AU613965B2 - New anthracycline derivatives, a process for the preparation thereof, and the use thereof as pharmaceuticals - Google Patents

Description

0-4F 4 COMMONWEALTH OF: AUSTRAL%~ b PATENTS ACT 1952-69 COMPLETE

SPECIFICATION

(ORIGINAL)

Class I nt. Class Application Number: Lodged: CdLbite Specification Lodged: Accepted: Published: PjT olrt Rolate3:Art Name of Applicant Adcres of Applicant: Actual Inventor: Address for Service HOECHST AKTIENGESELLSCHAFT 45 Bruningstrasse, D-6230 Frankfurt/Main Federal Republic of Germany HANS GERD BERSCHEID and HANS-WOLFRAM FEHLHABER EDWD. WATERS SONS, 50 QUEEN STREET, MELBOURNE, AUSTRAL.1A, J3000.

Complete Specification for the invention entitled: NEW ANTHRACYCLINE DERIVATIVES, A PROCESS FOR THE PREPARATION THEREOF, AND THE USE THEREOF AS PHARMACEUTICALS The following statement is a full description of this invention, including the best method of performing it known to d HOECHST AKTIENGESELLSCHAFT Description HOE 87/F 303 Dr.LA/je New anthracycline derivatives, a process for the preparation thereof, and the use thereof as pharmaceuticals The present invention relates to anthracycline derivatives of the formula I

,CH

2

CH

3 3u S 5 00

-C,

in which R 1 and R 2 are different and represent the sugar residue Roa (L-rhodosamine) of the formula

CH

3

CH

3 or the sugar combination -Roa-dF=D-Cin B (Roa: L-rhodosamine; dF 2-deoxy-L-fucose; 0-Cin B: D-cineruLose B) of the formula III

CH

3 CHg CH3

CH

3 as well as the pharmaceutically acceptable acid addition salts thereof.

The invention also relates to a process for the preparation of the compounds of the formula I, which comprises i 2 2 epimerization of the compounds of the formula I which can be obtained as specified in EP-A1 0,131,942 and in which

R

1 and R 2 are different and represent Roa (L-rhodosamine) or the sugar combination Roa-dF L-Cin B (Roa L-rhodosamine; dF 2-deoxy-L-fucose; L-Cin B: L-cineruLose This can be achieved by either a) direct treatment with suitable basic reagents of the said compounds which can be obtained as specified in EP-A1 0,131,942, or b) chromatography of the crude mixture which is obtained as specified in EP-A1 0,131,181 by cultivation of the microorganism strain Y-11,472 (DSM 2658), extraction of the mycelium and of the culture filtrate with an organic solvent, and treatment of the residue with a dilute S strong or moderately strong acid as specified in EP-A1 135 0,131,942, on silica gel which has been adjusted to a pH of 6.5-8.0.

S In the process mentioned under the starting compounds ("cytorhodin S" or "cytorhodin are treated with solvent mixtures which contain inorganic bases such as alkali metal hydroxides, for example NaOH, or basic ammonium salts such as, for example, ammonium acetate, or organic bases such as, for example, triethylamine, or tetraethyLammonium hydroxide, singly or else in a mixture. It is 2~ possible for the compounds according to the invention to be separated from one another and purified by customary chromatographic methods, for example column chromatography on silica gel which has been adjusted to a pH of 6.5-8.0, preferably In the process variant mentioned under the hydrolysis product obtained by acid treatment as specified in EP-A1 0,131,942 is chromatographed with solvent mixtures containing no acid component, preferably on silica gel which has been washed with acid and adjusted to a pH of 6.5-8.0, preferably 7.0-7.5, with an alkali metal hydroxide solution, for example sodium hydroxide solution, there being isolation of the compounds according to the invention.

3- An example of a compound obtained by the process according to the invention is the following: '-Epi-cytorhodin S: Red amorphous substance, readily soluble in methanol, ethyl acetate, chloroform and toluene, insoluble in water and hexane.

Thin-layer chromatography: Merck 60F 2 5 4 silica gel System: chloroform/methanol/glacial acetic acid/water 70/20/10/2, RF 0.54 UV maxima: 235, 255 290, 495 nm in methanol/10 1N HCL H NMR spectrum: Fig. 1 S C 4 8

H

6 4

N

2 0 17 M calc. 940 (confirmed by FAB-MS) The 5'''-epi-cytorhodin S has the formula I in which R 1 denotes Roa and R 2 denotes Roa-dF=D-Cin B.

