NO760873L - - Google Patents

Description

The present invention relates to methods for the production of a new naphthacene derivative with the formula;

as well as salts thereof.

The compound with formula I which is denoted by .no.

32,999 RP, is of very great interest•because of its action against tumors.

Compound 32,999 RP is a dark red basic compound which salts with acids.

The hydrochloride of 32,999 RP is soluble in methanol, pyridine, dimethylformamide and water in an amount of approx. 100'mg/cm-<5>, soluble in n-butanol, saturated with water at 20°C, in an amount of approx. 10 mg/cm^, insoluble in methanol in an amount of 5 rhg/cm^ and practically insoluble (solubility less than 0.1 mg/crr.^) in acetone, chloroform, methylene chloride, ethyl acetate, benzene, methyl isobutyl ketone, dioxane and hexar..

The hydrochloride of 32,999 RP contains carbon, hydrogen, oxygen, nitrogen and chlorine and has the following elemental analysis: C% = 56.6 E% = 5.7 0% = 29.41 Mf, = 2.39 Cl% = 6.0

The hydrochloride also has the following physico-chemical properties:

Appearance: orange-red, microcrystalline powder.

Melting point: approx. 200°C (splitting).

Specific twistability (determined in a 0.09% ± g ethanol solution

containing 0.1%- 1 N HC1):

UV spectrum (determined at a concentration of 10365 mg/l in ethanol containing 0.1% 1 N HC1): This spectrum is reproduced in fig. 1 in the accompanying drawings and absorption maxima are indicated in the following table below. Visible spectrum (determined at a concentration of 10.65 mg/l i ethanol containing 0.1% 1 N HC1): This spectrum is reproduced in fig. 2 in the accompanying drawings and absorption maxima are indicated in the table below.

IR spectrum (determined in a mixture with KEr): this spectrum is shown in fig. 3 in the accompanying drawings, where the abscissa indicates the wavelength in ym (the upper scale) and

the wave number in cm ^ (the lower scale) and on the ordinate the optical density.

The following table shows the most important absorption signals (the numerical values refer to the wave number in cm 1).

By thin-layer chromatography on silica gel, the hydrochloride of 32,999 RP shows an Rf value of approx. 0.2 when, at 24°C, a mixture of methylene chloride, formic acid and methanol is used as the developing solvent in a volume ratio of 80:17:3-By acid hydrolysis of 32,999 RP or one of its salts, a compound is obtained with the formula:

which is short of 32,999 RP..

.The aglycone of 32,999 RP is soluble in pyridine i

an amount of 20 mg/cm^, soluble in methanol, dioxane, chloroform and methylene chloride in an amount of 5 mg/cm^ and soluble in acetone, ethyl acetate, hexane and water in an amount of less. 0.1 mg/cm'<5>.

This compound also exhibits the following physicochemical properties.

Appearance: orange-red, microcrystalline powder.

Melting point: approx. l85°C (cleavage)-.

Elemental analysis:' C% - 61.37 M 5.31' 0% = 30.16.

Specific twistability (determined in a 0.1% dioxane solution):

UV spectrum (determined in the concentrations 50 and 5 mg/l respectively in

• ethanol containing 0.1% 1 N HC1):

This spectrum is reproduced in fig. 4 in the accompanying drawings where curves I and II correspond to the concentrations 50 and 5 mg/r respectively. Absorption maxima are indicated in the table below.

Visible spectrum (determined at a concentration of 10 mg/l in ethanol containing 0.1% 1 M HCl): this spectrum is reproduced in fig. 5 in the accompanying drawings and absorption maxima are indicated in the table below.

IR spectrum (determined in mixture with KBr): this spectrum is reproduced in fig. 6 in the accompanying drawings where the wavelength in ym (the upper scale) and the wave number in cm 1 (the lower scale) are indicated on the abscissa and the optical density on the ordinate.

The following table shows the most important absorption signals (the numerical values indicate the wave number in cm 1),

By thin-layer chromatography on silica gel, the aglycone of 32,999 RP shows an Rf value of approx. 0.7 when at 24°C a mixture of methylene chloride, formic acid and methanol is used as a developing solvent in a volume ratio of 80:17:3 - The new compound 32 999 RP with the formula I can according to the invention be prepared according to any of the following procedures..

(1) Through microbiological reduction of the antibiotic carminomycin with the formula:

Carminomycin and its production by cultivation of Actionomadura carminata Sp..nov. is described in the French patent application No. 74.00349, which is published under No. 2.235-700. The structure of carminomycin has been reported by M.G. Brazhnikova et al., "J. of Antibiotics", XXVII, No. 4, 254-259 (1974).

The microbiological reduction of carminomycin is usually carried out in an aqueous medium, either by means of cultures of micro-organisms which have reached a suitable stage of development, or by means of cells isolated from such cultures, or by means of enzymatic extracts obtained from these microorganisms.

The microorganisms that can be used in the method according to the present invention can belong to the genera Streptomyces or Bacterium. Examples of particularly suitable microorganisms are Streptomyces lavendulae (ATCC 8664), Streptomyces roseochromogenes (ATCC 13.400), Corynebacterium simplex (ATCC 6946) and Bacterium cykloxydans (ATCC 12.673-). The best results are obtained with Corynebacterium simplex (ATCC 6946).

The cultivation of the microorganism producing the enzymatic system capable of reducing carminomycin to 32,999 RP can be carried out according to any aerobic cultivation method. the surface or in.depth, but the latter is preferable, for reasons of convenience. For this purpose, the grafting and fermentation methods and the various types of apparatus that are generally used in the yeast industry are used.

The growing medium should mainly contain wedges for assimilable carbon and assimilable nitrogen, inorganic substances and weighting factors. All these substances can be used in the form of well-defined compounds or in the form of complex mixtures such as are encountered in biological products of different origins.

As sources of assimilable carbon, carbohydrates such as glucose or other carbon-containing compounds such as sugar alcohols or certain organic acids can be used.

Certain animal or vegetable oils such as lard oil or soya oil can advantageously replace these various carbonaceous compounds or be used as an addition to them.

The suitable sources for assimilable nitrogen are extremely variable. They can be very simple chemical compounds such as inorganic or organic ammonium salts, carbamide, certain amino acids. They can also be made up of complex substances that mainly contain nitrogen in protein form, such as casein, lactalbumin. gluten and hydrolyzate thereof, soya flour, peanut flour, fish meal, peptone, meat extract, iron extract, liquor, corn casting liquid.

Among the added inorganic substances, certain can have a buffering or neutralizing effect, such as alkali metal or non-alkali metal phosphates. Other inorganic substances provide the necessary ionic equilibrium for the development of the microorganism, such as chlorides and sulfates of alkali metals and alkaline 'j-word' alkali metals.

The weight gain factors are substances of a vitamin nature such as

. such as riboflavin, folic acid and pantothenic acid.

the p.H value. in the fermentation medium should be between 6.0 and 7.8 at the beginning of cultivation, preferably between 6.5 and 7.5'. The optimum temperature for fermentation is 29-31°C, but satisfactory development is achieved at temperatures between. 26 and .37°C.

The air supply during fermentation can vary within fairly wide limits. However, it has been found that an air supply of 0.3-3 1/1 culture broth per minute is particularly suitable. In order to achieve a culture with a good reducing effect, it is also suitable to stir the culture medium, e.g. using a stirrer whose rotation speed can vary between 100 and 250 revolutions per minute.

The microorganism culture usually achieves a satisfactory degree of development after a period of 24-48 hours.

The reducing effect mainly depends on the amount of cells that are formed during cultivation. - When using isolated cells, e.g. obtained by centrifuging the culture medium, the amount formed stands at 32,999 R? in direct proportion to the concentration of cells in the reaction medium. Reaction media with a high content of cells have excellent reducing power.

The reduction of carminomycin to 32,999 RP takes place by bringing an aqueous solution of carminomycin or a salt thereof into contact with a microorganism culture which has achieved a satisfactory degree of development and satisfactory reaction capacity, or with cells isolated from such a culture, or with enzymatic extracts obtained from such cells.

Enzymatic cell extracts can be obtained after destruction of the cell walls, either by chemical or biochemical methods or by physical methods. lighten the cell wall, such as lysozyme, or by subjecting the suspension to ultrasound treatment. The supernatant liquid obtained after centrifugation contains enzymatic extracts in solution. The solution of the enzymatic extracts is usually used in the reduction step without further treatment.

The reduction is usually carried out with stirring at a temperature between 32 and 37°C, preferably between 2.6 and 30°C, and the reduction is usually finished after 1-4 days.

The reduction can be carried out at a pH value between 5 and 10, but it is preferred to work at a pH value between 7 and 8.

The reaction medium can be stirred, e.g. using

a stirrer whose rotation speed can vary between 1.00 and 250 revolutions/minute.

One can reduce either pure carminomycin or a

of their pure salts, or an impure product, or the acid filtrate obtained from a culture medium in which carmin-

mycin is produced by cultivation. In order to achieve good yields, the concentration of carminomycin in the reaction medium should ideally be between 0.1 and 1 g/l - at the beginning of the reduction. (2) By aerobic cultivation of microorganisms belonging to the genus Streptomyces, more specifically any of the following microorganisms: Streptomyces coeruleorubidus .DS 8899 (NRRL 3046), Streptomyces coeruleorubidus DS 31-723 (NRRL 3045), Streptomyces coeruleorubidus DS 7126 (NRRL 8090) , .Streptomyces bifurcus DS 23. 219 (NRRL 3539)•

Cultivation is carried out in nutrient media suitable for Streptomyces strains in the presence of additives selected from the following: amethopterin, sulphanilamide, sulphathiazole, sulphapyridazine, barbital, phenobarbital, butobarbital and pentobarbital.

