Neuroinflammation and cholinergic deficit are key detrimental processes involved in Alzheimer's disease. Hence, in the search for novel and effective treatment strategies, the multi-target-directed ligand paradigm was applied to the rational design of two series of new hybrids endowed with anti-inflammatory and anticholinesterase activity via triple targeting properties, namely able to simultaneously hit cholinesterases, cyclooxygenase-2 (COX-2) and 15-lipoxygenase (15-LOX) enzymes. Among the synthesized compounds, triazoles 5b and 5d, and thiosemicarbazide hybrid 6e emerged as promising new hits, being able to effectively inhibit human butyrylcholinesterase (hBChE), COX-2 and 15-LOX enzymes with a higher inhibitory potency than the reference inhibitors tacrine (for hBChE inhibition), celecoxib (for COX-2 inhibition) and both NDGA and Zileuton (for 15-LOX inhibition). In addition, compound 6e proved to be a submicromolar mixed-type inhibitor of human acetylcholinesterase (hAChE). The anti-neuroinflammatory activity of the three most promising hybrids was confirmed in a cell-based assay using PC12 neuron cells, showing decreased expression levels of inflammatory cytokines IL-1β and TNF-α. Importantly, despite the structural resemblance to tacrine, they showed ideal safety profiles on hepatic and murine brain cell lines and were safe up to 100 μM when assayed in PC12 cells. All three hybrids were also predicted to have superior BBB permeability than tacrine in the PAMPA assay, and good physicochemical properties, drug-likeness and ligand efficiency indices. Finally, molecular docking studies highlighted key structural elements impacting selectivity and activity toward the selected target enzymes. To the best of our knowledge, compounds 5b, 5d and 6e are the first balanced, safe and multi-target compounds hitting the disease at the three mentioned hubs.

Biomedical Research Center University Hospital Hradec Kralove Czech Republic

City of Scientific Research and Technological Applications Egypt

Departamento de Química Orgánica y Química Inorgánica Universidad de Alcalá 28805 Alcalá de Henares Madrid Spain

Departamento de Química Orgánica y Química Inorgánica Universidad de Alcalá 28805 Alcalá de Henares Madrid Spain; Instituto de Investigación Química Andrés M del Río Universidad de Alcalá 28805 Alcalá de Henares Madrid Spain

Department of Biochemistry and Molecular Genetics Faculty of Medicine American University of Beirut Lebanon; Program for Neurotrauma Neuroproteomics and Biomarkers Research Department of Emergency Medicine University of Florida Gainesville FL 32611 USA

Department of Biochemistry Faculty of Science Alexandria University Alexandria Egypt

Department of Pharmaceutical Chemistry Faculty of Pharmacy Alexandria University Alexandria 21521 Egypt

Department of Pharmacology and Toxicology Faculty of Medicine and Medical Centre American University of Beirut Beirut Lebanon

Department of Pharmacology and Toxicology Faculty of Pharmacy Alexandria University Alexandria 21521 Egypt; Department of Pharmacology and Toxicology Faculty of Medicine and Medical Centre American University of Beirut Beirut Lebanon

Department of Pharmacology and Toxicology Faculty of Pharmacy Alexandria University Alexandria 21521 Egypt; Hypertension and Vascular Research Center School of Medicine Wake Forest University Winston Salem NC 27157 USA

Department of Pharmacy and Biotechnology Alma Mater Studiorum University of Bologna via Belmeloro 6 40126 Bologna Italy

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