The compounds of the formula I prepared in this way are 'istinguished oy high cytostatic activity and low toxicity, especially nephrotoxicity, and can therefore be used for the treatment of oncoses, for example for the therapy of malignant tumors.

The cytostatic activity of the compounds according to the invention was determined in the cell proliferation test on three different tumor cell lines, as described hereinafter: L1210, A 549 or HT 29 tumor cells in the exponential phase of growth are incubated in RPMI 1640 at a cell density of 5x10 3 cells/ml in a 96-well microtiter plate for 72 hours (370C, 5 C0 2 95 relative humidity) with various concentrations of the test substance. Cells incubated with fresh medium in place of the test substance are used as controls. Quadruplicate determinations are performed for each substance concentration and for the 4 controls each time. After 65 hours, 50 pL of a MTT solution (MTT: 3-(4,5-dimethylthiazolyl)-2,5-diphenyLtetrazoLium bromide) in a concentration of 2.5 mg/mL (dissolved in PBS) are added. MTT is reduced by Live ceLLs to a red insoluble formazan. After incubation with MTT for 7-24 hours (depending on the nature of the cell used) the supernatant medium is carefully removed by aspiration. The initially insoluble formazan dyestuff is dissolved by adding 100 iL of DMSO to each well, followed by cautious shaking. The extinction of the resulting colorea solution at a wavelength of 492 nm is determined using a Flow 340 CC multiscan photometer.

The results are reported as the ratio of the extinctions after incubation with test substance relative to the untreated control. The coefficient of variation for repeat determinations is less than 15 The following values were determined for 5" '-epi-cytorhodin S, for example: (pg/ml) L 1210 HT 29 A 549 8x10 3 2.1x10 3 3.4x10 3 The compounds and the acid addition salts thereof can therefore be used as medicaments for the treatment of tumors. The compounds can be administered in a variety of ways depending on the dosage form.

Normally, the compounds or the acid addition salts thereof, for example with D-gluconic acid, are administered mixed with pharmaceutically customary vehicles or diluents.

Thus, for example, they can be administered singly or mixed together with vehicles such as maltose or lactose or as non-toxic complexes, for example as deoxyribonucleic acid complex.

A typical mode of administration is injection of a solution of the compound according to the invention in distilled I I_ ~_C 5 water or in physiological saline. The solutions can be injected intraperitoneally, intravenously or intraarterially.

The daily dose and single dose can be established from animal experiments and from in vitro tests in such a way that the total dose which is administered continuously or at intervals does not exceed a previously defined range. Thus, the total dose for a treatment cycle is about 0.5-5 mg/kg of body weight. This dose can be administered in appropriate fractions over a period of 7 days. However, it is clear that specific doses for the treatment of human or animal can be defined individually as a function of the particular situation of the patient, for example age, body weight, sex, sensitivity, diet, time of administration, other medicaments administered, physical condition of the patient and severity of the disease.

The preparation of the compounds according to the invention is described in the examples which follow, with the components being identified under the measurement conditions described hereinafter: The proton resonance spectra H NMR spectra) were recorded at 270 MHz in a BRUKER HX-270 Fourier transform nuclear magnetic resonance spectrometer. The concentrations were 2-4 mg/0.5 ml of 99.8 CDCL 3 immediately after preparation, the solutions were shaken with 0.1 ml of 5 Na 2

CO

3 in 99.5 D 2 0.

The signals provided with an asterisk in the figures derive from low molecular weight impurities in the 0.1% range and from solvent residues.

The mass spectra were recorded in a AEI MS-902 S mass spectrometer using a FAB (fast atom bombardment) ion source. The substances were introduced in a matrix of thioglycerol into the ion source, in some cases with the addition of ammonium chloride.

6 The absorption spectra were recorded in the range 200-700 nm in: 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; the absorption maxima are reported in nm.

Example 1: Hydrolysis 750 g of crude basic glycoside mixture from a fermentation with Streptomyces purpurascens DSM 2658 (in accordance with EP-A 0,131,942) were dissolved in 4 L of 0.1 N HCL at 200C with stirring, and the pH was adjusted to 1.1 with 2 N HCL. After 16 hours, the reaction solution was adjusted to pH 1.8 with 5 N NaOH, and extracted by shaking 3 times with 6 L of toluene each time; thereafter the pH was successiveLy adjusted to pH 3, 4, 5 and 7 with 2 N NaOH and, at each of these pH values, the solution was extracted 5 times with 2 1 of CHCL 3 each time. The pH extract provided after evaporation in vacuo a residue of 81.6 g.

