GB2048245A - A biosynthetic anthracycline glycoside - Google Patents

Abstract

Cultivation of the microorganism Streptomyces peucetius var. caminatus (1524 DSM, 31502 ATCC, 4929 FRI, DR 81 FI Farmitalia microbiological collection) under aerobic conditions in an aqueous culture medium including sources of assimilable carbon and nitrogen and mineral salts leads to 13- deoxycarminomycin. The microorganism is obtainable by mutagenic treatment with N-methyl- N'-nitro-N-nitrosoguanidine of Streptomyces peucetius var. caesius. 13-Deoxycarminomycin has antitumour properties; it is also active against gram positive and gram negative bacteria. 13-deoxycarminomycin has the following structure: <IMAGE>

Description

SPECIFICATION A biosynthetic anthracycline glycoside The invention relates to a novel anthracycline glycoside and to its salts, to biosynthetic processes for their production, and to compositions containing them.

The invention provides 1 3-deoxywarminomycin and pharmaceuically acceptable acid addition salts thereof. 1 3-Deoxycarminomycin has the formula

The invention includes 1 3-deoxycarminomycin in the form of crude concentrates obtained from the microbiological preparation process described hereinbelow, in the pure crystalline form (base or salt) as isolated from the crude concentrates and in the form of pharmaceutical compositions containing it or its salts in admixture with a pharmaceutically acceptable diluent or carrier.

The invention further provides a microbiological process for the preparation of 13deoxycarminomycin, the process comprising cultivating Streptomyces peucetius var. carminatus (Farmitalia Carlo Erba collection of microorganisms No. DR81 F.l., Deutsche Sammlung Mikroorganismen No. 1524 DSM; American Type Culture Collection No. 31502 A.T.C.C.; Fermentation Research Institute, Japan, No. 4929 F.R.I.) under aerobic conditions in an aqueous cultural medium containing assimilable sources of carbon and nitrogen and mineral salts.

The microorganisms Streptomyces peucetius var. carminatus used in the process of the invention may be obtained by a mutagenic treatment with N-methyl-Nt-nitro-N-nitroso-guanidine of Streptomyces peucetius var. caesius (Arcamone et al. Biotechnol. Bioengeen. Xl, 1969, 1101-1110).

The new anthracycline glycoside according to the invention is useful as an antitumour agent in experimental animals; it is also active against gram positive and gram negative bacteria. The anthracycline glycoside according to the invention is referred to hereinafter as "antibiotic DOC".

THE MICROORGANISM Microscopic properties of strain DR 81 F.l.

The substrate mycelium is formed by fairly branched hyphae, 0.5-0.9 , in diameter, of variable length; the aerial mycelium which arises trom the former one is formed by fairly long, from straight to flexous hyphae 1.1-1.6,u in diameter; from these, by sympodial branching in a fasciculate fashion, spore bearing hyphae of variable length depart, which end in hooks or loops.

The spores are spherical and of the following size: 1-2 y in diameter, at first disposed in chains, then free. Under the electron microscope the spores appear nearly spherical, of irregular contours, with a warty-surface.

Macroscopic properties of strain DR 81 F.l.

The cultural characteristics of strain DR 81 Fl. are given in Table 1.

Growth is generally good on organic as well as on synthetic media; observations of growth on the media cited in Table 1 have been made from the 5th day of incubation at 280C onward till the end of growth.

The biochemical and physiological properties of strain DR 81 F.I. are given in Table 2.

No growth is observed at a temperature above 400 C. Sclerotia formation has not been observed on the media cited in Table 1.

Identification and classification of strain DR 81 F.l.

The over-all characteristics shown by strain DR 81 F.l. clearly correspond to those given for the genus Streptomyces Waksman et Henrici. Furthermore, the morphological, cultural and physical characeristics shown by strain DR 81 F.l. correspond to those described for the species Streptomyces peucetius var. caesius (U.S. Patent Specification No. 3,590,028; Arcamone et al. Biotechnol. Biogeen.

Xl, 1969, 1101-1110) from which it nevertheless differs because of its ability to form a terracotta to bright orange to carminic coloured substrate mycelium, because it utilizes m-inositol and L-arabinose and finally because it produces new anthracycline antibiotics.

