GB2108113A - Antibacterial agents LL- C23024 beta and iota and microorganism actinomadura yumaense NRRL 12515 - Google Patents
Antibacterial agents LL- C23024 beta and iota and microorganism actinomadura yumaense NRRL 12515 Download PDFInfo
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Abstract
Antibacterial and anti-coccidial agents (polyether antibiotics) designated LL-C23024 alpha , LL-C23024 beta and LL-C23024 iota, a novel microorganism Actinomadura yumaense sp. nov. and mutants thereof, and a novel process for the preparation of the same.
Description
SPECIFICATION
Antibacterial agents LL-C23024ss and iota and microorganism actinomadura yumaense NRRL 12515 This inovation relates to antibacterial and anticoccidial agents designated LL-C23024 α, ss, and iota, a novel microorganism Actinomadura yumaense sp. nov. or mutants thereof.
This invention further relates to a novel process for the production of antibiotic LL-C23024a, , and iota by fermentation under controlled conditions of the novel microorganism Actinomadura yumaense sp. nov.
The antibiotic LL-C22204a has the following structure:
The antibiotic LL-C23024p of the present invention is distinguished from prior art compounds by its physical and chemical characteristics, including but not limited to its microanalysis, optical rotation, infrared spectrum, 13C-nuclear magnetic resonance spectrum, and H-nuclear magnetic resonance spectrum.
Antibiotic LL-C23024p has the following postulated structure:
wherein R1 and R2 are different and are each selected from the group consisting of H and CH3.
The above postulated structure is based on the elemental analysis, optical rotation, field desorption mass spectroscopy noleculare weight, melting point, infrared (IR) spectrum, 13C-nuclear magnetic resonance (13C-NMR) spectrum, and proton magnetic resonance (H-NMR) spectrum, as detailed in the Examples and as shown in the accompanying figures:
Fig. I: IR Spectrum of LL-C12024ss in KBR
Fig. II:13C-NMR Spectrum of LL-C23024ss, 20 MHz in CDCI3, internal TMS reference equivalent.
Fig. Ill: H-NMR Spectrum of LL-C23024p. 80 MHZ in CDCI3, internal TMS reference equivalent.
TABLE I '3C-NMR SPECTRUM OF LL-C23024P (ppm relative to TMS)
Chemical Shift, (ppm) Number of Carbons 10.5 1 11.0 1 12.2 1 17.0 1 17.7 1 17.9 1 22.3 1 26.1 1 26.8 1 27.6 1 30.2 1 32.0 1 33.3 1 33.7 2 33.8 2 36.5 1 36.8 1 39.0 1 39.9 1 45.5 2 57.1 1 59.1 1 60.7 1 66.9 1 67.6 1 70.2 1 71.3 1 72.9 1 74.9 1 TABLE I (Continued)
Chemical Shift, (ppm) Number of Carbons 75.1 1 79.9 1 80.8 1 82.1 2 84.5 1 84.7 1 85.6 1 86.9 1 95.8 1 96.9 1 97.7 1 107.5 1 179.2 1 Total Carbons 46 LL-C23024 iota is a new polyether antibiotic with very potent anticoccidial activity. It is at least ten times more potent than most other known polyethers.Field desorption mass spectral data indicates it has a molecular weight of 930, which together with its carbon 3 nuclear magnetic resonance (13C-NMR) spectrum allows the proposal of a tentative molecular formula if C48H82017. LL-C23024 iota appears to have four methoxy groups, the same sugar observed in polyether antibiotic x-1 4868A [U.S. Patent 4,278,663], and one carboxyl group. The only other reported polyether antibiotic with a molecular weight of 930 is antibiotic A28695B (hydroxyseptamycin) [J. Antibiotics 33(2): 252 (1980)] which differs significantly in structure and biological properties.The above observations are based on the elemental analysis, optical rotation, calculated molecular weight by field desorption mass spectroscopy, infrared (IR) spectrum,'3C--NMR spectrum, and proton nuclear magnetic resonance (1H-NMR) spectrum, as detailed in the Examples and as shown in the accompanying figures:
Fig. IV: IR spectrum of LL-C23024 iota in KBR.
Fig. V:'H--NMR spectrum of LL-C23024 iota. 80 MHz in CDC13, internal TMS reference equivalent.
Fig. VI: 13C-NMR spectrum of LL-C23024 iota. 20 MHz in CDCI3, internal TMS reference equivalent.
Fig. VII A: 13C-NMR spectrum of LL-C23024 iota. (expanded scale: 0-50 ppm). 20 MHz iN
CDCI3, internal TMS
Fig. VIlIB: 13C-NMR spectrum of LL-C23024 iota (expanded scale: 50-110 ppm). 20 MHz in
CDCI3, internal TMS reference equivalent.
The 13C-NMR spectra of LL-C23024 iota were obtained in deuterated chloroform (CDCl3) at a field strength of 20 MHz. The peaks with their respective chemical shifts in parts per million relative to tetramethyl silane (TMS) and the estimated number of carbons per peak are listed in Table II. Peaks were observed for chloroform at 75.4, 77.0, and 78.6.
TABLE II
Chemical Shift No. of Carbons Chemical Shift No. of Carbons 10.6 1 59.9 1 10.9 1 60.8 2 11.7 1 68.5 1 16.4 1 68.8 1 17.6 1 71.3 1 18.0 1 72.2 1 21.7 1 76.1 1 22.9 1 76.9 1 24.1 1 78.5 -1 28.2 1 81.0 1 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 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 1 56.8 1 Total 48 Antibiotic LL--C23024a, P, and iota are organic carboxylic acids and thus are capable of forming salts with non-toxic pharmaceutically acceptable cations. Thus, salts formed by admixture of the antibiotic free acid with stoichiometric amounts of cations, suitably in a neutral solvent, may be formed with cations such as sodium, potassium, calcium, magnesium and ammonium, as well as organic amine cations such as tri(lower alkyl)amine (e.g., triethylamine, triethanolamine), procaine and the like. The cationic salts of antibiotic LL--C23024P are, in general, crystalline solids, relatively insoluble in water and soluble in most common organic solvents such as methanol, ethyl acetate, acetone, chloroform, heptane, ether and benzene. For the purposes of this invention, the antibiotic free acid is equivalent to its pharmaceutically acceptable non-toxic salts.
The antibiotic LL--C23024P and iota are active in vitro against gram-positive bacteria when tested by the standard agar dilution procedure. The results are reported as minimal inhibitory concentrations (MIC) in mcg./ml. in Tables Ill and IV. Antibiotic LL--C23024P and iota are not active against gram-negative organisms at concentrations of 256 mcg./ml. or less.
TABLE Ill
In Vitro Antibacterial Activity of LL-C23024ss
Minimal Inhibitory Organism Concentration (mcg./ml.) Staphylococcus aureus Smith 64 SSC 80-11 64 SSC 80-32 64 SSC 80-38 64 LL-14 64 LL-45 64 LL-27 128 ATCC 25923 64 Streptococcus pyogenes C203 1 ,e-hemolytic Keller T623 8 pneumoniae 78-1 | 8 Enterococcus SSC-80-62 64 " SSC-80-63 128 TABLE IV
In Vitro Antibacterial Activity of LL-C23024 iota
Minimal Inhibitory Concentration (mcg/ml) Organism LL-C23024 iota Enterococcus OSU 75-1 1 Enterococcus SMi 77-15 -1 Staphylococcus aureus SSC 79-18 1 Staphylococcus aureus FU 79-19-2 2 Micrococus lutea PC 1001 1 Staphylococcus aureus Smith 0.5 Staphylococcus aureus ATCC 25923 0.5 As demonstrated by the above data, antibiotic LL-C23024α, ss, ss, and iota inhibit the growth of certain gram-positive bacteria and are thus useful as a topical or surface antiseptic in wash solutions for skin, equipment, walls floors, etc.
