Design, synthesis, and mechanistic evaluation of propargylated salicylaldehyde derivatives as dual apoptosis–autophagy modulator for pancreatic cancer

In this study, eight novel propargylated salicylaldehyde derivatives and one propargylated indole derivative were successfully synthesized and evaluated for their cytotoxic activities against the human pancreatic cancer cell line PANC-1. Among the nine tested molecules, molecules 4 and 6 exhibited the most pronounced cytotoxic effects, with IC₅₀ values of 65.7 and 80.6 µM, respectively. Additionally, cytotoxicities of molecule 4 and 6 were also tested against prostate cancer (PC-3), lung adenocarcinoma (A549), and human dermal fibroblasts (hDF). Even though molecule 4 exhibited more moderate effects, gene expression analysis revealed that molecule 6 was associated with increased transcriptional expression of pro-apoptotic markers (Bax, Bad, Casp8, Casp9) and autophagy-associated genes (ATG6, ATG7, ATG8, AMBRA1), suggesting transcriptional engagement of apoptosis- and autophagy-related regulatory networks. These results were corroborated by complementary in silico docking studies, which demonstrated that molecule 6 had favorable binding interactions with the active sites of AMBRA1, BAX, ATG8, and Caspase-8. Despite having IC₅₀ values in the micromolar range, molecule 6 shows promise as an early-stage hit molecule for further optimization and preclinical investigation in pancreatic cancer models. In addition to all important results, in ovo HET-CAM analysis also revealed that both molecule 4 and molecule 6 were non-irritant, as no hemorrhage, vascular lysis, or coagulation was observed, resulting in an irritation score of zero. This favorable tolerability profile indicates the absence of acute membrane toxicity and supports the suitability of these molecules for further biological evaluation.