Synthesis and Antitumor Potency of 2E,21E-bis-(2-Pyridinylidene)-hollongdione in NCI-60 Panel and Zebrafish Model

Michael acceptors, such as chalcones and benzylidenes, are privileged scaffolds for the development of anticancer agents. Taking this into account, we developed a selective Claisen-Schmidt condensation of the dammarane-type triterpenoid hollongdione with pyridine-2-carbaldehyde, enabling controlled synthesis of mono- and bis-substituted triterpenes depending on the reaction conditions. The reaction demonstrated high temperature-dependent regioselectivity, providing C2-mono- <b>2</b> or 2,21-bis-substituted <b>3</b> triterpenes with yields up to 96% and 95%, respectively. The structures of the newly synthesized triterpene chalcones were elucidated by 1D and 2D NMR spectroscopy and unambiguously confirmed by a single-crystal X-ray diffraction, which established the <i>E</i> configuration of the exocyclic double bond. In biological studies, the bis-2-pyridylidene derivative <b>3</b> exhibited a pronounced and broad-spectrum antitumor activity in the NCI-60 panel, inducing cell death in 58 of 59 cancer cell lines. High selectivity toward melanoma, renal, and prostate cancer cell lines was observed, with selectivity indices (SI) of up to 18.82 for melanoma LOX IMVI. In MTT assays, compound <b>3</b> displayed a submicromolar cytotoxicity, particularly against the KRAS-mutant PANC-1 cell line (IC₅₀ = 0.22 µM). Anticancer activity was further confirmed in a zebrafish (<i>Danio rerio</i>) xenograft model of human HCT116 colon cancer, where tumor growth inhibition reached 72% without pronounced embryotoxicity (LC₅₀ = 1.4 µM). We have developed an efficient approach for the site-selective modification of hollongdione, providing access to potent anticancer dammarane-type chalcones. The bis-2-pyridylidene derivative <b>3</b> emerged as a promising lead compound, demonstrating submicromolar potency, high selectivity towards melanoma, and significant in vivo efficacy in a zebrafish xenograft model.