JPH05219980A - Antibiotic ll-c23024 beta and iota - Google Patents

Abstract

PURPOSE: To efficiently produce antibiotics LL-C23024 α, β and iota useful as an antimicrobial agent, an anticoccidial agent, etc., by carrying out fermentation with a new microbe, Actinomadura yumaense, aerobically in a liquid medium.

CONSTITUTION: A new microbe, Actinomadura yumaense sp. nov. (e.g. Actinomadura yumaense NRRL125l5) is subjected to aerobic fermentation in a liquid medium containing assimilable sources of carbon, nitrogen and inorganic salts until the medium is imparted with antibiotic activity, and subsequently antibiotics LL-C23024 α, β and iota are recovered from the medium. It is preferable that the fermentation is carried out by culturing a seed culture product of Actinomadura yumaense at 32°C under aeration for 24-72 hr.

COPYRIGHT: (C)1993,JPO

Description

Detailed Description of the Invention

The present invention relates to an antibacterial and anticoccidial agent called LL-C23024α, β and iota, and a novel microorganism Actinoma zurayumaense new species (Acti) for the same.
nomadura yumaense sp. nov.) or its mutants.

The present invention further relates to a novel process for the production of the antibiotics LL-C23024α, β and iota by fermentation under the controlled conditions of a novel microbial species Actinomadura umaensu.

The antibiotic LL-C23204α has the following structure:

[0004]

[Chemical 1]

The antibiotic LL-C23024β of the present invention
Is its microanalysis value, optical rotation, infrared spectrum, ¹³ C
Not limited to nuclear magnetic resonance spectra and ¹ H nuclear magnetic resonance spectra, but by virtue of their physical and chemical properties, they can be distinguished from known compounds.

The antibiotic LL-C23024β has the following putative structure:

[0007]

[Chemical 2]

In the above formula, R ₁ and R ₂ are different and are selected from the group consisting of H and CH _3, respectively.

The above deduced structure is described in detail in the examples and shown in the drawings. Elemental analysis, optical rotation, field desorption mass spectrometry, molecular weight, melting point, infrared (IR) spectrum, ¹³ C nuclear magnetic resonance. ( ¹³ C-NMR) spectrum and proton magnetic resonance ( ¹ H-NMR) spectrum.

[0010]

[Table 1] LL-C23024 iota is a novel polyether antibiotic with extremely potent anticoccidial activity.
It is at least 10 times more potent than most other known polyethers. Field desorption mass spectrometry data indicates that it has a molecular weight of 930, which indicates its carbon 13 nuclear magnetic resonance ( ¹³ C-NMR).
Along with the spectrum comes the proposal of a tentative molecular formula C ₄₈ H ₈₂ O ₁₇ . LL-C23024 iota is believed to have 4 methoxy groups, the same sugar and / carboxyl groups found in the polyether antibiotic X-14868A [US Pat. No. 4,278,663]. 9
The only other known polyether antibiotic with a molecular weight of 30 is the antibiotic A28695B (hydroxyceptamycin) (J. Antibiotics 33 (2), 252 (19
80)], which is markedly different in structure and biological properties. The above observations are, as described in detail in Examples and shown in the drawings, elemental analysis, optical rotation, calculated molecular weight by field desorption mass spectrometry, infrared (IR) spectrum, ¹³ C-NMR spectrum and proton nuclear magnetism. It is based on resonance ( ¹ H-NMR) spectra.

¹³ C-NMR of LL-C23024 iota
Spectra were acquired in deuterated chloroform (CDCl ₃ ) with a magnetic field strength of 20 MHz. Table 2 shows the peaks having the respective chemical shifts expressed in ppm relative to tetramethylsilane (TMS) and the estimated carbon number for each peak. 75.4, 7 for chloroform
Peaks were observed at 7.0 and 78.6 ppm.

[0012]

[Table 2] Antibiotics LL-C23024α, β and iota are organic carboxylic acids and are therefore capable of forming non-toxic pharmaceutically acceptable cation salts. That is, the salts formed by mixing the antibiotic free acid with a stoichiometric amount of cations, preferably in a neutral solvent, include cations such as sodium, potassium, calcium, magnesium and ammonium, and It can be generated, for example, by an organic amine cation such as tri (lower alkyl) amine (eg triethylamine, triethanolamine), procaine and the like. The cationic salts of the antibiotic LL-C23024β are generally crystalline solids, relatively insoluble in water and most common conventional compounds such as methanol, ethyl acetate, acetone, chloroform, heptane, ethers and benzene. It is soluble in organic solvents. For purposes of this invention, antibiotic free acid is equivalent to its pharmaceutically acceptable non-toxic salts.

The antibiotics LL-23024β and iota are active in vitro in vitro against Gram-positive bacteria when tested by standard agar dilution methods. The results are shown in Tables 3 and 4 as the lowest inhibitory concentration in mcg / ml. Antibiotics LL-C23024β and iota are not active against Gram-negative bacteria at concentrations below 256 mcg / ml.

[0014]

[Table 3]

[0015]

[Table 4] As the above data shows, the antibiotic LL-C2302
4α, β and iota exhibit inhibition of growth of certain Gram-positive bacteria and are thus useful as topical or surface disinfectants in cleaning solutions for skin, appliances, walls, floors, etc.

The antibacterial agents LL-C23024α, β and iota have been found to be active in vivo as anticoccidial agents for poultry, as shown by the tests below.

Two days before inoculation, diets supplemented with various concentrations of the drug were given to several groups of 1-day-old test chicks. The feed used during the test is prepared as follows: vitamin-amino acid premix 0.5% trace minerals 0.1% sodium chloride 0.3% dicalcium phosphate 1.2% ground limestone 0.5% stabilized fat 4.0% dehydrated alfa alfa , Protein 17% 2.0% corn gluten powder, protein 41% 5.0% herring powder, protein 60% 5.0% soybean oil meal powder, protein 44% 30.0% ground yellow corn 100% plus vitamins in the above feed composition The amino acid premix is prepared according to the following formulation. The quantities given below relate to units per kg of final feed composition.