It has also been found that antibaterial agent LL-C23024α, ss, and iota are active in viro as an anticoccidial agent for poultry as demonstrated by the following tests.
Two days before inoculation, medicated feed with several levels of drug was presented to the various groups of one-day-old test chicks. The poultry feed used through-out 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 alfalfa, 1 7% protein 2.0%
Corn gluten meal, 41% protein 5.0%
Menhaden fish meal, 60% protein 5.0%
Soybean oil meal, 44% protein 30.0%
Ground yellow corn, fine to 100%
The vitamin-amino acid premix in the above feed composition is prepared from the following formulation. The expressions of quantity relate to units per kilogram of the finished feed composition.
Butylated hydroxy toluene 125.0 mg.
dl-Methionine 500.0 mg
Vitamin A 3300.0 I.U.
Vitamin D3 1100.0 I.O.U.
Riboflavin 4.4 mg.
Vitamin E 2.2 lU.
Niacin 27.5 mg.
Panthothenic acid 8.8 mg.
Choline chloride 500.0 mg.
Folic acid 1.43 mg.
Menadione sodium bisulfate 1.1 mg.
Vitamin B,2 11.0 mcg.
Ground yellow corn, fine to 5.0 gm.
The test chicks were then inoculated by direct inoculation into the crop of each chick with a mixed
inoculum of 5000 sporulated oocysts of Eimeria acervulina and a sufficient number of oocysts of
Eimeria tenella to produce 85100% mortality in untreated controls. The chicks were given free access
to feed and water during the entire test period. Seven days after inoculation the tests were terminated
and the birds were weighed, necropsied and their intestinal tracts examined for lesions. The results
appear in Tables V and VI below and show that improved survival of infected chicks is obtained when
10 ppm or less of antibiotic LL-C23024ss or iota is administered in the diet. The results also show a
significant suppression of intestinal lesions due to Eimeria ten elba and Eimeria acervulina.
TABLE V
Evaluation of Antibiotic LL-C23024 ss as an Anticoccidial Agent in Chicks
Percent Birds with Reduced Lesions Concentration No. Birds Percent E. E.
Compound in Diet, ppm Started Survival tenella acervulina LL-C23024 ss 15 3 100 100 100 LL-C23024 ss 10 5 100 60 100 LL-C23024,K3 ss 5 5 80 0 100 NNC* 0 15 46.6 0 0 LL-C23024 ss ss 120 15 100 100 100 60 15 100 100 100 30 15 100 100 100 0 15 20 0 0 NNC 15 100 - LL-C23024 ss 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 = Nontreated, noninfected control.
TABLE VI
Evaluation of Antibiotic LL-C23024 lota as an Anticoccidial Agent in Chicks
Percent.Birds with Reduced Lesions Concentration No. Birds Percent in diet (ppm) Started Survival Eimeria tenella Eimeria acervulina 15 5 100 100 100 10 5 100 100 100 5 5 60 20 60 0 15 20 0 0 15 10 100 100 100 7.5 10 100 70 100 The following tests demonstrate the neomatocidal activity.
EXAMPLE A
Evaluation of test compositions for nematocidal activity using the free-living Hermaphroditic microbivorous nematode Caenorhabditis elegans //
In the following tests, C. elegans is used to determine the nematocidal activity of the fermentation broth and the harvest mash prepared by the procedure of this invention. This organism is also used to evaluate LL-O23024a and LL-C23024ss to determine the nematocidal activity thereof.
In these tests, fermentation broth is the fermentation liquid and solids mixture taken before the solids, i.e., the harvest mash, is separated from the liquid. Harvest mash is the solids remaining after separation.
For evaluation of harvest mash, the harvest mash solids are dispersed 1:1 in distilled water. The fermentation broth is used as is and contains both liquid and solids.
In the present evaluations, C. elegans is maintained in a C. briggsae Maintenance Medium. The
Maintenance Medium is commercially available from Grant Island Biological Company, Grant Island,
New York, and has the following compositions:
C. briggsae MAINTENANCE MEDIUM(1)
Component mg/L
INORGANIC SALTS CaCi2 2H20 220.50 CuOl2. 2H20 6.50
Fe(NH4)2 (SO4)2 6H20 58.80
KH2PO4 1225.50
KOH (a)
MnCI2 4H20 22.20
ZnCI2 10.20
OTHER COMPONENTS
N-Acetylglucosamine 15.00 Adenosine-3'-(2')-phosphoric acid.H20 365.00
Cytidine-3'-(2')-phosphric acid 323.00
D-Glucose 1315.00
Glutathione, reduced 204.00 Guanosine-3'-(2')-PO4Na2.H20 363.00
Magnesium citrate.5H20 (Dibasic) 915.00
Potassium citrate H20 486.00
DL-Thioctic acid 3.75 Thymine 126.00
Uridine-3'-(2')-phosophoric acid 324.00
AMINO ACIDS
L-Alanine 1395.00
L-Arginine 975.00
L-Aspartic acid 1620.00
L-Cysteine HCl.H20 28.00
L-Glutamate (Na).H20 550.00
L-Glutamine 1463.00
Glycine 722.00
L-Histidine 283.00
L-lsoleucine 861.00
L-Leucine 1439.00
L-Lysine HCI 1283.00
L-Methionine 389.00
L-Phenylalanine 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
Choline dihydrogen citrate 88.50
Cyanocobalamine (B12) 3.75
Folinate (Ca) 3.75
Myo-inositol 64.50
Niacin 7.50
Niacinamide 7.50
Pantethine 3.75
Pantothenate (Ca) 7.50
Pterolyglutamic acid 7.50
Pyridoxal phosphate 3.75
Pyridoxamine 2HCI 3.75
Pyridoxine HCI 7.50 Riboflavin-5'-PO4(Na) 7.50
Thiamine HCI 7.50
References: (1) Hansen, E.L., Proc.Soc.Exp.Bio.8 Med. 121:30-393 (1966).
Remarks: (a) As needed for adjustment to pH 5.9 + 0.1.
A well plate having a series of small wells is used for the evaluations. Twenty-five ml of fermentation broth, 1:1 water/harvest mash suspension, or an aqueous/acetone solution of LL-C23024a or LL-C23024ss containing from 31.25 to 500 ppm of test compound, is asceptically deposited in separate wells. To each well is then added 25 ml of the C. elegans maintenance medium containing 10-20 adult worms plus larvae of various ages plus eggs. The plates are then placed in a hood and examined at 48 hours post-inoculation to assess the speed and degree of nematocidal activity of the test compositions. Data obtained are reported below. Where activity is observed for the fermentation broth, said broth is further diluted to provide a 1:4 broth to C. elegans ratio.
TABLE VII
Concentration ppm or Nematocidal Activity Composition Dilution at 48 hours Fermentation Broth D= 1:1 8 (no motility) D=1:4 Harvest Mash LL-23024 @ 500 ppm 7 250 ppm 7 125 ppm 7 62.5 ppm 6 31.25 ppm 0 LL-23024 ,ss 500 ppm 7 250 ppm 0 The activity legend employed in these evaluations is as follows:
Activity Legend
8 = No Motility (Apparent Death)
7 = Markedly Reduced Motility
6 = Reduced Motility
0= Normal Motility for Species
EXAMPLE B
Evaluation of LL-C23O24a as a nematocidal agent against infective larvae** of ruminant nema to des Evaluation of LL-C23024α as a nematocidal agent against the infective larvae of ruminant nematodes: Haemonhmus contortus, Ostertagia circumcincta, Trichostrongylus axei, T.Columbriformis; and against the vinegar eel worm, Turbatrix acetiwas determined using the above-mentioned nematode species were substituted for C. elegans.
