GB2090243A - Polycyclic ethers "antibiotics" - Google Patents

Abstract

The polycyclic ether antibiotics designated UK-41,637 and UK-44,579, their free acids and non-toxic cationic salts in addition to sodium, mixtures of them and dried fermentation broths containing them, are produced by culturing a strain of Actinomadura cremea. UK-41,637 is a sodium salt of the formula:- <IMAGE> UK-44,579 is a sodium salt with a close structural relationship to UK-41,637. It has a melting point of 90-93 DEG C, an optical rotation of alpha <25>589=37.4 DEG (c, 0.1, CH3OH), and is also characterised by its i.r. (KBr), <1>H and <13>CC n.m.r. spectra.

Description

SPECIFICATION Polycyclic ethers This invention is concerned with new members of the acidic polycyclic ether group of antibiotics, a ciass of compounds characterized biologically by their effect on cation transport in mitochondria. This family of antibiotics includes monensin (J. Amer. Chem. Soc., 89:5737, 1967); nigericin (Biochem.

Biophys. Res. Comm., 33:29, 1968); grisorixin (J. Chem. Soc. Chem. Commun., 14211970); dianemycin (J. Antibiotics, 22:161, 1969); salinomycin (J. Antibiotics, 27:814, 1974); X-537A (J.

Chem. Soc. Chem. Commun., 967, 1972); X-206 (J. Chem. Soc. Chem. Commun., 927, 1971); and A204A (J. Amer. Chem. Soc., 95:3399, 1973).

The polycyclic ether antibiotics listed above are active against Gram-positive bacteria, fungi and protozoa. They exhibit potent anticoccidial activity.

U.S. Pat. No. 3,839,557 describes a process for the improvement of feed utilization by ruminants and monogastric animals fed on fibrous vegetable matter and administered monensin, dianemycin, nigericin or other polycyclic ether antibiotics. U.S. Patent Nos. 4,062,945, 4,129,578 and 4,150,152 describe various polycyclic ether antibiotics and their utilities.

According to the invention there are provided polycyclic ether antibiotics which Pfizer have designated UK-41637 and UK-44579. UK-41637 has the formula:

The structure of UK-44579 has not been completely elucidated at the present time. It is, however, a sodium salt with a close structural relationship to UK-41637.

The invention also provides the free acid forms of UK-41637 and UK-44579, and their non-toxic cationic salts in addition to sodium, such as the calcium, potassium, lithium, magnesium, copper, zinc, ammonium and silver salts.

The antibiotics are produced by the submerged aerobic propagation ofActinomadura cremea ATCC 31676 in an aqueous nutrient media and may be separated from each other by chromatography.

This culture was isolated from a soil sample from Japan. it was deposited with the A'american Type Culture Collection at Rockville, Maryland, U.S.A. on 18th August, 1980.

The compounds of the invention are active against a variety of microorganisms and stimulate the production of rumen propionic acid in ruminants, and thus have potential utility as improvers of feed conversion efficiency and growth promotants in ruminant species. The compounds also possess insecticidal and larvicidal activity.

The microorganism useful for the preparation of the antibiotics of the invention was isolated from a soil sample obtained from Japan. It was characterized by the white aerial mycelium, the cream substrate mycelium, the warty spores which are arranged in a flexuous, hooked, looped, or coiled (once) chains, and the presence of mesodiamino-pimelic acid and madurose in the whole-cell hydrolysates.

The culture was considered to represent a new strain of Actinomadura cremea.

An inoculum was prepared by planting the culture from a slant into ATCC &num; 172 broth and growing for 4 days at 280C on a shaker. It was then centrifuged for 20 minutes, washed three times with sterile distilled water, and planted on media commonly used for identification of members of the Actinomycetales. The culture was incubated at 280C and the results were read at varying time but most commonly were recorded at 13 or 14 days. The colours were described in common terminology, but exact colours were determined by comparison with colour chips from the ColourHarmonyManual, fourth edition.

The methods of whole-cell amino acid and sugar analysis were those described in Becker, B. et al, Appl. Microbiol., 12:421--423, 1964; and in Lechevalier, M. P., J. Lab. Clin. Med., 71:934-944, 1 968. Identification media used for the characterization of the culture and references for its composition were as follows: 1. Tryptone Yeast-Extract Broth -- (ISP &num; 1 medium, Difco).

