Fused Bicyclic Caspase-1 Inhibitors Assembled by Copper-Free Strain-Promoted Alkyne-Azide Cycloaddition (SPAAC)

Challenges exist in the development of potent and selective small-molecule inhibitors against caspase-1. Herein, by making use of the copper-free strain-promoted alkyne-azide cycloaddition (SPAAC) reaction between difluorinated cyclooctynes (DIFOs) and various azide-containing compounds, we showed for the first time that potential caspase-1 inhibitors could be rapidly synthesized. The resulting fused bicyclic compounds structurally resembled the central portion (P₂ -P₃ ) of Pralnacasan (a well-known small molecule caspase-1 inhibitor), with diversity at the P₄ -position of the parental inhibitor conveniently installed from the azide component. Since our SPAAC-assembled inhibitor library was synthesized by using a copper-free bioorthogonal chemistry, the resulting 52-membered library (2 DIFOs×26 azides) was immediately ready for subsequent cell-based screening for rapid identification of potential cell-permeable hits capable of effectively inhibiting endogenous caspase-1 activities. C1FS, a recently reported fluorogenic two-photon probe, which possesses improved live-cell imaging sensitivity against endogenous caspase-1, was used both in vitro and in LPS/ATP-induced macrophages (a well-established caspase-1-activated cell model) to screen against selected compounds from the above-mentioned library, leading to subsequent discovery of a novel caspase-1 inhibitor named b7-b.