TABLE 1 Cultural characteristics of strain DR 81 F.I.

Medium Substrate mycelium Aerial mycelium Soluble pigments Bennett's agar growth abundant slightly lichenoid absent orange-wine like to brown (Waksman, 1961) orange-rose to rose-wine like coloured Czapek's agar good growth, flat, compact, wine-like absent wine-like to violet (Waksman, 1961) to violet coloured Asparagine-glucose growth abundant, flat, compact, absent rose to wine-like agar (Waksman, 1961) orange to wine-like coloured Glycin-glycerol agar abundant growth, raised and ridged; absent from wine-like to brown to (Waksman, 1961) colonies sometimes crater-like; from dark brown orange to brown with violet tonalities coloured Emerson's agar abundant growth, raised and ridged; absent pale rose to wine-like brown (Waksman, 1961) crater-like; rose violet to brown coloured Inorganic salts - growth abundant, flat; orange to pale absent absent starch agar rose coloured (Pridham et al. 1957) Potato-glucose agar growth abundant, slightly raised; form absent brown violet (Waksman, 1961) wine-like violet to brown coloured Asparagine-glycerol agar growth abundant, raised and ridged absent bright orange to carmine (Waksman, 1961) colonies crater-like terracotta to bright orange to carmine coloured Yeast extract-glucose agar growth abundant, raised and ridged; absent from orange to pale wine (Waksman, 1961) colonies crater-like from orange to like carmine to wine-like coloured Starch-casein agar growth abundant flat; orange to absent from orange to rose (Waksman, 1961) carmine coloured wine-like SA-agar (see for growth abundant, raised and ridged; frequently absent, sometimes wine-like to violet opposition, maintenance terracotta to bright orange to present; gray with blue-green medium in example 1 carmine coloured tonalities TABLE 2. Physiological and biochemical properties of strain DR 81 F.l.* Utilization of glucose + " sucrose + " D-xylose + " mannitol + " m-inositol + " L-arabinose + " D-fructose + " adonitol " lactose + " d(+) mannose + maltose + " raffinose + " L-rhamnose " alpha-aipha-Trehalose + glycerol + " NH4-succinate + " glycogen + " paraffine + Negative control Liquefaction of gelatin Tyrosine decomposition + Melanin formation Hydrolysis of starch + H2S formation + Nitrate reduction Milk (pepton- and coag.) + Antibiotics produced: new anthracyclines + = positive reaction - - negative reaction * The medium for the carbohydrate utilisation test used is that described by R. D. Gordon and M. L. Smith: J. Bacteriology 69, 1955, pp. 147-150.

* The media used for the other physiological reactions are those reported by S. A. Waksman "The Actinomycetes" vol. Il, 1961. The Williams Wilkins Company, Baltimore.

We therefore consider strain DR 81 F.l. a variety of Streptomyces peucetius var. caesius, to which the designation Streptomycespeucetius var. carminatus is given.

Waksman S. A.: "The Actinomycetes" vol. II, 1961, William Wilkins Co. Baltimore.

Pridham T. G., Anderson P., Foley C., Lindenfelsen 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.

FERMENTATION PROCESS The production is carried out by the usual, well known methods and consists in culturing the mutated microorganism in a previously sterilized liquid culture medium under aerobic conditions at a temperature ranging from 250C to 370C (preferably at 280C) 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 consists of a carbon and a nitrogen source as well as of mineral salts.

The carbon source may, for example, be starch, dextrin, glucose, glycerin, mannitol, maltose, corn steep liquor, distillers solubles, soybean oil or soybean meal. The nitrogen source, besides the above mentioned complex substances containing nitrogen, may be for example dry yeast, meat peptone or casein. Good results are also obtained by using ammonium salts such as ammonium nitrate, ammonium sulphates, diammonium phosphates. The mineral salts useful for the production may vary according to the medium employed. In a medium 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 additions of micro-elements like iron, zinc, copper, magnesium and manganese salts are needed. The addition of sulphur containing compounds such as sulphanilamide, sulphathiazole, sulphapyridazine, penthiobarbital, ethionine and others may also be useful.

The fermentation may be carried out in Erlenmeyer flasks or in laboratory as well as in industrial fermenters of various capacities.

