DK161332B - BIOLOGICALLY CULTURE OF ACTINOMADURA YUMAENSE AND ITS APPLICATION BY A PROCEDURE FOR THE PREPARATION OF ANTIBIOTICS LL-C23024-ALFA, BETA AND IOTA - Google Patents

Description

The present invention relates to a biologically pure culture of Actinomadura yumaense which is characterized in that it is Actinomadura yumaense NRRL 12515 and that it can form antibiotics LL-C23024-α, β and iota by fermentation in a liquid nutrient medium containing assimilable sources of carbon, nitrogen and inorganic salts.

Antibiotics LL-C-23024-α, -β and -iota are known compounds having the structural formulas shown below and having antibacterial and anticoccidal activity. These antibiotics are known from e.g. DK patent no. 149,639, according to which they are produced by fermentation of a strain of the species Nocardia sp. X-14868, ATCC 31585.

The invention further relates to a process for the preparation of antibiotics LL-C23024-a, -β and -iota of the following structural formulas, which is characterized by aerobically cementing Actinomadura yumaense NRRL 12515 in a liquid medium containing assimilable sources of carbon, nitrogen and inorganic salts until substantial antibiotic activity is present in the fermentation broth, and the antibiotics are then recovered therefrom.

The antibiotic LL-C23024-a has the following structure: JQMe 25

Me * ^ V> 0 Me 0 ^ Me <^ V> Me i / —v / —v / —v γ 30 Æs O-jt C00H Me

The antibiotic LL-C23024- / J has the following structure: 35

ISLAND

DK 161332 B

2 OMe

MeO * Y

Oh 0H f COQH Me 10

The above structure for LL-C23024-6 is based on elemental analysis, optical rotation, field desorption, mass spectroscopic molecular weight, melting point, infrared (IR) spectrum, 13 13 15 C nuclear magnetic resonance (C-NMR) spectrum and proton magnetic resonance. (1 H-NMR) spectrum as indicated in detail in the examples and shown in the accompanying drawing, in which 1 is LL-C23024-6's IR spectrum in KBr; 2 is the 13C NMR spectrum of LL-C23024-6, 20 MHz in 20 CDCl 3, internal TMS reference equivalent, and FIG. 3 is 1 H-NMR spectrum for LL-C23024-8, 80 MHz in CDCl3, internal TMS reference equivalent.

Table I

25 NMR spectrum (ppm relative to TMS)

Chemical change (ppm) Number of carbon atoms 10.5 1 11.0 1 12.2 1 30 17.0 1 17.7 1 17.9 1 22.3 1 26.1 1 35 26.8 1

DK 161332 B

Table I (continued)

Chemical change (ppm) Number of carbon atoms 27.6 1 30.2 1 32.0 1 5 χ 33.3 1 33.7 2 33.8 2 36.5 1 10 36'8 1 39.0 1 39.9 1 45.5 2 57.1 1 15 59.1 1 60.7 1 66.9 1 67.6 1 70.2 1 20 71.3 1 72.9 1 74.9 1 75.1 1 79.9 1 25 80.8 1 82.1 2 84.5 1 84.7 1 85.6 1 30 86.9 1 95.8 1 96.9 1 97.7 1 107.5 1 35 179.2 1_

A total of 46 carbon atoms

DK 161332B

40 LL-C23024-iota is a polyether antibiotic with very potent anticoccidial activity. It is at least 10 times as strong as most other known polyethers. Field desorption mass spectral data show that it has a molecular weight of 930, which together with the 13C nuclear magnetic resonance 5 (13C NMR) spectrum allows a proposal for a preliminary molecular formula C48H82 ° 17. iota has been determined as having the formula OMe rY “10 fko> Me

CO₂H

The above observations are based on elemental analysis, optical rotation, molecular weight calculated by field 1300 desorption mass spectroscopy, infrared (IR) spectrum, C-NMR spectrum and proton nuclear magnetic resonance (1 H-NMR) spectrum, thus as detailed in examples and shown in the accompanying drawing, in which 4 is the LL-C23024-iota IR spectrum in KBr, FIG. 5 is LL-C23024-Itase 1 H NMR spectrum, 80 MHz in CDCl3 / internal TMS reference equivalent; 6 is the LL-C23024 iota 13C NMR spectrum, 20 MHz in CDCl3, internal TMS ref erence equivalent; 7 is LL-C23024-Itase JC-NMR spectrum, (extended scale: 0-50 ppm), 20 MHz in C0Cl3, internal TMS, and FIG. 8 is the LL-C23024 iotaase 3 C NMR spectrum, (extended scale: 50-110 ppm), 20 MHz in CDCl3, internal TMS reference equivalent.

13 LL-C23024-Itase C-NMR spectra were measured in deutero-35-chloroform (CDCl3) with a field strength of 20 MHz. The tips 5

DK 161332 B

° with their respective chemical shifts in parts per million to tetramethylsilane (TMS) and the estimated number of carbon atoms per million. tip is listed in Table II. The peaks are observed for chloroform at 75.4, 77.0 and 78.6.

Table II

Chemical change Number of C atoms_ Chemical change Number of C atoms 10.6 1 32.2 1 10.9 1 33.4 1 11.7 1 33.8 2 10 16.4 1 34.2 1 17.5 1 36.7 1 18.0 1 37.2 1 21.7 1 39.4 1 22.9 1 40.3 1 24.1 1 44.5 1 28.2 1 45.4 1 31.2 '2 47.6 1 56.8 1 81.8 1 20 59.9 1 84.8 1 60.8 2 85.3 1 68.5 1 85.6 1 68.8 1 86.8 1 71.3 1 88.5 1 25 72.2 1 95.9 1 76.1 1 97.9 1 76.9 1 99.6 1 78.5 1 107.2 1 81 , 0 1 173,0__1 30 81.3 _1_Total 48

Antibiotics LL-C23024-α, -β and -iota are organic carboxylic acids and are thus capable of forming salts with non-toxic pharmaceutically acceptable cations. Thus, salts formed by the antibiotic free acid 35 being mixed with stoichiometric amounts of cations can best

ISLAND

DK 161332 B

6 neutral solvent is obtained 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 cationic salts of the antibiotic LL-C23024- / J are generally crystalline solids which are relatively insoluble in water and soluble in most common organic solvents such as methanol, ethyl acetate, acetone, chloroform, heptane, ether and benzene. When used as an antibiotic against bacteria and cocci, the free acid is equivalent to the pharmaceutically acceptable, non-toxic salts.

