In Vitro Binding to a Mitochondrial G-Quadruplex-Forming Sequence and Exploratory Acute Tolerability Assessment of a Cyclic Anthraquinone Anticancer Agent

G-quadruplexes (G4s) have attracted considerable attention as molecular targets for the development of anticancer agents with improved selectivity and safety profiles. Cyclic anthraquinone derivatives have been developed as G4-binding ligands, and our previous studies demonstrated that cAQ-mBen preferentially binds to nuclear G4 structures, including telomeric and c-MYC G4s, and exhibits potent anticancer activity. In the present study, we examined the interaction of cAQ-mBen with the mitochondrial G4-forming oligonucleotide mt6363 and performed an exploratory single-dose tolerability assessment in rats. UV–visible absorption titration using the mitochondrial G4-forming sequence mt6363 revealed an association constant (Ka) of 7.5 × 105 M−1. CD spectroscopy showed that the overall CD spectral signature of mt6363 remained broadly similar following cAQ-mBen addition. Furthermore, CD melting analysis showed that cAQ-mBen increased the melting temperature of mt6363 by 8.5 °C, demonstrating pronounced stabilization of the mitochondrial G4 structure. These findings demonstrate that cAQ-mBen can bind to and stabilize the mitochondrial DNA-derived G4-forming sequence mt6363 in vitro. The animal experiment was designed as a preliminary exploratory acute tolerability assessment (n = 3 per group), not as a definitive toxicology study. Following a single intravenous dose of 1000 mg/kg administered at 10 mL/kg, no mortality or overt severe clinical abnormality was observed during the 14-day observation period; however, lower body-weight gain and intake and numerical increases in AST, BUN, and creatinine were observed in the treated animals, and the small sample size precludes statistical demonstration of absence of toxicity. Overall, the present results demonstrate the in vitro binding of cAQ-mBen to a mitochondrial DNA-derived G4-forming sequence, while mitochondrial localization, intracellular mtG4 target engagement, sequence selectivity, and definitive safety remain to be established in appropriately designed studies.