Example 2: Chromatography of the hydrolysis product on basic silica gel g of the hydrolysis product described in Example 1 were dissolved in 50 mL of a mixture of CHCL 3 /methanoL/ water 130/40/50 (Lower phase) and applied to a steeL column (70 x 6 cm, packed with 1.1 kg of silica gel of pH 8.0/20-45 p, 60 A, from Grace) and chromatographed with the above mixture at a flow rate of 15 mL/min under pressure. Fractions of 15 mL were coLLected, assessed by TLC (see above) and combined in a suitable manner.

The following yields were obtained after evaporation in vacuo: v a c 7 Fraction g TLC 1-59 1.9 60-80 81-100 2.4 Mainly 5 ''-epi-cytorhodin S and cytorhodin S 101-145 1.7 Cytorhodin S 146-250 3.8 to the end 1.2 with 7 L of methanoL Example 3: Chromatography on reversed phase materiaL 2 g of a mixture of cytorhodin S and 5' '-epi-cytorhodin S were dissolved in 90 ml of a solution of 0.5 (v/v) triethylamine in water (adjusted to pH 3.0 with H 3 P0 4 and applied to a steel column (250 x 32 mm, packed under pressure with 110 g of reversed phase Lichrosorb RP-18, p, (Merck)) and chromatographed with 2 L (then 3 L) of the mobile phases 10 (then 7) triethyLamine in water (pH 3.0 with H 3 PO4)/acetonitrile 84/16 under pressure. At a flow rate of 10 ml/min, and after a forerun of 1.5 L, fractions of 10 ml were collected. After 2 N NaOH had been added to pH 7.5, extraction with CHCL3 was carried out, and the fractions were tested by TLC and combined in a suitable manner. The following products were obtained after evaporation in vacuo: Fraction mg TL.C 1-59 40 Polar products 60-160 375 Mainly epi-cytorhodin S 161-304 860 Mainly cytorhodin S 305-320 460 Violet mixture, Rf similar to cytorhodin S Repetition of the chromatography with the product from fraction 60-160 and the mobile phase containing 7 (v/v) triethylamine in the manner described resulted in a total of 97 mg of 5'''-epi-cytorhodin S with a purity of more than 85 according to the 1H NMR spectrum.

Claims (8)

1. A compound of the formula I in which R 1 and R 2 are different and represent the sugar residue Roa (L-rhodosamine) of the formula OH CH3 C °CH 3 or the sugar combination Roa-dF=D-Cin B (Roa: L-rhodosamine; dF 2-deoxy-L-fucose; D-Cin B: D-cinerulose B) of the formula III CH 3 11 CH 3 0 acceptable acid addition as well as the pharmaceutically salts thereof.

2. A compound of the formula I as claimed in claim 1, in which RI represents the sugar residue Roa, and R2 represents the sugar combination Roa-dF=D-Cin B, as well as the pharmaceutically acceptable acid addition salts thereof.

3. A compound of the formula I as claimed in claim 1, in rl jrC h J't p p aid ~I II I 9 which R 1 represents the sugar combination Roa-dF=D-C in B, and R 2 represents the sugar residue Roa.

4. A process for the preparation of a compound as claimed in claim 1, which comprises epimerization of a compound of the formula 1 in which R 1 and R 2 are different and denote the sugar residue Roa or the sugar combination Roa-dF=C in B, where C in B represents L-cinerulose B. The process as claimed in claim 4, wherein either a) the said starting materials are directly treated with basic reagents, or b) the mixture which is obtained as specified in EP-A1 0,131,181 by cultivation of the micro-organism Y-11,472 (DSM 2658), extraction of the mycelium and of the culture filtrate with an organic solvent, and subsequent greatment of the resulting residue with a dilute strong or moderately strong acid as specified in EP-A1 0,131,942, is chromatographed on silica get which has been adjusted to a pH of 6.5-8.0.

6. The process as claimed in claim 5, wherein the basic reagents are alkali metal hydroxides, basic ammonium salts or organic bases.

7. The process as claimed in claim 5, wherein the silica gel is adjusted to a pH of 7 to with sodium hydroxide solution.

8. Method of treatment of oncoses in a patient comprising administrating an effective amount of the compound of claim 1 to said patient.

9. A method of preparation of a pharmaceutical having cytostatic activity comprising admixing in pharmacologically acceptable quantities the compound of claim 1 and pharmaceutically acceptable carriers or excipients. DATED this 16th day of May 1991. HOECHST AKTIENGESELLSCHAFT WATERMARK PATENT TRADEMARK ATTORNEYS 2ND FLOOR, "THE ATRIUM" 290 BURWOOD ROAD ~HAWTHORN VIC 3122 x^ v^,