Antibiotics LL-C23024- /? and iota are active in vitro against gram-positive bacteria when tested by a standard agar dilution method. The results are listed as minimum inhibitory concentrations (MIC) in µg / ml in Tables III and IV. Antibiotics LL-C23024- / 3 and -iota are not active against gram-negative organisms at concentrations of 256 Mg / ml or less.

Table III

LL-C23024 ~ 1 L antibacterial activity in vitro

Minimally inhibitory

Organism___ concentration (µg / ml) __

Staphylococcus aureus Smith 64 "" SSC 80-11 64 25 »" SSC 80-32 64 "" SSC 80-38 64 "" LL-14 64 "" LL-45 64 "· * LL-27 128 on" "ATCC 25932 64

Streptococcus pyogenes C203 1 "β-hemolytic Keller T623 8" pneumoniae 78-1 8

Enterococcus SSC-80-62 64 _ "SSC-80-63_ 128_ 35

ISLAND

7

DK 161332 B

Table IV

Antibacterial activity of LL-C23024-iota in vitro

Minimal inhibitory con-

Organism_centration (pg / ml) _ 5 Enterococcus OSU 75-1 1

Enterococcus SM 77-15 1

Staphylococcus aureus SSC 79-18 1

Staphylococcus aureus FU 79-19-2 2

Micrococcus lutea PCI 1001 1 10 Staphylococcus aureus Smith 0.5

Staphylococcus aureus ATCC 25923_0.5_

As shown in the above data, antibiotics LL-C23024 - / 3 and -iota inhibit the growth of certain gram-positive bacteria and thus can be used as a topical or surface antiseptic in skin solutions, equipment, walls , floors etc.

It has also been found that the antibacterial LL-C23024-α, -β and -iota are active in vivo as an anticoccidial agent for poultry, as shown in the following samples.

20

Two days before inoculation, feeds with several different levels of preparation are offered to different groups of one-day-old test chickens. The poultry feed used throughout the experiment is prepared as follows:

Vitamin amino acid premix 0.5%

Trace minerals 0.1%

Sodium chloride 0.3%

Dicalcium phosphate 1.2%

Painted limestone 0.5% 30. _

Stabilized fat 4.0%

Dehydrated alfalfa, 17% protein 2.0%

Corn gluten meal, 41% protein 5.0%

Herring fish flour, 60% protein 5.0%

Soybean Oil, 44% Protein 30.0%

Ground yellow corn up to 100.0% 35

ISLAND

DK 161332 B

8

The vitamin-amino acid premix in the above feed mix is prepared from the following composition. Quantity terms refer to units per unit. kg of finished compound feed.

5 Butylated hydroxytoluene 125.0 mg dl-Methionine 500.0 mg

Vitamin A 3300.0 I.E.

Vitamin 1100.0 I.C.E.

Riboflavin 4.4 mg Vitamin E 2.2 I.E.

Niacin 27.5 mg

Panthothenic acid 8.8 mg

Choline chloride 500.0 mg

Folic acid 1.43 mg Menadione sodium bisulfate 1.1 mg

Vitamin B. ^ 11.0 µg

Ground yellow corn up to 5.0 g

The test chickens are then inoculated by direct 20 inoculation into the body of each chicken with a mixed inoculum of 5000 spore-forming oocysts of Eimeria acervulina and a sufficient number of oocysts of Eimeria tenella to result in an 85-100% mortality in untreated control animals. The chickens have free access to feed and water throughout the trial period. Seven days after inoculation, the experiments are terminated and the birds are weighed, autopsied and the intestinal tract examined for lesions. The results are found in Tables V and VI below and show that improved survival for infected chicks is obtained when 10 ppm or less antibiotic is administered LL-C23024-8 or -iota with the feed. The results also show a significant decrease in lesions of the intestinal tract due to Eimeria tenella and Eimeria acervulina.

35

ISLAND

9

DK 161352 B

Table V

Assessment of antibiotic LL “C23024-g as an anticoccidial agent for chickens

Wife. Number Percent of birds with re- 5 in birds induced lesions feed from% E. E.

Compound ppm start survival tenella acervulina LL-C230243 15 3 100 100 100 LL-C230243 10 5 100 60 100 10 LL-C230248 5 5 80 0 100 _NNC * 15_46,6_0_0__ LL-C230243 120 15 100 100 100 60 15 100 100 100 30 15 100 100 100 15 0 15 20 0 0 _NNC * 15_100 _ “_— LL-C230248 20 · 15 100 87 100 15 15 100 60 100 10 15 60 0 0 20 5 15 47 7 0 0 15 27 0 0 _NNC * 15_100_ ~ _ NNC = non-treated, non-infected control

Table VI

Assessment of antibiotic LL-C23024-iota as an anticoccidial agent for chickens ,, ^ ..,. . ~. Ant. birds with red. read.

Concentration Number of birds% -2-- in feed (ppm) at start survival E. tenella E.acervulina 30 15 5 100 1 00 1 00 10 5 100 100 100 5 5 60 20 60 _0__15_20__0__0 15 10 100 100 100 35 7.5_10_100_70_100

DK 161332B

10 o'clock

The following samples show the nematodicidal activity.

5 Assessment of test compositions for nematodic activity using free-living hermaphroditic microbivore nematode Caenorhabditis elegans II

In the following tests, C. elegans is used to determine the nematodicidal activity in the fermentation broth and the harvested mass prepared by the method of the present invention. This organism is also used to evaluate LL-C23024-a and LL-C23024-3 for their nematodicidal activity.

In these tests, the fermentation nutrient liquid is the mixture of fermentation liquid and solids taken before the solids, ie. the harvested mass is separated from the liquid. Harvested pulp is the solids that remain after the separation.

For the purpose of assessing harvested mass, its solids are dispersed in a 1: 1 ratio in distilled water.

The fermentation broth is used as it is and contains both liquid and solids.

