Investigation of Premyrsinane and Myrsinane Esters in Euphorbia cupanii and Euphobia pithyusa with MS2LDA and Combinatorial Molecular Network Annotation Propagation

The species Euphorbia pithyusa and Euphorbia cupanii are two closely related Mediterranean spurges for which their taxonomic relationships are still being debated. Herein, the diterpene ester content of E. cupanii was investigated using liquid chromatography coupled to tandem mass spectrometry. The use of molecular networking coupled to unsupervised substructure annotation (MS2LDA) indicated the presence of new premyrsinane/myrsinane diterpene esters in the E. cupanii fractions. A structure-guided isolation procedure yielded 16 myrsinane (11a–h, 12, and 13) and premyrsinane esters (14a–c and 15a–c), along with four 4β-phorbol esters (16a–c and 17) that showed inhibitory activity against chikungunya virus replication. The structures of the 16 new compounds (11a–c, 11h, 12, 13, 14a–c, 15a–c, 16a–c, and 17) were characterized by NMR spectroscopy and X-ray crystallography. To further uncover the diterpene ester content of these two species, the concept of combinatorial network annotation propagation (C-NAP) w...