WO1982000587A1

WO1982000587A1 - New metabolites,processes for their production and their use

Info

Publication number: WO1982000587A1
Application number: PCT/EP1981/000121
Authority: WO
Inventors: Ag Sandoz
Original assignee: Tscherter H; Dreyfuss M
Priority date: 1980-08-15
Filing date: 1981-08-12
Publication date: 1982-03-04
Also published as: GR75310B; EP0057724A1; PT73516B; ES504747A0; IT1138149B; IL63571A0; DK168082A; YU198481A; IT8123488A0; PT73516A; JPS57501266A; ES8306506A1

Abstract

The antibiotics S 41062/F-1, S 41062/F-6 and S 41062/F-7 are obtained from a new Cryptosporiopsis strain.

Description

NEW METABOLITES, PROCESSES FOR THEIR PRODUCTION AND THEIR USE

The present invention relates to the compounds S 41062/F-1, S 41062/F-6 and S 41062/F-7.

In accordance with the invention S 41062/F-1, S 41062/F-6 and/or S 41052/F-7 are obtained by cultivating an S 41062/F-1 and/or S 41062/F-6 and/or S 41062/F-7 producing strain, e.g. of the fungal genus Crypto sporiopsis in the presence of a culture medium.

The compounds S 41062/F-1, S 41062/F-5 and S 41062/F-7 exhibit the following approximate characteristics.

S 41062/F-1

- white micro-crystalline powder

- m.p. 235-240°C (gradual decomposition, particularly at

>265°C) - = -6° (c = 0.5 in methanol)

- UV spectrum in methanol λ max 193 nm = 786

(222) nm „ = 140

279 nm „ = 14 - IR spectrum in KBr, see Figure 1;

- ¹H-NMR spectrum (90 mHz in DMSO) with tetramethyl - silane as internal standard, see Figure 4;

- ¹³C-NMR spectrum in D₄-methanol (36 mg in 0.3 ml) with tetramethylsilane as internal standard, see Table 2, column A;

- On field desorption mass spectrum peaks M(+li)⁺ 1055 and M(+Na)+ 1071 corresponding to empirical formula

C₅₀H₈₀N₈O₁₆ MW 1048; - Analysis after drying at room temperature in high vacuum

Calculated (C₅₀H₈₀N₈O₁₆) : C 57.3 : H 7.7 : N 10.7 : O 24.4 % Found: C 55.7 : H 7.7 : N 10.1 : O 24.7 % C 56.5 : H 7.8 : N 10.5 : O 24.1 %

- Solubility : easily soluble in methanol, ethanol, pyridine, dimethylsulfoxide (DMSO); difficulty soluble in water, chloroform, ethyl acetate, ether, benzene, hexane;

- the compound was stable in aqueous methanol at pH 4 to 7. Under alkaline or strongly acidic conditions decomposition with loss of anti-fungal activity occurred.

- acidic hydrolysis (6N HCl, 110°C, 24 hours) yielded as degradation product a saturated fatty acid which in the form of its methyl ester showed a mass spectrum peak at M⁺ 270.

Amino acid analysis (according to the method of Stein and Moore) Degradation products were obtained which had retention times the same as or similar to e.g. aspartic acid hydroxyproline, serine and ammonia as well as an unknown amino acid with a retention time between those of leucine and tyrosine.

S 41062/F-1 is a neutral cyclic hexapeptide, which bears a branched, amide linked, saturated (C-16) fatty acid radical.

On the basis of investigative chemistry (amino acid analysis, etherification) and spectral data ( ¹H- , ¹³C-

NMR) the following amino acids are believed to be present: serine, 4-hydroxyproline, 4-methyl-3-hydroxy-proline, 3-hydroxy-homo-tyrosine and probably 4,5-dihydroxy-ornithine; the sixth, unknown amino acid unit contains an H₂NCO group and, it is thought, a secondary hydroxyl function. S 41062/F-1 has the following structure:

Thin layer chromatogram - Table 1 Anti- fungal activity - Table 5.

S 41062/F-6

On the basis of the acid degradation products S 41062/F-6 is a mixture of at least 3 components.

