DE3404470A1 - Anthracyclinone glucuronides, process for their preparation and pharmaceuticals containing them - Google Patents

Abstract

An anthracyclinone glucuronide of the formula <IMAGE> in which R is H, lower alkyl or an alkali metal is described. This compound is prepared by cultivation of Streptomyces peucetius var. vinaceus (DSM 2597), a mutant strain of Streptomyces peucetius var. aureus, under aerobic conditions in an aqueous culture medium which contains assimilable sources of carbon and nitrogen as well as mineral salts, and subsequent workup by processes known per se. The anthracyclinone glucuronide has antitumour properties.

Description

Description

The invention relates to an anthracyclinone glucuronide and its salts and esters, biosynthetic processes for their production and medicaments containing them. It has been found that anthracyclinone glucuronide, hereinafter referred to as FCE 22724, is effective to inhibit the growth of malignant tumors such as P 388 leukemia in test animals. The structural formula of FCE 22724 has been determined and can be represented as follows The invention further relates to a biosynthetic process for the production of FCE 22724. The process is characterized in that Streptomyces peuceti-Us var. Vinaceus (DSM 2597) is cultivated under aerobic conditions in an aqueous culture medium containing assimilable sources of carbon and nitrogen and mineral salts . The microorganism Streptomyces peucetius var. Vinaceus is a mutant strain of Streptomyces peucetius var. Aureus (ATCC 31428). The invention further relates to a method for obtaining FCE 22724 from the fermentation broths, the concentration from crude solutions and the purification. The invention also relates to the new anthracyclinone glucuronide FCE 22724 in the form of the raw concentrates that are obtained in the biosynthetic processes, in pure form, as free acid or alkali salts, isolated from the raw concentrates and in the form of pharmaceutical preparations in which FCE 22724 or its salts or its esters mixed with pharmaceutically acceptable diluents and / or carriers are included.

The microorganism The one used in the method of the invention Microorganism was induced by a reverse mutation after the mutagenic treatment with UV rays from Streptomyces peucetius var. aureus strain of 416 F.I.

(ATCC 31428; DSM 1367; F.R.I.4622) as obtained from Cassinelli et al. (J. Antib., Vol. 35, No. 2, pp. 176-183, 1982). The so obtained, new strain was with the code number M83 FCE from the Farmitalia Carlo Erba collection of Microorganisms and at the Deutsche Sammlung for Microorganisms deposited under the number DSM 2597.

The new microorganism differs from the stock culture and of all other mutants obtained from Streptomyces peucetius mainly in that it forms a pigment of the substrate mycelium, which in young cultures is pink to red to wine-like on all solid media and deep purple in color as it ages will. This change in tone is also observed in the excessively present, soluble pigment, which is found on all solid media normally used for the Classification of Streptomycetes used forms. Such media include Bennet's Agar, Czapek's Agar, Asparagine-Glucose-Agar, Glycine-Glycerin- Agar, Potato glucose agar, asparagine-glycerin agar (Waksman S.A .: "The Actinomycetes". Volume II, 1961, William Wilkins & Co. Baltimore) and inorganic salts-starch-agar (T.G. Pridham, P.Anderson, C.Foley, L.A. Lindenfelser, CoA Hesseltine and ROG.Benedict: A selection of media for maintenance and taxonomic studies of Streptomyces. Antibiotics Ann. 1956/1957, 947-953).

It is therefore believed that the strain M83 FCE is a variant of Streptomyces peucetius is called Streptomyces peucetius var. Vinaceus is given. The microorganism Streptomyces peucetius var.

vinaceus is also a subject of the present invention.

Fermentation process Production takes place according to the usual, an well known methods and comprises culturing the microorganism in one previously sterilized, liquid culture medium under aerobic conditions and a temperature in the range from 25 to 370 (preferably at 28 ° C) over a period of time ranging from 3 varies to 7 days (preferably from 5 days), and at a pH that is initially in the range of 6.5 to 7.0 and towards the end of the fermentation process in the range is from 6.5 to 8.0.