ANALYTICAL METHOD When samples of fermentation broths and crude mixtures are subjected to paper chromatography, by using Whatman No. 1 paper, buffered with M/1 5 phosphate buffer at pH 1 5.4, employing as eluent a mixture of 7:1:2 n-propanol/ethylacetate/water and the paper strip are bioautographed against Bacillus subtilis, the new anthracycline glycoside is found to occur at a Rf medium value of 0.77, together with other active components at different Rf values.

A quantitative estimation of the total red constituents present in the fermentation broths can be evaluated by the following method.

To a sample of broth, adjusted to pH 8.6, two volumes of 9.1 chloroform/methanol are added and the resulting mixture is sonicated for 1 minute at R.T. then, on a sample of the organic phase, diluted with acidic methanol, total content of the anthracycline-like constituents and of their aglycones can be spectrophotometrically determined at 495 nm: On a sample of the organic phase, concentrated under reduced pressure, quantitative determination of the antibiotic DOC can be obtained by paper chromatography using the above reported system. The red coloured zone is cut off and eluted with a 80:20 mixture of methanol and 0.01 N hydrochloric acid and the solution used for the spectrophotometrical determination at 495 nm.

ISOLATION PROCEDURE After the fermentation is completed, the active compounds are contained in the mycelia and in the fermentation liquors. The anthracycline antibiotic can be extracted at pH 8.5-9.0 as free bases from the culture broth "in toto" with a water immiscible organic solvent such as butanol, methyl isobutyl ketone, chloroform, methylene dichloride and ethyl acetate. Preferably the mycelia and the fermentation liquors are separated by filtration at pH4 with the aid of diatomaceous earth, then extracted separately.

The filtration cake is extracted with a mixture of a water-soluble solvent, such as acetone and lower alcohols, and of a 0.1 N acqueous solution of organic or inorganic acids; preferably a 4:1 (v/v) mixture of acetone: 0.1 N hydrochloric acid is employed.

The mycelial extracts are collected adjusted to pH 4 then concentrated under reduced pressure.

The concentrate is combined with the filtered broth, adjusted to pH 8.5-9, then extracted with a water immiscible organic solvent, preferentially chloroform or n-butanol. The extracts are concentrated under reduced pressure and a crude mixture is precipitated by addition of a fivefold volume ofn-hexane.

The constituents of this crude mixture are then fractionated and purified by column chromatography methods.

PURIFICATION PROCEDURE Purification of the antibiotic DOC and its separation may be affected by using silicagel and cellulose column chromatography. The crude red purple powder may be dissolved in a 300:55:6 chloroform:methanol:water mixture and subjected to siiica-gel column chromatography with the same mixture. The active eluates containing glycoside DOC are concentrated to dryness under vacuum and the residue is further purified by column chromatography on pH 5.4 buffered cellulose powder with a 9:1 n-butanol:ethyl acetate mixture. The active fractions containing pure glycoside DOC are extracted with acidic water, then the aqueous phase is adjusted to pH 8.5 and extracted with chloroform. The extract is washed with water, dried on sodium sulphate, and concentrate under reduced pressure to a small volume.Addition of an equivalent of methanolic hydrogen chloride gives a crystalline precipitate of pure antibiotic DOC as the hydrochloride.

CHEMICAL AND PHYSICAL PROPERTIES The novel anthracycline antibiotic, isolated as hydrochloride, is a red solid having a melting point of 1710--1750C (with decomposition). The calculated molecular formula C2eH29NOs.HCI for its hydrochloride is confirmed by field desorption mass spectrometry giving the following peaks corresponding to the molecular weight, 499, of the antibiotic as free base and its fragmentation products: m/e 500 (MH+), 499 (M+), 481 (M-H20), 370 (Aglycone) and 334 (bisanhydroaglycone).

Specific rotation: [CL]3" +275 (c = 0.1, methanol).

Solubility; as free base the antibiotic DOC is soluble in chloroform, methylene dichloride, dioxan, acetone, n-butanol, methanol and water, slightly soluble in ethyl acetate and insoluble in benzene and petroleum ether. As the hydrochloride it is soluble in water, lower alcohols and dimethylsulphoxide, slightly soluble in acetone and ethyl acetate and insoluble in chloroform, diethyl ether and petroleum ether.

