ent-Kaurane Diterpenoids from Chinese Liverworts and Their Antitumor Activities through Michael Addition As Detected in Situ by a Fluorescence Probe.

It is generally accepted that the origin of the cytotoxicity of ent-kaurane diterpenoids is due to the formation of reactive oxygen species (ROS) and that the α,β-unsaturated carbonyl is a pivotal moiety. Herein we demonstrate the isolation of 32 new and 12 known ent-kaurane diterpenoids from two Chinese liverworts. These compounds and three semisynthesized derivatives were screened against human cancer cell lines. The results revealed that their anticancer activities are caused by ROS formation through Michael modification of the protein thiols and depletion of glutathione unselectively. We also found that N-acetylcysteine reverses the cytotoxicity of these diterpenoids by forming Michael adducts, not through a well-recognized ROS scavenging pathway as previously reported. In situ intracellular thiol detection helped us visualize the intracellular distribution of the diterpenoids and determine the potency of their cytotoxicity. An alkaline analogue was found to be more selective because of the altered subcellular distribution.