GB2138805A - An anthracycline glucuronide - Google Patents

Abstract

An anthracyclinone glucuronide of formula <IMAGE> (R = H, lower alkyl, alkali metal> is prepared by cultivating Streptomyces peucetius var. vinaceus (DSM 2597), a mutant strain of Streptomyces peucetius var. aureus, under aerobic conditions in an aqueous culture medium containing assimilable sources of carbon and nitrogen and mineral salts, followed by appropriate work- up procedures. The anthracycline glucuronide has antitumour properties. The claims extend to the anthracyclinone glucuronide, its fermentive preparation, the microorganism Streptomyces peucetius var. vinaceus used therein, and pharmaceutical compositions containing the anthracyclinone glucuronide.

Description

SPECIFICATION An anthracyclinone glucuronide The invention relates to an anthracyclinone glucuronide and to its salts and esters, to biosynthetic processes for their production, and to compositions containing them. The anthracyclinone glucuronide, hereinafter referred to as FCE 22724, has been found to be effective in inhibiting the growth of malignant tumours such as P 388 leukemia in experimental animals.

The structural formula of FCE 22724 has been determined and may be represented as

The invention provides a biosynthetic process for the production of FCE 22724 the process comprising cultivating Streptomyces peucetius var. vinaceus (DSM 2597) under aerobic conditions in an aqueous culture medium containing assimilable sources of carbon and nitrogen and mineral salts. The microorganism Streptomyces peucetius var. vinaceus is a mutant strain of Streptomyces peucetius var. aureus (ATCC 31428). The invention includes processes for recovery of FCE 22724 from the fermentation broths, concentration from crude solutions and purification.The invention further includes within its scope the new anthracyclinone glucuronide FCE 22724 in the form of crude concentrates, obtained from the biosynthetic processes, in the pure form, as free acid or alkaline salts, isolated from the crude concentrates and in the form of pharmaceutical compositions in which FCE 22724 and its salts or esters are admixed with a pharmaceutically acceptable diluent or carrier.

THE MICROORGANISM The microorganism used in the process of the invention was obtained as an induced back mutation after a mutagenic treatment with U.V. rays of Streptomyces peucetius var. aureus strain of 416 F.l. (ATCC 31428; DSM 1367; F.R.I. 4622) described by CASSINELLI et al.

(J.Antib. vol. 35, n. 2, pp. 176-183, 1982). The new strain, thus obtained, has been given the code number M83 FCE of the Farmitalia Carlo Erba collection of Microoganisms and has been deposited at the Deutsche Sammlung für Mikroorganismen under the number DSM 2597.

The new microorganism differs from its parent culture and from all the other mutants obtained from Streptomyces peucetius mainly because it produces a pigment of the substrate mycelium which in young culture on all solid media is pink to red to vine-like, and becomes deep violet on aging. This tonal change is also observable for the abundant soluble pigment which is formed on all solid media commonly used for the classification of streptomycetes. Such media include Bennet's agar, Czapek's agar, asparagine-glucose agar, glycin-glycerol agar, potato glucose agar, asparagine-glycerol agar (Waksman S.A.: "The Actinomycetes" vol. Il, 1961, William Wilkins 8 -Co.Baltimore) and inorganic salts-starch agar (Pridham T.G., Anderson P., Foley C., Lindenfelser L.A., Hesseltine C.A. and Benedict R.G.: A selection of media for maintenance and taxonomic studies of Streptomyces. Antibiotics Ann. 1956/1957, 947-953).

We therefore consider strain M83 FCE a variety of Streptomyces peucetius to which the designation of Streptomyces peucetius var. vinaceius is given. The micoorganism Streptomyces peucetius var. vinaceius is itself within the scope of the invention.

