NO852758L - 1-HYDROXY-CYTORODINES, A MICROBIOLOGICAL PROCEDURE FOR THEIR PREPARATION AND THEIR USE AS CYTSTATICS - Google Patents

Abstract

1. Claims for the Contracting States : BE, CH, DE, FR, GB, IT, LI, LU, NL, SE Compounds of the formula I see diagramm : EP0167954,P12,F1 wherein a) R1 and R2 are identical and each represent the sugar combination Roa-Rod-Rod or Roa-dF=CinB, or b) R1 and R2 are different and each represent the sugar combination Roa-Rod-Rod and Roa-dF-Rod, or c) R1 and R2 are different, R1 representing the sugar combination Roa-dF-CinA and R2 representing the sugar combination Roa-Rod-Rod or Roa-Rod-Acu, or R1 representing the sugar combination Roa-Rod-Acu and R2 representing the sugar combination Roa-Rod-Rod and their physiologically acceptable acid addition salts. 1. Claims for the Contracting State : AT A process for the preparation of a compound of the formula I see diagramm : EP0167954,P12,F2 wherein a) R1 and R2 are identical and each represent the sugar combination Roa-Rod-Rod or Roa-dF=CinB, or b) R1 and R2 are different and each represent the sugar combination Roa-Rod-Rod and Roa-dF-Rod, or c) R1 and R2 are different, R1 representing the sugar combination Roa-dF-CinA and R2 representing the sugar combination Roa-Rod-Rod or Roa-Rod-Acu, or R1 representing the sugar combination Roa-Rod-Acu and R2 representing the sugar combination Roa-Rod-Rod, and their physiologically acceptable acid addition salts, which comprises fermenting the microorganism Streptomyces purpurascens (DSM 2658) in the presence of a nutrient medium with an elevated oxygen supply, otherwise in a customary manner under aerobic conditions, and isolating the compounds formed from the culture liquid and the mycelium by extraction with organic solvents followed by chromatography or partition in a customary manner.

Description

The invention relates to 1-hydroxy-cytorodines of formula I

in which means hydrogen or the radical OR^ and R2 means the radicals -OR4or. -COOR5, wherein R5 means C^-C-^-alkyl and R3 and R4 are the same or different and mean hydrogen or a sugar combination of the following composition: Roa-dF-Rod, Roa-dF-CinA, Roa-dF<=>CinB, Roa -Rod-Rod, Roa-dF-Acu, Roa-Rod-CinA, Roa-Rod-Acu.or Rod-Rod-Rod, where Roa means rhodosamine, dF means deoxyfucose, Rod means rhodinose, Acu means aculose, CinA means cinerulose A and Cin B mean cinerulose B and dF = Cin B means that the two sugar building blocks are linked by an extra ether bridge next to the usual glycosidic bond.

The invention further relates to a method for producing compounds of formula I, the method being characterized in that the strain Streptomyces purpurascens DSM 2658 is fermented at a pH value of 6.5 - 8.5, a temperature of 24-40°C and a high degree of aeration in a suitable nutrient and compound of formula I is isolated from the culture liquid of mycelium according to usual methods.

The isolation and identification of Streptomyces purpurascens DSM 2658 is described in the German patent application P 332 325.0. Microorganisms were deposited on 24.5.1983 at DSM (Deutsche Samm-lung von Mikroorganismen)" in D-3400 Gottingen (Grisebachstr.8).

The said patent application also mentions a method for producing 1-dehydroxyanthracycline derivatives of formula I, the so-called cytorhodins, by fermentation of the strain Streptomyces purpurascens-DSM 2658 under aerobic conditions.

