Design, synthesis, and evaluation of novel andrographolide derivatives as anti-pancreatic cancer therapeutics.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease, with TP53 mutations in 50-70% of patients. Current treatments for PDAC remain mostly ineffective, underscoring the need for innovative therapeutic agents. While wild-type p53 functions as a tumor suppressor, missense mutations in its DNA-binding domain, such as p53R273H and p53R248W, confer oncogenic gain-of-function properties. Andrographolide, a natural compound derived from Andrographis paniculata, exhibits notable anticancer properties. This study aimed to develop novel andrographolide derivatives with enhanced anti-PDAC activity. We present a novel strategy to attach a 7-nitrobenz-2-oxa-1,3-diazol (NBD) fluorophore to andrographolide via acetal formation, preserving the C-14 hydroxyl for further modification with groups such as azido and COX-2 inhibitors. The anti-PDAC and mutant p53-targeting activity of these derivatives was evaluated. Among them, compound 4, a fluorescent andrographolide derivative, exhibited potent cytotoxicity and reduced the levels of multiple oncogenic p53 proteins. The autophagy/lysosome inhibitor bafilomycin A1 restored p53R273H protein levels in compound 4-treated PANC-1 cells. Compound 4 suppressed the migration and proliferation of PDAC cells. Furthermore, compound 4 downregulated the transcription of cancer-related genes downstream of oncogenic p53R273H, including CXCL1, CXCL2, PCNA, CCNA2, TIGAR, and MYC. Fluorescent signals of compound 4 were detectable within 5 min and remained stable for 48 h after incubation with PDAC cells. In vitro labeling experiments revealed that compound 4 covalently bound to the p50 subunit of NF-κB, a known target of andrographolide. In conclusion, compound 4 acts as both a promising anti-pancreatic cancer agent and a fluorescent probe for tracking cellular distribution and identifying target proteins.