For these assessments, C. elegans is kept in a C. briggsae maintenance medium. This medium, Maintenance 25 Medium, is sold by Grant Island Biological Company, Grant Island, New York, and has the following composition: 30 35 11

DK 161332 B

0 (1) C. briggsae Maintenance Medium

Bestanddel_mg / liter_

Inorganic salts

CaCl2.2H20 220.50 5 CuC12.2H20 6.50

Fe (NH 4) 2 (SO 4) 2 * 6H 2 O 58.80 KH 2 PO 4 1225.50 KOH (a)

MnCl2.4H20 22.20 ZnCl2 10.20

Other constituents N-Acetylglucosamine 15.00

Adenosine-3 '- (2') - Phosphoric Acid. H2 O 365.00

Cytidine-3 (21} -phosphoric acid 323.00 D-Glucose 1315.00

Glutathione, reduced 204.00

Guanosine-3 (2 ') - PO4Na2.H2 O 363.00

Magnesium citrate.5H 2 O (dibasic) 915.00

Potassium citrate.H 2 O 485.00 DL-1,2-Dithiolane-3-pentanoic acid 3.75

Thymin 126.00

Uridine-3 (2 ') - phosphoric acid 324.00

Amino Acids L-Alanine 1395.00 L-Arginine 975.00 L-Aspartic Acid 1620.00 L-Cysteine.HCl.H20 28.00 L-δlutamate (Na) .H20 550.00 L-Glutamine 1463.00 30 Glycine 722 , 00 L-Histidine 283.00 L-Isoleucine 861.00 L-Leucine 1439.00 L-Lysine.HCl 1283.00 L-Msthionine 389.00 L-Phenylalanine 803.00 L-Proline 653.00

ISLAND

DK 161332 B

12

Ingredient_mg / liter L-Serine 788.00 L-Threonine 717.00 L-Tryptophan 184.00 5 L-Tyrosine 272.00 L-Valine 1020.00

Vitamins p-Aminobenzoic Acid 7.50

Biotin 3.75 10 Choline dihydrogen citrate 88.50 12

Cyanocobalamin (B) 3.75

Folinate (Ca) 3.75

Myo-inositol 64.50

Niacin 7.50 Niacinamide 7.50

Pantethine 3.75

Panto thenat (Ca) 7.50

Pterolyglutamic acid 7.50

Pyridoxal phosphate. 3.75 20 Py r idoxamine. 2HCl1 3.75

Pyridoxine.HCl1 7.50

Riboflavin-51 - (PO 2 (Na) 2 O 7.50)

Thiamin.HC1 7.50 References: (1) E.L. Hansen, Proc. Soc. Exp. Bio. &. With. 121: 30-393 (1966).

Note: (a) As needed- to adjust to pH 5.9 + 0.1.

30 A rating plate with a number of small recesses is used for the assessments. 25 ml of fermentation broth, a water / harvested pulp suspension of 1: 1 or an aqueous / acetone solution of LL-C23024-0C or LL-23024-8 containing from 31.25 to 500 ppm test compound is aseptically deposited in each 35 wells. . To each well is then added 25 ml of C.elegans maintenance medium containing 10-20 adult worms plus larvae of varying ages plus eggs. Then apply

ISLAND

13

DK 161332 B

the plates in the fume cupboard and examined 48 hours after inoculation to assess the rate and degree of nematodicidal activity of the test compositions. The results obtained are listed below. When activity is observed for the fermentation nutrient liquid, it is further diluted to obtain a 1: 4 ratio of nutrient liquid to C. elegans.

Table VII Wives.

10 ppm el. Nematodicide ac-

Medium dilute. Activity for 48 hours.

Fermentation level = 1: 1 8 (no lubricant F. = 1: 4 lithium) Harvested mass 15 LL-23024-α 500 ppm 7 250 ppm 7 125 ppm 7 62.5 ppm 6 31.25 ppm 0 20 LL-23024-β 500 ppm 7 250 ppm 0

The activity scale used in these assessments is as follows: 25 Activity scale: 8 = no motility (apparent death) 7 = severely reduced motility 6 = reduced motility 0 = normal motility of the species 30

Assessment of LL-C23024-a as nematodicide for infectious ruminant nematode larvae

Assessment of LL-C23024-a as nematodicide against 35 infectious larvae of the ruminant nematodes: Haemonhmus contortus, Ostertagia circumcincta, Trichostrongylus axei, T. colubriformis, and against the vinegar worm Turbatrix aceti. (x) pupated third-stage larvae.

ISLAND

DK 161332 B

14 is voted by the above-mentioned nematode species instead of C. elegans.

The results obtained are listed in the table below.

Table VIII

Wife. in vitro of activity at 48 hours against LL-C23024-g H. contort. O.circum. T. axei T.colub._T. aceti 500 ppm 8 8 ^ 8 ^ 8 6 10 250 ppm 7 8 ^ 8 ^ 7 6 125 ppm 7 7 7 6 6 62.50 ppm 76 76 6 31.25 ppm 66 06 6 0 (control) 0_0_0_0_0 a 15 Made in 96-well tissue culture plates, LL-C23024-a prepared in double distilled water: 25 µl LL-C23024-a solution and 25 µl nematode culture are added in each well.

H

Within 24 hours.

20 Activity scale: as listed in Table VII, page 13.

Assessment of antibiotic LL-C23024-a's nematodicidal activity against Nematospiroides dubius and Aspicularis tetraptera in 25 mice

In the following samples, white Swiss-Webster female mice are infected with Nematospiroides dubius and Aspicularis tetraptera and kept for three weeks to allow the infections to mature.

After this period, the mice are divided into random 30 groups of four, and the groups are placed in separate cages. Feed containing from approx. The 31.25 to 4000 ppm test compound is then offered to the mice ad libitum for one week. Water is also provided ad libitum during the trial period. During the treatment period, the mouse excrement is examined to determine if worms are passing. All treatment groups show siq to allow worms to pass, thus showing nematodic activity at all compound concentrations against both nematodes.

DK 161332 B

15 ° species. After 1 week of treatment with compound feed, the mice are autopsied and the contents of their gastrointestinal tract examined for worms. The results obtained are listed below as percent reduction of worms in comparison to infected control animals that have not received the preparation.

5

In these experiments, crude fermentation culture liquids obtained prior to harvesting of the pulp and containing 1000-4000 ppm of the nematodicidal antibiotic are also assessed. Also, mouse feed treated with from 31.25 to 500 ppm LL-C23024-a dissolved in a 1: 1 mixture of acetone and water is evaluated.

10

Activity (% reduction) ^^ _, opposite

Trial Wife. in feed _ preparation_ppm_N. dubius A. tetraptera x fermentation 4000 73 nutrient liquid 2000 44 53 with LL-C23034a 1000 65 33 500 70 SA ** 20

250 63 SA

125 61 SA

62.5 29 SA

_31,25_32_SA_ * = amount of LL-C23024-a not determined 25 XX = light activity: increased number of child worms when examining faeces XXX = compared to infected control animals not receiving the preparation 30 The antibacterial and anticoccidial compounds are formed during cultivation under controlled conditions by Actinomadura yumaense NRRL 12515.

This microorganism is isolated from a soil sample collected in Yuma Couty, Arizona, and stored in the collection of 35 Lederle Laboratories Division, American Cyanamid Company,

Pearl River, New York, with No. LL-C23024. A living culture

DK 161332 B

16 of this strain are deposited with the Culture Collection Laboratory, 0 Northern Regional Center, U.S. Department of Agriculture, Peoria, Illinois 61604 and is part of its permanent collection with No. NRRL 12515. The strain is freely available and can be obtained from this collection.

