3D Electron Diffraction Elucidates the Structure of a 1,3,5-Triazin-2,4-dione Cinnamamide Covalent Inhibitor of SARS-CoV‑2 3CL^pro

Eleven covalent 1,3,5-triazin-2,4-dione cinnamamides (<b>1a</b>–<b>1k</b>) bearing a Cys145-directed α,β-unsaturated Michael acceptor were synthesized and evaluated against SARS-CoV-2 3CL^pro. <b>1a</b> showed the highest potency (IC₅₀ = 92.45 nM) and was further characterized by structural and docking analyses.

To determine its crystal structure, we applied three-dimensional electron diffraction (3DED), which enabled atomic-resolution analysis of nanocrystals under ultralow dose conditions. Integration of three data sets improved completeness from 58.2% to 91.0%, yielding a refined structural model at 0.85 Å resolution. The resulting structure provides an experimentally derived model of the molecular conformation and solid-state organization of compound <b>1a</b> and illustrates the geometry of the α,β-unsaturated cinnamamide moiety in the crystalline state.