The amount of antibiotic 32,999 RP that is formed during cultivation varies with the composition of the culture medium, with the amount of additive added and with the time of its addition, as will be shown later.

The microorganisms Streptomyces coeruleorubidus DS 8899 (NRRL 3046) and Streptomyces coeruleorubidus DS 31.723

(NRRL 3045) is described in French Patent No. I.380.810, and Streptomyces bifurcus DS 23 219 (NRRL 3539) is described in French Patent No. 1,593-235.

Streptomyces coeruleorubidus DS 7126 is a new strain isolated from a soil sample'. A sample of this organism has been deposited at the Norther Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Illinois (USA), where it is registered under the designation NR 8098. Samples of the microorganism can be obtained from this laboratory.

The isolation of this strain has taken place according to a common method whereby a small amount of soil is suspended in sterile distilled water, the suspension is diluted to different concentrations and a small volume of each dilution is spread on the surface of Petri dishes containing a nutrient agar medium. After incubation for a few days at 26°C, during which the microorganism develops, the colonies that you wish to isolate for further study are taken out and these are transplanted onto slant cultures of nutrient agar so that

shall achieve more abundant cultures.

The strain Streptomyces coeruleorubidus DS 7126 belongs to the species Streptomyces coeruleorubidus, which is described by G.F. Gauze et al. (Problems in the classification of.'Antagon-istic Actinomycetes - The American Institute of -Biological Sciences, Washington, 1959, p. 100) according to which it exhibits the same main characteristics as this species. The new strain exhibits a blue to light greenish-blue sporulating aerial mycelium, and on inorganic medium No. 1 according to Gauze it develops a vegetative mycelium which has a reddish color and which gives a soluble pigment of the same color, which pigment diffuses into the agar. All these properties are characteristic of the species Streptomyces coeruleorubidus. The new strain is therefore called Streptomyces ceoruleo-rubidus, strain DS 7126.

Streptomyces coeruleorubidus DS 7126 forms short, spherical to oval spores measuring 0.7-0.9 ym/0.8-1.1 ym. The stem develops simple or tufted sporophores. Chains of spores which are quite long and which can include ■ up to several tens of spores, are rolled together to form more or less open spirals which often contain 2-5 or 6 spiral windings. Because of its mode of spore formation, Streptomyces coeruleorubidus DS 7126 is classified in Section Spira in Pridham's classification system.

Streptomyces coeruleorubidus DS 7126 develops well at 25°C, somewhat less well at 57UC, and not at all at 50°C. When cultivated at 26°C, the strain exhibits the following biochemical properties:

The following table shows the cultivation characteristics of Streptomyces coeruleorubidus DS 7126 on a number of media which are usually used to determine the morphological characteristics of Streptomyces strains. Cultivations on agar media have been carried out on slant cultures. The stated data has been obtained by studying cultures that have reached a good stage of development, i.e. after cultivation for approx. 3 weeks at 26°C unless otherwise stated. The references given in the table are as follows.

Through the blue to greenish blue coloring of

the spore-forming aerial mycelium belongs to the strain Streptomyces coeruleorubidus DS 7126 of the series "Coerulescens", which is described by Gauze et al. Similarly. like the strains S. coeruleorubidus and S. bicolor described in this series, the strain DS 7126 exhibits a red-coloured vegetative mycelium. The strain DS

However, 7126 differs from S. bicolor in that, in contrast to S. bicolor, it utilizes cellulose and does not coagulate milk and, above all, in that it can form a red soluble pigment on synthetic media that diffuses into the agar, a property which strain DS 7126 shares only with the species S. coeruleorubidus. The latter characteristic in combination with the other characteristics shows that strain DS 7126 belongs to the species S. coeruleorubidus.

The ability of Streptomyces coeruleorubidus DS 7126 to

to utilize various carbon sources and nitrogen sources to ensure their development, is determined according to the method described by Pridham and Gottlieb in ('J. of Bact', 56, 107-114, 1948).

The degree of development is observed on the base medium specified by the authors, whereby the glucose has either been replaced with various investigated carbon sources or (NH^)2 SO^ has been replaced with various investigated nitrogen sources. The results are indicated in the table below.

The procedure for making 32,999 R? essentially consists in growing the above-mentioned Streptomyces strains on a culture medium under suitable conditions in the presence of a sufficient amount of additives and then separating the product formed during the cultivation.

The cultivation of the micro-organisms can take place according to any method of aerobic cultivation on the surface or in the depth, but the latter is preferable for reasons of convenience. For this purpose, different types of equipment are used which are in common use in the yeast industry.

In particular, the following breakdown of the work steps can be used:

The yeast medium should mainly contain a source of assimilable carbon and a source of assimilable nitrogen, inorganic substances, especially chlorides or carbonates, and possibly weighting factors. All these substances can be used in the form of well-defined compounds or in the form of complex mixtures, such as are encountered in biological products of different origins.

As sources of assimilable carbon, carbohydrates such as glucose, lactose, dextrins and starch can be used, or other carbonaceous substances such as sugar alcohols (glycerol) or certain organic acids, e.g. lactic acid or citric acid. Certain animal or vegetable oils such as lard or soya oil can advantageously replace these various carbonaceous compounds or be used as an addition to them.

.-The suitable sources for assimilable nitrogen are

extremely variable'. They can be very simple chemical compounds such as inorganic or organic ammonium salts, carbamide

■and certain amino acids. They can also be made up of complex substances that mainly contain nitrogen in protein form, such as

casein, lactalbumin, gluten and hydrolyzate thereof, soya flour, ground nut flour, fish meal, meat extract, yeast extract, liquor and corn-molding liquid.

Among the added inorganic substances, some may have a buffering or neutralizing effect, such as alkali metal or alkaline earth phosphates, or calcium and magnesium carbonates. Other inorganic substances provide the necessary ionic equilibrium for the development of the microorganisms and the formation of 32,999 RP, such as chlorides and sulphates of alkali metals and alkaline earth metals. Finally, certain substances act more specifically as activators for the metabolic reactions of the microorganisms, such as salts of zinc, cobalt, iron, copper and manganese.

The growth factors are usually substances of a vitamin nature such as thiamiric acid, riboflavin and pantothenic acid.

The additives can be added during different stages of cultivation, e.g. during the graft cultivation in a 'shaken flask' or during the production cultivation. ' However, the best results are obtained when the additives are added either at the beginning of the production cultivation or during this.

The very best additives are sulphanilamide, sulphathiazole and sulphapyridazine.

Concentration of the additive in the mud

can vary within free limits depending on the composition of the culture medium and the nature of the additive. Concentrations of between 0.01 and 1 g/l culture medium are usually most appropriate;

At the start of cultivation, the pH value in the fermentation medium should be between 5.6 and 7.4, preferably between 5.8 and 7.2. The optimum temperature for fermentation is between 25 and 30°C, but

satisfactory production is achieved at temperatures between 23 and 33°C. The air supply during fermentation can vary within wide limits. However, it has been found that an air supply of 0.3-3 1/1 culture broth per minute is particularly suitable. The maximum yield of 32,999 RP is achieved after 2-10 days of cultivation, whereby the time essentially depends on the medium used.

From the above it appears that. the general conditions for growing the specified microorganisms in the presence of the specified additives can vary within wide limits and be adapted to special requirements. (3) Cultivation of a new microorganism belonging to the genus Streptomyces and designated Streptomyces atroviolaceus DS 8938. A specimen of this organism has been deposited with the United States Department of Agriculture, Peoria, 111., where it is registered under the designation NRRL 314 8. Samples of microorganisms may be obtained from this laboratory.

This microorganism which is isolated from a soil sample exhibits such characteristics that it cannot be identified with any previously described species. It should therefore be considered as

a new species.

The isolation of this microorganism has taken place according to a common method where a small amount of soil is suspended in sterile distilled water, the suspension is diluted to various concentrations and a small volume of each dilution is spread on. surface of Petri dishes containing a nutrient agar medium. After incubation for a few days at 26°C during which the microorganism develops, the colonies that you wish to isolate for further study are taken out, and these are transplanted onto slant cultures of nutrient agar so that

shall achieve more abundant cultures.

Streptomyces atroviolaceus DS 8938 forms oval spores measuring 0.4-0.6 ym/0.6-0.9 yrn. The microorganism does not develop branching sporophores on the aerial mycelium. These spore chains, which are long and usually contain dozens of spores, are rolled together in several, usually tight spiral windings. -Due to its mode of spore formation, the strain is classified in Section Spira in Pridham's classification system.

Streptomyces atroviolaceus DS 8938 'develops well at 25°C' and at 30°C, less well at 37°C and not at all at 50°C. On the majority of culture media, the microorganism forms a vegetative mycelium which is at first brownish-yellow to brownish-red and which quickly assumes a dark colour, a color which goes from very dark brown to b.run violet or black-brown. The spore formation • only occurs on a limited number of media and then only slowly, whereby a very light gray aerial mycelium is formed. On the other hand, you can notice. that the aerial mycelium on a certain number of culture media can be colored light pink or light violet when the culture forms abundant dark violet soluble pigment.