Data obtained are reported in the Table below.
** Third Stage Sheathed Larvae
TABLE VIII
In Vitroa
Concentration Activity Against (at 48 hours) of#LL-C23024α ppm H. Contort. O. Circum. T. Axei T. Colub T. Aceti
500 8 8b 8b 8 6
250 7 6b 8b 7 6
125 7 7 7 6 6
625 7 6 7 6 6
31.25 6 6 0 6 6 0 (Control) 0 0 0 0 0
aConducted in 96-well tissue culture plates. X 14858 A prepared in double-distilled water 25y1 of LL-C'23024a solution and 25 l of nematode culture were added per well.
Activity Legend:
8 = No Motility (Apparent Death)
7 = Markedly Reduced Motility
6 = Reduced Motility
0 = Normal Motility for Species
bWithin 24 hours.
EXAMPLE C
Evaluation of nematocidal activity of antibiotic LL-C23O24cx againstNematospiroides dubius and
Aspicularis tetraptera in mice
In the following tests, Swiss-Webster female white mice are infected with Namatospiroides dubius and Aspicularis tetraptera and held for three weeks to permit the infections to mature.
After the holding period, the mice are randomly divided into groups of four, and the groups placed in separate cages. Medicated feeds containing from about 31.25 ppm to 4,000 ppm of test compound are then offered at libitum to the mice for one week. Water is also provided ad libitum throughout the test period. During the treatment period, the mouse droppings are examined to determine whether works are being passed. All treatment groups were found to be passing worms, thus indicating nematocidal activity at all compound concentrations against both nematodes. At the end of the oneweek medicated feed treatment, the mice are necropsied, and the contents of the intestinal tracts thereof examined for worms. Data obtained are reported below as percent reduction of worms as compared with infected, unmedicated controls.
In these tests, crude fermentation broths obtained before harvest of the mash, and containing from 1,000 ppm to 4,000 ppm of the nematocidal antibiotic were evaluated. Also evaluated was mouse feed treated with from 31.25 ppm of LL-C23024α dissolved in an acetone/water 1:1 mixture.
Activity
(% Reduction)***
Against
Test Dietary
Composition Conc. ppm N. dubius A. tetraptera
*Fermentation Broth 4,000 73
with LL-C23034a 2,000 44 53
LL-C23024a 1,000 65 33
500 70 SA* 250 63 SA
125 61 SA
62.5 29 SA
31.25 32 SA
* = Amount of LL-C23024a undetermined.
** = Slight activity: Increased number of pinworms recovered in fecal
examination.
*** = Compared to infected unmedicated controls.
The new antibacterial and anticoccidial agents of this invention are formed during the cultivation under controlled conditions of Actinomadura yumaense sp. nov.
A representative strain of this microorganism was isolated from a soil sample collected in Yuma
County, Arizona and is maintained in the culture collection of the Lederle Laboratories Division,
American Cyanamid Company, Pearl River, New York as culture number LL-O23024. A viable culture of this representative strain has been deposited with the Culture Collection Laboratory, Northern
Regional Center, U.S. Department of Agriculture, Peoria, Illinois 61604, and has been added to its permanent collection under the accession number NRRL 12515, deposited on August 10, 1981.
Taxonomic Characterization of NRRL 12515
The strain NRRL 1 251 5 has been taxonomically characterized and identified as the type strain of a new species of the genus Actinomadura to be known as Actinomadura yumaense sp. nov.
Observations were made of the cultural, physiological and morphologal features of representative strain NRRL 12515 using methods detailed by E. B. Shirling and D. Gottlieb, "Methods for characterization of Streptomyces species", Internat. J. Syst Bacteriol. 16:313-340(1966), and R. E.
Gordon et al., "Nocardia coeliaca, Nocardia autotrophica, and the nocardin strain," Internat. J. Syst.
Bacteriol. 24:54-63 (1974). Media used in this study were selected from those recommended by T. G.
Pridham et al., "A selection of media for maintenance and taxonomic study of Streptomycetes,"
Antibiotics Ann., pp. 947-953 (1 956/1957); G. F. Gauze etc!., "Problems in the classification of antagonistic actinomycetes," State Publishing House for Medical Literature, Medgiz, Moscow (1 957); and R. E. Gordon et al., supra, for the taxonomic study of actinomycetes and soil bacteria. Chemical composition of the cell walls of the microorganism was determined using the method of H. A.
Lechevalier etc!., "Chemical composition as a criterion in the classification of actinomycetes," Adv.
Appl. Microbiol. 14:47-72 (1971). Phospholipid patterns were determined using the method of M. P.
Lechevalier et awl., "Chemotaxonomy of aerobic actinomycetes: phopholipid compostion," Biochem.
Syst. Ecol. 5:249-260(1977). Details are recorded in Tables IV-lX, and a general description of the culture is given below. Underscored descriptive colors are taken from K. L. Kelly and D. B. Judd, "Color.
Universal Language and Dictionary of Names," U.S. Nat. Bur. Stand., Spec. Publ. 440, Washington D.C.
(1976) and the accompanying Inter-Society Color Council, Natl. Bur. Stand. Centroid Color Charts.
The data observed for this novel species as represented by strain NRRL 12515 were compared with the data published for the known species of the genus Actinomadura [M. Goodfellow et al., "Numerical Taxonomy of Actinomadura and related actinomycetes," J. Gen. Microbiol. 122:95-111 (1979); L. H. Huang, "Actinomadura macra sp. nov., the producer of antibiobic CP47,433 and CP-47,434," Internat. J. Syst. Bacteriol. 30:565-568 (1980); J. Meyer, "New species of the genus
Actinomadura," Z. Allgem. Mikrobiol. 19:37-44 (1979); H. Nomura and Y. Ohara, "Distribution of actinomycetes in soil. Xl. Some new species of the genus Actinomadura, Lechevalier, etna!.," J. Ferment.
Technol. 49:904-91 2 (1 971); and T. P. Preobrazhenskaya et a/., "Key for identification of the species of the genusActinomadura," The Biology of Actinomycetes and Related Organisms 12:30-38(1977)].
Culture NRRL 1 251 5 bears a slight resemblance to Actinomadura pelletieri, but resembles no other described species and differs from A. pelletieri in a number of characteristics. Therefore, strain
NRRL 1 251 5 has been designated the type strain of a new species to be known as Actinomadura yumaense, sp. nov., named for the site of collection of the soil sample from which the type strain was isolated.
Micromorphology Spores are formed in short spiral chains (maximum length approximately 20 spores per chain) on branched, almost verticillate aerial sporophores. The spores are ovoid, 0.6 to 0.8 micron by 1.0 to 1.4 micron, with a smooth surface.
Cell Wall Composition
Whole cell hydrolysates of this culture contain madurose (3-O-methyl-D-galactose) and the meso isomer of diaminopimelic acid (DAP). The culture also has a Type P-1 phospholipid pattern and no other diagnostic phospholipid other than some phosphatidyl glycerol. These characteristics are all very typical of members of the genus Actinomadura.
