A strategy for improving blood-brain barrier permeability of ChE inhibitors through blood-brain barrier (BBB) transporter.

Overcoming the blood-brain barrier (BBB) remains a pivotal challenge in central nervous system (CNS) drug development. Here, we present an active CNS-targeted strategy based on BBB endogenous transporters, specifically through the rational design of novel tacrine derivatives. This study pioneers the design and synthesis of two novel series of centrally-targeted tacrine derivatives, achieved by incorporating a quaternary ammonium group targeting the choline transporter and an amino acid fragment targeting the L-type amino acid transporter 1 (LAT1). These compounds maintained potent inhibitory activity against both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Using an in vitro BBB model (bEnd.3 cells), compound C4 demonstrated a significantly higher apparent permeability coefficient (Papp) of (19.58 ± 1.17) × 10-6 cm s-1 compared to tacrine ((13.41 ± 0.58) × 10-6 cm s-1), indicating enhanced BBB penetration. Interestingly, compound L3 showed a Papp value ((11.25 ± 3.12) × 10-6 cm s-1) similar to tacrine in vitro but exhibited unexpectedly high brain exposure in subsequent in vivo experiments. Its poor passive diffusion, confirmed by parallel artificial membrane permeability assays and poor lipophilicity, suggests the involvement of an LAT1-mediated active transport pathway. Furthermore, competitive inhibition assays demonstrated the underlying mechanism that the transport specificity of the preferred compounds L3 and C4 is mediated by LAT1 and CHT, respectively. Induced-fit molecular docking experiments further validated the high affinity of L3 for LAT1. In conclusion, this work not only provides lead compounds with high BBB permeability but also advances our understanding of utilizing endogenous transporters for brain-targeted delivery, offering a solid foundation for developing new CNS therapeutics.