Streptomyces atroviolaceus DS 8938 does not produce black soluble melanin pigment on Waksman tyrosine yeast extract agar medium (melanin formation medium). However, the micro-organism can, on numerous media, both synthetic and organic, form a soluble pigment which colors the agar dark and which colors the same violet-brown, violet-black, black-brown or black..

When cultivated at 26°C, the microorganism exhibits the following biochemical properties.

The cultivation characteristics for Streptomyces atroviolaceus DS 8938 are compiled in the following table below.' 3 weeks at 26°C unless otherwise stated. The properties have been observed on such nutrient agar media and broths as are usually used to determine the morphological properties of Streptomyces strains. Cultivation on agar media is carried out on slant cultures. A certain number of the culture media used are prepared according to the formulas given in "The Actinomycetes", S.A. Waksman, pp. 193-197, Chronica Botanica Company, Waltham, Mass., USA, 1950. In these cases, the media are designated by the letter W followed by the number assigned to the media in "The Actinomycetes". The other culture media have the following references or compositions.

Streptomyces. atroviolaceus DS '8938 exhibits a number of characteristics that do not exactly correspond to those of. previously described strains,, and the microorganism should therefore be considered to be a new species.

With regard to the species described 'in "The Actinomycetes" (vol. 2, S.A. Waksman, The Williams and Wilkins Company, Baltimore 1961) and in Bergey's "Manual- of Determinative Bacteriology" (7- edition, The Williams and Wilkins Company, Baltimore, 1957), then one should almost equate the new microorganism with Streptomyces violaceoniger, since the new microorganism, in the same way as Streptomyces violaceoniger, does not

■ produces black, soluble melanin pigment, but on the other hand-on synthetic agar according to Czapék with sucrose (sucrose-nit.rat-agar) produces a brown-violet soluble pigment' which very quickly turns black. S. atroviolaceus DS 8938 also gives -on numerous media brown violet to black violet soluble pigments, in certain cases even black pigments. In the same way as S. -violaceoniger, S. atroviolaceus DS 8938 also exhibits a spore-forming aerial mycelium which is grey-coloured, and its spore chains are rolled up into spirals. S. atrioviolaceus DS 8938 and ,S. violaceoniger should, however, be considered two different species. In spite of the above-mentioned similarities, it can be noted that S. violaceoniger does not produce any soluble pigment on potatoes, develops a gray vegetative mycelium on gelatin and, above all, when aging shows a black coloring of the aerial mycelium, which means that this species included, in the group of S. hygroscopicus. The strain of S. atroviolaceus DS 8938, on the other hand, produces a brown to dark brown-violet soluble pigment on potatoes and develops a brownish to violet vegetative mycelium on gelatin, nor do very old cultures ever show any blackening of the aerial mycelium. It may also be noted that, especially on synthetic agar according to Czapek with sucrose (sucrose-nitrate agar), the soluble pigment formed by S. violaceoniger at the beginning of cultivation has a bluish tint, -which is never the case with those of S .'atroviolaceus DS 8938 formed soluble pigment which, before darkening, shows a purplish-violet color and not a blue-violet color.

The ability of Streptomyces atroviolaceus DS 8938 to

to .use different carbon sources and nitrogen sources to

ensure its development is determined according to the method described by Pridham and Gottlieb (' U. of. Bact., 56., 107-114, 1948). The degree' of development is observed on the base medium specified by the authors, whereby .has either replaced the glucose with various investigated carbon sources or replaced ■(NH^^ SO^with various investigated nitrogen sources. The results are compiled in the following table.

The method for producing 32,999 RP essentially consists of growing Streptomyces atroviolaceus DS 8938 or its mutants on a culture medium under suitable conditions, and then separating the product formed during cultivation.

The cultivation of Streptomyces atrovilaceus DS 8938 can be carried out according to any method of aerobic cultivation at the surface or at depth, but the latter is preferred for reasons of convenience. Different types of equipment are used for this purpose, which are usually used in the yeast industry.

In particular, the following breakdown of the work steps can be used:

The yeast medium should mainly contain a source of assimilable carbon and a source of assimilable nitrogen, inorganic substances, especially chlorides or carbonates, and possibly growth factors. All these substances can be used in the form of well-defined compounds or in the form of complex mixtures, such as those encountered in biological products of different origins.

As sources of assimilable carbon, one can use carbohydrates such as glucose, lactose, dextrins and starch, or other carbon-containing compounds such as sugar alcohols.

(glycerol) or certain organic acids, e.g. lactic acid or citric acid. Certain animal or vegetable oils, such as lard or soya oil, can advantageously replace these various carbonaceous compounds or be used as an addition to them.

The. suitable sources for assimilable nitrogen are extremely variable. They can be very simple chemical compounds such as inorganic or organic ammonium salts,

■bamide and certain amino acids. They can also be made from complex substances which mainly contain nitrogen in protein form, such as casein, lactalbumin, gluten and their hydrolysates, soya flour, groundnut flour, fish meal, meat extract, yeast extract, liquor and corn liquor.

Among the added inorganic substances, these can have a buffering or neutralizing effect, such as alkali metal or alkaline earth metal phosphates or calcium or magnesium carbonates. Other inorganic substances in the necessary' ionic equilibrium for the development of Streptomyces atroviolaceus DS 8938 and the formation of 32 999.RP, such as chlorides and sulphates of alkali metals and alkaline earth metals." Finally, certain substances act more specifically as activators of the metabolic reactions of Streptomyces atroviolaceus DS 8938, 'such as salts of zinc^cobalt, iron, copper and manganese."

The growth factors are substances of a vitamin nature such as riboflavin, folic acid, pantothenic acid and thiamine. ■

The pH value in the fermentation medium at the beginning of cultivation should be -between 5.8 and 7.3.- The optimum temperature for fermentation is between 25°C and 30°C, but satisfactory production is achieved at temperatures between 23 and 33°C .' The air supply during fermentation can vary within fairly wide limits. However, it has been found that an air supply of 0.3-3 1/1 culture broth per minute is particularly suitable. medium used.

From the above, it appears that the general conditions for cultivation of Streptomyces atroviolaceus DS -8938 for the purpose of producing the compound 32 999 RP can vary within wide limits and can be adapted to special requirements.

The compound 32,999 RP can be separated from the isolation medium according to methods which are common for the isolation of antibiotics of this type.

The connection 32,999 RP can e.g. is extracted from the reaction medium or the fermentation medium at a pH value close to 9 with the help of an organic solvent such as chloroform, methylene chloride, n-butanol or a mixture of these solvents in a suitable ratio.

The fermentation medium can also be filtered in the presence of a filtration aid at a pH value of -between 1.5 and 9- It is appropriate to carry out this operation in an acidic medium, especially a medium that is acidified to a pH value between .1 ,5 and 2 using pksalic acid. It is also possible to carry out the filtration at a pH value of between 2 and 8 in the presence of an aliphatic alcohol containing 1-3 carbon atoms. 32,999 RP can be extracted from the filtrate, after concentration, using an organic solvent such as chloroform, methylene chloride, n-butanol or a mixture of these solvents in suitable proportions. -32,999 RP. can be isolated from the organic extracts after successful washings and extractions, by precipitation after concentration of the extracts to a small volume or by the addition of a miscible solvent in which 32,999 RP is practically insoluble, such as hexane, possibly after conversion of the antibiotic to an acid addition salt, e.g. the hydrochloride.

The connection 32 999 R? can possibly be purified by applying different classic purification methods such as crystallization or chromatography on different absorbents.

The compound 32 999 RP and its non-toxic, i.e. pharmaceutically acceptable, salts have been shown to be very effective against tumours. The compound and its salts have thus been shown to be effective in mice against leukemia L 1210, leukemia P 388 and leukosarcomatosis by intraperitoneal administration in doses of between 0.125 and 0.250 mg/kg.

The toxicity of the hydrochloride of 32,999 RP has been studied mainly in mice. The lethal dose 50% (LD^Q)

eir close to 0.4 mg/kg by intraperitoneal administration for four consecutive days.

The invention shall be illustrated by the following non-limiting examples. In the examples, the compound is 32,999

RP identified by means of thin-layer chromatography on silica gel, whereby the Rf values for the products obtained have been compared with the Rf values for carminomycin or 32,999 RP in the same solvent system. The products of 32,999 RP are determined by means of thin-layer chromatography, either by comparing the intensity of the spot obtained with the extract with the spot obtained with pure 32,999 RP, or by comparing the areas of the t.uppers which is recorded by means of a Chromoscan plate, or by colorimetric determination of the compound 32,999 RP, eluted from the product spot on the chromatographic carrier.

The pH value of a solution of the first three components in 1000 cm^ of distilled water is adjusted to 6.9 by adding 1N sodium hydroxide solution, after which the solution is distributed into flasks of 300 cm 3 in an amount of .50 cm 3/flask. These flasks are sterilized at 122 C for 20 minutes and then cooled. The cultivation medium is then completed with

that 1.5 cm^ of a sterile glucose solution containing 500 g of glucose per litres. The medium then has a pH value of 7.05.

80 of these flasks are inoculated by adding 2.5 cm^ of a seed culture of a strain of Corynebacterium' to each flask. simplex (ATCC 6946). The culture is developed on a table rotating at 220 revolutions/minute in a room with a temperature of 30°C. After 48 hours the culture is well developed and the pH is 7.70. -To each flask, 2.5 cm^ of a water solution containing 10 mg of carminomycin hydrochloride per; cm^. The reduction is carried out under the same rotation conditions at 26°C.

