - 28 -

Each tank is inoculated with 3 - 10 # (v / v) inoculum prepared as described above. The batch is stirred at a rate of 0.5-2.0 dm of stiff air per dm of broth per minute and the fermentation medium is agitated by a stirrer at 200-500 rpm. The temperature is kept at 25 - 35 ° C, preferably at 32 ° C. Fermentation is continued until antibiotic builds up in the fermentation medium, usually after 100-150 urea, at which time the antibiotic is harvested.

The antibiotic may be harvested and purified according to the specifications described in U.S. Patent No. 1, 278,663, or according to the following procedure:

The crude fermentation broth containing the cellular cells prepared in the manner described above is mixed with an equal volume amount of any non-hydrocarbon water-immiscible organic solvent. Preference is given to methylene chloride or ethyl acetate. The organic phase is separated and concentrated under reduced pressure to an oily syrup.

The oily syrup is dissolved in methylene chloride and added to a column of silica gel, alumina, Sephadex LH-20® 20 (Pharmacia Fine Chemicals Div. Of Pharmacia, Inc., Piscataway, H.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: ethyl ether, 10 - 10% acetone in methylene chloride, 2 - 10 # lower alcohol (the preferred is methanol) 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 checked for the presence of antibacterial activity by bioanalysis against a susceptible organism, e.g. Bacillus subti-30 lis. Active fractions are combined and concentrated under reduced pressure to a residue. This residue is dissolved in an organic solvent, e.g. tert-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: 8204069-29 - ethyl acetate, diethyl ether, hexane: ethyl acetate, hexane: chloroform, or hexane: ether. Diethyl ether is preferred. This solution is shaken with water and the pH is adjusted to a pH of 1.5-1.0, preferably about 2.5, with any dilute mineral acid. The organic phase is separated, washed with water to remove any excess acid, dried over an appropriate desiccant, filtered, and concentrated under reduced pressure to a residue.

This residue is dissolved in a suitable solvent and the solution is allowed to evaporate slowly, preferably at about 1 + ° 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 °0 ° C, with any hydrocarbon boiling at moderate temperature, e.g. hexane or heptane, and air dried to obtain the antibiotic X-11868a in the form of the free acid as the final product,

If the product is in the form of a salt, the free acid can be reacted by treatment with the appropriate cation, preferably in the form of a dilute inorganic base, according to procedures known to those skilled in the art.

The following examples illustrate the invention. The media A, B, C etc. are as defined above. Unless otherwise indicated, all procedures were performed at room temperature (approximately (22 ° C) and at a pressure of 1 atm.

Example I

Washed traces of an agar slope of Actinomadura yumaense 3 3 NERL 12515 were used to: inoculate a 500 cm bottle containing 100 cm sterile medium B. The bottle was incubated on a rotary shaker at 28 ° C for 2 days.

A 5% inoculum from this culture was then transferred 3 3 in 100 cm sterile medium C in a 500 cm bottle and incubated on a rotary shaker at 28 ° C for 5 days.

The presence of antibiotic activity was monitored daily by bioanalysis against Staphylococcus aureus ATCC 6538 P, Bacillus 8204069-30 subtilis, and Streptococcus faecalis and anthelmintic activity was followed by bioanalysis against the free-living nematode Caenorhabditis elegans.

Example II

• 3 3 5 Seven 500 cm bottles, each containing 100 cmJ, were prepared from one of the following sterile media: “3

Bottle 1: 100 cur medium C o bottle 2: 100 cm medium C with 1.5? cottonseed flour instead of soy flour

10 bottle 3: 100 cm medium C with 1.5% soluble meat ingredients instead of soy flour bottle U: 100 cm ^ medium D

3

bottle 5: 100 cm medium E

bottle 6: 100 cmr medium F • 3 15 bottle 7: 100 cm medium G.

These bottles were each seeded with a 5% seed material of Actinomadura yumaense NRBL 12515 grown in medium B. The bottles were then incubated on a rotary shaker at 28 ° C for k - 6 days.

