GB2232668A - Antiparasitic compounds - Google Patents

Abstract

Compounds obtainable by fermentation of Saccharothrix sp N814-103 (ATCC 59301) are active against endo- and ectoparasites. They are of formula (I): <IMAGE> wherein R is a residue of formula A, B or C: <IMAGE>

Description

ANTIPARASITIC MACROLIDE COMPOSITIONS This invention relates to compositions and compounds which may be used as antiparasitic agents and in particular to novel macrolide compounds and to processes for their preparation and compositions thereof.

These compounds may be produced by fermentation of a hitherto unknown microorganism of the genus Saccharothrix, designated herein as N814-103. A sample of this culture has been deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville Maryland 20852 under the accession number ATCC 59301 on 3 May 1989. The microorganism was isolated from a soil sample taken from Fukui City, Fukui Prefecture, Japan and is assigned to the genus Saccharothrix based on cell morphology and chemistry.

The morphological and cultural characteristics of Saccharothrix sp N814-103 have been determined as follows: Culture N814-103 was inoculated from a slant into ATCC #172 broth and grown for four days at 28"C on a shaker. It was then centrifuged for 20 minutes, washed three times with sterile distilled water, and inoculated on media commonly used for identification of members of the Actinomycetales.

The plates and tubes were incubated at 28"C and the results might be read at varying times but most commonly were taken at two weeks and four weeks. The colours were described in common terminology, but exact colours were determined by comparisons with colour chips from.the Color Harmony Manual, fourth edition. The methods of whole-cell amino acid and sugar analyses are those described in Becker, B. et al., Appl. Microbiol., 12: 421-423, 1964 and in Lechavalier, M. P., J. Lab. Clin. Med., 71: 934-944, 1968. About 50 grams of autoclaved, wet mycelia of culture N814-103 were used for mycolate analyses, using the method described by Lechavalier, M.P. et al. in J Bacteriol., 105: 313-318, 1971. For phospholipid analyses, the method described by Minnikin, D. E. et al., in J. Microbiol. Method, 2: 233-241, 1984, is used.

Menaquinones were isolated from 57 grams of cells killed by addition of formalin to a final concentration of 0.5t and standing at room temperature for one hour, then the cells extracted with chloroform-methanol (2:1) and (1:2) overnight with shaking. The crude extracts were combined, taken to dryness in vacuo at 50"C, spotted on PTLC plates and developed in a hexane:ethyl ether (85:15) system. Ultraviolet-absorbing bands comigrating with a standard sample of vitamin K1, were scraped off and eluted with chloroform. The components of these mixtures were determined by the molecular ions present in their mass spectra, using the method described by Carlone, G. M. and Anet, F. A. L., in J Gen.

Microbiol., 129: 3385-3393, 1983.

Identification media used for the characterization of the culture and references for their composition are as follows: 1. Tryptone-Yeast Extract Broth - (ISP #1 medium, Difco).

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

3. Oatmeal Agar - (ISP #3 medium, Difco).

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

5. Glycerol-Asparagine Agar - (ISP #5 medium, Difco).

6. Peptone-Yeast Extract Iron Agar - (ISP #6 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. 328.

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. Gordon and Smith's Tyrosine Agar - R. E. Gordon and M. M.

Smith, J. Bacteriol. 69: 147-150, 1955.

13. Casein Agar - Ibid.

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

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

16. Starch - Ibid.

17. Organic Nitrate Broth - Ibid.

18. Dextrose Nitrate Broth - S. A. Waksman, The Actinomycetes, Vol. 2, medium no. 1, p. 328, 1961, with 3 g dextrose substituted for 30 g sucrose and agar omitted.

19. Potato Carrot Agar - M. P. Lechavalier, J. Lab. and Clinical Med. 71: 934-944, 1968, but use only 30 g potatoes, 2.5 g carrots and 20 g agar.

20. 2% Tap Water Agar.

21. Skim Milk - Difco.

22. 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.