It was now surprisingly found that by increasing the oxygen supply during the fermentation, 1-hydroxy-cytorodines are formed. These new, above all, cytostatically active compounds lead to the group of 1-hydroxy-anthracyclines, of which the following representatives have already been mentioned:

1-hydroxy-3-rhodomycins - also called 6-iso-rhodomycins:

e.g. violamycins A and B I - k (W. Fleck et al., Z Allgem. Mikrobiologie 14, 551 (1974) and g-isorodomycins (H. Brock-menn et al., Chem. Ber. 98, 3145 (1965).

e-pyrromycins (cinerubins): e.g. cinerubin A (W. Keller-Schierlein, ANtimicrob. Agents Chemother. 68 (1970)), various rhodirubins (H. Umezawa et al., J. Antib. 30, 616

(1977) and aclacinomycins A2, B2, M2, S2 and T2 (T. Oki et al., J. Antibiot. 28, 830 (1975) and 32, 801 (1979), as well as Musetta-, Marcello-, Rudolfomycin et al. (D.E. Nettleton et al., J. Nat. Prod. 43, 242 (1980)).

The process according to the invention for the preparation of compounds of the general formula I is carried out by fermentation of Streptomyces purpurascens-DSM 26 58 at a pH value of 6.5 - 8.5 and a temperature of 24-40°C under aerobic conditions a sufficiently high oxygen supply, e.g. at a high degree of aeration in a nutrient medium containing carbon

and nitrogen sources such as inorganic nutrient salts and trace elements and subsequent isolation of the compounds from the culture liquid and the mycelium according to usual methods as discussed below.

Suitable carbon sources are glucose, starch, dextrin and glycerol. Suitable nitrogen sources are soy flour, yeast extract, meat extract, malt extract, corn-swell water, peptone and casein. As inorganic nutrient salts, it is suitable, e.g. sodium chloride, .. magnesium sulphate or calcium carbonate. Iron, magnesium, copper, zinc and cobalt can be used as trace elements.

Streptomyces purpurascens - DSM 2658 can be fermented at temperatures of 24-40°C at a pH of 6.5 - 8.5 and an efficiency of 0.8 - 1.2 vvm. Preferably, the fermentation of Streptomyces purpurascens - DSM 2658 takes place under aerobic conditions at 30°C and at a pH value of 7.0. After 70 - 130, preferably 90 - 110 hours, when the highest yield has been achieved, the fermentation is interrupted. Preferably, the fermentation may involve a submerged fermentation.

The progress of the fermentation and the formation of the anthracycline compounds can be followed by means of the antibacterial action against S. aureus 209 P and Bae. subt., as well as directly by extracting the total culture solution (Mycel and culture filtrate) with an organic solvent and measuring the absorption intensity of the violet compounds at 495, 525 and 568 nm. Ethyl acetate is preferably used as organic solvents.

Anthracycline compounds in the culture filtrate and in the mycelium are isolated according to scheme and arrangement I (cf. also ex. 3).

The anthracycline compounds in the mycelium are extracted with an organic solvent, preferably with aqueous acetone, which was adjusted to a pH value of 3.5. After removal of the acetone, the pH value of the aqueous phase is adjusted to 7.5, and then extracted at a pH value of 7.5 in an organic solvent such as butyl acetate, methyl acetate or chloroform, preferably with ethyl acetate. The ethyl acetate extracts from the mycelium and the culture filtrate are combined or worked up separately, concentrated and taken up in an organic solvent such as benzene or toluene. Preferably, toluene is used. The toluene solutions are then extracted with an acetate buffer of a pH value of 3.5. At this stage, the mixture of anthracycline derivatives is divided into two fractions. The mixture remaining in the toluene phase is designated as fraction A, the glycoside mixture remaining in the aqueous phase is designated as fraction B.

Fraction B is further purified according to arrangement II. As shown

in arrangement II one obtains from fraction B 1-hydroxy-cytorodine A, B, C and P and the mixture N + 0 (1:1). Fractions I, II, III and IV refer to undivided mixtures of the anthracycline compounds of formula I.

Those from the culture solution of Streptomyces purpurascens - DSM 2658 isolated and according to the statement I purified compounds of the general formula I.

Arrangement I

Arrangement II

The preferred compounds according to the invention have the structure shown in formula II, with R 4 and R 1 having the following meanings:

Anthracycline compounds according to the invention are distinguished by a strong effect against gram-positive bacteria as well as a pronounced cytostatic effect.

