GB2131793A

GB2131793A - A daunorubicin derivative

Info

Publication number: GB2131793A
Application number: GB08235165A
Authority: GB
Inventors: Giuseppe Cassinelli; Sergio Merli; Segio Penco
Original assignee: Farmitalia Carlo Erba SRL
Current assignee: Pfizer Italia SRL
Priority date: 1982-12-09
Filing date: 1982-12-09
Publication date: 1984-06-27
Also published as: GB2131793B

Abstract

The microbial stereoselective reduction of 4-demethoxy- daunorubicin to one of the stereo- isomers of 4-demethoxy-13-dihydro- daunorubicin is described. The claims relate to the stereoisomer, its preparation, pharmaceutical preparations containing it and the microorganism used in the preparation.

Description

SPECIFICATION A daunorubicin derivative The invention relates to a stereoisomer of 4-demethoxy-1 3-dihydrodaunorubicin, to its preparation by microbial stereoselective reduction of 4-demethoxydaunorubicin, to pharmaceutical compositions containing it the stereoisomer and to the microorganism.

A non-stereoselective reduction of the 1 3-keto group of 4-demethoxy-daunorubicin to give a mixture of the two C-13 stereoisomeric alcohols is described in our British Patent Application No.

8211692. We have now found that certain micro-organisms, in particular Corynebacterium mediolanum (ATCC 14004), Mycobacterium fortuitum (NRRL B-8119) and a novel mutant of the species Streptomyces peucetius, designated strain M 87 F.l., deposited at the Deutsche Sammulung für Microorganismen where it is registered under the accession number DSM 2444, are able stereoselectively to reduce the 1 3 keto group of 4-demethoxy-daunorubicin (a known compound, see United States Patent Specification No. 4046878) and accumulate the new stereoisomer of 4 demethoxy-13-dihydro-daunorubicin (hereinafter FCE 22723) in fermentation broths.

The invention provides the compound FCE 22723, being a stereoisomer of 4-demethoxy-1 3- dihydro-daunorubicin and having the chemical and physical properties described herein, and pharmaceutically acceptable salts thereof.

The invention further provides a method for the preparation of compound FCE 22723, the method comprising the microbial stereoselective reduction of 4-demethoxy-daunorubicin carried out by cultivating Streptomyces peucetius subs. aureus strain M 87 F.l. (DSM 2444), Mycobacterium fortuitum (NRRL 8-8119) or Corynebacterium mediolanum (ATCC 14004) under aerobic conditions in an aqueous culture medium containing an assimilable source of carbon and an assimilable source of nitrogen in the presence of 4-demethoxy-daunorubicin, and recovering the resultant FCE 22723 from the culture medium.

The microorganism Streptomyces peucetius subs. aureus strain M 87 F.l. (DSM 2444) as characterised herein is also within the scope of the invention.

The invention also provides a pharmaceutical composition comprising the compound FCE 22723 or a pharmaceutically acceptable salt thereof in admixture with a pharmaceutically acceptable diluent or carrier.

The microorganisms Mutants which are characterized by their ability stereoselectively to reduce the 13-keto group of 4-demethoxy-daunorubicin and accumulate the new compound FCE 22723 in the fermentation beer can be obtained by mutating different genera of bacteria including Streptomyces.

Mutation of S. peucetius subs. aureus (ATCC 31428), using nitrosoguanidine, has resulted in the production of a novel mutant, which selectively transforms 4-demethoxy-daunorubicin into FCE 22723. This mutant microorganism M 87 F.l. of S. peucetius subs. aureus has been given the accession number DSM 2444 by the Deutsche Sammlung für Microorganism, West Germany, where it has been deposited in the permanent collection.

The morphology of mutant M 87 F.l. is indistinguishable from that of the parent S. peucetius (ATCC 31428), whereas both cultures are clearly distinguishable in their cultural and biochemical characters. In fact mutant M 87 F.l. does not produce on agar media the straw yellow to lemon yellow soluble pigment which characterizes its parent S. peucetius (ATCC 31428).

Moreover mutant M 87 F.l. can selectively transform 4-demethoxy-daunorubicin into FCE 22723 whereas the parent S. peucetius (ATCC 31428) is not selective in this respect. This property of mutant M 87 F.l. makes it highly useful, as disclosed herein.

