Nano-TiO₂ Catalyst-Based Method Development to Synthesize Pyrano-Chromenes Targeting TFF3 in Breast Cancer

Trefoil Factor 3 (TFF3) is an oncogenic protein implicated in breast cancer progression through regulation of PI3K-AKT, MAPK, STAT3, and NF-κB signaling pathways. Targeting TFF3 therefore represents a promising therapeutic strategy. In this study, chromene-3-carbonitrile derivatives were synthesized using nano-TiO₂ as an efficient catalyst and evaluated for their anticancer potential against TFF3 in MCF-7 breast cancer cells through combined in vitro and in silico approaches. Thermodynamic feasibility of the synthetic process was assessed using free energy calculations. Cytotoxicity of the synthesized compounds was determined in MCF-7 cells, while molecular docking studies were performed to investigate binding interactions with TFF3. Protein expression analysis was performed using western blotting to confirm TFF3 inhibition. Several derivatives demonstrated significant cytotoxic activity, with lead compounds F8, F10, F12, and F13 exhibiting IC₅₀ values of 0.751, 1.70, 0.68, and 0.52 µM, respectively. Docking studies revealed strong binding affinity and favorable interactions with TFF3 and energy profiling supported the thermodynamic viability of synthetic route. Western blot results further confirmed downregulation of TFF3 expression. Overall, these findings identify chromene-3-carbonitrile derivatives as potent TFF3-targeting agents with potential therapeutic relevance for breast cancer.