Design, synthesis and evaluation of 4,7-disubstituted 8-methoxyquinazoline derivatives as potential cytotoxic agents targeting β-catenin/TCF4 signaling pathway

Overactivation of Wnt/beta-catenin signaling by accumulated beta-catenin in the nucleus has been shown to play a crucial role in the etiology of cancer. Interaction of beta-catenin with Transcription factor 4 (TCF4) is a key step for the activation of Wnt genes in response to upstream signals of the Wnt/beta-catenin pathway. Hence, down regulation of Wnt/beta-catenin signaling or targeting downstream events by selective beta-catenin/TCF4 protein-protein interaction inhibitors could be a potential therapeutic strategy against such cancers. In this study structure-based drug design approach was followed to design novel 4,7-disubstituted 8-methoxyquinazoline-based derivatives which could act as potential cytotoxic agents inhibiting the beta-catenin/TCF4 protein-protein interactions. Fifteen compounds possessing 4,7-disubstituted 8-methoxyquinazoline scaffold were synthesized. Cytotoxic potential of the synthesised derivatives were determined against constitutively activated beta-catenin/TCF4 signaling pathway cancer cells (HCT116 and HepG2) using the sulforhodamine B assay. The most potent compound (18B) was selected for detailed biological evaluation. Cell morphology, Hoechst 33342 and Annexin V/PI staining were used to detect apoptosis, while inhibition of cell migration was assessed by in vitro wound healing assay against HCT116 and HepG2 cells. Effect on beta-catenin/TCF mediated transcriptional activity was assessed by TOPFlash/FOPFlash assay, TCF4 and beta-catenin protein expression by immunocytofluorescence, and Wnt target genes (like c-MYC and Cyclin D1) mRNA levels by RT-PCR against HCT116 cells. Cytotoxic potency of the most potential compound (18B) against primary human gallbladder cancer cells was also evaluated. The derivatives showed interactions with active site residues of beta-catenin and were capable of hindering the TCF4 binding, thereby disrupting beta-catenin/TCF4 interactions. Cytotoxic potencies (IC50) of these derivatives ranged from 5.64 +/- 0.68 to 23.18 +/- 0.45 mu M against HCT116 and HepG2 cells respectively. Compound (18B), the most potent compound among the series, induced apoptosis and inhibited cell migration against HCT116 and HepG2 cells. Mechanistic studies indicated that compound (18B) downregulated beta-catenin/TCF4 signaling pathway, beta-catenin and TCF4 protein expression, and mRNA levels of c-MYC andCyclin D1 in HCT116 cells and showed cytotoxicity against primary human gallbladder cancer cells with IC50 value of 8.50 +/- 1.44 mu M. Thus, novel 4,7-disubstituted 8-methoxyquinazoline derivatives were identified as potential cytotoxic agents with potencies comparable to that of imatinib mesylate. Compound (18B) represents a promising lead molecule as anticancer agent against colon, hepatocellular and gallbladder cancers targeting beta-catenin/TCF4 signaling pathway.

Automated summary

In this study, novel 4,7-disubstituted 8-methoxyquinazoline derivatives were designed as potential cytotoxic agents against cancers by inhibiting the protein-protein interactions of beta-catenin/TCF4. The most potent compound (18B) showed significant cytotoxicity, inducing apoptosis and inhibiting cell migration in HCT116 and HepG2 cells. Mechanistic studies revealed that compound (18B) downregulated the beta-catenin/TCF4 signaling pathway, protein expression, and mRNA levels of c-MYC andCyclin D1. It also exhibited cytotoxicity against primary human gallbladder cancer cells. These findings suggest that compound (18B) is a promising lead molecule for anticancer therapy targeting the beta-catenin/TCF4 signaling pathway.