Isolation and Characterization of Unusual Terpene Derivatives from the Fungus Echinodontium tinctorium

The North American fungus Echinodontium tinctorium has not been explored for its small-molecule constituents for 60 years. Herein, we describe chemical investigation of its organic extracts and bioactivity-guided fractionation, which led to the isolation of two new diterpenoids, echinodin A (1) and B (2). Both 1 and 2 contain an unprecedented tetracyclic 5/3/7/5 carbon ring system with the highly constrained cyclopropane motif. Bis-(2,4-dihydroxy-6-methylphenyl) methane (4) was isolated for the first time in a natural source together with four other known compounds (3, 5-7). The structures of 1-7 were determined by a combination of NMR, ESI-MS, and single-crystal X-ray diffraction analyses. Cytotoxic assays revealed that 1, 2, and 4 have weak cytotoxic activity against human cervical adenocarcinoma (HeLa) and human glioblastoma (U251). Echinodol (5) and echinodone (6), investigated for the first time for their bioactivity, exhibited strong cytotoxic activity with IC₅₀ values ranging from 1.2 to 5.5 μM against a panel of human cancer cell lines. A mechanism of action investigation showed that echinodol (5) induced apoptosis and arrested the cell cycle at the S phase in U251 cells. These findings not only highlight the new chemistry and biology of isolated compounds but also position E. tinctorium as a source for further pharmacognostic exploration.