In vitro and computational investigations of novel synthetic carboxamide-linked pyridopyrrolopyrimidines with potent activity as SARS-CoV-2-MPro inhibitors

Abstract

An essential target for COVID-19 is the main protease of SARS-CoV-2 (M^pro). With the objective of targeting this receptor, a novel set of pyrido[1,2-a]pyrrolo[2,3-d]pyrimidines with terminal carboxamide fragments was designed, synthesized, and considered as an initial motif for the creation of effective pan-coronavirus inhibitors. Accordingly, nine derivatives (21-29) have been introduced for in vitro assay to evaluate their antiviral activity and cytotoxicity effect against COVID-19 virus using Vero cells. The obtained data revealed that the majority of these derivatives showed potent cellular anti-COVID-19 activity and prevent viral growth by more than 90% at two different concentrations with weak or even no detectable cytotoxic effect on Vero cells. Extensive molecular docking simulations highlighted proper non-covalent interaction of new compounds within the binding pocket of M^pro as a potential target for their antiviral activity. In vitro assay for all the synthesized derivatives against the viral M^pro target indicated that compounds 25 and 29 have promising inhibitory activity with IC₅₀ values at low micromolar concentrations. The molecular dynamic simulation results predicted the stability of compound 29 in the binding cavity of SARS-CoV-2 M^pro and hence supported the high inhibitory activity shown by the In vitro assay. These results suggested that compounds 25 and 29 merit further investigations as promising drug candidates for the management of SARS-CoV-2.

Fig. 1. Frequently prescribed antiviral drugs and recently identified molecules with prominent activity against SARS-CoV-2.

Fig. 2. Carboxamide, pyridine, and pyrimidine incorporating molecules with potent SARS-CoV-2-M^Pro antagonistic activity.

Fig. 3. Molecular design and rationale analysis for the binding interactions of novel pyridopyrrolopyrimidines within active pocket of SARS-CoV-2-M^Pro.

Scheme 1. Synthetic protocol of pyrido[1,2-a]pyrrolo[2,3-d]pyrimidine derivatives (21–29).

Fig. 4. SAR analysis of target compounds correlated with M^pro protease activity.

Fig. 5. 2D and 3D binding interaction data of active 29 analog (A and B) compared to Co-Crystalized ligand (C and D) in active site of SARS-CoV 2 M^pro enzyme with PDB entry code 7L11. Color-coding scheme for interaction includes polar hydrogen bonding interaction as blue or green arrows and non-polar aromatic type as green lines mediated with small aromatic ring.

Fig. 6. MD simulation analysis of 29–SARS-CoV-2 M^pro (A) RMSD (Protein RMSD is presented in grey while RMSD of compound 29 are presented in red color) (B) protein amino acids RMSF (C) 2D ligand interaction diagram and (D) protein–ligand contact analysis of MD trajectory.