Selective Covalent Targeting of SARS-CoV-2 Main Protease by Enantiopure Chlorofluoroacetamide

28 September 2021, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


The pandemic of COVID-2019 has urged the development of antiviral agents against its causative pathogen SARS-CoV-2. The main protease (Mpro), a cysteine protease essential for viral replication, is a promising protein target. Here we report an irreversible SARS-CoV-2 Mpro inhibitor possessing chlorofluoroacetamide (CFA) as the warhead for covalent modification of Mpro. Ugi multi-component reaction employing chlorofluoroacetic acid allowed rapid generation of CFA derivatives, of which diastereomers displayed significantly different inhibitory activity against Mpro. We established a practical protocol for the optical resolution of chlorofluoroacetic acid, which enable the isolation of the stereoisomers of the best CFA compound. Kinetic analysis revealed that (R)-CFA is crucial for both binding affinity and the rate of irreversible inactivation of Mpro. Our findings highlight the prominent influence of the CFA chirality on the covalent modification of cysteine, and provide the basis for improving the potency and selectivity in the development of novel CFA-based covalent inhibitors.


main protease
Ugi multi-component reaction

Supplementary materials

Supporting Information
Supplementary Figures, synthetic procedures and spectral data for compounds.


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