Synthesis and Biological Evaluation of O6C-Salvinorin Alkyne Probes.

The salvinorins represent a unique class of polyoxygenated, non-nitrogenous diterpenes that potently and selectively modulate the kappa-opioid receptor (KOR). We recently disclosed a de novo asymmetric synthesis of this scaffold that enabled the discovery of highly potent, biased analogs, including C11-alkynes designed for the CATCH (Clearing-Assisted Tissue Click Chemistry) assay. Here, we report an optimized, scalable synthetic route that substitutes a problematic 1,4-conjugate addition of a Grignard reagent with a robust organozinc protocol in DMA, and improves a samarium-mediated Reformatsky reaction via the addition of LiBr. These methodological improvements facilitated the scalable synthesis of two highly active positive-control probes and three tailored negative-control probes, including functionally silent and cross-linking deficient analogs. A screen of >300 GPCRs confirmed the exceptional KOR selectivity of the novel alkyne probe, establishing it as a promising tool poised to validate target-specific engagement in situ, an application currently under investigation.