2. Yeast Extract-Malt Extract agar -- (ISP # 2 medium, Difco).

3. Oatmeal Agar - (ISP &num; 3 medium, Difco).

4. Inorganic Salts-Starch agar - (ISP # 4 medium, Difco).

5. Glycerol-Asparagine Agar - ISP &num; medium, Difco).

6. Peptone-Yeast Extract Iron Agar -- (ISP &num; medium, Difco).

7. Czapek-Sucrose Agar - S. A. Waksman, The Actinomycetes, Vol. 2, medium No. 1, p. 328, 1961.

8. Glucose-Asparagine Agar - Ibid, medium No. 2, p. 238.

9. Bennett's agar - Ibid, medium No. 30, p. 331.

10. Emerson's agar - Ibid, medium No. 28, p. 331.

11. Nutrient Agar - Ibid, medium No. 14, p. 330.

12. cordon and Smith' Tyrosine Agar -- R. E. Gordon and M. M. Smith, Jr. Bact. 69:147-150, 1955.

13. Cesein Agar - Ibid.

14. Calcium Malate Agar - S. A. Waksman, Bact. Rev. 21:1-29, 1957.

15. Gelatin - R. E. Gordon and J. M. Mihm, Fr. Bact. 73: 5-27, 1957.

16. Starch -- Ibid.

1 7. Organic Nitrate Broth - Ibid.

18. Dextrose Nitrate Broth - S. A. Waksman, The Actinomycetes, Vol. 2, medium No. 1, p. 328, 1961, with dextrose substituted for 30 g sucrose and agar omitted.

19. Potato Carrot agar - M. P. Lechevalier, J. Lab. and Clinical Med. 71: 934-944, 1 968 but use only 30 g potatoes, 2.5 g carrots and 20 g agar.

20. 2% Tap Water Agar.

21. Gauzes &num; 1 Mineral Agar - G. G. Gauze et a/., Problems in the Classification of Antagonistic Actinomycetes. English Ed., p. 13, 1957.

22. Gauze's # 2 Organic Agar - Ibid.

23. Skim Milk -- Difco.

24. Cellulose utilization- (a) H. L. Jensen, Proc. Linn. Soc. N.S.W. 55:231-248, 1930.

(b) M. Levine and H. W. Schoenlein, A. Compilation of Culture Media, medium No. 2511, 1930.

25. Carbohydrates -- C-2 Medium, H. Nonomura and Y. Ohara, J. Ferment. Technol.

49(11)887-894,1971.

26. Temperature Range - ATCC medium 172 in ATCC Culture Collection Catalogue. 14th ed., p. 518, 1980.

The culture was described as follows on the various media: Yeast Extract-Malt ExtractAgar- Growth good, white to pale yellowish (1 2 ca), raised, wrinkled, with white aerial mycelium; reverse pale yellowish (2 ea); no soluble pigment.

OatmealAgar- Growth moderate, off-white to very pale yellowish (near gray series I ba to 2bal, thin, emooth, with sparse, white aerial mycelium; reverse same as-surface; no soluble pigment.

Inorganic Salts-Starch Agar -- Growth moderate, white to pale yellowish (1 i ca), thin, smooth, with whirs aerial mycelium; reverse cream to pale yellowish (2 ca to 2 ea); no soluble pigment.

Glycérole-Asparagine Agar-Growth moderate to good, pale cream (near gray series 1 ba), slightly raised, smooth, sparse aerial mycelium observed only under the microscope; reverse same as surface no soluble pigment Gordon and Smith'Tyrosine Agar--Growth moderate to good grayish brown to brown (41 g to 51 g), slightly raised, smooth to slightly wrinkled, no aerial mycelium; reverse yellowish brown (31 c); soluble pigment dark brown (4 pl to pn).

Czapek-SucroseAgar- Growth moderate, very pale yellowish (near gray series 1 ba), thin, smooth, sparse aerial mycelium observed only under the microscope; reverse same as surface; no soluble pigment.

Glucose-AsparagineAgar- Growth moderate to good, off-white to pale yellowish 1 ca), thin to slightly raised, smooth but may be wrinkled near the end of the streak, no aerial mycelium; reverse pale cream (1+ ca); no soluble pigment.

Calcium MalateAgar- Growth poor, colourless to off-white, thin, smooth, no aerial mycelium; reverse same as surface; no soluble pigment.

Casein Agar-- Growth good, pale cream (2 ca), slightly raised, smooth to slightly wrinkled, no aerial mycelium; reverse pale yellowish (2 ea); soluble pigment gold (21 c).