- white micro-crystallline powder;

- m.p. 240-244°C (gradual decomposition);

- (c = 0.5 in methanol);

- UV spectrum in methanol λ max 193 nm = 861

(222) nm „ = 340 278 nm „ = 15 - IR spectrum in KBr, see Figure 2;

- 1H-NMR spectrum (90 mHz in DMSO) with tetramethyl silane as internal standard - see Figure 5;

- ¹³C-NMR spectrum in D₄-methanol (44 mg in 0.3 ml) with tetramethylsilane as internal standard, see Table 2 column B;

- Analysis after drying at room temperature in high vacuum:

Found C 56 . 3 : H 7. 7 : N 10 . 9 : O 26 . 0 % C 56 . 8 : H 7 . 9 : N 10 . 7 : O 24 . 8 %

- Solubility: easily soluble in methanol, ethanol, pyridine, dimethylsulfoxide (DMSO) ; difficulty soluble in water, chloroform, ethyl acetate, ether, benzene, hexane; - the compound was stable in aqueous methanol at pH 4 to 7. Under alkaline or strongly acidic conditions decomposition with loss of anti-fungal activity occurred.

- acidic hydrolysis (6N HCl, 110°C, 24 hours) yielded as degradation product a saturated fatty acid which in the form of its methyl ester showed a mass-spectrum peak at M⁺270;

- Amino acid analysis (according to the method of Stein and Moore) Degradation products were obtained which had retention times the same as or similar to e.g. aspartic acid, hydroxyproline, allo-hydroxyproline, serine and ammonia as well as an unknown amino acid with a retention time between those of leucine and tyrosine;

- Thin-layer chromatogram - Table 1; - Anti-fungal activity - Table 5.

S 41062/F-7

- White powder;

- M.P. from 205°C (sinter with gradual decomposition);

- = -6° (c = 0.5 in methanol)

- UV spectrum in methanol λ max 194 nm = 890

(222) nm " = 339

278 nm " = 15

- IR spectrum in KBr, see Figure 3; - ¹H-NMR spectrum (90 mHz in DMSO) with tetramethylsil ane as internal standard, see Figure 6; -¹³ C-NMR spectrum in D₄-methanol (38 mg in 0.25 ml) with tetramethyl silane as internal standard, see Table 2, column C; - Analysis after drying at room temperature in high vacuum : Found C 54.5 : H 7.5 : N 9.9 : O 25.0 % C 55.1 : H 7.5 : N 10.2 % A sample was dissolved in ethyl acetate/isopropanol shaken with very dilute hydrochloric acid, evaporated and precipitated from methanol with acetone. Subsequent analysis gave the following results: Found : C 57.4 : H 8.0 : N 11.0 : O 24.6 % C 56.0 : H 7.9 : N 10.3 : O 24.8 %

- Solubility : easily soluble in methanol, ethanol, pyridine, dimethyIsulfoxide (DMSO) ; difficulty soluble in water, chloroform, ethylacetate , ether, benzene, hexane;

- Acidic hydrolysis (6N HCl, 110°C, 24 hours) yielded as degradation product a saturated fatty acid which on the basis of gas chromatographical retention times and mass spectroscopical molecule and fragment peaks is postulated as dimethyl-myristic acid;

- Amino acid analysis Degradation products were obtained with retention times the same as or similar to e.g. aspartic acid, hydroxyproline/allo-hydroxy- proline, serine and ammonia as well as a further amino acid with retention time between those of leucine and tyrosine.

Thin layer chromatogram - Table 1 Antifungal activity - Table 5

Iodine vapour or other suitable spray reagents can be used to detecr the substances. A suitable spray reagent can for example be prepared by dissolving 100 mg of ferric chloride in 1 ml of ethyl acetate and adding 100 ml of concentrated nitrate-free sulphuric acid.

The completely solvent-free thin layer chromatogram was sprayed with the above-mentioned spray reagent and then heated using an incandescent heater until strong brown spots appeared.

Table 2

¹³ C-NMR spectra (apparatus : Bruker HX-90-E ; recording at 22. 63 mHz ; sweep width : 6000 Hz)

The process according to the invention may be effected by known methods. A sub-culture of the S 41062/F-1, S 41062/F-6 and/or S.41062/F-7 producing strain was deposited on 2.1.80 at the American Type Culture Collection (ATCC) , Rochville, Maryland, USA and is available to the public under the culture number ATCC 20594. The culture is also available from Sandoz Ltd., Basle, Switzerland.

A further sub-culture was deposited on 16.6.80 at the United States Department of Agriculture

(Northern Utilization Research and Development Division), Peoria, Illinois, USA and is available to the public under the culture number NRHL 12192.