23. Utilization of Organic Acids - R. E. Gordon et al., Int. J.

Syst. Bacteriol. 24: 54-63, 1974.

24. Hydrolysis of Hippurate and Esculin - Ibid.

25 Decomposition of Adenine, Hypoxanthine, Xanthine, and Urea Ibid.

26. Resistance to Lysozyme - Ibid.

27. Survival at 500C - Ibid.

28. Acid Production from (and Utilization of? Carbohydrates Ibid.

29. Temperature Range - ISP #2 medium plus 50 ml per litre of coconut milk.

Yeast Extract-Malt Extract Agar - Growth good, dark yellowish (3 Ic) with some white aerial mycelium, raised, smooth to wrinkled; reverse yellowish to dark yellowish (3 ia, 3 lc) but may turn red (6 1/2 pc, 6 1/2 nc) at 16 days; soluble pigment yellowish (2 Ic).

Oatmeal Agar - Growth moderate, pale pink-yellow (3 ca), slightly raised, smooth, aerial mycelium same as surface; reverse yellowish (2 ga, 3 ga,); soluble pigment cream to pale yellowish (2 ca, 2 ea).

Inorganic Salts-Starch Agar - Growth moderate; white, cream to yellowish orange (2 ca, 3 ga, 3 ia); moderately raised, smooth, aerial mycelium white to cream; reverse yellowish orange (3 ia); soluble pigment cream (2 ca).

Glycerol-Asparagine Agar - Growth poor to moderate, pale pink-yellow (3 ca), slightly raised, smooth, aerial mycelium same as surface; reverse pale pink-yellow to yellow (3 ca, 3 ea); no soluble pigment.

Glucose-Asparagine Agar - Growth moderate to good; white, cream to yellowish (2 la, 2 lc, 2 ca); moderately raised, smooth to granular, aerial mycelium white to cream (2 ca); reverse dark yellowish (2 lc); soluble pigment pale yellowish (2 ea).

Czapek-Sucrose Agar - Growth moderate; white, cream to yellowish orange (2 ca, 3 ga); slightly raised, smooth, aerial mycelium white to cream (2 ca); reverse yellowish (2 3c); no soluble pigment.

Emerson's Agar - Growth good, white to dark yellowish orange (3 lc), raised, wrinkled, aerial mycelium white; reverse dark yellowish orange to brown (3 nc, 3 ne); soluble pigment brown (3 ne).

Nutrient Agar - Growth moderate, yellowish brown to brown (3 gc, 3 ie) with some white aerial mycelium, slightly raised, smooth; reverse yellowish to yellowish brown (2 ga, 3 gc); no soluble pigment.

Bennett's Agar - Growth good, white to yellowish orange (3 la, 3 lc), raised; smooth, granular to wrinkled; aerial mycelium white; reverse yellowish brown (3 lc); soluble pigment yellowish brown (3 nc).

Gordon and Smith's Tyrosine Agar - Growth moderate, brown (3 le) with some white aerial mycelium, slightly raised, smooth to granular; reverse yellowish brown to brown (3 lc, 3 le); soluble pigment brown (3 ne).

Calcium Malate Agar - Growth moderate; white, cream to yellowish brown (3 lc, 2 ca); slightly raised to raised, smooth to wrinkled, aerial mycelium white to cream; reverse yellowish brown to brown (3 lc, 3 le) but may turn red (6 1/2 lc) at. 16 days; no soluble pigment.

Casein Agar - Growth good; yellowish orange, yellowish brown to brown (3 la, 3 lc, 3 le); moderately raised to raised, slightly wrinkled to wrinkled, no aerial mycelium; reverse yellowish brown (3 lc, 3 le); soluble pigment brown (3 le).

Gelatin Agar - Growth moderate to good, cream to yellowish orange (2 ca, 3 ga), slightly raised, smooth, aerial mycelium cream (2 ca); reverse yellowish to yellowish orange (2 ga, 3 ga); soluble pigment yellowish (2 ga).

