FI75187B - ETT NYTT FOERFARANDE FOER FRAMSTAELLNING AV KAENDA ANTIBIOTIKA LL-C23024ALFA, LL-C23024BETA OCH LL-C23024IOTA. - Google Patents

Description

, 75187

The present invention relates to a novel process for the preparation of the known antibiotics LL-C23024alpha (= X-14868A), LL-C23024beta (^) (= X-148) and = X-148. LL-C23024 for preparation of (s) (= X-14868B), OMe

MeO xx \ Me

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X-14868 A = LL-C23024alpha (jt) OMe 20 MeO \

Me OH OH = m ~ OMe O Me

Me H ° Me h 1 Me C00H X-14868C = LL-C23024beta (p1) OMe ”.. rV“ CO ^ H Me X-14868B = LL-C23024 wait (s) 2 75187

Said three antibiotics are known from FI patent 70247.

The novel process according to the invention for the preparation of said known antibiotics is characterized by aerobically fermenting the microorganism Actinomadura yumaense NRRL 12515 in a liquid medium containing assimilable sources of carbon and nitrogen and inorganic salts, maintaining the fermentation culture at 25-35 ° C. 150 hours, after which the antibiotic is isolated in a manner known per se.

The structure of the antibiotic LL-C23024beta is based on elemental analysis, optical rotation, field desorption, mass spectroscopic molecular weight, melting point, IR spectrum, C-nuclear magnetic resonance spectrum (C-NMR) and proton-magnetic resonance (H-NMR) as detailed in the examples and the accompanying figures:

Figure I; IR spectrum of LL-C23024p in KBr.

Figure II; 13 C-NMR spectrum of LL-C23024 / S, 20 MHz in CDCl 3, internal TMS reference equivalent.

Figure III: 1 H-NMR spectrum of LL-C23024, 80 MHz in CDCl 3, internal TMS-25 reference equivalent.

3 75187

Table I 13 C-NMR spectrum of LL-C230248 (ppm with respect to TMS)

Chemical transfer. . ,,. ..,. Carbon number (pom) ......

-3 1 _ maara_ 10.5 i n, o, 12.2 17.0 17.7} 17 9 10 2 2 ^ 3] 26.1; 26.8} 27.6! 30.2 | 32.0} 33.3} 15 33.7 y 33.8 t 36.5 f 36.8 t 39.0 7 39.9, 45.5 2 20 57.1 ^ 59.1 i 60.7 n 66.9 7 67.6 i 70.2 7 71.3 7 72.9 1 25 74.9 7 75.1 7 79.9 7 80.8 -) 82.1; 84.5 i 84.7 i 30 85'6 1 86.9 i 95.8 i 96.9 i 97.7 i 107.5 i 179.2 i 35 46 4 75187 LL-C23024 io is a polyether antibiotic with a very strong anticoccidial effect. It is at least ten times more potent than most known polyethers. The values given by field desorption mass spectroscopy show that it has a molecular weight of 930. Based on this and the carbon-13 nuclear magnetic resonance spectrum (C-NMR), its tentative molecular formula can be suggested as C48H82 ^ 17 * that the antibiotic LL-C23024 is the sugar species is found in the polyester antibiotic X-14868A (U.S. Patent No. 4,278,663) and a carboxy group. The only other polyether antibiotic reported with a molecular weight of 930 is the antibiotic A29695B (hydroxyceptamycin) (J. Antibiotics 33 (2): 252 (1980)), which differs significantly in structure and biological properties. The findings are based on elemental analysis, optical rotation, calculated molecular weight by field desprotection mass spectroscopy, IR spectrum, 13 C-NMR spectrum, and proton nuclear magnetic resonance (1 H-NMR) spectra, as detailed in the Examples and the accompanying drawings.

Figure IV: IR spectrum of LL-C23024 iota in KBr.

Figure V: 1 H-NMR spectrum of LL-C23024 iota, 80 MHz in CDCl 3, internal TMS reference equivalent.

13

Figure VI: C-NMR spectrum of LL-C23024 iota, 20 MHz in CDCl 3, internal TMS reference equivalent.

Figure VIIA: 13 C-NMR spectrum of LL-C23024 iota (spread scale: 0-50 ppm), 20 MHz CDCl 3 internal TMS reference equivalent.

13

Figure VIIIB: C-NMR spectrum of LL-C23024 iota (spread scale: 50-110 ppm), 20 MHz in CDCl 2, internal TMS reference equivalent-35 lent.

The C-NMR spectrum of the LL-C23024 lot was obtained in deuterated chloroform (CDCl 3) at a field strength of 20 MHz. Table II shows the peaks with their corresponding chemical shifts in parts per million relative to tetramethylsilane (TMS) and the estimated number of carbon atoms per peak.

Chloroform peaks were observed at 75.4, 77.0 and 78.6.

δ 75187

Table II

chemical number of carbon atoms chemical number of carbon atoms displacement number of displacements 5 10 »6 1 59i9 1 10i9 1 00.8 2 11.7 1 68.5 1 16.11 1 68.8 1 17.6 1 71.3 '1 10 18.0 1 72 , 2 1 21 <7 1 76.1 1 22t9 1 76.9 1 24 <1 1 78.5 1 28.2 1 81.0 1 15 31.2 2 81.3 1 32.2 1 81.8 1 33.4 1 84.8 1 33.8 2 85.3 1 · 34.2 1 85.6 1 20 36.7 1 86.8 1 37.2 1 88.5 1 39.4 1 95.9 1 40.3 1 97.9 1 44.5 1 99, 6 1 45.5 1 107.2 1 47.6 1 173.0 _i_ 56.8 1 Amount 1j8 il 7 75187

The antibiotics LL-C23024 - <-, / 'and iota are organic carboxylic acids and can thus form salts with non-toxic, pharmaceutically acceptable cations. Salts formed by mixing an antibiotic in the form of a free acid and stoichiometric amounts of cations can be formed with cations such as sodium, potassium, calcium and ammonium and organic amine cations such as tri (lower alkyl) amine (e.g. triethylamine, triethanolamine), procaine and the like. . The cationic salts of the anti-10 biotic LL-C230243 are generally crystalline solids, relatively insoluble in water and soluble in most common organic solvents such as methanol, ethyl acetate, acetone, chloroform, heptane, ether and benzene. For the purposes of the invention, the antibiotic in free acid form and its pharmaceutically acceptable, non-toxic salts are equivalent.