The stereoselective transformation can also be brought about by Corynebacterium mediolanum (ATCC 14004) and by Mycobacterium fortuitum (NRRL B-81 19).

The transformation process The stereoselective transformation of the invention can be effected in a growing culture of S.

peucetius M 87 F.l. by adding 4-demethoxy-daunorubicin as substrate to the culture during the incubation period. The 4-demethoxy-daunorubicin can be added, as its hydrochloride, after dissolution in sterile distilled water. The preferred, but not limiting, range of concentration of 4-demethoxydaunorubicin in the culture is about 100-400 micrograms per litre. The culture is grown in a nutrient medium containing an assimilable carbon source, for example a carbohydrate, and an assimilable nitrogen source, for example a proteinaceous material. Preferred carbon sources include glucose, sucrose, glycerol, starch, cornstarch, dextrin, molasses and the like.Preferred nitrogen sources include corn steep liquor, yeast extract, brewer's dry yeast, soy bean meal, cotton seed meal, corn meal, casein, fish meal, distiller's solids, animal peptone, meat extract, ammonium salts and the like. Combinations of these carbon and nitrogen sources can be used advantageously. Trace metals, for example, zinc, magnesium, manganese, cobalt, iron and the like, need not necessarily be added to the fermentation media, since tap water and unpurified ingredients are used as components of the medium prior to sterilization of the medium.

The transformation process may take place over a period of from about 72 hours to 8 days. The incubation temperature during the transformation process may be from about 250C to about 370C, with 290C being preferred. The contents of the transformation vessels are aerated by shaking at about 250 r.p.m. or by agitating with sterilized air, to facilitate growth of the microorganism, and thus enhance the effectiveness of the transformation process.

Analytical methods The progress of the microbial transformation reaction may be monitored by withdrawing samples of fermentations at various time intervals, and extracting at pH 8.2 with 9:1 by volume dichloromethane:methanol mixture. When a sample of the organic extract is subjected to thin layer chromatography (TLC), using as eluent a mixture of chloroform:methanol:acetic acid:water 80:20:7:3 (by volume), compound FCE 22723 is found to occur at Rf medium value of 0.25, while 4-demethoxydaunorubicin is found at Rf 0.35. A quantitative estimation of the two anthracyclines may be performed after TLC using the above mentioned eluting systems by scraping off and eluting with methanol the corresponding red coloured zones and finally spectrophotometric determination at 482 nm.

Isolation procedure The whole fermentation broth, in which 4-demethoxy-daunorubicin has been subjected to conversion into FCE 22723, is filtered over diatomaceous earth. The orange-red mycelial cake is extracted with a water miscible organic solvent, such as methanol or another lower alcohols, dioxan, acetonitrile or acetone. Methanol is preferred. The mycelial extracts are collected, concentrated under reduced pressure, combined with the filtered fermentation liquors, adjusted to pH 8.2 and then extracted with a water-immiscible organic solvent such as n-butanol, chloroform, dichloromethane or, preferably, a dichloromethane:methanol 9:1 by volume mixture. The organic extracts contain FCE 22723 along with 4-demethoxy-daunorubicin and some minor degradation products.

Purification procedure The organic extract istconcentrated under reduced pressure to dryness and the residue, dissolved in dichloromethane, is chromatographed on a column of silica gel, buffered at pH 7, with a gradient of dichloromethane:methanol:water mixture. 4-Demethoxy-daunorubicin is eluted first with a 89.5:10:0.5 (by volume) mixture, followed by FCE 22723 with a 30:5.5:0.6 (by volume) mixture. From the pooled fractions, after washing with water, concentration to a small volume in the presence of npropanol, addition of an equivalent of hydrochloric acid and of an excess of diethyl ether, a precipitate of pure FCE 22723, as its hydrochloride, is obtained.

Chemical and physical properties FCE 22723 as free base is soluble in polar organic solvents and aqueous alcohols, while its hydrochloride is soluble in water and lower alcohols but insoluble in organic solvents. The hydrochloride of FCE 22723 has the following physicochemical properties: melting point: 1 69- 1700C (with decomposition) specific rotation: []D3w+ 177 (c=0.05, methanol) U.V. and visible absorption spectrum Arnnetnanof 252, 286, 482, 514 nm.