The culture medium contains carbon and nitrogen sources as well Mineral salts. Suitable carbon sources are starch, dextrin, glucose, glycerin, Mannitol, maltose, corn steep liquor9 or other vinasse produced during distillation Products, soybean oil and soybean meal. Suitable nitrogen sources are those such as the above carbon sources containing nitrogen, dry yeast, meat peptone or casein. Quality Results are obtained by using ammonium salts, such as ammonium nitrate, ammonium sulfate and diammonium phosphate are used. The mineral salts, which are useful for production can according to the medium used vary. In media containing different substances, such as different flours and fermentation residues included is the addition of calcium carbonate and sodium or potassium phosphates useful. Media containing glucose or ammonium salts have higher contents of mineral salts, such as potassium, sodium or calcium salts, and small additions Iron, zinc, copper, magnesium and manganese salts required. The fermentation can be placed in conical flasks or in laboratory or industrial fermentation devices be carried out with different capacities.

Analytical procedures will take samples of the fermentation broths and raw Mixtures of thin layer chromatography (TLC) on precoated with silica gel Plates using a mixture of chloroform-methanol-acetic acid-water (80/20/7/3, by volume) as an eluent, one finds FCE 22724 with an Rf mean value of 0.22 as the red colored main component. A quantitative one Determination of FCE 22724 which is present in the fermentation broths can be performed using the following procedure. Add to a sample of the broth add 2 Vol.Acetonitrile and the resulting mixture is 1 min at room temperature sonicated and then filtered.

When a sample of the filtrate is subjected to silica gel TLC analysis, using the above elution system, a spectrophotometric Determination of FCE 22724 at 493 nm can be performed by scratch off the corresponding red-colored zone and with a mixture of chloroform Ethanol (9/1 by volume) eluted.

Isolation Procedure After fermentation is complete, it becomes FCE 22724 found mainly in the myeella, which emerged from the fermentation fluids separated by filtration at pH 7.5 with the aid of diatomaceous earth. The mycelium cake is mixed with water, organic solvent such as acetonitrile, Dioxane, methanol and other lower alcohol, extracted; methanol is preferred used. The mycelium extracts are collected and concentrated under reduced pressure and the aqueous concentrate contains FCE 22724 in the form of an alkali salt. FCE 22724 can not except the filter broth by means of extraction at pH 4 with a with water miscible organic solvents such as methyl isobutyl ketone, methyl propyl ketone and n-butanol, are recovered; Methyl isobutyl ketone is preferred.

From the organic extracts, FCE 22724 can be used as a pH 7 in water Sodium salt re-extracted with aqueous sodium hydroxide or sodium hydrogen carbonate will.

FCE 22724 can be extracted from the filtered solids and aqueous concentrates on hydrophobic, polymeric, crosslinked divinylbenzene resins, such as Amberlite XAD 2 or XAD 4 and ER 180 (Rohm and Haas Co. * Inc.), are absorbed; preferred is ER 180 used. The absorbed FCE 22724 is easily mixed with aqueous lower alcohols, preferably 10% by volume n-propanol in water, eluted.

Reinizunzsverfahren Raw, aqueous concentrates, whose pH value through Addition of sodium chloride (5%) was adjusted to 7, be passed through a column made of ER 180 resin. After washing with water, it is eluted with 10 n-propanol by volume in water and the eluate is collected in fractions. the end the selected fractions, which are checked by silica gel TLC, are obtained after concentrating in vacuo and freeze drying, raw FCE 22724 in the form its sodium salt. To obtain pure FCE 22724, the pH is an aqueous Solution of the crude sodium salt adjusted to 4 and then with methyl isobutyl ketone extracted. The organic extract gives after concentration in vacuo to one low volume, precipitation with n-hexane and crystallization from methyl isobutyl ketone pure FCE 22724 as a free acid in the form of fine, red, crystalline needles. the Treatment of the alcoholic solution of pure FCE 22724 (I) with one equivalent of alcoholic sodium hydroxide and subsequent concentration and precipitation with diethyl ether gives the corresponding, pure sodium salt (II). The ones with acid catalyzed esterification of FCE 22724 in methanol yields the corresponding methyl ester (III).