The antibiotics LL-C230234 / 'and iota are effective in vitro against gram-positive bacteria when tested according to a standardized agar dilution method. In Tables III and IV, the results are reported as the minimum inhibitory concentration (MEK) in micrograms per milliliter. Antibiotics LL-C230234 and iota do not work against gram-negative concentrations of 256 ug / ml or less.

8 75187

Table III

Antibacterial potency of LL-C23024 f '- in vitro

Minimum inhibitory concentration _ Organism (thio / ml) _ 5 Staphylococcus aureus Smith 64 "" SSC 80-11 64 "" SSC 80-32 64 "" SSC 80-38 64 "" LL-14 64 10 "" LL-45 64 " "LL-27 128" "ATCC 25923 64

Streptococcus pyogenes C203 1 "/ ^ - hemolytic. _ Keller T623 8 15" pneumoniae 78-1 8

Enterococcus SSC-80-62 64 "SSC-80-63 128 20

Table IV

LL-C23024 iootan antibacterial potency in vitro LL-C23024 iootan

Minimum inhibitory concentration of the organism 25___centration (μg / ml)

Enterococcus OSU 75-1 1

Enterococcus SM 77-15 1

Staphylocccus aureus SSC 79-18 1

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

Staphylococcus aureus Smith 0.5

Staphylococcus aureus ATCC 25923 0.5

II

9 75187

As can be seen from the above evaluations, the antibiotics LL-C23024 ^ io inhibit the growth of certain Gram-positive bacteria and are thus useful as topical antiseptics or surface-5 antiseptics in solutions for washing the skin, utensils, fungi, floors, etc.

It has also been shown that the antibiotics LL-C23024 r1, Jb and ioota are effective in vivo as drugs against poultry coccidiosis, as demonstrated by the following 10 tests.

Two days before inoculation, different groups of day-old chicks were fed a diet supplemented with varying amounts of drugs. The poultry feed used was prepared as follows: 15 vitamin-amino acid ready mixes 0.5% trace minerals 0.1% sodium chloride 0.3% dicalcium phosphate 1.2% ground limestone 0.5% 20 stabilized fat 4.0% dehydrated alpha-alpha, 17% protein 2 .0 »% corn gluten meal, 41% protein 5.0% menhaden meal, 60% protein 5.0% soybean meal, 44% protein 30.0% 25 ground yellow corn ad 100%

The vitamin-amino acid preparation of the feed mixture described above was prepared according to the following composition. The amounts are calculated per kilogram of ready-to-eat compound feed, butylated hydroxytoluene 125.0 mg 30 dl-methionine 500.0 mg vitamin A 3300.0 I.U.

Vitamin D 1 1100.0 I.C.U.

riboflavin 4.4 mg vitamin E 2.2 I.U.

35 niacin 27.5 mg 10 751 87 pantothenic acid 8.8 mg choline chloride 500.0 mg folic acid 1.43 mg menadione sodium bisulphate 1.1 mg vitamin 5 B ^ 2 ”vitamin 11.0 μg ground yellow maize ad 5.0 g

The test chickens were then inoculated by inoculating directly into each chick's dome an inoculum mixture of 5,000 Eimeria acervulina spore-forming oocysts and a sufficient number of Eimeria tenella oocysts to cause 85-100% mortality in untreated controls. Throughout the test period, the chicks were free to ingest feed and water. After seven days, testing was stopped and the birds were weighed, killed, and examined. The gastrointestinal tract was examined for signs of damage. The results are shown in Tables V and VI below and show that the number of infected surviving chickens increased when the antibiotic LL-C23024 / * or iota was 10 ppm or less in the diet.

20 The results also show that gastrointestinal damage caused by Eimeria tenella and Eimeria acervulina was significantly reduced.

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The following tests show efficacy as an anthelmintic.

Example A

Efficacy Evaluation of Test Compositions as an Anthelmintic 5 Using a Free-Live, Bisexual, Microbial Cancer Gaenorhabditis elegans II

In the tests below, the C. elegans nematode was used to determine the drug efficacy in the fermentation broth and bacterial mass prepared according to the invention.

This organism was used to determine the nematode efficacy of the antibiotics LL-C23024Ö.

In these tests, the fermentation broth forms a fermentation broth in which various solids dissolve before the bacterial mass in solid form is separated from the broth. A solid bacterial mass remains after separation.

To evaluate the bacterial mass, the solid therein was dispersed in distilled water in a ratio of 1: 1. The fermentation broth was used as such and contained both 20 liquids and solids.

