Structure–Activity Relationships of Pyrrolyl-Containing Diketo Acid and Non-Diketo Acid Derivatives as Inhibitors of SARS-CoV-2 nsp13-Associated Activities

Discovery of a new class based on 6-methoxypyrimidine as efficient low weight reversible inhibitors of SARS-CoV-2 main protease (Mpro) with antiviral activity.

The main protease Mpro of SARS-CoV-2 is an important enzyme for the viral cycle and its inhibition affects replication and infection propagation. In this way, it has been reported as an excellent target for drug design. In this work, we discovered a low molecular weight non-peptide, heterocyclic inhibitor, that inactivates Mpro. Initially, we screened a focused chemical library of heterocyclic compounds against Mpro that included several cysteine protease inhibitors. A compound based on a 6-methoxypyrimidine scaffold was identified as an inhibitor of this enzyme and structure-activity relationship studies revealed all the important groups for Mpro inhibition. Optimization of the hit compound provided a small (306 g/mol) competitive inhibitor that inactivated Mpro activity, being non-promiscuous to other cysteine proteases such as PLpro, human CatL, and cruzain. PH521 (4f) presented increased potency against SARS-CoV-2 infection of cells being 20-fold more potent than the initial hit compound.

Development of SARS-CoV-2 papain-like protease inhibitors with a thiadiazole and oxadiazole linker

The integrated rational design-based synthesis, biological evaluation, and computational investigations collectively identified thiadiazole/oxadiazole scaffolds as promising candidates for the development of SARS-CoV-2 inhibitors, offering valuable insights for next-generation antiviral agents targeting coronavirus proteases.

Design and synthesis of 1,3,4-oxadiazole-tethered N-substituted isatin hybrids as SARS-CoV-2 main protease inhibitors: biological evaluation, kinetic investigation, and in silico studies

The ongoing emergence of SARS-CoV-2 mutations underscores the urgent need for new antivirals that target key viral proteins. This study describes the design, synthesis, and evaluation of two series of 1,3,4-oxadiazole-tethered N-substituted isatin hybrids as inhibitors of the SARS-CoV-2 main protease (Mpro): 1,2,3-triazole-linked derivatives (9a–h) and pyrazole-linked derivatives (15a–d). Compounds 15a–c were identified as the most active derivatives in initial FRET-based screening. With an IC50 of 15.38 µM, 15c was the most effective inhibitor, as determined by subsequent enzymatic assays. 15a and 15b had IC50 values of 24.43 and 30.55 µM, respectively. The active chemicals inhibit Mprovia a noncompetitive/mixed mechanism (α = 0.41–0.44), at a KI value of 13.24 µM, according to enzyme kinetic studies. Additionally, the compounds exhibited favorable physicochemical and ADMET profiles and minimal cytotoxicity against normal IMR-90 cells. The stable predicted binding of the most active compound, 15c, within the Mpro active site was further supported by molecular docking and molecular dynamics simulations. Collectively, these findings identify 15c as the most active derivative in the present series and provide preliminary SAR insights that may guide further optimization of this scaffold as a potential class of SARS-CoV-2 Mpro inhibitors.

Synthesis of Novel Pyrazolopyranopyrimidine Acyclic Nucleosides Disubstituted at N2/C5 as Potential Anti-SARS-CoV-2 Inhibitors: Structural Characterization, and In Silico Studies

Identifying and Evaluating Flavonoids as Potential Inhibitors of SARS-CoV-2 Main Protease (Mpro/3CL) Through Docking and Molecular Dynamics

Findings highlight glycosylated flavonoids as promising scaffolds for future structure-based optimization and provide structural insights to guide experimental validation.

Discovery of piperazine-based multi-site occupying coronavirus Mpro inhibitors with potent and broad-spectrum antiviral activity.

The continuous evolution of SARS-CoV-2 and the emergence of drug-resistant variants underscore the urgent need for broad-spectrum antiviral agents targeting conserved viral proteins. The main protease (Mpro) represents a promising target due to its essential role in coronavirus replication. In this study, we report the discovery and optimization of a novel series of piperazine-based Mpro inhibitors using a multi-site binding strategy guided by analysis of conserved residues within the coronavirus Mpro active sites. Starting from the noncovalent lead GC-14, systematic optimization of substituents occupying the S1', S1, S2, and S4 subsites of Mpro led to the development of the noncovalent inhibitor GY-e2, which showed improved inhibitory efficacy against both SARS-CoV-2 and SARS-CoV Mpro. To further enhance its antiviral efficacy in cellular models, reactive warheads targeting C145 were incorporated into the scaffold to generate covalent inhibitors. This strategy yielded the isomeric compounds Y-U0-R and Y-U0-S, which displayed potent Mpro inhibition and markedly enhanced antiviral activity in SARS-CoV-2-infected Calu-3 cells. Moreover, both compounds exhibited broad-spectrum antiviral activity against other human coronaviruses, and notably remained effective against the two major Nirmatrelvir-resistant strains evaluated in this study. Mechanistic studies further confirmed kinetically stable binding and time-dependent inhibition of Y-U0-R, supporting the rationale of covalent inhibitor design. These findings highlight the utility of structure-based design for the development of promising broad-spectrum anti-coronavirus agents.