Starch Agar - Growth moderate to good, yellowish brown (3 ic, 3 lc), moderately raised, slightly wrinkled but finely wrinkled toward edge; aerial mycelium none to sparse, white; reverse yellowish brown to brown (3 lc, 3 ne); soluble pigment yellowish (2 Ic).

Potato Carrot Agar - Growth moderate, cream (2 ca), slightly raised, smooth, aerial mycelium cream (2 ca); reverse colourless to yellowish (2 ga, 2 inc); no soluble pigment.

Tap Water Agar - Growth poor, cream (2 ca), thin, smooth, aerial mycelium cream (2 ca); reverse colourless to cream (2 ca); no soluble pigment.

Morphological Properties: The following morphologic observations were made on oatmeal agar after 16-day incubation: aerial mycelium pale pink-yellow, may fragment into rods or filaments of varying lengths; the fragments straight, curved to flexuous, 2-30 (or longer) x 0.6-1.0 Mm, smooth as revealed by scanning electron microscopy.

Biochemical Properties: I. Melanin production negative; production of hydrogen sulfide positive; gelatin liquefaction positive; nitrate reduction negative; hydrolysis of esculin, hippurate, and starch positive; decomposition of casein, hypoxanthine, tyrosine and urea positive; decomposition of adenine and xanthine negative; no growth and no decomposition on both cellulose broths; coagulation and peptonization on milk; resistance to lysozyme positive.

II. Utilization of organic acids: acetate, citrate, lactate, oxalate, propionate, pyruvate, and succinate utilized; benzoate, dextrin, malate, mucate, and phenol not utilized.

III. Acid production from carbohydrates: Acid produced from adonitol, arabinose, cellobiose, fructose, glucose, glycerol, inositol, lactose, mannitol, maltose, mannose, melibiose, alpha -methyl-D-glucoside, raffinose, rhamnose, ribose, saliin, starch, sucrose, trehalose, and xylose; acid not produced from dulcitol, erythritol, melezitose, sorbitol, and sorbose.

IV. Carbohydrate utilization: Adonitol, arabinose, cellobiose, fructose, glucose, glycerol, inositol, lactose, mannitol, maltose, mannose, melibiose, alpha-methyl-D-glucoside, raffinose, rhamnose, ribose, starch, sucrose, trehalose, and xylose utilized; dulcitol, erythritol, melezitose, salicin, sorbitol, and sorbose not utilized.

Temperature Relation: The culture showed good growth at 210C, excellent growth at 28C and 370C, and no growth at 50C. It failed to survive at 500C for 8 hours.

Cell Wall Analysis: The whole-cell hydrolysates contained mesodiaminopimelic acid, galactose, and traces of mannose and rhamnose.

Nycolate Analysis: There were no mycolates of any kind present in the cell wall.

Phospholipid Analysis: The extracts of the cell membrane contained phosphatidylglycerol, phosphatidylinositol, and unknown glucosamine containing phospholipids.

Menaquinone Analysis: The predominant menaquinone present in cells was MR-9(H4) with traces of MK-5 (H4), MK-6 (H4), blK-7 (H4), MK-8(H4), and MK-l0(H4).

The culture N814-103 is characterized by the white to cream aerial mycelium; the yellowish, yellowish brown to brown substrate mycelium; and the cream, yellow or brown soluble pigment. The aerial mycelium might fragment into rods or filaments of varying lengths. The whole-cell hydrolysates contained mesodiaminopimelic acid, galactose, traces of rhamnose and mannose.

The absence of arabinose indicates that it has type III cell walls rather than type IV cell walls. No mycolic acids were present in the cell walls. The phospholipid composition of this culture included unknown glucosamine-containing phospholipids as the characteristic phospholipids, placing it in the phospholipid pattern group PIV. MK-9 (H4) was the predominant menaquinone in the cells. Except that the phospholipid pattern is type PIV rather than type PII, it fits into the description of the genus Saccharothrix, as defined by Labeda et al. (Int. J. Syst.