Chemical and physical properties FCE 22724 as a free acid (I) is in alkaline, aqueous media, lower alcohols and polar, organic Solvents soluble in acetone, ethyl acetate, diethyl ether, methylene dichloride and chloroform somewhat soluble and hardly soluble in water, benzene and n-hexane. FCE 22724 as the sodium salt (II) is in water, lower alcohols and polar, organic solvents and soluble in other organic solvents something soluble or insoluble. FCE 22724 methyl ester (III) is in lower alcohols, Acetone, ethyl acetate, methylene dichloride and chloroform soluble, a little in diethyl ether soluble, but insoluble in water and n-hexane. FCE 22724 has the following chemical and physical properties: Melting point: 168 to 1690C (dec.) UV and VIS absorption spectrum: in methanol: # max 233, 252, 288, 360, 474, 493 t525 Sch.) nm (E1cm1% 490, 495, 144, 47, 184, 184, 100) in 0.01N methanolic Sodium hydroxide: A max 233, 252, 290 (350 Sch.), 540 and 580 nm in M / 15 phosphate buffer pH 7: h max 232, 252, 288, 352, 474, 488 nm (E1cm1% 562, 526, 175, 55, 193, 187).

IR spectrum (KBr): peaks at the following frequencies: 3420, 2920, 1730, 1620, 1585, 1430, 1405, 1330, 1245, 1210, 1175, 1120, 1060, 940, 910, 870, 830 and 8N cm Molecular Formula: C28H28O5, M.W. 604, 53, based on the elemental analysis and 13 C-NMR spectrum 1 H-NMR spectrum (CDCl3 + 1 drop DMSO-d6) at 200 MHz: 0.99 (t, J = 7.3 Hz, 3H, CH3CH2); 1.40, 1.66 (two m, 2H, CH3CH2); 1.9-2.3 (m, 2H, H-8); 3.56 (s, 3H, COOCH3); 3.5-3.8 (mD 3H, H-2 ', H-3', H-4 '); 3.94 (d, J = 9Hz, IH, H-5 '); 4.14 (s, 1H, H-10); 4.85 (d, J = 7Hz, 1H, H-1 '); 5.14 (br.s, 1H, H-7); 7.65 (d, J = 4.4Hz, 2H, H-1, H-3); 8.00 (t, J = 4.4Hz, 1H, H-2); 13.30 and 13.60 # (two s, 2H, OH-6, OH-11) 13C-NMR spectrum (CDCl3 / CD30H 1: 1, vol.) At 50 MHz: 6.08 (C-14); 32.38 (C-13); 3442 (C-8); 51.52 (C-10); 51.89 (COOCH3); 61.64 (C-4 '); 71.22 (C-7); 71.56 (C-9); 72-89 (C-2 '); 7514 (C-3 ', 5'); ; 101.50 (C-1 '); 122.03 (C-1); 124.44 (C-3); 133.09 (C-12a); 134.92 (C-6a); 135.81 (C-2); 138.16 (C-10a); 155.47 (C-11); 156.24 (C-6); 158.36 (C-4); 170.61 (C5, -COOCH3); 171.52 (C10, -COOCH3); 186.07 (C-5) and 187.13 # (C-12).

Structure determination The structure assigned to FCE 22724 and corresponding to 4-0- (ß-D-glucuronyl) - £ -rhodomycinone (I), 0 OH COOCH3 m 5 = 3c u e, m 3su 1 acr to B "B 0 3: R = H II 4 \ -8: R II = Na III R = CH 3 acidic or °> ° n @ 0 <A0> COOJ! CH2Cli2 HO 1 '+ O 0 O is determined as follows: Aqueous, acidic hydrolysis gives # -rhodomycinone (IV) as insoluble, purple-colored aglycone and D-glucuronic acid (V), both of which were identified by direct comparison with authentic samples. The incubation of the FCE-22724 sodium salt with different glucuronidases enzymatically confirms the presence of a part of the molecule of D-glucuronic acid (V), which is bound to # -rhodomycinone (IV). The position and the ß-configuration of the glycosidic bond in FCE 22724 was assigned based on the UV-VIS, IR, 1H and 13C-RER spectra.