However, S 41062/F-1, S 41062/F-6 and/or S 41062/F-7 producing strains may be used which may be obtained from the parent strain of ATCC 20594 or NRRL 12192 by mutation e.g. by radiation, treatment with conventional mutagenic substances, or by selection. Characteristics of the strain ATCC 20594 (and NRRL 12192)

This strain was isolated from a plant root collected near Soyhières (Switzerland) and forms in pure culture compact open conidioma (acεrvuli). This characteristic allows the strain deposited as ATCC 20594 and NRRL 12192 to be positively attributed to the Melanconiaceae. On the basis of the microscopic charactsristics and employing the identification key according to B.C. Sutton (in Ainsworth, Sparrow & Sussman: The Fungi, v. IVa., Academic Press 1973, chapter 11, p.555) the fungus can be attributed to the genus Cryptosporiopsis Bub & Kab. With the exception of a few indeterminable characteristics this classification shows good conformity with that of J.A. von Arx (A Revision of the Fungi classified as Gloeosporium, Bibliotheka Mycologica, v.24, J. Cramer, 1970, pp. 5, 6, 27-29). In view of the fact that no fructifications were observed on the original substrate and that so far no perfect state has been observed together with the fact that taxonomically relevant culture descriptions are very sparse in the literature (J.W. Groves 1939) : Some Pezicuϊa species and their conidial stages. Can. J. Research 17; 125-143; J.W. Groves 1939: Some Pezicula species occurring on alnus. Mycologia 32; 112-123; J.W. Groves 1941: Pezicula carnea and Pezicula subcarnea. Mycologia 33; 510-522; J.W. Groves 1947: Pezicula Morthieri on Rhamnus. Mycologia 39; 328-333), a more precise classification does not seem to be cur rently possible.

The following physiological and morphological features are characteristic for the S 41062/F-1, S 41062/F-6 and/or S 41062/F-7 producing Cryptosporiopsis strain ATCC 20594: Growth Occurs well on agar media preferably at lower temperatures. The minimal and maximal temperatures for growth are 0° and 31-33°C respectively with optimal growth between 15-24 °C.

Table 3 shows the growth characteristics dependent on incubation temperature and medium.

Colony diameters (average of 6 colonies in mm) were measured after a 14-day incubation period on petri dishes seeded punctate and serve as a measure of growth rate.

The vigorously developing fluffy to woolly fluffy, aerial mycelium of white to creamy appearance found in the young culture on the MA-medium gradually becomes light grey to grey-brown or light brown during futher incubation. From underneath the colonies are ochre-brown to chestnut-brown and cultures incubated at approximately 27°C excrete a dark brown pigment into the agar.

In a liquid MA-medium (MA-mediuia without agar), the pH of which can be adjusted with hydrochloric acid or sodium hydroxide, ATCC 20594 will grow quite well at pH 2.0 with shaking (180 rpm) and at a temperature of 21-22°C whereas at a pH above 9.3 no growth takes place. Good growth occurs throughout the pH region of 2.3 to 8.0.

Lower temperatures are also preferable for sporulation of the Cryptosporiopsisstrain ATCC 20594.

After 7 to 10 days incubation on an MA medium at 18°C numerous micro- and macroconidia have already formed. These form in cream to light brown disc shaped conidioma (acervuli) with a diameter of 50 to 200 μ , or in aggregates of conidioma of up to 3 mm, and accumulate in slime on the conidioma's surface. On the basis of light microscopy the conidio genous cells present in the upper conidioma layer are judged to be phialides. Frequently two micro-conidia forming phialides are situated directly behind each other whereby the flask-shaped terminal phialides' bases are inflated and measure 6-10 x 3-5 μ. The lower phialides have a lateral porus. The phialides forming macroconidia are often considerably longer (8-26 x 3-5 μ ) and are not generally arranged behind each other.

The hyaline single celled microconidia are very small and. cylindrical to slender clavate, whereby the broader end is rounded, the narrower truncate. The colourless to weakly amber coloured single celled macroconidia on the other hand are large and normally allantoid. One end is rounded and the other has a protruding truncate base.

The culture conditions can have a marked influence on the formation and. size of the micro- and macro-conidia as summarised in the following Table 4.

