Synthesis and In Vivo Antifungal Evaluation of 3-Acyl-bromoindole Regioisomers: A Multi-Targeting Study on Postharvest Pathogen Control and Molecular Dynamics

Background/Objectives: Postharvest fungal decay caused by Botrytis cinerea and Monilinia fructicola poses major threats to global fruit security. Driven by the need for sustainable crop protection agents, this work presents the systematic synthesis, biological evaluation, and computational modeling of a comprehensive 33-compound library of 3-acyl-bromoindole regioisomers (series 4a–k, 5a–k, and 6a–k) to establish clear structure–activity relationship (SAR) design rules. Methods: The regioisomeric library was assembled via a microwave-assisted catalytic protocol in an ionic liquid, expanding the known chemical space with seven newly synthesized 4-bromoindole derivatives (4d–f, 4h–k). Primary in vitro data were modeled using Hansch QSAR and Principal Component Analysis (PCA). Postharvest in vivo efficacy was evaluated on fresh ‘Lapins’ sweet cherries inoculated with M. fructicola. Molecular docking and 100 ns molecular dynamics (MD) simulations were performed against succinate dehydrogenase (SDH) and M. fructicola catalase 2 (MfCat2). Results: In vitro screening demonstrated marked target selectivity: parent core 4 displayed high mycelial suppression against M. fructicola (EC50 = 7.05 µg/mL), whereas C3-acylation with a four-carbon linear chain (4c) achieved optimal broad-spectrum dual action (98% and 86% spore germination inhibition). In vivo cherry bioassays proved that bromoindoles 4, 6a, and 6d significantly suppressed Brown Rot severity to 44–47% (a 20–27% reduction vs. untreated control). Docking and MD trajectories confirmed stable multi-target binding within SDH and MfCat2 active sites (RMSD < 2.0 Å). Conclusions: Bromine regiochemistry dictates pathogen selectivity and life-stage targeting. The novel 4-bromoindole derivatives and multi-target profile establish these scaffolds as promising leads for postharvest crop protection.