The aqueous or alcoholic solutions are red but turn to reddish purple in alkaline media.

Adsorption spectrum: ultraviolet and visible absoption maxima are seen at: 236,256,293,464 (sh), 495,512 (sh) and 529 nm (E11cm 620,520,164,200,260,197 and 186). The IR spectrum in KBr ahows peaks at the following wavelengths in cm-1: 3420,2930,1600,1400,1410,1290,1250, 1240,1200,1160,1130,1120,1085,1060,1040,1010,985,920,870,820 and 435.

The PMR spectrum of the antibiotic as the hydrochloride in DMSO shows "inter alia" the following peaks (ppm): 0.96 (t, CH3-C(H2)), 1.18 (d, CH3-C(5')), 4.76 (broad s, C(7)-H), 5.24 (broad s, C-(1 ')-H) and 7.20 N 7.85 (m, aromatic protons).

On the base of its paper chromatographic behaviour the new anthracycline glycoside can be also distinguished from other known anthracyclines: Antibiotic DOC Rf 0.77 Carminomycin Rf 0.65 13-dihydrocarminomycin Rf 0.60 Daunorubicin Rf 0.50 Doxorubicin Rf 0.30 by using Whatman No 1 paper buffered with M/1 5 phosphate buffer at pH 5.4 and a mixture of npropanol: ethyl acetate: water 7:1:2 (by volume) as eluent by descending method.

STRUCTURE DETERMINATION The structure of the antibiotic DOC was determined as follows: aqueous acid hydrolysis gave an insoluble purple red alglycone.

After neutralization the aqueous layer was freeze-dried and the residue crystallized giving an aminosugar as the hydrochloride identified as daunosamine by direct comparison with an authentic sample.

The structure of the aglycone was determined from its chemical and physical properties, reported in Table 4, and by direct comparision with an authentic sample of 1 3-deoxycarminomycinone prepared from 1 3-deoxydaunomycinone as described in Example 8 hereinbelow.

TABLE 4 Chemical and physical properties of the aglycone m.p.: 240 C (with decomposition) # MeOH 235, 525, 293, 467, 481 (sh), 495, 516 (sh) and 530 nm U.V. and VIS spectra: max E 1% 1 cm 1020, 770, 298, 320, 363, 410, 300 and 290 I.Rl spectrum: 3250, 2970, 2930,1600, 1450, 1430, 1400, 1375, 1350,1310, 1250, 1200, 1180, in KB cm-1 1160, 1140, 1125, 1100, 1080, 1060, 1040, 1020, 1000, 990, 915, 875, 830, 820, 745, 720, 695, 680, 660, 585, 490, 470, 460, 425 and 395.

Empirical formula: C20H16O7 Molecular weight: calculated 370 found by mass spectrometry: m/e 370 (M+) BIOLOGICAL ACTIVITY DATA a) Antibacterial activity The in vitro minimum inhibitory concentrations (MIC) of antiobiotic DOC were determined for some microorganisms using the standard tube dilution procedure and are reported in Table 5.

TABLE 5

MIC in g/ml Test organism Antibiotic DOC Staphylococcus aureus 209P 12.5 Sarcina lutea ATCC 9341 1.56 Bacillus subtilis ATCC 6633 6.25 Escherichia coli B # 3.12 Staphylococcus aureus 153 25 b) Antitumor activity The new antibiotic DOC has been tested against HeLa cells in vitro (time of exposure to the drug: 24 hrs), and on P388 leukemia in mice in comparison with daunorubicin (daunomycin). The results are reported in Table 6.

TABLE 6

P388 (b) Effect of HeLa Cell (a) viability in vitro Dose T/C Toxic (c) Compound ID 50 mg/ml mg/kg % deaths Daunerubicin HCl 9.6 2.9 170 2/8 (daunomycin) 4.4 175 6/8 6.6 160 7/8 13-deoxocarmino- 3.2 1 154 1/10 mycin.HCl 1.5 109 10/10 2.25 54 10/10 a) HeLa cells were exposed to the drugs for 24 hrs, then plated.

b) Mice were treated i.p. on day 1 after tumor cell inoculation.

c) evaluated on the base of macroscopic findings.