(E:=730, 150, 188 and 1 15).

I.R. Spectrum (KBr): peaks at the following frequencies: 3400, 2970, 2930, 1 620, 1 585, 1 500, 1475,1430,1410,1375,1340,1310,1280,1230,1200,1110,1080,i065,1000,980,930, 910,890, 870,850, 820, 785, 765, 730, 700,450 and and 430 cm-'.

13C-NMR Spectrum: (D2O) at 50 MHz: 16.7 (C-14, C-6') 28.7 (C-2'), 32.8 (C-10). 34.2 (C-8), 47.8 (C-3'), 67.2 (C-5'), 68.0 (C-4'), 70.7 (C-7), 72.6 (C-13), 73.4 (C-9), 100.1 (C-i'), 110.5 and 111.3 (C-5a,C-1 la), 127.3 and 127.5 (C-i,C-4), 132.8 and 132.9 (C-4a, C-12a) 135.1 (C-lOa), 136.2 and 136.4 (C-2, C-3), 138.0 (C-6a), 156.1 and 157.1 (C-6, C-11),186.5 and 186.8 a (C-5, C12).

Molecular formula: C26H2909N. HCI m/z in FD equivalent to the free base 499 (M+).

A selective high pressure liquid chromatography (HPLC) method allows the separation of the two C-1 3 stereoisomeric alcohols, present in a sample of synthetic 4-demethoxy-1 3-dihydro-daunorubicin, prepared as described in our British Patent Application No. 8211 692. The stereoisomers display two peaks with retention times of 32.7 and 34 minutes.

Using the same HPLC method, FCE 22723 appears as a single peak with a retention time of 34 minutes corresponding to that of the slower moving constituent of synthetic 4-demethoxy-1 3-dihydrodaunorubicin.

*HPLC Method Column: Lichrosorb RP 18 (5 m) Hibar Merck.

Mobile phase A: aqueous solution of KH2PO4 (10 g/l), made to pH 2.6 with citric acid 0.5 M/CH3CN=90/10 (by vol.) Mobiie phase B: CH3CN Elution: isocratic for 40 minutes (80%A+20%B), there for 20 minutes 40%A+60%B.

Flow rate: 0.6 ml/min.

Detection: 254 nm.

Biological activity The antiproliferative activity of FCE 22723 was tested in vitro on HeLa cells colony formation. As reported in Table I, FCE 22723 resulted 3 times more potent than daunorubicin and as potent as 4demethoxy-daunorubicin.

The antitumour activity of FCE 22723 was tested in vivo against ascitic P388 leukemia. CDF-1 mice were transplanted intraperitoneally with 106 cells/mouse; the treatment with compounds under study was carried out 24 hours after. Table II shows the results of two experiments. At the optimal dose tested, FCE 22723, was more potent (N10 times) than daunorubicin and as potent as 4demethoxy-daunorubicin. The antitumour activity of FCE 22723, evaluated as increase of life span, was equal to that of daunorubicin and the 4-demethoxy-daunorubicin at their optimal doses.

Table 1 Colony-forming ability of cultured HeLa cells after treatment with FCE 22723 Inhibition of Compound Dose (ng/ml) colonies { /OJ /Dso (ng/ml) Daunorubicin 25 98 12.5 53 i2 6.2 3 4-Demethoxy- 12.5 100 daunorubicin 6.2 96 N3 3.1 50 1.5 16 FCE 22723 12.5 100 6.2 59 3.1 55 N4 1.5 28 'HeLa cells were exposed to the drugs for 24 hrs.

Table 2 Antitumour activity of FCE 22723 against P388 ascitic leukemia' Compound Dose(mg/kg) MST (T/CO%) Toxic deaths Daunorubicin 2.9 160,163 0/20 4.4 140,165 8/20 6.6 118,155 10/20 4-Demethoxy- 0.33 145,150 0/20 daunorubicin 0.5 1 50,1 60 1/20 0.75 122,165 9/20 FCE 22723 0.14 136 0/10 0.22 140 0/10 0.33 140,154 0/20 0.5 145,150 4/20 0.75 113,165 7/20 1Treatment, i.p., on day 1 2Median survival time expressed as percentage of untreated controls; data from 2 experiments.

