NO137904B - PROCEDURES FOR THE PREPARATION OF A NEW ANTRACYCLING LYCOSIDE ANTIBIOTIC MIXTURE, VIOLAMYCIN, ITS SUB-COMPONENTS, AND MIXTURES OF ITS AGLYCON COMPONENTS - Google Patents

Description

The invention relates to a method for the production of a hitherto unknown pigment antibiotic with cancerostatic, virostatic and especially with new and surprising effects against various agents of mycoplasma infections, which is designated as violamycin. In the face of tumor diseases and virus-caused diseases in humans and animals, there are only a few resp. insufficient funds.

Cultivation methods are already known

of Streptomyces violacens strains and also the antibiotics produced thereby, for example in R. Hiitter "Systematik der Streptomy-ceten unter besonderer Beriicksichtigung der von ihnen gebildeten Antibiotika", Bibliotheca Microbiologica, Fase. 6, S. Karger AG, Basel, 1967. A comparison of the antibiotics mentioned there with the general formula of the one produced according to the invention

only the antibiotic violamycin allows the inference that identity can be ruled out.

-Furthermore, British patent no. 1,257,821 mentions-

spoken an antibiotic mitocromin, which is admittedly a representative

tant for the daunomycin group, which derives from ^t-methoxy-6,9-dihydroxy-anthraquinone. • In British patent no. I.OO3.383, the antibiotic F.I. has become known. 1762, an antibiotic identical to daunomycin. The antibiotic trypanomycin mentioned in British patent no. 1,290,910 also differs clearly from violamycin,

for trypanomycin is a representative of the pyromycin group, which derives from 1,4,6-trihydroxy-anthraquinone. Violamycin is also not identical to the antibiotic rhodomycin, which was

disclosed in British Patent No. 708,749. Admittedly, rhodomycin can also be derived from 4,6,9-trihydroxy-anthraquinone, but rhodomycin is only mentioned with the components B-rhodomycinone and the glycosidically bound sugars rhodosamine and deoxy-L-fucose. ' In violamycin, on the other hand, 3-rhodomycinone is only one of six different components

(violamycinones A-^.-.Ag) and violamycin also contains the sugar rhodinose. In "The Journal of Antibiotics", series A, volume XIII, no. 1/1960, chapter "Actinomycetes synthesizing Antibiotics" violarin can of course also be derived from polyhydroxyanthraquinones, as they do not, however, contain polyhydroxyanthraquinones of the type e-isorodomycinone and c ^-rhodomycinone, no identity remains. Nor does the antibiotic mycetin contain the aglycones e-isorodomycinone and a^-rhodomycinone. Other antibiotics such as ciro-lerosus according to British patent No. 1. 084 . 381, danubomycin according to British patent no. 901,830, virusin and luridin (UdSSR patents) also have clear differences compared to violamycin. In addition, there are antibiotics based on streptomyces violaceus, which e.g. of Mil, 719, D 5199 and D 5366, which have not yet been discussed in more detail. Only because of the related differences in physical properties (solubility) and effects (D 5199/D 5366 are anthelminthically active) can it be said that there is no identity with violamycin in these cases either.

It is a disadvantage that the chemotherapeutics used against mycoplasma diseases do not show any bactericidal effect on the producer.

The purpose of the invention is to provide a drug for tumor, viral and mycoplasma diseases.

The invention is based on the task of providing a suitable manufacturing method.

It has been found that in fermentation solutions of the strain IMET JA 6844, a new antibiotic suitable for the therapy of neoplastic, viral and especially mycoplasma-induced diseases is formed, which was designated as violamycin in good yield and can be isolated using the methods described below eb et inge Iser.

The microorganism used in the production of violamycin is deposited in the strain collection of the Central Institute for Microbiology and Experimental Therapy, Jena, German Academy of Sciences in Berlin under strain collection number IMET JA 6844. The wild type of the former was isolated in 1958 from a soil sample from Schwellenburg near Elxleben (Erfurt ). The strain belongs to the genus Streptomyces and corresponds to the description of Streptomyces violaceus (Rossi-Doria 1891, Kraszilnikov 1941, Waksman 1953), as given in International Streptomyces after examination of the neo-type strain ISP 5082 (=INMI-1, RIA 656, ATCC 15888) Project by Shirling and Gottlieb (Int ..J.. syst. Bact. 19:^97, ( 1969)). The strain has the following features: The substrate mycelium is colored red-violet or blue on the different media and at different times. The pigment, which can also diffuse in agar media, has indicator properties: acid-red, alkaline-blue. The aerial mycelium is pale pink, pink or grey-pink in colour. The spore chains are arranged in more or less regular, mostly open spirals on sometimes long main hyphae. The spore surface is prickly. On peptone or tyrosine-containing medium' diffuses a brown to blue-black pigment in agar. The tribe is growing

on basal medium with the addition of D-glucose, L-arabinose, D-

xylose, m-inositol, D-mannitol, D-fructose, rhamnose, sucrose and raffinose.