Taxonomic Characteristics of NRRL 12515 5

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

Observations of the culture, physiological and morphological features of strain NRRL 12515 have been made using methods that have been described in detail by E.B. Shirling and D. Gottlieb in "Method for characterization of Streptomyces species", Internat. J. Syst. Bacteriol. 16: 313-340 (1966), and by R.E. Gordon et al. in "Nocar-15 dia celiaca, Nocardia autoyrophica and the nocardin strain", Internat. J. Syst. Bacteriol. 24: 54-63 (19 74). The media used for this study is selected from those recommended by T.G. Pridham et al. in "A selection of media for maintenance and taxonomic study of Streptomycetes," Antibiotics Ann., pp. 947-953 (1956/1957), by G.P. Gauze et al. i "Problems in the classification of antagonistic actinomycetes",

State Publishing House for Medical Literature, Medgiz, Moscow (1957), and by R.E. Gordon et al. above, for the purpose of taxonomic study of Actinomycetes and soil bacteria. The chemical composition of the cell walls of the microorganism is determined by that of H.A. Lechevalier et al. described method, "Chemical composition as a criterion in the classification of actinomycetes", Adv. Appl. Microbiol. 14: 47-72 (1971). Phospho-lipid patterns are determined by M.P. Lechevalier et al's method, "Chemotaxonomy of aerobic actinomycetes: phospholipid composition", Biocehm. Syst. Ecol. 5: 249-260 (1977). Details are given in Tables IV-IX, and a general description of the culture is given below. The underlined descriptive colors are taken from K.L- Kelly and D.B. Judd's "Color. Univer-o5 Will Language and Dictionary of Names", U.S. Night. Cage. Stand., Spec. Publ. 440, Washington, D.C. (1976), and the accompanying

ISLAND

DK 161332 B

17

Inter-Society Color Council, Natl. Cage. Stand. Centroid Color Charts.

The data observed for this new species represented by the strain NRRL 12515 are compared with published data for known species of the strain Actinomadura [M. Goodfellow et al., "Numerical Taxonomy of Actinomadura and Related Acti-Nomycetes", J. Gen. Microbiol. 122: 95-111 (1979), L.H. Huang, "Actinomadura macra sp. Nov., The producer of antibiotic CP-47,433 and CP-47,434", Internat. J. Syst. Bacteriol. 30: 10 565-568 (1980), J. Meyer, "New species of the genus Actinoma dura", Z. General. 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. in J. Ferment. Technol. 49: 904-912 (1971), and T.P. Preobrazhen-15 skaya et al., "Key for identification of the species of the genus Actinomadura", The Biology of Actinomycetes and Related Organisms 12: 30-38 (1977)]. The culture NRRL 12515 bears a slight resemblance to Actinomadura pelletieri, but does not resemble any other described species and varies from A. pelletieri in terms of a number of characteristics. Therefore, the strain NRRL 12515 has been designated as the strain type of a new species named Actinomadura yumaense, sp. Nov., named after the site of the collection of the soil sample from which the strain type is isolated. Micromorphology 25 Spores are formed in short spiral chains (maximum length about 20 spores per chain) on branched almost coronary air-sporophore. The spores are egg-shaped, 0.6-0.8 ^ u x l, 0-l, 4 ^, u, with a smooth surface.

Cell Wall Composition 30 Whole cell hydrolysates of this culture contain madurose (3-O-methyl-g-galactose) and the meso-isomer of diamino-pimelic acid (DAP). The culture also has a phospholipid pattern type P-1 and no other detectable phospholipid than a little phosphatidyl-glycerol. These characteristics are all very typical of members of the genus Actinomadura.

growth Stock

Good growth is observed on Bennett's agar, Gauze # 2

ISLAND

DK 161332 B

18 agar, NZ amine starch glucose agar (ATCC Medium 172), tomato puree oatmeal agar and yeast extract malt extract agar; moderate fluid is observed on Benedict's agar, Czapek's sucrose agar, glycerol asparagine agar, Hickey-Tresner agar and oatmeal agai; 5 slight growth is observed on calcium malate agar, Gauze No. 1 agar and inorganic salt starch agar.

Vegetative mycelium In media where good growth occurs, it is observed that the vegetative mycelium is produced and rolled and in the rule of color with yellowish-gray shades.

The color of aerial mycelium and spores

Air mycelium and / or trace masses are white to 264: light gray in color. Air mycelium reproduction is poor on most media.

15 Soluble pigments

Missing on many media; yellow pigment on Benedict's agar and glycerol-asparagine agar; yellow-green pigment on calcium malate agar; greenish-brown pigment on NZ-amine starch-giucose agar; orange pigment on Bennett's agar and yeast extract malt text-20 funnel agar.

Physiological reactions

No meian pigments on peptone iron agar and tyrosine agar (ISO-7); strong peptonization of litmus milk; strong proteolysis of nutrient gelatin, moderate reduction of nitrate, inorganic hydrolysis of adenine or guanine, strong hydrolysis of hypoxanthine, tyrosine and xanthine, weak starch hydrolysis, esculin hydrolysis, urea variable hydrolysis. No growth at 4, 10 or 55 ° C, moderate growth at 25 and 45 ° C, good growth at 32 and 37 ° C. Carbohydrate utilization according to T.G. Pridham and D. Gottlieb's, "The utilization of carbon compounds by some actinomycetales as an aid for species determination," J. Bac-teriol. 56: 107-114 (1948): good utilization of glucose, glycerol and trehalose, moderate utilization of maltose and sucrose; poor utilization of fructose, galactose, inositol, mannose and 35 melezitosis; no utilization of adonitol, arabinose, dulcitol, lactose, mannitol, mayibiosis, raffinose, rhamnose, salicin, sorbitol and xylose. Acid production from carbohydrates by Gor-

ISLAND

DK 161332 B

19 don et al's method, above: good acid production from glucose, glycerol, maltose, sucrose and trehalose; low acid production from galactose, inositol and mannose. Utilization of organic acids by means of that of Gordon et al.

5 described above: utilization of acetate, malate, propionate, pyruvate, succinate and tartrate; no utilization of bone zoat, citrate, lactate, mucate and oxalate.

Table IX

Cultivation data for Actinomadura yumanese NRRL 12515

Incubation: 14 days__Temp: 28 ° C

Growth- Dissolve. rear

Medium_mount Air mycelium and / or traces pigment color

Benedict's Moderate Flat Powdered. colonies; Cute 89.

