Since 2002, no clinical candidate against Alzheimer’s disease has reached the market; hence, an effective therapy is urgently needed. We followed the so-called “multitarget directed ligand” approach and designed 36 novel tacrine phenothiazine heterodimers which were in vitro evaluated for their anticholinesterase properties. The assessment of the structure−activity relationships of such derivatives highlighted compound 1dC as a potent and selective acetylcholinesterase inhibitor with IC₅₀ = 8 nM and 1aA as a potent butyrylcholinesterase inhibitor with IC₅₀ = 15 nM. Selected hybrids, namely, 1aC, 1bC, 1cC, 1dC, and 2dC, showed a significant inhibitory activity toward τ(306−336) peptide aggregation with percent inhibition ranging from 50.5 to 62.1%. Likewise, 1dC and 2dC exerted a remarkable ability to inhibit self-induced Aβ₁₋₄₂ aggregation. Notwithstanding, in vitro studies displayed cytotoxicity toward HepG2 cells and cerebellar granule neurons; no pathophysiological abnormality was observed when 1dC was administered to mice at 14 mg/kg (i.p.). 1dC was also able to permeate to the CNS as shown by in vitro and in vivo models. The maximum brain concentration was close to the IC₅₀ value for acetylcholinesterase inhibition with a relatively slow elimination half-time. 1dC showed an acceptable safety and good pharmacokinetic properties and a multifunctional biological profile.

Keywords: Alzheimer’s disease; acetylcholinesterase; phenothiazine; tacrine; multitarget directed ligands