Pyridine-Fused C ₂₅ <i>abeo</i> -Steroid Alkaloids from a Fungus: Biomimetic Semisynthesis and Anti-HIV Mechanistic Study

With the employment of feature-based molecular networking-guided screening of a refined reference library, four unconventional conjugates, pyricitristerols A-D (<b>1</b>-<b>4</b>), pyridine-fused C₂₅ <i>abeo</i>-steroids featuring a novel 6/7/7/6/5-fused framework, were isolated from a plant-sourced fungus <i>Penicillium citrinum</i> YSC-1. To account for the pyridine moiety, a biosynthetical route was proposed and substantiated by a concise four-step biomimetic semisynthesis from readily prepared precursors <b>5</b>-<b>8</b>, highlighted by a defining gold-catalyzed one-pot condensation that forged the core architecture. Biological evaluation revealed that <b>1</b>-<b>4</b> potently inhibit HIV-1 replication (IC₅₀ = 0.9-3.6 μM), and structure-activity analysis showed that the pyridine moiety served as the core pharmacophore, while C-23/C-24 stereochemistry finely tuned activity. Mechanistic investigations integrating time-of-addition/washout assays and molecular docking simulations demonstrated these compounds as early stage HIV-1 entry inhibitors that target host-cell surface components, potentially including receptors, such as CCR5, with the pyridine core and C-23 hydroxy group directly engaging in specific binding interactions.