Uncoupling DNA binding in solution from cellular efficacy: Structure–distribution relationships of indolo[3,2-b]quinolines in cancer cells

Understanding how small-molecule DNA binders distribute within cells is crucial for the rational design of effective anticancer agents. This study elucidates critical structure-distribution relationships governing the subcellular localization and anticancer activity of indolo[3,2- b ]quinolines in cancer cells. Through systematic analysis of four derivatives using Two-Photon Fluorescence Microscopy, DNA interaction and antiproliferative assays, we studied the effect of lipophilicity of the compounds at physiological pH, influenced by aromatic chlorination status and alkylamine side chain length at position 11. The mono-chlorinated derivative with the longest alkylamine side chain ( 6 ) showed superior nuclear targeting and antiproliferative potency (IC₅₀ values between 1.8 ± 0.1 and 2.4 ± 0.3 μM), attributed to its lower lipophilicity (Log D 7.4 = 0.6 ± 0.1) facilitating nuclear membrane permeation. Di-chlorinated analogs 3 and 4 , with higher lipophilicity (Log D 7.4 ≥ 3.2), exhibited higher cytoplasmic accumulation and lower antiproliferative activity, despite comparable DNA-binding affinity. Our results reveal a critical disconnect between DNA binding in solution and the efficacy of intracellular DNA targeting. We demonstrate that nuclear accumulation, essential for potent cytotoxicity, is determined by the pH-dependent lipophilicity, which in turn, is governed by aromatic chlorination status, alkylamine side chain length and basicity. These findings provide a structural blueprint for designing DNA-targeting indoloquinolines, prioritizing less lipophilic compounds. The demonstrated correlation between subcellular distribution patterns, cytotoxic activity and lipophilicity, resolves previous discrepancies in structure-activity relationships, offering new strategies to optimize chemotherapeutic agents. • Indoloquinolines (IQs) easily permeate cytoplasmatic membrane of breast cancer cells. • Accumulation of IQs in the nucleus correlates inversely with their lipophilicities. • Antiproliferative activity of IQ correlates positively with nuclear accumulation. • Compounds´ binding affinity to DNA does not govern nuclear accumulation. • Anticancer activity does not correlate straightforwardly with binding affinity to DNA.