FERMENTATION PROCESS The production is carried out by the usual, well known methods and comprises culturing the microorganism in a previously sterilized liquid culture medium under aerobic conditions at a temperature ranging from 25 C to 37"C (preferably at 28 C) over a period of time varying from 3 to 7 days (preferably 5 days) and at a pH which initially is from 6.5 to 7.0 and at the end of the fermentation process ranges from 6.5 to 8.0. The culture medium comprises carbon and nitrogen sources as well as mineral salts. Suitable carbon sources include starch, dextrin, glucose, glycerin, mannitol, maltose, corn steep liquor, distillers solubles, soybean oil and soybean meal. Suitable nitrogen sources include such of the above carbon sources as contain nitrogen, dry yeast, meat peptone or casein.Good results are also obtained by using ammonium salts such as ammonum nitrate, ammonium sulphates and diammonium phosphates. The mineral salts useful for the production may vary according to the medium employed. In media containing complex substances such as various meals and fermentation residues, the addition of calcium carbonate and sodium or potassium phosphates have proved useful. In media containing glucose, or ammonium salts, much higher levels of mineral salts such as potassium, sodium or calcium, and trace additions of iron, zinc, copper, magnesium and manganese salts are needed. The fermentation may be carried out in Erlenmeyer flasks or in laboratory or industrial fermenters of various capacities.

ANALYTICAL METHODS When samples of fermentation broths and crude mixtures are subjected to thin layer chromatography (TLC) on silica gel pre-coated plates, using as eluent a mixture of chloroform; methanol: acetic acid: water (80:20:7:3 by volume), FCE 22724 is found to occur at RF medium value of 0.22 as the major red coloured constituent. A quantitive estimation of FCE 22724 present in the fermentation broths can be performed by the following method. To a sample of broth, two volumes of acetonitrile are added and the rsulting mixture is sonicated for 1 minute at room temperature then filtered.When a sample of the filtrate is subjected to silicagel TLC analysis, using the above mentioned eluting system, a spectophotometric determination of FCE 22724 at 493 nm can be performed by scraping off and eluting with a mixture of chloroform: ethanol (9:1 by volume) the corresponding red coloured zone.

ISOLATION PROCEDURE After the fermentation is completed FCE 22724 is mainly found in the mycelia which are separated from the fermentation liquors by filtration at pH 7.5 with the aid of diatomaceous earth. The mycelial cake is extracted with a water-miscible organic solvent such as acetonitrile, dioxan, methanol and other lower alcohols; preferentially methanol is employed. The mycelial extracts are collected and concentrated under reduced pressure and the aqueous concentrate contains FCE 22724 in the form of an alkaline salt.FCE 22724 can be recovered from the filtered broths by extraction at pH 4 with a water-immiscible organic solvent such as methyl isobutyl ketone, methyl propyl ketone and nbutanol; methyl isobutyl ketone is preferably employed. carom the organic extracts FCE 22724 can be reextracted at pH 7 into water as sodium salt with aqueous sodium hydroxide or sodium hydrogen carbonate. From the filtered broths and aqueous concentrates FCE 22724 can be absorbed on hydrophobic cross-linked divinylbenzene polymeric resins such as Amberlites (R.T.M.) XAD 2, XAD 4 and ER 180 (Rohm and Haas Co., Inc), preferentially ER180 is employed. The absorbed FCE 22724 is readily eluted with aqueous lower alcohols, preferably 10% (by volume) npropanol in water.

PURIFICATION PROCEDURE Crude aqueous concentrates, adjusted to pH 7, after addition of sodium chloride (5%), are passed through a column of ER 180 resin. After washing with water, the elution is carried out with 10% (by volume) npropanol in water and the eluate is collected in fractions. From the selected fractions, monitored by silica-gel TLC, after concentration in vacuo and freeze-drying, crude FCE 22724 as its sodium salt is obtained. In order to obtain pure FCE 22724, an aqueous solution of the crude sodium salt is adjusted to pH 4 and extracted with methyl isobutyl ketone. The organic extract, after concentration in vacuo to a small volume, precipitation with nhexane and crystallization from methyl isobutyl ketone gives pure FCE,22724 as free acid in the form of fine red crystalline needles.Treatment of an alcoholic solution of pure FCE 22724 (1) with an equivalent of alcoholic sodium hydroxide, followed by concentration and precipitation with diethyl ether gives the corresponding pure sodium salt (II). Acid catalyzed esterification of FCE 22724 in methanol gives the corresponding methyl ester (III).

CHEMICAL AND PHYSICAL PROPERTIES FCE 22724, as free acid (I), is soluble in alkaline aqueous media, lower alcohols and polar organic solvents, slightly soluble in acetone, ethyl acetate, diethyl ether, methylene dichloride and chloroform, and barely soluble in water, benzene and nCexane. FCE 22724 as sodium salt (II) is soluble in water, lower alcohols and polar organic solvents, and slightly soluble or insoluble in other organic solvents. FCE 22724 methyl ester (III) is soluble in lower alcohols, acetone, ethyl acetate, methylene dichloride and chloroform, slightly soluble in diethyl ether but insoluble in water and nhexane. FCE 22724 has the following chemical and physical properties: Melting point: 168-1 69 C (with decomposition).