Butylated hydroxytoluene 12
5.0 mg dl-Methionine 500.0 mg Vitamin A 3,300.0 IU vitamin D ₃ 1,100.0 ICU Ribofurapin 4.4 mg Vitamin E 2.2 IU Naiamin 27.5 mg pantothenic acid 8.8 mg was Choline chloride 500.0 mg Folic acid 1.43 mg menadione sodium bisulfite 1.1 mg vitamin B ₁₂ 11.0 mcg grinding Yellow corn was added to 5.0 mg, and then each group of chicks was given a sufficient number of Eimeria tenella to give 85-100% mortality to Eimeria acervulina 5000 sporulated oocysts and untreated controls. (Eimeria te
nella) oocysts were directly inoculated with the mixed inoculum.
The chicks were allowed free access to feed and water throughout the entire test period. The study was discontinued 7 days after inoculation, the birds were weighed, necropsied and the intestine examined for lesions. The results are shown in Tables 5 and 6 below. These results show that the antibiotic LL-C230 at 10 ppm or less in the feed.
It has been shown that the incorporation of 24β or iota improves the survival rate of infected chicks. These results also indicate a significant inhibition of intestinal lesions by E. tenella and E. acervulina.

[0019]

Table 5 Table 5 chicks percentage of compounds ppm beginning% of birds with several viability decreased lesions concentrations birds evaluation in feed antibiotics LL-C23024β as coccidiostats E. tenella E. acervulina LL-C23024β 15 3 100 100 100 LL-C23024β 10 5 100 60 100 LL-C23024β 5 5 80 0 100 NNC * 0 15 46.6 0 0 ────────────────── ────────────────── LL-C23024β 120 15 100 100 100 60 15 100 100 100 30 15 100 100 100 0 15 20 0 0 NNC 15 100 ---- ── ────────────────────────────────── LL-C23024β 20 15 100 87 100 15 15 100 60 100 10 15 60 0 0 5 15 47 7 0 0 15 27 0 0 NNC 15 100 ---- ──────────────────────────────── ───── * NNC =, untreated, uninfected controls

[0020]

[Table 6]

[0021]

Examples Example A Free-living hermaphroditic nematode Kenorha b. Ditis elegan
Nematicidal activity using Caenorhabditis elegans II
Evaluation of Test Compositions for Sex In the following tests, Kenorhabdistis elegans is used to determine the nematicidal activity of the fermentation process prepared according to the method of the present invention and the introduced Matsushima. This microorganism is LL-C23024α and LL-C230
For the evaluation to examine the nematicidal activity of 24β,
use.

Fermentation pros in these tests means
It is a mixture of fermentation liquid and solid collected before separating the solid from the liquid, that is, the introduced mash.

For the evaluation of the incorporated mash, the mash solids are dispersed in distilled water in a ratio of 1: 1. Fermentation pros is used as is and contains both liquids and solids.

In this evaluation, the Kenor Habbsitis
The elegance of Kenor Hab Sitsis briggs
ae) Keep in maintenance medium. This medium is commercially available from Grant Island Biological Company, Grant Island, New York and has the following composition.

C. Brygsae maintenance medium ⁽¹⁾ Component mg / L Inorganic salts CaCl ₂・ 2H ₂ O 220.50 CuCl ₂・ 2H ₂ O 6.50 Fe (NH ₄ ) ₂ (SO ₄ ) ₂・ 6H ₂ O 58.80 KH ₂ PO ₄ 1225.50 KOH ( a) MnCl ₂・ 4H ₂ O 22.20 ZnCl ₂ 10.20 Other components N-acetylglucosamine 15.00 Adenosine-3 ′-(2 ′)-phosphate ・ H ₂ O 365.00 Cytidine-3 ′-(2 ′)-phosphate 323.00 D− Glucose 1315.00 Reduced glutathione 204.00 Guanosine-3 '-(2')-PO ₄ Na ₂ .H ₂ O 363.00 Magnesium citrate 5H ₂ O (dibasic) 915.00 Potassium citrate H ₂ O 486.00 DL-thioctic acid 3.75 Thymine 126.00 Uridine-3 ′-(2 ′)-phosphate 324.00 Amino acid L-alanine 1395.00 L-Arginine 975.00 L-Aspartic acid 1620.00 L-Cysteine HCl ・ H ₂ O 28.00 L-Glutamate (Na) ・ H ₂ O 550.00 L- glutamine 1463.00 glycine 722.00 L-histidine 283.00 L-isoleucine 861.00 L-leucine 1,439.00 L-lysine · HCl 1,283.00 L-methionine 389.00 L-Hue Ruaranin 803.00 L-proline 653.00 L-serine 788.00 L-threonine 717.00 L-tryptophan 184.00 L-Tyrosine 272.00 L-Valine 1020.00 vitamins p- aminobenzoic acid 7.50 Biotin 3.75 dihydrogen citrate Choline 88.50 cyano cobalt amine (B ¹²⁾ 3.75 Folate (Ca) 3.75 Myo-inositol 64.50 Niacin 7.50 Niacinamide 7.50 Pantethine 3.75 Pantothenate (Ca) 7.50 Butellolyglutamic acid 7.50 Pyridoxal phosphate 3.75 Pyridoxamine 2HCl 3.75 Pyridoxine HCl 7.50 Riboflavin-5′-PO ₄ (Na) ・ 2H ₂ O 7.50 Thiamine HCl 7.50 References: (1) Hansen EL, Proc.Soc.Exp.Bio. & Me
d. 121 , 30-393 (1966) Note: (a) Necessary amount for adjusting pH to 5.9 ± 0.1 as needed For evaluation, use a plate with a series of small holes.
LL-C23024α or LL-2302 containing 2.5 ml of fermentation broth, 1: 1 water / incorporated Matsushita suspension, or 31.25-500 ppm of test compound.
Place the 4β water / acetone solution in separate holes. Each well is then supplemented with 25 ml of Kenorhabditis elegans maintenance medium containing 10-20 adults and larvae of various ages and eggs. The plates are then placed in a hood and examined 48 hours after inoculation for evaluation of the rate and extent of the nematicidal activity of the test composition. The results are shown in the table below. If activity is observed against the fermentation pros, the pros is further diluted to a ratio of 1: 4 pros to kenorha bzytis elegans.

