Antitumor aza-anthrapyrazoles: biophysical and biochemical studies on 8-aza and 9-aza regioisomers.

Aza-bioisosteres of anthrapyrazoles (Aza-APs) bearing the C–N substitution at position 9 are powerful anticancer agents now in clinical trials. In contrast, their 8-substituted regioisomers are practically devoid of chemotherapeutic effects. To understand the molecular basis for a dramatically different response by otherwise very similar compounds, we performed a detailed investigation on the physico-chemical properties of several aza-APs belonging to the two families, on their DNA-binding affinity and specificity as well as on their capacity to impair the activity of the two isoforms of human Topoisomerase II (top2a and top2b). Our results indicate that molecular size and shape, electronic distribution, redox properties, lipophilicity and protonation equilibria are essentially the same when comparing 9- with 8-substituted congeners. Although no major difference could be picked up when comparing the DNA binding properties of corresponding members of the 8- and 9-aza families, interestingly the affinity and specificity for the nucleic acid is modulated by the nature of the sidearms linked to the aza-AP scaffold, suggesting structural motifs that may determine DNA sequence recognition by the studied drug. Topoisomerase II poisoning activity was much higher for 9-aza derivatives than 8-aza analogues as shown by a cleavage assay with purified recombinant top2 isoforms. The difference appears to account for the divergent anticancer potential exhibited by different aza-AP regioisomers and suggests a specific molecular recognition of the cleavage complex by the studied drugs.