Alpha-ketoamides as broad-spectrum inhibitors of coronavirus and enterovirus replication

Linlin Zhang; Daizong Lin; Yuri Kusov; Yong Nian; Qingjun Ma; Jiang Wang; Albrecht von Brunn; Pieter Leyssen; Kristina Lanko; Johan Neyts; Adriaan de Wilde; Eric J. Snijder; Hong Liu; Rolf Hilgenfeld

Determination of the cell toxicity of candidate compounds. The CellTiter 96Aqueous One Solution Cell Proliferation Assay (MTS test, Promega), the CellTiter Glo assay kit (Promega), the Non-Destructive Cytotoxicity Bio-Assay (ToxiLight (measuring the release of adenylate kinase from damaged cells), Lonza Rockland), or the AlamarBlue™ Cell Viability Reagent (ThermoFisher) were used to determine the cytotoxic effect of compounds towards host cells according to the manufacturers' recommendations. 29, 65 Chemical synthesis of a-ketoamides General procedure. Reagents were purchased from commercial sources and used without purification. HSGF 254 (0.15 -0.2 mm thickness) was used for analytical thin-layer chromatography (TLC). All products were characterized by their NMR and MS spectra. 1 H NMR spectra were recorded on 300-MHz, 400-MHz, or 500-MHz instruments. Chemical shifts were reported in parts per million (ppm, δ) down-field from tetramethylsilane. Proton coupling patterns were described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and broad (br). Mass spectra were recorded using a Bruker ESI ion-trap HCT Ultra. HPLC spectra were recorded by LC20A or LC10A (Shimadzu Corporation) with Shim-pack GIST C18 (5 µm, 4.6x150mm) with three solvent systems (methanol/water, methanol/ 0.1% HCOOH in water or methanol/0.1% ammonia in water). Purity was determined by reversed-phase HPLC and was ≥95% for all compounds tested biologically.