The following Examples illustrate the invention.

Example 1 A culture of Streptomyces peucetius, strain M 87 F.l. (DSM 2444), was grown for 14 days at 28 C on agar slants of the maintenance medium SA. Medium SA is glucose, 3%; brewer's dry yeast, 1.2%; NaCI, 0.1%; KH2P04, 0.05%; CaCO3, 0.1%; MgSO4,0.005%; FeSO4. 7H2O, 0.0005%; ZnS047H20, 0.0005%; CuSO4. 5H20, 0.0005%; agar, 2%; tap water up to 100 ml; pH 6.7; sterilization is carried out by heating in an autoclave at 11 50C for 20 minutes.

The spores of the resultant culture were collected and suspended in 3 ml of sterile distilled water.

The suspension was inoculated in 300 ml Erlenmeyerflasks containing 60 ml of the following liquid growth medium: brewer's dry yeast, 0.3%; peptone, 0.5%; Ca(NO3)2. 4H20, 0.05%; tap water up to 100 ml; sterilization by heating in autoclave at 1200C for 20 minutes; pH after sterilization 6.8-7.0.

The inoculated flasks were shaken for 2 days at a temperature of 280C on a rotary shaker running at 250 r.p.m. and describing a circle of 7 cm diameter. 1.5 ml of the culture grown as described above were inoculated in 300 ml Erlenmeyerflasks containing 50 ml of the following transformation medium; yeast extract,1.5%; KH2PO4, 0.25%; glucose, 1.5%; tap water up to 100 ml; pH 6.9; sterilization by heating in autoclave at it 500 for 20 minutes. The glucose solution was sterilized separately and added to each sterilized flask at the proper concentration.

The flasks were thus incubated at 280C under the conditions described for the seed phase, for 24 hours. At this time 1.25 ml of a solution of 4-demethoxy-daunorubicin in sterile distilled water at a concentration of 10 mg/ml was added to each flask. The shaken flasks were incubated for a further 2 days to obtain an 85% conversion of 4-demethoxy-daunorubicin into FCE 22723.

Examples 2 A culture of S. peucetius strain M 87 r.l. was grown on solid medium as described in Example 1.

The spores of three slants were pooled and collected in 10 ml of sterile distilled water; the suspension so obtained was inoculated in a 2 litre baffled round-bottomed flask containing 500 ml of the seed medium described in Example 1. The flask was incubated for 48 hours on a rotary shaker running at 120 r.p.m. and describing a circle of 7 cm diameter at a temperature of 280 C. The whole seed was inoculated in a 10 litre stainless-steel fermenter containing 7.5 litres of the biotransformation medium described in Example 1 and sterilized by vapour at 1 200C for 30 minutes, the glucose solution being sterilized separately and added at the proper concentration to the sterilized fermenter. The culture was allowed to grow at 280C under stirring at 230 r.p.m. and aerated with an air flow of 0.7 litre/litre of the medium/minute.After 48 hours the 4-demethoxy-daunorubicin substrate was added at the concentration described in Example 1, and the culture was incubated for a further 3 days. There was obtained a 60% conversion of 4-demethoxy-daunorubicin into FCE 22723.

Example 3 A culture of Mycobacterium fortuitum (NRRL B 8119) was grown for 5 days at 300C on the following medium: NaCI, 0.8%; peptone, 0.8%; meat extract, 0.3; dried yeast, 0.3%; agar, 2%; distilled water up to 100 ml; sterilization in an autoclave at 1 1 OOC for 20 minutes. The cell suspension so obtained was inoculated in the biotransformation medium described in Example 1 and allowed to grow for 3 days at 300C in 300 ml Erlenmeyerflasks containing 50 ml of medium under shaking at 220 r.p.m.

The substrate 4-demethoxy-daunorubicin was then added at the concentration described in Example 1, and the culture was incubated under the same conditions for a further 3 days to obtain a 60% conversion of 4-demethoxy-daunorubicin into FCE 22723.