U.V. and VIS absorption spectrum: in methanol :#may 233, 252, 288, 360, 474, 493, (525 sh) nm 1% E 490, 495, 144, 47, 184, 184, 100) in 0.01 N methanolic sodium hydroxide :max 233, 252, 290, (350 sh), 540 and 580 nm jn M/1 5 phosphate buffer pH 7: Amax 232, 252, 288, 352, 474, 488 nm 1% (E 562, 526, 175, 55, 193, 187) 1 cm I.R. spectrum (KBr): peaks at the following frequencies: 3420, 2920, 1730, 1620, 1585, 1430, 1405, 1330, 1245, 1210, 1175, 1120, 1060, 940, 910, 870, 830 and 800 cm-.

Molecular formula: C28H28O5, MW. 604, 53, based on the elemental analysis and 13 C NMR spectrum 1H NMR spectrum: (CDCI3 + 1 drop DmSO-d6) at 200 MHz: 0.99 (t, J = 7.3 Hz, 3H, CH3CH2); 1.40, 1.66 (two m, 2H, CH3CH2); 1.9-2.3 (m, 2H, H-g); 3.56 (s, 3H, COOCH3); 3.5-3.8 (m, 3H, H-2', H-3', H-4'); 3.94 (d, J = 9Hz, 1H, H-5'); 4.14 (s, 1H, H-10); 4.85 (d, J = 7Hz, 1 H, H-1'); 5.14 (bs, 1 H, H-7); 7.65 (d, J = 4.4 Hz, 2H, H-1, H-3); 8.00 (t, J = 4.4, 1H, H-2); 13.30 and 13.60 # (two s, 2H, OH-6, OH-li).

'3C NMR spectrum (CDCI3/CD30H 1:1 by volume) at 50 MHz: 6.08(0-14); 32.38(0-13); 34.42(0-8); 51.52 (C-10); 51.89 (COOCH3); 61.64(0-4'); 71.22(0-7); 71.56(0-9); 72-89 (C-2'); 75.14 (C 3', 5'); 101.50 (C-1'); 122.03 (C-1); 124.44(0-3); 133.09 (C-12a); 134.92 (C-6A); 135.81(0-2); 138.16 (C-1 Oa); 155.47 (C-11); 156.24(0-6); 158.36(0-4); 170.61 (C5-COOCH3); 171.52 (010-COOCH3); 186.07 (0-5) and 187.138 (C-12).

STRUCTURE DETERMINATION The structure assigned to FCE 22724 and corresponding to 4-0-(ssD-glucuronyl)-rhodomyci- none (I)

I:R = H Il:R = Na III:R = CH3 acid or enzymatic hydrolysis

was determined as follows: aqueous acid hydrolysis gives Erhodomycinone (IV), as insoluble purple red aglycone and D-glucuronic acid (V), both identified by direct comparison with authentic samples.Incubation of FCE 22724 sodium salt with different glucuronidases enzymatically confirmed the presence of a moiety of D-glycuronic acid (V) linked to e-rhodomycinone (IV). The position and the ss-configuration of the glycosidic linkage in FCE 22724 has been assigned on the basis of UV., VIS, IR., 1H and '3C-NMR spectra. While e-rhodomycinone (IV) and its 7-o-glycosides are known constituents of several anthracycline complexes produced by different species of the genus Streptomyces, such as pyrromycin (Chem. Ber. 92, 1904, 1959), cinerubin A and B (United States Patent Specification No. 3,864,480), musettamycin and marcellomycin from bohemic acid complex (United States Patent Specification No.

4,039,736),FCE 22724 (I) is the first biosynthetic anthracycline like antitumour compound containing in its molecule a ss-D-glucuronyl moiety linked to the phenolic hydroxy group at the 4 position of -rhodomycinone.

The following Examples illustrate the invention.

