Ingol and Ingenol-Type Diterpenes from Miller with Keratinocyte Inhibitory Activity.

Ingenol mebutate, isolated from , is an ingenane-type diterpenoid, primarily used for the topical treatment of actinic keratosis, a premalignant skin condition. The aim of our work was to investigate other species to find structurally similar diterpenes that can be used as alternatives to ingenol mebutate. Pharmacological investigation of , , , and revealed the potent keratinocyte (HPV-Ker cell line) inhibitory activity of these spurge species. From the methanolic extract of the aerial parts of Miller, the most active species, five ingol (-) and four ingenane-type diterpenoids (-) were isolated by various chromatographic separation techniques, including open column chromatography, vacuum liquid chromatography, thin-layer chromatography, and high-performance liquid chromatography. The structures of the compounds were determined by NMR spectroscopic analysis and by comparison of the assignations with the literature data. The cytotoxic activity of the compounds against keratinocytes was tested in vitro by using ingenol mebutate as a positive control. Among the isolated compounds, two ingenane derivatives ( and ) exhibited remarkably stronger cytotoxic activity (IC values 0.39 μM and 0.32 μM, respectively) on keratinocytes than ingenol mebutate (IC value 0.84 μM). These compounds could serve as starting materials for further investigations to find alternatives to Picato (with active substance ingenol mebutate), which was withdrawn from marketing authorization in the European Union.