A series of biologically unexplored substituted 1,3,4-subtituted-pyrrolo[3,2-c]quinoline derivatives (PQs) was evaluated against a panel of about 60 tumor cells (NCI). Based on the preliminary antiproliferative data, the optimizations efforts permitted us to design and synthesize a new series of derivatives allowing us to individuate a promising hit (4g). The insertion of a 4-benzo[d] [1,3]dioxol-5-yl moiety on increased and extended the activity towards five panel tumor cell lines such as leukemia, CNS, melanoma, renal and breast cancer, reaching IG50 in the low ?M range. Replacement of this latter with a 4-(OH-di-Cl-Ph) group (4i) or introduction a Cl-propyl chain in position 1 (5), selectively addressed the activity against the entire leukemia sub-panel (CCRF-CEM, K-562, MOLT-4, RPMI-8226, SR). Preliminary biological assays on MCF-7 such as cell cycle, clonogenic assay, ROS content test alongside a comparison of viability between MCF-7 and non-tumorigenic MCF-10 were investigated. Among the main anticancer targets involved in breast cancer, HSP90 and ER receptors were selected for in silico studies. Docking analysis revealed a valuable affinity for HSP90 providing structural insights on the binding mode, and useful features for optimization.