Example 1 A culture of Streptomyces peucetius var. vinaceius, strain M83 FCE, was grown for 14 days at 28"C on agar slants of the medium SA (Glucose 3%; brewer's dry yeast 1.2%; NaCI 0.1%; KH2PO4 0.5%; CaCO3 0.1%; MgSO4 0.005%; FeS04.7H2O 0.0005%; ZnS04.7H20 0.0005%; CuSO4.5H20 0.0005%; agar 2%; tap water up to 100 ml; pH 6.7; sterilization carried out by heating in an autoclave at 11 5 C for 20 minutes).

The mycelial fragments scraped off from the agar culture were collected in 3 ml of sterile distilled water and homogenized on a vortex homogenizer. The suspension so obtained was inoculated in 300 ml Erlenmeyer flasks containing 60 ml of the following liquid growth medium: brewer's dry yeast 0.3o/o; peptone 0.5%; Ca(N03)2.4H20 0.05%; tap water up to 100 ml; sterilization carried out by heating in autoclave at 1 20 C for 20 minutes; pH after sterilization between 6.8 and 7.0.The inoculated flasks were shaken for 2 days at a temperature of 28"C on a rotary shaker running at 250 rpm and describing a circle of 7 cm diameter. 1.5 ml of the culture grown as described above were inoculated in 300 ml Erlenmeyer flasks containing 50 ml of the following production medium; glucose 6%; brewer's dry yeast 2.5% soy bean meal 1%; NaCI 2%; KH2PO4 0.1%; ZnSO4. 7H20 0.001%; CuSO4.5H20 0.001%; tap water up to 100 ml; pH 6.7; sterilization carried out by heating in autoclave at 11 5 C for 20 minutes. The flasks were thus incubated at 28"C for 7 days in the identical conditions as described for the seed phase. The maximum concentration of FCE 22724 was reached between the sixth and seventh days of fermentation with a production of 50 mcg/ml.

EXAMPLE 2 The whole fermentation procedure was carried out as described for Example 1, the only difference being the utilization of the following production medium: glycerol 6%; brewer's dry yeast 2.5%; soy bean meal 1%; NaCI 2%; KH2PO4 0.1%; CaCO3 0.2%; MgSO4 0.01%; FeSO4.7H20 0.001%; ZnS04.7H2O 0.001% OuS04.5H20 0.001%; tap water up to 100 ml; pH 6.7; sterilization carried out by heating in autoclave at 11 5 C for 20 minutes.

The maximum concentration of FCE 22724 was reached between the sixth and sevenths days of fermentation with a production of 100 mcg/ml.

Example 3 The culture of strain M83 FCE was obtained as described in Example 1. The mycelial fragments of three slants were pooled and collected in 10 ml of sterile distilled water; the suspension, after homogenization carried out as described in Example 1, was inoculated in a 2 1 buffled round-bottomed flask containing 500 ml of the seed medium described in Example 1.

The flask was incubated for 48 hours on a rotary shaker running at 120 r.p.m. and describing a circle of 7 cm diameter, at a temperature of 28 C. The whole seed was inoculated in an 80 1 stainless steel fermenter containing 50 1 of the seed medium described in Example 1 and sterilized by vapour pressure at 1 20 C for 30 minutes. The seed phase was carried out at 28 C, under stirred conditions at 230 r.p.m. and an air flow of 0.5 litre/litre of medium per minute.

After 30 hours, this seed was inoculated in a 800 1 stainless steel fermenter containing 500 1 of the production medium described in Example 2, and sterilized by vapour pressure at 1 20to for 30 minutes. The fermentation was performed at 28"C, stirred at 250 r.p.m. and aereated with an air flow of 0.7 litre/litre of medium per minute. The maximum concentration of the FCE 22724 was reached between the sixth and seventh days of fermentation with a production of 70 mcg/ml.

Example 4 The whole broth (3 I) from a fermentation obtained according to Example 1 was filtered at pH 7 using 3% diatomaceous earth as filter aid. The wet filter cake was extracted with methanol (3 1). After filtration, two additional extractions (respectively with 3 1 and 2 1 of methanol) were effected to ensure a complete recovery of the red pigments. The combined extracts were concentrated under reduced pressure to 300 ml. The filtered broth obtained above was adjusted to pH 4 with sulphuric acid and extracted with methyl isobutyl ketone (3 1). After two additional extractions, the organic extracts were combined, concentrated and re-extracted with 3% aqueous sodium hydrogen carbonate.To the aqueous layer combined with the aqueous concentrate of the mycelial extract was added sodium chloride (5%), and the whole was absorbed on a column (3.2 x 27 cm) filled with resin Er 1 80 (200 ml). After discarding the initial washing with water, elution was carried out with 10% by volume nprnpanol in water.