Bennett's Agar -- G rowth good, white to pale yellowish (1 2 ca); slightly to moderately raised, wrinkled, with white aerial mycelium); reverse pale yellowish (12 ca); soluble pigment pale yellowish (1+ ea).

Ernerson'sAgar- Growth good, white to pale yellowish (1+ ca); 2 ca to 2 ea), raised, wrinkled; aerial mycelium white, sparse; reverse pale yellowish (2 ea to 2 ga); no soluble pigment.

NutrientAgar Growth moderate to good, pale yellowish (1 2 ca to 2 ca), slightly raised, smooth to slightly wrinkled, no aerial mycelium; reverse same as surface; no soluble pigment.

elatThAgar- Growth good, pale yellowish, slightly raised, smooth but wrinkled near the edge; aerial mycelium white, sparse; reverse same as surface; soluble pigment pale yellowish (12 ca).

SterchAgar- Growth good, cream (1 - ca), raised, wrinkled, aerial mycelium white, sparse; reverse pale yellowish (12 ca); soluble pigment very pale yellowish (12 ca).

Potato CzrrotAgar -- Growth poor to moderate, off-white to very pale yellowish (near gray series 1 ba), thin, smooth, with off-white aerial mycelium; reverse same as surface; no soluble pigment.

Tap WaterAgar- Growth poor, colourless to off-white, thin, smooth, aerial mycelium off-white, sparse; reverse same as surface; no soluble pigment.

Gauze's # 1 Mineral - Growth moderate to good, white to cream (1 T ca), thin, smooth, with white aerial mycelium; reverse cream; soluble pigment very pale yellowish (near gray series 1 ba).

Gauze # 2 Organic Agar -- Growth good, pale yellowish (1 + ca to 1 2 ea), raised, wrinkled, no aerial mycelium; reverse same as surface; no soluble pigment.

The culture's morphological properties were as follows:- The morphological observations were made on inorganic salts-starch agar after 1 6 days of incubation: spore colour in mass in White colour-series; spore chains flexuous, hooked, looped, or coiled once, rarely coiled twice, 3 to 1 2 spores per spore chain, with the upper portion often coiled into a globoid mass; sporophores monopodially branched; spores globose, oval to elliptical, 1--1.4 FLm in diam., of 1.2-1.8 (-2.0) x 0.9-1.2 um, warty, as revealed by a scanning electron microscopy.

The culture's biochemical properties were as follows:- 1. Melanin not produced; hydrogen sulfide produced; gelatin liquefied; starch hydrolyzed; nitrate reduced to nitrite on both media; very poor growth and no disintegration on both cellulose media; clearing and coagulation on milk; casein digestion positive; digestion of calcium plate negative; tyrosine digestion positive.

2. Carbohydrate utilization - Glucose, arabinose, fructose, glycerol, mannitol, rhamnose, sucrose, and xylose utilized; inositol, melibiose, and raffinose not utilized.

3. Cell wall analysis -- The whole-cell hydrolysates contain mesodiamino-pimelic acid, madurose, galactose, mannose and ribose.

Temperature relations of culture growth: 210C 280C 370C 450C Poor Excellent Excellent Good to growth growth growth excellent growth The culture was similar to Actinomadura helvata, A. pusilla,and A. cremea in the production of white aerial mycelium. It differed from A. helvata ATCC 27295 in the absence of pseudosporangia; warty rather than smooth spores; positive utilization of sucrose; and negative utilization of inositol, melibiose and raffinose. It differed from A. pusilla ATCC 27296 in the absence of pseudosporangia, warty rather than smooth spores, cream rather than brown to dark red substrate mycelium on some media, positive utilization of mannitol, and negative utilization of inositol. A comparison of the description of the culture with the of A. cremea INA 292 (T. P. Preobrazhenskaya, N..V. Lavrova, R. S.

Ukholina and N. P. Nechaeva, Antibiotiki 5: 404-409, 1975) indicated a close resemblance between the two cultures. The white aerial mycelium, the cream substrate mycelium, the morphology of the spore chains, the surface features of the spores, and failure to produce melanin were the same with both cultures. The culture was therefore considered as a new strain of Actinomadura cremea.

(Preobrazhenskaya, Lavrova, Ukholina and Nechaeva 1 975). The culture has been deposited with the American Type Culture Collection with the accession number ATCC 31 676.