The mature macro-conidia are filled with globular material which turns intensively red with Sudan III dye and deep black with Sudan-black B. Micro-conidia do not stain. The germination of macro-conidia is optimal at 24°C on MA-medium in the narrow pH range of 3.1 to 3.8 (70-80% germination). Above pH 5.5 the germination rate is under 5%. The germination rate of the micro-conidia was less than 1% under all conditions tested.

The new strain ATCC 20594 can be cultivated at a suitable temperature on various conventional nutrient media as aerobic surface or immersion cultures. As soon as a sufficient amount of the new compound S 41062/F-1, S 41062/F-6 and/or S 41062/F-7 has been produced in the culture which may e.g. be ascertained by the activity towards Candida sp., the mycelium may be separated from the culture broth and extracted in conventional manner. A preferred isolation procedure comprises homogenising the mycelium with methanol and separating and concentrating the liquid phase. Subsequent extraction is effected with a water-immiscible organic solvent e.g. ethyl acetate followed by removal of the solvent in vacuum, dissolution of the residue in methanol and after scouring, e.g. with hexane, evaporation to dryness in vacuum.

S 41062/F-1, S 41062/F-6 and/or S 41052/F-7 can be isolated and purified in conventional manner, e.g. employing chrpmatographic techniques or countercurrent partitioning. The invention also concerns fermentation broths which are obtained during the cultivation of the S 41062/F-1, S 41062/F-6 and/or S 41062/F-7 producing strains of Cryptosporiopsis.

The compounds S 41062/F-1, S 41062/F-6 and S 41062/F-7 exhibit antibiotic activity. They exhibit a growth inhibiting effect towards micr-organisms such as Yeasts and fungi, but no significant activity towards gram positive and gram negative bacteria.

The wide activity against yeasts and fungi is particularly noticeable against-various human pathogenic Candida strains.

The following Table 5 indicates the minimum inhibition concentration (MIC) of S 41062/F-1, F-6 and F-7 against various micro-organisms determined in known manner in a series dilution test in a malt extract medium, incubation temperature 27°C over 43 to 72 hours.

This activity is also observed in vivo in genital model infection test. In one such test mice and rats which have been pre-treated with estradiol-benzoate are infected intra-vaginally and then treated on 5 successive days either orally or parenterally (mouse) or intravaginally (rat) with test substance. The success of treatment is determined by the presence or absence of fungus in the vagina or from the uterus.

Antimycotic activity is observed on local application at concentrations of 2% and upward.

Systemic activity is observed in vivo on sub-cutaneous application in a dosage range of from ca. 5 to 50 mg/kg animal body weight.

The compounds S 41062/F-1, S 41062/F-6 and S 41062/F-7 are therefore useful as antibiotics.

For the above-mentioned use the dosage will, of course, vary depending on the compound employed, mode of administration and condition to be treated. However, in general, satisfactory results are obtained when administered at a daily dosage of from 5 mg to about

50 mg per kg animal body weight, conveniently given in divided doses 2 to 4 times a day or in sustained release form. For the larger mammal, the total daily dosage is in the range from about 300 to about 3000 mg, e.g. up to 500 mg. Unit dosage forms suitable for oral admini stration comprise from about 75 mg to about maximally 1500 mg, e.g. 250 mg, of the compounds admixed with a solid or liquid pharmaceutical carrier or diluent.

The invention therefore also concerns a method of treating diseases or infections caused by yeasts and fungi using the compound S-41062/F-1, S-41062/F-6 and/or S-41062/F-7 and also these

compounds for use as pharmaceuticals, e.g. antibiotics and for use in the treatment of the human or animal body by therapy.

The compounds may be administered preferably orally or parenterally suitably in admixture with conventional pharmaceutically acceptable diluents and carriers, and, optionally, other excipients.

Such compositions also form part of the invention. The following examples illustrate the invention. All temperatures are in degrees Centigrade.

EXAMPLE 1: Cultivation of Strain ATCC 20594 in a Glass Fermenter

a) Agar Starting Culture

The spore and mycelium suspension for inoculation is produced from a slant culture of strains ATCC 20594 which is obtained after 21 days incubation at 21° on an agar medium of the following composition.

Malt extract (syrup) 20 g

Yeast extract 4 g Agar 20 g

Demineralised water to 1 litre

b) Preculture

Spores and mycelia of the culture obtained under a) are suspended in physiological common salt solution and used to inoculate 1 litre of the following preculture medium in a 2 litre Erlenmeyer flask. Malt extract (syrup) 20 g Yeast extract 4 g

Demineralised water to 1 litre.

