**2.2.4 Benzo-acronycine derivatives**

Acronycine is a natural alkaloid extracted from the bark of an Australian ash scrub that presented interesting antitumor activities but was poorly soluble and, consequently, too toxic in first clinical trials. The discovery of an unstable acronycine epoxide opened the way to the rational drug design of S23906-1 (Figure 4), that appeared to be a highly active compound (Guilbaud et al., 2001) with an original mode of action (David-Cordonnier 2002; 2005; Depauw et al., 2009) and consequently entered phase I clinical trials in 2006. As for the clinically used drug Ecteinascidine 743 (ET-743, Trabectedin, Yondelis TM from Pharmamar), S23906-1 alkylates the exocyclic NH2 group of guanines in the minor groove. But, in contrast with ET-743, S23906-1 does not reinforce the stability of the ds-DNA helix but destabilizes it, generating portions of ss-DNA (David-Cordonnier et al., 2005; Depauw et al., 2009). Various spectral and biochemical approaches convinced with this conclusion. Indeed, classical DNA melting temperature studies evidenced a strong decrease of the Tm values upon alkylation with S23906-1 or other biologically active benzo-acronycine derivatives. Similarly, spectral analysis of the ratio of fluorescence properties of picogreen (a ds- and ss-DNA interacting dye) and BET (a ds-DNA specific dye) evidenced an increase of picogreen *vs*. BET fluorescence which enlightens the generation of single-stranded portions of the DNA upon S23906-1 alkylation. Biochemical approaches like digestion of the alkylated DNA by singlestrand specific nuclease S1 and electrophoretic mobility shift assays (EMSAs) confirmed the opening of the DNA. The destabilization was relatively wide since mapping with nuclease S1 evidenced locally opened DNA portions within a 117 bp DNA fragment alkylated by S23906-1 whereas EMSAs, performed with oligonucleotides as long as 24 bp, evidenced fully single-stranded alkylated oligonucleotides in the presence of S23906-1 or derivatives (David-Cordonnier et al., 2005; Depauw et al., 2009).