The invention will be explained in more detail with the help of some examples.

Example 1

Production of 1-hydroxy-cytorodines by fermentation of Streptomyces purpurascens - DSM 2658.

Streptomyces purpurascens - DSM 2 658 was grown on yeast-malt agar of the following composition:

In addition, the medium was distributed on Roux bottles and sterilized for 30 minutes at 121°C, cooled, inoculated with spore suspension, and incubated for 7-14 days at 25°C. Subsequently, good growth and good spore formation could be determined. From this stock culture, a spore suspension was produced with 10 7 spores per ml, in sterile NaCl (0.8%-ig). 10 ml served as inoculum of 500 ml of the following pre-culture medium:

Each time 500 ml of the above-mentioned medium was distributed in 2000 ml Erlenmeyer flasks, sterilized for 30 minutes at 121°C.

The inoculated flasks were incubated for 2 days at 25°C and 200 rpm. minute on a rotary shaker.

500 ml of the growing pre-culture was used as inoculum for the following main culture:

9 liters of the above medium were filled in a 12 liter fermenter and sterilized for 50 minutes at 121°C. The fermentation was carried out at 28°C under a stirring speed of 60 rpm. minute and an aeration rate of 8-9 liters of air per minute.

The fermentation duration was 90 - 110 hours. To fix the time of harvest, the formation of an absorption maximum at 568 nm was followed. In addition, samples of the culture solution were extracted with ethyl acetate and the absorption spectrum recorded between 400 and 600 nm.

Example 2

Fermentation of Streptomyces purpurascens - DSM 2 6 58 on a larger scale.

According to example 1, Streptomyces purpurascens - DMS 2658 was grown in 200 ml Erlenmeyer flasks with 500 ml of pre-culture medium, and for 2 days inoculated into a 30 liter fermenter with 20 1 of pre-culture medium. Fermentation took place at 28°C with stirring at 160-180 rpm. minute with an aeration rate of 6-7 liters per minute. After 48 hours, this pre-culture serves as inoculum for 300 1 ferments with 200 1 main culture medium (Example 1). The fermentation took place for 90-110 hours at 28°C, a stirring peripheral speed of 3.5 m/sec. and an aeration rate of 1 vvm (200 ml/min). The harvesting criteria corresponded to those from example 1.

Example 3

Isolation of the crude mixture of anthracycline compounds..

190 liters of culture solution were mixed with 0.5% formalin and with acetic acid adjusted to pH 5.2. The mycelium was separated using the usual methods (solids separator, filter press) and washed several times with a little water.

The culture filtrate (approx. 185 litres) was combined as the wash water, adjusted with NaOH to pH 7.5-8.0, and extracted twice with 50 ml of ethyl acetate each time. The aqueous phase was discarded.

The organic phases were combined, evaporated in vacuo to 8 liters and then extracted 4 times with 3 liters of sodium acetate buffer solution (p.H 3.5). The combined buffer solutions were then washed 3 times with 3 liters of toluene.

The separated mycelium (approx. 3.4 kg) was stirred 3 times with 15 liters of acetone, the decanted acetone solution evaporated in vacuo, mixed with 5 liters of N-acetate buffer solution of pH 3.5, and washed 3 times with 2.5 liters of toluene.

The aqueous buffer phase of the culture filtrate preparation was adjusted to pH = 7.9 with NaOH, and extracted 3 times with 3 liters of ethyl acetate. The combined ethyl acetate extracts were evaporated in vacuo.

The aqueous buffer phase from the mycelium preparation was adjusted to pH 8.0 with NaOH, and extracted 3 times with 2.5 1 ethyl acetate each time. The combined organic extracts were evaporated in vacuo.

The remaining aqueous phase was discarded.

Example 4

Isolation of 1-hydroxy-cytorodin A by means of "Reversed phase" HPLC.