Example 4 A culture of Corynebacterium mediolanum (ATCC 14004) was grown for 5 days at 300C on the following medium: peptone, 0.8%; NaCI,0.8%; dried yeast,0.6%; agar, 2%; distilled water up to 100 ml; sterilization in autoclave at 11000 for 20 minutes. The cell suspension so obtained was inoculated in the biotransformation medium described in Example 1 and allowed to grow for 3 days at 300C in 300 ml Erlenmeyer flasks containing 50 ml of medium, under shaking at 220 r.p.m. The 4-demethoxydaunorubicin substrate was then added at the concentration described in Example 1, and the culture was incubated under the same conditions for a further 3 days to obtain a 32% conversion of 4demethoxy-daunorubicin into FCE 22723.

Example 5 The whole beer (5 litres) from a fermentation according to Example 2 was filtered using 2% diatomaceous earth as filter aid. The wet filter cake was extracted with methanol (3 litres). After filtration two additional extractions with methanol were effected to ensure a complete recovery of the orange red pigments. The combined methanolic extracts were concentrated under reduced pressure and the concentrate (1 litre) was combined with the filtered broth and exhaustively extracted at pH 8.2 with a dichloromethane:methanol 9:1 by volume mixture. The organic extract, containing 4demethoxy-daunorubicin and FCE 22723 with minor amounts of degradation products, was concentrated under reduced pressure to dryness. The residue, dissolved in dichloromethane, was chromatographed on a column of silica gel buffered at pH 7 with a gradient of dichloromethane:methanol:water mixture. 4-Demethoxy-daunorubicin was eluted first with a 89.5:10:0.5 by volume mixture followed by FCE 22723 with a 30:5.5:0.6 by volume mixture.

From the pooled fractions, after washing with water, concentration to a small volume in the presence of n-propanol, addition of an equivalent of hydrochloric acid and of an excess of diethyl ether, pure FCE 22723 (0.72 g, 60%), as the hydrochloride (m.p. 1 69--1700C, with decomposition) was obtained. Following the same procedure untransformed 4-demethoxy-daunorubicin (0.48 g, 40%), as the hydrochloride was also recovered.

Example 6 The whole beer (500 ml), from a fermentation obtained according to Example 3, was extracted and the extract, submitted to column chromatography following the procedure reported in Example 5, gave pure FCE 22723 (0.07 g) and 4-demethoxy-daunorubicin (0.05 g) as the hydrochlorides.

Example 7 The whole beer (500 ml), from a fermentation obtained according to Example 4, was processed as described in Example 5 to give pure FCE 22723 (0.035 g) and 4-demethoxy-daunorubicin (0.075 g), as the hydrochlorides.

Claims

1. The compound identified herein as FCE 22723, being a stereoisomer of 4-demethoxy-i 3- dihydro-daunorubioin and having the chemical and physical properties described herein, or a pharmaceutically acceptable salt thereof.

2. A method for the preparation of the compound identified herein as FCE 22723, the method comprising the microbial stereoselective reduction of 4-demethoxy-daunorubicin carried out by cultivating Streptomyces peucetius subs. aureus strain M 87 F.l. (DSM 2444), Mycobacterium fortuitum (NRRL B-81 19) or Corynebacterium mediolanum (ATCC 14004) under aerobic conditions in an aqueous culture medium containing an assimilable source of carbon and an assimilable source of nitrogen in the presence of 4-demethoxy-daunorubicin, and recovering the resultant FCE 22723 from the culture medium.

3. A process according to claim 2 in which the cultivation is carried out for from 3 to 8 days at a temperature of from 250otto 370C.

4. A process according to claim 2 or claim 3 in which the mycelium is removed from the culture medium by filtration through diatomaceous earth and the crude FCE 22723 is solvent extracted from the mycelium and/or the filtered broths.

5. A process according to claim 4 in which the crude FCE 22723 is purified by chromatography on silica gel and isolated as its hydrochloride.

6. A process for the preparation of the compound identified herein as FCE 22723, the process being substantially as described herein with reference to Example 1, Examples 2 and 5, Examples 3 and 6 or Examples 4 and 7.

7. A pharmaceutical composition comprising the compound FCE 22723 as claimed in claim 1 or a pharmaceutical!y acceptable salt thereof in admixture with a pharmaceutically acceptable diluent or carrier.

8. The microorganism Streptomycespeucetiussubs. aureus strain M 87 F.l. (DSM 2444) as characterised herein.