Inciibation is performed on a rotary shaker at 180 rpm for 7 days at 21°. c) Production Culture

The preculture obtained under b) is used to inoculate 10 litres of the following production culture medium in a 14 litre glass fermenter.

Malt extract (liquid) 20 g Yeast extract 4 g

Citric acid 12 g

NaOH (IN) 112 ml

HCl (IN) 44 ml

Demineralised water to 1 litre Prior to sterilisation the pH is adjusted to 3.8 to 4.1 with IN HCl/NaOH.

Inciibation is performed for 5 days at 21° with stirring (150 rpm) and aeration (1 litre/min/litre medium).

EXAMPLE 2: Cultivation of Strain ATCC 20594 in a

Steel Fermenter a) Agar Starting Culture

Cultivation is carried out for 21 days at 21° in 400 ml Roux flasks using 150 ml of the same agar medium as in Examnle 1a). b) Preculture

To prepare the suspension required for inoculation, 50 ml of physiological salt solution are added and the spore and mycelia suspension is obtained by using a sterile scraper.

250 ml of an inoculum thus prepared are used for inoculation of 50 litres of preculture medium of the same composition as described in Example 1b) in a 75 litre steel fermenter.

Incubation is carried out for 5 days at 21° , air rate 0.5 litre/min/litre medium at 0.5 bar and 150rpm. c) Intermediate Culture

500 litres of the following intermediate culture medium in a 750 litre steel fermenter are inocul- ated with 100 litres of the above preculture:-

Malt extract (syrup) 50 g Yeast extract 10 g

FeSO₄ . 7H₂O 16.68 mg

ZnSO₄ . 7H₂O 6.88 mg Demineralised water to 1 litre

Incubation conditions: 4 days at 21°, 100 rpm. 0.5 bar and aeration of 0.5 litre/min/litre medium. d) Main Culture

3,000 litres of production culture medium composed as in Example 1c) in a 4,500 litre steel ferm enter are inoculated with the intermediate culture. Inoculation is carried out for 5 days at 21°, 0.5 bar and an air rate of 1.0 litre/min/litre medium.

EXAMPLE 3; Isolation of the Compounds S 41062/F-1, S 41062/F-6 and S 41062/F-7

300 litres of fermentation broth (obtained according to Example 2) are adjusted to pH 6-7 with dilute sulphuric acid or sodium hydroxide and the mycelium precipitate separated from the culture filtrate with a Westfalia separator.

The wet mycelium is homogenised in a Dispax reactor with 10 times the quantity of methanol and the liquid phase separated off. This procedure is repeated twice with 90% aqueous methanol. The filtrates are combined, freed from methanol in vacuum and then extracted three times with ethyl acetate and once with ethyl acetate/isopropanol (4:1). The extracts are freed from solvent in vacuum and the residue dissolved in 10 times the quantity of 90% aqueous methanol. After scouring with hexane the polar phase is again evaporated to dryness in vacuum. This residue is dissolved in 1.5 litres of a mixture of methylene chloride/methanol/water (78:20:2) and separated by chromatography on 20 kg of kieselgel - Merck (0.05 - 0.2 mm) using the same mixture of solv ents as eluant. Fractions of 13 litres are obtained.

The fractions active against Candida albicans, nos. 8 and 9 , which contain primarily the compounds S 41062/F-1 and S 41062/F-7 are combined and chromatographed on 1.5 kg of Sephadex LH 20 in methanol (at 25 ml per fraction) whereby the two metabolites were considerably enriched in the fraction 42 to 62.

The concentrated residues of these fractions are divided into 4 portions which are each chromatographed on 1.2 kg of kieselgel H Merck (in columns of 10 cm diameter) using methylene chloride/methanol (4:1) whereby 100 ml fractions are collected.

The fraction residues are tested for purity by thin layer chromatography and the pure fractions combined. Pure S 41062/F-7 was found in the fractions between 40 to 65 and pure S 41Q62/F-1 in those from about 75 to 85.

The concentrated residues containing S 41062/F-1 are dissolved in ethanol, combined, treated with active charcoal and filtered to clarity through a layer of talc. The almost colourless filtrate is concentrated under a mild vacuum and slowly mixed with acetone, whereupon S 41062/F-1 is obtained as a white and, damp with solvent, as a highly hygroscopic compound in microcrystalline form. M.p. 235-240° (gradual decomposition).