Rhodomycinone (IV) and its 7-0-glucosides are known ingredients different anthracycline complexes by different species of genus Streptomyces such as pyrromycin (Chem. Ber. 92, 1904, 1959), cinerubin A and B (U.S. Pat 3,864,480), musettamycin and marcellomycin from bohemic acid complex (US Pat. No. 4,039,736), can be obtained. FCE 22724 (I) is the first biosynthetic, anthracycline-like Anti-tumor compound that contains a ß-D-glucuronyl group in its molecule, the bound to the phenolic hydroxyl group in the 4-position of the & -rhodomycinone is.

The following examples illustrate the invention.

B e i s »i e l 1 A culture of Streptomyces peucetius var. Vinaceus, Strain M83 FCE is grown on agar slanting plates of the medium SA (glucose 3%; Brewer's dry yeast 1.2%; NaCl 0.1%; KH2P04 0.5%; CaCO 3 0.1%; MgSO4 0.005%; FeS04.7H20 0.0005%; ZnS04.7H20 0.0005%; CuS04.5H20 0.0005%; Agar 2%; tap water up to 100 ml; pH 6.7; the sterilization is carried out by heating grown in an autoclave at 115 ° C for 20 min).

The mycelium fragments scraped from the agar culture are in 3 ml of sterile, distilled water and collected using a vortex homogenizer The suspension obtained in this way is used for inoculating 300 ml Erlenmeyer flasks, which contain 60 ml of the following liquid growth medium is used: dry Brewer's yeast 0.3%; Peptone 0.5%; Ca (N03) 2.

4H20 005%; Tap water up to 100 ml; the sterilization takes place by heating in an autoclave at 1200C for 20 minutes; the pH value is after Sterilization between 6.8 and 7.0. The inoculated flasks are kept for 2 days at a Temperature of 28 ° C on a rotary shaker operating at 250 rpm and describes a circle 7 cm in diameter, shaken. 1.5 ml of the like The grown cultures described above are used to inoculate 300 ml Erlenmyer flasks used containing the 50 ml of the following production medium: glucose 6%; dry brewer's yeast 2.5%; Soybean meal 1%; NaCl 2X; KH2P04 0.1%; ZnSO4.7H2O 0.001%; CuSO4.5H2O 0.001%; Tap water up to 100 ml; pH 67; the sterilization takes place by heating in an autoclave at 1150 ° C. for 20 minutes 7 days at 280C under the same conditions as described above for the vaccination phase, incubated.

The maximum concentration of FCE 22724 was between the 6th and Reached the 7th day of fermentation with a production of 50 mcg / ml.

Example 2 The entire fermentation process was carried out according to Example 1, the only difference being the use of the following production medium consisted of: glycerin 6%; dry brewer's yeast 2.5%; Soybean meal 1%; NaCl 2%; KH2PO4 0.1%; CaCO 3 0.2%; MgS04 0.01%; FeS04.7H20 0.001% ZnS04.7H20 0.001%; CuS04.5H20 0.001%; Tap water up to 100 ml; pH 6.7; the sterilization was done by heating in an autoclave at 1150C for 20 min.

The maximum concentration of FCE 22724 was between the 6th and Reached the 7th day of fermentation with a production of 100 mcg / ml.

B e i s n ie l 3 The culture of the strain M83 FCE was carried out according to the example 1.

The mycelium fragments from the three slants were collected and collected in 10 ml of sterile, distilled water. The suspension was after implementation the homogenization according to example 1 for inoculating 2 1 round bottom flasks with baffles, which contained 500 ml of the germination medium described in Example 1 was used. the Flasks were rotated for 48 hours on a rotary shaker operating at 120 rpm and describes a circle with a diameter of 7 cm at a temperature of 280C incubated. All inoculum was used to inoculate an 80 liter stainless Steel fermentation vessel containing 50 l of the inoculation medium described in Example 1 contains, used and sterilized by steam pressure for 30 min at 1200C. The vaccination phase he follows at 280C with stirring at 230 rpm and an air flow from 0 * 5 1/1 medium / min. After 30 hours, this inoculum was used to inoculate 500 l of the production medium described in Example 2, stainless 800 1 fermentation tanks used. It was under steam pressure for 30 min Sterilized at 120 ° C.