The invention therefore relates to a method for producing a new anthracycline glycoside antibiotic mixture, violamycin, its subcomponents violamycin A,31 and BII as well as mixtures of its aglycon components violamycin A^...Ag resp. Bj---.Bgwith the general formula

with the meaning

as the violamycinones A-^ Ag resp. B-j_ Bg in violamycin A is glycosidically connected with the sugar rhodosamine, in violamycin BII glycosidically with the sugars rhodosamine and desoxy-L-fucose, and in violamycin-BI glycosidically with. sugars rhodosamine, desoxy-L-

fucose and rhodinose, when cultivating a streptomycete under aerobic cultivation conditions in a liquid nutrient medium containing carbon and nitrogen sources as well as mineral salts, the method being characterized by the use of Streptomyces violaceus IMET JA 6844, the cultivation is carried out in a manner known per se in suitable nutrient media at temperatures from 25 to 37°C for 2 to 6 days under aeration and stirring,

the antibiotic formed is extracted from the culture filtrate with n-butanol and from the mycelium with methanol, the methanol extract of the mycelium is concentrated, transferred to the aqueous phase and re-extracted at pH 8 with n-butanol and then combined with the n-butanol extract of the culture filtrate, and from the extract concentrate precipitates by mixing with petroleum ether while stirring, the violamycin crude product is freed from solvent, after which this crude product is optionally dissolved in chloroform, filtered off from insoluble residue and the formed solution is washed with water and then the organic violamycin fraction A formed on one side is treated and on the other on the other hand, the aqueous violamycin B-containing phase is further separated, the aqueous phase being extracted with n-butanol at pH 8.5 and then the chloroform extract and the n-butanol extract are mixed separately with petroleum ether and the precipitates formed are similarly dissolved separately in methanol , these solutions are subjected to gel filtration on "Sephadex" LH 20, as it violamycin A and violamycinone A are recovered from the chloroform extract and violamycin BI, violamycin BII and violamycinone B from the n-butanol extract.

The cultivation of the strain IMET JA 6844 takes place under aerobic conditions. On soil, lyophilized dried spore material of the stem is inoculated into a suitable agar nutrient medium and then into liquid,

pre-sterilized nutrient media and the formed mycelium are grown in a manner known per se at a temperature between 25 and 37°C (preferably 28°C) over a period of 2 to 6 days (preferably 4 days) at an acidity level as at the beginning of the fermentation process lies between pH 6.2 and 7.0 and at the end between pH 7.5 and 8.3 - The nutrient medium consists of carbon and nitrogen sources as well as inorganic salts. As carbon sources, starch, glucose, glycerol, mannitol, dextrin, sucrose, soya oil and soya flour can be used. As nitrogen sources, in addition to the nitrogen-containing substrates mentioned above, dry yeast, meat peptone, casein can also be mentioned.

Good fermentation results can be achieved by adding mineral salts. The latter favors the fermentation process depending on the nutrient medium used. In complex media containing different flours, additions of calcium carbonate, Na phosphate or K-phosphate. The fermentation of the organisms can be carried out in inclined culture flasks and round flasks of different contents, in glass fermenters and in V2A tanks.

The isolation of the pigment antibiotic violamycin is carried out in such a way that the antibiotic is extracted from the culture filtrate with butanol and from the mycelium with methanol. Violamycin is transferred from the methanol extract to the butanol phase by evaporation and re-extraction. This butanol extract is combined with that extracted and evaporated from the culture filtrate and evaporated again. By mixing with petroleum ether and separating the solvent, a violamycin crude product is obtained.

This violamycin raw product is dissolved in chloroform, insoluble residue is filtered off, the resulting solution is washed with water and concentrated. By mixing the chloroform extract with petroleum ether, violamycin fraction A is precipitated. This violamycin fraction A is dissolved in methanol, purified by column chromatography, separated into the biologically inactive aglycon violamycinone A and into the biologically active antibiotic complex violamycin A.