In the reviews described below, C. elegans was maintained in C. briggsae maintenance medium. This medium is sold by Grant Island Biological Company, Grant Island,

New York and has the following composition: 25 "C. brjqgsae MAINTENANCE MEDIUM" (1)

Component

Inorganic salts mg / l

CaCl2. 2H 2 O 220.50 30 CuCl 2 * 2H 2 O 6.50

Fe (NIl4) 2 <SO4) 2. rm20 58.80 KH2PO4 1225.50 Κ0Η (a)

MnCl2 '. 41120 22.20 35 ZnCl2 10.20 14 751 8 7

Other components N-acetylglucosamine 15.00 adenosine-3 '- (2') - phosphoric acid. 1 ^ 0 365.00 cytidine-3 '- (2') - phosphoric acid 323.00 5 D-glucose 1315.00 glutathione, reduced 204.00 guanosine-3 '- (2') - PO 00 magnesium magnesium citrate.5H2O (dibasic) 915.00 potassium citrate. DL-thioctic acid 3.75 thyrine 126.00 uridine-31- (21) -phosphoric acid 324.00

Amino acids L-alanine 1395.00 15 L-arginine 975.00 L-aspartic acid 1620.00 L-cysteine. HCl / O 28.00 L-glutamate (Na) .1 ^ 0 550.00 L-glutamine 1463.00 20 glycine 722.00 L-histidine 282.00 L-isoleucine 861.00 L-leucine 1439.00 L- lysine.HC1 1183.00 25 L-methionine 389.00 L-phenylalanine 803.00 L-proline 653.00 L-serine 788.00 L-threonine 717.00 30 L-tryptophan 184.00 L-tyrosine 272.00 L-valine 1020.00

Vitamins P-aminobenzoic acid 7.50 35 biotin 3.75 choline dihydrogen citrate 88.50

II

is 75187 cyanocobalamin (Β. ^) 3.75 folinate (Ca) 3.75 myoinositol 64.50 niacin 7.50 5 niacinamide 7.50 pantethine 3.75 pantothenate (Ca) 7.50 pyridoxal phosphate 3.75 pyridoxane 2HCl 3.75 10 pyridoxine.HCl 7.50 ribofavin-5'-PO2 (Na). 21 ^ 0. 7.50 thiamine.HCl 7.50

References: (1) Hansen, E.L., Proc. Soc. Exp. Bio. & Med. 121: 30-393 15 (1966).

Note. (a) If necessary to adjust the pH to 5.9 + 0.1.

The reviews used a corrugated plate equipped with a series of small waves. 25 ml of fermentation broth, water-bacterial-20 pulp suspension in a ratio of 1: 1 or an aqueous acetone solution of LL-C23024 <A or LL-C23024 / 5 with 31.25-500 ppm of the test compound were aseptically added to the different wave conditions. Then, 25 ml of C. elegans maintenance medium with 10 to 20 adult worms plus larvae of different ages plus 25 eggs was added to each wave space. The plates were then placed under cover and examined 48 hours after inoculation for the rate and efficacy of the test compositions as an nematode drug. The values obtained are shown below. If potency was observed in the fermentation broth, this was further diluted to achieve a 1: 4 ratio of broth to C. ele-30 ganc.

16,751 87

Table VII

Composition Concentration (ppm) Anthelmintic or dilution ratio ho at 48 hours

Fermentation- D = 1: 1 8 (no motility) lie, nl o = 1: 4

Bacterial mass LL-23024-V 500 ppm 7 250 ppm 7 10 125 ppm 7 62.5 ppm 6 31.25 ppm 0 LL-23024 ^ 500 ppm 7 _250 ppm_0_ 15 The following rating scale was used in these reviews:

Power scale 8 = no motility (worms appear dead) 7 = markedly reduced motility 2 0

6 = decreased motility 0 = species-specific motility Example B

Review of LL-C23024oC as an effective nematode drug against ineffective larvae of ruminant nematodes-25 (third obscure larval stage).

Evaluation of LL-C230241 as an ineffective larval effective against nematode Haemonhmus contortus, Ostertagia circumcincta, Trichostrongylus axei, T. colubriformis and Turbatrix 30 aceti in ruminant nematodes was performed using the above-mentioned nematode species C. elegans.

The results obtained are shown in the table below.

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Example C

Evaluation of antibiotic LL-C2302400 as an effective nematode against nematospiroides budiuks and Aspicularis tetraptera in mice ^ In the following experiments, white female Swiss-Webster mice were infected with Nematospiroides dubius and Aspicularis tetrapter and kept infected for three weeks.

After this time, the mice were divided into groups of four animals and the groups were placed in different cages. The mice were then fed a diet of about 31.25 to 4000 ppm of the test compound as a drug during the week. Throughout the test period, the mice also received plenty of water. During the treatment period, the faeces of the mice were examined for possible worms. It turned out that the worms passed through the animals of all treatment groups. This thus indicates that the substances at all concentrations were effective as anthelmintics for both species of nematodes. After a week of treatment with drugs within the 2Q interval, the mice were sacrificed and worms were searched for gastrointestinal contents. The results are shown below as a percentage reduction in worms compared to infected and untreated controls.

These tests evaluated the fermentation broth obtained before bacterial pulp recovery and containing 1000-4000 ppm of nematode antibiotic. Similarly, mice fed with 31.25-500 ppm antibiotic LL-C23024OC dissolved in acetone-water (1: 1) were evaluated.

II

19 751 87

Test composition Concentration Percentage reduction in potency in food *** ppm N. dubius A. tetraptera * LL-C23024 <+ 'contains 4 000 7 3 5. fermented 2000 44 53 broth 1 000 65 33 500 70 SA **

250 63 SA

125 61 SA

10 ^.

62.5 29 SA

_______ 31,25_32_RA

* = Amount of LL-C23024 specified ton ** = somewhat effective: increased ^ 5 number of nematodes was found in faeces *** = compared to infected, untreated controls.

The above-mentioned antibacterial and anticoccidial drugs are formed by culturing the microorganism Actinomadura yumaense sp.nov under controlled conditions.

This representative strain of the microorganism was isolated from a soil sample found in Yuma County, Arizona and maintained in the culture collection of Lederle Laboratories Division, American Cyanamid Company, Pearl River, New York, under culture number LL-C23024. A viable culture of this representative strain has been deposited with the Culture Collection Laboratory, Northern Regional Center, U.S. Pat. Department of Agriculture, Peoria, Illinois 61604 and has been included in the permanent collection of this laboratory under accession number NRRL 12515.

