Azapeptide-based SARS-CoV-2 Main Protease Inhibitors: Design, Synthesis, Enzyme Inhibition, Structural Determination, and Antiviral Activity

The main protease (Mpro) of SARS-CoV-2 plays a crucial role in the replication and pathogenesis of the virus. The relatively high conservation within the Coronaviridae family makes it an attractive therapeutic target for developing broad-spectrum agents to prevent and control SARS-CoV-2 and potentially future coronavirus-associated diseases. This study describes the design, synthesis, and structure-activity relationships of azapeptide-based SARS-CoV-2 Mpro inhibitors, leading to several compounds with low nanomolar IC50 values. Examples include 14r (IC50 = 13.3 nM), 14s (IC50 = 30.6 nM), 20a (TPG-25-1, IC50 = 34.7 nM), 20g (IC50 = 30.4 nM), and 20h (IC50 = 31.3 nM). Some compounds inhibit MERS-CoV and SARS-CoV-1 Mpro but not the human protease cathepsin L. Several inhibitors, particularly 20a and 20f, exhibit antiviral activity with potencies comparable to nirmatrelvir and were highly potent against the E166V-carrying SARS-CoV-2 variant (SARS-CoV-2E166V). An Mpro co-crystal structure with 20a shows a covalent adduct with the catalytic Cys145. Overall, these new inhibitors are promising chemical tools that may contribute to the identification of future pan-anti-coronaviral drugs.