[reaction: see text] Asymmetric aldol addition reactions have been conducted with (1R,2S)-ephedrine-derived 3,4,5,6-tetrahydro-2H-1,3,4-oxadiazin-2-one (2). Diastereoselectivities range from 75:25 to 99:1 for the formation of the crude non-Evans syn adducts 8a-h. The facial selectivity of the enolate is directed by the stereogenic N(4)-methyl substituent. Aldol adduct 8a is readily cleaved by acid hydrolysis to afford (2S,3S)-3-hydroxy-2-methyl-3-phenylpropionic acid (9) in >95% ee.

Arenaviruses are a significant cause of hemorrhagic fever, an often-fatal disease for which there is no approved antiviral therapy. Lassa fever in particular generates high morbidity and mortality in West Africa, where the disease is endemic, and a recent outbreak in Nigeria was larger and more geographically diverse than usual. We are developing LHF-535, a small-molecule viral entry inhibitor that targets the arenavirus envelope glycoprotein, as a therapeutic candidate for Lassa fever and other hemorrhagic fevers of arenavirus origin. Using a lentiviral pseudotype infectivity assay, we determined that LHF-535 had sub-nanomolar potency against the viral envelope glycoproteins from all Lassa virus lineages, with the exception of the glycoprotein from the LP strain from lineage I, which was 100-fold less sensitive than that of other strains. This reduced sensitivity was mediated by a unique amino acid substitution, V434I, in the transmembrane domain of the envelope glycoprotein GP2 subunit. This position corresponds to the attenuation determinant of Candid#1, a live-attenuated Junín virus vaccine strain used to prevent Argentine hemorrhagic fever. Using a virus-yield reduction assay, we determined that LHF-535 potently inhibited Junín virus, but not Candid#1, and the Candid#1 attenuation determinant, F427I, regulated this difference in sensitivity. We also demonstrated that a daily oral dose of LHF-535 at 10 mg/kg protected mice from a lethal dose of Tacaribe virus. Serial passage of Tacaribe virus in LHF-535-treated Vero cells yielded viruses that were resistant to LHF-535, and the majority of drug-resistant viruses exhibited attenuated pathogenesis. These findings provide a framework for the clinical development of LHF-535 as a broad-spectrum inhibitor of arenavirus entry and provide an important context for monitoring the emergence of drug-resistant viruses.

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Previously we reported the optimization of antiviral scaffolds containing benzimidazole and related heterocycles possessing activity against a variety of arenaviruses. These series of compounds were discovered through an HTS campaign of a 400,000 small molecule library using lentivirus-based pseudotypes incorporated with the Lassa virus envelope glycoprotein (LASV GP). This screening also uncovered an alternate series of very potent arenavirus inhibitors based upon an acylhydrazone scaffold. Subsequent SAR analysis of this chemical series involved various substitutions throughout the chemical framework along with assessment of the preferred stereochemistry. These studies led to an optimized analog (ST-161) possessing subnanomolar activity against LASV and submicromolar activity against a number of other viruses in the Arenaviridae family.