Amount of Growth
Good growth is observed on Bennett's agar, Gauze No. 2 agar, NZ-amine-starch-glucose agar (ATCC Medium 172), tomato paste-oatmeal agar, and yeast extract-malt extract agar; moderate growth is observed on Benedict's agar, Czapek's sucrose agar, glycerol-asparagine agar, Hickey-Tresner agar, and oatmeal agar; poor growth is observed on calcium malate agar, Gauze No. 1 agar, and inorganic salts-starch agar.
Vegetative Mycelium
On media where good growth occurred, the vegetative mycelium was observed to be raised and convoluted and was generally yellowish-gray shades in color.
Aerial Mycellum and Spore Color
Aerial mycelia and/or spore masses were white to 264. light gray in color. Aerial mycelia production is light on most media.
Soluble Pigments
Absent on many media; yellow pigment on Benedict's and glycerol-asparagine agars; yellowgreen pigment on calcium malate agar; greenish brown pigment on NZ-amine-starch-glucose agar; orange pigment on Bennett's and yeast extract-malt extract agars.
Physiological Reactions
No melanin pigments on peptone-iron agar and tyrosine agar (ISO--7); strong peptonization of litmus milk; strong proteolysis of nutrient gelatin; moderate reduction of nitrate; no hydrolysis of adenine or guanine; strong hydrolysis of hypoxanthine, tyrosine, and xanthine; weak hydrolysis of starch; hydrolysis of esculin; variable hydrolysis of urea. No growth at 40C., 1 OOC.. or 550C.; moderate growth at 250C. and 450C; good growth at 320C and 370C. Carbohydrate utilization as per the method of T. G. Pridham and D. Gottlieb, "The utilization of carbon compounds by some actinomycetales as an aid for species determination," J.Bacteriol. 56:107-114 (1948): good utilization of glucose, glycerol and trehalose; moderate utilization of maltose and sucrose; poor utilization of fructose, galactose, inositol, mannose, and melezitose; no utilization of adonitol, arabinose, dulcitol, lactose, mannitol, melibiose, raffinose, rhamnose, salicin, sorbitol and xylose. Acid production from carbohydrates by the method of Gordon, et al., supra: Good acid production from glucose, glycerol, maltose, sucrose and trehalose; weak acid production from galactose, inositol and mannose. Utilization of organic acids by the method of Gordon et al., supra: utilization of acetate, malate, propionate, pyruvate, succinate and tartrate; no utilization of benzoate, citrate, lactate, mucate and oxalate.
TABLE VII
Cultural Characteristics of Actinomadura yumaense NRRL 12515
Incubation: 14 days Temperature: 28 C
Amount of Soluble Medium Growth Aerial Mycelium and/or Spores Pigment Reverse Color Benedict's Moderate Flat, powdery colonies; aerial mycelia white Yellowish 89. pale yellow Agar to poor to 264. light gray Bennett's Good No aerial mycelia; convoluted vegetative Orange 81. dark grayish Agar growth 93. yellowish gray to 80. grayish yellowish brown yellowish brown Calcium Poor Flat growth; sparse white aerial mycelia Yellow-green 90. greenish yellow Malate Agar Czapek's Poor to Flat growth; moderate aerial mycelia vegetative None 89. pale yellow Sucrose Moderate growth 90. grayish yellow Agar Gauze No. 1 Poor Colorless flat growth; sparse white aerial None Clorless Agar mycelia Gauze No. 2 Good Raised convoluted colonies; vegetative mycelia None 72. dark organge Agar 93. yellowish gray; moderate aerial mycelia 92. yellow yellowish white Glycerol- Poor to Flat, powdery colonies; white aerial mycelia Yellow 89. pale yellow Aspargine Moderate Agar Hickey- Moderate Flat waxy colonies, 90. grayish yellow None 90. grayish yellow Tresner Agar sparse aerial mycelia, white to 264, light gray Inorganic Poor Flat, colorless, powdery colonies; None Colorless Salts- white aerial mycelia Starch Agar TABLE VII (Continued)
Amount of Soluble Medium Growth Aerial Mycelium and/or Spores Pigment Reverse Color NZ-amine- Good Heavy convoluted growth, 61. grayish yellowish Greenish 78. drak yellowish Starch- brown to 65. brownish black; moderate aerial brown brown Glucose Agar mycelia, white to 264, light gray Oatmeal Moderate Flat waxy growth, 90. brayish yellow; None 90. grayish yellow Agar moderate aerial mycelia, white Tomato Paste Good Flat waxy growth, 91. dark grayish yellow; None Oatmeal trace of white aerial mycelia Agar Yeast Extract Good Raised, waxy, convoluted colonies, Orange 78. dark yellowish Malt Extract 93. yellowish gray to 80. grayish yellowish brown Agar brown; no aerial mycelia TABLE VIII
Micromorphology of Actinomadura yumaense NRRL 12515
Aerial Mycelium and/or Spore Medium Sporiferous Structure Spore Shape Spore Size Surface Czapek's Aerial sporophores; branched, almost 0.6 - 0.8 micron Sucrose verticilate; carrying relatively ovoid x Smooth Agar short spiral chains of mature spores 1.0 - 1.4 micron TABLE IX
Physiological Reactions of Actinomadura yumaense NRRL 12515
Incubation Amount of Physiological Medium Period Growth Reaction Peptone- 7 Days Good Slight browning Iron Agar 14 Days Good Slight browning Tyrosine 7 Days Moderate No pigment Agar 14 Days Good Yellowish pigment Litmus 14 Days Good Good peptonization Milk 28 Days Good Strong peptonization Nutrient 14 Days Good Slight proteolysis Gelatin 28 Days Good Total proteolysis Nitrate 14 Days Good Very weak reduction Broth 28 Days Good Moderate reduction Adenine 14 Days Good No hydrolysis Agar 21 Days Good No hydrolysis Guanine 14 Days Good No hydrolysis Agar 1 21 Days Good No hydrolysis Hypoxanthine 14 Days Good Total hydrolysis Agar 21 Days Good Total hydrolysis Tyrosine 14 Days Good Strong hydrolysis Agar 21 Days Good Strong hydrolysis Xanthine 14 Days Good Moderate hydrolysis Agar 21 Days Good Strong hydrolysis .NZ-amine 5 Days Poor or no growth at 4 C., 10 C and 55 CC.; with Soluble moderate growth at 25 C., 28 C. and Starch and 45 C.; good growth at 3Z-C. and 37 C.
Glucose Agar (ATCC Med.