Bacteriol. 34: 426-431, 1984).

The culture N814-103 closely resembles Saccharothrix aerocolonigenes (Labeda, D. P., Int. J. Syst. Bacteriol. 36: 109-110, 1986) in morphologic, cultural and biochemical properties. The former has a phospholipid pattern of type PIV rather than type PlI as found in the latter. The former differs from the latter in its ability to utilize alpha-methyl-D-glucoside and its failure to utilize malate. All of the other biochemical properties are identical.

It has been found that fermentation of microorganism N814-103 in an appropriate fermentation medium produces three fermentation factors which are useful as antiparasitic agents.

Thus according to one aspect of the present invention, there is provided a composition having antiparasitic properties obtainable by fermentation of micro-organism N814-103 or a mutant or recombinant form thereof having the ability to produce said composition.

According to another aspect of the present invention, there is provided a process for producing a composition having antiparasitic properties which comprises fermenting micro-organism N814-103 or a mutant or recombinant form thereof having the ability to produce said composition in a fermentation medium, and isolating said composition from the medium.

The invention also extends to the individual compounds having antiparasitic properties obtained from the fermentation.

A suitable fermentation medium is an aqueous or solid agar culture medium containing an assimilable source of carbon, nitrogen and inorganic salts. The fermentation may be carried out under submerged aerobic fermentation conditions or surface agar fermentation until a recoverable amount of the desired compounds are obtained.

The term mutant includes any mutant strain of the micro-organism which arises spontaneously or by the application of known techniques, such as exposure to ionising radiation, ultraviolet light, and/or chemical mutagens such as N-methyl-N-nitroso-urethane, nitrosoguanidine and ethane methane sulphate. Genetically transformed and recombinant forms include mutants and genetic variants produced by genetic engineering techniques, including for example recombination, transformation, Cultivation and isolation of the antiparasitic composition may be conducted under conditions similar to those generally employed to produce antibiotics by fermentation. Cultivation may take place in an aqueous nutrient medium containing suitable sources of carbon, nitrogen and trace elements for a period of several days under aerobic conditions at a temperature in the range of 20 to 370C.The pH of the nutrient medium can vary from about 5.0 to 9.0 with a preferred range of 6.0 to 7.5. Examples of media suitable for culturing strains of Saccharothrix sp N 814-103 are described below as "Medium A" and "Medium B".

Medium A Soyabean flour 10.0g Cerelose lO.0g Starch 10.0g Distillers solubles 5.0g NaCl 5.0g CaCO3 l.0g CoCl2. 6H20 0.005g Distilled water 1.0 litre p11 7.2 Medium B Glucose 10.0g Soluble.starch 5.0g Corn steep powder 2.5g Blood meal 5.0g Cacao3 3.0g Distilled water 1.0 litre pH 6.2 As with the majority of fermentations the amounts and proportions of N814-103 factors obtained will vary with changing fermentation conditions especially with regard to nutrient components, aeration conditions and pH. The recovery of the instant compounds is facilitated by their greater solubility in organic solvents than in water.Thus in one recovery method the whole fermentation broth is agitated with an equal volume or less of water-immiscible solvent such as ethyl acetate, dichloromethane, methyl isobutyl ketone or n-butanol followed by evaporation of the solvent under vacuum. This procedure may be repeated several times to achieve maximum recovery.

Alternatively the mycelium may be separated by centrifugation or filtration and extracted with a water-miscible solvent such as acetone or methanol. The solvent extract is concentrated and the instant compounds are extracted into a water-immiscible organic solvent such as ethyl acetate, dichloromethane, methyl isobutyl ketone or n-butanol, followed by evaporation of the solvent under vacuum.