[0026]

TABLE 7 7 Table group formed concentration, ppm or dilution (D) killing line after 48 hours insect activity fermentation prosulfuron D = 1: 1 8 (no motility) D = 1: 4 Harvest mash LL-23024α 500 ppm 7 250 ppm 7 125 ppm 7 62.5 ppm 6 31.25 ppm 0 LL-23024β 500 ppm 7 250 ppm 0 The activity ratings used in these assessments are: activity rating 8 = no mobility (clear Death = 7 = significantly reduced motility 6 = reduced motility 0 = normal motility for species Example B As nematode control agent against infectious larvae of ruminant nematodes **
Evaluation of LL-C23024α in Nematodes: Haemonhmus contort
us), Ostertagia c
ircumcincta), Trico Stron Gils Axey (Tri
chostrongylus axei), an infectious larva of Tricolongillus colubriformis, and a larva of Scenteus, Turbatrixacet
LL-C23024α as a nematode control agent for i)
Was evaluated using the above C. elegans species instead of Kenorha Habthistis elegans.

The obtained data is shown in the table below. ** Third instar larvae

[0028]

TABLE 8 Test Concentration of Table 8 LL-C23024α See below nematodes paired to that activity ^a H. Control O. Shirukumu T. malignant T. Koruburi T. acetylide ppm Rutsusu think-Horumisu 500 8 8 ^b 8 ^b 8 6 250 7 8 ^b 8 ^b 7 6 125 7 7 7 6 6 6 25 7 6 7 6 6 31.25 6 6 0 6 6 0 (control) 0 0 0 0 0 ─────────────── ─────────────────────── a 96 well tissue culture plate, X14868A, prepared in double distilled water.

25 μl LL-C230 per hole
The 24α solution and 25 μl nematode culture were added.

Activity rating: 8 = no motility (obvious death) 7 = markedly reduced motility 6 = reduced motility 0 = normal motility to species b within 24 hours Example C Nematode in mice. Spyroides Zubius (Nemato
spiroides dubius) and Aspiclaris tetraptera
Antibiotics LL-C against (aspicularis tetraptera)
Evaluation of 23024α nematicidal activity In the following test, Swiss Webster female white mouse is infected with Nematospiroides zubius and Aspiclaris tetraptera and kept for 3 weeks to mature the infection.

After this holding period, the mice are randomly divided into 4 groups and the groups are placed in separate cages. Then about 31.25ppm to 4,000ppm
The drug-containing feed containing the test compound of 1. was arbitrarily given to the mice for 1 week. Water is also given ad libitum during the test period. During the treatment period, examine the mud dung to see if the larvae pass. Larvae were observed to pass through in all treatment groups, indicating exterminating activity against both nematodes at all compound concentrations. At the end of the one week treatment with the drug-containing feed, the mice are necropsied and their intestinal contents are examined for larvae. The data obtained are shown in the table below as a percentage reduction in larvae compared to infected untreated controls.

In these tests, 1,000 ppm to 4000 ppm obtained before the intake of Matsushiu
Crude fermentative pros containing the nematode control antibiotics were evaluated. 3 dissolved in a 1: 1 mixture of acetone / water
1.25-500 ppm LL-C23024
Mus murine diets treated with α were also evaluated.

[0033]

[Table 9] Activity (decrease rate%) *** Concentration in feed, N. zubius A. tetraptera Test composition relative to ppm LL-C23034α 4,000 73-containing fermentation process 2,000 44 53 LL-C23024α 1,000 65 33 500 70 Low activity ** 250 63 Low activity 125 61 Low activity 62.5 29 Low activity 31.25 32 Low activity * = Undetermined amount of LL-C23024α ** = Increased amount of worms collected in feces test ** * = Comparison to untreated infected controls The novel antibacterial and anticoccidial agents of the present invention are produced during culture under controlled conditions of Actinomadura umaens spp.

A representative strain of this microorganism was isolated from a soil sample collected in the Yuma group, Arizona, and cultured under the culture number L.
L-C23024 is preserved during the culture collection of the Rederley Research Institute branch office of American Cyanamide Company, Pearl River, New York. A viable culture of this representative strain is Illinois 61604,
Pelia is deposited at the US Department of Agriculture, Northern Center, Culture Collection and is added to the permanent collection at that location under Accession Number NRRL 12515.

Taxonomic Characterization of NRRL12515 Strain NRRL12515 is characterized and identified as a strain of a new species of Actinomadura that should be identified as a new Actinomadura yumaense sp. Nov.

Shearing and Gottlieb [EB Shir
Ling, D. Gottlieb, “Method of characterization of matreptomyces species”, Internat. J. Syst. Bacteriol. 1
6 , 313-340 (1966)], and Gordon et al. [REGordon et al., “Nocardia coeliaca (Nocardi
a coeliaca) Nocardia a
utotrophica) and Nocardin strain ”, Internat.J.Sys
t. Bacteriol. 24 , 54-63 (1974)], using the method described in detail in the representative strain NRRL12515.
Observations were made on the culture, physiology and morphological characteristics of. The medium used in this study was Puridam et al. [T.
G. Pridham et al, “Selection of media for conservation and taxonomic studies of matreptomycestes”, Anntibiotics Ann., 9
47-953 (1956/1957)]; Gauze et al. [G.
F.Gauze et al, "Problems in the classification of antagonistic actinomycetes", State Publishing House for Medical Literatur
e, Medghiz, Moskoh (1957); and Gordon et al., cited above for their taxonomic study of actinomycetes and soil fungi. The chemical composition of the cell membranes of microorganisms is described by Rekebarille et al. [(HALechevalier et al.
"Chemical composition as a criterion in the classification of Actinomycetes", Adv. Appl. Microbiol. 14 , 47-72 (1
971)]. The phospholipid pattern is described by Rekebaril et al. [“Chemical taxonomy of aerobic actinomycetes: phospholipid composition”, Biochem.Syst.Ecol. 5 , 249-
260 (1977)]. Details are given in Tables 4-9, and a general description of the culture is given below. The underlined descriptive color is Kelly (K.
L. Kelly) and DBJudd, "Color. Universal Language and Dictionary of Names", USNat.Bur.Stand., Spec.Publ.
440, Washington D.C. (1976) and accompanying I
nter-Society Color Council, Natl.Bur.Stand.Centroid
Taken from the color chart.