No. 172) Urea Broth 28 Days Good Decomposition variable Esculin 14 Days Good Hydrolysis Broth 28 Days Good Hydrolysis Starch 5 Days Good No Hydrolysis Agar 10 Days Good No Hydrolysis TABLE X
Carbon Source Utilization of Actinomadura yumaense NRRL 12515
on ISP-9 Carbohydrate Utilization Medium
Incubation: 28 days Temperature: 28tC
Carbon Source Utilization Adonitol /-Arabi nose Dulcitol Fructose Poor d-Galactose Poor d-Glucose Good Glycerol Good i-Inositol Poor Lactose Maltose Fair d-Mannitol d-Mannose Poor d-Malezitose Poor d-Mel ibiose d-Raffinose 1-Rhamnose Sal loin Sorbitol Sucrose Fair d-Trehalose Good d-Xy lose Negative Control TABLE XI
Acid Production from Various Carbohydrates
by Actinomadura yumaense NRRL 12515
on Gordon's Basal Inorganic Nitrogen Medium
Incubation: 28 days Temperature: 28^C
Acid Production* Carbon Source 7 Days 28 Days Adonitol l-Arabinose Dulcitol Fructose - d-Galactose - + d-Glucose +++ +++ Glycerol ++ +++ i-Inositol - + Lactose Maltose - +++ d-Mannitol ~ d-Mannose - + d-Melezitose - d-Melibiose - d-Raffinose /-Rhamnose - - - - Salicin - -- Sorbitol - Sucrose - +++ d-Trehalose - +++ d-Xylose - Negative Control - +++ = Strong positive response
++ = Moderate positive response
+ = Slight positive response
- = Negative response
TABLE XII
Utilization of Organic Acids by Actinomadura
yumaense NRRL 12515 on Gordon's Modification
of Koser's Basal Agar (Koser's Citrate Agar)
Incubation: 28 days Temperature: 28oC
Carbon Source Utilization* Acetate + Benzoate Citrate Lactate Malate + Mucic Acid Oxalate Propionate + Pyruvate + Succinate + Tartrate .*: + = Positive response -:= Negative response
It is to be understood that the term Actinomadura yumaense is not limited to strain Actinomadura yumaense NRRL 1 251 5 or to strains fully answering the above growth and microscopic characteristics, which are given for illustrative purposes only. Actinomadura yumaense described herein includes all strains of Actinomadura yumaense which produce the antibiotic LL-O23024a, , and iota and which cannot be differentiated from Actinomadura yumaense NRRL 1251 5 and its subcultures, including mutants and variants thereof.The term "mutants" includes the natural (spontaneous) mutants of this organism as well as induced mutants produced from this organism by various mutagenic means known to those skilled in the art, such as exposure to x-ray radiation, ultraviolet radiation, nitrogen mustard, actinophages, nitrosamines, and the like. It is also desired and intended to include inter- and intraspecific genetic recombinants produced by genetic techniques known to those skilled in the art, such as for example conjugation, transduction and genetic engineering techniques.
Cultivation of Actinomadura yumaense may be carried out with a wide variety of solid or liquid culture media. Media which are useful for the production of antibiotic LL-O23024a, ,3 and iota include an assimilable source of nitrogen such as protein, protein hydrolysate, polypeptides, amino acids, corn steep liquor, etc., and inorganic anions and cations, such as potassium, sodium, ammonium, calcium, sulfate, carbonate, phosphate, chloride, etc. Trace elements such as boron, molybdenum, copper, etc., are supplied as impurities of other constituents of the media. Aeration in tanks and bottles is provided by forcing sterile air through or onto the surface of the fermenting medium. Further agitation in tanks is provided by a mechanical impeller. An antifoaming agent such as lard oil or silicone defoamer may be added as needed.
Actinomadura yumaense is grown and maintained on agar slants, for example Bennett's agar,
Yeast Malt Agar, or ATCC Medium #172. ATOC Medium #1 72 is preferred. The slant is inoculated with a culture ofActinomadura yumaense and incubated at 28--370C, preferably at about 320C, for approximately 7 days. These stock cultures may be maintained by serial transfers to fresh agar slants, or a plug of the agar containing mycelia from the well-grown agar slant may be used to inoculate liquid media.
Shake flask inoculations of Actinomadura yumaense are prepared by inoculating 100 ml. of sterile liquid medium in 500 ml. flasks with scrapings or washings of spores from an agar slant of the culture.
Examples of suitable seed media are:
Medium A
Beef extract 0.3% BactoB tryptonel 0.5%
Glucose 1.0%
Yeast extract 0.5%
Water qs 100% [1A peptone, registered trademark of Difco Laboratories, Detroit, Michigan]
The pH is adjusted to 6.8-7.2 with dilute base, e.g. sodium hydroxide.
Medium B
Glucose 1% Starch 2%
Yeast extract 0.5% N-Z Amine A2 0.5%
Calcium carbonate 0.1%
Water qs 100% [2Casein digest, registered trademark of Sheffield Chemical Co., Div. Nat'l. Dairy Products Corp.,
Norwich, N.Y.]
Medium B is preferred.
The flasks are incubated at a temperatre of 25--350C., preferably at 320C., and agitated vigorously on a rotary shaker for 1-4 days. This seed inoculum is then used to inoculate fermentation culture, or this culture may be frozen and stored to provide inoculum for subsequent seed cultures.
The following media are examples of those suitable for the fermentation of Actinomadura yumaense to produce antibiotic LL-C23024a, , and iota.
Medium C
Glucose 1.5%
Glycerol 1.5%
Soy flour3 1.5%
Calcium carbonate 0.1% Sodium chloride 0.3%
Water qs 100% [3May be substituted by cottonseed flour or meat solubles with equal effect.]
Medium D
Glucose 3%
Soy flour 1.5%
Calcium carbonate 0.1%
Sodium chloride 0.3%
Water qs 100% Medium E
Starch 1% Molasses 2%
Soy flour 1.5%
Calcium carbonate 0.1%
Water qs 100% Medium F
Glucose 3%
Meat solubles 2.5%
Sodium chloride 0.2%
Calcium carbonate 0.1%
Water qs 100% Medium G
Glucose 3%
Soy flour 0.5% Ammonium sulfate 0.3%
Sodium chloride 0.2%
Calcium carbonate 0.1%
Water qs 100% Medium H
Glucose 3%
Ammonium sulfate 0.3%
Dibasic potassium phosphate 0.1%
Calcium carbonate 0.2%
Sodium chloride 0.1%
Water qs 100% Medium D is preferred.
The fermentation may be carried out in 100 mis. of media in a 500-ml. flask inoculated with 310% (v/v) of a seed culture prepared as described above and incubated at 25 C-35 C, preferably at about 32 C, for 24-72 hours with aeration. Samples of the fermentation culture may be frozen and stored for later use as inoculum for seed cultures.
Alternatively, the fermentation may be carried out in larger fermentation tanks equipped with aeration and agitation means. Each tank is inoculated with 3-1 0% (v/v) of inoculum prepared as described above. Aeration is supplied at the rate of 0.5 to 2.0 liter of sterile air per liter of broth per minute and the fermenting medium is agitated by an impeller driven at 200-400 rpm. The temperature is maintained at 25-35 C, preferably at 32 C. The fermentation is continued until antibiotic accumulates in the fermentation medium, usually after 100-1 50 hours, at which time the antibiotic is harvested.
The antibiotic may be harvested and purified according to the methods described in U.S. Patent 4,278,663, supra, or according to the following procedure:
The crude fermentation broth containing the whole cells, prepared as described above, is mixed with an equal volume of any non-hydrocarbon water-immiscible organic solvent. Methylene chloride or ethyl acetate is preferred. The organic phase is separated and concentrated in vacuo to an oily syrup.
The oily syrup is dissolved in methylene chloride and added to a column of silica gel, alumina,
Sephadex LH-20 (Pharmacia Fine Chel licals-Div. of Pharmacia, Inc., Piscataway, N.J.), or magnesium aluminum silicate. Examples of suitable solvents for developing the column are diethyl ether, ethyl acetate, a 1:1 to 1:7 (v/v) mixture of methylene chloride:ethyl ether, 1 00% acetone in methylene chloride, 2-10% lower alcohol (methanol is preferred) in methylene chloride, 2-15% acetonitrile in methylene chloride, or 2-1 5% dioxane in methylene chloride. Methylene chloride: ethyl acetate 1:1 (v/v) is preferred. Fractions are collected and checked for the presence of antibacterial activity by bioassay against a susceptible organism, e.g. Bacillus subtilis.Active fractions are combined and concentrated in vacuo to a residue. This residue is dissolved in an organie solvent, e.g. t-butanol (preferred):benzene, orp-dioxane, and freeze-dried.