Either recovery procedure generally provides a crude mixture of N814-103 factors, the proportions of which will vary depending on the particular fermentation conditions employed. A suitable technique for determining the precise proportions of each component is high pressure liquid chromatography (HPLC) using conditions described below and monitoring of the fractions by ultraviolet (W) absorbance at a fixed wavelength, preferably in the region of 250 nm. The presence of the desired compounds may also be determined by analysing the various chromatographic fractions using alternative spectroscopic techniques or by determination of a specific biological activity possessed by the N814-103 factors.

Enrichment and separation of the instant compounds can be achieved by a variety of techniques, including column chromatography with silica gel, alumina or various polystyrene resins, or preparative layer chromatography, familiar to those skilled in the art. Final purification is often achieved by preparative normal or reverse phase HPLC. The individual N814-103 factors, herein designated factors A-C may be recognised by their characteristic spectroscopic properties and their HPLC retention time using an Ultrasphere-ODS (5Mm) (Trademark-Beckman) column (4.6 x 250mm) eluting with a water-methanol mixture thermostated at 400C and using W absorbance detection at 243nm, as detailed in Table 1 below.

Table 1 Retention times of N814-103 Factors A-C Factor Retention Time, min * A 8.03 B 10.52 C 15.54 * Eluting with water : methanol 15 : 85 at 1.0 ml per minute The N814-103 factors obtained as above have been studied by detailed analysis of the spectroscopic characteristics of the various components, in particular their ultraviolet, infra-red, nuclear magnetic resonance and mass spectra.

Based on the data obtained factors having antiparasitic properties are believed to have the general formula (I) below.

in formula (I) the identity of R varies according to the factor considered as given in Table 2 below.

Table 2

Factor R Me O OH A HO Me Me Me Me Me Me OH Me OH > OH B Mew Me Me Me Me OH Me OH C Me 0eO Me Me Me Me Thus, according to another aspect of the invention, there is provided a compound having formula (I) above wherein R is a residue given in Table 2 above.

According to another aspect of the invention, there is provided a compound of formula (I) obtainable by fermentation of micro-organism N814-103.

According to yet another aspect of the invention, there is provided a composition comprising at least one compound of formula (I), the composition having antiparasitic properties.

Compounds are obtained by fermentation of N814-103 and subsequent isolation of the fermentation factors as described above are antiparasitic agents having particular utility as anthelmintics, insecticides, acaricides, flukicides, taenicides and anticoccidial agents.

Thus the compositions and compounds are effective in treating a variety of conditions caused by endoparasites including, helminthiasis which is most frequently caused by a group of parasitic worms described as nematodes and which can cause severe economic losses in swine, sheep, horses and cattle as well as affecting domestic animals and poultry. The compounds are also effective against liver flukes and tapeworms which affect various species of animals.

The compounds are also of value in treating ectoparasite infections including in particular arthropod ectoparasites of animals and birds such as ticks, mites, lice, fleas, blowfly, biting insects and migrating dipterous larvae which can affect cattle and horses.

The compounds are also insecticides active against household pests such as the cockroach, clothes moth, carpet beetle and the housefly as well as being useful against insect pests of stored grain and of agricultural plants such as spider mites, aphids, caterpillars and against migratory orthopterans such as locusts.

It has further been found that the compounds exhibit anti-coccidial activity and accordingly are useful for treating coccidiosis, especially in poultry.

The compounds of formula (I) are administered as a formulation appropriate to the specific use envisaged and to the particular species of host animal being treated and the parasite or insect involved. For use as an anthelmintic the compounds may be administered by injection, either subcutaneously or intramuscularly, alternatively they may be administered orally in the form of a capsule, bolus, tablet or liquid drench, or they may be administered as a pour-on formulation or as an implant. Such formulations are prepared in a conventional manner in accordance with standard veterinary practices. Thus capsules, boluses or tablets may be prepared by mixing the active ingredient with a suitable finely divided diluent or carrier, additionally containing a disintegrating agent and/or binder such as starch, lactose, talc, or magnesium stearate.A drench formulation may be prepared by dispersing the active ingredient in an aqueous solution together with dispersing or wetting agents and injectable formulations may be prepared in the form of a sterile solution or emulsion. These formulations will vary with regard to the weight of active compound depending on the species of host animal to be treated, the severity and type of infection and the body weight of the host. Generally for oral administration a dose of from about 0.001 to 10 mg per Kg of animal body weight given as a single dose or in divided doses for a period of from 1 to 5 days will be satisfactory but of course there can be instances where higher or lower dosage ranges are indicated and such are within the scope of this invention.