Represented by strain NRRL 12515
The accepted data for this new species as
Published day for known species of Lepidoptera
Ta [M.Goodfellow et al., “Actinoma
Mathematical taxonomy of chives and related actinomycetes ", J. Gen. Microbiol.
122, 95-111 (1979); Juan (L.H.Huan
g), new species of Actinomasura macula, antibiotic CP-47,
433 and CP-47,434 producing bacteria ”, Internat. J. Sy
st.Bacteriol.Thirty, 565-568 (1980);
J. Meyer, "New Species of the Genus Actinophylla" Z.All
gem.Mikrobiol.19, 37-44 (1979); Nomura
And O'Hara, “Distribution of actinomycetes in soil XI. Actino
Some new species of the genus Mazura, Requebarir et al., “J.Ferm
ent.Technol.49, 904-912 (1971); and
Preobrazhenskaya et al.,
"A Guide to the Identification of Actinomycetes Species", The Bi
oligy of Actinomycetes and Related Organisms1
Two, 30-38 (1977)]. Culture NR
RL12515 is a type of A. pell.
etieri) and has a few similarities, but
There are no similarities to the Seed of Knowledge and in many characteristics A.
It is different from Rechieri. Therefore, the strain NRRL12
515 is the collection site of the soil sample from which this representative strain was isolated.
As a new species of actinomycedra umaens, named after
It represents a new representative bacterial species to be identified.

[0038] Micro morphology spores branched, mostly produced as short helical chain on whorled of aerial sporophores (1 chain per best length of about 20 spores). Spores have a smooth surface,
It has an oval shape of 0.6 to 0.8 micron x 1.0 to 1.4 micron.

Cell Membrane Composition The whole cell hydrolyzate of this culture consisted of mazulose (3-O
-Methyl-D-galactose) and the meso isomers of diaminopimelic acid (DAP). This culture is P-
It has a type 1 phospholipid pattern and no other characteristic phospholipids other than some phosphatidylglycerins.
All of these characteristics are highly typical of the Actinomycetes genus.

Amount of growth good growth can be achieved by using Veneto culture medium, Gauze No. 2 culture medium,
It was observed on NZ-amine-starch-glucose culture medium (ATCC medium 172), tomato paste-oatmeal culture medium, and yeast extract-malt extract culture medium. Moderate growth was observed in Benedict's culture medium, Zapeck's sucrose culture medium, glycerin-asparagine. Found on media, Hitzky-Tresner media, and oatmeal media; inadequate growth is found on calcium maleate, Gauze No. 1 media, and inorganic salt-starch media.

On the medium in which favorable growth of vegetative mycelia occurs, swelling and rotation of the vegetative mycelium are recognized, and generally, it has a yellowish gray color tone.

The color of aerial mycelia and spores of a color aerial mycelia and / or spores thereof are white to 264 light gray <br/>. The aerial mycelial product is pale on most media.

Soluble pigment Not present on many media; yellow pigment on Benedict's and glycerin-asparagine media; NZ
-Amine-starch-a brown pigment with a rim on glucose medium; an orange pigment on Veneto medium and yeast extract-malt extract medium.

Physiological Reactions Peptone-Iron and Tyrosine Media (ISO-7)
No melanin pigment above; strong peptization of litmus milk; strong proteolytic hydrolysis of nutritional gelatin; slow reduction of nitrates; no hydrolysis of adenine or guanine; strong hydrolysis of hypoxanthine, tyrosine and xanthine; weak starch. Hydrolysis; Esculin hydrolysis; Urea indefinite hydrolysis. No growth at 4 ° C, 10 ° C or 55 ° C; slow growth at 25 ° C and 45 ° C;
Good growth at 32 ° C and 37 ° C. Puri Dam (TG
Pridham) and D. Gottlieb ["Use of carbon compounds by several actinomycetes as a tool for species determination", J. Bacteriol. 56 , 107-114 (19).
48)] Utilization of carbohydrates: good utilization of glucose, glycerin and trehalose; moderate utilization of maltose and sucrose; inadequate utilization of fructose, galactose, inositol, mannose and melezitose; adonitol, arabinose, dulcitol, No use of lactose, mannitol, melibiose, raffinose, rhamnose, salicin, sorbitol and xylose. Acid production from hydrolysis by the method of Gordon et al .: Good acid production from glucose, glycerin, maltose, sucrose and trehalose; Weak acid production from galactose, inositol and mannose. Utilization of organic acids by the method of Gordon et al .: Utilization of acetic acid, maleic acid, propionic acid, pibric acid, succinic acid and tartrates; benzoic acid, citric acid,
No use of lactic acid, mucus acid and oxalates.

[0045]

[Table 10]

[0046]

[Table 11]

[0047]

[Table 12]

[0048]

[Table 13]

[0049]

[Table 14] ++ = Strong positive response ++ = Moderate positive response + = Slight positive response − = Negative response

[0050]

[Table 15] The term Actinomys lacuma NRRL12515 refers to the strain Actinoma muras NRRL1251.
It is to be understood that it is not limited to strains 5 or those which fully match the growth and microscopic characteristics mentioned above, and these are given solely for the purpose of illustration. The actinomysella yumaens described herein is the antibiotic L.
L-C23024 Includes all strains of Actinoma sulayumaens that are indistinguishable from Actinomadura umaenus NRRL12515 and its subcultures, including mutants and mutants that produce iota and β .. The term “mutant” refers to natural (spontaneous) mutants of this organism as well as, for example, X-ray irradiation,
Induced mutants produced from this organism by a variety of mutagenic means known in the art, such as exposure to UV light, Nitrogen mustard, actinophage, nitrosamines, etc. Includes. Those familiar with the field, such as, for example, conjugation, transduction and genetic engineering techniques, preferably also include inter- and intra-species genetic recombination products produced by known genetic techniques. And it is intended.