Cultivation of Actinomadura cremea ATCC 31676 preferably takes place in aqueous nutrient media at a temperatura of 28-360C., and under submerged aerobic conditions with agitation. Nutrient media which are useful for such purposes include a source of assimilable carbon such as agars, starches and glycerol: a source of organic nitrogen such as casein, enzymatic digest of casein, soybean meal, cotton seed meal peanut meal, wheat gluten, soy flour, meat meal and fish meal. A source of growth substances such as grain solubles and yeast extract as well as salts such as sodium chloride and calcium carbonate and trace elements such as iron, magnesium, zinc, cobalt and manganese may also be utilized with advantageous results.If excessive foaming is encountered during fermentation, antifoam agents such as vegetable oils or silicones may be added to the fermentation medium. Aeration of the medium in tanks for submerged growth is preferably maintained at the rate of about T to 2 volumes of free air per volume of broth per minute. Agitation may be maintained by means of agitators generally familiar to those in the fermentation industry. Aseptic conditions must, of course, be maintained through the transfer of the organism and throughout its growth.

Inocululum for the preparation of the antibiotic may be obtained by employing growth from a slant of the culture. The growth may be used to inoculate either shake flasks or inoculum tanks or the inoculum tanks may be seeded from the shake flasks. Growth in shaken flasks will generally have reached its maximum in 3 to 5 days whereas inoculum in submerged inoculum tanks will usually be at the most favourable period in 2 to 3 days. Substantial antibiotic activity is obtained in the final fermentor stage in approximately 3 to 5 days. The antibiotic levels range from 10 to 100 mg. per litre.

The process of antibiotic production is conveniently followed during fermentation by biological assay of the broth employing a sensitive strain of Bacillus subtilis. Standard plate assay technique is employed in which the zone of inhibition surrounding a filter paper disc saturated with the broth is used as a measure of antibiotic potency.

Thin-layer chromatography employing silica gel is a useful tool for analyzing the antibiotics produced in fermentation media and the composition of crude and purified materials extracted from the fermentation broths. The Merck Kieselgel 60 F 254 silica gel chromatograms are developed with ethyl acetate. Antibiotic compound UK-41637 may be visualized by exposure to 254 nm light or by spraying with 3% vanillin in ethanolic sulphuric acid (97:3, v/v) where it shows up as a red spot on a white background after warming on a steam bath or a hot plafe. Bio-overlay with agar seeded with a sensitive strain of Staphylococcus aureus or Bacillus subtilis is a further procedure for detection of the antibiotic.

The antibiotic compound UK-41637 may be separated and recovered from the fermentation broth by several methods.

Compound UK-41637 can be separated and recovered from clarified fermentation broth by extraction with organic solvents such as chloroform, n-butanol, ethyl acetate or methylisobutylketone. A significant portion of the antibiotic resides on the separated mycelium and is conveniently extracted therefrom by slurrying with solvents such as methanol or butanol.

The antibiotic compound UK-41 637 can also be recovered by extraction of the whole (unfiltered) broth with water-immiscible solvents such as butanol, chloroform, ethyl acetate or methyl isobutyl ketone. Alternatively the whole (unfiltered) broth can be freeze-dried to remove water and the antibiotic compound obtained by extraction of the resulting freeze-dried solids with these solvents.

The preferred method of isolation of compound UK-41637 and the minor component UK-44579 from solvent extracts (obtained by any of the above mentioned processes) is as follows:- The solvent extract is concentrated under vacuum to an oily residue which is treated with petroleum ether (boiling range 6080C C) to remove insoluble contaminants. The petroleum ether solution is concentrated in vacuo to produce an oily residue which is then charged on a filter bed of silica gel (Merck Art. 7731) covering a further bed of silica gel (Malinkrodt CC7). The resulting silica gel column is successively developed with petroleum ether (60--800C range) then mixtures of petroleum ether (60--80"C range) containing increasing amounts of ethyl acetate.From the antibiotic -- rich fractions compound UK-41637 is obtained as a single entity by further chromatography on silica using a Waters Prep. 500 high pressure liquid chromatograph with hexane: ethyl acetate (1:1) as eluant. By this procedure UK-44579, the second structurally-related antibiotic, and present as a minor component (~10% of UK-41 637 titre) is also obtained. Antibiotic compound UK-41637 can then be crystallized from hexane.