As an alternative the compounds may be administered with the animal feedstuff and for this purpose a concentrated feed additive or premix may be prepared for mixing with the normal animal feed.

For use as an insecticide and for treating agricultural pests the compounds are applied as sprays, dusts, pour-on formulations, emulsions and the like in accordance with standard agricultural practice.

For human use the compounds are administered as a pharmaceutically acceptable formulation in accordance with normal medical practice.

The invention is illustrated by the following Examples which describe the preparation, isolation and identification of the factors of formula (I).

In the following Examples: Oxoid peptone and Oxoid Lab Lemco were supplied by Oxoid Limited, Wade Road, Basingstoke, Hampshire, UK.

Ultraviolet spectra were recorded on line using a Hewlett-Packard HP 1090 diode-array detector.

Electron impact mass spectroscopy was performed using a VG model 7070F mass spectrometer.

Fast atom bombardment mass spectroscopy was performed using a VG model 7070E mass spectrometer. Samples were introduced using a matrix consisting of glycerol, thioglycerol, sodium chloride and water.

ACE mass spectroscopy was performed using a VG model 7070E mass spectrometer (CH4 reagent gas).

Nuclear magnetic resonance spectral data were obtained using a Nicolet QE300 or a General Electric GN500 spectrometer.

EXAMPLE 1 The microorganism Saccharothrix sp N814-103 was preserved on agar slants of the following composition: Glucose 10.0 g/l, Nadex starch 20.0 g/l, Bacto-Casitone (supplied by Difco Co., Detroit, USA) 5.0 g/l, Oxoid Yeast extract 5.0 g/l, calcium carbonate 1.0 g/l, agar 20.0 g/l adjusted to pH 7.1.

Aerial growth was rubbed from a slant with 10 ml of sterile distilled water. This suspension was used to inoculate two 300 ml Erlenmeyer flasks each containing 50 ml of a medium comprising glucose 1.0 g/l, starch 24.0 g/l, Oxoid peptone 5.0 g/l, yeast extract 5.0 g/l, Oxoid Lab Lemco 3.0 g/l, calcium carbonate 4.0 g/l with the pH ajusted to 6.8. These flasks were incubated at 28"C for 48 hours with agitation at 180 rpm after which time the contents of one of these flasks were transferred to 700 ml of the same medium contained in a 3 litre Fernbach flask. This was incubated as above before being used to inoculate a fermenter containing 15 litres of Medium A as described above. The fermenter was operated at 280C with agitation at 350 rpm and an aeration rate of 15 litres per minute.

After 187 hours the broth was recovered by stirring with 15 1 methyl isobutyl ketone (MIBK) and the two layers separated. The aqueous layer was re-extracted with 8 1 MIBK and the combined organic layers were concentrated to dryness under vacuum to give a mobile oil. This material was extracted with 350 ml methanol and the supernatant was decanted off. Water (35 ml) was added and the solution extracted with 300 ml petroleum ether (boiling range 40-600C).

The methanolic solution was separated, a further 100 ml water added and re-extracted with petroleum ether (500 ml). The methanolic layer was separated and concentrated to dryness under vacuum to give a mobile, clear red oil (10.33 g) containing the N814-103 factors obtained during the fermentation.