A strong violet charge of cytorhodin A from crude, polar cytorhodin mixture (complex II), recovers ordinary column chromatography on silica gel with acetic acid mobile phase, and subsequent further enrichment by preparative HPLC on SiO 2 was by means of preparative HPLC further divided into a " reversed phase" adsorbent. Thus, e.g. 250 mg dissolved in 4 ml of a mobile phase CHCl^-methanol-10% ammonium acetate in water, 150 : 1050 : 375 and placed on a steel column (3.2 x 25 cm), filled with approx. 120 g "LiChrosorb", RP-18, 25-40 y (Merck) under pressure chromatographed at a flow rate of 4 ml per minute.

The separation was monitored with two sequentially coupled flow-through photometers at wavelengths of 490 and 560 nm. After summarizing normal processing resp. of the red and violet fractions, the following results emerged:

About. 150 mg of 1-hydroxy-cytorodin A prepared in the above-mentioned manner was further purified by renewed preparative HPLC on RP-18, 25-40 u under the above-mentioned conditions. 60 mg of 1-hydroxy-cytorodin A was formed with less than 2% cytorodin A remaining.

<C>60H88<N>2°21' Molecular weight calculated 1172 (FAB-MS confirmed).

Example 5

Isolation of l-hydroxy-cytorodin V by preparative medium pressure and layer chromatography. 7 g of crude cytorhodin mixture recovered by extraction of the culture solution as. mentioned in statement I, is chromatographed on 120 g of silica gel 31 u (Grace) in two radially compressed Silica cartridges (Waters, "Prep LC/System 500") with the stirring agent mixture CHCl₂/methanol/96% acetic acid/water = 80 : 10 : 10 : 2. The sample was loaded into 65 ml of the mobile phase on the equilibrated column and divided at a flow rate of 21 ml/minute into fractions of 23 ml. The assessment took place by thin-layer chromatography and by means of analytical HPLC.

In fractions 29 - 34, 195 g of a mixture of cytorhodin appeared

V and l-hydroxy-cytorodine V at a purity of 80% (Rf 0.38

in system A).

60 mg of this sample was purified by preparative thin-layer chromatography (Merck, DC preformed plate silica gel 60).

Elution of the scraped bands took place with CHCl 2 /Methanol 1:1. The elution solution was neutralized with aqueous disodium hydrogen phosphate solution, mixed with water to separate the CHCl 2 phase. The chloroform phase was separated, washed once

with a little water, dried over anhydrous Na 2 SO 4 and evaporated. After absorption in a little chloroform, it was smeared with 10 times the volume amount of petroleum ether and the precipitate dried in a vacuum. Next to 12 mg of cytorodin C, 17 ml of 1-hydroxy-cytorodin V was formed.

V: identified by "<*>"H-NMR.

C60<H>84<N>2°20'<m>olecYlweight'1152 (FAB-MS. confirmed) .

<C>60<H>84<N>2°21' molecular weight calculated: 1168 (FAB-MS confirmed).

Example 6

Isolation of l-hydroxy-cytorodin B by means of "reversed phase" HPLC.

A violet Charge of cytorhodin B recovered by ordinary preparative column chromatography on silica gel (acetic acid mobile phase) was chromatographed under pressure on a steel column (1.6 x 25 cm) filled with 30 g 10 y "Lichrosorb" RP 18 (Merck). In addition, 120 mg were dissolved with 1 ml of the mobile phase CHCl3-MeOH-10% ammonium acetate in water, 150:1050:375 and injected. At a flow rate of 4 ml/minute, fractions of 4 ml were collected and assessed by measuring the extinction at 490 and 570 nm.

Cohesive factions were united and worked up as usual.

It was formed:

B: Identification by "4-I-NMR, absorption spectrum by FAB-MS.

<C>60<H>86<N>2°22</>Molecular weight, calculated 1186, (FAB-MS confirmed).

Example 7

Isolation of l-hydroxy-cytorodin C by "reversed phase" HPLC.