30 Taxonomic characterization of NRRL 1215

Strain NRRL 12515 has been taxonomically characterized and identified in a new species of Actinomadura, Actinomadura yumaense sp. Nov., as a type stock.

The culture, physiological and morphological characteristics of the representative strain NRRL 12515 have been observed by the methods described by E. B. Shirling and D. Gottlieb, 20 751 87 "Methods for the characterization of Streptomyces species", Internat. J. Syst. Bacteriol. 16: 313-340 (1966), and R.E. Gordon et al., "Nocardia coeliaca, Nocardia autotrophica, and the nocardine strain," Internat. J. Syst. Bacteriol 5 24: 54-63 (1974). The media used in this study were those recommended by T.G. Pridham et al., "A selection of media for maintenance and taxonomic study of Streptomycetes", Antibiotics Ann., Pp. 947-953 (1956/1957), G.F. Gauze et al., "Problems in the classification of antagonistic actinomycetes", State Publishing House for Medical Literature, Medgiz, Moscow (1957) and R.E.

Gordon et al., Supra, for taxonomic studies of Actinomycetes species and terrestrial bacteria. The chemical composition of the cell wall of microorganisms was determined by the method described in H.A. Lechevalier et al., "Chemical Composition as a Criterion in the Classification of Actinomycetes", Adv. Appl. Microbiol. 14: 47-72 (1971). Phospholipid structures were determined by the method described in M.P. Lechevalier et al., "Chemotaxo-20 nomy of aerobic actinomycetes; phospholipid composition",

Biochem. Syst.Ecol. 5: 249-260 (1977). Details are shown in Tables IV-IX and an overview of the culture is given below. The underlined descriptive colors are taken from K.L. Kelly and D.B. Judd, "Color, Universal Language and 25 Dictionary of Names," U.S. Pat. Nat. Bur. Stand., Spec. Pub.

440, Washington D.C. (1976) and the related International Society Color Council, Natl. Bur. Stand. Centroid Color Charts.

The values observed in this new species, represented by strain NRRL 12515, were compared with those published for 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 35 of antibiotic CP-47,433 and CP-47,434", Internat J. Syst.

Bacteriol. 30: 565-568 (1980), J. Myer, “New species of the

II

2i 75187 genus Actinomadura ", Z. Allgem, Mikrobiol. 19: 37-44 (1979), H. Nomura and Y. Ohara," Distribution of actinomycetes in soil. XI Some new species of the genus Actinomadura, Lechevalier et al. "J. Ferment. Technol. 49: 904-912 (1971) 5 and TP Preobrazhenskaya et al.," Key for identification of the species of the genus Actinomadura ", The Biolody of Actinomycetes and Related organism 12: 30-38 (1977 The culture NRRL 12515 is somewhat similar to Actinomadura pelletieri, but does not resemble any of the other 10 species described and differs in many respects from A. pelletieri, so strain NRRL 12515 is considered to be a new species, Actinomadura yumaende sp. from the place where the type stock was isolated from the soil sample found.

15 Micromorphology

Spores form short helical chains (maximum length approximately 20 spores per chain) in branched, nearly circular spore carriers. The spores are ovoid, 0.6-0.8 microns times 1.0-1.4 microns and the surface is smooth.

Cell wall composition The whole cell hydrolyzate of this culture contains modulose (3-O-methyl-D-galactose) and diaminopimelic acid (DAP) mesoisomers. The culture also has a type 25 P-1 phospholipid structure pattern and no diagnostic phospholipids other than some phosphatidylglycerol. All these characteristics are very typical of the genus Actinomadura.

Growth rate 30 Growth was found to be good on Bennett agar, Gauze No. On 2 agar, NZ-amine-starch-glucose agar (ATCC Medium 172), tomato paste-oatmeal agar and yeast extract-malt extract agar, moderate growth was observed on Benedict agar. Czapek sucrose agar-35, glycerol-asparagine agar, Hickey's and Tresner's agar, and oat agar, and growth was observed to be weak on all 22 751 87 sump malate agar, Gauze No. agar and starch agar containing inorganic salts.

Vegetative mycelium

In media with good growth, vegetative mycelium was found to increase and convolve. It was usually yellowish gray in color.

Air mycelium and spore color

Air mycelium and / or spore masses ranged in color from white to light gray 264. Air mycelium growth 10 was readily performed on most media.

Soluble pigments

Many media without pigment, yellow pigment with Benedict's glycerol-asparagine agar, yellow-green pigment with calcium malate agar, greenish-15 brown pigment with NZ-amine starch glucose agar, and orange pigment with Bennett's and yeast extract.

Physiological reactions

No melanin pigment on peptone-iron agar or 20 tyrosine agar (ISO-7), litmus milk causes strong peptonization, medium gelatin causes strong proteolysis, nitrate is moderately reduced, adenine and guanine are not hydrolyzed, hypoxanthine, tyrosine and xanthine hydrolyzate , esculin is hydrolyzed and urea hydrolysis varies. No growth at 4 ° C, 10 ° C and 55 ° C, moderate growth at 25 ° C and 45 ° C and good growth at 32 ° C and 37 ° C. Utilization of Carbohydrates T.G. Pridham and D. Gottlieb, 30 "The ulitization of carbon compounds by some actinomycetes as an aid for species determination", J. Bacteriol. 56: 107-114 (1948); glucose, glycerol and trehalose are used well, maltose and sucrose are used moderately, fructose, ga-35 lactose, inositol, mannose and melecitose are used poorly; and adonitol, arabinose, dulsitol, lactose, mannitol, melibiose, raffinose, rhamnose

II

23 751 8 7 salicin, sorbitol and xylose are not used at all. Production of acids from carbohydrates Gordon et al. according to the method described above: acids are well formed from glucose, glycerol, maltose, sucrose and trehalose and acids are weakly formed from galactose, inositol and mannose. Utilization of Organic Acids Gordon et al. according to the method described above: acetate, malate, propionate, pyruvate, succinate and tartrate are utilized, benzoate, citrate, lactate, mucate and oxalate are not utilized.