Cultivation of Actinomadura umaemens can be carried out using a wide variety of solid or liquid culture media. Media useful for the production of the antibiotics LL-C23024α, β and iota include assimilable nitrogen sources such as proteins, protein hydrolysates, polypeptides, amino acids, corn exudates and potassium, eg potassium,
Sodium, ammonium, calcium, sulfuric acid, carbonic acid,
It contains inorganic anions and cations such as phosphoric acid, chlorine and the like. Trace elements, such as boron, molybdenum, copper, etc., are provided as impurities in other components of the medium. Aeration into tanks and bottles is provided by blowing sterile air into or on the surface of the fermentation medium. Further movement in the tank is provided by mechanical impellers. If necessary, an antifoaming agent such as lard oil or silicone antifoaming agent is added.

Actinomys communis may be used on a slant medium such as Venetian medium, yeast malt medium, or AT medium.
Grows and is maintained on CC medium # 172. ATCC medium # 172 is preferred. The slant medium is inoculated with a culture of Actinomadura umaenense and cultured at 28 to 37 ° C, preferably about 32 ° C, for example, for about 7 days. These stock cultures may be maintained by sequential transfer to fresh slants, or plugs of mycelium-containing culture medium from fully grown slants may be used to inoculate liquid medium.

A shake flask inoculum of Actinomadura umaenus was prepared by inoculating 100 ml of sterile liquid medium in a 500 ml flask with spore scraping or washing from the slant of the culture. To do.
Examples of suitable seed media are as follows.

Medium A Beef extract 0.3% Bactryptone ¹ 0.5% Glucose 1.0% Yeast extract 0.5% Add water to 100% [ ¹ Peptone, a registered trademark of the Difco Institute of Detroit, Michigan] Dilute alkali, for example sodium hydroxide. p
Adjust H to 6.8-7.2.

Medium B Glucose 1% Starch 2% Yeast extract 0.5% NZ amine A ² 0.5% Calcium carbonate 0.1% Water added to 100% [ ² National D'ary, Sheffield Chemical Company, New York, New York, NY] A registered trademark of casein digested product of Products Corporation] Medium B is preferred.

The flask is heated to 25 to 35 ° C., preferably 32.
Incubate at a temperature of ° C and shake vigorously on a rotary shaker for 1-4 days. This seed culture may then be used to inoculate a fermentation culture, or the culture may be frozen and stored to serve as an inoculum for a later seed culture.

The following medium contains the antibiotic LL = C2302
It is an example of what is suitable for the fermentation of Actinomadura umayens to produce 4α, β and iota.

Medium C Glucose 1.5% Glycerin 1.5% Soybean flour ³ 1.5% Calcium carbonate 0.1% Sodium chloride 0.3% 100% by adding water [The same effect can be achieved by replacing with ³ cottonseed flour or meat solubles] Medium D Glucose 3% Soybean flour 1.5% Calcium carbonate 0.1% Sodium chloride 0.3% Water added 100% Medium E Starch starch 1% Molasses 2% Soybean flour 1.5% Calcium carbonate 0.1% Water added 100% Medium F Glucose 3 % Meat solubles 2.5% Sodium chloride 0.2% Calcium carbonate 0.1% Water added 100% Medium G glucose 3% Soybean powder 0.5% Ammonium sulfate 0.3% Sodium chloride 0.2% Calcium carbonate 0.1% Water added 100% Medium H glucose 3% ammonium sulfate 0.3% dibasic potassium phosphate 0.1% calcium carbonate 0.2% sodium chloride 0.1% 100% medium D with water added is preferred.

Fermentation was carried out as described above in 3 to 1
Inoculated with 0% (vol / vol) seed culture and 25-
It can be carried out in 100 ml of medium in a 500 ml flask cultivated at 35 ° C., preferably about 32 ° C., with aeration for 24-72 hours. A sample of the fermentation culture can be frozen and stored for later use as an inoculum for the seed culture.

Alternatively, the fermentation may be carried out in a large fermentor equipped with aeration and shaking means. 3-1 for each tank
Inoculate with 0% (vol / vol) of the inoculum prepared above. Aeration is 0 for 1 liter broth per minute.
The fermentation medium is fed with a speed of sterile air of 5 to 2.0 l and the fermentation medium is agitated by an impeller driven at 200 to 400 rpm. The temperature is maintained at 25-35 ° C, preferably 32 ° C.
Until the accumulation of antibiotics in the fermentation medium, usually 100 ~
Fermentation is continued until 150 hours, at which point the antibiotic is harvested.

Antibiotics are described in the aforementioned US Pat. No. 4,278,6.
It can be harvested and purified according to the method described in No. 63, or according to the following procedure: A crude fermentation broth containing whole cells prepared as described above is treated with an equal volume of non-hydrocarbonaceous material. Mix with an organic solvent that is immiscible with water. Methylene chloride or ethyl acetate are preferred. The organic phase is separated and concentrated under reduced pressure to an oily syrup.

The oily syrup was dissolved in methylene chloride, and silica gel, alumina, Sephadex LH-20 was used.
^R (Pharmacia Huaine Chemicals Branch, Pharmacia Inc., Piscataway, NJ) or magnesium aluminum silicate column. Examples of suitable solvents for column development are diethyl ether, ethyl acetate, 1: 1 to 1: 7 (vol / vol) mixture of methylene chloride: ethyl ether, 10-40% acetone in methylene chloride, methylene chloride. 2-10% lower alcohol (methanol is preferred), 2-15% acetonitrile in methylene chloride or 2-15% dioxane in methylene chloride. Methylene chloride: ethyl acetate 1: 1 (volume / volume) is preferred. Fractions are collected and tested for the presence of antibacterial activity by bioassay against susceptible organisms such as Bacillus subtilis. The active fractions are combined and concentrated under reduced pressure to dryness. This residue is dissolved in an organic solvent such as t-butanol (preferred), benzene or p-dioxane and freeze dried.

