A series of 21 novel coumarin–triazole–isatin hybrids was synthesized and evaluated for their potential as multitarget agents in Alzheimer’s disease (AD). The compounds featured variations in alkyl linker length that connects coumarin and triazole and substitution at the 5-position of the isatin ring. Several derivatives showed potent butyrylcholinesterase (BChE) inhibition with selectivity over acetylcholinesterase (AChE). The lead compound, 6c1, exhibited strong BChE inhibition (IC₅₀ = 1.74 μM), surpassing donepezil. Enzyme kinetics revealed a mixed-type mechanism, while molecular docking studies confirmed dual binding at catalytic and peripheral sites. Structure–activity relationship (SAR) analysis highlighted the influence of linker flexibility and steric/electronic effects of substituents. The observed BChE selectivity, combined with favorable in vitro profiles, identifies these hybrids as promising leads for AD drug development.

Background/Objectives: Oxidative metabolism can influence the development of bioactive compounds by affecting metabolic stability and cytochrome P450 (CYP) interactions. This study aimed to assess the CYP-related properties and oxidative susceptibility of 21 coumarin–1,2,3-triazole–isatin hybrids by integrating in silico prediction with online electrochemistry–mass spectrometry (EC–MS). Methods: All 21 derivatives were evaluated in silico for CYP substrate and inhibitor profiles and CYP-mediated transformations. Four structurally representative derivatives (6a1, 6b1, 6c1, and 6c2) were selected for analysis by directly coupled online electrochemistry–mass spectrometry (EC–MS). Compounds 6a1, 6b1, and 6c1 formed a C2–C4 homologous series, whereas 6c2 was the 5-fluoro analogue of 6c1. Results: CYP3A4 was the isoform most consistently associated with substrate classification, while CYP3A4 inhibition probabilities remained close to the classification threshold. GLORY identified aromatic hydroxylation as the predominant top-ranked transformation across the series. Linker length had little influence on the principal predicted oxidation sites. EC–MS revealed a conserved principal oxidation pattern across the C2–C4 homologues, including +16 Da ions and two lower-mass ion series showing systematic 14 Da shifts with each additional methylene group, compatible with monooxygenation, oxidative ether cleavage, and N-dealkylation. In the 6c1/6c2 pair, 5-fluoro substitution removed the second-ranked isatin 5-position hydroxylation predicted for 6c1, leaving the three highest-ranked predictions for 6c2 on the coumarin ring, and was accompanied by a selective 18 Da shift in an isatin-containing electrochemical oxidation product. Conclusions: The combined in silico and EC–MS approach identified structurally dependent predicted CYP-related properties and electrochemical oxidative susceptibility within this series. The findings support further evaluation of 6c1 and related analogues using biological metabolism models to establish their metabolic stability and CYP-mediated transformation pathways.