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.

SARS-CoV-2 Mpro Inhibitors: Achieved Diversity, Developing Resistance and Future Strategies

“…A lead derivative from this series, 7, with a p-pentafluorosulfanyl phenyl group in P2 and a 5-pyrimidinyl group in P1, has an IC50 of 0.056 µM (Ki = 1.34 µM) and acts selectively only in its (R,R)-cis configuration. More recently, novel pyrazoline skeleton derivatives bearing a chloroacetamide warhead also showed promising SARS-CoV-2 M pro inhibition [35]. A lead compound from this series, 8, has an IC50 of 0.53µM in its R-configuration, while the S-enantiomer is five times less active.…”

Targeting SARS-CoV-2 Main Protease for Treatment of COVID-19: Covalent Inhibitors Structure–Activity Relationship Insights and Evolution Perspectives

“…Extensive SAR analyses on C-4-substituted compounds led to the isolation of the optimized trisubstituted cis derivative HW-2-010B (IC 50 against the target protein in the nanomolar range, 14 nM) ( Figure 36 ). 84 …”

“…Chemical structures of pyrazoline-based compounds ( R )-EN82, HW-2-010B, and QUB-00006-Int-07 as SARS-CoV-2 M PRO inhibitors. , …”

“…Extensive SAR analyses on C-4-substituted compounds led to the isolation of the optimized trisubstituted cis derivative HW-2-010B (IC 50 against the target protein in the nanomolar range, 14 nM) (Figure 36). 84 The application of advanced in silico techniques, such as highresolution all-atom molecular dynamics simulations and absolute binding free energy calculations applied on SARS-CoV-2 M PRO , enabled the design of the unusual cyclic α,α- difluoro-amide QUB-00006-Int-07 as a covalent inhibitor, which showed an IC 50 of 0.83 μM (Figure 36). 85 2.3.…”

Large library docking for novel SARS-CoV-2 main protease non-covalent and covalent inhibitors

“…Among them is the orally active MPro inhibitor S-217622 7 , which has entered clinical trials. Other inhibitors show much promise 4,[8][9][10][11][12][13][14][15][16] , including a non-covalent MPro inhibitor from the international Covid-19 Moonshot consortium that may be characterized as an advanced pre-clinical candidate [17][18][19] , and more experimental molecules that are relatively potent but have not proceeded far from hit to lead 20 . Notwithstanding these successes, both the resistance that may be expected to emerge 21,22 , and the inevitable liabilities of the early drugs support the discovery of new scaffolds.…”