Design, synthesis and antiviral evaluation of a new thio-aminophenyl-bromopyrimidinone as an inhibitor of the SARS-CoV-2 viral enzyme Nsp15

The coronavirus disease 2019 (COVID-19) pandemic underscored the need for new antiviral agents effective against emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. Beyond the main protease (Mpro) and RNA-dependent RNA polymerase (RdRp), the uridine-specific endoribonuclease non-structural protein 15 (Nsp15, also termed NendoU) is an attractive, underexplored target due to its role in viral RNA processing and innate immune evasion. Here we report the design, synthesis and evaluation of a new tipiracil-inspired uracil derivative, 6-((2-aminophenyl)thio)-5-bromopyrimidine-2,4(1H,3H)-dione (compound 5), as a candidate Nsp15 inhibitor. Compound 5 was synthesized in three steps from 2,4,6-trichloropyrimidine, characterized by nuclear magnetic resonance (NMR) spectroscopy, and evaluated by molecular docking against Nsp15 (PDB ID: 9HH5 ), showing a more favorable interaction energy (−65.4 kcal mol −1 ) than tipiracil (−56.4 kcal mol −1 ). In silico toxicology (Deep-PK and ProTox 3.0) predicted low mutagenicity, carcinogenicity, hERG-channel blockade and eye-irritation risk, and a lower probability of respiratory toxicity than tipiracil. In Vero E6 cells, compound 5 was non-cytotoxic and protected both uninfected and SARS-CoV-2-infected cells, maintaining 72.6% and 68.1% viability, respectively, versus 22.0% in infected untreated controls, approaching the protection afforded by chloroquine. These results identify compound 5 as a promising, low-toxicity Nsp15-targeting scaffold that warrants further structure-activity relationship (SAR) optimization as a next-generation anti-coronavirus agent.