After 48 hours, the reduction is finished and the culture medium's pH value is 8.5 - The contents of the -80 flasks are combined. A homogeneous sample of 50 cm<J> of culture medium is taken out and this sample is treated with 25 cmJ of a borate buffer so that the pH value is raised to 9.0. It is then extracted with 2 x 50 cm<J> of a mixture of methylene chloride and n-butanol in the volume ratio 70:30. The organic extracts are dried over anhydrous sodium sulfate. The production of 32,999 RP is determined by thin-layer chromatography of the extract on silica gel. The deposited extract volumes are 10-20 yl. It is developed with a mixture of methylene chloride, formic acid and methanol in the volume ratio 80:17:3 in order to

could achieve a good spearation of carminomycin and '32,999 RP. The Rf values of the products obtained are compared with the Rf values of reference compounds under the same conditions.

The quantitative determination of the production of 32,999 RP shows that the culture of Corynebacterium simplez (ATCC 6946) reduces the carminomycin to 32,999 RP in a yield of 80%, calculated on the amount of added carminomycin. Example 2

A culture medium A is prepared and distributed in flasks of 300 cm^ in the same way as in example 1.

Cultivation medium B is also prepared and this is also distributed in 300 cm flasks. The cultivation medium B has the following composition:

The medium's pH value is set to 7.2 by adding 1N sodium hydroxide solution, after which the medium is sterilized at 122°C for 20 minutes.

A flask containing 50 cm^ of medium A with a pH value of 7.05 and a flask containing 50 cm^ of medium B with a pH of 6.5 are inoculated with each 2.5 cm^ of one inoculation culture of the strain Corynebacterium simplex (ATCC 6946) .

The cultures are developed on a table rotating at 220 revolutions/minute in a room with a temperature of 30°C. After a suitable cultivation time (48 hours for medium A and 72 hours for medium B), 2.5 cm^ of a water solution containing 10 mg of carminomycin hydrochloride is added to each flask. cm'). This results in a carminomycin concentration of 0.5 g/l.

After these additions, media A and B are incubated for two and four days respectively on a table that rotates at 220 revolutions/minute in a room with a temperature of 26°C.

The contents of the flasks are then treated in the same way as in example 1.

The table below shows the yields of 32,999 RP, calculated on the amount of added carminomycin.

Example 3

In 300 cm^ flasks, cultures of Corynebacterium simplex (ATCC 6946) are prepared in culture medium A in the same way as in example -1. The cultures are developed on a table that rotates at 220 revolutions/minute in a room with a temperature of 30°C.

After 30 hours' incubation, the contents of 5 flasks (250 cm^ culture medium) are combined, and the cells present are separated by centrifugation. The amount of cells obtained is then suspended in a 300 cc flask containing 50 cc of a M/15 phosphate buffer of pH 8.0. In the same way, another cell suspension is also prepared by suspending the cells in 50 cm 2 of a m/15 phosphate buffer with pH 5.0. To each flask, 2.5 cm^ of a water solution containing 4 mg of carminomycin hydrochloride per cm'<1>. The carminomycin concentration becomes thereby 0.2 g/l.

The flasks are incubated for 48 hours under the conditions specified in example 1, and the contents of the flasks are then treated in the same way as in example 1.

The following table shows the yields of the antibiotic 32,999 RP, calculated on the amount of carminomycin added.

Example 4

. A culture medium A with a pH value of 6.8 is prepared and this is distributed in 300 cm 3 flasks in an amount of 50 cm -z per

flask in the same way as in example 1.

Into several flasks, 2.5 cm^ of an inoculum culture of the strain Bacillus cyciooxydans (ATCC 12,673) is added - The culture is developed for 48 hours on a table rotating at 220 revolutions/minute in a room with a temperature of 30°C.

The reduction of carminomycin is then carried out either by using the entire culture medium whose pH value is 7.65- or by using a cell suspension in a buffer with pH 8.0, prepared in the same way as in example 3-

To each flask, add 1 cm 3 of a water solution - containing 10 mg of carminomycin per cm^. This results in a carminomycin concentration of 0.2 g/l.

The reduction of the carminomycin is carried out over four days on a table rotating at 220 revolutions/minute in a room with a temperature of 26°C. The contents of the flasks are then treated in the same way as in example 1. The following table shows the obtained yields of 32,999 RP, calculated on the amount of carminomycin added.

Example 5

In the same way - as in example 4, cultures of Bacillus cyciooxydans (ATCC 12.673) are prepared in culture medium .A. After incubation of the cultures for 48 hours, -carminomycin is added to the culture media whose pH value is 7.65- To each flask, either 1 cm^ of a water solution containing 10 mg of pure compound/cm^ (0.2 g carminomycin/1 ) or 4 cm^ of an aqueous solution containing '10 mg/cm"<3>of an impure product containing ±6% carminomycin' (0y13 g carmiriomycin/1)■

The flasks are then incubated for four days under the conditions described in example 1, and the contents of the flasks are then treated in the same way as in example 1. The following table shows the yields of 32,999 RP, calculated on the amount of added carminomycin.

Example 6

A cultivation medium C is prepared with the following composition:

The above-mentioned ingredients except the glucose are dissolved or suspended in 900 cm^ of distilled water.

After setting the pH value to 7.20 by adding 1N sodium hydroxide solution, the solution is distributed into 300 cm^ flasks in an amount of 45 cm^/flask. The flasks are sterilized at 122°C for 20 min. and then cooled. The culture medium is completed by sterilely adding 5 cm^ of a sterile glucose solution containing 500 g glucose/l to each flask. The medium ready for grafting has a pH value of 7.0.

A 300 cc tube containing 50 cc of the above-described culture medium at pH 7.0 is inoculated with 2.5 cc of a seed culture of the strain Streptomyces roseochrompgenes (ATCC 13-400). The culture is developed for 48 hours on a table rotating at 220 rpm. in a room with a temperature of 30°C. The cells produced during the cultivation are then collected. The reduction of carminomycin to the antibiotic 32,999 RP is carried out with a suspension of the cells in 50 cm of an M/15 phosphate buffer with pH 8.0 containing 1 cm of an aqueous solution containing 10 mg of carminomycin hydrochloride per cm^. The original carminomycin concentration is therefore 0.2 g/l. The reduction is carried out for 48 hours on one table that rotates at 220 rpm. in a room with a temperature of 26°C. The suspension is treated in the same way as described in example 1. The yield of 32,999 RP is 57%, calculated on the amount of carminomycin added.

Example 7'

A culture medium C prepared in the same way as in example 6 is distributed in 300 cm^ flasks.

Flasks containing 50 cm" of this medium with a pH value of 6.90 are inoculated with 2.5 cm"<5> of an inoculum culture of the strain Streptomyces lavendulae (ATCC 8664). The culture is developed for 40 hours on a table rotating at 220 rpm in a room with a temperature of 26° C. To each flask's culture medium, whose pH value is 7.50, 1 cm"' of a ■ water solution containing 10 mg of carminomycin hydrochloride per . cm^. This results in a carminomycin concentration ratio of 0.2 g/l. Karminomycin is then reduced for four hours under the same stirring conditions and at the same temperature as indicated above. The contents of the flasks are then treated in the same way - as in ex. 1. The yield of 32,999 RP is 61%, calculated on the amount of carminomycin added.

Example 8 (A) Fermentation.

In a fermentation container with a volume of 170 litres, the following substances are added:

After setting the pH value to 6.8 with 40 cm^ 10N sodium hydroxide solution, 300 g of calcium carbonate are added.

The medium's pH value is then approx. 6.8. The medium is sterilized by bubbling through water vapor at 122°C for 40 min. After cooling, the volume of the broth is 120 1 and the pH value is 8.3 as a result of condensation of water vapor during the sterilization'.

One inoculates with 250 cm" of an inoculum culture of the strain Corynebacterium simplex (ATCC 6946), which inoculum culture is prepared by cultivation in a shaken flask. The culture • is developed for 32 hours at 30°C with stirring and aeration with sterile air. is then suitable to be used for inoculation of the production medium.

The production cultivation is carried out in a fermentation container with a volume of 800 1 to which the following substances are added:

After setting the -pH-value-to 7.0 by adding 460 cm of ION sodium hydroxide solution, the medium is sterilized by recirculation of steam at 122°C for 40 min. After cooling, the broth has a volume of 385 1 as a result of condensation of water vapor during sterilization. The volume of the medium is then adjusted to 400 1 by adding 15 1 of a sterile water solution containing 6 kg of glucose monohydrate.