15 agar - low aerial mycelium white to 264, pale yellow light gray

Bennett's God No aerial mycelia; rolled up Orange 81. dark agar vegetative growth, 93. yellowish to greyish-yellow 80. greyish-yellowish-brown corpse-brown 20--

Calcium ma- Low flat growth; sparse white air- Yellowish- 90. green- lat-agar mycelia green-yellow-yellow

Czapek's Poor Flat Growth; moderate aerial mycelia- No 89. pale sucrose to moss, vegetative growth, 90. grayish yellow 25 seagar yellow

Gauze No. Rings Colorless flat wax &st; sparse Nothing Colorless 1 agar white aerial mycelia

Gauze No. Good Raised Rolled Colonies; ve- Nothing 72. dark 30 2 agar getative mycelia 93. yellowish gray; orange-yellow moderate aerial mycelia, 92. yellowish-white

Glycerol-a- Rings Flat, powdery colonies; white- Yellow 89th pale sparagin- to fashion aerial mycelia yellow 35 agar therein

ISLAND

DK 161332 B

20

Table IX (cont.) Growth- Dissolve. Bagside-

Medium_mount Air mycelium and / or traces pigment color

Hickey- Moderate Flat waxy colonies, 90. Nothing 90. gray 5 Tresner greyish yellow sparse aerial yellow agar clay, white to 264 light gray

Uorg. sal- Rings Flat, colorless, powdery No Colored starch colonies, white aerial mycelia loose se-agar ________ 10 NZ-amine trail- Good Heavy rolled growth, 61 gray- Green- 78. dark velvet-yellowish yellow brown to 65. brownish corpse yellow cuddle-agar black, moderate aerial mycelia, white-brown brown those to 264. light gray

Oatmeal- Fashion- Flat waxy growth, 90. greyish Nothing 90. greyish 15 agar steed yellow, traces of white aerial mycelium yellow

Ifcmatpuré- Good Flat waxy growth, 91. dark No oatmeal-grayish yellow, traces of white air agar_mycelier_ 20 Yeast-Good Raised, waxy rolled Orange 78. dark funnel colonies, 93. yellowish-gray to 80th yellow malt text-grayish yellowish brown, nothing aerial brown tract mycelium

Table X

The micromorphology of Actinomadura yumanese NRRL 12515

Air mycelium and / or Spore- Spore-

Medium_spore-bearing structures shape Trace size overf1.

Czapek's Luftporporophore, branch- Egg shape- 0.6 - 0.8 ^ u Smooth 30 saccane, almost wreath-styled x roseagar de, bearing front. short 1.0 - 1.4 µl spiral chains of mature _spores__ 35

ISLAND

21 DK 161332 B

Table XI

The physiological reactions of Actinomadura yumaense NRRL 12515

Medium_Inkubationst. Amount of growth Physiological reaction

Pepton Iron 7 days good Light tan 5 agar_14 days_good_Light tan

Tyrosine agar 7 days moderate No dye _14 days_good_Gullig pigment_

Litmus milk 14 days good Good peptonization _28 days_good_Vigorous peptonization 10 Nutrition gela- 14 days good Easy proteolysis thaw_28 days_good_Total proteolysis_

Nitrate Nutrition 14 days good Very weak reduction fluid_28 days_good_Moderate reduction

Adeninagar 14 days good No hydrolysis 15__21 days_good_No hydrolysis_

Guaninagar 14 days good No hydrolysis __21 days_good_No hydrolysis_

Hypoxanthine- 14 days good Total hydrolysis agar_21 days_good_Total hydrolysis_ 20 Tyrosine agar 14 days good Heavy hydrolysis _21 days_good_High hydrolysis_

Xanthinagar 14 days good Moderate hydrolysis _21 days_good_Powerful hydrolysis_ NZ amine with dissolution 5 days little or no growth at 4, 25 starch and 10 and 55 ° C, moderate growth v. 25, glucose agar 28 and 45 ° C, good growth at 32 and (ATCC Med. 172) _ 27 ° C.

Urinstofnær, v. 28 days_good_Decomposition, variable

Esculin Near- 14 days good Hydrolysis 30 liquid_28 days_good_Hydrolysis_

Starch agar 5 days good No hydrolysis _10 days_good_No hydrolysis_ 35

ISLAND

DK 161332 B

22

Table XII

Actinomadura yumaense NRRL 12515 carbon source utilization on ISP-9 carbohydrate utilization esmedium Incubation; 28 days Temperature; 28 ° C

5 Carbon source Exploitation

Adonitol 1-Arabinose -

dulcitol

Fructose rings 10 d-Galactose rings d-Glucose good

Glycerol good i-Inositol rings

Lactose 15 Maltose fairly good d-Mannitol - d-Mannose rings d-Melezitose rings d-Melibiosis 20 d-Raffinose 1-Rhamnose Saiicin Sorbitol

Sucrose fairly good 25 d-Trehalose good d-Xylose Negative control 30 35

ISLAND

DK 161332 B

23

Table XIII

Acid production by actinomadura yumaense NRRL 12515 from various carbohydrates on Gordon's inorganic nitrogenous medium

5 Incubation: 28 days_Temperature: 28 ° C

Acid production_ _Carbon source_7 days_2S days

Adonitol 1-Arabinose - - 10 Duicitol

Fructose d-Galactose - + d-Glucose +++ +++

Glycerol ++ +++ 15 i-Inositol - +

lactose

Maltose - +++ d-Mannitol d-Mannose - + 20 d-Melezitose d-Melibiosis d-Raffinose l-Khamnose Salicin Sorbitol

Sucrose - +++ d-Trehalose - +++ d-Xylose Negative control 30 *: +++ = strong positive reaction ++ = Moderate positive reaction + = Slight positive reaction - = Negative reaction 35 24 DK 16 1332 Β

Table XIV

Exploitation of Organic Acids by Actinomadura Yuma Ense NRRL 12515 on Gordon's Modification of Koser's Basic Agar (Koser's Citrate Agar)

Incubation: 28 days_Temperature: 28 ° C

5 _Carbon source_Utilization * _

Acetate +

benzoate

citrate

Lactate 10 Malate +

Mucous acid -

oxalate

Propionate +

Pyruvate + Succinate +

Tartrate + i: + = positive reaction - = negative reaction · 20

Cultivation of Actinomadura yumaense can take place in a wide variety of solid or liquid culture media. Media useful in the preparation of antibiotics LL-C23024-a, -β and -iota comprise an assimilable nitrogen source such as protein, protein hydrolyzate, polypeptides, amino acids, maize molding liquid, etc., and inorganic anions and cations such as potassium, sodium, ammonium, calcium, sulfate, carbonate, phosphate, chloride, etc. Substances such as boron, molybdenum, copper, etc., are supplied by impurities from other constituents of the media. Aeration in tanks and flasks is done by forcing sterile air through or towards the surface of the fermentation medium. Stirring in the tanks takes place with mechanical impeller. An anti-foaming agent, such as blubber oil or silicone foaming agent, can be added as needed.