The wash water formed by chloroform extraction of the violamycin crude product was subjected to re-extraction with n-butanol. The butanol re-extract is concentrated and, by adding petroleum ether, violamycin fraction B is obtained. This violamycin fraction B is dissolved in methanol, then purified by column chromatography and separated into the biologically inactive aglycon violamycinone B and the biologically active antibiotic complex violamycin B.

The column chromatographic purification of violamycin A and violamycin B as well as the further division of violamycin B into violamycin BI and violamycin BII and into the aglycone fraction takes place from the antibiotic fractions A and B. For this purpose, in contrast to the hitherto usual purification methods for anthracycline antibiotics, gel filtration over "Sephadex" LH 20 is used for the first time. The pre-purified violamycin fraction A or B is dissolved in a little methanol and separated using the method of column chromatography on " Sephadex" LH 20. Depending on the material used, the following eluate fractions are thereby obtained in the order of their exit from the column.

When using the pre-purified violamycin fraction A:-

Violamycin A and the aglycone fraction violamycinone A.

When using the prepurified violamycin-

y-fraks.ion B: Violamycin BI and violamycin BII as well as the aglycone fraction B.

Violamycin A and violamycin B differ by

, -the content of sugar compounds and can be divided paper chromatographically into six different components. The hydrolyzate of violamycin A, the 'aglycon mixture A^...'A6 and the hydrolyzate of violamycin B, .aglycon mixtures; B-^.....Bg is qualitatively, i.e. chemically, identical. In quantitative terms, there are admittedly minor differences in the quantitative composition of the two aglycon mixtures.

The aglycone mixtures produced from the hydrolysates were separated by column chromatography, the aglycones violamycinone A^... Ag and violamycinone B-^.... Bg compared with each other and their belonging to the chemical structural class of anthracyclinones determined. With the help of UV spectra and analytical methods, the exact structure of the individual aglycones was established. The result of the structure clarification is shown in the following table.

Violamycin A is a complex amorphous red-brown

which dissolves well in chloroform, acetone and alcohols, slightly in benzene and not in ether and water. The antibiotic violamycin A has indicator properties and is red in acidic solutions, blue in alkaline solutions.

The antibiotic violamycin B is a complex of amorphous red-brown substances, which dissolve well in water, lower alcohols, acetone and chloroform, but not in benzene, petroleum ether and ether. Violamycin B also has indicator properties and is red in acidic solutions and blue in alkaline solutions.

Violamycinones Aj (resp. B1)....Ag (resp. Bg) var

in the result of the assessment of the UV spectra as well as the analytical investigations all to assign the rhodomycinones resp. the isorhodomycinones. Violamycinone A-^ (B^), A2 (B2) and Ag (Bg) have an OH group in the <C>1Q position and thus correspond to the type of beta-rhodomycinone resp. beta-isorhodomycinone. The violamycinones Aj (B-j), A^ (Bjj) and A^ (B5) have a CO-O-CHj group in the C10 position and thereby correspond to the type epsilon-rhodomycinone resp. epsilon-isorhodomycinone, which was previously unknown as a component of an antibiotic. Violamycinone A^ (B^) also has a peculiarity, as no OH group is present in the C^ position, this aglycon therefore corresponds to the type of zeta-isorhodomycinone. The Violamyc inones Aj (Bj), Aj (Bj) and A^ (B^) are, according to their UV absorption, the isorhodomycinone compounds, A^ (B^)-, A2 (<B>2) and Ag (Bg) rhodomycinone- the connections.

Both fresh fermentation fluids of the violamycin former and the antibiotic isolated from it have a growth-inhibiting effect on gram-positive bacteria and mycobacteria, to a lesser extent on representatives of gram-negative bacteria, but do not affect antibiotic yeast and fungi. Against mammalian cells in vitro and against animal experimental tumors on mice, violamycin exerts cytostatic resp. cancerostatic activities.

Investigation of the cancerostatic effect of the antibiotic violamycin.

In the first stage of the cancerostatika test (area of experimental therapy of ZIMET Jena), the raw product of violamycin unfolds on mice, to which the experimental leukemia L 1210 and LAJI resp. of Sarcoma l80, effect against Sarcoma 180. With one-time i.p. application of 5 resp. 2.5 meg violamycin raw product per 20 g mice, the growth of the solid tumor is inhibited compared to untreated controls around 48.9 resp. 53.6?.

Bacterial viruses are similarly inhibited intracellularly by the antibiotic as representatives of animal viruses in their reproduction without simultaneously blocking the host cell metabolism. Violamycin exerts a particularly strong effect on cell division and the resting metabolism of mycoplasma types.