The medium's pH value is 6.8. One grafts with. 40 1 inoculum culture from the above-described cultivation in the fermentation vessel with a volume of 170<*>1. The culture is developed at 30°C under agitation with an agitator rotating at 100 rpm, and under aeration with 20 cm^ of sterile air per minute. hour. After 30 hours, the culture is ready for use for the biochemical conversion of carminomycin to 32,999 RP. Four 1 of a water solution containing 40 g of carminomycin hydrochloride are introduced into the culture medium. ;The incubation is continued for 17 hours at 26°C under the same stirring and aeration as described above. The carminomycin is reduced to 32,999 RP in. a yield of close to 100%. ;(B) Extraction. To .420 1 of the reaction solution obtained above, 42 1 n-butanol and 2.9 1 6N sodium hydroxide solution are added - the active ingredient is extracted countercurrently with a butanol (60% by volume) using two countercurrent centrifuges -is. The extract, which has a volume of 330 1, is concentrated under a reduced pressure of 5-10 mm Hg at 40°C to a volume of 20 1. To the concentrated extract is added 20 1 of methylene chloride, and the resulting mixture is washed with 10 1 of water if The pH value is set to 10 by adding sodium hydroxide solution. The wash water containing 32,999 RP is extracted twice with 10 1 of a mixture of methylene chloride and n-butanol in a volume ratio of 80:20. The three organic extracts are combined and then concentrated under a reduced pressure of 5-10 mm Hg to a volume of 15 1. 0.2 1 of a mixture of n-butanol and 11N hydrochloric acid is then added while stirring in a volume ratio of 9.0:10, after which one concentrates under a reduced pressure of 5-10 mm Hg to a volume of 5 1; The antibiotic 32,999 RP in the form of hydrochloride is precipitated by adding the concentrate to 35 1 of hexane. The precipitate is filtered off, washed and dried. 52 g of impure hydrochloride of 3.2 999 RP is thereby obtained. (C) Purification. ;(a) To a suspension of 50 g of the impure hydrochloride of 32 999 RP obtained above in 130 cm 3 of water is added 520 cm^ of dioxane. The solution obtained is filtered. The hydrochloride of 32,999 RP is then crystallized by adding 4 l of dioxane to the filtrate at a temperature close to 20° C. The crystals formed are filtered off, washed with dioxane and dried. 27 g of semi-pure hydrochloride of 32,999 RP is thereby obtained. (b) 4.2 g of the impure hydrochloride obtained above, of 32,999 RP, is dissolved in 0.2 1 of water. The pH value of the obtained solution is adjusted to 4.5 by adding 10N sodium hydroxide solution. The antibiotic in this solution is fixed on a column of 0.4 1 "Amberlite . IRC 50" in acid form.' The column is washed with 1 l of water, with 2 l of 50% methanol, with 2 l of 90% methanol and finally with 3 l of 90% methanol containing 1% sodium chloride. The eluate is concentrated under a reduced pressure of 5-10 mm Hg at 40°C to a volume of 0.5 1. The concentrate, whose pH value is adjusted to 10 by adding 11N sodium hydroxide solution, is extracted with twice 0.5 1 chloroform and twice 0.5 L of a mixture of chloroform and n-butanol in a volume ratio of 80:20. The organic extracts are combined and concentrated under a reduced pressure of 5-10 mm Hg at 40 °C. During the concentration, 10 cm^ of n-butanol containing 1% by volume of 11N hydrochloric acid and 5% by volume of water are added. The concentrate, whose volume is set to 30 crrP, is slowly added to 500 cm^ of hexane. -The precipitate obtained is filtered off , washed and dried. 1.26 g of semi-pure hydrochloride of 32,999 RP is obtained. solution is filtered. The hydrochloride of 32,999 RP is crystallized by adding 2.7 1 acetone to the filtrate at a temperature close to 20°C. The obtained NOK The whey is filtered off, washed and dried. 17 g of pure hydrochloride of 32,999 RP; Example 9; 50 cm^ of a culture of Corynebacterium simplex (ATCC 6'946), developed during 30 hours under the same conditions as described above in ex. 8 A, is sterile centrifuged at a temperature close to 0°C. After the centrifugation, the cells obtained are suspended in 50 cm 3 M/15 phosphate buffer with pH 7.5. The cells are then subjected to an ultrasound treatment at 25 kHz. 15 min. at a temperature close to 0°C. After centrifugation, add 10 mg of carminomycin hydrochloride to the supernatant liquid. The solution thus obtained is incubated under the same conditions; conditions as described in ex. 1. After 40 hours, the carminomycin has been reduced to 32,999 RP in a yield of approx. 60%. Example 10 50 cm 3 of a culture of Corynebacterium simplex (ATCC 6946), developed during 30 hours under the conditions indicated in ex. 8 A, is sterile centrifuged at a temperature close to 0°C. After the centrifugation, the cells obtained are suspended in 50 ml of tris(hydroxymethyl)aminomethane M/15 HCl buffer with a pH of 8.1, after which the suspension is transferred to a 300 ml E-flask. 25 mg of ethylenediaminetetraacetic acid, 25 mg of cetyltrimethylammonium bromide and 200 mg of lysozyme are then added. The flask is then kept for 1 hour at 37°C on a table rotating at 220 rpm. After centrifugation, 5 mg of carminomycin hydrochloride is added to the supernatant liquid. The solution thus obtained is incubated under the same conditions as described in ex. 1. After 40 hours, the carminomycin has been reduced to 32,999 RP in a yield of approx. 30%. ;Example 11 ;0.5 g of pure hydrochloride of 32,999 RP is hydrolysed by dissolving it in 50 cm<:>>water containing 10 cnr<3>11N hydrochloric acid. The obtained solution is heated to boiling for two hours. A crystalline precipitate of the aglycone is formed during this hydrolysis. The formed precipitate is filtered off, washed and dried. 0.3 g of pure aglycone is obtained. ;Example 12 ;A cultivation medium A is prepared with the following composition: ; The pH value of the obtained suspension is adjusted to 6.5 by adding 1 cm of ION sodium hydroxide solution. 19 g of glycerol (d = 1.26), 2 g of potassium carbonate and distilled water are then added to a total of 1000 cm^. ;The culture medium obtained is distributed in 300 cm^ flasks in an amount of 50 cm'5 per flask. The flasks are sterilized at 122°C for 20 min. After cooling, the medium's pH value is 5.9 - A sterile solution containing 10 mg of sulfanilamide per cm^ and has a pK value of 9.0, by dissolving sulfanilamide in a mixture of 5N sodium hydroxide solution and distilled water and subsequent filtration through "Milli-pore membranes" (0.45 µm). The flasks with the -sterile medium A are divided into six groups of two flasks each. No sulfanilamide is added to the flasks in group A-. Add sulfanilamide in the following quantities to the flasks in each of the other five groups: ;Group A-: 1 cm^ of the sulfanilamide solution (0.2 g/l); Group A,: 2 cm of the sulfanilamide solution (0.4 g/l)-; Group A^: 3 cm of the sulfanilamide solution (0.6 g/l);. Group A,5 -: 4 cm^ v of the sulf anilamide solution (0.8 g/l); Group Ag: 1 cm of the sulf anilamide solution (0.2 g/l); All flasks in the six groups are inoculated with 2, 5 cm^ of a 72-hour-old inoculum culture of Streptomyces coeruloe-rubidus DS 7126 (NRRL 8098). The cultures are developed on a table that rotates at 2.20 rpm. in a room with a temperature of 26 C. An additional 1 cm 3 of sulfanilamide solution is added to the flasks in group Ag after 48 and 72 hours of incubation respectively (the total sulfanilamide concentration is therefore 0.6 g/l). After each of these additions, the incubation of the flasks is continued under the conditions stated above. After seven days' incubation, one flask from each group is treated at the same time to determine the amount of 32,999 RP. This treatment takes place by adding 4 g of oxalic acid to each flask (thereby the pH of the medium is adjusted to 1.5-) and 50 cm absolute ethanol. The flask is then hermetically sealed and placed for one hour on a table rotating at 220 rpm. The mixture is then filtered through filter paper, and the filtrate is evaporated under reduced pressure at 50°C to half of its original volume (50 cm ). The pH value of the concentrate is adjusted to 4.5 by adding 5N sodium hydroxide solution and then 50 cm 3 of a mixture of methylene chloride and n-butanol'in the volume ratio 80:20 and 25 cm^ 0.5 M\ b.oratpuf f is with pH 9.0. "After stirring in a flask, the colored solvent phase is collected" by decantation, and the treatment is repeated with the aqueous mother liquors, which are extracted with 50 cm 3 of the same solvent mixture. The organic phases are combined and dried over anhydrous sodium sulfate. The volume of the organic phase is finally adjusted to 100 cm" by adding the same solvent mixture. The production of 32,999 RP is determined by thin-layer chromatography of these extracts: (1) A qualitative assessment of the production is carried out by placing on silica gel plates ("Merck 60" - glass substrate without fluorescence indicator) and depositing 100 pl of extract and 2.5- 10 ug pure 32,999 RP as reference material. The plates are developed with a mixture of methylene chloride, ethanol, acetic acid and water in a volume ratio of 80:20:10:4. The possible presence of 32,999 RP in the extract is detected after development by examining the chromatogram either in daylight or in ultraviolet light with a wavelength of 254 nm. (2) The production of 32,999 R? is measured quantitatively by depositing extract volumes between 10 and 100 µl on silica gel plates ("Merck 60 F 254" - glass substrate with fluorescence indicator), whereby the deposited volume is determined on the basis of qualitative thin-layer chromatography. On other such plates, a mere 32,999 RP is also deposited on a scale of 0.5-1.5 ug. The plates are developed with the solvent system described above. After development, the plates are examined with the aid of a "Chromoscan" plate, which registers peaks corresponding to the amount of 32,999 RP - One compares the areas of the peaks corresponding to the amounts of 32,999 RP in the extracts with the areas of the peaks corresponding to the deposits amounts of pure 32,999 RP, and in this way the amount of 32,999 RP formed in the culture solutions can be determined. The results obtained are listed in the following table. ; Example 13 ;A culture medium A is distributed in the same way as in ;3 3 ;ex. 12 in 300 cm flasks in a quantity of 50 cm per flask. A corresponding sulfanilamide solution (10 mg/cm^) is also prepared as in example 12. Flasks containing sterile medium A are divided into four; groups (A-^, A' ^, A^ and A'^) of two flasks each. To each flask in groups A^ and A'^ add 4 cm^ of the sulfanilamide solution (0.8 g of sulfanilamide per 1). No sulfanilamide is added to the flasks in groups A-, and A'. ;3 The flasks in groups A1 and Aq are then inoculated with 2.5' cm of a 72-hour-old inoculum culture of Streptomyces coeruleorubidus DS 8899 (NRRL 3046), and the flasks in groups A^ and A'5 are inoculated with 2.5 cm^ of a 72-hour-old inoculum culture of Streptomyces coeruleorubidus DS 31-723 (NRRL 3045). The cultures are developed on a table that rotates at 220 rpm. in a room with a temperature of 26°C. After 7 days of incubation under these conditions, the amount of formed'32,999 RP is determined in the same way as in ex. 12. The results achieved are; Example 14: A cultivation medium A is distributed in the same way to germinate in ;3 o 3 ;ex. 12 in 300 cm flasks in a quantity of 50 cm per flask. A sulfanilamide solution (10 mg/cm 3 ) is also prepared in the same way as in ex. 12. Flasks containing sterile medium A are divided into four groups of two flasks each. To the flasks in'group A^. no sulfanilamide is added, but sulfanilamide is added to the flasks in the other groups in the quantities indicated below: Group Ap: 1 cm 3 sulfanilamide solution. (0.2 g/l) ;Group A^.: ' 2 cm 3sulfanilamide solution (0.4 g/l) ;Group A^: 3 cm sulfanilamide solution (0.6 g/l) ;After these additions, inoculate all the flasks in all groups A-^-A^ with 2.5 cm."5 of a 72-hour-old inoculum culture of Streptomyces bifurcus DS 23.219 (NRRL 3539). ;The cultures.are.developed on a table.rotating at 220 rpm. in a room with a temperature of 26°C. After incubation for 7 days under these conditions, the amounts formed are determined; 32,999 RP in the same way as in ex'. 12. The results obtained are compiled in the following table below. ; Example 15 ; Man prepares a culture medium B with the following composition: This medium whose pH value is adjusted to 7.20 by adding 1N sodium hydroxide solution, distributed in 300 cm^ flasks in an amount of 50 cm^ per flask and sterilized at 122°C for 20 min. After cooling, the medium has a pH value of 6.60. ;An equivalent aqueous solution of sulfanilamide is prepared as described in ex. 12. ;Flasks containing sterile medium B is divided into two groups B and B in two flasks each. To each flask in group B, 2 cm3 of the sulfanilamide solution (0.4 g sulfanilamide per liter) is added. No sulfanilamide is added to the flasks in group B. All flasks in groups Bn and B are then inoculated with 2, 5 cm of a 72-hour-old inoculum culture of Streptomyces coeruleorubidus DS 31-723 (NRRL 3045).' The culture is developed on a table rotating at 220 rpm in a room with a temperature of 26° C. After 7 days of inoculation under these conditions, the contents of the flasks are treated in the same way as in example 12 to determine the amounts formed - 32,999 RP The results obtained are compiled in the following table. ; ; Example 16 ;In the same way as in example 12, a cultivation medium A is distributed in 300 cm 3 flasks in an amount of 50 cm 3 per flask. ;Man also prepare a sulfathiazole solution (10 mg/cm^) under the same conditions as stated in ex. 12 for the preparation of the sulfamylamide solution. Flasks containing sterile, medium A are divided into four groups of two flasks each. To the flasks in group A^ are added no sulphathiazole, but sulphathiazole is added to the flasks in the other groups in the following amounts: ;Group A7: 0.25 ctn^ sulphathiazole solution (0.05 g/l) ;Group Ag: 1 cm sulphathiazole solution (0.2 g/l) ; Group A^: 0.5 cm^ sulfathiazole solution (0.1 g/1); All flasks are then inoculated with -2, 5 cm^ of a 73-hour-old inoculum culture of Streptomyces coeruleorubidus; DS 7126 (NRRL 8098). The cultures are developed on a table that rotates at 220 rpm. in a room with a temperature of 26°C. An additional 0.5 cmJ of sulfathiazole solution is added to the flasks in group Ag after 48 or 72 hours of incubation (the total sulfathiazole concentration is therefore 0.3 g/D- After each of these additions, the incubation is continued under the conditions stated above. ;After incubation for 7 days, the formed amounts of 32,999'RP are determined in the same way as in example 12. The results obtained are compiled in the table below. ;Example 17 ;In the same way as in example 12, a culture medium A is distributed in 300 cm^ flasks in an amount of 50 cm^ per flask. A sulfathiazole solution (10 mg/cm^) is also prepared. <p>in the same way as in example 16. ; Flasks containing sterile medium A distributed into four groups, each with two flasks. "No sulfathiazole is added to the flasks in group A-^, but sulfathiazole is added to the flasks in the other groups in the quantities indicated below: ;Group A^Q: 0.5 cm^ sulfathiazole solution (0 .1 g/l) ;Group A-^: 2 cm^ sulfathiazole solution (0.4 g/l) ;Group A12: 2.5 cm^ sulfathiazole solution (0.5 g/l); All flasks are then inoculated with 2.5 cm'1 of a 72-hour-old inoculum culture of Streptomyces coeruleorubidus DS 31.723 (NRRL 3045). The cultures are developed on a table that rotates at 220 rpm. in a room with a temperature of 26°C. ;After seven days' incubation under these conditions, the formed amounts of 32,999 RP are determined in the same way as in ex. 12. The results obtained are compiled in the following table.1; ; Example 18: In the same way as in example 12, a culture medium A' is distributed in 300 cm^ flasks in a volume of 50 cm^ per flask. A sulfathiazole solution (10 mg/cm<1>) is also prepared in the same way as in example 16. Flasks containing sterile medium A are divided into four groups of two flasks each. No sulfathiazole is added to the flasks in group A^, but sulfathiazole is added to the flasks in the other groups in the following amounts: ;Group A1Q: 0.5 cm x sulfathiazole solution (0.1 g/l); ;Group Ag: 1 cm'5 sulf atiazole solution- (0.2 g/l) ;Group 2 cm^ sulf atiazole solution (0.4 g/l) ;All flasks are then inoculated with 2.5 cm; of a 72-hour-old inoculum culture of Streptomyces bifurcus DS 23-219 (NRRL 3539). The cultures are developed on a table that rotates at 220 rpm. in a room with a temperature of 26°C. ;After seven days' incubation under these conditions, the formed amounts of 32,999 RP are determined in the same way as in ex. ;12. The results obtained are compiled in the table below. ; Example 19 A cultivation medium C containing the following substances is prepared: ; The medium is produced in the following way. The beans are first boiled in an autoclave with 50 cm of distilled water for 45 minutes at 122°C, after which a puree is made from the beans. The liquor, soy oil and cobalt chloride and distilled water are then added to a total volume of 900 cm"<*.>

