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

Abstract 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 (P 2 –P 3 ) of Pralnacasan (a well‐known small molecule caspase‐1 inhibitor), with diversity at the P 4 ‐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 .