The free acid forms of UK-41637 and UK-44579, and their other cationic forms, can be prepared by conventional procedures. The free acids are typically prepared by acidifying UK-41 637 or UK-44579 in a suitable solvent, e.g. chloroform, with dilute hydrochloric of phosphoric acid. After separation and drying of the organic phase, it is evaporated in vacuo to leave the acid. Fairly mild acidification conditions are Freferred to avoid degradation of the molecule. Similarly, cationic salts can be prepared by treating a solution of the appropriate free acid in a suitable organic solvent with e.g. potassium hydroxide, silver nitrate, etc.

Compounds UK-41637 and UK-44579 exhibit inhibitory action against the growth of a number of microorganisms. The results for UK-41637 are given in Table I below. Minimal inhibitory concentrations (MICs) were determined by using a standard agar plate technique. Organisms were grown in Iso sensitestbroth (Oxoid Ltd.) at 370C for 18 hours. Cultures were diluted 100-fold in Iso-sensitest broth and inoculated onto Iso-sensitest agar containing serial doubling dilutions of antibiotic, using a multipoint inoculator (Denley Instruments), and gave an inoculum of approximately 104 colony-forming units. After incubation at 370C for 1 8 hours, the MIC was recorded as the lowest concentration of antibiotic which completely inhibited visible growth of the organism.

TABLE I

j C.(gmI) Organism (Cpd. UK--41637) Strep. pyogenes strain 18 0.098 S. Lutea 0.195 Bacillus subtilis 0.195 Bacillus cereus 0.098 Comamonas terrigena j 1.56 Staph. aureus strain 222 0.39 strain 223 0.39 strain 267 0.39 strain 450 0.39 strain 451 0.39 strain 453 0.39 E. Coli strain 116 > 100 The insecticidal and larvicidal activity of the compounds can be illustrated by the following tests: Twenty females of the World Health Organization standard fully-susceptible strain of Musca domestic, 2 4 days old, are anaesthetized with carbon dioxide. One microliter of a solution containing the test compound is applied to the dorsal surface of the thorax. The flies are then maintained in gauzecovered pots at 250C. and approximately 50% relative humidity for 24 hours with a cotton wool pad moistened with sugar solution and placed on the gauze as food. Twenty flies are treated with solvent only and serve as controls. At the end of this period the mortality is noted and recorded as a percentage after correction for any mortality among the controls.

0.5 ml of compound test solution is pipetted evenly on to Whatman No. 1 filter paper (8 cm. x 6.25 cm) to give a deposit of 50 to 100 mg/m2. When dry, the paper is rolled and slid into a test tube to which added 1.5 ml of calf serum which is absorbed by the filter paper and serves as food.

Fifteen Lucilia sericata larvae, approximately 36 hours old, are introduced and the tube closed with a cotton wool plug and stored with the top part only in a strong light to keep the larvae in the iower part of the tube and thus in contact with the filter paper. The insects are maintained for 24 hours, and mortalities calculated. Fifteen larvae confined with untreated filter paper served as controls.

The test results for UK-41637 are tabulated in Table Il:- TABLE ll

Musca domestica | Lucilia sericata (adult) (larvae) Dose Dose (y)/female % Kill (mgm2) % Kill 1 80 100 100 50 50 100 25 87 12.5 17 The value of animal feeds generally has been determined directly by feeding the animal.Great Britain Patent No. 1,197,826 details an in vitro rumen technique whereby the changes occurring in feeds brought about by microorganisms are measured more readily and with great accuracy in the evaluation of animal feeds. This technique involves the use of an apparatus in which the digestive processes of the animals are conducted and studied in vitro. The animal feeds, rumen inoculum and various growth promotants are introduced into and withdrawn from a laboratory unit under carefully controlled conditions and the changes taking place are studied critically and progressively during the consumption of the feed by the microorganisms. An increase in the propionic acid content in the rumen fluid indicates that a desirable response in overall ruminant performance has been brought about by the growth promotant in the feed composition.The change in propionic acid content is expressed as percent of the propionic acid content found in the control rumen fluid. Long term in vivo feeding studies are used to show a reliable correlation between propionic acid increase in the rumen fluid and improved animal performance.

Rumer fluid is collected from a fistulated cow which is fed on a commercial fattening ratio plus hay. The rumen fluid is immediately filtered through cheese cloth, and 19 ml added to a 50 ml conical flask containing 400 mg. of standard substrate (68% corn starch + 17% cellulose + 1 5% extracted soybean meal), 10 ml of a pH 6.8 buffer and the test compound. The flasks are gassed with oxygen-free nitrogen for about two minutes, and incubated in a shaking water bath at 39?C for about 6 hours. All tests are conducted in triplicate.