The pH value of the mixture is adjusted to 7.2 by adding 5N sodium hydroxide solution. The mixture is distributed in 300 cm"<i>flasks in an amount of 4 5 cm'' per flask. The flasks are sterilized at 122°C for 20 min. After cooling, 5 crn^ of a sterile solution containing 15% glucose The resulting sterile medium's pH value is 6.70.

A water solution of sulfathiazole is also prepared in the same way as in ex. 16.

Flasks containing sterile medium C are dispensed into

two groups C^and Cp of two flasks each. For the flasks in group C^. no sulfathiazole is added, but 0.5 cm^ is added to each flask in group C0. sulfathiazole solution (0 .• 1 g sulfathiazole per 1).

All flasks are then inoculated with 2.5 omJ of one

72-hour-old inoculum culture of Streptomyces bifurcus DS 2-3.

219 (NRRL 3539). The cultures are developed on a table that rotates at 220 rpm. in a room with a temperature of 26°C. After 10 days of incubation under these conditions, the amounts formed 32,999 RP are determined in the same way as in example 12. The results obtained

are compiled in the table below.

Example 20

In the same way as in ex. 12 one distributes a culture medium A in 300 cm^ flasks in an amount of 50 cm^ per flask.

A sulfapyridazine solution (10 mg/cm^) is then prepared by dissolving sulfapyridazine in a mixture of distilled water and 5N hydrochloric acid. The obtained solution whose pH value is 2.0 is sterilized by filtration through "Millipore" membranes (0.45 µm).

Flasks containing sterile medium A are divided into three groups of two flasks each. No sulfapyridazine is added to the flasks in group A, but sterile sulfapyridazine is added to the flasks in the other groups in the following amounts:

Group A, • 0.5 cm'' sulfapyridazine solution (0.1 g/l)

Group A-j^: 1 om<3>sulf apyridazine solution (0.2 g/l)

All flasks are inoculated there. t• is with 2.5 cm 3 of a 72-hour-old inoculum culture of Streptomyces coeruleorubidus DS

7126 (NRRL 8098) .

The cultures are incubated for seven days under the same conditions as described in example 12. The amounts formed, 32,999 RP, are then determined in the same way as in ex. 12.

The results obtained are compiled in the table below.

Example 21-

In the same way as in example 12, a cultivation medium A is distributed in 300 cm'' flasks in an amount of 50 cm^/flask.

An amethopterin solution (20 mg/cm^) is also prepared by dissolving amethopterin in a mixture of distilled water and 5N sodium hydroxide solution. The obtained solution whose pH value is 10.0 is sterilized by filtration through. "Millipore" membranes (0o 4 5 ym).

