Discovery and Characterization of 7,8-Dihydropyrido[4,3-d]pyrimidines as SARS-CoV-2 Entry Inhibitors

Background/Objectives: We have established a robust, cell-based high-throughput screening platform capable of identifying SARS-CoV-2 entry inhibitors within a BSL-2 facility. Methods: Using a curated compound library, we conducted a screening campaign that led to the discovery of potent viral entry inhibitors active in both pseudoviral and infectious SARS-CoV-2 inhibition assays. Results: Among those, Hit-1 exhibited submicromolar antiviral activity across all tested SARS-CoV-2 strains, including the highly transmissible Omicron subvariants. Biophysical binding assays confirmed that Hit-1 and related compounds directly engage the prefusion-stabilized SARS-CoV-2 spike proteins of both authentic WA1/2020 and Omicron viral strains. To elucidate potential binding orientations and interactions of the hit compounds with the SARS-CoV-2 spike protein, molecular docking studies were performed targeting two putative binding sites. Conclusions: Preliminary structure–activity relationship studies identified a promising subset of drug-like 7,8-dihydropyrido[4,3-d]pyrimidine-based inhibitors with potential for further development as novel therapeutic agents aimed at blocking viral entry and thereby preventing or mitigating SARS-CoV-2 infection. Among these, compound 13 stands out due to its superior in vitro potency and favorable pharmacokinetic properties, positioning it as a strong candidate for in vivo efficacy evaluation.