EXAMPLE 2 A part of an enlarged colony of the microorganism Saccharothrix Sp N814-103 was suspended in a 500 ml Erlenmeyer flask containing 100 ml of a medium comprising glucose 1.0 g/l, starch 24.0 g/l, Oxoid peptone 5.0 g/l, yeast extract 5.0 g/l, Oxoid Lab Lemco 3.0 g/l, calcium carbonate 4.0 g/l with the pH adjusted to 6.8. This flask was incubated at 28"C with agitation at 200 rpm for 4 days after which time 7.5 ml of this inoculum was transferred into a 500 ml side-armed flask containing 150 ml of the medium as described above. This flask was incubated at 28"C with agitation at 200 rpm for 3 days. The contents of this flask were then used to inoculate a pot fermenter (total volume 6 1) containing 3 litres of Medium B as described above.The fermenter was operated at 280C with agitation at 1,730 rpm and an aeration rate of 3 litres per minute.

After 7 days the broth was freeze dried and extracted with methanol-water (4:1). After filtration the solution was concentrated to aqueous solution under vacuum and extracted with ethyl acetate. The organic layer was then concentrated to dryness under vacuum to give a dark coloured gum containing the N814-103 Factors obtained during the fermentation.

In alternative procedures the pot fermentations were conducted under identical conditions except that the time prior to freeze drying could be varied between 4 and 10 days.

Example 3 Half of the aerial growth from a slant culture of the microorganism Saccharothrix sp N814-103 (maintained as described in Example 1) was suspended in sterile distilled water (5 ml) and added to a 300 ml Erlenmeyer flask containing 50 ml of a medium comprising glucose 1.0 g/l, starch 24 g/l, Oxoid Peptone 5g/l, Yeast extract 5 g/l, Oxoid Lab Lemco 3 g/l, calcium carbonate 4 g/l with the pH adjusted to 6.8. This flask was incubated at 28"C for 48 hours with agitation at 180 rpm. 2 ml of this inoculum was transferred to a 300 ml Erlenmeyer flask containing 50 ml of the medium described above and the incubation was carried out as before. After 48 hours the contents of this flask were used to inoculate a pot fermenter (total volume 5 litres) containing 2.5 litres of Medium B described above. The fermenter was operated at 280C with agitation at 1000 rpm and an aeration rate of 2.5 litres per minute.

After 12 days the broth from four fermentations carried out under identical conditions was combined and 375 g of a diatomaceous earth filter aid was added with stirring. The suspension was suction-filtered through a bed of diatomaceous earth. The moist filter cake was suspended in 2 1 methanol and filtered. The filter cake was again suspended in 2.1 1 methanol, filtered and the spent cake was discarded. The combined methanolic extracts were evaporated under vacuum to give an aqueous suspension. This was extracted twice with ethyl acetate (250 ml) and the organic solution was concentrated under vacuum to give a gum (2.35 g) containing the N814-103 Factors obtained during the fermentation.

Example 4 Isolation of N814-103 Factors A, B and C Twelve pot fermentations were carried out according to the procedure described in Example 2. Four pots were freeze dried each on days 4, 7 and 10 and the combined solids extracted as described in Example 2. The combined extracts, totalling 11.5 g were divided into two equal halves and each was chromatographed on a Waters Prep 500 A chromatograph, using a reverse phase cartridge (45 x 300 mm) and eluting with a water-methanol mixture 15 : 85 at a flow rate of 150 ml per minute. Fractions were monitored for the N814-103 factors contained by the analytical HPLC conditions detailed above (Table 1). Factors A, B and C began to elute after about 500 ml had passed through the column. Fractions containing the desired Factors were combined and evaporated to dryness under vacuum.This material was further purified by HPLC using a Zorbax ODS (8 Mm) column. (Trademark, Dupont) (21.2 x 250 mm) eluting with water-methanol 15 : 85 at a flow rate of 4 ml per minute.