The following Examples illustrate the invention.

EXAMPLE 1 A culture of Streptomyces peucetius var. carminatus, strain DR 81 F. l. was grown for 14 days at 280C on agar slants of the following maintenance medium (Medium SA): Glucose 3% Brewer's Dry Yeast 1.2% Sodium Chloride 0.1% Potassium Dihydrogen Orthophosphate 0.05% Calcium Carbonate 0.1% Magnesium Sulphate 0.005% Ferrous Sulphate Heptahydrate 0.0005% Zinc Sulphate heptahydrate 0.0005% Cuprice Sulphate Pentahydrate 0.0005% Agar 2% Tap water 100 ml % pH 6.7 Sterilisation autoclave/i 1 50C/20 minutes The spores of the culture so obtained were collected and suspended in 3 ml of sterile distilled water; the suspension so obtained was inoculated in 300 ml Erlenmeyer flasks containing 60 ml of the following liquid growth medium: Brewer's Dry Yeast 0.3% Peptone 0.5% Calcium Nitrate Tetrahydrate 0.05% Tap water to 100 ml Sterilisation autoclave/1200C/20 minutes pH (after Sterilisation) 6.8 to 7.0 The inoculated flasks were shaken for 2 days at a temperature of 280C 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 was inoculated in a 300 ml Erlenmeyer flask containing 50 ml of the following production medium:: Glucose 6% Brewer's Dry Yeast 3% Sodium Chloride 0.2% Potassium Dihydrogen Orthophosphate 0.1% Calcium Carbonate 0.2% Magnesium Sulphate 0.01% Ferrous Sulphate Heptahydrate 0.0010/0 Zinc Sulphate Heptahydrate 0.001% Cupric Sulphate Pentahydrate 0.001% Tap water to 100 ml pH 6.7 Sterilisation autoclave/i 1 50C/20 minutes The flasks were thus incubated at 280C for 7 days in the identical conditions as described for the seed phase.

The maximum concentration of the active compounds is reached between the sixth and seventh days of fermentation with a production of 20 mcg/ml.

EXAMPLE 2 The culture of strain DR 81 F.l. was obtained as described in Example 1. The spores of three slants were pooled and collected in 10 ml of sterile distilled water; the suspension so obtained is inoculated in a 2 litre baffled round-bottomed flask containing 500 ml of the seed medium described in Example 1.

The flask was incubated for 48 hours at 280C on a rotary shaker running at 1 20 r.p.m. and describing a circle of 7 cm diameter.

The whole seed was inoculated in an 80 litre stainless steel fermentation vessel containing 50 litres of the seed medium described in Example 1 and sterilized by vapour pressure at 1 2O0C for 30 minutes. The seed phase was carried out at 280C, under stirred conditions at 230 r.p.m. and an air flow of 0.5 litre per litre of medium per minute. After 30 hours, this seed was inoculated in a 800 litre stainless steel fermentation vessel containing 500 litres of the production medium described in Example 1, and sterilized by vapour pressure at 1 200C for 30 minutes.

At the 24th hour of fermentation, an addition of sulphonylamide at a concentration of 0.5 g/l was made. At the 48th hour of fermentation, a further addition of this substance at a concentration of 1 g/l was made. The fermentation was performed at 280C, under stirring at 250 r.p.m. and aereation with an air flow of 0.7 litre per litre of medium per minute.

The maximum concentration was reached between the sixth and seventh days of fermentation with a production of 70 mcg/ml of active compounds.

EXAMPLE 3 The whole beer (4 litres) from a fermentation obtained according to Example 1 was adjusted to pH 4 with hydrochloric acid and filtered using 3% diatomaceous earth as filter aid, to yield a cake and a filtrate which were extracted separately. The wet filter cake was treated with about 4 litres of a mixture of acetone and 0.1 N aqueous hydrochloric acid (80:20 by volume). After filtration a second treatment with an additional 3 litres of acidified aqueous acetone was effected to ensure complete extraction of the active compounds. The combined aqueous acetone extracts were adjusted with ammonium hydroxide to pH 4, concentrated under reduced pressure to about one litre and combined with the filtered broth that was extracted at pH 8.5 N 9.0 with half a volume of chloroform.The combined organic extracts were washed with water, dried on anhydrous sodium sulphate and concentrated under reduced pressure to a volume of about 50 ml. By addition of 200 ml of n-hexane the crude complex precipitated as a red-purple-brown powder (50 mg).