The fermentation took place at 28 ° C with stirring at 250 rpm and Aeration with an air flow of 0.7 1/1 medium / min. The maximum concentration of the FCE 22724 was produced between the 6th and 7th day of fermentation with a production of 70 mcg / ml is achieved.

Example 4 All of the broth (3 1) from fermentation obtained according to Example 1, was at a pH of 7 using 3% diatomaceous earth filtered as a filter aid. The wet filter cake was extracted with methanol (3 liters). After filtering, two additional extractions (with 3 1 or 2 1 methanol) to ensure that all of the red pigments have been recovered. The combined extracts are concentrated to 300 ml under reduced pressure. The pH of the filtered broth obtained as described above was adjusted with sulfuric acid adjusted to 4 and then extracted with methyl isobutyl ketone (3 1). To two additional extractions the organic extracts were combined, concentrated and extracted again with 3% aqueous sodium hydrogen carbonate. To the watery one Layer that was combined with the aqueous concentrate of the mycelial fume extract, gave sodium chloride (5%) and the whole was on a column (3.2 x 27 cm) which filled with resin Er 180 (200 ml) was absorbed.

First was washed with water, which was discarded, and then the elution took place with 10 vol-, 'n-propanol in water. The selected factions, found by TLC1 was concentrated under reduced pressure and then freeze-dried to give crude FCE 22724 as the sodium salt (1 g).

Example 5 The pH of an aqueous solution (200 ml) of crude FCE 22724 as the sodium salt (1 g), obtained according to Example 4, is treated with sulfuric acid 4 and then extracted with methyl isobutyl ketone. The organic Extracts are concentrated under reduced pressure and almost pure FCE 22724 (0.3 g) was precipitated with n-hexane. Crystallization from methyl isobutyl ketone gave pure, red needles of FCE 22724 (0.25 g) as the free acid; M.p. 1680C (dec.).

Example 6 A methanolic solution (20 ml) of FCE 22724 in the form of the free acid (0.12 g), obtained according to Example 5, was with one equivalent (0.2 mmol) methanolic sodium hydroxide. After the concentration and Precipitation with diethyl ether gives pure FCE 22724 in the form of its sodium salt (0.11 g); M.p. 190 to 1910C (dec.).

EXAMPLE 7 A solution of FCE 22724 (0.1 g) in anhydrous Methanol (100 ml) is treated with 1N methanolic hydrochloric acid (1 ml). After 2 h at room temperature the reaction mixture is washed with ethyl acetate (100 ml) and concentrated to 50 ml under reduced pressure. After this Washing with aqueous sodium hydrogen carbonate and water becomes the organic phase dried over anhydrous sodium sulfate and concentrated to low volume. Addition of n-hexane gives the pure methyl ester of FCE 22724 (0.09 g) as red, microcrystalline powder; M.p. 150 to 1520C; UV and VIS spectrum: N MeOH 223, 252, 288, 474, 493 (525 sh.) Nm (E1cm1% 505, 500, 147, 48, 178, 178, 96).

Its molecular formula C29H30O15, which is based on elemental analysis, was confirmed by field desorption mass spectrometry (m / e 618) and the assigned Structure (III) was confirmed by IR and NMR spectroscopy: 1 NMR spectrum (CDCl3) at 200 MHz: 1.12 (t, J ~ 7.2 Hz, 3H, CH3CH2); 1.12, 1.52 (two m, 2H, CH2CH3); 2.0-2.4 (m, 2H H-8), 3.69 (s, 3H, C10-COOCH3); 3.82 (s, 3H, C5 * COOCH3); 3.8-4.0 (m, 3H, H-3 ', H-4t, H-2'); 4.20 (s, 1H, H-10), 4.28 (d, J = 8Hz, 1H, H-5 '); 5.06 (br.s, 1H, H-1 '), 5.19 (br.s, 1H, H-7); 7.2-7.6 (m, 3H, H-1, H-2, H-3); 13.21, 13.47 # (two s, 2H, OH-6, OH-11).

