Methoxy topology controls potency and erythrocyte compatibility of 4′-hydroxychalcones: Integrated profiling in two lymphoma-derived cell lines, PBMCs, hemoglobin, and red blood cells

Chalcones are tunable electrophiles whose bioactivity depends on aryl substitution and membrane interactions. We profiled eight 4'-hydroxychalcones with differing in methoxy topology on ring B in primary human PBMCs (24 h, XTT) and canine lymphoma/leukemia cell lines (CLBL-1, CLB70; 72 h, MTT), integrating RBC compatibility assays (hemolysis, morphology, osmotic fragility C50, transmembrane potential, and reduced glutathione), hemoglobin tryptophan-fluorescence quenching, and cell-free antioxidant assays. In canine tumor models, chalcones were more cytotoxic to neoplastic than to normal cells, indicating selectivity. Among them, 3-methoxy-4'-hydroxychalcone showed the most favorable profile, combining strong anticancer activity in CLBL-1 and CLB70 with lower toxicity toward normal cells, whereas 2,5-dimethoxy-4'-hydroxychalcone, although potent, showed a less favorable safety profile. RBC studies revealed marked echinocytosis after 1 h at 100 µM without acute hemolysis and no changes in osmotic resistance, transmembrane potential, or GSH levels. After 24 h, hemolysis was observed only for two compounds at concentrations ≥ 200 µM. Hb quenching was static (Kq above the diffusion limit), with the highest Ksv values for 3-methoxy-4'-hydroxychalcone and 4'-hydroxychalcone, but the highest Kb for 3,4,5-trimethoxy-4'-hydroxychalcone, followed by 4'-hydroxychalcone, indicating that fluorophore proximity and complex stability are not directly coupled. DPPH and CUPRAC assays showed no detectable direct antioxidant activity, whereas ABTS•+ revealed weak and comparable radical-scavenging activity across the series. A concise SAR emerged: meta- and ortho/meta-methoxy substitution enhanced biological activity, whereas para-methoxy and 3,4,5-trimethoxy substitution attenuated it; the unsubstituted parent compound retained substantial activity. Overall, methoxy topology shapes both cytotoxicity and interactions with blood components, with 3-methoxy-4'-hydroxychalcone emerging as the most promising lead for early preclinical evaluation.