EXAMPLE 4 A chloroform solution of crude complex (50 mg), obtained according to Example 3, was chromatographed on a silica gel column prepared in chloroform. The column was washed with chloroform followed by elution with a 300:55:6 by volume mixture of chloroform:methanol:water. The active eluates containing antibiotic DOC were concentrated to dryness and further purified on a column of pH 5.4 buffered ceilulose powder by elution with a 9:1 by volume n-butanol:ethyl acetate mixture.

The active fractions containing pure antibiotic DOC were extracted with acidic water and then the aqueous phase, adjusted to pH 8.5, was extracted with chloroform. The extract, washed with water and dried on anhydrous sodium sulphate, was concentrated under reduced pressure to a small volume.

Addition of an equivalent of methanolic hydrogen chloride gave a crystalline precipitate of pure antibiotic DOC (10 mg) as the hydrochloride: m.p. 171 0..-1750C (with decomposition); EXAMPLE 5 The whole beer (500 litres) from a fermentation obtained according to Example 2 was extracted as described in Example 3. The excess of sulphanylamide contained in the extract was separated by crystallization from n-propanol: acetone and the crude complex (30 g) was precipitated from the mother liquors with n-hexane.

EXAMPLE 6 Antibiotic DOC was separated and purified from the crude complex obtained in Example 5 fcllowing the procedures described in Example 4, and isolated as the crystalline hydrochloride (1 g): m.p. 171-175 0C (with decomposition).

EXAMPLE 7 A 100 mg sample of antibiotic DOC was dissolved in 25 ml of 0.2 N aqueous hydrochloric acid and the solution was heated for 1 hour at 950C. A crystalline purple-red precipitate of aglycone (60 mg) was collected by filtration, washed with water and dried: m.p. 2400C (with decomposition) m/e 370 (M+). The almost colourless aqueous acidic solution was adjusted to pH 5 with an anion exchange resin and then freeze-dried. The residue (20 mg) crystallized from methanol:acetone, gave a crystalline compound, m.p. 1 660C (with decomposition), identified as daunosamine hydrochloride by direct comparision with an authentic sample.

EXAMPLE 8 A sample of 20 mg of 1 3-deoxydaunomycinone (prepared from daunomycinone following the method described by T. H. Smith et al. in J. Med. Chem., 2, 280, 1978) in dichloromethane (10 ml) was treated with aluminium tribromide (20 mg) and refluxed for 15 minutes under nitrogen. The reaction mixture was treated with a 10% aqueous solution of oxalic acid (20 ml) and the organic phase was separated, washed with aqueous sodium bicarbonate and water.

After evaporation the organic phase gave a crystalline residue of 1 3-deoxycarminomycinone, m.p.

2400C, m/e 370 (M+), the melting point, l.R., U.V. and visible spectra of which were indistinguishable from those of a sample of the aglycone of antibiotic DOC.

Claims (6)

1. 1 3-Deoxycarminomycin or a pharmaceutically acceptable acid addition salt thereof.

2. A pharmaceutical composition comprising 13-deoxycarminomycin or a pharmaceutically acceptable acid addition salt thereof in admixture with a pharmaceutically acceptable diluent or carrier.

3. A microbiological process for the preparation of 1 3-deoxycarminomycin, the process comprising cultivating, under aerobic conditions in an aqueous cultural medium containing an assimilable source of carbon, an assimilable source of nitrogen and mineral salts, the microorganism Streptomyces peucetius var. carminatus (1524 DSM, 31502 ATCC, 4929 FRI, DR81 Fl Farmitalia microbiological collection).

4. A process according to claim 3 which is carried out at a temperature of from 250C to 370C, for a period from 3 to 7 days and at a pH which initially is from 6.5 to 7.0 and at the end of the fermentation is from 6.5 to 8.0.

5. A process according to claim 3 or claim 4 further comprising extracting 1 3-deoxycarminomycin from the fermentation mass, the separated mycelium or the filtered broth.

6. A microbiological process according to claim 3 substantially as described herein with reference to the Examples.