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

Abstract

Background

Lower respiratory tract infections are a major global cause of death, with around 2.5 million deaths recorded in 2021. A substantial proportion of these infections are viral in origin, primarily caused by pathogens such as influenza virus, respiratory syncytial virus (RSV), and SARS-CoV-2.

Objective

The present study aimed to design and synthesize novel pyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidine acyclonucleosides bearing HBG-derived pseudosugar moieties as potential antiviral agents targeting key SARS-CoV-2 proteins.

Methods

A series of novel derivatives 2–8 was synthesized in satisfactory yields through a multi-step synthetic strategy involving the construction of a fused pyrazolo-pyrano-pyrimidine scaffold incorporating a pyran ring as a spacer between the pyrazole and pyrimidine fragments. All synthesized compounds were structurally characterized using advanced spectroscopic techniques, including ¹H, ¹³C, and DEPT-135 NMR spectroscopy, and high-resolution mass spectrometry (HRMS). The compounds were computationally evaluated against MPro, PLPro, and RdRp using molecular docking, molecular dynamics simulations, MM-GBSA binding free energy calculations, and AD-MET analysis.

Results

The computational results revealed stable ligand–protein interactions and favourable binding profiles compared with the co-crystallized ligands (CCLs). In MPro, ligands 3S and 7S formed key interactions with several active-site residues (N142, M49, M165, G143, and E166), among which N142, M49, and G143 were common to both ligands. Notably, N142 contributed most significantly to ligand binding. In PLPro, both ligands (2S and 8R) formed stable π–π interactions with Y268, enhancing complex stability and contributing more than 7 kcal/mol to the binding free energy. Other major contributing residues included Y264, P247, and P248. In RdRp, residues Y38, F45, and Y728 were critical for ligand stabilization, contributing more than 8 kcal/mol to the binding free energy. MM-GBSA energies confirmed strong binding of 3S and 7S to MPro, outperforming the CCL, while 2S and 1R exhibited binding affinities comparable to those of the CCLs in PLPro and RdRp, respectively.

Conclusion

The ADMET profiles were favourable, suggesting that these ligands are promising candidates for further experimental validation as potential SARS-CoV-2 antiviral agents.