The investigation of the bacteriostatic and bactericidal effect of the antibiotic violamycin on mycoplasmas.

The effect of the antibiotic was tested on two significant animal-infection producers in agriculture, especially in breeding and livestock keeping. Violamycin A and B are distinguished by a surprisingly strong bacteriostatic and at the same time also bactericidal activity against Mycoplasma gallisepticum (strain S 6) and Mycoplasma hyorhinis. The comparative investigation of the biological activity of the antibiotic complex violamycin A and B gives, after assessment of the dilution sample on a broth nutrient base, the following spectrum of action:

Those in table. The results listed in 1 show that violamycin A is more bacteriostatically effective against Mycoplasma gallisepticum (strain S 6) than violamycin B, while however violamycin B exhibits a stronger bactericidal activity against Mycoplasma hyorhinis than violamycin A. Both violamycins differ from the previously known natural substances with effects against mycoplasmas by their several hundred times stronger bactericidal effect. Such a biological activity against representatives of mycoplasmas that can produce disease symptoms in beneficial animals has not been described for anthracycline antibiotics so far and is surprising.

The new antibiotic violamycin and its derivatives can be used as potential chemotherapeutics against mycoplasma diseases in humans and farm animals, against tumor and viral diseases.

The following examples shall explain the invention in more detail.

Example 1.

Two 500 ml slant culture bottles each contain 80 ml of the following culture medium:

Sterilization: 35 minutes at 110°C. After sterilization, the acidity is at pH 6.3.

Each slant culture bottle is inoculated with a spore and mycelial suspension, which is prepared with sterile, isotonic sodium chloride solution from a 10- to 14-day-old culture of the violamycin producer grown in a test tube on the following starter nutrient medium:

Sterilization: 35 minutes at 120°C, natural acid.

The inoculated enrichment cultures are grown for -48 hours

at 28°C on a shaker with a frequency of 180 revolutions per minute. 8 ml of a growth culture grown in this way is used to inoculate 500 ml slant culture flasks, each containing 80 ml of the following production medium:

Sterilization: 35 minutes at 115°C. The degree of acidity after sterilization is pH 6.3 - The temperature during fermentation is 28°C and the shaking frequency at 180 revolutions per minute.

After 72 hours of fermentation, the highest content of violamycin (125 mcg/ml) is achieved. The activity was determined using a number of known and new microbiological agar diffusion test methods. In the latter, the inhibitory effect of violamycin on the multiplication of bacterial viruses in their host cell serves as a measure of the activity of the antibiotic (Fleck, W. 1968, Allg. Mikrobiol. 8 (2), 139).

Example 2.

You proceed as in example 1 with the difference that the production medium has the following composition:

Sterilization: 40 minutes at 116°C. The degree of acidity is after sterilization at pH 6.2. The highest content of violamycin is achieved after 90 minutes (100 mcg/ml).

Example 3.

With a culture of the violamycin-former from a solid nutrient medium, as described in example 1, 80 ml of the liquid culture medium listed in example 1, which is contained in a 500 ml inclined culture bottle, is inoculated. Cultivation is carried out for 24 hours at 28°C on a shaker with a frequency of 180 revolutions per minute. minute. 12 ml of the thus formed culture liquid serves to inoculate 400 ml of the same culture medium which is contained in a 2 liter glass flask. Cultivation is carried out for a further 24 hours at 28°C and at a shaking frequency of 180 revolutions per minute. minute before the 400 ml culture liquid thus formed is used to inoculate 20 liters of the production medium mentioned in example 1. The latter is contained in a 32 liter glass fermenter. During the fermentation, which was carried out with a stirring speed of 400 revolutions per minute and with an air supply of 15 liters per minute, foam formation is controlled by adding small amounts of sunflower oil or other silicone-containing foam protection agents.

20 liters of culture solution are adjusted with hydrochloric acid to pH 5.0

and after separating the mycelium, 18 liters of culture filtrate are obtained. The butanol extract of the culture filtrate is evaporated in vacuum to<* >1:10 of the initial volume and combined with the butanol extract of the mycelium extract concentrate. The mycelium is stirred beforehand

methanol twice in a ratio of 1:6. The combined extracts are evaporated under reduced pressure to 1/20 of the methanol volume and the resulting aqueous solution is extracted completely with butanol. The combined butanol extracts are brought together with the butanol extract of the culture filtrate to 1/25 of the culture solution volume. From the final concentrate, 300 g of biologically active raw product (A 6844) per m^ culture dissolution.