The chromatogram fractions in which only S 41062/F-7 was shown on thin-layer chromatography are combined, filtered in methanol through a talc layer, concentrated in vacuum and slowly mixed with acetone whereupon S 41062/F-7 is obtained as an amorphous precipitate. M.p. from 205°C (sinter with gradual decomposition).

The fractions 10 and 11, which were obtained from the first chromatogram and which contain predominantly S 41062/F-6, are combined and then separated on 500 g of kieselgel Merck (0.04 - 0.06 mm) in methylene chloride/methanol (4:1) with collection of 80 ml fractions.

The fractions active against Candida albicans are evaporated to dryness in vacuum and the residues tested for purity by thin-layer chromatography.

The fractions which consist almost entirely of S 41062/F-6 are combined, dissolved in methanol, filtered clear through a. talc layer and after concentration in vacuum, slowly mixed with acetone. S 41062/F-6 precipitates as a white microcrystalline compound, m.p. 240-244° (gradual decomposition).

Claims

WE CLAIM :

1. A compound chosen from the following related compounds:- S 41062/F-1 fitting the following approximate characterising features when in the form of a white microcrystalline powder:

(i) = -6º (c = 0.5 in methanol);

(ii) analysis:

C 55.7 : H 7.7 : N 10.1 : O 24.7 %;

(iii) IR spectrum in KBr, see Figure 1; (iv) ¹H-NMR spectrum (90 mHz in DMSO) with tetra methylsilane as internal standard, see Figure 4; (v) UV spectrum in methanol λ max 193 nm

(222) nm _" = 140 279 nm " = 14;

(vi) antibiotic activity, see Table 5; (vii) molecular weight 1048.

S 41062/F-6 fitting the following approximate characterising features when in the form of a white microcrystalline powder: (i)

(ii) analysis :

C 56.3 : H 7.7 : N 10.9 : O 26.0 %; (iii) IR spectrum in KBr, see Figure 2; (iv) ¹H-NMR spectrum ( 90 mHz in DMSO) with tetra methylsilane as internal standard, see Figure 5;

(v) UV spectrum in methanol λ max 193 nm

(222) nm _" = 340 278 nm " = 15

(vi) antibiotic activity, see Table 5; and

S 41062/F-7 fitting the following approximate characterising features when in the form of a white powder;

(i)

(ii) analysis:

C 54.5 : H 7.5 : N 9.9 : O 25.0 %;

(iii) IR spectrum in KBr, see Figure 3; (Iv) ¹H-NMR spectrum (90 mHz in DMSO) with tetra methylsilane as internal standard, see Fig. 6; (v) UV spectrum in methanol

(222) nm _" = 339 278 nm _" = 15 (vi) antibiotic activity, see Table 5.

2. S 41062/F-1 having the formula

3. S 41062/F-1 as defined in Claim 1 or Claim 2.

4. A process for the production of a compound according to Claim 1 or 2 which comprises cultivating a S 41062/F-1, and/or S 41062/F-6 and/or S 41062/F-7 producing strain in a nutrient medium.

5. A process according to Claim 4 wherein the strain is a Cryptosporiopsis strain.

6. A process according to Claim 5 wherein the strain is ATCC 20594 or NRRL 12192.

7. A compound according to Claim 1 whenever prepared by a process according to any one of Claims 4 to 6 or 15.

8. A pharmaceutical composition which comprises a compound according to any one of Claims 1 to

3, 7 or 15 in association with a pharmaceutically acceptable diluent or carrier.

9. A S 41062/F-1 and/or S 41052/F-6 and/or

S 41062/F-7 producing strain.

10. A strain according to Claim 10 which is a Cryptosporiopsis strain.

11. The strain known as NRRL 12192 or ATCC 20594.

12. Fermentation broth prepared by cultivating a strain according to any one of Claims 9 to 11.

13. A method of combatting micro-organisms in or on a mammal in need of such treatment which comprises administering an antibiotically effective amount of a compound according to any one of Claims 1 to 3, 7 or 15.

14. A compound according to any one of Claims 1 to 3, 7 or 15 for use as an antibiotic.

15. A compound according to Claim 1, a process according to Claim 4 or a strain according to Claim 9 substantially as hereinbefore described.