Selected fractions, monitored by TLC, were concentrated under reduced pressure and then freeze dried to give crude FCE 22724 as its sodium salt (1 9).

Example 5 An aqueous solution (200 ml) of crude FC 22724 as its sodium salt (1 9), obtained according to Example 4, was adjusted to pH 4 with sulphuric acid and extracted with methyl isobutyl ketone. The organic extracts were concentrated under reduced pressure and almost pure FCE 22724 (0.3 9) was precipitated with nhexane. Crystallization from methyl isobutyl ketone gave pure red needles of FCE 22724 (0.25 g) as free acid: m.p. 168"C (with decomposition).

Example 6 A methanolic solution (20 ml) of FCE 22724 free acid (0.12 g), obtained according to Example 5, was treated with an equivalent (0.2 m moles) of methanolic sodium hydroxide. After concentration and precipitation with diethylether, pure FCE 22724 as its sodium salt (0.11 9, m.p. 190-191 C with decomposition) was obtained.

Example 7 A solution of FCE 22724 (0.1 9) in anhydrous methanol (100 ml) was treated with 1 N methanolic hydrochoric acid (q ml). After 2 hours at room temperature, the reaction mixture diluted with ethyl acetate (100 ml) was concentrated to 50 ml under reduced pressure. After washing with aqueous sodium hydrogen carbonate and water, the organic phase, dried on anhydrous sodium sulphate, was concentrated to a small volume. Addition of nhexane gave the pure methyl ester of FCE 22724 (0.09 9), as a red microcrystalline powder; m.p. 150-152"C; U.V. and VlS spectrum: MeOH X 223, 252, 288, 474, 493 (525 sh) nm Max 1% (E 505, 500, 147,48, 178, 178, 96).

1 cm Its molecular formula C29H30O,5, based on elemental analysis, was confirmed by field desorption mass spectrometry (m/e 618) and the assigned structure (III) was conformed by l.R. and NMR spectroscopy: INMR spectrum (CDCI3) at 200 MHz: 1.12 (t, J = 7.2 Hz, 3H, CH3CH2); 1.12, 1.52 (two m, 2H, CH2CH3); 2.0-2.4 (m, 2H, H-8); 3.69 (s, 3H, C,o~COOCH3); 3.82 (s, 3H, C5, -COOCH3); 3.8-4.0 (m, 3H, H-3, H-4', H-2'); 4.20 (s, 1 H, H-10); 4.28 (d, J = 8Hz, 1 H, H5'); 5.06 (bs, 1H, H-l'); 5.19 (bs, 1H, H-7); 7.2-7.6 (m, 3H, H-l, H-2, H-3); 13.21, 13.47 8 (two s, 2H, OH-6, OH-11).

Example 8 A solution of FCE 22724 (0.1 9) in dioxan (2 ml) and 1% aqueous sulphuric acid (10 ml) was heated for 2 hours at 100 C. The reaction mixture, containing a red coloured precipitate, was diluted with water (20 ml) and extracted with ethyl acetate. The extract, washed with 3% sodium hydrogen carbonate and water and dried on anhydrous sodium sulphate, was concentrated to a small volume under reduced pressure. Addition of an excess of n-hexane gave a red crystalline compound (0.045 g m.p. 210QC) identified as e-rhodomycinone (IV) after comparison with an authentic sample.On the other hand, the acidic constituent present in the water soluble fraction of the above hydrolysate, was identified by silica-gel TLC as D-glucuronic acid (V) after comparison with an authentic sample.

Example 9 A solution of FCE 22724 (0.05 9) in M/1 5 phosphate buffer at pH 7 (50 ml) was treated with an aqueous solution (50 ml) of lD-glucurnnidase (50 mg of "Bacterial type VII" enzyme Sigma) and incubated for 1 hour at 37"C.

From the ethyl acetate extract was isolated e-rhodomycinone (IV, 0.022 g), while on the aqueous phase was reconfirmed the presence of D-glucuronic acid (V).