The lyophilized solid is dissolved in a suitable organic solvent such as methylene chloride, hexane, methylene chloride: ethyl acetate, diethyl ether, hexane: ethyl acetate, hexane: chloroform, or hexane: ether. Diethyl ether is preferred. The solution is shaken with water and adjusted to pH 1.5-4.0 with dilute mineral acid.
It is preferably adjusted to about 2.5. The organic phase is separated, washed with water to remove excess acid, dried over a suitable desiccant, filtered and then evaporated to dryness under reduced pressure.

The residue is dissolved in a suitable solvent and the solution is slowly evaporated, preferably at about 4 ° C. Examples of suitable solvents are methylene chloride, hexane, methylene chloride: ethyl acetate, diethyl ether, hexane: ethyl acetate, hexane: chloroform or hexane: ether. Hexane: ether 5: 2 (volume / volume) is preferred. The crystals thus obtained are collected and washed, preferably at about 40 ° C., with a hydrocarbon of a suitable boiling point, such as hexane or heptane, and then air dried to give the final product, the antibiotic X-in the form of the free acid.
Acquires 14868A.

If it is desired that the product be in the form of a salt, then a suitable cation, preferably in the form of a dilute inorganic base, may be prepared according to procedures known to those familiar with the art. Can be converted into the free acid by treating with.

The following example illustrates the invention. The media A, B, C, etc. are as described above. Unless otherwise noted, all procedures were performed at room temperature (approximately 22
℃) at a pressure of 1 atm.

Example 1 Washed spores from the slant culture of Actinomadura yumaenth NRRL 12515 were used to inoculate a 500 ml flask containing 100 ml of sterile medium B. The flask was cultivated on a rotary shaker at 28 ° C. for 2 days.

A 5% inoculum of this culture was then added to 500 m
It was transferred to 100 ml of sterile medium C in a 1-liter flask and cultured on a rotary shaker at 28 ° C. for 5 days.

The presence of antibiotic activity indicates the presence of Staphylococcus aureus (St
aphylococcus aureus) ATCC 6538P, Bacillus subtilis (B
acillus subtilis) and Streptococcus faecalis (Streptococcus
faecalis), and the anthelmintic activity was tested by a bioassay for the free-living nematode Kenorhabdistis elegans.

Example 2 Containing 100 ml of the following sterile medium each,
Seven 500 ml flasks were prepared: Flask 1: 100 ml medium C Flask 2: 100 ml medium C with 1.5% cottonseed flour instead of soybean flour C Flask 3: 1.5 instead of soybean flour % 100% Meat Soluble Media C Flask 4: 100 ml Medium D Flask 5: 100 ml Medium E Flask 6: 100 ml Medium F Flask 7: 100 ml Medium G These flasks are each in medium B Inoculated with a 5% inoculum of Actinomadura yumaenth NRRL 12515 grown in. Then shake the flask on a rotary shaker 28
Culturing was carried out at ℃ for 4 to 6 days.

Each of the above cultures was tested for antibiotic activity as in Example 1 and in chick kidney tissue cultures with Eimeria tenell.
Activity was observed when assaying for anti-scale insect activity against a).

Example 3 Frozen fermented cultures of Actinomadura umaensu NRRL 12515 were used to inoculate a 500 ml flask containing 100 ml of sterile medium B. The flask was then cultivated on a rotary shaker at 32 ° C. for 4 days.

A 5% inoculum of this culture was then added to 500 m
Transferred to 100 ml of sterile medium H in a 1 liter flask.

The antibacterial activity was confirmed in the same manner as in Example 1,
Also, the anticoccidial activity was confirmed in the same manner as in Example 2.

The presence of antibiotic activity was also confirmed by thin layer chromatography on silica gel plates developed with ethyl acetate: chloroform 70:30 (vol / vol).
It was calibrated.

Example 4 100 ml of sterile medium A in a 500 ml flask was inoculated with washed spores from the slant culture of Actinoma murarum NRRL 12515. This flask was cultivated on a rotary shaker at 32 ° C. for 2 days.

A 5% inoculum of this culture was then added to 500 m
It was transferred to 100 ml of sterile medium H in a 1-liter flask and cultured on a rotary shaker at 32 ° C. for 2 days.

A 5% inoculum of this culture was then added to 100 m
It was transferred to a 500 ml flask containing 1 liter of fresh sterile medium H and cultured at 28 ° C. for 6 days on a rotary shaker.

The antibacterial activity was confirmed in the same manner as in Example 1.

Example 5 Two 500 each containing 100 ml of sterile medium
Actinomysula umaensu NRRL12 in ml flask
515 frozen seed cultures were inoculated and cultivated on a rotary shaker for 2 days at 32 ° C.

The contents of both flasks were then combined and
12 liters of fresh sterile medium A in 1 liter bottle were added. This culture was then aerated for 2 days at 28 ° C with aeration.

The contents of the bottle were then transferred to a 300 l seed tank containing 288 l of sterile core A and the culture was stirred at 32 ° C. for 25 hours with aeration.

After culturing for 25 hours, 300 l of seed culture was transferred to a 1500 l fermentor containing 1200 l of sterile medium C. The culture is aerated and agitated with 28
The cells were cultured at 115 ° C for 115 hours.

Fermentation broth was assayed for antibiotic activity by thin layer chromatography on silica gel plates developed in ethyl acetate: chloroform 70:30 (vol / vol). Separately, a bioassay of Bacillus subtilis on several developed plates showed antibiotic activity.