Flasks containing sterile medium A are divided into four groups (A-^A-^, A-^and A^) of two flasks each. No amethopterin is added to the flasks in group A-^. Add amethopterin in the following amounts to the flasks in the other three groups:

Group A,,-: 0.25 cm^ amethopterin solution (0.1 g/l)

Group A-^g: 0.6 cm 3 amethopterin solution (0.24 g/l)

Group A1^: 1 cm^ amethopterin<p>solution (0.4 g/l)

3' All flasks are then inoculated with 2.5' cm of a 72 hour old inoculum culture of Streptomyces coeruleorubidus DS 7126 (NRRL 8098). The cultures are incubated for 7 days with stirring in the same way as in ex. 12. The formed amounts of 32,999 RP are then determined in the same way as in ex. 12. The results obtained are compiled in the following table.

Example 22.

In the same way as in example 12, a culture medium A is distributed in 300 cm flasks in an amount of 50 cm"' per flask. A barbital solution (50 mg/cm^) is also prepared by dissolving barbital in a mixture of distilled water

■and 5N sodium hydroxide solution. The obtained solution whose pH value is 11.0 is sterilized by filtration through "Millipore" membranes (0.45 µm).

Flasks ■ containing sterile medium A are divided into four groups (A-^, A-^g, A^and A?Q) of two flasks each. No barbital is added to the flasks in group A1. Add barbital in the following quantities to the flasks in groups A-j^q and A^g:

Group A-^g: 0.5 cm^ barbital solution (0.5'g/l)

Group A-^: 2 cm^ .barbital solution (2 g/l)

Barbital is added to the flasks in group A2q

■the cultivation as indicated below...

Simultaneous flasks are inoculated with 2.5 cm^ of a 72-hour-old inoculation culture of Streptomyces coeruleorubidus DS 7126

(NRRL 8098)

The flasks are incubated in a room with a temperature of 26°C on a table rotating at 22 rpm. After 24 hours of incubation, 1.5 cc of barbital solution (1.5 g/l) is added to each flask in group AgQ, after which the incubation of these flasks is continued under the conditions stated above.

After seven days' incubation, the contents of the flasks are treated in the same way as in ex. 12 and the formed quantities 32,999 RP are determined. The results obtained are indicated in the table below.

One works in the same way as indicated above and distributes flasks containing culture medium A into five groups (A-^,<A>21'<A>225A23°s A24^ P^ each two flasks- To the flasks in' group A- nothing is added. To the flasks in group A?1 add 0.5 g of phenobarbital sodium per 1, to the flasks in group A^ add 0.5 g butobarbital sodium (sodium salt of 5-buty1-5-ethylbarbituric acid), and to the flasks in the groups A?- and A'^ h, 0.5 g' and 2 g respectively of pentobarbital [5~ethyl-5-(1-methylbutyl)thiobarbituric acid] are placed per 1. After inoculation in the same way as above and incubation in 9 days in the same way as above, the amounts formed are determined 32,999 RP • The results obtained are compiled in the table below.

Example 23 (a) Fermentation.

The following substances are added to a fermentation container with a volume of 75 1:

Soya flour 1500 g

Beverage 250 g Partially hydrolyzed starch 1750 g

Soybean oil 750 cn<r5>

Sodium chloride 500 g Ordinary tap water to

altogether 45 1

The pH value is set to 7.50 by adding ■

"40 cm^ 10N sodium hydroxide solution. The medium is sterilized by bubbling through water vapor at 122°C for 40 min. After cooling, the volume of the broth is 50 1 as a result of condensation of water vapor during sterilization, and the pH value is 6.70. then graft with 200 cm"<5->of er. culture of Streptomyces coerul-erorubidus DS 7126 (NRRL 8098), which culture is prepared by cultivation in a shake flask. The culture is developed at 30°C for 25 hours with stirring and aeration with sterile air. It is then suitable for use for inoculation of the production medium.

The production cultivation is carried out in a fermentation container with a volume of 800 1, to which the following substances are added:

After setting the pK value to 6.50 by adding 210 cm 2 ION sodium hydroxide solution is added. 7.6 kg glycerol, 0.800 kg calcium carbonate and ordinary tap water to a total volume of 360 1.

The medium's pH value is then equal to 6.45. The medium is sterilized by bubbling through steam at 122°C for 40 min. After cooling, the broth has a volume of 400 1 as a result of condensation of steam during sterilization, and the pH value is 6.25. ,

10 1 of a sterile water solution containing 308 g of sulfanilamide is then added.

The medium's pH value is then equal to 6.80. One inoculates with 40 1 of the inoculum culture from the cultivation described above in the fermentation vessel with a volume of 75 1 - The temperature of the medium is kept at 27°C. The medium is stirred with a stirrer that rotates at 205 rpm, and aerated with 20 m of sterile air per hour. After 24 hours of cultivation, 3 1 of a sterile water solution containing 4 4 g of sulfanilamide is added, after which the fermentation is continued.

After 140 hours of cultivation, the medium has a pH value of 6.75 and the volume is 350 1. The cultivation medium then contains 35 mg 32,999 RP per 1.

(b) Extraction.

To 350 1 of the culture broth obtained above, 350 1 of methanol and 6N sodium hydroxide solution are added until the pH value is 7.5. The whole thing is stirred for 1 hour. After adding 25 kg of filtration aid, the suspension is filtered in a filter press. The filtrate is collected. The filter cake is washed with 100 1 50% methanol. The filtrate and washing solution are combined and concentrated under reduced pressure to a volume of 250 L.

After alkalization by the addition of 220 cm'' oN sodium hydroxide solution, the antibiotic in the concentrate is extracted with 2 x' 150 1 of a mixture of methylene chloride and butanol in a volume ratio of 80:20. The combined organic extracts, which have a volume of 238 1, are concentrated under reduced pressure to a volume of 30-1. 100 crP n-butanol saturated with 11N hydrochloric acid is then added. After concentrating to a volume of 3 1, the concentrate obtained is slowly added to 21 1 of hexane. -The precipitate obtained is filtered off, washed and dried. This gives 137 g of impure hydrochloride of 32,999 RP.

(c)"Cleaning..'■''-

(1) 112 g of the above obtained impure hydrochloride of

32,999 RP is dissolved in 1:1 water at 50°C. The obtained solution whose pH value is 2.9 is filtered through fritted glass... The colored impurities in the obtained filtrate are extracted with 2 x 500 cm^ of chloroform and then once with 1 1 of chloroform. After adding 75 g of sodium chloride to the aqueous phase, the antibiotic is extracted with 3 x 0.75 l of n-butanol. The pH value of the water phase is set to 9.5 by adding concentrated sodium hydroxide solution, and the remaining antibiotic is extracted with 0.5 1 n-butanol.

The obtained butanol extracts containing the antibiotic are combined and concentrated under reduced pressure to a volume of 1 1. One then adds 1 1 of chloroform to the obtained concentrate. The obtained solution is washed with 2 x 500 cm 3 of water that has been alkalized to pH 9.5. The washing solutions are extracted with 2 x 500 cm 3 of a mixture of chloroform and n-butanol in a volume ratio of 80:20 at pH 9.5. The organic extracts are combined and concentrated under reduced pressure.

During the concentration, 300 cm^ of a mixture of n-butanol, water and 11N hydrochloric acid is added in a volume ratio of 94:5:1 - The concentration is continued until the volume is 150 cm^, after which -

the obtained solution is slowly added to 2 1 hexane. The precipitate obtained is filtered off, washed and dried. This results in 30 g of a product containing the hydrochloride of 32,999 RP.-

(2) 13 g. of it. product obtained above is dissolved in

26 cm^ of a mixture of water and dioxane in a volume ratio of 20:80 at 35°C. The antibiotic is brought to crystallize by slow addition of 624 cm-<5>dioxane. After stirring for 3 hours, the crystals are filtered off, after which they are washed and dried. This gives 2 g of a crystallized product containing the hydrochloride of '32'999 RP.

(3) - 4.47 g of a crystallized product, obtained on

the same way as described under (2) above is dissolved in 40 cm^

water. The solution obtained is filtered. That in the filtrate to-

present hydrochloride of the antibiotic 32,999 RP is brought to crystallisation by adding 450 cc of acetone with stirring. The crystals obtained are filtered off, washed and dried. This results in 2.47 g of a product containing crystallized hydrochloride of 32,999 RP. (4) 2.84. g of a product, obtained in the same way as described under (3) above, is dissolved in 28.4 cm"5 of a mixture of equal volumes of methanol and acetone. To the obtained solution is added 16.9 cm^ of a mixture of acetone and water in a volume ratio of 84:16.- The hydrochloride of 3 2 999 RP is brought to crystallization by heating to 30°C and subsequent cooling. The crystals obtained are filtered off, washed and dried. This gives 2.33 g crystallized, semi-purified hydrochloride of 32. 999_.._RP... (5) 1.85 g of the semi-purified hydrochloride obtained above is dissolved in 400 cm of water'with a pH value of' 5.5-.The colored impurities in the solution obtained is extracted with 10 x 400 cm^ of a mixture of n-butanol" and chloroform in a volume ratio of 20:80. The water phase is concentrated and n-butanol is added during the concentration. A butanol phase with a volume of 75 cm"5 is thereby obtained, which is slowly added to 1.5 1 of hexane. The resulting precipitate is filtered off, washed and dried: 1.4 g of pure hydrochloride of 32,999 RP, which is identical to the product described in example 8.