Each of the above cultures was found to be active in antibiotic activity analysis as in. Example I and analysis for anticoccidial activity against Eimeria tenella in tissue cultures of chick kidneys.

Example III

25 Frozen fermentation culture cells of Actinomadura yumaense 3. 3 NEEL 12515 were used to inoculate a 500 cm bottle containing 100 cm sterile medium B. The bottle was then incubated on a rotary shaker at 32 ° C for 1+ days.

A 5% inoculum from this culture was then transferred 3 · 3 30 m 100 cm sterile medium H m a 500 cm bottle and incubated on a rotary shaker at 28 ° C for 5 days.

Antibacterial activity was confirmed in the manner described in Example I and anticoccidial activity was confirmed in the manner described in Example II.

The presence of antibiotic activity was also demonstrated by thin layer chromatography over silica gel plates, de-plated in ethyl acetate: chloroform TO: 30 (v / v).

Example IV

~ 3 3

100 cm stem medium A was inoculated into a 500 cm bottle with faded spores from an agar slope of Actinomadura yumaense NREL 12515.

The bottle is incubated for 2 days at 32 ° C on a rotating shaker.

A 5 / S's inoculum from this culture was then transferred. 3 3 m 100 cm stem medium H in a 500 cm bottle and incubated on a rotary shaker for 2 days at 32 ° C.

A 5 # 's inoculum from this culture was then transferred to a 500 cm flask containing 100 cmJ of fresh sterile medium H and incubated on a rotary shaker at 28 ° C for 6 days.

The antibacterial tack is confirmed in the manner described in Example I.

Example V

3 · 3

Two 500 cm bottles each containing 100 cm stem medium A were seeded with an inoculated culture of Actinomadura yumaense NRRL 12515 and incubated on a rotary shaker for 2 days at 32 ° C.

The contents of the bottles are then combined and added 3 ... 3 to 12 dm fresh stem medium A in a 20 dm bottle. This culture is then incubated for 2 days at 28 ° C under aeration.

The contents of this bottle are then transferred to a 300 dm ^

O

Grafting tank containing 288 dnr sterile medium A and this culture was aerated and agitated for 25 hours at 32 ° C.

At the end of the 25 hour incubation, the 300 dm 3 3 seed culture transferred to a 1500 dm fermentor containing 1200 dm 1 sterile medium C. This culture was incubated at 28 ° C for 115 hours under aeration and agitation.

The fermentation broth was analyzed for antibiotic clearance by thin layer chromatography on silica gel plates, which were thickened in ethyl acetate: chloroform TO: 30 (v / v). Alternatively, several of the desalinated plates were subjected to bioanalysis against Bacillus subtilis, demonstrating the presence of antibiotic activity.

Example VI

A typical medium used to grow the primary inoculum was formulated according to the following formulation 5: beef extract 0.3%

Bacto® -trypton 0.5% glucose 1.0% yeast extract 0.3% 10 Bacto®-agar 0.15 # water q, .s. 100%

Spores and mycelia obtained by washing or scraping from an agar slope of Actinomadura yumaense NRRL 12515 were used to inoculate a 500 cm flask containing 100 cm of the above stenlized medium 15. The bottle was placed on a rotary shaker and agitated vigorously at 32 ° C for b hours. The resulting inoculum (100 cm 3) in the bottle was used to inoculate 1 dm 2 of the same sterile medium in a 2 dm bottle. This seed material was aerated with sterile air while growth was continued at 28 ° C for 20 hours. This 1 dm-3 culture was then used to inoculate a 30 cm fermentor tank containing the same sterile medium, and this tank was aerated with sterile air and incubated at 28 ° C for 2 hours.