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Table X

The ability of Actinomadura yumaense NRRL 12515 to utilize a carbon source in a carbohydrate recovery vessel-5 with ISP-9

Incubation time: 28 days Temperature: 28 ° C

Carbon source_approval adonitol 1-arabinose 10 dulcitol fructose bad d-galactose bad d-glucose good glycerol good 15 i-inositol bad lactose maltose moderate d-mannitol d-mannose bad 20 d-melecitose bad d-melibinose d-raibiosis d-raibinose sorbitol sucrose moderate d-trehalose good d-xylose negative control 30 75187

Table XI

Ability of Actinomadura yumaense NRRL 12515 to produce acids from various carbohydrates on Gordon's basic inorganic nitrogen medium

5 Incubation time: 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-melecitose - - d-melibiose - - d-raffinose - - 1-rhamnose sorbitol 25 sucrose - +++ d-trehalose - +++ d-xylose negative control * 30 + + + = strongly positive response ++ = moderately positive response + = slightly positive response - = negative response Tue 31 75187

Table XII

Ability of Actinomadura yumaense NRRL 12515 to utilize organic acids by Gordon's transformation from Koser's basic agar (Koser's citrate agar)

5 Incubation time: 28 days Temperature: 28 ° C

Carbon source Utilization * acetate + benzoate - citrate lactate malate + mucic acid oxalate + pyruvate + 15 succinate + tartrate + * + = positive response - = negative response 20

It is clear that the expression Actinomadura yumaense is not limited to the strain Actinomadura yumaense NRRL 12515 or to strains which fully correspond to the microscopic characterization of the growths described above, which is given by way of illustration only. The Actinomadura yumaense described herein comprises all strains of Actinomadura yumaense capable of forming the antinotes LL-C23024oi, β and io and which are absolutely indistinguishable from Actinomadura yumaense NRRL 12515 and its subcultures.

Actinomadura yumaense can be cultured using a wide variety of solid or liquid culture media. Substrates useful in the production of antibiotics LL-C23024.Αφ and ioota include 35 assimilable nitrogen sources such as protein, protein hydrolyzate, polypeptides, amino acids, corn steep liquors, etc., and inorganic anions and potassium, ammonium, sodium, potassium, sodium, carbonate, phosphate, chloride, etc. Trace elements such as boron, molybdenum, copper, etc. are added to the medium in the form of impurities of other ingredients. The tanks and flasks are aerated by supplying sterile air through or onto the fermentation medium. In addition, the tank is agitated with a mechanical stirrer.

10 If necessary, a defoamer such as lard oil or silicone type oil can be added.

Actinomadura yumaense is cultured and maintained on oblique agar, e.g., Bennett's agar on yeast malt agar or ATCC Medium No. 172.ATCC Medium No. 172 is recommended. The oblique culture is inoculated

Actinomadura yumaensen culture and incubated at 28-37 ° C, preferably at about 32 ° C for about seven days. These stock cultures can be maintained by serial inoculation into fresh stock slant cultures, or a mycelium-containing agar plug from well-grown slant agar can also be used to inoculate liquid media.

Actinomadura yumaensen shake flask inocula were prepared by inoculating 100 ml aliquots of sterile liquid medium in 500 ml flasks with scraped or washed spores from an oblique agar culture. Examples of suitable seed media are 11 33 7 5 1 87

Medium A

meat extract 0.3% R 1

Bacto tryptone 0.5% glucose 1.0% 5 yeast extract 0.5% water q.s. 100% peptone, registered trademark Difco Laboratories, Detroit, Michigan The pH was adjusted to 6.8-7.2 with a dilute base solution, e.g., sodium hydroxide solution.

Medium B

glucose 1% starch 2% yeast extract 0.5% 1 -5 R 2 N-Z-amino A 0.5% calcium carbonate 0.1% water q.s. 100% 2 casein extract, registered trademark Sheffield Chemi-20 cal Co., Div. Nat'l. Dairy Products Corp. Norwich, N.Y.

Medium B is recommended.

The flasks were incubated at 25-35 ° C, preferably 32 ° C and mixed vigorously on a rotary shaker for 1-4 days. This inoculum was then used to inoculate the fermentation culture, or alternatively this culture can be frozen and stored for use as an inoculum for subsequent inoculations.

The following are examples of suitable fermentation media for Actinomadura yumaensen for the preparation of antibiotics LL-C23023 ^ (P and 30).

Medium C

glucose 1.5% glycerol 1.5%, 3 soi3a3auho 1.5% 35 calcium carbonate 0.1% sodium chloride 0.3% water q.s. 100% 34 3 75187 can be replaced by cottonseed meal or soluble meat ingredients with equally good results.

Medium D

glucose 3% 5 soy flour 1.5% calcium carbonate 0.1% sodium chloride 0.3% water q.s. 100%

Medium E

10 starches 1% syrup 2% soy flour 1.5% calcium carbonate 0.1% water q.s. 100%

15 Medium F

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

Medium G

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

Medium H

glucose 3% ammonium sulphate 0.3% dibasic potassium phosphate 0.1% calcium carbonate 0.2% sodium chloride 0.1% water q.s. 100%

Medium D is recommended.

11 35 7 5 1 87

The fermentation can be performed in a 100 ml flask of a 500 ml medium, inoculated with 3-10% w / v of an inoculum prepared as described above, and incubated at 25-35 ° C, preferably about 32 ° C: at 5 to 24-72 hours with aeration. Fermentation culture samples can be frozen and stored for later use as an inoculum for inoculum cultures.

