The choice of target reflects a deliberate strategy to move beyond the two viral proteins that dominate the current antiviral arsenal. Approved and emergency-authorized drugs against COVID-19 largely fall into two mechanistic classes: inhibitors of the main protease Mpro, such as nirmatrelvir, and agents that attack the RNA-dependent RNA polymerase, including remdesivir and molnupiravir. While these drugs remain valuable, the continued evolution of SARS-CoV-2 has produced variants that partially escape vaccine-induced and infection-induced immunity and reduce the effectiveness of some monoclonal antibodies. Diversifying the antiviral toolkit by targeting additional conserved viral enzymes has therefore become a pressing priority for the field.

Nsp15, the uridine-specific endoribonuclease also known as NendoU, has attracted growing attention as exactly such a complementary target. The enzyme is conserved across nidoviruses and cleaves both single- and double-stranded RNA at uridine residues, generating 2′,3′-cyclic phosphodiester and 5′-hydroxyl termini. Beyond its role in viral RNA processing, Nsp15 helps the virus evade innate immune sensors by degrading viral double-stranded RNA intermediates that would otherwise trigger antiviral signaling. Genetic and biochemical studies indicate that this activity contributes to optimal viral replication and pathogenesis, which makes disabling it an attractive proposition for small-molecule drug design.

The design of compound 5 drew on structural work showing that tipiracil, an approved thymidine phosphorylase inhibitor, binds to the uridine recognition site of SARS-CoV-2 Nsp15 and suppresses its endoribonuclease activity. Although tipiracil alone does not fully block viral replication at clinically relevant concentrations, it provides a validated structural starting point. The Brazilian team, building on prior QSAR-guided virtual screening and on literature describing phenylthio-uracils as potent non-nucleoside inhibitors of HIV-1 reverse transcriptase, added a 2-aminophenylthio group at the C-6 position of the uracil core and a bromine atom at C-5. The rationale was that the bulky aromatic thioether would extend into a hydrophobic subpocket adjacent to the uracil-binding site, adding van der Waals and possible π-stacking contacts beyond those available to tipiracil’s pyrrolidinylmethyl substituent, while the electron-withdrawing bromine would tune the electronic character and hydrogen-bonding capacity of the pyrimidinedione core.

Synthesis proceeded in three steps from commercially available 2,4,6-trichloropyrimidine. Controlled hydrolysis under basic conditions selectively yielded 6-chlorouracil in 76 percent yield. Bromination at C-5 with elemental bromine then furnished 5-bromo-6-chlorouracil, albeit in a modest 31 percent yield. The final step, a nucleophilic aromatic substitution of 2-aminothiophenol onto the C-6 chloride in the presence of triethylamine under nitrogen, delivered compound 5 in 59 percent yield, for an overall yield of roughly 14 percent. Nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry confirmed the structure, with the observed molecular ion at m/z 313.9590 matching the calculated value of 313.9593 for the protonated molecule. The authors note that the route uses inexpensive reagents and is amenable to scale-up and diversification, for example by varying the arylthio group at C-6.

Computational docking against the crystal structure of Nsp15 deposited under Protein Data Bank entry 9HH5, resolved at 2.08 angstroms, supported the design hypothesis. Compound 5 achieved a total interaction energy of −65.4 kilocalories per mole, compared with −56.4 kilocalories per mole for tipiracil, with the advantage coming primarily from stronger van der Waals contacts (−54.4 versus −49.1 kilocalories per mole) and hydrogen bonding (−10.9 versus −7.3 kilocalories per mole). At the residue level, the compound engaged His235, Gly248, Trp333, Glu340, Thr341 and Tyr343, residues previously implicated in catalysis and uracil recognition. Trp333 in particular contributed a substantial van der Waals interaction of −12.5 kilocalories per mole plus a hydrogen bond of −3.5 kilocalories per mole with the uracil moiety. Visualization of the best-ranked poses confirmed that both ligands occupy the uracil-binding pocket in similar orientations, with the aminophenylthio substituent of compound 5 reaching into the adjacent hydrophobic subpocket. Docking runs were repeated in triplicate and poses were cross-checked by an independent evaluator.

