Synthesis, DNA interactions and activation of novel cytotoxic anthraquinones.
Mitoxantrone is a dihydroxyanthracenedione derivative with significant clinical activity against advanced breast cancer, lymphoma and several types of leukaemia. However, as with all conventional anticancer agents, its non-specific nature results in dose-limiting systemic toxicity. The pharmacological (and toxicological) effects of mitoxantrone are thought to be mediated through its interaction with DNA and the DNA processing enzyme, topoisomerase II. Hence, an investigation of the DNA binding properties of mitoxantrone and related bis-substituted alkylaminoanthraquinones was undertaken with a view to developing agents with reduced toxicity to normal cells. DNA intercalation was evaluated using spectrophotometric and DNA thermal denaturation (Tm) techniques with calf thymus DNA. Overall, compounds that were hydroxylated at the 5 and 8 positions of the anthraquinone chromophore had increased DNA binding affinity compared to their non-hydroxylated analogues. The ~ Tms of mitoxantrone and its non-hydroxylated analogue ametantrone, were 26.4 °C and 21.5°C respectively. The affinity constants (K) for the chromophore-hydroxylated compounds were between 3.94 and 4.95 xl06M-1 while K values for their non-hydroxylated analogues were between 1.63 and 3.25 xl06M-l. For the di-N-oxide, AQ4N, intercalation was not detectable. The mono-N-oxide, AQ6N, showed modest DNA binding activity, with a ~Tm of7.0°C and an affinity constant of 3.64 Xl06M-l. Several of the alkylaminoanthraquinones were further investigated for their ability to inhibit decatenation of kDNA by topoisomerase II. Mitoxantrone and its analogues, AQ4 and AQ6, inhibited decatenation at concentrations ofO.75IlM, 1.51lM and 1.0llM respectively. In contrast, total inhibition of decatenation by the N-oxides, AQ4N and AQ6N, required concentrations of 50llM and 1 OIlM respectively. Hence modification of the terminal nitrogen on both alkylamino side chains to form a di-N-oxide resulted in a large decrease in DNA binding affinity and topoisomerase II inhibition. In view of the importance of the cationic alkyl amino side chains in intercalative binding and the enhanced ability of mitoxantrone to inhibit topoisomerase II, a series of acetalanthraquinones were synthesized. These compounds, referred to as YCG7 (1- substituted), YCG8 (l,4-bis-substituted), and YCG9 (l,5-bis-substituted), possessed dimethoxy groups in place of the alcohol groups of mitoxantrone, and were designed to be converted to their respective aldehydes, which should increase their cytotoxic activity due to their potential to form Schiffs bases with intracellular targets. The acetalanthraquinones were relatively poor DNA intercalators compared to the parent compound, ametantrone. The ~Tm values for YCG7, YCG8 and YCG9 were 3.6 °C, 14.2 °C and 15.7 °C respectively. The bathochromic shifts for the acetalanthraquinones in the presence of calf thymus DNA and 0.5M NaCl/0.008M Tris at a DNA:drug ratio of 10:1 were 1.4nm, 3.5nm and 3.6nm respectively for YCG7, YCG8 and YCG9 (ametantrone=12.2nm). Oxidative metabolism by NADPH-fortified mouse liver micro somes provided an effective route for the oxidation of acetalanthraquinones, producing two polar metabolites for YCG7 and three polar metabolites for both YCG8 and YCG9. The metabolites ofYCG7 and YCG8 were considerably more cytotoxic against the V79 Chinese hamster lung cell line than their respective acetalanthraquinones (4.4-fold for both compounds).YCG9 was found to be relatively cytotoxic per se, possibly due to its different mode of DNA binding, involving 'straddling' of the DNA helix. The increase in cytotoxicity of the metabolized products compared with the acetalanthraquinones supports the concept t?at they were conv.e~ed to their corresponding aldehydes. This leads the way to the deSIgn of acetal-contammg cytotoxic agents which can be selectively activated in tumours.
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