Alternatively, the fermentation can be performed in a larger fermentation tank equipped with il-10 masting and mixing equipment. Each tank is inoculated with 3-10% w / v of the inoculum prepared as described above. Aeration is performed at a rate of 0.5 to 2.0 liters of sterile air per liter of culture medium per minute and the medium is mixed with a stirrer at a rotation speed of 200 to 400 rpm. The temperature is maintained at 25-35 °, preferably 32 ° C. The fermentation is continued until the antibiotic is enriched in the fermentation medium, i.e. usually 100-150 hours, at which time the antibiotic is recovered.

The antibiotic can be recovered and purified by the methods described in U.S. Patent No. 4,273,663 or the following method.

The crude fermentation broth containing whole cells prepared as described above and an equal volume of a water-immiscible, non-hydrocarbon type organic solvent are mixed. Methylene chloride and ethyl acetate are preferred. The organic phase is separated and concentrated in vacuo to an oily syrup.

the oily syrup is dissolved in methylene chloride and the solution is applied to a column packed with silica gel,

R

alumina, Sephadex LH-20 (Pharmacia Fine Chemicals, a subsidiary of Pharmacia, Inc. Piscataway, N.J.) or magnesium aluminosilicate. Examples of suitable solvents for developing the column include diethyl ether, ethyl acetate, a mixture of methylene chloride and ethyl ether in a volume / volume ratio of 1: 1 to 1: 7, 10-40% acetone in water, Ä 75187

Already in ethylene chloride, 2-10% lower alcohol (methanol is recommended) in methylene chloride, 2-15% acetonitrile in methylene chloride or 2-15% dioxane in methylene chloride. Methylene chloride-ethyl acetate 5 v / v 1: 1 is preferred. The fractions are collected and tested for antibacterial activity by bioanalysis against the susceptible organism, e.g. Bacillus subtilis. The active fractions are combined and evaporated in vacuo to a residue which is dissolved in an organic solvent, e.g. t-butanol (preferred), benzene or p-dioxane and lyophilized.

The lyophilized solid is dissolved in a suitable organic solvent, e.g. methylene chloride, hexane, methylene chloride-ethyl acetate, diethyl ether, hexane-ethyl acetate, hexane-chloroform or hexane-ether. Diethyl ether is recommended. Shake this solution and water and adjust the pH to 1.5-4.0, preferably about 2.5 with slightly dilute mineral acid. The organic phase is separated, washed with water to remove any 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 at about 4 ° C. Examples of suitable solvents include methylene chloride, hexane, methylene chloride-ethyl acetate, diethyl ether, hexane-ethyl acetate, hexane-chloroform and hexane-ether. Hexane-ether in a volume / volume ratio of 5: 2 is recommended. The resulting crystals are isolated and washed, preferably at about 40 ° C, with a moderately high boiling hydrocarbon, e.g. hexane or heptane, and air-dried to give the final antibiotic X-14868A in the form of the free acid.

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

The following examples illustrate the invention. Media A, B, C, etc. are as above. Unless otherwise indicated, all methods are performed at room temperature (about 22 ° C) 5 and 1 atm.

Example 1

Washed spores recovered from Actinomadura yumaense NRRL 12515 vino agar were inoculated into a 500 ml flask containing 100 ml of sterile medium B. Flask 10 was incubated on a rotary shaker for two days at 28 ° C.

This culture was then inoculated with 5% inoculum in 100 ml of sterile medium C in a 500 ml flask and incubated for five days at 28 ° C on a rotating 15 'shaker.

The antibiotic efficacy was examined daily by bioanalysis against Staphylococcus aureus ATCC 6538 P, Bacillus subtilis and Streptococcus faecal is, and the efficacy as an anthelmintic was monitored by bioassay for free live nematode Caenorhabbitis elegans.

Example 2

Seven 500 ml flasks were prepared, each containing 100 ml of the following sterile media: Flask 1: 100 ml of medium C

25 Flask 2: 100 ml of medium C with 1.5% cottonseed meal instead of soy flour Flask 3: 100 ml of medium C with 1.5% soluble meat instead of soy flour Flask 4: 100 ml of medium D 30 Flask 5: 100 ml of medium E Flask 6: 100 ml of medium F Flask 7: 100 ml of medium G

Each of these flasks was inoculated with 5% inoculum of Actinomadura yumaens NRRL 12515 cultured in medium B. The flasks were then incubated on a rotary shaker for 4-6 days at 28 ° C.

38 751 87

Each of the cultures described above was found to be active when tested for their abbiotic efficacy as described in Example 1 and for their anti-coccidiosis activity in tissue cultures prepared from chicken kidney.

Example 3

A 500 ml flask containing 100 ml of sterile medium B was inoculated with frozen fermentation culture cells of Actinomadura yumaense NRRL 12515. Flask 10 was then incubated for four days at 32 ° C on a rotary shaker.

A 5% inoculum from this culture was inoculated into 100 ml of sterile medium H in a 500 ml flask and incubated for five days at 28 ° C on a rotary shaker.

The antibacterial potency was found as in Example 1 and the anticoccidial potency as in Example 2.

The antibiotic potency was also analyzed by thin layer chromatography on silica gel plates developed in 20 ethyl acetate-chloroform (volume to volume ratio 70:30).

Example 4 100 ml of sterile medium A in a 500 ml flask was inoculated with washed spores recovered from vinoaga-25 of Actinomadura yumaense NRRL 12515. The flask was incubated for two days at 32 ° C on a rotary shaker.

A 5% inoculum from this culture was then inoculated into 100 ml of sterile 50 medium H in a 500 ml flask and incubated for two days at 32 ° C on a rotary shaker.