Example 8 A solution of FCE 22724 (0.1 g) in dioxane (2 ml) and 1% aqueous sulfuric acid (10 ml) is heated at 1000 ° C. for 2 h. The one colored red The reaction mixture containing precipitate is diluted with water (20 ml) and diluted with Extracted ethyl acetate. The extract is made with 3% sodium hydrogen carbonate and Washed with water and dried over anhydrous sodium sulfate and reduced dertem Pressure concentrated in a small volume. The addition of an excess of n-hexane gives a red, crystalline compound (0.045 g; m.p. 2100C) which is known as ε-rhodomycinone (IV) identified after comparison with an authentic sample. on the other hand that present in the water-soluble fraction of the above hydrolyzate became acidic Constituent by silica gel TLC as D-glucuronic acid (V) after comparison with a authentic sample identified.

Example 9 A solution of FCE 22724 (0.05 g) in M / 15 phosphate buffer at pH 7 (50 ml) is mixed with an aqueous solution (50 ml) of B-D-glucuronidase (50 g of "Bacterial type VII" Enzyme-Sigma) and incubated at 37 ° C. for 1 h.

@ -Rhodomycinone (IV; 0.022 g), while in the aqueous phase the presence of D-glucuronic acid (V) again has been confirmed.

Biological activity FCE 22724 shows cytotoxic activity in the HeLa cell cloning efficacy in vitro; it is 40 / ug / ml as the mean concentration, which inhibits 50% of the growing cells (IDSo).

FCE 22724 also shows antitumor activity in vivo in P 388 leukemia in mice, as shown in Table 1.

Table 1 in vivo effect of FCE 22724 on P-388 murine leukemia a) Dose T / Cb) Deathsc) mg / kg% 4.4 135 0/10 6.6 160 0/10 15.0 165 0/10 225 175 1/10 a) treatment i.p. one day after tumor inoculation (106 cells / mouse) b) mean survival time; % compared to untreated comparison animals (c) evaluation based on the autopsy examinations of the dead mice.

Claims (8)

Anthracyclinonglucuronide, process for their preparation and medicaments containing them Patent claims 1. Anthracyclinonglucuronide with anti-tumor effect of the general formula A. wherein R represents a hydrogen atom, a lower alkyl group or an alkali metal atom. 2. Process for the preparation of an anthracyclinone glucuronide of the general Formula A, in which R denotes a sodium atom, characterized in that microorganisms of the strain Streptomyces peucetius var. vinaceus (DSM 2597) under aerobic conditions in an assimilable Sources of aqueous culture medium containing carbon and nitrogen and mineral salts breeds. 3. The method according to claim 2, characterized in that the cultivation for 3 to 7 days at a temperature of 25 to 37 ° G and a pH value of at the beginning in the range from 6.5 to 7.0 and towards the end of the cultivation in the range from 6.5 to 8.0. 40 The method according to claim 2 or 3, characterized in that one the mycelium removed from the culture medium, the mycelium with a water-miscible, organic solvent extracted and the extracts concentrated under reduced pressure, the crude Anthracyclinonglcuronid of the general formula A is obtained, wherein R represents a sodium atom. 5. Process for the purification of the crude anthracyclinone glucuronide of general formula A, wherein R denotes a sodium atom, which according to one of the claims 2 to 4 has been obtained, characterized in that a concentrated, aqueous solution of the crude compound on a hydrophobic, crosslinked, polymeric Divinylbenzene resin absorbed, eluted with an aqueous lower alcohol, the pH value of the eluate for the release of the anthracyclinone glucuronide of the general Formula A, in which R is a hydrogen atom, adjusts to 4.0, the Eluate extracted with methyl isobutyl ketone, the anthracyclinone glucuronate in pure Form precipitates by adding n-hexane and turning it into the pure sodium salt by treatment converted again with one equivalent of methanolic sodium hydroxide and then precipitates with diethyl ether. 6. The method according to claim 5, characterized in that the aqueous, lower alcohol 10% by volume n-propanol in water is used. 7. Pharmaceutical preparation, characterized in that it is a therapeutically effective amount of a compound of claim 1 admixed with one pharmaceutically acceptable diluents and / or carriers. 8. The microorganism Streptomyces peucetius var. vinaceus (DSM 2597).