Synthesis, in-vitro and in-silico bioactivity and computational studies of pyrene and p-methoxycinnamaldehyde linked...

4-Hydroxycoumarin is a well-known pharmacophore of significant relevance in medicinal and pharmaceutical chemistry. The pursuit of multifunctional therapeutic agents continues to inspire the exploration of coumarin-based scaffolds. The derivatives of 4-hydroxycoumarin have been intriguing to researchers for drug design, synthesis, and the investigation of their therapeutic potential particularly against microbial infections and cancer. This study reports the design, synthesis, structural characterization and comprehensive in-vitro and in-silico evaluation of two novel 4-hydroxycoumarin derivatives: 3,3'-(pyren-1-ylmethylene) bis(4-hydroxy-2 H-chromen-2-one) (PBHC) and (E)-3,3'-(3-(4-methoxyphenyl) prop-2-ene-1,1-diyl) bis(4-hydroxy-2 H-chromen-2-one) (MPBHC). The antibacterial and anti-cancer activity of these derivatives are evaluated. The cytotoxicity was assessed on HEK-293 normal cells to evaluate selectivity. PBHC and MPBHC were synthesized and structurally confirmed through, FT-IR, NMR, HRMS and UV-Vis spectroscopic techniques. Quantum chemical investigations employing density functional theory (DFT, B3LYP/6-311 + G(d, p)) and time-dependent DFT (TD-DFT) accurately reproduced the electronic absorption spectra and provided insights into electronic transitions Molecular electrostatic potential (MESP) mapping, Frontier molecular orbital (FMO) analysis and topological investigations consisting of ELF, LOL, ALIF, NCI-RGD, and QTAIM analyses were conducted to identify the reactive centers and characterize the non-covalent interaction networks. As per the biological evaluation studies it was observed that PBHC displayed significant antibacterial activity against Staphylococcus aureus and exhibited selective cytotoxicity toward HepG2 hepatocellular carcinoma cells (IC₅₀ = 46.69 ± 0.021 µM, the cytotoxicity of PBHC towards HEK-293 normal cells shows an IC₅₀ of 10.24 ± 1.2 µM. In contrast, MPBHC displayed enhanced cytotoxic potency against HepG2 cells (IC₅₀ = 9.407 ± 0.300 µM) with comparatively lower cytotoxicity toward HEK-293 normal cells (IC₅₀ = 47.59 ± 2.5 µM), moderate activity toward MCF-7 breast carcinoma cells (IC₅₀ = 47.33 ± 0.19 µM), and strong anti-biofilm efficacy against S. aureus (MBIC = 0.1 µg/mL). Pharmacokinetic predictions and toxicity profiling further indicated favourable drug-likeness and safety (oral LD₅₀ ≈ 3000 mg/kg). This study reveals that structural alteration of 4‑hydroxycoumarin derivatives can substantially affect the drug‑like properties and their bioactivity, offering vital direction for the development of future antibacterial and anticancer medicines.