Carltonine B derivatives: Synthesis and structure-activity relationship insights for selective butyrylcholinesterase inhibition

Butyrylcholinesterase (BChE) is recognized as a promising therapeutic target for the late stages of Alzheimer's disease (AD) due to its role in the hydrolysis of acetylcholine (ACh), while acetylcholinesterase (AChE) activity declines during disease progression. Here, we have reported an efficient chemistry procedure for the naturally occurring Amaryllidaceae alkaloid carltonine B, along with the design and synthesis of 36 novel carltonine-based analogues to determine structure-activity relationship (SAR). Most of the synthesized compounds exhibited potent and selective human BChE ( h BChE) inhibition, with IC 50 values ranging from low micromolar to nanomolar concentrations. The drug-like properties of the molecules were assessed by in silico tools, using the blood-brain barrier (BBB) score algorithm, and subsequently validated by in vitro permeability assessment via parallel artificial membrane permeability assay (PAMPA). The derivatives exhibited potent h BChE inhibition in the low micromolar to submicromolar range, while their cytotoxicity against human neuroblastoma (SH-SY5Y) cells was observed only at higher micromolar concentrations, indicating a favorable safety profile. The synthesized alkaloid carltonine B ( 37 ) and its N -ethyl derivative ( 38 ) emerged as the most potent and selective h BChE inhibitors, with IC 50 values of 0.014 ± 0.002 μM and 0.013 ± 0.001 μM, respectively. Enzyme kinetic studies were conducted to elucidate the inhibition mechanism toward h BChE enzyme. Compound 37 demonstrated competitive inhibition with K i value of 0.055 μM. In contrast, compound 38 showed a noncompetitive inhibition profile, with a K i value of 0.067 μM. Molecular modeling suggested that the superior potency of compounds 37 and 38 arises from their more optimal engagement of the BChE active-site gorge compared to compound 33 . For the additional safety assessment, CYP inhibition assay revealed that compounds 37 and 38 may pose a risk of CYP3A4-mediated drug-drug interactions during chronic administration. • A scalable synthesis of carltonine B and 36 novel analogues was developed. • SAR analysis identified key structural features for selective h BChE inhibition. • Carltonine B derivatives showed micromolar to nanomolar h BChE inhibition with high selectivity. • Lead compounds displayed good BBB permeability and low neuronal cytotoxicity. • Kinetic studies revealed competitive and non-competitive h BChE inhibition modes.