The SARS-CoV-2 3CL protease is a critical drug target for small molecule COVID-19 therapy, given its likely druggability and essentiality in the viral maturation and replication cycle. Based on the conservation of 3CL protease substrate binding pockets across coronaviruses and using screening, we identified four structurally distinct lead compounds that inhibit SARS-CoV-2 3CL protease. After evaluation of their binding specificity, cellular antiviral potency, metabolic stability, and water solubility, we prioritized the GC376 scaffold as being optimal for optimization. We identified multiple drug-like compounds with <10 nM potency for inhibiting SARS-CoV-2 3CL and the ability to block SARS-CoV-2 replication in human cells, obtained co-crystal structures of the 3CL protease in complex with these compounds, and determined that they have pan-coronavirus activity. We selected one compound, termed coronastat, as an optimized lead and characterized it in pharmacokinetic and safety studies in vivo. Coronastat represents a new candidate for a small molecule protease inhibitor for the treatment of SARS-CoV-2 infection for eliminating pandemics involving coronaviruses.

We describe a mammalian cell-based assay to identify coronavirus 3CL protease (3CLpro) inhibitors. This assay is based on rescuing protease-mediated cytotoxicity and does not require live virus. By enabling the facile testing of compounds across a range of 15 distantly related coronavirus 3CLpro enzymes, we identify compounds with broad 3CLpro inhibitory activity. We also adapt the assay for use in compound screening and in doing so uncover additional SARS-CoV-2 3CLpro inhibitors. We observe strong concordance between data emerging from this assay and those obtained from live virus testing. The reported approach democratizes the testing of 3CLpro inhibitors by developing a simplified method for identifying coronavirus 3CLpro inhibitors that can be used by the majority of laboratories, rather than the few with extensive biosafety infrastructure. We identify two lead compounds, GC376 and compound 4, with broad activity against all 3CL proteases tested including 3CLpro enzymes from understudied zoonotic coronaviruses. Importance Multiple coronavirus pandemics have occurred over the last two decades. This has highlighted a need to be proactive in the development of therapeutics that can be readily deployed in the case of future coronavirus pandemics. We develop and validate a simplified cell-based assay for the identification of chemical inhibitors of 3CL proteases encoded by a wide range of coronaviruses. This assay is reporter-free, does not require specialized biocontainment, and is optimized for performance in high-throughput screening. By testing reported 3CL protease inhibitors against a large collection of 3CL proteases with variable sequence similarity, we identify compounds with broad activity against 3CL proteases and uncover structural insights that contribute to their broad activity. Furthermore, we demonstrate this assay is suitable for identifying chemical inhibitors of proteases from families other than 3CL proteases.

We have developed a formal [4+1] approach to pyrrolidines from readily available unactivated terminal alkenes as 4-carbon partners. The reaction provides a rapid construction of various pyrrolidine containing structures, especially for the diastereoselective synthesis of spiro-pyrrolidines. Mechanistic investigation suggests a Rh(III)-catalyzed intermolecular aziridination of the alkene and subsequent acidpromoted ring expansion for the pyrrolidine formation.

Herein we report a Rh(III)-catalyzed three-component carboamination of alkenes from readily available aryl boronic acids as a carbon source and dioxazolones as nitrogen electrophiles. This protocol provides facile access to valuable amine products including a-amino acid derivatives in good yield and excellent regioselectivity without the need for a directing functionality. A series of experiments suggest a mechanism in which the Rh(III) catalyst undergoes transmetalation with the aryl boronic acid followed by turnover limiting, alkene migratory insertion into the Rh(III)-aryl bond. Subsequently, fast Rh-nitrene formation provides the syn-carboamination product selectively after reductive elimination and proto-demetalation. Importantly, the protocol provides 3-component coupling products in preference to a variety of 2-component undesired by-products. File list (2) download file view on ChemRxiv article_ChemRxiv.pdf (1.36 MiB) download file view on ChemRxiv SI_ChemRxiv.pdf (8.32 MiB) 1 Rh(III)-Catalyzed Three-Component Syn-Carboamination of Alkenes Using Arylboronic Acids and Dioxazolones

We report a RhIII‐catalyzed regio‐ and diastereoselective synthesis of δ‐lactams from readily available acrylamide derivatives and unactivated alkenes. The reaction provides a rapid route to a diverse set of δ‐lactams in good yield and stereoselectivity, which serve as useful building blocks for substituted piperidines. The regioselectivity of the reaction with unactivated terminal alkene is significantly improved by using Cpt ligand on the RhIII catalyst. The synthetic utility of the reaction is demonstrated by the preparation of a potential drug candidate containing a trisubstituted piperidine moiety. Mechanistic studies show that the reversibility of the C−H activation depends on the choice of Cp ligand on the RhIII catalyst. The irreversible C−H activation is observed and becomes turnover‐limiting with [CptRhCl2]2 as catalyst.