Chiral Separation, X-ray Structure, and Biological Evaluation of a Potent and Reversible Dual Binding Site AChE Inhibitor

Abstract

Acetylcholinesterase (AChE) inhibitors (AChEIs) still remain the leading therapeutic options for the symptomatic treatment of cognitive deficits associated with mild-to-moderate Alzheimer's disease. The search for new AChEIs benefits from well-established knowledge of the molecular interactions of selective AChEIs, such as donepezil and related dual binding site inhibitors. Starting from a previously disclosed coumarin-based inhibitor (±)-cis-1, active as racemate in the nanomolar range toward AChE, we proceeded on a double track by (i) achieving chiral resolution of the enantiomers of 1 by HPLC and (ii) preparing two close achiral analogues of 1, i.e., compounds 4 and 6. An eudismic ratio as high as 20 was observed for the (-) enantiomer of cis-1. The X-ray crystal structure of the complex between the (-)-cis-1 eutomer (coded as MC1420) and T. californica AChE was determined at 2.8 Å, and docking calculation results suggested that the eutomer in (1R,3S) absolute configuration should be energetically more favored in binding the enzyme than the eutomer in (1S,3R) configuration. The achiral analogues 4 and 6 were less effective in inhibiting AChE compared to (±)-cis-1, but interestingly butylamide 4 emerged as a potent inhibitor of butyrylcholinesterase (BChE).

Figure 1

Ligand-based design of dual binding site inhibitors of AChE.

Scheme 1

Reagents and conditions: (a) 4-chlorobutyryl chloride, THF, triethylamine, rt; (b), benzylamine, KI, acetone, rt; (c), N-Boc protected isonipecotic acid, HOBt, DIC, CH₂Cl₂, rt; (d), TFA, CH₂Cl₂, 0 °C. (e), benzyl bromide, K₂CO₃, acetone, rt; (f) Boc₂O, THF, rt; (g) semipreparative chiral HPLC.

Figure 2

HepG2 cells viability, measured by the MTT assay, in the absence (black bar) and presence (gray bars) of (±)-cis-1. The percentage of MTT reduction observed is relative to control cells (DMEM). Values are expressed as mean ± SEM from six replicates, being significantly different from the control (untreated cells) as estimated by the Student’s t test (*p < 0.01).

Figure 3

Chromatograms of enantiopure samples obtained by chiral resolution of (±)-cis-7.

Figure 4

Michaelis–Menten plot for inhibition of hAChE by MC1420 at various inhibitor concentrations. The inset displays the corresponding Lineweaver–Burk plot.

Figure 5

X-ray structure of the MC1420/TcAChE complex (PDB ID: 6TT0). Data refined for the 1R,3S-cis- (A) and 1S,3R-cis- (B) configurations. The ligands and relevant amino acid residues are rendered as sticks, the water molecule W1 responsible for a water-mediated interaction with Phe288 is shown as a red sphere, while protein is represented as a cartoon.

Figure 6

Top-scored docking poses for the (1R,3S)-cis- (A) and (1S,3R)-cis- (B) configurational isomers of 1 within the binding site of TcAChE. The ligand itself, relevant amino acid residues, and the water molecule W1 responsible for water-mediated interaction with Phe288, are all rendered as sticks, while protein is represented as a cartoon. H-bonds are depicted by dotted lines.