The inherent complexity of Alzheimer's disease (AD) and failed clinical trials have spiked the interest in multifunctional ligands that target at least two key disease-associated macromolecules in AD pathology. Here we present a focused series of pleiotropic N-carbamoylazole prodrugs with dual mechanism of action. Pseudo-irreversible inhibition of the first therapeutic target, human butyrylcholinesterase (hBChE), enhances cholinergic transmission, and thereby provides symptomatic treatment, same as the standard therapeutics in use for AD. Simultaneously, this step also functions as a metabolic activation that liberates a nanomolar selective α ₂-adrenergic antagonist atipamezole, which blocks pathological amyloid β (Aβ)-induced and noradrenaline-dependent activation of GSK3β that ultimately leads to hyperphosphorylation of tau, thus achieving a disease-modifying effect. Lead compound 8 demonstrated long-term pseudo-irreversible hBChE inhibition, metabolic activation in human plasma, blood-brain barrier permeability, and p.o. bioavailability in mice. Multi-day in vivo treatment with 8 in an Aβ-induced AD murine model revealed a significant alleviation of cognitive deficit that was comparable to rivastigmine, the current drug of choice for AD therapy. Furthermore, decreased GSK3β activation and lowered tau phosphorylation were observed in APP/PS1 mice. This surpasses the symptomatic-only treatment with cholinesterase inhibitors, as it directly blocks an essential pathological cascade in AD. Therefore, these multifunctional α ₂-adrenergic antagonists-butyrylcholinesterase inhibitors, exemplified by lead compound 8, present an innovative, small molecule-based, disease-modifying approach to treatment of AD.

We describe the development of quinolylnitrones (QNs) as multifunctional ligands inhibiting cholinesterases (ChEs: acetylcholinesterase and butyrylcholinesterase-hBChE) and monoamine oxidases (hMAO-A/B) for the therapy of neurodegenerative diseases. We identified QN 19, a simple, low molecular weight nitrone, that is readily synthesized from commercially available 8-hydroxyquinoline-2-carbaldehyde. Quinolylnitrone 19 has no typical pharmacophoric element to suggest ChE or MAO inhibition, yet unexpectedly showed potent inhibition of hBChE (IC₅₀ = 1.06 ± 0.31 nmol/L) and hMAO-B (IC₅₀ = 4.46 ± 0.18 μmol/L). The crystal structures of 19 with hBChE and hMAO-B provided the structural basis for potent binding, which was further studied by enzyme kinetics. Compound 19 acted as a free radical scavenger and biometal chelator, crossed the blood-brain barrier, was not cytotoxic, and showed neuroprotective properties in a 6-hydroxydopamine cell model of Parkinson's disease. In addition, in vivo studies showed the anti-amnesic effect of 19 in the scopolamine-induced mouse model of AD without adverse effects on motoric function and coordination. Importantly, chronic treatment of double transgenic APPswe-PS1δE9 mice with 19 reduced amyloid plaque load in the hippocampus and cortex of female mice, underscoring the disease-modifying effect of QN 19.