Discovery of Potent Pyrazoline‐Based Covalent SARS‐CoV‐2 Main Protease Inhibitors**

Abstract

While vaccines and antivirals are now being deployed for the current SARS‐CoV‐2 pandemic, we require additional antiviral therapeutics to not only effectively combat SARS‐CoV‐2 and its variants, but also future coronaviruses. All coronaviruses have relatively similar genomes that provide a potential exploitable opening to develop antiviral therapies that will be effective against all coronaviruses. Among the various genes and proteins encoded by all coronaviruses, one particularly “druggable” or relatively easy‐to‐drug target is the coronavirus Main Protease (3CLpro or Mpro), an enzyme that is involved in cleaving a long peptide translated by the viral genome into its individual protein components that are then assembled into the virus to enable viral replication in the cell. Inhibiting Mpro with a small‐molecule antiviral would effectively stop the ability of the virus to replicate, providing therapeutic benefit. In this study, we have utilized activity‐based protein profiling (ABPP)‐based chemoproteomic approaches to discover and further optimize cysteine‐reactive pyrazoline‐based covalent inhibitors for the SARS‐CoV‐2 Mpro. Structure‐guided medicinal chemistry and modular synthesis of di‐ and tri‐substituted pyrazolines bearing either chloroacetamide or vinyl sulfonamide cysteine‐reactive warheads enabled the expedient exploration of structure‐activity relationships (SAR), yielding nanomolar potency inhibitors against Mpro from not only SARS‐CoV‐2, but across many other coronaviruses. Our studies highlight promising chemical scaffolds that may contribute to future pan‐coronavirus inhibitors.

Targeted Protein Degradation through Recruitment of the CUL4 Complex Adaptor Protein DDB1

“…33 As such, we also tested whether a more metabolically suitable warhead could be accommodated. While warhead swapping often compromises the activity of covalent ligands, 34 we found that the acrylamide warheadbearing counterpart of MM-02-08, MM-04-09, still bound to DDB1 and degraded BRD4 in cells (Figure 5D,F). AR Degradation Using MM-02-57-Based PROTACs.…”

On the origins of SARS-CoV-2 main protease inhibitors

“…The strong SARS-CoV-2 M pro binding inhibitor and vinylsulfoxide-bearing PM-2-071 ( 144 ) was reported following a screen of 582 acrylamide or chloroacetamide-bearing compounds and some hit optimization. 400 However, the toxicity issue of vinylsulfoxide-bearing compounds should be kept in mind for further development of this type of Michael acceptor. 184 The α-chloroamide-bearing benzodiazepine 145 was also found to be a modest inhibitor of SARS-CoV-2 M pro following the screening of 5000 compounds.…”

Recent Advances in SARS-CoV-2 Main Protease Inhibitors: From Nirmatrelvir to Future Perspectives

“…Afterwards, Moon and co-workers carried out an exhaustive SAR analysis of this class of derivatives by exploring the effect of different substituents at the C-4 pyrazoline-ring. This work led to the discovery of compound 68, called cis-HW-2-010B, which showed inhibitory activity against SARS-CoV-2 M pro in the nanomolar range (IC 50 = 14 nM; Figure 42) [79]. In another study, El Khoury Léa et al applied advanced in silico techniques for the design and synthesis of Other interesting non-peptidyl SARS-CoV-2 M pro inhibitors containing an α-chloroacetamide warhead are represented by the pyrazoline-based compounds 67 and 68 and the difluorinated amide 69 (Figure 42).…”

“…In another study, El Khoury Léa et al applied advanced in silico techniques for the design and synthesis of Other interesting non-peptidyl SARS-CoV-2 M pro inhibitors containing an α-chloroacetamide warhead are represented by the pyrazoline-based compounds 67 and 68 and the difluorinated amide 69 (Figure 42). Compound 67, called (R)-EN82, was developed by Moon et al and underwent activity-based protein profiling, a preliminary screening test applied on a library of 582 chloroacetamides and acrylamides [79]. From this first screening, four compounds emerged as promising antiviral candidates.…”