Synthesis, Anticancer Evaluation, mRNA Expression Analysis, and Molecular Docking of Fluorinated Picolinamide–Benzothiazole Derivatives Against Breast Cancer-Related Genes CDK2, ERK2, and BCL2

Abstract

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.