Example 6 A typical medium used to grow a primary inoculum was
Prepared according to the following formulation: Beef extract 0.3% Bacto ^R -tryptone 0.5% Glucose 1.0% Yeast extract 0.5% Bacto ^R agar 0.15% Water was added to wash 100% Actinomadura Yumaens NRRL 12515 slant culture medium and 500 ml containing 100 ml of the above sterile medium using scraped spores and hyphae
Flask was inoculated. The flask was placed on a rotary shaker and shaken vigorously for 48 hours at 32 ° C. The flask inoculum (100 ml) thus obtained was used for 2 l
1 liter of the same sterile medium in a bottle was inoculated. The inoculum was grown for 48 hours at 28 ° C. with sterile air. This 1 liter culture was then used to inoculate a 30 liter fermentor containing the same sterile medium and cultivated at 28 ° C. for 42 hours while passing sterile air through the fermenter.

Example 7 A fermentation medium was prepared according to the following formulation: Dextrose 1.5% Glycerin 1.5% Soy flour 1.5% Calcium carbonate 0.1% Sodium chloride 0.3% Water was added to 100% pH with 6N sodium hydroxide. After adjusting to 7.0, the medium was sterilized. A 30 liter portion of the inoculum prepared as described in Example 1 was used to inoculate 250 liters of the above medium in a 300 liter fermentor. The mash was stirred with blades operating at 220 rev / min while supplying aseptic aeration to the mash. After fermentation was carried out at 32 ° C for 97 hours, Matsushiu was introduced.

Example 8 A fermentation medium was prepared according to the following formulation: Dextrose 3.0% Soybean flour 1.5% Calcium carbonate 0.1% Sodium chloride 0.3% Water was added and the pH was adjusted to 7.0 with 100% 6N sodium hydroxide. After that, the medium was sterilized. 3000 in a fermentor with a 300 l portion of the inoculum prepared as described in Example 1
1 of the above medium was inoculated. Aseptic aeration was supplied to the mash. The mash was agitated by a blade driven at 100 rpm. After fermentation was carried out at 32 ° C for 115 hours, Matsushiu was introduced.

Example 9 1. Fermented Matsushiyu (100 l) was added with 6N HCl.
Adjusted to H4.0. The acidic mash was then stirred with an equal volume of methylene chloride for 1-2 hours. The mixture of mash and methylene chloride was then filtered using diatomaceous earth as a filter aid. The methylene chloride extract was removed and concentrated under reduced pressure to about 21 of partially purified antibiotic.

2. The partially purified antibiotic silica gel.
Chromatography on a glass column with a diameter of 7 cm was packed up to a height of 91 cm by means of worm silica gel TSC. The crude concentrate (see above) having a volume of about 21 was impregnated into a column of silica gel. The column was then developed first with 3 l methylene chloride and then with methylene chloride: ethyl acetate (1: 1 by volume) to give 80 ml each.
A total of 126 fractions were obtained with a volume of. The column was then further developed with ethyl acetate-ethanol (7: 3) to give an additional 87 fractions. Fractions 58-87 rich in C23024α were combined and concentrated under reduced pressure to give 90.4 g of residue. The residue was dissolved in a mixture of 600 ml diethyl ether and 600 ml hexane. The suspension thus obtained was filtered to give a clear filtrate. The clear filtrate was concentrated under reduced pressure until the onset of crystal formation. The suspension was left overnight for aging. The crystalline C23024α was collected on a funnel, washed with cold ether and then air-dried to give 33.1 g of crystalline C23024α.
Got By further reducing the combined volume of wash and filtrate, 12.4 g of additional C23024α was obtained.

Fractions 177-203 enriched in C23024β from elution with ethyl acetate-ethanol were combined, concentrated under reduced pressure and 6.7 g of partially purified C.
23024β was obtained and processed as described above.

LL-C23024 Iota-rich fractions 204-213 from elution with ethyl acetate-ethanol
Were collected, concentrated under reduced pressure into a paste, and then repeatedly extracted with methanol. The methanol extract was extracted with methylene chloride and loaded onto a column containing well silica gel. The column was washed with methylene chloride and then eluted with methylene chloride: ethyl acetate (2: 3). Each fraction was examined by thin layer chromatography to collect active fractions, treated with activated carbon and concentrated,
Repeated extraction with heptane. The final heptane extract was kept at 0 ° C. for 48 hours, after which the crystals were separated from the mother liquor by filtration.

This mother liquor was dissolved in methylene chloride and allowed to enter a glass column packed with well silica gel.
The column was then eluted sequentially with 2 l methylene chloride, 8 l methylene chloride: ethyl acetate (1: 1) and 4 l ethyl acetate: methanol (9: 1). The eluate was collected and each fraction was examined for antibiotic composition. The active fractions were collected and concentrated under reduced pressure to give a residue containing LL-C23024 iota.

Example 10 C23024 from chromatography on silica gel
Further purified Sephadex LH20 of β was swollen in a mixture of hexane-methylene chloride-methanol (10: 5: 1). 5
A glass column having a diameter of cm was packed with the gel to a height of 86 cm. A charge consisting of 3 g of purified LL-C23024β was dissolved in 10 ml of methylene chloride, 1 ml of methanol and 10 ml of hexane and impregnated into the gel column. The column was then developed with a solvent having the same composition as used to put the antibiotic in the column. Many fractions were obtained using a collector. The first 23 fractions each had a volume of 17 ml. Fractions 17 to 26 were C23 according to thin layer chromatography
It contained 024β. These fractions were combined and concentrated under reduced pressure to give pure amorphous LL weighing 1269 mg.
-C23024β was obtained.

Example 11 Crystallization of LL-C23024β as sodium salt Purified LL-C23 prepared as described in section B and C
024 (2.4 g) to diethyl ether (500 m
l), hexane (160 ml) and methylene chloride (25
ml). 30 times the solution thus obtained
Transferred into a separatory funnel containing 0 ml of water. After shaking and settling, dilute HCl (1N) was added dropwise until the pH was 2.0-2.5. The acidic aqueous layer was discarded, and the acid-treated organic layer was washed with 400 ml of water three times. The washed organic layer was stirred with 1 teaspoonful of Darkco G60 powder for 15 minutes and then filtered through Celite. Place the decolorized organic layer on 500 ml of distilled water and shake to adjust the pH to 1
5N NaOH was added dropwise until it reached 1.0-11.5 and then allowed to settle. The alkaline aqueous phase was discarded and the remaining organic layer was washed twice with 400 ml of water each time. The washed organic layer is dried over sodium sulfate and then dried under reduced pressure for 150 m.
It was concentrated to l. The concentrate was left in the hood for several hours. The crystalline LL-C23024β was collected on a funnel, washed with cold hexane and air dried to obtain 402 mg of crystalline salt of LL-C23024β. Microanalysis was performed on this sample and some spectral data was determined.