The freeze-dried solid is dissolved in an appropriate organic solvent, e.g. methylene chloride, hexane, methylene chloride:ethyl acetate, diethyi ether, hexane:ethyl acetate, hexane:chloroform, or hexane:ether. Diethyl ether is preferred. This solution is shaken with water and the pH is adjusted to DH 1.5-4.0, preferably about 2.5, with any dilute mineral acid. The organic phase is separated, washed with water to remove any excess acid, dried over an appropriate drying agent, filtered, and concentrated to a residue in vacuo.
This residue is dissolved in an appropriate solvent and the solution is allowed to evaporate slowly, preferably at about 4 C. Examples of suitable solvents are methylene chloride, hexane, methylene chloride:ethyl acetate, diethyl ether, hexane:ethyl acetate, hexane:chloroform, or hexane:ether.
Hexane:ether 5:2 (v/v) is preferred. The resulting crystals are collected and washed, preferably at about 40 C. with any moderately boiling hydrocarbon such as for example hexane or heptane, and air dried to yield as the final product the antibiotic X14868A in the free acid form.
If the product is desired in the form of a salt, the free acid may be converted by treatment with the appropriate cation, preferably in the form of a dilute mineral base, according to procedures well-known to those skilled in the art.
The following Examples illustrate the invention. The Media A, B, C, etc. are those defined above.
Unless otherwise specified, all procedures were performed at room temperature (approximately 220C) and at 1 atm pressure.
EXAMPLE 1 Washed spores from an agar slant ofActinomadura yumaense NRRL 1251 5 were used to inoculate a 500 ml. flask containing 100 ml. of sterile Medium B. The flask was incubated on a rotary shaker at 280C. for 2 days.
A 5% inoculum of this culture was then transferred to 100 mls. sterile Medium C in a 500 ml.
flask and incubated at 28 C for 5 days on a rotary shaker.
The presence of antibiotic activity was monitored daily by bioassay against Staphylococcus aureus ATOC 6538 P, Bacillus subtilis, and Streptococcus faecalis, and anthelmintic activity was monitored by bioassay against the free-living nematode Caenorhabditis elegans.
EXAMPLE 2
Seven 500 ml. flasks were prepared, each containing 100 mls. of one of the following sterile
media:
Flask 1: 100 ml. Medium C
Flask 2: 100 ml. Medium C with 1.5% cottonseed flour substituted for the soy flour
Flask 3:100 ml. Medium C with 1.5% meat solubles substituted for the soy flour
Flask 4: 100 ml. Medium D
Flask 5: 100 ml. Medium E
Flask 6: 100 ml. Medium F
Flask 7: 100 ml. Medium G
These flasks were each inoculated with a 5% inoculum of Actinomadura yumaense NRRL 12515
growth in Medium B. The flasks were then incubated on a rotary shaker at 280C for four to six days.
Each of the above cultures was found to be active when assayed for antibiotic activity as in
Example 1 and when assayed for anticoccidial activity against Eimeria tenella in chick kidney tissue
cultures.
EXAMPLE 3
Frozen fermentation culture cells of Actinomadura yumaense NRRL 12515 were used to
inoculate a 500 ml. flask containing 100 ml. sterile Medium B. The flask was then incubated at 320C.
For 4 days on a rotary shaker.
A 5% inoculum of this culture was then transferred to 100 ml. sterile Medium H in a 500 ml. flask
and incubated at 280C for 5 days on a rotary shaker.
Antibacterial activity was confirmed as in Example 1 and anticoccidial activity as in Example 2.
The presence of antibiotic activity was also assayed by thin layer chromatography on silica gel
plates developed in ethyl acetate:chloroform 70:30 (v/v).
EXAMPLE 4
One hundred milliliters sterile Medium A in a 500 ml. flask was inoculated with washed spores from an agar slant of Actinomadura yumaense NRRL 1251 5. The flask was incubated at 320C for 2 days on a rotary shaker.
A 5% inoculum of this culture was then transferred to 100 ml. sterile Medium H in a 500 ml. flask
and incubated on a rotary shaker at 320C for 2 days.
A 5% inoculum of this culture was then transferred to a 500 ml. flask containing 100 ml. fresh sterile 'Medium H and incubated at 280C for 6 days on a rotary shaker.
Antibacterial activity was confirmed as in Example 1.
EXAMPLE 5
Two 500 ml. flasks each containing 100 ml. sterile Medium A were inoculated with a frozen seed
culture of Actinomadura yumaense NRRL 1251 5 and incubated at 320C for 2 days on a rotary shaker.
The contents of the two flasks were then combined and added to 12 liters of fresh sterile Medium .A in a 20 liter bottle. This culture was then incubated for 2 days at 280C with aeration.
The contents of this bottle were then transferred to a 300 liter seed tank containing 288 liters
sterile Medium A and this culture was aerated and agitated for 25 hours at 320C.
At the end of 25 hours' incubation, the 300 liters of seed culture were transferred to a 1 500 liter
fermentor containing 1 200 liters of sterile Medium C. This culture was incubated with aeration and
agitation for 11 5 hours at 280C.
The fermentation broth was assayed for antibiotic activity by thin-layer chromatography on silica
gel plates developed in ethyl acetate:chloroform 70:30 (v/v).Alternatively, several of the developed
plates were subjected to bioassay against Bacillus subtilis which showed the presence of antibiotic
activity.
EXAMPLE 6
A typical medium used to grow the primary inoculum was prepared according to the following
formula:
Beef extract 0.3% BactoB-trytone 0.5%
Glucose 1.0%
Yeast extract 0.5% Bactoagar 0.15% Water qs 100%
Washed or scraped spores and mycelia from an agar slant of Actinomadura yumaense
NRRL 1251 5 were used to inoculate a 500 ml. flask containing 100 ml. of the above sterilized medium.
The flask is placed on a rotary shaker and agitated vigorously for 48 hours at 320C. The resulting flask
inoculum (100 ml.) is used to inoculate one liter of the same sterile medium in a 2 liter bottle. This
inoculum is aerated with sterile air while growth is continued for 48 hours at 28"C. This one-liter
culture was then used to inoculate a 30-liter fermentor tank containing the same sterile medium, and this tank was aerated with sterile air and incubated at 280C for 42 hours.
EXAMPLE 7
A fermentation medium was prepared according to the following formula:
Dextrose 1.5%
Glycerol 1.5%
Soy flour 1.5%
Calcium carbonate 0.1% Sodium chloride 0.3%
Water qs 100%
The pH was adjusted to 7.0 with 6N sodium hydroxide and the medium was sterilized. A 30 liter portion of inocullum prepared as described in Example 1 was used to inoculate 250 liters of the above
medium in a 300 liter fermentor. Sterile aeration was supplied to the mash and the mash was agitated
by a impeller driven at 220 rpm. Fermentation was carried out at 320 C. for 97 hours at which time the
mash was harvested.
EXAMPLE 8
A fermentation medium was prepared according to the following formula:
Dextrose 3.0 /0 Soy flour 1.5%
Calcium carbonate 0.1%
Sodium chloride 0.3%
Water qs 100%
The pH was adjusted to 7.0 with 6N sodium hydroxide and the medium was sterilized. A 300 liter portion of inocuium prepared as described in Example 1 was used to inoculate 3000 liters of the above medium in a fermentor. Sterile aeration was supplied to the mash. The mash was agitated by an impeller driven at 100 rpm. Fermentation was carried out at 320C for 11 5 hours at which time the mash was harvested.
