Inhibiting Acetylcholinesterase to Activate Pleiotropic Prodrugs with Therapeutic Interest in Alzheimer's Disease

Abstract

Alzheimer's disease (AD) is a multifactorial neurodegenerative disease which is still poorly understood. The drugs currently used against AD, mainly acetylcholinesterase inhibitors (AChEI), are considered clinically insufficient and are responsible for deleterious side effects. AChE is, however, currently receiving renewed interest through the discovery of a chaperone role played in the pathogenesis of AD. But AChE could also serve as an activating protein for pleiotropic prodrugs. Indeed, inhibiting central AChE with brain-penetrating designed carbamates which are able to covalently bind to the enzyme and to concomitantly liberate active metabolites in the brain could constitute a clinically more efficient approach which, additionally, is less likely to cause peripheral side effects. We aim in this article to pave the road of this new avenue with an in vitro and in vivo study of pleiotropic prodrugs targeting both the 5-HT₄ receptor and AChE, in order to display a neuroprotective activity associated with a sustained restoration of the cholinergic neurotransmission and without the usual peripheral side effects associated with classic AChEI. This plural activity could bring to AD patients effective, relatively safe, symptomatic and disease-modifying therapeutic benefits.

Keywords: 5-HT4 receptors; Alzheimer’s disease; MTDL; acetylcholinesterase; prodrug.

Figure 1

Illustration of the pleiotropic prodrug concept.

Figure 2

Structure of Ladostigil (1), Donecopride (2), Rivastigmine (3), phenolic derivatives (4–6) of Donecopride and targeted pleiotropic prodrugs (7–9).

Scheme 1

Synthetic pathways for access to compounds 11–14. Reagents and conditions: (a) CDI, THF, 8 h, rt; (b) KO₂CCH₂CO₂Et, MgCl₂, THF, 40 °C, 2 h, 42%; (c) N-Boc 4-iodomethylpiperidine, K₂CO₃, DMF, rt, 1 night; (d) KPH, EtOH/H₂O, reflux, 3 h, 56%; (e) N-Boc 4-hydroxymethylpiperidine, NaH, THF, rt, 3 h, 10%; (f) HOBT, EDC.HCl, N-Boc 4-aminomethylpiperidine, DMF, rt, 1 night, 15%.

Scheme 2

Synthetic pathways for access to compounds 17–19. Reagents and conditions: (a) Et(Me)NCOCl, K₂CO₃, DMF, 70 °C, 2 h, 87%; (b) NaBH₄, MeOH, 0 °C, 30 min, 84%; (c) PBr₃, Et₂O, 0 °C, 30 min, 44%.

Scheme 3

Synthesis pathways for access to compounds 4–6 and 7–9. Reagents and conditions: (a) TFA, DCM, rt, 1 h; (b) 15, K₂CO₃, DMF, 110 °C, 1 h, 49–69%; (c) 3-hydroxybenzyl iodide, K₂CO₃, DMF, 110 °C, 2 h 30 min, 51–67%.

Scheme 4

Synthesis pathways for access to 19, 20. Conditions and reagents: (a) fumaric acid, iPrOH, reflux, 1 h 15 min, 66–68%.

Figure 3

Donepezil position from the X-ray structure (A) compared to the compound 7 positioned in (h)AChE binding sites using the docking studies, cluster 1 (B) and cluster 2 (C). The compound and the selected side chains of the binding site residues are in stick and the protein in ribbon representation. This figure was made with PyMOL (DeLano Scientific, 2002, San Carlo, USA).

Figure 4

Pharmacological profile of RS67333 and compound 19. Representative experiment illustrating agonist activities towards (h)5-HT_4(a) receptors.

Figure 5

Lineweaver-Burk plots of inhibition kinetics show that 19 acts as a non-competitive AChE inhibitor (A) and 20 as a mixed-type AChE inhibitor (B).

Figure 6

Decarbamoylation of 20 by (ee)AChE; (A): 20 (5 µM) and 4 (5 µM) without (ee)AChE; (B): 20 (5 µM) in the presence of 250 U/mL (ee)AChE (C): 20 (5 µM) without (ee)AChE (negative control).

Figure 7

Effect of compound 20 on spontaneous locomotor activity. Data are expressed as the mean ± standard error of mean (SEM, n = 8 per group). Drugs were administered intraperitoneally (IP) 30 min before the behavioral test. Compound 20: 1–3–10 mg/kg; CPZ: chlorpromazine 3 mg/kg (*** p < 0.05 versus NaCl, SNK test).

Figure 8

Effect of compound 20 on scopolamine impairment during the spontaneous alternation test. Data are expressed as the mean ± standard deviation (SEM, n = 8–10 per group) NaCl and compound 20 (1 mg/kg) were administered IP 30 min before the test, scopolamine (SCOP, 0.5 mg/kg)) was administered SC 20 min before the test. (# p < 0.05, ### p < 0.001 versus 50%; univariate t-test). (* p < 0.05, *** p < 0.001 versus other groups, SNK test).

Figure 9

Effect of compound 20 on MK 801 induced impairment during the spontaneous alternation test. Data are expressed as the mean ± standard deviation (SEM, n = 8–10 per group). NaCl and compound 20 (1 mg/kg) were administered IP 30 min before the test, MK801 (0.1 mg/kg) was administered SC 20 min before the test. (# p < 0.05, ### p < 0.001 versus 50%; univariate t-test). (*** p < 0.05 versus MK801, SNK test).