A by usual preparative column chromatography on silica gel recovered from crude violet cytorhodin C-containing product (25 mg) was chromatographed on a steel column (1.6 x 25 cm) filled with 30 g 10 y "Lichrosorb" RP-18 (Merck) with the mixture CHCl₂-methanol-10% ammonium acetate in water 150 : 1050 : 375 at a flow rate of 2 ml/min. Fractions of 2 ml were collected and pooled after assessment by measuring the extinctions at 490 and 570 nm. After normal processing, it was formed:

Fraction 80 - 142 10 mg cytorhodin G 1

15 9 - 175 7 mg 1-OH-cytorodine C

<C>60<H>80<N>2°23'<m>olecYlwt' calcd, 1196 (FAB-MS confirmed).

Example 8

Isolation of l-hydroxy-cytorodine N + 0 by means of "re-ver sed phase" - HPL.

By ordinary multi-step column chromatography on 31 μg silica gel, pH 7.5 and then on 10 μg "Lichrosorb" RP-18 (see example 4), a strong violet charge was formed from a product which behaves according to DC and analytical HPLV on silica gel with system B as cytorhodin N + 0 and which could be further divided by repeated preparative HPLC on "reversed phase" adsorbents

in a red and violet part.

In addition, e.g. 230 mg of the violet product dissolved in

4 ml of the mobile phase CHCl3-methanol-10% ammonium acetate in water 150:1050:375, and chromatographed on approx. 100 g 10 y Lichrosorb" RP-18 (Merck) in a steel column 3.2 x 25 cm under pressure at a flow of 4 ml/minute. Fractions of 8 ml were collected and summarized after assessment by analytical "reversed phase"- HPLC with dual wavelength detection at 490 and 560 nm. It was formed after normal work-up:

From two corresponding chromatographic runs, it was prepared

a total of 73 mg of 1-hydroxy-cytorodine N + 0 and rechromatographed in the above-mentioned manner. 18 mg of l-hydroxy-cytorodin N + 0 was formed with less than 2% residual cytorodin N + 0.

<C>60<H>88<N>2°22' molecular weight calculated 1188, (FAB-MS confirmed). The isolated product is a 1:1 mixture of the two structurally isometric components 1-OH-N + 1-OH-O. This is evident from the H-NMR spectrum, decomposition experiments (hydrogenolysis) and determination of the individual sugar building blocks after hydrogenolysis and total hydrolysis.

Example 9

Isolation of l-hydroxy-cytorodin P was "reversed phase"-HPLC.

By ordinary column chromatography on 31 u silica gel at pH 7.5,

a strong violet product was formed, whose main components by DC (system A) and analytical HPLC on silica gel showed a slightly higher retention than l-hydroxy-cytorodin C. By repeated preparative "reversed phase" HPLC the new compound could be isolated. In addition, e.g. 175 mg of the violet crude product dissolved in 2.5 ml of the mobile phase CHCl^-methanol 15% ammonium acetate in water 500 : 1100 : 300, and chromatographed on approx. 100 g 10 u Lichrosorb" RP-18 (Merck), in a steel column 3.2 x 25 cm under pressure at a flow of 4 ml/min. It was collected in fractions of 8 ml and contiguous fractions combined after assessment by means of analytical "reversed phase" HPLC with dual wavelength detection at 490 and 560 nm,

It was formed:

In several similar chromatographic runs, highly enriched 1-OH-cytorodin is produced. The charger was combined and chromatographed again under the above-mentioned conditions. Of approx. 80 mg, 20 mg of pure l-hydroxy-cytorodin P was formed.

<C>60<H>82<N>2°22' molecular weight calculated 1182 (FAB-MS confirmed).

Experimental conditions:

1. High pressure liquid chromatography (HPLC) on silica gel:

Steel columns (4.6 x 250 mm), packed with 7 y (Lichrosorb" Si 60 (Merck) or 7 y silica gel 60 (Grace), mobile phase: CHCl^-methanol-86% acetic acid-triethylamine-water 80 : 10 : 10 : 2 : 0.01 Flow: 0.5 - 1.5 ml/min detection at 26 0 or 490 nm with flow photometer.