Example VII

A fermentation medium according to the following formulation was prepared: dextrose 1.3% glycerol 1.5% soybean meal 1.3% calcium carbonate 0.1 # sodium riumchlori 0.3% water q.s. 100%

The pH was adjusted to 7.0 with 6 N sodium hydroxide and the medium was stenlized. A 30 dm portion of the inoculum prepared according to Example 35 I was used to make 250 dm 2 of the medium described above 8204069-33-q in 300 dm 2? fermenter. Sterile air was left at the well and agitated with a stirrer at 220 rpm. Fermentation was carried out at 32 ° C for 97 hours, after which the mash was harvested.

Example VIII

A fermentation medium was prepared according to the following formulation: dextrose 3.0 # soybean meal 1.3% 10 calcium carbonate 0.1 # sodium chloride 0.3 # water q.s. 100%

The pH was adjusted to 7> 0 with 6 N sodium hydroxide and the medium was stenlized. A 300 dm serving of it according to example • ·. Grafts prepared were used to inoculate 3000 dm of the above medium into a fermentor. Sterile air was admitted to the pot. The mash was agitated by a stirrer at 100 rpm. Fermentation was carried out at 32 ° C for 115 hours after which the mash was harvested.

Example IX

1. The fermentation broth (100 dm ^) was adjusted to pH 0.0 with 6 N HCl. The acidic mash was then stirred with an equal volume of methylene chloride for 1-2 hours. The mixture of mud and methylene chloride was then filtered using diatomaceous earth as a filter aid. The methylene chloride extract was withdrawn and concentrated to about 2-3 under reduced pressure. ...

dm of partially purified antibiotics.

2. Chromatography of the partially purified antibiotics on silica gel.

A glass column with a diameter of 7 cm was packed to a height of 91 cm with Woelm silica gel TSC. The crude concentrate (see above under 1) with a volume of about 2 dm 2 was left in the column .... 3 seeping from silica gel. The column was then first developed with 3 dm methylene chloride followed by methylene chloride: ethyl acetate (1: 1 by volume ratio) to give a total of 126 fractions q each with a volume of 80 cm. This was then further developed using 8204069-3 using ethyl acetate ethanol (7: 3) to yield an additional 87 fractions.

The fractions 58-87 * rich in O2302 ^ ¾ were combined and concentrated under reduced pressure to a residue weighing 90, kg. This residue was dissolved in a mixture of 600 cm3 of diethyl ether and 600 cm3 of hexane. The resulting suspension was filtered to yield a clear filtrate. The clear filtrate was concentrated under reduced pressure until crystals started to form. This suspension was allowed to age overnight. The crystalline C2302U was collected on a dropping funnel, washed with cold ether and air dried to obtain 33.1 g of crystalline C2302U. An additional amount of C2302U of 12, ug was obtained by further reducing the volume of the combined washing liquid and filtrate.

The fractions 177-203 of the elution with ethyl acetate-ethanol enriched in C2302k / S were combined and concentrated under reduced pressure to obtain 6.7 g of partially purified C2302U / 3, which was described as described above treated.

The fractions 20k-213 of the elution with ethyl acetate-ethanol, enriched in LL-C2302U iota, were combined and concentrated to paste under reduced pressure and extracted repeatedly with methanol. The methanol extract was extracted with methylene chloride, which was applied to a column containing Woelm silica gel.

The column was washed with methylene chloride and then eluted with methylene chloride: ethyl acetate (2: 3). The drained materials were followed by thin-day 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 b8 hours and the crystals were removed by filtration of 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 successively eluted with 2 dm methylene o 8 dmr methylene chloride: ethyl acetate (1: 1) and U dmJ ethyl acetate: methanol (9: 1). The eluate was collected in fractions and fractions 8204069-35 were tested for their antibiotic composition. The active fractions were combined and concentrated under reduced pressure to give a residue containing LL-C23024 iota.

Example X.

3. Further purification of C2302y3 from the chromatography on s-gel.

Sephadex ^ LH 2 O was allowed to swell in a mixture of hexane-methylene chloride-methanol (10: 5: 1). A 5 cm diameter glass column was packed with the gel to a height of 86 cm. The batch, 3 g of purified LL-C23024 / tf, 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 used to deliver the antibiotic to the column. Fractions were collected using a collector. The first 23 fractions each had a volume of 17 cm 2. The fractions 17-26 contain, according to thin layer chromatography, C23024 / 3.