Calculated for C ₄₆ H ₇₇ O ₁₇ Na: C, 5
9.70; H, 8.33; O, 29.46; ash, 24
9. Analytical value C, 61.55, H, 8.79; ash content, 3.3
1; N, O. Melting point (Fishier Jones equipment) =
174 FD mass spec = 924. [Α] _D ²⁶ = + 3.2 (in methanol) Example 12 Purification of LL-C23024 Iota A Waters Preparation 500A high performance liquid chromatograph was packed with silica gel cartridges under a pressure of 550 psi. A charge of 5 g of crude material from Example 3 was added to 50 ml of heptane: ethyl acetate (45: 5).
5) Dissolved in and injected onto a column of silica gel.
The column was then developed with the same solvent mixture at a flow rate of 200 ml / min, yielding 40 fractions of 200 ml each.
Fractions 21-32 were combined and concentrated, the residue was triturated with hexane and evaporated to give pure LL-C230.
I got 24 Jotas. The above procedure was repeated 4 times to give a total of 280 mg of amorphous LL-C23024 iota.

Amorphous LL-C23024 Iota 90m
g portion was dissolved in 10 ml of diethyl ether and
After mixing with 0 ml of water, pH was adjusted with 0.1N hydrochloric acid.
Adjusted to 2.5. The ether layer was washed with water, the pH was adjusted to about 11 with 0.1N sodium hydroxide and separated,
It was washed again with water. The ethereal phase was dried over sodium sulphate, filtered and concentrated to give a residue. A solution of this residue in ether-hexane was slowly evaporated to give an amorphous white solid.

This amorphous white solid has the following properties: Elemental analysis: C, 61.79; H, 8.84; ash, 0.3
2; [α] _D ²⁶ = + 27 (C 0.724, methanol); F
D mass spectrometry: (M + Na) ⁺ = m / e 953, hence calculated molecular weight is 930.

[Brief description of drawings]

FIG. 1 is an IR of LL-C23024β in KBr.
It is a spectrum.

FIG. 2 is ¹³ C-NM of LL-C23024β at 20 MHz in CDCl ₃ with TMS as an internal standard.
It is an R spectrum.

FIG. 3 is ¹ H-NM of LL-C23024β at 80 MHz in CDCl ₃ with TMS as an internal standard.
It is an R spectrum.

FIG. 4 is an IR spectrum of LL-C23024 iota in KBr.

FIG. 5: ¹ H-of LL-C23024 iota at 80 MHz in CDCl ₃ with TMS as internal standard.
It is an NMR spectrum.

FIG. 6 shows ¹³ C-of LL-C23024 iota at 20 MHz in CDCl ₃ with TMS as internal standard.
It is an NMR spectrum.

FIG. 7 shows ¹³ C-of LL-C23024 Iota at 20 MHz in CDCl ₃ with TMS as internal standard.
NMR spectrum (enlarged scale: 0-50 pp
m).

FIG. 8 shows ¹³ C-of LL-C23024 iota at 20 MHz in CDCl ₃ with TMS as internal standard.
It is an NMR spectrum (enlarged scale: 50-110).
ppm).

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical indication C12N 1/20 A 7236-4B (C12P 19/60 C12R 1:03) (C12N 1/20 C12R 1 : 03) (72) Inventor Donald Bruce Borders New York, USA 10901 Suffern Heatherhill Lane 13 (72) Inventor Raymond Thomas Testa New Jersey, USA 07009 Cedar Grove Rotskridge Place 55 (72) Inventor Jim Young Henry Edward James Martein New York, USA 10956 New York Carolina Drive 14 (72) Inventor Sydney Canter New Jersey, USA 08876 Summer Bill Burtuk Saidrain 410 (72) Inventor Robert El Kennett Junia 08525 Hopewell Township (no street number) (72) Inventor Erwin Bee Woods United States New Jersey 08534 Pennington Boxes 182A · Earl number 1

Claims (6)

[Claims] 1. Actinomysella yumaens sp. In a liquid medium containing assimilable carbon, nitrogen and inorganic salt sources. n
ov. Antibiotic LL-C23024α, characterized in that the fermentation medium is aerobically fermented until it imparts substantial antibiotic activity, and then the antibiotic is recovered from it.
β and iota manufacturing method. 2. The method according to claim 1, wherein the temperature of the fermentation culture is maintained at 25-35 ° C. for 24 to 150 hours. 3. The actinomysella yumaens sp. no
v. The method according to claim 1 or 2, wherein is actinomycema yumaens NRRL 12515. 4. Actinomysella yumaenth sp. In a liquid medium containing assimilable carbon, nitrogen and inorganic salt sources. n
ov. Of the antibiotic LL-C23024α according to claim 5, characterized in that the fermentation medium is aerobically fermented until it imparts a substantial antibiotic activity, and then the antibiotic is recovered. Production method. 5. A poultry edible carrier and about 0.0000.
A method for suppressing coccidiosis of poultry, which comprises orally administering 25 to 0.06% by weight of an antibiotic LL-23024α. 6. A nematicidally effective amount of compound LL-C2.
3024α or a pharmaceutically acceptable salt of these compounds or a mixture of these compounds or salts or a fermentation medium from which a nematicidal antibiotic is obtained or a harvested Matsushiu solid is orally introduced into a warm-blooded animal Or a method of controlling nematodes in soil containing warm-blooded animals and nematodes by applying to soils that have been infected with nematodes.