2. "reversed phase" HPLC.

Steel columns (4.6 x 250 mm) packed with 10 y "Lichrosorb" RP-18

(Merck)

mobile phase: CHCl3-methanol-10% ammonium acetate in water 250 : 1050 : 375.

Flow: 0.5 - 1.5 ml/min, detection at 490 nm and 560 nm with a flow photometer.

The analytical preparative HPLC columns used were with the methods recommended by the company "Merck" (Darmstadt) themselves using a pneumatic pump (Haskel).

3. Preparative column chromatography.

For ordinary preparative column chromatography, 31 μg of silica gel 60 (Grace) or 15-40 μg of silica gel 60 (Merck) were used in open glass columns or steel columns packed under pressure. The solvent mixture of similar compositions as in the above-mentioned analytical HPLC system served as mobile phase, possibly with reduced amounts of methanol, acetic acid and water.

For chromatography of other strong polar components, 31 μg of silica gel 60 (Grace) was washed with 2 n metal-free HCL and, after washing, treated with acid in an aqueous suspension with 5 n NaOH until a pH of 7.5 had been reached. After decanting, the modified silica gel was dried at 130°C, sieved and adjusted with the mobile phase in the column. Solvent mixtures CHCl3/water/methanol 13:4:3-7 were used as mobile phase. The load ratio amounted to approx. 1 : 100 - 1 : 300.

4. Thin layer chromatography (DC).

Silica gel plates F254(<M>erck) were developed with system A: CHCl^-methanol-96% acetic acid-water-triethylamine 80 : 10 : 2 : 0.01 or system B: CHC 13Tmethanol-99% acetic acid 75 : 15 : 10 : 2.

5. Normal processing.

The usual work-up was carried out as follows: Combined fractions of "reversed phase" HPLC were mixed with approx. half its volume of water and added chloroform for phase separation. The separated chloroform phase was washed with water, dried over anhydrous Na 2 SO 4 and evaporated in water. The products thus formed were freed of accompanying fat, plasticizers and metal ions in the following way: The product,

e.g. 30 mg was dissolved in 20 ml of sodium acetate buffer, pH 3.5, mixed with 1 ml of 0.001 molar aqueous ethylenediaminetetraacetic acid (EDTA), adjusted with NaOH to pH 3.5), and shaken with 5 ml of toluene. The toluene phase was discarded, the aqueous phase adjusted with 2 N NaOH to pH 7.5, extracted with 20 ml of CHCl2 with good shaking. After drying over anhydrous Na 2 SO 4 , it was filtered and evaporated in vacuo. If necessary, the residue was dissolved in a little

CHCl^ and the solution was aspirated through a glass frit

and evaporated by addition of heptane to cloudiness in vacuo.

6. Spectroscopic investigations.

The identification of the components in the above examples took place under the following measurement conditions: The proton resonance spectra (<1>H-NMR spectra) were measured on a HX-270 Bruker Fourier-transform nuclear resonance spectrometer

"at 270 MHZ. The concentrations were 2-4 mg/0.5 ml

99.8% CDCl 2 , the solutions were shaken immediately after preparation with 0.1 ml of 5% Na 2 CO 3 in 99.5% D 2 O.

The signals marked with a star in the figures originate from low-molecular impurities in the %-0 range and from solvent residues.

The mass spectra were measured on mass spectrometers MS-902 S, AEI, using a FAB (solid atom bombardment) ion source. The substances were brought into a matrix of thioglycerol in the ion source partly with the addition of ammonium chloride.

The absorption spectra were measured in the range 200 - 700 nm:

a) water-methanol 1 : 9

b) 10% 1 n HCl in methanol

c) 10% 1 n NaOH in methanol.

The substance concentration was 10 - 30 mg/l, absorption maxima are given in nm and the molar extinction coefficient (log e).

