Rapid HPLC-Based ADME Profiling of Novel Donepezil and Rivastigmine Analogues for Potential Application in Alzheimer’s Disease

Abstract Multitarget ligands derived from established cholinesterase inhibitors offer a strategy for addressing the multifactorial pathology of Alzheimer’s disease, but biological potency must be considered together with developability. In this study, a rapid biomimetic high-performance liquid chromatography-based workflow was used to characterize key absorption, distribution, metabolism and excretion-related properties of 26 donepezil- and rivastigmine-derived hybrid compounds, including plasma stability, plasma protein binding, membrane affinity, biomimetic distribution parameters, blood–brain barrier permeability, intestinal absorption, experimental log P and aqueous solubility. Plasma stability was reassessed using first-order regression across seven time points with 95% confidence intervals. Most profiles showed limited degradation during the 24 h experiment; accordingly, many apparent half-lives required extrapolation and are interpreted as comparative stability parameters rather than estimates of in vivo persistence. Human serum albumin and α1-acid glycoprotein binding was generally high, emphasizing the need to balance protein binding with the unbound fraction, while immobilized artificial membrane-based models predicted central nervous system-positive classification and high intestinal absorption across the series. Importantly, the blood brain barrier permeability and CNS-distribution estimates are derived from biomimetic models and should be regarded as predictive descriptors that require confirmation in dedicated in vivo pharmacokinetic studies. Solubility was more variable and emerged as an important differentiating property, with compound (26) showing the most favorable profile. An exploratory desirability-based ADME score placed compounds (15), (8), (26), (4), (10) and (24) in the highest tier. Integration with previously reported cholinesterase, fatty acid amide hydrolase and monoamine oxidase activities, which were not remeasured in the present study, further refined lead selection: (24) provided the clearest potency-developability compromise and was prioritized as the principal lead, whereas (26) emerged as a developability-oriented colead. (3) and (18) remained potency-driven secondary candidates. Overall, combining biomimetic ADME profiling with existing pharmacological evidence provides a transparent framework for early lead prioritization and supports confirmatory pharmacokinetic, exposure and in vivo efficacy studies.