ISLAND

DK 161332 B

25

Actinoxnadura yumaense is grown and stored on oblique agar, e.g. Bennett's agar, yeast malt agar or ATTC medium # 172. ATCC medium # 1/2 is preferred. The inclined agar is inoculated with a culture of Actinomadura yuma ense and incubated at 28-37 ° C, preferably at ca. 32 ° C, for approx.

7 days. These stem cultures can be maintained by serial transfer to freshly inclined agar or a plug of the agar containing mycelia from a road-grown inclined agar can be used to inoculate liquid media.

Shake flask inoculations of Actinomadura yumaense are prepared by incubating 10U ml of sterile liquid medium in 500 ml flasks with scraping or washing liquids of spores from an oblique styled agar with the culture. Examples of suitable seed media are:

Medium A

Meat extract 0.3% "Bacto" tryptone1 0.5%

Glucose 1.0% Yeast tract 0.5%

Water up to 100%

A peptone from Difco Laboratories, Detroit, Michigan).

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

Medium B

Glucose 1%

Starch 2% Yeast extract 0.5% "N-Z Amine A" 2 0.5% 30

Calcium carbonate 0.1%

Water up to 100% 2 (Casein digest product from Sheffield Chemical Co., D iy. Nat'l. Dairy Products Corp., Norwich, N.Y.).

Medium B is preferred.

35

DK 161332 B

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 fermentation culture, or the culture can be frozen and stored as inoculation for later seed cultures.

The following media are examples of those which can be used for the fermentation of Actinomadura yuma.ense for the preparation of antibiotics LL-C23024-ot, -3 and -iota.

Medium C

Glucose 1.5% 10 Glycerol 1.5% 3

Soybean 1.5%

Calcium carbonate 0.1%

Sodium chloride 0.3%

Water up to 1U0% o 15 (Can be replaced with cotton seed meal or soluble meat with similar effect)

Medium D

Glucose 3%

Soybean 1.5% Calcium Carbonate 0.1%

Sodium chloride 0.3%

Water up to 100%

Medium E

Starch 1% 25

Molasses 2%

Soybean 1.5%

Calcium carbonate 0.1%

Water until 100% 30

Medium F

Glucose 3%

Soluble Meat 2.5%

Sodium chloride o, 2% 35

Calcium carbonate 0.1%

Water up to 100%

DK 161332 B

27

Medium G

Glucose 3%

Soybean 0.5%

Ammonium sulfate 0.3%

Sodium Chloride 0.2% 5

Calcium carbonate 0.1%

Water up to 100%

Medium H

Glucose 3% 10

Ammonium sulphate 0.3%

Dibasic potassium phosphate 0.1%

Calcium carbonate 0.2%

Sodium ciloride U, 1%

Water up to 100%

Medium D is the preferred one.

The fermentation is 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 ca. 32 ° C, for 24-72 hours under aeration. Specimens of fermentation culture can be frozen and stored for later use as inoculum for seed cultures.

Alternatively, the fermentation may be carried out in larger fermentation tanks equipped with aeration and stirring means.

Each tank is inoculated with 3-10% (v / v) graft prepared as described above. Air supply is carried out at a rate of 0.5-2.0 liters of sterile air per day. liter of nutrient liquid per liter. The fermentation medium is stirred with a forest wheel driven at 200-30 400 rpm. The temperature is maintained at 25-35 ° C, preferably at 32 ° C. The fermentation is continued until the antibiotic accumulates in the fermentation medium, usually after 100-150 hours, at which time the antibiotic is harvested.

The antibiotic can be harvested and purified as described 23

DK 161332 B

0 in U.S. Patent No. 4,278,663 above or by the following method:

The crude fermentation liquid containing the entire cells prepared as described above is mixed with an equal volume of any organic water-immiscible solvent other than a hydrocarbon. Methylene chloride or ethyl acetate is preferred. The organic phase is separated and evaporated in vacuo to an oily syrup.

The oily syrup is dissolved in methylene chloride and added to a column of silica gel, alumina, "Sephadex® 10 LH-20" (Pharmacia Fine Chemicals Div. Of Pharmacia, Inc.,

Piscataway, N.J.) or magnesium aluminum silicate. Examples of suitable solvents for eluting the column are diethyl ether, ethyl acetate, a 1: 1-1: 7 (v / v) mixture of methylene chloride / 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 checked for the presence of antibacterial activity by bioassay against a susceptible organism, e.g. Bacillus subtilis. Active fractions are combined and evaporated in vacuo for a residue. This residue is dissolved in an organic solvent, e.g. t - butanol (preferred), benzene or p-dioxane, and freeze-dried.

The freeze-dried 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. Diethyl ether is the preferred one.

This solution is shaken with water and the pH is adjusted to 1.5-4.0, preferably approx. 2.5 with any dilute mineral acid. The organic phase is separated, washed with water to remove excess acid, dried over a suitable desiccant, filtered and evaporated to a residue in vacuo.

This residue is dissolved in a suitable solvent and the solution is allowed to evaporate slowly, preferably 35

ISLAND

DK 161332 B

29 at approx. 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 (vo / v) is preferred. The crystals obtained are collected and washed, preferably at ca. 40 C, with any hydrocarbon having a moderate boiling point such as e.g.

hexane or heptane and air dried to yield the final product antibiotic LL-C23024 in the form of free acid.

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

The invention will now be further illustrated by way of example. The media a, b, c etc. are the above defined. Unless otherwise stated, all procedures are carried out at room temperature (about 22 ° C) and a pressure of 1 atm.

Example 1

Washed spores from an obliquely styled Actinomadura Yuuman NRRL 12515 agar are used to inoculate a 500 ml flask containing 100 ml of sterile medium B. The flask is incubated on a rotary shaker at 28 ° C for 2 days.

A 5% seed of this culture is then transferred to 100 ml of sterile medium C in a 500 ml flask and incubated for 5 days on a rotary shaker.

Presence of antibiotic activity is monitored daily with bioassay against Staphylococcus aureus ATCC 6538 P, Bacillus subtilis and Streptococcus faecalis, and anthelmintic activity is monitored by bioassay against the free-living nematode.

UV

tode Caenorhabditis elegans.