7. Determination of cytotoxic activity.

The determination of the cytostatic effect of the compounds mentioned here took place on L1210 leukemia cells in mice. In detail, the following test systems were used:

a) Proliferation equipment.

This method determines in vitro after incubation of

the cells with different concentrations of the test substance, the extent to which the cells can absorb radioactive DNA precursors (e.g. C14 labeled thymidine). Untreated Ll210 cells are subjected to the same test conditions and serve as a control. In the following, the method is briefly described.

Ll210 cells in exponential growth phase (5.x 10 /ml in RPMI 1640) are incubated in a microtiter plate for 72 hours with different concentrations of the test substance (37°C, 5% C02,

95% relative humidity). The control consists of cells

which are only incubated with free medium. All provisions are carried out as four-time provisions. After 65 hours, 50 µl of C-1 14 thymidine (1.5 µc/ml) is added to mark the DNA of the cells radioactively. After 7 hours of incubation, the cells are aspirated, the DNA is separated with 5% trichloroacetic acid and washed in sequence with water or methanol.

After drying at 50°C, a DNA built-in radioactivity is determined after adding 5 ml of scintillation liquid.

The results are given as a ratio of scintillation index after incubation with the test substance as a percentage of untreated controls. From the measured values obtained in this way, the dose-response curve is established, and IC^q? is determined graphically. i.e. the concentration which, under the test conditions, lowers the incorporation of radioactive thymidine by 50% compared to the controls. The IC5q values of the compounds mentioned here compared to adriamycin (ADM) are summarized in table 1.

b) Colony formation of L1210 leukemia cells in soft agar.

This method serves to detect an impact of the test substances on the growth conditions of the cells over several generations (with a cell cycle time of 10-12 hours, approximately 14 consecutive generations are observed in the test period of 7 days).

In this sample, cytostatically active substances cause a reduction in the number of colonies thus observed compared to an untreated control. In detail, the test is carried out as follows: 500 leukemia cells per plate is incubated with different concentrations of test substance for 1 hour at approx. 30°C. Then the cells are washed twice with McC.oy5A medium and finally "poured out into Petri dishes after the addition of 0.3% agar. The controls are only incubated with free medium. Instead of 1 hour incubation, in many cases different concentrations and test substance of upper agar layer in order to calculate a continuous exposure of the cells over the total incubation time. After the agar's effect, the plates are incubated in an incubator for 7 days at 37°C (5% C02, 95% relative humidity). The number of colonies formed with a diameter of 60 y. The results are given as colony numbers in treated agar plates as a percentage of untreated controls. From the thus formed dose-effect curve, ^Cj-q is determined as a measure of the effect of the substance. The results of the compounds mentioned here compared to adriamycin are summarized in table 1.

As stated above, the compounds according to the invention have a cytostatic effect, i.e. a therapeutic effect against tumors, especially malignant tumors in animals and humans.

The compounds and acid addition salts can consequently be used as drugs for the treatment of tumors. The compounds can be administered in different ways, depending on the dosage form. Normally, compounds are administered mixed with pharmaceutically common carriers or diluents. Thus, it can e.g. administered singly or in mixture together with carriers such as maltose or lactose, or as non-toxic complexes, e.g. as deoxyribonucleic acid complex.

A typical type of administration is injection of a solution of the compounds according to the invention in distilled water or in physiological saline solution. The solutions can be injected intraperitoneally, intravenously or intraarterially.

Daily dose and unit dose can be determined by animal experiments and recording in vitro samples so that the total dose can be administered continuously or at intervals that do not exceed a predetermined range. Thus, the total dose for a treatment cycle amounts to approx. 0.5-5 mg/kg body weight. This dose can be administered in equivalent fractions over a period of 7 days. It is clear, however, that specific doses for the treatment of humans or animals can be determined individually depending on the eventual situation, e.g. age, body weight, sex, sensitivity, nutrition, administration, time, additional drugs administered, the patient's physical condition against the severity of the disease.

