DNA-interactive anticancer aza-anthrapyrazoles: Biophysical and biochemical studies relevant to the mechanism of action

The physicochemical and DNA-binding properties of anticancer 9-aza-anthrapy razoles (9-aza-APs) were investigated and compared with the carbocyclic ana logs losoxantrone (LX) and mitoxantrone (MX). Unlike their carbocyclic coun terparts, the tested 9-aza-APs do not undergo self-aggregation phenomena. T he pyridine nitrogen at position 9, missing in the carbocyclic derivatives, is involved in protonation equilibria at physiological pH. In addition, 9- aza-APs are electrochemically reduced at a potential intermediate between L X and MX. These data fully agree with quantum mechanical calculations. Bind ing to nucleic acids was examined by spectroscopic, chiroptical, and DNase I footprinting techniques as a function of ionic strength and base composit ion. The 9-aza-APs exhibit prominent affinity for DNA, with an important el ectrostatic contribution to the binding free energy. A very remarkable sequ ence preference pattern dramatically favors GC steps in double-helical DNA, whereas the carbocyclic reference compounds show a substantially lower sel ectivity for GC. A common DNA complexation geometry, considerably differing from that of MX, characterizes all anthrapyrazoles. Hence, bioisosteric su bstitution and ring-hydroxy deletion play an important role in defining the physicochemical properties and in modulating the affinity of anthrapyrazol es for the nucleic acid, the geometry of the intercalation complex, and the sequence specific contacts along the DNA chain. Drug stimulation of topois omerase II-mediated DNA cleavage is remarkably attenuated in the aza-bioiso steric derivatives, suggesting that other non-enzyme-mediated cytotoxic mec hanism(s), possibly connected with free radical production, are responsible for efficient cell killing. The biophysical and biochemical properties exh ibited by 9-aza-APs contribute to clarifying the peculiar pharmacological p rofile of this family of compounds.