35

ISLAND

DK 161332 B

30

Example 2 7,500 ml cuber is prepared, each containing 100 ml of one of the following sterile media:

Flask 1: 10U ml of medium C

5

Flask 2: luO ml of medium C with 1.5% cotton flour instead of the soybean meal

Flask 3: 100 ml medium C with 1.5% soluble meat instead of soybean flask 4: 100 ml medium D 10 Flask 5: 100 ml medium E

Flask 6: 100 ml medium F Flask 7: 100 ml medium G

These flasks are inoculated with a 5% graft of Acinthomadura yumaense NRRL 12515 grown in medium B. The flasks

O

then incubate on a rotary shaker at 28 ° C for 4-6 days.

Each of the above cultures is found to be active when tested for antibiotic activity as in Example 1 and when tested for anticoccidial activity against Eimeria tenella in chicken kidney tissue cultures.

20

Example 3

Frozen fermentation culture cells of Actinomadura yumaense NRRL 12515 are used to inoculate a 500 ml flask containing

contains 100 ml of sterile medium B. The flask is then incubated 25 °

at 32 C for 4 days on a rotary shaker.

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

Antibacterial activity is confirmed as in Example 1 and anticoccidial activity as in Example 2.

The presence of antibiotic activity is also tested by thin layer chromatography on silica gel plates eluted in ethyl acetate / chloroform at 70:30 (v / v) ratio.

35

ISLAND

DK 161332 B

31

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

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

A 5% seed of this culture is then transferred 10 to a 500 ml flask containing 100 ml of fresh sterile medium H and incubated at 28 ° C for 6 days on rotary shaker.

Antibacterial activity is confirmed as in Example 1.

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

The contents of the two flasks are then combined and added to 12 liters of fresh sterile medium A in a 20 liter flask. This culture is then incubated for 2 days at 28 ° C under aeration.

The contents of this bottle are then transferred to a 300 liter graft tank containing 288 liters of sterile 25 A medium and this culture is aerated and stirred for 25 hours at 32 ° C.

After the 25 hour incubation, the 300 liter seed culture is transferred to a 1500 liter fermentation tank containing 1200 liters of sterile medium C. This culture is incubated under aeration and stirring for 115 hours at 28 ° C.

The fermentation liquid is tested for antibiotic activity by thin-layer chromatography on silica gel plates eluted with ethyl acetate / chloroform at 70:30 (v / v). Alternatively, several evoked plates are subjected to bioassay against Bacillus subtilis showing the presence of antibiotic activity.

ISLAND

DK 161332 B

32

Example 6

A typical medium used for the cultivation of primary graft is prepared with the following composition: Meat extract 0.3% 5 "BactoH tryptone 0.5%

Glucose 1.0% Yeast Extract 0.5% Bacto Agar 0.15%

Water up to 100% 10 Washed or scraped spores and mycelia from an oblique Actinomadura yumaense NR3RL 12515 agar are used to inoculate a 500 ml flask containing 100 ml of the above sterilized medium. Place the flask on a rotary shaker and stir vigorously for 48 hours at 32 ° C. The resulting flask graft (100 ml) is used to inoculate one liter of the same sterile medium into a 2 liter bottle. This inoculum is aerated with sterile air while cultivation is continued for 48 hours at 2b ° C. This 1-liter culture is then used to inoculate a 30-liter fermentation tank containing 20 of the same sterile medium and this tank is aerated with sterile air and incubated at 28 ° C for 42 hours.

Example 7

A fermentation medium having the following composition is prepared:

Dextrose 1.5%

Glycerol 1.5%

Soybean 1.5%

Calcium Carbonate 0.1% Sodium Chloride 0.3%

Water up to 100% pH is adjusted to 7.0 with 6N sodium hydroxide and the medium is sterilized. A 30 liter portion of inoculum is prepared as described in Example 1 and this is used to inoculate 250 liters of the above medium into a 300 liter fermentation tank. Sterile aeration is applied to the mass which is stirred.

ISLAND

DK 161332 B

33 res with a impeller driven at 220 rpm. The fermentation takes place at 32 ° C for 97 hours, after which the mass is harvested.

Example 8 A fermentation medium is prepared having the following composition:

Dextrose 3.0%

Soybean 1.5%

Calcium Carbonate 0.1% Sodium Chloride 0.3%

Water up to 100% pH is 7.0 with 6N sodium hydroxide and the medium is sterilized. A 300 liter portion of inoculum prepared as described in Example 1 is used to inoculate 15,000 liters of the above medium into a fermentation tank. Sterile aeration is applied to the mass, which is stirred with a impeller driven at 100 rpm. The fermentation is carried out at 32 ° C for 115 hours, after which the mass is harvested.

Example 9 1. The fermentation mass of Example 8 (100 liters) is adjusted to pH 4.0 by means of 6N HCl. The acidic mass is then stirred for 1-2 hours with the same volume of methylene chloride. The mixture of mass and methylene chloride is then filtered, using diatomaceous earth as filter aid. The methylene chloride extract is extracted and evaporated in vacuo to ca. 2 liters of partially purified antibiotics.

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

30 A 7 cm diameter glass column is packed to a height of 91 cm with "Woelm silica Gel TSC". The crude concentrate (cf. above) with a volume of approx. 2 liters are allowed to seep into the silica gel column. Then, the column is first developed with 3 liters of methylene chloride, then methylene chloride / ethyl acetate (1: 1 by volume), giving a total of 126 fractions, each with a volume of 80 ml. It is then further developed with ethyl acetate / ethanol (7: 3),

ISLAND

DK 161332 B

34 ket gives an additional 87 fractions.

Fractions 58-87, rich in LL-C23024-a, are combined and evaporated in vacuo to a residue weighing 90.4 g. This residue is dissolved in a mixture of 600 ml diethyl ether and 5 600 ml hexane. The resulting suspension is filtered to give a clear filtrate. The clear filtrate is evaporated in vacuo until crystals begin to form. The suspension is allowed to ripen overnight. The crystalline LL-C23024-a is collected on a funnel, washed with cold ether and air-dried, yielding 33.1 g of crystalline LL-C23024-a. An additional 12.4 g of LL-C23024-a is obtained after further reducing the volume of the combined washing liquid and the filtrate.

Fractions 177-203 from elution with ethyl acetate / e-thanol and enriched with LL-C23024-6 are combined and evaporated in vacuo to give 6.7 g of partially purified LL-C23024-8 treated as above.

Fractions 204-213 from elution with ethyl acetate / e-thanol and enriched with LL-C23024 iota are combined and evaporated in vacuo to a paste and extracted repeatedly with methanol.

The methanol extract is extracted with methylene chloride which is charged to a column containing "Woelm" silica gel. The column is washed with methylene chloride and then eluted with methylene chloride / ethyl acetate (2: 3). The fractions are monitored by thin layer chromatography and the active fractions are combined, treated with 25 charcoal, evaporated and extracted repeatedly with heptane.