In silico toxicology profiling using two independent platforms, Deep-PK and ProTox 3.0, suggested a generally favorable preliminary safety profile. Both compounds were predicted to be inactive or low-risk for Ames mutagenicity and carcinogenicity, and neither was flagged for significant eye irritation or blockade of the hERG potassium channel, a common cause of cardiac toxicity. Notably, tipiracil was predicted to carry a high probability of respiratory toxicity, whereas compound 5 was assigned a low probability for that endpoint. The authors caution that such machine-learning predictions do not replace experimental safety testing, but they argue that this kind of early computational triage can help minimize late-stage attrition in antiviral drug development, where rapid progression from hit to lead is critical.

The decisive test came in cell culture. Working with a Brazilian clinical isolate of SARS-CoV-2 in Vero E6 cells, the team used MTT-based viability assays to measure both cytotoxicity and antiviral protection. In uninfected cells treated with compound 5 at 20 milligrams per milliliter, viability was 72.6 percent, marginally below the commonly accepted 80 percent non-cytotoxic threshold and indicating low to moderate cytotoxicity at this high concentration. In infected cells, however, the picture was striking: treatment raised viability to 68.1 percent, roughly three times the 22 percent seen in infected vehicle controls and far above the 17 percent in PBS controls. Chloroquine diphosphate at 20 micromolar, the positive control, achieved 82 percent viability under the same conditions. The protective effect of compound 5 therefore approached, though did not match, that of the reference antiviral, consistent with an antiviral mechanism potentially linked to interference with Nsp15 function, although direct enzymatic inhibition was not measured in this study.

The authors are careful to delineate the limits of the work and the steps needed before the scaffold can advance. Antiviral activity was assessed at a single concentration in Vero E6 cells, a line widely used for coronavirus propagation but lacking certain components of the human innate immune response, so validation in human-derived systems such as Calu-3 cells, A549-ACE2 cells or primary airway epithelial cells will be essential. Quantitative enzymatic IC50 values against Nsp15, dose-response curves and selectivity indices remain to be determined, as do preliminary ADME studies and in vivo safety and efficacy testing. The field context, however, is encouraging: recent first-in-class thiazolidinedione and rhodanine Nsp15 inhibitors such as KCO237 have achieved sub-micromolar enzymatic potency, confirming that the uridine active site is druggable and that structurally diverse chemotypes are needed. With its accessible synthesis, favorable predicted binding to key catalytic residues, low-risk toxicological profile and demonstrable cell protection, compound 5 offers other laboratories a convenient, reproducible template for structure-activity relationship exploration, spanning modifications of the aryl ring, the amino group and the C-5 halogen, as new variants of the virus continue to emerge.

Subject of Research: Design and antiviral evaluation of a thio-aminophenyl-bromopyrimidinone inhibitor of the SARS-CoV-2 endoribonuclease Nsp15

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

Article References: Collin, H. P., Delevati, M. A., dos Santos, P. R., da Silva da Fonseca, G., Tasso, L., Reisdorfer, F., Gonzalez, L. C., Machado, P., Gonçalves, R. S. B., & Moura, S. (2026). Design, synthesis and antiviral evaluation of a new thio-aminophenyl-bromopyrimidinone as an inhibitor of the SARS-CoV-2 viral enzyme Nsp15. Results in Chemistry, 31, Article 103895. https://doi.org/10.1016/j.rechem.2026.103895

Image Credits: AI Generated

DOI: 10.1016/j.rechem.2026.103895

Keywords: SARS-CoV-2, Nsp15, endoribonuclease, uracil derivatives, tipiracil, antiviral drug discovery, molecular docking, Vero E6 cells, bromopyrimidinone, COVID-19, drug design, in silico toxicology