Compound 7 h exerts its anti-oncogenic effects on colorectal cancer cells by inducing death-receptor-mediated apoptosis, promoting DNA damage, and obstructing autophagic flux.

BACKGROUND AND AIM: Previous research described a catalyst-free, post-Ugi synthesis of two distinct series of highly substituted fused pyrazolo-pyrazines. One of these series demonstrated antitumor activity against colorectal cancer (CRC) cells. However, the precise mechanistic pathways remain poorly understood, particularly with respect to effects on autophagy regulation, DNA repair processes, and death-receptor-mediated extrinsic apoptosis. METHODS: The cytotoxic properties of compound 7 h were assessed through MTT and colony formation assays, while apoptosis was evaluated by flow cytometry and western blotting. Immunofluorescence, GFP-LC3 transfection and mCherry-GFP-LC3B transfection were performed to assess autophagic activity. Synergistic interactions between derivative 7 h and 5-fluorouracil (5-FU) were investigated to assess enhanced CRC chemosensitivity. Molecular docking simulations were conducted to predict the binding conformation of compound 7 h with its molecular target and to identify potential interaction sites contributing to its inhibitory activity. RESULTS: Compound 7 h exhibited significant cytotoxic effects, inhibiting the proliferation and growth of CRC cells in vitro. Mechanistic investigations demonstrated that the cytotoxic activity of compound 7 h was mediated by impeding autophagic flux, thereby inducing DNA damage, inhibiting DNA repair, and ultimately promoting death receptor (DR)-mediated extrinsic apoptosis in CRC cells. Furthermore, the combination of compound 7 h with 5-FU exhibited synergistic inhibition of CRC cell proliferation, suggesting that compound 7 h could enhance CRC sensitivity to 5-FU. CONCLUSIONS: Our results suggest that impaired autophagic flux progression following treatment with compound 7 h leads to DNA damage and death-receptor-mediated apoptosis, underscoring the potential of compound 7 h as a novel therapeutic approach for CRC. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s40360-026-01087-2.