Collybolide

Collybolide was first isolated from R. maculata in 1911,^[4][5][6] but its structure remained unsolved until the 1970s, when X-ray crystallography yielded the structure of a collybolide epimer, isocollybolide,^[5] and ¹H and ¹³C NMR elucidated the structure and relative stereochemistry of collybolide.^[7] Importantly, early reports were unable to confidently assign the absolute configuration of collybolide; a 1986 isolation of a collybolide congener noted that the absolute configuration of the series "remains to be determined",^[8] and a 2001 circular dichroism study was only able to tentatively infer which enantiomer naturally occurred based on density functional theory calculations.^[9] A 2016 report claimed to have conclusively assigned the absolute configuration of collybolide by X-ray crystallography,^[1] but a following 2022 report noted that the Flack parameter accompanying the 2016 crystal structure was inconclusive,^[2] and could not be used to confidently assign its absolute stereochemistry.

Collybolide is a sesquiterpene that contains a furyl-δ-lactone, a structural feature shared with the diterpene natural product salvinorin A. Salvinorin A is a hallucinogen that acts via high-potency agonism of the human kappa-opioid receptor (KOR), and collybolide's structural similarity to salvinorin A prompted a 2016 team to investigate collybolide's activity at the KOR, in the hopes of discovering a new, non-nitrogenous opioid.^[1] Radioligand displacement and functional assays showed collybolide binding to (K_i = 0.9 nM) and activating the human KOR, and an in vivo assay described collybolide inhibiting chloroquine-induced itch in mice at an extremely low dose (IC₅₀ = 0.08 mg/kg). These results attracted widespread attention in the biomedical community, as collybolide appeared to be a potent and selective KOR agonist that might be developed into a new treatment for pain or pruritus,^[10][11] lacking the adverse effects of typical mu-opioid receptor agonist pain treatments. These claims of KOR agonism also attracted the attention of the recreational psychedelic community.^[12]

Independent chemical synthesis and biological assay of collybolide in 2022 found that it was devoid of opioid activity.^[2] Radioligand displacement assays showed only weak (K_i = 794 nM) binding of collybolide to the human KOR, and functional assays showed that collybolide does not activate KOR signaling at concentrations up to 10 μM (measured by [³⁵S]GTPγS binding, cAMP accumulation, and beta-arrestin recruitment assays). Shevick et al. note the presence of surface-modifying agents in the 2016 assay procedures, in addition to low percent stimulation in the 2016 [³⁵S]GTPγS assay, that may have caused noise in the data to be mistaken as signal.^[2] The source of the false positive result for KOR agonism in the 2016 study has yet to be rigorously identified. However, the findings and conclusions of the 2022 study – that collybolide was incorrectly assigned as a KOR agonist – explain why no credible reports of collybolide's psychoactivity have surfaced.^[13][14]

The 2022 reevaluation of collybolide's KOR activity leveraged access to both natural and unnatural enantiomers of collybolide via total synthesis.^[2][15] Key features of the synthesis included an enantioselective Diels-Alder reaction using the Hayashi-Jørgensen proline organocatalyst, and an enamine [3,3]-sigmatropic rearrangement to stereoselectively install a late-stage benzoyloxy (BzO) group.