Structural Analysis of HRF-4467, a Potent Second-Generation HIV-1 Maturation Inhibitor

HRF-4467 is a betulinic acid-derived HIV-1 maturation inhibitor that targets the canonical CA-SP1 region and blocks the final proteolytic step of viral maturation. Here, we characterize HRF-4467 and its para-fluoro analog using solution and solid-state NMR spectroscopy, supported by quantum-mechanical calculations. Using this approach, we established the molecular connectivity and conformation of HRF-4467 in solution and in the solid state. Introducing a para-fluorine substitution produces localized changes within the aromatic moiety without substantially altering the rest of the molecular scaffold. This work establishes the para-fluoro derivative as a structurally representative analog of HRF-4467 and highlights 19 F NMR spectroscopy as a powerful tool for directly investigating inhibitor conformation and molecular recognition within the HIV-1 capsid maturation intermediate assemblies.