These fractions were combined and concentrated under reduced pressure to obtain pure amorphous LL-C2302U / 3 in an amount of 1269 mg.

Example XI

Crystallization of LL-C2302ly3 as the sodium salt.

Purified LL-C23024 (2.4 g), prepared as described in sections B and C, was dissolved in a mixture of 500 ml diethyl ether, 160 car hexane and 25 car methylene chloride. The resulting solution was transferred to a separatory funnel containing 300 cm of distilled water. Diluted HCl (1N) was added dropwise until the pH after shaking and settling reached 2.0-2.5. The acidic aqueous phase was discarded and the acid-treated organic layer was washed three times in succession with 400 cur water. The washed organic layer was stirred with a teaspoon of Darco G βθ powder for 15 minutes and filtered through Celite. The decolorized organic 3 layer was brought to 500 cm distilled water and 5 N NaOH was added dropwise until the pH after shaking and settling reached 11.0 - 11.5. The basic aqueous phase was discarded and the remaining organic layer was washed twice with 400 cm portions of water. The washed organic layer was dried over sodium sulfate 8204069-36 and then concentrated under reduced pressure to a volume 3 of 150 cm. This concentrate was left in a fume hood for several hours. The crystalline LL-C2302k / 3 was collected on a trexter, washed with cold hexane and air-dried to give CO2 mg of 5 the crystalline salt of LL-C2302U / 3. This sample was used for microanalyses and selected spectral data.

Analysis: Calculated for C 14 H 20 O Na: C 59.70; H 8.33; 0 29, k6; axis 2, k9 $; found: C 61.55; H 8.79; axis 3.31 ^; N, 0.

10 MP. (Fisher-Johns apparatus = 17k (FDMass Spec) = 92k s + 3.2, in methanol.

Example XII

Purification of LL-C2302k iota.

A Waters Prep. 500A h.p.l.c. (high performance liquid chromatography) instrument was provided with a silica gel cartridge under a pressure of 38 bar. The batch, which was 5.0 g of the crude material from Example III, was dissolved with 50 cm heptane: ethyl acetate (k5: 55) and injected into the silica gel column. The column was then developed with the same solvent mixture at a flow rate of 200 cm / min, collecting kO fractions of k00 cm. Fractions 21-32 were combined, concentrated to a residue, triturated with hexane and evaporated to give pure LL-C2302k iota. The above procedure was repeated four times to obtain an overall combined amount of 280 mg amorphous LL-C2302k iota 25.

A 90 mg portion of amorphous LL-C2302k iota was dissolved in 3 ml of diethyl ether, mixed with 10 cm of water, and adjusted to pH 2.5 with 0.1 N hydrochloric acid. The ether layer was washed with water, adjusted to a pH of about 11 with 0.1 N 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 allowed to evaporate slowly to yield an amorphous white solid.

This amorphous white solid had the following properties: elemental analysis: C 61.79; H 8.8k; axis 0.32; Jp1-q-+27 (C 0.72k, 8 2 0 a 0 6 9 - 37 - methanol); field desorption mass spectrometry: (M + Na) + = m / e 953, so the calculated molecular weight is 930.

820 4 06 9

Claims (14)