Example 24 (A)' Fermentation.

In a fermentation container with a volume of 170 1, the following substances are added:

The resulting medium whose pH value is '6.0 is sterilized by bubbling through steam at 122°C for 40 min. After cooling, the volume of the broth is 120 1 due to condensation of water vapor during sterilization, and the pH value is 6.65. Mon

■ inoculate with 250 cm^ of a culture of Streptomyces atroviolaceus

DS 8938., which culture is prepared by cultivation in a shaken E-flask. The culture is developed for 35 hours at 20°C with stirring and aeration with sterile air. It is then suitable for inoculation of the production medium.

The production cultivation is carried out in a fermentation container with a volume of 800 l, to which the following substances are added:

The medium's pH value is adjusted to 7.5 by adding 500 cm"' ION' sodium hydroxide solution, after which the medium is sterilized by bubbling through steam at 122°C for 40 min. After cooling, the broth has a volume of 550 1 as a result of condensation of water vapor during the sterilization. The pH value of the medium is 6.60. Man'pods with 55 1 of inoculation culture from, the above indicated cultivation in the fermentation container with the volume of 170 1. The culture is developed for 117 hours at 26°C while stirring with a stirrer that r. aerate at 205 rpm, and during aeration with 15 m"5 sterile air per hour. After completion of cultivation, the medium has a pH value of '7.60.

(B) Recovery.

28 kg of oxalic acid is added to 560 1 of the culture solution obtained above. After stirring for one hour at 40°C and adding 25 kg of filtration aid, the solution is filtered in a filter press. The mycelium cake is washed with 120 1 water, which has a temperature of 50°C and which contains 3 kg of oxalic acid. The filtrate and the washing solution are combined to form a solution whose pH value is equal to 1. This solution is cooled to 10°C. The pH value is then adjusted to 4.5 by adding 50 1 6N sodium hydroxide solution. The antibiotic present in the solution obtained is fixed on a column of 24 1 "Amberlite IRC 50" in H+<-> form. the ion exchanger is washed with water until the effluent is colourless, then with 100 1 of a mixture of equal volumes of methanol and water and finally with 100 1 of a mixture of methanol and water in a volume ratio of 90:10. The antibiotic is then eluted with 250 1 of a mixture of methanol and water in a volume ratio of 90:10, which mixture contains 1 g of sodium chloride per 100 ml. The eluate is concentrated

under a reduced pressure on. 5-10 mm Hg at kO°C to a volume of

20 1. The concentrate's pH value is set to 9 by adding 11N sodium hydroxide solution, after which the concentrate.extra is washed once with 20 1 chloroform and then twice with 10 1 chloroform. A total of 35 1 of extract is obtained, which is concentrated under a reduced pressure of 5-10 mm Hg at 40°C to a volume of 2.1. "The remaining solution is made acidic by the addition of 50 c'm^ n-butanol saturated with 11N hydrochloric acid and then concentrated to a volume of 1 1. The solution thus obtained is poured slowly into 7 1 of hexane. The precipitate formed filtered off, washed and dried. This gives 13 g of impure hydrochloride of the antibiotic 32,999 RP..

(C) Cleansing..

(a) The impure hydrochloride of 32,999 RP is purified by

countercurrent distribution in three flasks, A, B and G with a volume of 2 1.

A mixture of -methylene chloride, n-butanol and M/15 phosphate buffer with pH 7.38 is prepared in a volume ratio of 8:2:10. When the mixture has been brought to equilibrium, the two phases are separated.

In flask A pour one. 500 cm"' of a mixture of phosphoric acid (85% by weight) and water in a volume ratio of 1:125 as well as 500 cm"<1>, of the heavy organic phase obtained above. In this phase mixture, 13 g of the impure hydrochloride of the antibiotic 32,999 RP obtained above are dissolved. After 15 minutes of stirring and removal of an insoluble substance by filtration, the pH value is adjusted to .7.5 by adding 1N sodium hydroxide solution.

3

In each of flasks B and C, add 5.00 cm of the light liquid phase obtained above, whereby the pH value in flask B is set to 7 and the pH value in flask C is set to 6.5 using 85% phosphoric acid.

In the direction flask A-flask B-flask C, a resistance distribution is carried out with five transfers, whereby the heavy phase (solvent) is used as the mobile phase, and the light phases (water phases) with pH 7.5, 7 and 6.5 as stationary phases. 500 cm is used each time; mobile phase, and the pH value is kept constant in the stationary phases.

The antibiotic in the water phase in flask C is extracted

at pH 9.5 with twice 500 cm'5 of a mixture of chloroform and

Claims (2)

n-butanol in the volume ratio 8; 2. The organic extracts are combined and concentrated under a reduced pressure of .5-10 mm Hg at 40°C. During the concentration, 300 cm'5 of n-butanol containing 6% by volume of 2N hydrochloric acid is added. The volume of the concentrate is set to 5 cm'<5>, after which the concentrate is slowly poured into 500 cm'5 of hexane. The formed precipitate is filtered off, washed and dried. This gives 275 mg of semi-purified hydrochloride of 32,999 RP. (b) 80 mg of the semi-purified hydrochloride of 32,999 RP obtained above is dissolved in a mixture -of 1' cirr5 of water and •1 cm 3 of methanol. The solution obtained is divided into • four equal parts, which are deposited linearly on four analytical chromatography plates equipped with an even layer of silica gel (Merck). The plates are prepared for one hour in a container whose walls are covered with filter paper containing a mixture of methylene chloride, formic acid and methanol in a volume ratio of 80:17--3- The zone containing 32,999 RP is removed after drying by scraping off the silica gel . The powder obtained is suspended in 40 cc of water and the antibiotic is extracted at pH 9.5 with twice 20 cc of chloroform. The organic extracts are combined and concentrated under a reduced pressure of 5-10 mm Hg at 40°C. During the concentration, 1 cm-3 of n-butanol containing 6 volume-? 2N hydrochloric acid. The volume of the concentrate is set to 2 cm"<5>, after which the concentrate is slowly poured into 50 cm^ of hexane. The precipitate formed is filtered off, washed and dried. This gives 4 mg of pure hydrochloride of 32,999 RP in the form of an orange-red, microcrystalline powder which melts at a temperature of up to 200°C during cleavage. The product obtained is identical to the product obtained in example 8. Patent claim 1..Procedure for the production of a new naphthacene derivative designated with No. 32 999 RP and with the formula: either in the form of a free base or■in the form of addition salts with an acid, whereby the hydrochloride of the compound has the following properties: it is an orange-red, microcrystalline powder which is soluble in methanol, pyridine, dimethylformamide and water in an amount of about 100 mg/cm 3 , soluble in butanol saturated with water at 20°C in an amount of 10 mg/cm-<3>, soluble in ethanol in an amount of 5 mg/cm^ and soluble in acetone, chloroform, methylene chloride, ethyl acetate, benzene, menthyl isobutyl ketone, diox an and hexane in an amount of less than 0.1 mg/cm^; it has the following elemental analysis: C% = 56.6; E% = 5.7; 0% - 29.41; M = 2.39; Cl% = 6.0; the specific rotatability (determined in a 0.09% solution in ethanol containing 0.1% IN HCI) is: its UV spectrum and its visible spectrum (determined in solutions containing 10.65 mg/l of the hydrochloride in ethanol containing 0.1% IN HCI) exhibit absorption maxima at 233 nm, 254 nm, 292 nm, 493 nm, 512 nm and 527 nm; its IR spectrum exhibits absorption signals at 3410, 3250, . 3220. . , 695, 665, 640, 625, 600, 580, 565., 540, 530, 485, 475, 465, 450,- 438, 415, 390 and 320 cm<-1>; it exhibits an Rf value of 0.2 by thin-layer chromatography on silica gel when a mixture of methylene chloride, formic acid and methanol in a volume ratio of 80:17:3; in mice, it is effective against leukemia L 1210, leukemia P 388 and leukosis chromatosis in intraperitoneal doses of between 0.12.5 and 0.250 mg/kg; characterized by (a) microbiologically reducing carminomycin with the formula: or a salt thereof; or that one; (b) cultivates one of the following microorganisms: Streptomyces coeruleorubidus DS 7126 (NRRL 8098) Streptomyces coeruleorubidus DS 31 723 (NRRL 3045) Streptomyces coeruleorubidus DS 8899 (NRRL 3046) Streptomyces bifurcus DS 23 219 (NRRL 3539) in a aerated nutrient medium suitable for Streptomyces strains and in the presence of additives selected from amethopterin, barbital, phenobarbital, butobarbital, pentobarbital, sulfanilamide, sulfapyridazine and sulfathiazole, after which the desired end product is recovered from the culture medium; or that 'one' (c) grows Streptomyces atroviolaceus DS 89.38 (NRRL 8l48) or its producing mutants in an aerated nutrient medium containing sources of assimilable carbon and assimilable nitrogen and inorganic salts at an initial pH value of 5.8-7 ,8 and at a temperature of between 23" to 33°C, after which the desired end product is extracted from the culture medium. 2. Method according to claim 1, alternative (a), vessel, characterized in that the microbiological reduction is carried out using a culture of one of the following microorganisms: Streptomyces lavendulae (ATCC 8664), , • Streptomyces rosecchromogen.es (ATCC 13.400), Corynebacterium simplex (ATCC 6946), Bacterium cyciooxydans (ATCC 12.673), or by means of cells' isolated from such a culture, or by means of enzymatic extracts obtained from cells of these microorganisms.