Flavescensines A-L: Novel azaspiro[4.4] heterodimeric alkaloids from Sophora flavescens protect against liver injury by inhibiting DUSP2-mediated mitochondrial apoptosis.

By employing a targeted strategy integrating a building blocks-based molecular network (BBMN), network annotation propagation (NAP), ultraviolet spectroscopy (UV), and mass spectrometry (MS), twelve novel spirocyclic heterodimeric alkaloid flavescensines A-L (1-12) with 5/6/6/6/5 and 6/5/6/6/5 pentacyclic skeletons were isolated from Sophora flavescens. Their structures were unambiguously elucidated through comprehensive spectroscopic data, quantum chemical calculations, and single-crystal X-ray diffraction. Structurally, these compounds represent the first examples of azaspiro[4.4] alkaloids formed through the inert ring A or B of the C(15) matrine-type alkaloid and C(9) units. Notably, the NMR signals of C-9' methylene can serve as diagnostic indicators to determine the absolute configuration of the spiro carbon. A plausible biosynthetic pathway involving an unusual pattern of [3 + 2] cycloaddition was proposed. The hepatoprotective activities of 1-12 were evaluated in vitro, and 10 exhibited the most significant activity. Further in vivo experiments demonstrated that 10 dramatically inhibited the APAP-induced increase in the serum ALT, AST, and LDH levels, reversed the depletion of hepatic GSH, and attenuated hepatic centrilobular necrosis and hemorrhage. Mechanistically, 10 exhibits a potential interaction with DUSP2 and inhibits its expression, thereby suppressing DUSP2-mediated mitochondrial apoptosis via PI3K/Akt/JNK pathway. This represents the first discovery of DUSP2 involving in the hepatoprotection.