After incubation, 2.5 ml of the sample are mixed with 0.5 ml of 25% metaphosphoric acid, and the mixture centrifuged at 10;000 r.p.m. for 10 minutes. Samples are then analyzed by gas-liquid chromatography by the method of D. W. Kellog, J. Dairy Science 52, 1690 (1969). Peak heights for acetic, propionic and butyric acids are determined for samples from untreated and treated incubation flasks.When tested by this in vitro method compound UK-41637 and the minor component UK-44579 also formed in the fermentation using the culture ATCC 31676 gave rise to increased levels of production of propionic acid as shown in Table Ill:- TABLE Ill

Compound Dose % Propionic Acid Produced UK-41637 20 ppm 156 5 ppm 153 UK-44579 20 ppm 149 (Minor Component) 5 ppm 156 Untreated Controls - 100 Similar results may be obtained with other non-toxic salts or the free acid of compound UK41637, also for UK-44579 salts or the free acid.

Based on these data it can be projected that these antibiotics, when administered to ruminants such as cattle and sheep as the free acid, sodium salt, potassium salt etc. or mixtures thereof, will result in improved utilization of feed. It can also be projected that such compositions will also improve feed utilization in monogastric animals in which fermentation plays a role in digestive processes e.g. in pigs, rabbits and horses.

Crude compound or dried fermentation broth containing the antibiotic or antibiotics may be incorporated in feed compositions at the desired potency concentrations.

The following Examples illustrate the invention.

EXAMPLE 1 A vegetative inoculum was prepared by growing celis of Actinomadura cremea ATCC 31676 on agar slopes containing the following sterile medium ["(a)"]: Ingredient Grams/Litre Glucose 10 Starch 20 Enzymatic digest of casein 5 Yeast Extract 5 Calcium carbonate 1 Agar 20 pH 7.1 to 7.2 These slope cultures were grown for 5-7 days at 300C and cells were then harvested from the slope and transferred to flasks containing 1 litre of the following sterile aqueous inoculum medium ["(b)"]: Ingredient Grams/Litre Glucose 10 Starch 20 Enzymatic digest of casein 5 Yeast extract 5 K2HPO4 0.5 CoCI2 0.002 CaCO3 4 pH 7.1 to 7.2 The flasks were shaken for 3-4 days at 300C on a rotary shaker.An aliquot of this grown inoculum, sufficient to give 24% v/v was then transferred to a four litre fermenter containing 2.5 litres of the following sterile production medium ["(c)"]: Ingredient Grams/Litre Glucose 10 Starch 20 Enzymatic digest of casein 5 Yeast extract 5 CaCO3 1 pH 7.0 The fermentation was then carried out at 30OC for 3-4 days, with stirring at 1 500 revolutions per minute and aeration at about one volume of air per volume of broth per minute.

The whole broth was then freeze-dried and the resulting solids extracted with ethanol. The separated solvent extract was concentratad to a dark oil containing UK-41637 and UK-44579.

EXAMPLE II The inoculum medium (b) of Example I was used to prepare a vegetative inoculum of Actinomadura cremea ATCC 31676 in shaken culture. After 3 days incubation at 300C a 3% v/v inoculum was transferred to 60 litres of the inoculum medium (b) of Example I in a 100 litre fermenter.

After 40-48 hours incubation at 300C with stirring at 450 r.p.m. and aeration at one volume of air per volume of broth per minute, a 4% v/v inoculum was transferred to 1 500 litres of the following production medium in a 2000 litre fermenter [medium "(d)"]: Ingredient Grams/Litre Sucrose 10 Starch 5 Soya bean flour 5 Corn steep powder 5 Calcium carbonate 3 pH 7.0 Fermentation was carried out at 300 C, with stirring at 1 65 r.p.m. and an aeration rate of 0.8 volumes of air per volume of broth per minute. The fermentation was terminated after 4 days, and the whole broth was then filtered on a plate and frame press.

The fiitrate was divided in two portions and these were separately concentrated to 50 litre and 227 litre volumes. The 50 litre fraction was then extracted with 3 x 50 litres of methyl isobutyl ketone and the 227 litre fraction extracted with 2 x 200 litres of methylisobutyl ketone. The combined methylisobutyl ketone extracts were then concentrated to a dark viscous oil, To 120 g of this oil was added 1 litre of 60--800C petroleum ether and the resulting insoluble solids filtered off and discarded.