A 5% inoculum from this culture was then inoculated into 100 ml of fresh sterile medium H in a 500 ml flask and incubated for six days at 28 ° C on a rotary shaker.

The antibacterial potency was found as in Example 1.

li 39 751 8 7

Example 5

Two 500 ml flasks, each containing 100 ml of sterile medium A, were inoculated with a frozen inoculum of Actinomadura yumaense NRRL 12515 and incubated for two days at 32 ° C in a rotating medium.

The contents of both flasks were then combined and added to 12 liters of fresh sterile medium A in a 20 liter flask. This culture was then incubated for two 10 days at 28 ° C with aeration.

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

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

The fermentation broth was analyzed for antibiotic activity by thin layer chromatography on silica gel plates developed in ethyl acetate-chloroform (70:30 v / v). Alternatively, several of these developed plates were bioassayed for Bacillus subtilis and found to have antibiotic activity.

Example 6

A typical medium used for culturing the primary inoculum was prepared according to the following recipe: 30 meat extracts 0.3%

(S

Bacto tryptone 0.5% glucose 1.0% yeast extract 0.5%

Bacto agar 0.15% 35 water q.s. 100% 40 7 5 1 87

Washed or mycelium recovered from Actinomadura yumaense NRRL 12515 vino agar in a 500 ml flask was inoculated with 100 ml of the above sterilized medium. The flask was placed on a rotary shaker and stirred vigorously for 48 hours at 32 ° C.

The obtained flask inoculum (100 ml) was inoculated with 1 liter of the same sterile medium in a 2 liter flask. This inoculum was aerated with sterile air while culturing for 48 hours at 28 ° C. This single 10 liter culture was then inoculated with a 30 liter fermentation tank containing the same sterile medium, and this tank was aerated with sterile air and incubated for 42 hours at 28 ° C.

Example 7 A fermentation broth was prepared according to the following recipe: dextrose 1.5% glycerol 1.5% soy flour 1.5% calcium carbonate 0.1% sodium chloride 0.3% water q.s. The 100% pH was adjusted to 7.0 with 6 N sodium hydroxide solution and the medium was sterilized. A thirty liter 25 inoculum aliquot prepared as described in Example 1 was inoculated with 250 liters of the above medium in a 300 liter fermentor. Sterile air was blown onto the surface and the pulp was mixed with an impeller mixer at 220 rpm. Fermentation 30 lasted 97 hours at 32 ° C, after which the pulp was recovered.

Example 8

A fermentation broth was prepared according to the following recipe:

II

35 41 75187 dextrose 3.0% soy flour 1.5% calcium carbonate 0.1% sodium chloride 0.3% water q.s. The 100% pH was adjusted to 7.0 with 6 N sodium hydroxide solution and the medium was sterilized. With 300 liters of inoculum prepared as described in Example 1, 300 liters of the above-mentioned medium were inoculated in 10 fermentors. Sterile air was blown onto the surface.

The mass was mixed with an impeller mixer at a rotation speed of 100 rpm. The fermentation lasted 115 hours at 32 ° C, after which the pulp was recovered.

Example 9 1. The pH of the fermentation broth (100 liters) was adjusted to 4.0 using 6 N HCl. The acidified mass and an equal volume of methylene chloride were then stirred for 1-2 hours. The mixture of pulp and methylene chloride was then filtered using diatomaceous earth as a filter aid. The methylene-20 chloride extract was isolated and concentrated in vacuo to about two liters of purified antibiotics.

2. Chromatography of partially purified antibiotics on silica gel.

A glass column with a diameter of 7 cm was packed to a height of 91 cm with a Woelm Silica Gel TSC. Impure concentrate (see point 1 above) with a volume of approx.

2 liters, was allowed to seep onto a silica gel column. The column was then developed first with three liters of methylene chloride and then with methylene chloride-ethyl acetate (1: 1 by volume) to give a total of 126 fractions, each with a volume of 80 ml. This was further developed with ethyl acetate-ethanol (7: 3) to give a further 87 fractions.

Fractions 58-87 rich in antibiotic C23024 were combined and concentrated in vacuo to a residue of 42,751,87, which weighed 90.4 g. This residue was dissolved in a mixture of 600 ml of diethyl ether and 600 ml of hexane. The resulting suspension was filtered to give a clear filtrate. This was concentrated in vacuo until crystals began to form. This suspension was allowed to stand overnight. The crystalline C23024 was collected in a funnel, washed with cold ether, air dried to give 33.1 g of crystalline C23024. In addition, 12.4 g of C23024ck were obtained by further concentrating the combined filtrate and washings.

Fractions 177-203 eluted with ethyl acetate-ethanol enriched in C23024 / 3 were combined and concentrated in vacuo to give 6.7 g of partially pure C23024 / 3 and continued as above.

Fractions 204-213 obtained by eluting with ethyl acetate-ethanol and enriched in L-C23024 were combined and concentrated in vacuo to a paste which was extracted several times with methanol. The methanol extract was extracted with methylene chloride, and this extract was poured onto a column containing Woelm's silica gel. The column was washed with methylene chloride and then eluted with methylene chloride-ethyl acetate (2: 3). Fractionation was controlled by thin layer chromatography and the active fractions were combined, treated with charcoal, concentrated and extracted several times with heptane. The final heptane extract was kept at 0 ° C for 48 hours and the crystals were isolated from the mother liquor by filtration.

This mother liquor was dissolved in methylene chloride and the resulting solution was allowed to seep onto a glass column packed with Woelm silica gel. The column was then eluted successively with 2 liters of methylene, 8 liters of methylene chloride-ethyl acetate (1: 1) and 4 liters of ethyl acetate-methanol (9: 1). The eluate was collected as fractions, the antibiotic composition of which was examined. The active fractions were combined and concentrated in vacuo to give 35 residues containing LL-C23024.