Claims (15)

1. Compounds of formula I where means hydrogen or the residue OR^ and R2 means the residues -0R4 or -COOR5, where C5 means C-^-C^ alkyl and R3 and R4 are the same or different and mean hydrogen or a sugar combination of the following composition: Roa-dF-Rod, Roa-dF-CinA, Roa-dF=CinB, Roa-Rod-Rod, Roa-dF-Acu, Roa-Rod-CinA, Roa-Rod-Acu or Rod-Rod-Rod, in which Roa means rhodosamine, dF means deoxyfucose, Rod means rhodinose, Acu means aculose, CinA means cinerulose A and CinB cinerulose B, and dF = Vin B means that the two sugar building blocks are joined by an extra ether bridge next to the usual glycosidic bond, as well as their physiologically tolerable acid addition salts. 2. Compound with formula I according to claim 1, in which R 1 means the residue -OR 3 , and R 2 means the residue OR 4 , wherein R 3 and R 4 are the same or different and mean one of the aforementioned sugar combinations. 3. Compound according to claim 2, wherein R3 means the sugar combination Roa-Rod-Rod, and R4 the sugar combination Roa-Rod-Rod, as well as their physiologically tolerable acid addition salts. 4. Compound according to claim 2, wherein R3 means sugar combinations -Roa-Rod-Rod, and R4 means the sugar combination Roa-dF-Cin A, as well as their physiological acid addition salts. 5. Compound according to claim 2, wherein R3 and R4 mean sugar combinations Roa-dF=C inB, as well as their physiologically tolerable acid addition salts. 6. Compound according to claim 2, wherein R3 means sugar combinations Roa-dF-Rod, and R4 means sugar combinations Roa-Rod-Rod, as well as their physiologically tolerable acid addition salts. 7. Compound according to claim 2, wherein R 1 means sugar combinations Roa-Rod-Rod, and R 4 means sugar combinations Roa-dF-Rod, as well as their physiologically tolerable acid addition salts. 8. Compound according to claim 2, wherein R 1 means the sugar combination Roa-Rod-Rod, and R 2 means the sugar combination Rod-Rod- Acu, as well as their physiologically tolerable acid addition salts. 9. Compound according to claim 2, wherein R^ means the sugar combination Roa-Rod-Acu, and R^ means the sugar combination Roa-dF-Cin A, as well as their physiologically tolerable acid addition salts. 10. Process for the preparation of a compound of formula I according to claim 1, characterized in that microorganisms Streptomyces purpurascens (DSM 2658) are fermented by elevated oxygen supply in an otherwise usual manner under aerobic conditions in the presence of a nutrient medium and the compounds formed are isolated in the usual manner from the culture liquid and the mycelium by extraction with organic solvents and subsequent chromatography or partitioning. 11. Method according to claim 9, characterized in that microorganisms Streptomyces purpurascens DSM 2658 are fermented in a nutrient medium containing carbon and nitrogen sources as well as inorganic nutrient salts and trace elements at temperatures of 24-40°C and a pH of 6.5-8.5 in formed compounds are extracted from the culture liquid with ethyl acetate and chloroform and from the mycelium firstly with aqueous acetone from the separated aqueous phase, then with ethyl acetate, the combined ethyl acetate extracts are concentrated and taken up with benzene or toluene, the formed solution is extracted with an acetate buffer at a pH value of 3.5, and from the solution formed the compounds of formula I are isolated by chromatography or distribution. 12. Method according to claim 10, characterized in that the chromatographic isolation of the compounds of formula I takes place by means of silica gel columns, "reversed phase" adsorbents, drop-counterflow chromatography or distribution on subsequent thin-layer or high-pressure liquid chromatography. 13. Process for the production of medicinal products, characterized in that a compound of formula I according to claim 1 is brought into a therapeutically suitable administration form, optionally with conventional pharmaceutical carriers and/or stabilizers. 14. Medicinal product, characterized by a content of a compound of formula I according to claim 1. 15. Compound of formula I according to claim 1 for use as a medicine.