EXAMPLE 9 1. The fermentation mash (1001.) was adjusted to pH 4.0 using 6N HCI. The acidic mash was then stirred for 1 to 2 hr. with an equal volume of methylene chloride. The mixture of mash and methylene chloride was then filtered using diatomaceous earth as a filter aid. The methylene chloride extract was drawn off and concentrated in vacuo to approximately 2 I. of partially purified antibiotics.
2. Chromatography of the Partially Purified Antibiotics on Silica Gel
A glass column with a diameter of 7 cm. was packed to a height of 91 cm. with Woelm Silica Gel
TSC. The crude concentrate (See 1 above) with a volume of approximately 2 1. was allowed to seep into the column of silica gel. The column was then first developed with 3 I. of methylene chloride, followed by methylene chloride: ethyl acetate (1 :1 by volume) to give a total of 126 fractions, each with a volume of 80 ml. This was then developed further using ethyl acetate-ethanol (7:3) to give an additional 87 fractions.
Fractions, 58 to 87, rich in C23024a, were combined and concentrated in vacuo to a residue weighing 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. The clear filtrate was concentrated in vacuo until crystals started to form. This suspension was allowed to age overnight. The crystalline C23024 was collected on a funnel, washed with cold ether and air dried to give 33.1 g. of crystalline C23024a. An additional 12.4 g. of C23024ar was obtained upon further reduction in volume of the
combined was and filtrate.
Fractions 1 77 to 203 from elution with ethyl acetate-ethanol and enriched in C23024 were combined and concentrated in vacuo to give 6.7 g. of partially purified C23024P and treated as above.
Fractions 204 to 21 3 from elution with ethyl acetate-ethanol and enriched in LL-O23024 iota were combined and concentrated in vacuo to a paste and repeatedly extracted with methanol. The methanol extract was extracted with methylene chloride which was charged on a column containing
Woelm silica gel. The column was washed with methylene chloride and then eiuted with methylene chloride:ethyl acetate (2:3). The cuts were monitored by thin layer chromatography and the active fractions were pooled, treated with charcoal, concentrated and repeatedly extracted with heptane. The final heptane extract was chilled at OOC. for 48 hours and the crystals removed by filtration from the mother liquor.
This mother liquor was dissolved in methylene chloride and allowed to seep into a glass column packed with Woelm silica gel. The column was then eluted in succession with 2 liters of methylene, 8 liters of methylene chloride:ethyl acetate (1 :1) and 4 liters of ethyl acetate:methanol (9:1). The eluate was collected in fractions and monitored for their antibiotic composition. The active fractions were combined and concentrated in vacuo to give a residue containing LL-O23024 iota.
EXAMPLE 10 3. Further Purification of C23024P from Chromatography on Silica Gel SephadexB LH20 was allowed to swell in a mixture of hexane-methylene chloride-methanol (10:5:1). A glass column with a diameter of 5 cm. was packed to a height of 86 cm. with the gel. The charge, 3 g. of purified LL--C23024P was dissolved in 10 ml. of methylene chloride, 1 ml. of methanol and 10 ml. of hexane and allowed to seep into the column of gel. The column 3 was then developed with solvent with the same composition as that used to deliver the antibiotic to the column. Fractions were collected using a coilector. The first 23 fractions had a volume of 1 7 ml. each. Fractions 17 to 26 contained C23024p by TLC.These fractions were combined and concentrated in vacuo to give pure amorphous LL-C23024p, weighing 1269 mg.
EXAMPLE 11
Crystallization ofLL--C23024P. as Sodium Salt
Purified LL-C23024 (2.4 g.) prepared, as described in sections B and C was, dissolved in a mixture of diethyl ether (500 ml.) hexane (160 ml.) and methylene chloride (25 ml.). The resulting solution was transferred to a separating funnel containing 300 ml. of distilled water. Dilute HCI (IN) was added dropwise until the pH reached 2.0 to 2.5 after shaking and settling. The acidic aqueous phase was discarded and the acid treated organic layer was washed three successive times with 400 ml.
volumes of water. The washed organic layer was stirred with a teaspoon to Darco G-60 powder for
1 5 min. and filtered through celite. The decolorized organic layer was placed over 500 ml. of distilled water and 5N NaOH was added dropwise until the pH reached 11.0 to 11.5 after shaking and settling.
The alkaline water phase was discarded and the remaining organic layer was washed twice with portions of water having a volume of 400 ml. The washed organic layer was dried over sodium sulfate and then concentrated in vacuo to a volume of 1 50 ml. This concentrate was allowed to stand in a hood for several hours. The crystalline LL-C23024p was collected on a funnel, washed with cold hexane and air dried to give 402 mg. of the crystalline salt of LL--C23024P. This sample was sent for microanalyses and selected spectral data.
Anal. Calculated for C46H770,7Na.C,59.70; H,8.33; 0,29.46; Ash, 2.49. C, 61.55,H,8.79; Ash, 3.31; N,O. MP.(Fisher-Johns Apparatus= 174 (FDMass Spec) = 924 [a]26 = +3.2 in methanol).
EXAMPLE 12
Purification of LL-C23O24 iota
A Water Prep. 500A high performance liquid chromatography instrument was fitted with a silica gel cartridge under a pressure of 550 psi. The charge, which was 5.0 g. of the crude material from
Example 3, was dissolved in 50 ml. of heptane:ethyl acetate (45:55) and injected onto the column of silica gel. The column was then developed with the same solvent mixture at a flow rate of 200 ml/minute with collection of forty 200 ml. fractions. Fractions 21-32 were combined, concentrated to a residue, triturated with hexane and evaporated, giving pure LL-C23024 iota. The above procedure was repeated four times giving a combined total of 280 mg. of amorphous LL-C23024 iota.
A 90 mg. portion of amorphous LL-C23024 iota was dissolved in 10 ml. of diethyl ether, mixed with 10 ml. of water, and adjusted to pH 2.5 with 0.1 N hydrochloric acid. The ether layer was washed with water, adjusted to about pH 11 with 0.1 N sodium hydroxide, separated, and again washed 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 an amorphous white solid.
This amorphous white solid has the following properties:
Elemental analysis: C, 61.79; H, 8.84; ash, 0.32; [a]26 = +27 (C 0.724, methanol);
Field desorption mass spectrometry: (M+Na)+ = m/e 953, therefor the calculated molecular weight is
930.
Claims (11)
1. A biologically pure culture ofActinomadura yumaense sp. nov., said culture being capable of producing the antibiotic LL-O23024a, , and iota in recoverable quantities upon fermentation in a liquid nutrient medium containing assimilable sources of carbon, nitrogen, and inorganic salts.
2. The biologically pure culture of Claim 1, wherein saidActinomadura yumaense sp. nov. is
Actinomadura yumaense NRRL 1251 5.
3. A biologically pure culture of the microorganism Actinomadura yumaense sp. nov. according to
Claim 1 or 2, wherein said microorganism has spontaneously mutated such that the microorganism is genetically altered but still retains the ability to synthesize antibiotic LL-C23024a, , and iota.
4. A biologically pure culture of the microorganism Actinomadura yumaense sp. nov. according to
Claim 1 or 2, wherein said microorganism has been subjected to mutagenic means such that the microorganism is genetically altered but still retains the ability to synthesize antibiotic LL-C23024α, ss, and iota.