The final heptane extract is cooled at 0 ° C for 48 hours and the crystals removed by filtration from the mother liquor.

This mother liquor is dissolved in methylene chloride and allowed to seep into a glass column packed with "Woelm" silica gel.

Then the column is first eluted with 2 liters of methylene chloride, then 8 liters of methylene chloride / ethyl acetate (1: 1) and finally with 4 liters of ethyl acetate / methanol (9: 1). The eluate is collected in fractions and monitored for their antibiotic composition. The active fractions are combined and evaporated in vacuo to give a residue containing LL-C23024 iota.

ISLAND

DK 161332 B

35

Example 10 3. Further purification of LL-C23024-6 from chromatography on silica gel.

"Sephadex ® LH 2 O" is allowed to swell in a mixture of 5 hexane, methylene chloride and methanol in a ratio of 10: 5: 1. A 5 cm diameter glass column is packed at a height of 86 cm with the gel. The batch, 3 g of purified LL-C23024-8, is dissolved in a mixture of 10 ml of methylene chloride, 1 ml of methanol and 10 ml of hexane and allowed to seep into the gel column. The column is then developed with solvent of the same composition as that used when the antibiotic is applied to the column. The fractions are collected by means of a collector. The fractions each have a volume of 17 ml. Fractions 17 to 26 contain LL-C23024-6 as determined by TLC. These fractions are combined and evaporated in vacuo to give pure amorphous LL-C23024 weighing 1269 mg.

Example 11

Crystallization of LL-C23024g as sodium salt Purified LL-C23024-8 (2.4 g) prepared as described in Example 10 is dissolved in a mixture of 500 ml of diethyl ether, 160 ml of hexane and 25 ml of methylene chloride. The resulting solution is transferred to a separatory funnel containing 300 ml of distilled water. Dilute 1N hydrochloric acid is added dropwise until the pH reaches a value of 2.0-2.5 after shaking and settling.

The acidic aqueous phase is discarded and the acid-treated organic layer is washed three times in succession with 400 ml portions of water. The washed organic layer is stirred with a teaspoon of "Dar-co G-60" powder for 15 minutes and filtered through "Celite®" · The decolorized organic layer is placed over 500 ml of distilled water and 5N NaOH is added dropwise until the pH reaches 11 , 0-11.5 after shaking and settling. The alkaline aqueous phase is discarded and the remaining organic layer is washed twice with 400 ml portions of water. The washed organic layer is dried over sodium sulfate and then evaporated in vacuo to a volume of 150 ml. This concentrate is left in the fume hood for several hours. The crystalline Na salt of LL-C23024-6 is collected on a funnel, washed

ISLAND

DK 161332 B

36 with cold hexane and air dried to give 402 mg of crystalline Na salt of LL-C23024-3. This sample is used for microanalyses and selected spectral data.

Analysis: Calculated for C c cHH OO-Na: δ C 59.70, H 8.33, O 29.46, ash 2.49.

Found: C 61.55, H 8.79, N O ash 3.31

Mp. (Fisher-John's apparatus) = 174 ° C.

(Field desorption mass spectrometry) = 924, [α] D = + 3.2 ° (in methanol).

Example 12

Purification of LL-C23024-iota

And Waters prep. 500A high performance liquid chromatography instrument is equipped with a silica gel cartridge under a pressure of approx. 39 kg / cm. The batch, which is 5.0 g of the crude material from Example 9, is dissolved in 50 ml of heptane / ethyl acetate (45:55) and injected onto the column of silica gel. The column is then developed with the same solvent mixture at a flow rate of 200 ml / min, collecting 40 200 ml fractions. Fractions 21-32 are combined, evaporated to a residue, triturated with hexane and evaporated to give pure LL-C23024-iota. The above procedure is repeated 4 times, giving a combined total of 280 mg of amorphous LL-C23024 iota.

A portion of 90 mg of amorphous LL-C23024 iota is 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 is washed with water, adjusted to pH approx. 11 with 0.1N sodium hydroxide, is separated and washed again with water. The ether phase is dried over sodium sulfate, filtered and evaporated to a residue. A solution of this residue in ether / hexane is allowed to slowly evaporate to give an amorphous white solid.

This amorphous white solid has the following properties:

Elemental analysis: C 61.79, H 8.84, ash 0.32.

[α] 25 = + 27 ° (C 0.724, methanol).

Field desorption mass spectrometry: (M + Na) = m / e 35 953, calculated molecular weight is 930.

Claims (4)

1. Biologically pure culture of Act withinadura yumaense, characterized in that the culture is Actinomadura 5 yumaense NRRL 12515 and that it can form antibiotic LL-C23024-Q! with the structural formula Ml «10? * · M j [*„ ii. Trr ^ Me H Me H HH OH 15 COOH M * antibiotic LL-C23024-) 3 having the structural formula 20 qh OH g foH Η 1 H Me H Me H HR OH COOH 25 wwn and antibiotic LL-C23024 iota, which was later found to have the following structural formula OM «r €. OM «OH | S.QS ** * Μ · α ^ αΟΜ · Ϊ O 35 \ ΗΜλΕ H it i cf COjH DK 161332B in recoverable amounts by fermentation in a liquid nutrient medium containing assimilable sources of carbon, nitrogen and inorganic salts. 2. Process for Preparation of Antibiotics LL- C23024-a, -β and -iota of formulas OM «~ ir QMe S Me4A / M * JL m * YYM'fV \ nKyf | ^ ° ΤγΤ ° Me 15. OH Η i H Me H Me Η Η H OH COOH Me OMe o QH O «£ 25 [^ οη ηΪη Me H Me H HH OH COOH Me OMe rV“ 30 OMe OH Μβ | £ ΤϊΤ Me H Η Η H OMe C02H DK 161332 B characterized by aerobically fermenting Actino-madura yumaense NRRL 12515 in a liquid medium containing assimilable sources of carbon, nitrogen and inorganic salts until significant antibiotic activity is to 5 present in the fermentation broth and then the antibiotics are recovered therefrom. Process according to claim 2, characterized in that the temperature of the fermentation culture is kept 10 at 25-35 ° C for a period of 24-150 hours. Method according to claim 3 for the preparation of antibiotic LL-C23024-a, characterized in that the aerobic culture of Actinomadura yumaense NRRL 12515 is aerobically cultured in a liquid medium containing assimilable sources of carbon, nitrogen and inorganic salts until a substantial antibiotic activity is present. in the fermentation broth, and then extracting the antibiotic therefrom.