The progress of the separation was again monitored by analytical HPLC. Final separation of Factors A, B and C was achieved by repeating the chromatography on the Zorbax column under identical conditions. Final purification of N814-103 Factor A was achieved by normal phase HPLC using a Zorbox Sil (21.2 x 250 mm) column (Trademark, Dupont) eluting with a mixture of dichloromethane and ethyl acetate (3 : 1) at a flow rate of 4 ml per minute, monitoring by W absorption at 261 mm, to give pure N814-103 Factor A. Final purification of Factors B and C was achieved by reverse phase chromatography using an Ultrasphere -ODS 5 Mm (Trademark, Beckman) column (10 x 250 mm) eluting with a 23 methanol-water gradient beginning at 4 : 1 and finishing at 20 : 1 over a 70 minute period with a flow rate of 2.5 ml per minute to give pure N814-103 Factors B and C.

These individual components were distinguished and characterised by the spectroscopic characteristics shown below: Factor A Factor B Factor C FAB Mass Spectrum 683 (M+Na+) 715 (MfNa+) Spectrum, 242 242 242 max (nm) 275 275 275 NMR spectrum, 7.3 (d )(1H) 7.3 (d )(1H) 7.3 (d )(1H) (CDCl3 solution) 6.8 (dd)(lH) 6.6 (dd)(lH) 6.6 (dd)(lH) (in part) 6.6 (dd)(lH) 5.75(d )(1H) 5.75(d )(1H) 6.3 (d )(1H) 5.2 (dd)(lH) 5.25(dd)(lH) 4.8 (dd)(2H) 5.7 (d )(IH) 3.25(s )(3H) 4.8 (dd)(2H) 5.2 (dd)(lH) 2.0 (bs)(3H) 3.3 (s )(3H) 4.8 (dd)(2H) 1.9 (bs)(3H) 3.05(s )(3H) 3.3 (s )(3H) 2.0 (bs)(3H) 2.0 (bs)(3H) 1.85(bs)(3H) 1.9 (bs)(3H)

Claims (10)

1. A composition having antiparasitic properties obtainable by fermentation of micro-organism N814-103 or a mutant or recombinant form thereof having the ability to produce said composition.

2. A process for producing a composition having antiparasitic properties which comprises fermenting micro-organism N814-103 or a mutant or recombinant form thereof having the ability to produce said composition in a fermentation medium, and isolating said composition from the medium.

3. A compound of general formula (I)

wherein R is a residue having one of the following formulae:

4. A compound of the general formula (I):

wherein R is an organic residue, obtainable by fermentation of micro-organism N814-103.

5. A compound obtainable by fermentation of micro-organism N814-103 having spectroscopic characteristics (a), (b) or (c):

(a) (b) (c) FAB Mass Spectrum 683 (M+Na+) 715 (N+Na+) UV Spectrum, 242 242 242 max (nm) 275 275 275 NMR spectrum, 7.3 (d )(1H) 7.3 (d )(1H) 7.3 (d )(1H) (cDCl3 solution) 6.8 (dd)(lH) 6.6 (dd)(lH) 6.6 (dd)(lH) (in part) 6.6 (dd)(lH) 5.75(d )(1H) 5.75(d )(1H) 6.3 (d )(1H) 5.2 (dd)(lH) 5.25(dd)(lH) 4.8 (dd)(2H) 5.7 (d )(1H) 3.25(s )(3H) 4.8 (dd)(2H) 5.2 (dd)(lH) 2.0 (bs)(3H) 3.3 (s )(3H) 4.8 (dd)(2H) 1.9 (bs)(3H) 3.05(s )(3H) 3.3 (s )(3H) 2.0 (bs)(3H) 2.0 (bs)(3H) 1.85(bs)(3H) 1.9 (bs)(3H)

6. An antiparasitic composition, comprising a compound according to claim 3, 4 or 5.

7. A compound according to claim 3, 4 or 5 for use in veterinary or human medicine.

8. Use of a compound according to any one of claims 3 to 5 for making a medicament for treatment of parasitic diseases.

9. An antiparasitic compound composition, substantially as hereinbefore described with reference to the foregoing Examples.

10. Saccharothrix sp N814-103.