Design and fluorescence-guided optimization of styryl-benzonitrile cytotoxic chromophores: DFT and molecular docking studies

• Three styryl-benzonitrile chromophores 3, 4 , and 5 were synthesized and characterized by IR, NMR, and MS tools. • The styryl-benzonitrile compounds display clear intramolecular charge-transfer behavior, which helps separate charge but also tends to suppress their fluorescence output. • The absorption spectra of styryl-benzonitriles disclosed the piperidinyl 3 had the highest Δ E max , while the emission showed the morpholino 4 . • The cytotoxic activity demonstrated compound 5 as the safest one toward normal cells. • In docking studies against Escherichia coli DNA gyrase B, chromophore 5 displayed the strongest binding energy Styryl-Benzonitrile chromophores 3, 4 , and 5 were synthesized from diethyl 4-cyanobenzyl phosphonate ( 1 ) and benzaldehyde derivatives ( 2 ) via the Horner-Wadsworth-Emmons reaction. The trans configuration of the synthesized chromophores 3 - 5 has been verified by the distinctive nitrile stretching band (at ∼2210 cm⁻¹) and coupling constants (J ≈ 15.5 Hz). The DFT studies indicated that the HOMO was located on the donor fragment and the LUMO was spread on the benzonitrile acceptor fragment, confirming the important intramolecular charge transfer (ICT). Chromophore 5 displayed the smallest HOMO-LUMO gap = 2.82 eV, which correlated well to red-shifted absorption (λ max = 393 nm) and emission (λ em = 561 nm). The absorption spectra of these probes in varied solvents revealed that the piperidinyl 3 had the highest Δ E max (+3.09 kcal.mol -1 ), however, their emission spectral data presented hybrid 4 as the most polarity-induced (Δ E max +3.56 kcal·mol -1 ). Biologically, chromophore 5 unveiled the highest cytotoxic effectiveness towards HepG2 cells (IC₅₀ = 19.04±0.13 μM), with a higher selectivity index (SI = 4.6). Docking studies on DNA gyrase B (PDB: 1HNY, resolution 1.59 Å) showed a good binding (S = -5.97 kcal·mol -1 ) with important H-bonding bindings with Arg195 and Asp300. As well, the pharmacokinetic profiles of the synthesized chromophores indicated that all probes have auspicious drug-likeness properties, rendering them suitable for further evaluation.