BIOLOGICAL ACTIVITY FCE 22724 shows cytotoxic activity on HeLa cells cloning efficiency "in vitro", having an ID50 of 40 yg/ml. ID50 is the median concentration inhibiting 50% of growing cells.

FCE 22724 displays also "in vivo" antitumour activity on P 388 leukemia in mcie, as shown in the Table.

Table--"ln vivo" effect of FCE 22724 on P-388 murine leukemia) Dose T/Cb) mg/kg % Toxic deaths ", 4.4 135 0/10 6.6 160 0/10 15.0 165 0/10 22.5 175 1/10 a) Treatment i.p. on day 1 after tumour inoculation (106 cells/mouse) b) Median survival time; % over untreated controls c) Evaluated on the basis of autoptic findings on dead mice.

Claims (9)

1. An antitumoural anthracyclinone glucuronide of the general formula A:

wherein R represents a hydrogen atom, a lower alkyl group or an alkali metal atom.

2. A process for the production of an anthracyclinone glucuronide of the general formula A wherein R represents a sodium atom, the process comprises cultivating the microorganisms Streptomyces peucetius var. vinaceus (DSM 2597) under aerobic conditions in an aqueous culture medium containing assimilable sources of carbon and nitrogen and mineral salts.

3. A process according to claim 2 wherein the cultivation is carried out from 3 to 7 days at a temperature of from 25 to 37"C and at pH which is initially from 6.5 to 7.0 and which is from 6.5 to 8.0 at the end of the cultivation.

4. A process according to claim 2 or claim 3 further comprising removing the mycelium from the culture medium, extracting the mycelium with a water-miscible organic solvent and concentrating the extracts under reduced pressure to obtain the crude anthracyclinone glucuronide of the general formula A wherein R represents a sodium atom.

5. A process for the purification of the crude anthracyclinone glucuronide of the general formula A wherein R represents a sodium atom, obtained according to any of claims 2 to 4, which process comprises absorbing a concentrated aqueous solution of the crude compound on a hydrophobic cross-linked divinylbenzene polymeric resin, eluting with an aqueous lower alcohol, adjusting to 4.0 the pH of the eluate to liberate the anthracyclinone glucuronide of the general formula A wherein R represents a hydrogen atom, extracting the eluate with methyl isobutyl ketone, precipitating the anthracyclinone glucuronate in pure form by addition of nhexane, and re-transforming it, into the pure sodium salt by treatment with an equivalent of methanolic sodium hydroxide followed by precipitation with diethyl ether.

6. A process according to claim 5 wherein the aqueous lower alcohol is 10% by volume npropanol in water.

7. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to claim 1 in admixture with a pharmaceutically acceptable diluent or carrier.

8. The microorganism Streptomyces peucetius var. vinaceus (DSM 2597).

CLAIMS (26 Mar 1984)

1. An antitumoural anthracyclinone glucuronide of the formula A:

or a pharmaceutically acceptable salt or esters thereof.

2. A process for the production of the anthracyclinone glucuronide of the formula A, the process comprising cultivating the microorganisms Streptomyces peucetius var. vinaceus (DSM 2597) under aerobic conditions in an aqueous culture medium containing assimilable sources of carbon and nitrogen and mineral salts.

4. A process according to claims 2 or claim 3 further comprising removing the mycelium from the culture medium, extracting the mycelium with a water-miscible organic solvent and concentrating the extracts under reduced pressure to obtain the crude anthracyclinone glucuronide of the formula A.

5. A process for the purification of the crude anthracyclinone glucuronide of the formula A, obtained according to any of claims 2 to 4, which process comprises absorbing a concentrated aqueous solution of the crude compound on a hydrophobic cross-linked divinylbenzene polymeric resin, eluting with an aqueous lower alcohol, adjusting to 4.0 the pH of the eluate to liberate the-anthracyclinone glucuronide of the general formula A wherein R represents a hydrogen atom, extracting the eluate with methyl isobutyl ketone, precipitating the anthracyclinone glucuronide in pure form by addition of n-hexane.

7. A process according to claim 5 or claim 6 further comprising transforming the precipitated anthracyclinone glucuronide into its pure sodium salt by treatment with an equivalent of methanolic sodium hydroxide followed by precipitation with diethyl ether.

8. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to claim 1 in admixture with a pharmaceutically acceptable diluent or carrier.

9. The microorganism Streptomyces peucetius var. vinaceus (DSM 2597).