Synthesis, Anticancer Evaluation, mRNA Expression Analysis, and Molecular Docking of Fluorinated...

The present study focuses on the design, synthesis, characterization, and biological evaluation of two new series of derivatives 4-(4-amino-3-fluorophenoxy)-N-methylpicolinamide (3a-e) and 2-aminobenzothiazole (5a-e) as potential antioxidant and anticancer agents. The compounds were synthesized via a one-pot condensation reaction of 4-(4-amino-3-fluorophenoxy)-N-methylpicolinamide and 2-aminobenzothiazole with various phenyl isocyanates, isothiocyanates, and sulfonamides in the presence of triethylamine at 60°C. Reaction progress was monitored by TLC, and products were purified using column chromatography. Structural elucidation was achieved using FT-IR, ¹H NMR, ¹³C NMR, HRMS, and CHN analyses. Antioxidant activity, evaluated by DPPH and ABTS assays, revealed that compounds 3d and 5b exhibited the highest radical scavenging potential, with IC₅₀ values of 21.45 μg/mL and 20.05 μg/mL (DPPH) and 22.24 μg/mL and 21.98 μg/mL (ABTS), comparable to ascorbic acid. Cytotoxicity assessment by MTT assay demonstrated strong antiproliferative activity of 3d and 5b against MDA-MB-231 breast cancer cells (IC₅₀ = 22.13 μg/mL and 20.11 μg/mL), with minimal toxicity toward normal 3T3-L1 cells. Fluorescence microscopy and flow cytometry confirmed apoptosis profile induction and G2/M phase arrest. The qRT-PCR analysis showed significant down-regulation of Bcl-2, CDK2, and ERK2 gene expression, correlating with apoptotic activation. Molecular docking and MM-GBSA analyses revealed stable and high-affinity binding of 3d and 5b with Bcl-2, CDK2, and ERK2 proteins, supported by multiple hydrogen bonding and π-π interactions. Molecular dynamics simulations (150 ns) confirmed the structural stability of these complexes. Overall, compounds 3d and 5b emerged as promising dual-function 'antioxidant and anticancer' candidates and in vivo evaluations for therapeutic development.