5. The antibiotic LL-C32024ss wherein the substantially pure form: (a) has an optical rotation [ g]a5 of +320 + 2 (0.495%, methanol) and [ar]o25 of +460 + 2 (0.440%, chloroform);
(b) has an elemental analysis (percent) of about: C, 61.55; H, 8.79;
(c) has a melting point of about 171173aC; (d) has a molecular weight by field desorption mass spectroscopy of 902;
(e) has a carbon-1 3 nuclear magnetic resonance spectrum in deuterated chloroform at 80 MHz substantially as shown in Figure II.
(f) has an infrared absorption spectrum in KBr substantially as shown in Figure l; and
(g) has a proton nuclear magnetic resonance spectrum in deuterated chloroform at 20MHz substantially as shown in Figure III.
6. A pharmaceutically acceptable acid-addition salt of antibiotic LL-C23024ss according to Claim 5.
7. The antibiotic LL-C23024 iota wherein the substantially pure form:
(a) has an optical rotation [aiD26 = +27 (C 0.724, methanol);
(b) has an elemental analysis (percent of about: C, 61.79; H, 8.84;
(c) has a molecular weight by field desorption mass spectroscopy of 930; and
(d) has a 13C-nuclear magnetic resonance spectrum in deuterated chloroform at 80 MHz substantially as shown in Figures VI, VII, and VIII.
(e) has a infrared adsorption spectrum in KBr substantially as shown in Figure IV; and
(f) has a proton nuclear magnetic resonance spectrum in deuterated chloroform at 20MHz substantially as shown in Figure V.
8. A pharmaceutically acceptable acid-addition salt of antibiotic LL-C23024 iota according to
Claim 7.
9. A process for the preparation of antibiotic LL-C23024a, ,3 and iota which comprises aerobically fermenting Actinomadura yumaense sp. nov. or a mutant thereof in a liquid medium containing assimilable sources of carbon, nitrogen, and inorganic salts, until substantial antibiotic activity is
imparted to the fermentation broth and then recovering the antibiotic therefrom.
1 0. A process according to Claim 9 wherein the temperature of the fermentation culture is
maintained at 25 35 C. for a period of 24-1 50 hours.
11. A process according to Claim 9 or 10 wherein the.Actinomadura yumaense sp. nov. is
Actinomadura yumaense NRRL 1251 5.
1 2. A method for the control of protozoan infections in warm-blooded animals comprising orally
administering to the animals a protozoacidally effective amount of the compound LL-C23024ss or LL-C23024 iota.
1 3. A method according to Claim 12 wherein the warm-blooded animas are meat-producing
animals, the infection is coccidiosis and the anticoccidial agent is administered to the animals in the
feed or drinking water containing 2.5 to 120 ppm of the anticoccidial agent.
1 4. A composition for the control of coccidiosis infections in poultry comprising an edibie carrier
and about 0.000025% to about 0.06% by weight of the compound LL-C32024, LL-C23024 iota or
the pharmaceutically acceptable salts thereof.
1 5. A composition according to Claim 14, wherein the edible carrier is drinking water and it
contains from 2.5 to 120 ppm of the anticoccidial agent.
1 6. A method of controlling nematodes in warm-blooded animals and in soil containing the
nematodes by introducing orally into the animals or applying to nematode-infested soil, a nematocidally
effective amount of a compound LL-C32024a, LL-C23024 or pharmaceutically acceptable salts of
the compounds, mixtures of the compounds or salts, or fermentation broths or harvest mash solids from
which the nematocidal antibiotics are obtained.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US06/313,849 US4407946A (en) | 1981-10-22 | 1981-10-22 | Process for producing antibiotic X-14868A |
US37278482A | 1982-04-28 | 1982-04-28 |
Publications (2)
Publication Number | Publication Date |
---|---|
GB2108113A true GB2108113A (en) | 1983-05-11 |
GB2108113B GB2108113B (en) | 1985-08-07 |
Family
ID=26979078
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
GB08225339A Expired GB2108113B (en) | 1981-10-22 | 1982-09-06 | Antibacterial agents ll-c23024b and iota and microorganism actinomadura yumaense nrrl 12515 |
GB08426115A Expired GB2147808B (en) | 1981-10-22 | 1984-10-16 | Controlling nematodes in soil or animals |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
GB08426115A Expired GB2147808B (en) | 1981-10-22 | 1984-10-16 | Controlling nematodes in soil or animals |
Country Status (17)
Country | Link |
---|---|
JP (1) | JPH0824593B2 (en) |
AR (1) | AR229058A1 (en) |
AU (1) | AU558914B2 (en) |
CA (1) | CA1198386A (en) |
CH (1) | CH661283A5 (en) |
DE (1) | DE3238316A1 (en) |
DK (1) | DK161332C (en) |
ES (1) | ES516718A0 (en) |
FI (1) | FI75187C (en) |
FR (1) | FR2515207B1 (en) |
GB (2) | GB2108113B (en) |
HU (1) | HU190814B (en) |
IE (1) | IE54813B1 (en) |
IL (1) | IL66671A (en) |
IT (1) | IT1197433B (en) |
NL (1) | NL194396C (en) |
SE (2) | SE456588C (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GR78648B (en) * | 1982-07-26 | 1984-09-27 | Bristol Myers Co | |
DE102006028817A1 (en) * | 2006-06-21 | 2007-12-27 | Evonik Degussa Gmbh | Processing of Reaction Solutions from Whole Cell Biotransformations |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4278663A (en) * | 1980-01-30 | 1981-07-14 | Hoffmann-La Roche Inc. | Antibiotic X-14868A, B, C and D |
-
1982
- 1982-08-18 CA CA000409648A patent/CA1198386A/en not_active Expired
- 1982-08-29 IL IL66671A patent/IL66671A/en not_active IP Right Cessation
- 1982-09-06 GB GB08225339A patent/GB2108113B/en not_active Expired
- 1982-10-15 DE DE19823238316 patent/DE3238316A1/en active Granted
- 1982-10-18 DK DK462082A patent/DK161332C/en active
- 1982-10-20 AR AR291028A patent/AR229058A1/en active
- 1982-10-20 IT IT49317/82A patent/IT1197433B/en active
- 1982-10-21 FI FI823603A patent/FI75187C/en not_active IP Right Cessation
- 1982-10-21 IE IE2542/82A patent/IE54813B1/en not_active IP Right Cessation
- 1982-10-21 NL NL8204069A patent/NL194396C/en not_active IP Right Cessation
- 1982-10-21 AU AU89653/82A patent/AU558914B2/en not_active Expired
- 1982-10-21 SE SE8205992A patent/SE456588C/en not_active IP Right Cessation
- 1982-10-21 CH CH6216/82A patent/CH661283A5/en not_active IP Right Cessation
- 1982-10-21 ES ES516718A patent/ES516718A0/en active Granted
- 1982-10-21 SE SE8205992D patent/SE8205992L/en not_active Application Discontinuation
- 1982-10-21 HU HU823370A patent/HU190814B/en not_active IP Right Cessation
- 1982-10-22 FR FR8217727A patent/FR2515207B1/en not_active Expired
-
1984
- 1984-10-16 GB GB08426115A patent/GB2147808B/en not_active Expired
-
1992
- 1992-04-08 JP JP4114181A patent/JPH0824593B2/en not_active Expired - Lifetime
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Legal Events
Date | Code | Title | Description |
---|---|---|---|
732E | Amendments to the register in respect of changes of name or changes affecting rights (sect. 32/1977) | ||
PE20 | Patent expired after termination of 20 years |
Effective date: 20020905 |