Discovery of C5-Substituted 2′-Fluorinated Pyrimidine Nucleosides as Potent and Orally Efficacious Anti-HSV Agents

Herpes simplex virus (HSV) infections remain a significant global health concern. Through phenotypic screening of a nucleoside-focused library followed by systematic structure−activity relationship (SAR) explorations, we identified C5-substituted 2′-fluorinated pyrimidine nucleosides as promising anti-HSV scaffolds. We found that introducing five-membered heteroaryls at the uracil C5 position significantly enhanced antiviral potency, whereas cytosine and alkynyl derivatives showed limited efficacy. The optimized lead, B10, exhibited potent in vitro inhibition against HSV-1 (EC50 = 0.60 μM) and HSV-2 (EC50 = 7.32 μM) with minimal cytotoxicity. Importantly, B10 demonstrated a highly favorable oral bioavailability of 48.0% in rats. In a murine intranasal HSV-1 infection model, oral administration of B10 significantly improved survival rates, alleviated weight loss, suppressed acute viral replication in the central nervous system, and reduced pulmonary viral titers. These findings highlight the 2′-fluoro-C5-heteroaryl uracil framework as a robust chemotype and establish B10 as an orally efficacious candidate for HSV therapy.