Il 43 751 87

Example 10 3. Further purification of C23024P purified on silica gel

SephadexRLH20 was allowed to swell in a 10: 5: 1 mixture of hexane, methylene chloride and methanol. A glass column with a diameter of -5 and 5 cm was filled with gel to a height of 86 cm. A batch of 3 g of purified LL-C23024P was dissolved in a mixture of 10 ml of methylene chloride, 1 ml of methanol and 10 ml of hexane, and the solution was allowed to seep onto a gel column.

Column 3 was then developed with a solvent mixture of the same composition as when the antibiotic was added to the column.

Fractions were collected on a fraction collector. The volume of each fraction in the twenty-three first fraction was 17 ml. According to thin layer chromatography, fractions 17-26 contained C23024p. These fractions were combined and concentrated in vacuo to give 1269 mg of pure, amorphous LL-C23024 / 3.

Example 11 Crystallization of LL-C23023β as the Sodium Salt 2.4 g of purified LL-C23024 P prepared as described in B and C above were dissolved in a mixture of 500 ml of diethyl ether, 160 ml of hexane and 25 ml of ml of methylene chloride. The resulting solution was transferred to a separatory funnel with 300 mL of distilled water. Dilute HCl (1-n) was added dropwise until the pH became 2.0-2.5 after shaking and settling. The second phase of acidic water was discarded and the acid-treated organic layer was washed three times in succession with 400 ml portions of water. The washed organic layer and a teaspoon of Darco G-60 powder were mixed for 15 minutes and filtered through celite.

The decolorized organic layer was poured onto 500 ml of distilled water and 5 N sodium hydroxide solution was added dropwise until the pH became 11.0-11.5 after shaking and settling. The alkaline aqueous phase was discarded and the remaining organic layer was washed twice with 400 ml portions of water. The washed organic layer was dried over sodium sulfate and then concentrated in vacuo to a volume of 150 mL. This concentrate was allowed to stand protected for several hours. The crystalline substance LL-C23024 / 3 was collected in a funnel, washed with cold benzene, air-dried to give 402 mg of the crystalline salt of LL-C23024p. Microanalyses were performed on this sample and selected spectral values were determined.

Analysis: C ^ H ^ O ^ Na

Calculated: C 59.70 H 8.33 0 29.46 ash 2.49 10 Found: C 61.55 H 8.79 ash 3.31 N 0

Melting point (Fisher-John apparatus): 174 ° C FDMS = 924, - + 3.2 ° (in methanol)

Example 12 Purification of LL-C23024 Iota High Performance Liquid Chromatography Apparatus (Waters Prep.

500A) was equipped with a silica gel cartridge at a pressure of 38.5 kp / cm. A batch of 5.0 g of the crude material obtained in Example 3 was dissolved in 50 ml of heptane-ethyl acetate (45.55) and sprayed onto a silica gel column. The 20 columns were then developed with the same solvent mixture at a flow rate of 200 ml / min and 40 fractions were collected, each with a volume of 200 ml. Fractions 21-32 were combined, concentrated to a residue and the residue triturated in hexane and evaporated to dryness to give LL-C23024 25 ions pure. The procedure described above was repeated four times to give a total of 280 mg of amorphous LL-C23024.

A 90 mg portion of the amorphous LL-C23024 iota was dissolved in 10 ml of water and the pH was adjusted to 2.5 with 0.1 N hydrochloric acid. The ether layer was washed with water, the pH was adjusted to about 11 with 0.1 N sodium hydroxide solution, 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 slowly evaporate to give a white solid.

li 45 75187 The properties of this white solid were: Elemental analysis: C 61.79 H 8.84 ash 0.32 = + 27 ° (c = 0.724 in methanol)

Field desorption mass spectrometry: (M + Na) + = m / e 5,953, so the calculated molecular weight is 930.

Claims (7)

New method for the known antibiotics LL-C23024-alpha (o6) (= X-14868A), LL-C23024beta (/ *) (= X-14868C) and LL- 5 C23024 to prepare (L) (= X-14868B), OMe Me O- Me U OMe OH '= * · 0 Me * ^ OMe O j, je O-IyJo 0H H Me11 Me H Me H H H COOH 15 X-14868 A = LL-C23024alpha (oi) OMe 20 OH OH 1 Me 1 OMe f O Me Me 25. OH ί i * Me H Me H H H OH COOH Me X-14868C = LL-C2 302 4beta (β) OMe 3. A5Me iin> Me (¾ Hi H Me H Me HHH OMe CO 2 H Me X-14868B = LL-C23024ioota (£) II 47 7 5 1 87 characterized by aerobic fermentation of microorganisms Actinomadura yumaense NRRL 12515 in a liquid medium which contains assimilable sources of carbon and nitrogen, as well as inorganic salts, by maintaining the fermentation culture at 25-35 ° C for 24-150 hours, after which the antibiotic is isolated in a manner known per se. ) (= X-14868A), LL-C23024beta-5 (/ 3) (= X-14868C) and LL-C23024io (I) (= X-14868B), OMe Me ° 0, OMe 0H 10 VV "^ \« e. ... Me I OH H kh Me H He HJJ ° nJ COOH Me HH OH 15 X-14868 A = LL-C23024alpha (tC) OMe -VV 20 \ / 0 OH OH 1 Me Ξ * OMe O Me. ΧτΏτντΰγΤΤΓ / rotyTo ^ o ^ ^ • o / = £ 'o't JvoT e (OH, iu Me H Me HHH qh ^ J 1 Me COOH' X-14868C = LL-C23024beta (/?) OMe / ^ V ^ OMe? Me? Ο ™ »® 35. OH H li H Me H Me H H H OMe C02H Me X-14868B = LL-C23024ioota (L) II