The petroleum ether filtrate was then concentrated in vacuo to yield a viscous oil (70 g) which was further purified by chromatography on silica gel using a Waters Prep. 500 liquid chromatograph as follows. Using a flow rate of 200 minute and ethyl acetate: 40-600C petroleum ether (1:1) as eluting solvent, a 25 g portion of the oil in 100 ml eluant was applied to the column and the ensuing fractionation was monitored byt.I.c. on silica gel using ethyl acetate as developing solvent.

Concentration of the antibiotic-rich fraction afforded a yellow viscous oil which a treatment with hexane afforded compound UK-41637 as a colourless solid (900 mg).

The remainder of the methylisobutylketone concentrate was treated as described above to yield a total of 13.3 g of compound UK-41637. This was re-crystallized from HPLC grade hexane to give compound Us41 637 as colourless rhombs, m.p. 196-1 980C; CKs825g + 500 (c, 0.43, CH30H); i.r. V max (KBr disc) 3560, 3200, 1715, 1585, 1400, 1365, 1090, cm-l. (see Fig. 1 for the i.r. spectra).

The 1H and 13C nmr spectra for compound UK-41 637 are shown in Figs. 2 and 3 respectively.

Elemental analysis indicated the following percentages by weight: C, 62.0: H, 8.7%.

which are fully consistent with the formula C4BH,7015Na (obtained from X-ray studies) which requires C, 61.8; H, 8.7%.

An X-ray crystal analysis of compound UK-41637 established the structural formula to be that specified on page 2 above.

Compound UK-41637 was also obtained by extracting the mycelial cake with methylisobutyl ketone (2 x 200 litres, 1 x 100 litres), concentrating the combined extracts to a viscous oil and chromatographing the oil as described above. HPLC analysis indicated 30.2 g of compound UK-41637 to be present in the mycelial extract.

EXAMPLE Ill The inoculum medium (b) of Example I was used to prepare a vegetative inoculum of Actinomadura cremea ATCC 31676 in shaken culture. After incubation for 3 days at 300C a 4% v/v inoculum was transferred to a 70 litre fermenter containing 60 litres of the following aqueous medium: Ingredient Grams/Litre Sucrose 10 Maize starch 5 Corn steep powder 5 Lactic casein 5 CaCO3 3 pH 7.2 The fermentation was carried out at 300C for 94 hours, with stirring at 450 revolutions per minute and an aeration rate of one volume of air per volume of broth per minute.

The whole broth (70 litres) was then extracted with n-butanol (70 litres) without pH adjustment.

The butanol extract was then concentrated under vacuum to yield an oily concentrate. Addition of 750 ml petroleum ether (boiling range 60-800C) caused precipitation of insoluble solids which were removed by filtration. The resulting filtrate was then concentrated to yield an orange viscous oil (70 g) which was dissolved in 300 ml petroleum ether (60--80"C), and poured on to a bed of 500 g of t.l.c.

grade silica gel (Merck Art 7731) covering a further bed of column grade t.l.c. silica gel (Malinkrodt CC7-500 g) in a sintered glass funnel. The filter bed was then eluted under suction with 3 x 1 litre of petroleum ether (60--800C), 4 x 1 litre of ethyl acetate: 60-800C petroleum ether (1:19), 2 x 1 litre of ethyl acetate: 60-800C petroleum ether (1 :9) 3 x 1 litre of ethyl acetate: 80--800C petroleum ether (1 :3) and finally 3 x 1 litre of ethyl acetate: 6O-800C petroleum ether (1 :1).The eluates were examined by t.l.c. and compound UK-41637 and the second (minor) antibiotic component UK-44579 were found to reside primarily in the ethyl acetate: 60--800C petroleum ether (1:1) fractions.

The antibiotic containing fractions were evaporated in vacuo to yield viscous oils, and one of these oily fractions (1 :1 g) was further purified by preparative liquid chromatography on silica gel, using a Waters Prep. 500 liquid chromatograph. Ethyl acetate: hexane (1 :1) was used as eluant at a flow rate of 200 ml/minute. Evaporation of the antibiotic contairling fractions gave a glassy solid (600 mg) containing compound UK-41637 and UK-44579 in the ratio 10:1. These two antibiotics were separated into pure components by further chromatography on silica gel using a Waters prep. 500 chromatograph, a flow rate of 100 ml/minute and hexane: ethyl acetate (1:1) as eluent.