Example 4.

The procedure differs from this according to example 3 in that the violamycin-former, after cultivation in a 2 liter glass flask instead of in a 32 liter glass fermenter, is grafted into a 400 liter V2A tank containing the production medium mentioned in example 1. The stirring speed amounts to 280 revolutions per minute and the air supply is set to 400 liters per minute.

From 300 liters of culture solution, 300 g of raw product of 6844/ is produced according to the method mentioned in example 3. The raw product A 6844 is used for the production of the biologically active complexes violamycin A and B.

Example 5-

200 g of raw product A 6844 are suspended in 5 liters of chloroform and after stirring for one hour, the solution is separated from the insoluble residue. The chloroform solution is washed 5 to 6 times with half the volume of water and thereby completely freed from water-soluble pigments.

After drying, the chloroform solution is concentrated under reduced pressure to 1/50 of the initial volume and stirred with 10 times the amount of petroleum ether. Thereby the biologically active violamycin fraction A is precipitated. This violamycin fraction A is filtered off, washed with pure petroleum ether and dried under reduced pressure in a desiccator over concentrated sulfuric acid. Yield of violamycin fraction A amounts to 16 g.

Violamycin fraction A was then dissolved in methanol and the resulting solution was subjected to gel filtration on "Sephadex" LH 20, methanol was used as eluent for violamycin A. The eluate of violamycin A was evaporated, transferred into the butanol phase and caused to precipitate by adding petroleum ether. Thereby, 5 g of the biologically active complex violamycin-A was formed from 10 g of violamycin fraction A. The eluate of the biologically inactive aglycon mixture, violamycinone A, was evaporated to dryness.

Violamycin fraction B is to be recovered from the washing water of the chloroform extract after adjusting the acidity to pH'8.5 by extraction with n-butanol. After concentration of the butanol extracts and after their stirring with petroleum ether, the corresponding precipitate is filtered off and dried. The yield of violamycin fraction B amounts to 25 g per 200 g violamycin raw product.

Violamycin fraction B was dissolved in methanol and subjected to gel filtration on "Sephadex" LH 20, methanol served as eluent for violamycin B. The first exiting eluate of violamycin BI was likewise evaporated, transferred into the n-butanol solution and precipitated by the addition of petroleum ether. Thereby, 8.55 g of violamycin BI were formed from 10 g of violamycin fraction B.

In the same way, violamycin BII was isolated from the eluate of violamycin BII. 0.95 g of violamycin BII was formed from the 10 g of fraction B.

Claims (1)

Process for the preparation of a new anthra-cycline glycoside antibiotic mixture, violamycin, its subcomponents violamycin A, BI and BII as well as mixtures of its aglycon^components violamycinone Aj....Ag resp. Bj....Bg with the general formula with the meaning ir.rt violamyc inonene Aj....Ag resp. Bj....Bg in violamycin A is glycosidically linked to the sugar rhodosamine, in violamycin BII glycosidically to the sugars rhodosamine and desoxy-L-fucose, and in violamycin BI glycosidically to the sugars rhodosamine, desoxy-L-fucose and rhodinose, in the cultivation of a streptomycete under aerobic cultivation conditions in a liquid nutrient medium containing carbon and nitrogen sources as well as mineral salts, characterized in that Streptomyces violaceus IMET JA 684<4>,' is used. cultivation is carried out in a manner known per se in suitable nutrient media at temperatures from 25 at 37°C for 2 to 6 days under aeration and stirring, the antibiotic formed is extracted from the culture filtrate with n-butanol and from the mycelium with methanol, the methanolic extract of the mycelium is concentrated, transferred to the aqueous phase and re-extracted at pH 8 with n-butanol and is then combined with the n-butanol extract of the culture filtrate, and from the extract concentrate the violamycin crude product is precipitated by mixing with petroleum ether while stirring and freed from solvent, after which this crude product is optionally dissolved in chloroform, filtered from insoluble residue and the resulting solution is washed with water and then treated on on the one hand the organic violamycin fraction A and on the other hand the aqueous violamycin B-containing phase separated further, the aqueous phase being extracted with n-butanol at pH 8.5, and then the chloroform extract and the n-butanol extract are mixed separately with petroleum ether and the formed precipitates are likewise dissolved separately in methanol, these solutions und is subjected to gel filtration on "Sephadex" LH 20, whereby violamycin A and violamycinone A are recovered from the chloroform extract and violamycin BI, violamycin BII and violamycinone B from the n-butanol extract.