Novel zerumbone-amide-triazole hybrids as potential NF-κB pathway inhibitors: design, synthesis, cytotoxicity evaluation, computational studies, and mechanistic insights.

A series of fifteen novel zerumbone-amide-triazole hybrids 12a-i and 15a-f were successfully designed and synthesized from azazerumbone II (2) via click reaction. The cytotoxicity of these derivatives was evaluated against four human cancer cell lines: gastric carcinoma (AGS), hepatocellular carcinoma (HepG2), lung carcinoma (A549), and acute leukemia (HL-60). Most derivatives displayed significantly improved cytotoxicity compared with the parent compound 9, and several hybrids showed low-micromolar potency. These results highlight the crucial role of the simultaneous incorporation of secondary amide and 1,2,3-triazole pharmacophores into the zerumbone scaffold in enhancing anticancer activity. In particular, compound 12g was the most active derivative, exhibiting IC50 values of 3.25 μM (AGS), 2.21 μM (HepG2), 3.84 μM (A549) and 2.43 μM (HL-60), while 15e also demonstrated consistently strong activity across all cell lines. Molecular docking suggested that the hybrids preferentially associate with non-canonical surface regions within the Rel homology domain (RHD) of NF-κB p65, rather than occupying the DNA-binding groove, with key contacts involving residues such as Phe298, Phe301, Pro303, Lys337 and Lys318. To further assess interaction stability, 50 ns molecular dynamics simulations and MM-GBSA calculations were performed, supporting productive surface binding for the most active ligands and helping discriminate less active candidates. Mechanistically, Western blot analysis in HepG2 cells showed that compound 12g reduced NF-κB p65 and phospho-p65 (Ser536) levels, together with phospho-IκB-α (Ser32), predominantly at 2× IC50, suggesting attenuation of NF-κB activation at higher effective concentrations. Overall, the combined experimental and computational results support zerumbone-amide-triazole hybrids as promising cytotoxic leads, with NF-κB p65 modulation likely contributing to their biological effects.