Compound UK-41637 thus obtained (63 mg) was identical to that described in example ll above.

The minor component UK-44579 was obtained as a microcrystalline solid (40 mg), m.p. 90--930 with infra red absorption (KBr disc) at Vmax. 3560, 3180, 171 5, 1580, 1460, 1400, 1090 cm-' (for the i.r. spectra see Fig. 4), a258% + 37.4 (c, 0.1, CH3OH).

1H and '3C n.m.r. spectra for UK-44579 are shown in Figs. 5 and 6 respectively, and indicate the close structural relationship between this compound and antibiotic UK-41637.

It will be appreciated from the foregoing that what we will claim may comprise any novel feature described herein, for example:-- (1) Compound UK-41637 having the formula (I) defined herein, its free acid form, and its non-toxic cationic salts in addition to sodium; (2) Compound UK-44579, its free acid form, and its non-toxic cationic salts in addition to sodium said compound UK-44579 having a melting point of 90-930C, an optical rotation of ct2589 = + 37.40 (c, 0.1, CH3OH), and i.r. (KBr), 1H and '3C n.m.r. spectra as specified, respectively, in Figures 4, 5 and 6 hereinafter; (3) A mixture of compounds UK-41637 and UK-44579; (4) Dried fermentation broth containing compounds UK-41637 and UK-44579;; (5) A process for producing antibiotics UK-41637 and UK-44579, which comprises cultivating the microorganism Actinomadura cremea ATCC 31 676 in aqueous culture media containing an assimilable source of carbon, nitrogen and inorganic salts until substantial antibiotic activity is obtained, followed by, if desired, separating the antibiotics from the fermentation medium, or, taking the fermentation medium to dryness; (6) A composition comprising a compound, mixture or dried fermentation broth as defined in (1), (2), (3) or (4) above, together with a non-toxic diluent or carrier; (7) A method of treating a bacterial infection in a non-human animal, which comprises administering to the animal an antibacterially effective amount of a compound or mixture as defined in (1), (2) or (3) above either alone or in admixture with a non-toxic diluent or carrier; ; (8) A method of killing insects or their larvae, which comprises contacting them with, respectively, an insecticidal or larvicidal amount of a compound or mixture as defined in (1), (2) or (3) above either alone or in admixture with a non-toxic diluent or carrier.

(9) A method of improving feed utilization efficiency in a ruminant, which comprises feeding the ruminant with an effective amount of animal feed containing a compound, mixture or dried fermentation broth as defined in (1), (2), (3) or (4) above.

Claims (9)

1. An antibiotic of the formula:

its free acid form, and its non-toxic cationic salts in addition to sodium.

2. The antibiotic UK-44,579, its free acid form, and its non-toxic cationic salts in addition to sodium, said compound UK-44,579 being a sodium salt having a melting point of 9O-930C, an optical rotation of a2589 = +37.4 (c, 0.1, CH3OH), and i.r. (KBr), 1H and '3C n.m.r. spectra as shown, respectively, in Figures 4, 5 and 6 of the drawings.

3. A mixture of the antibiotics claimed in claims 1 and 2.

4. Dried fermentation broth containing a mixture of the antibiotics claimed in claims 1 and 2.

5. A process for producing the antibiotics claimed in claims 1 and 2, which comprises cultivating the microorganisms Actinomadura cremea ATCC 31676 in aqueous culture media containing an assimilable source of carbon, nitrogen and inorganic salts until substantial antibiotic activity is obtained, followed by, if desired, separating the antibiotics from the fermentation medium, or, taking the fermentation medium to dryness.

6. A composition comprising a compound, mixture or dried fermentation broth as claimed respectively in claims 1 or 2, 3 and 4, together with a non-toxic diluent or carrier.

7. A method of treating a bacterial infection in a non-human animal, which comprises administering to the animal an antibacterially effective amount of a compound, mixture or dried fermentation broth as claimed in, respectively, claims 1 or 2, 3 and 4 either alone or in admixture with a non-toxic diluent or carrier.

8. A method of killing insects or their larvae, which comprises contacting them with, respectively, an insecticidal or larvicidal amount of a compound or mixture as claimed in claims 1 or 2 and 3 respectively, either alone or in admixture with a non-toxic diluent or carrier.

9. A method of improving feed utilization efficiency in a ruminant, which comprises feeding the ruminant with an effective amount of animal feed containing a compound, mixture or dried fermentation broth as claimed in claims 1 or 2, 3 and 4 respectively.