Free-Energy and Position-Dependent Diffusion Profiles Shape Passive Permeation of a Semisynthetic Naphthoquinone across a Model Lipid Bilayer

Abstract Passive membrane permeation depends on both solute partitioning and coordinate-dependent mobility, but their separate and spatial-coupling contributions are rarely quantified within a common resistance framework. CNFD (6b,7-dihydro-5H-cyclopenta[b]naphtho[2,1-d]furan-5,6(9aH)-dione), a semisynthetic naphthoquinone with low-micromolar cytotoxicity and tumor-growth inhibition in preclinical models, was examined in a four-component model lipid bilayer. Established simulated tempering-enhanced umbrella sampling, WHAM, the Hummer positional-autocorrelation formalism, and the inhomogeneous solubility-diffusion model were combined. Three independent replicas yielded favorable interfacial partitioning (−4.66 kJ mol–1) and a positive membrane-center free energy (+12.36 kJ mol–1). Controlled profile substitutions showed that PMF heterogeneity and position-dependent diffusion amplified CNFD resistance by factors of 10.54 and 14.89, respectively, whereas their residual spatial-coupling factor was 1.134. The PMF maximum and diffusion minimum were separated by 2.14 nm. The primary intrinsic Peff was 9.09 × 10–2 cm s–1. Applying the same analysis to β-lapachone and plumbagin identified contrasting resistance regimes, with plumbagin combining a favorable center PMF with much lower central resistance localization. These results show how spatially separated free-energy and mobility landscapes govern intrinsic lipid-phase permeation.