A new class of methuosis inducers via PI3K/Akt-ENC1 signaling axis from Daphne genkwa: Structure characterization, total synthesis and antitumor evaluation.

Methuosis is one of the new types of cell deaths identified in recent years and has been considered to be a promising alternative strategy for cancer treatment. Thus, the discovery and study of small-molecule methuosis inducers has become an emerging research topic in the last decade. In this study, an in vivo antitumor bioassay-guided chemical exploration on the twigs and leaves of Daphne genkwa yielded a series of meroterpenoids comprising ten previously unreported ones (1a/1b-6), and the new compounds were structurally characterized by comprehensive spectroscopic means particularly quantum chemical NMR calculation, electronic circular dichroism and modified Mosher's method. The first total synthesis of (±)-3 was accomplished using a highly efficient bio-inspired strategy in 20.3% overall yield, with the two key steps involving construction of the chromene unit via an intramolecular oxa-[3 + 3] annulation and formation of three consecutive chiral centers through a biomimetic acid-catalyzed ring closure. A preliminary cytostatic assay established that compounds 1a, 3a and 3b exhibited significant growth inhibition against four carcinoma cell lines, and subsequent evaluation identified them as a new class of methuosis inducers toward breast cancer cells, with the in vivo antitumor efficacy validated for rac-3 in mice. Further mechanistic investigations revealed that compound 3 triggered methuosis via regulation of PI3K/Akt-ENC1 signaling axis both in vitro and in vivo. Also, it is the first report to uncover that ENC1 might play an important role in methuosis. In conclusion, a new class of natural methuosis inducers has been obtained and characterized in the present work, and they could be used as template molecules for the design of better methuosis-inducing agents in cancer intervention and as new molecular probes for the further investigation of this intriguing cell death modality.