1. Biologically pure culture of Actinomadura yumaense sp. nov. which culture is capable of producing the antibiotics LL-C2302 and iota in collectable amounts after fermentation in a liquid nutrient medium containing assimilable sources of carbon, nitrogen and inorganic salts- The biologically pure culture of claim 1, wherein said Actinomadura yumaense sp. nor. Actinomadura yumaense NRBL 12515 is. 3. Biologically pure culture of the microorganism Actinomadura yumaense sp. nov. according to claim 1 or 2, wherein the microorganism is mutated spontaneously so that the microorganism is genetically altered but still has the ability to synthesize the antibiotics LL-C2302 ^ a (en iota. U. Biologically pure culture of the microorganism Actinomadura yumaense sp Nov according to claim 1 or 2, wherein the microorganism has been mutagenized so that the microorganism is genetically altered but still has the ability to synthesize the antibiotics LL-C2302 ct / 3 and iota. 5, The antibiotic LL-C2302U in which the nearly pure form (a) has an optical rotation / + 7 ° of + 32 ° + 2 (0, ^ 95 $, methanol) 20 and fjxj ^ of + U6 ° + 2 (0, Uh-O 2, chloroform), (b) an elemental analysis {%) of about C 61.55; H has 8.79; (c) has a melting point of about 171-173 ° C; (d) has a molecular weight according to field desorption mass spectroscopy of 902; (e) has a carbon-13 nuclear magnetic resonance spectrum in dechlorinated chloroform at 80 MHz which is substantially as shown in Figure 2; (f) has an infrared absorption spectrum in KBr, which is substantially 30 as shown in Figure 1; and (g) has a proton ceramic magnetic resonance spectrum in deuterated chloroform at 20 MHz which is substantially as shown in 8204069-39 - Figure 3. · The pharmaceutically acceptable acid addition salt of the antibiotic LL-C23024 / 3 according to claim 5. 7. The antibiotic LL-C2302U iota, in the substantially pure form 26 (a) has an optical rotation%%] £ - +27 (C 0.72U, methanol); (b) an elemental analysis (%) of about C 61.79; H has 8.8U; (c) has a molecular weight by field desorption mass spectroscopy of 930; and 13 (d) has a C nuclear magnetic resonance spectrum in deuterated chloroform at 80 MHz which is substantially as shown in Figures 6, 7 and 8; (e) has an infrared absorption spectrum in KBr that is substantially as shown in Fig. U; and 15 (f) has a proton nuclear magnetic resonance spectrum in deuterated chloroform at 20 MHz, which is substantially as shown in Figure 5. Pharmaceutically acceptable acid addition salt of the antibiotic LL-C2302 iota according to claim 7 9. Method for the preparation of the antibiotics LL-C2302k <1/3 and iota, comprising an aerobic fermentation of Actinomadura yumaense sp. nov. or a mutant thereof in a liquid medium containing assimilable sources of carbon, nitrogen and inorganic salts, until substantial antibiotic dilution has been imparted to the fermentation broth, and then recovery of the antibiotic therefrom. The method according to claim 9, wherein the temperature of the fermentation culture is maintained at 25-35 ° C for 2k-150 hours. A method according to claim 9 or 10, wherein the Actinomadura yumaense sp. nov. Actinomadura yumaense NRRL is 12515. 12. A method of management of protozoan infections in intestinal blood animals, comprising an oral administration to the animals of a protozoacidally effective amount of the compound LL-C2302U / or LL-02302 / Iota. The method of claim 12, wherein the warm-blooded animals are meat-producing animals, the infection is coccidiosis and the anti-coccidial agent is administered to the animals in the diet or the 8204069 1 -> 0 - (drinking water containing 2.5 ~ 120 ppm of the anticoecidial agent. Lk, Composition for controlling coccidiosis infections in poultry, comprising an edible carrier and about 0.000025 wt.% To about 0.06% by weight of the compound LL-C3202lyS, LL-C2302U iota. or the pharmaceutically acceptable salts thereof. The composition of claim 1U, wherein the edible carrier is drinking water and contains 2.5-120 ppm of the anticoecidial agent. 16. Method of controlling nematodes in venomous animals and in the soil containing the nematodes, by oral introduction into the animals or application to nematode-infested soil, of a nematocidically amount of a compound LL-C2302kec , LL-C2302 ^ 2 or pharmaceutically acceptable salts of these compounds, mixtures of the compounds or salts, or fermentation broths or solid harvesting pulp from which the nematocidal antibiotics are obtained. 8204069