Alzheimer’s disease (AD) is the most common type of dementia syndrome typically affecting individuals over the age of 65. It is a multifactorial disease that lacks effective treatment[1]. Alzheimer’s disease patients may experience a prolonged period of suffering with the potential of death due to associated issues such as lung infections, urinary tract infections, pressure mattresses, and systemic failure[2]. Despite the fact that the exact pathogenic mechanism of AD is still unknown, scientists have proposed several theories, including the Tau Protein Theory[3], Amyloid-β (Aβ) Protein Theory[4], Calcium Ion Dysregulation Theory, Central Cholinergic Damage Theory[5], [6], Inflammatory Immunity Theory, and Mitochondrial Cascade Theory[7].

In 2021, the United States Food and Drug Administration (FDA) approved several drugs (Fig. 1) for the treatment of AD, including AChE inhibitors (e.g. Donepezil, Galantamine, and Rivastigmine), N-methyl-D-aspartate receptor (NMDA) antagonists (e.g., Memantine)[8], [9], [10], [11], and the first monoclonal antibody drug, Aduanumab in 2021[12]. Additionally, in 2019, China released Sodium Oligomannate, a novel treatment for AD that targets the brain-gut axis. On July 6, 2023, lecanemab was the first Alzheimer's drug to receive full FDA approval within 20 years.

In the field of biomedicine, the successful treatment of AD has long been a challenge for medical professionals[13]. The central cholinergic injury hypothesis is one of the more commonly accepted theories, however, the FDA has not approved any medications that solely target AChE in recent years. It has been revealed through clinical studies that numerous single-target therapies are unsuccessful in treating multifactorial Alzheimer’s symptoms or disease progression[14]. Targeting several pathogenic factors is crucial given the intricacy of this condition. Ladostigil, a dual inhibitor of AChE and brain selective monoamine oxidase-B (MAO-B), is safe and well-tolerated Clinically, which could reduce volume loss in the brain and hippocampus[15]. Correspondingly, some clinical studies have suggested that multidrug combinations of AChE inhibitors may be beneficial for patients with AD[16]. A recent open pilot study found that the combination of an NMDA antagonist and a cholinesterase inhibitor was well-tolerated in dementia treatment. Therefore, we adopted a molecular hybridization strategy to facilitate the realization of multiple targets for a single molecule.

Oxidative stress is a reactive process caused by the accumulation of free radicals and is often induced by changes in the surrounding environment[17]. ROS are free radicals that exacerbate stress responses[18]. It has been established that an excessive amount of ROS in the brain environment leads to the production of Aβ proteins. These proteins are a common feature in the brains of individuals with neurodegenerative diseases[19], [20], such as AD. Conversely, neuroinflammation is a frequent disorder related to the central nervous system, which is regulated by the blood–brain barrier (BBB). When glial cells and astrocytes are activated by Aβ, inflammatory cytokines are produced in the body, initiating neuroinflammation. Additionally, inflammatory cytokines can lead to accelerating neuronal dysfunction and neuronal degeneration, eventually hastening the exacerbation of various neurodegenerative diseases[21]. Thus, enhancing the anti-inflammatory capacity of these compounds favors the suppression of AD[22].

Tacrine (Fig. 1), the first approved drug for the treatment of AD[23], [24], was eventually removed from the market[25], [26] because of severe cholinergic side effects and hepatotoxicity. This has prompted medicinal chemists to synthesize tacrine derivatives such as 7-methoxytacrine[27], [28] and 6-chlorotacrine[29] which have shown more favorable inhibitory effects on AChE than their parent compounds[29], [30]. Donepezil is one of the most successful AChE inhibitors available[31], [32] and is used to treat mild to severe AD[33], [34].

Donepezil and AChE have two distinct binding sites[35] such as a catalytic anion center (CAS) comprising of the amino acid residues Trp86, Tyr133, and Phe338; and a peripheral anion center (PAS) composed of Trp286, Tyr341, and Asp74. As shown in Fig. 2A and B, the benzocyclopentanone of donepezil binds to Trp286 and Tyr341 of PAS, whereas its tailed benzene ring undergoes Pi-Pi stacking with Trp86 of CAS. The basic nitrogen atom of donepezil forms mutual reciprocities with residues Trp86, Asp74, and Tyr337, which can enhance the overall affinity of AChE for the ligand. Moreover, the 1,2,3,4-tetrahydroacridin structure in the tacrine formed a Pi-Pi Stacked interaction with Trp86 of CAS and the primary amine group formed hydrogen bond interaction with Tyr337 (Fig. 2C and D).

Based on the results from reported study, the tacrine scaffold could interact within CAS as well as PAS regions of AChE and the propane-1,3-diamine structure enhances AChE inhibition among tacrine derivatives[36], [37], [38]. Some research demonstrated that the incorporation of a hydroxyl group could lead to the acquisition of antioxidant activity[39], [40] and N-benzylpiperidine[41] or its isostere N-benzylpiperazine[42], [43] moieties are indeed CAS ligands. To obtain a superior drug for AD treatment, we employed a molecular fusion design strategy to combine the active structures of two AChE inhibitors, tacrine and donepezil, and link with the β-amino alcohol section.

As shown in Fig. 3, N-benzylpiperazine was successfully applied in some agents with anti-dementia activities[44], [45], [46], [47]. Choubdar et al. found that replacing the piperidine ring with piperazine could enhance the activity against ChE[48]. The benzylpiperazine moiety is involved in several ChEIs, acting as an appropriate binding group that simultaneously interacts with the PAS and CAS of the enzyme[49]. Therefore, these ChEIs may alleviate cognitive deficits in AD by restoring cholinergic function. Moreover, these ChEIs can act as disease-modifying agents by decreasing Aβ levels, as the direct interaction between the ChEIs and the PAS of AChE slows down Aβ assembly[37].

Some studies have suggested that the basic nitrogen atom in the N-benzyl piperidine ring, which gets protonated at physiological pH, leads to increased affinity for AChE[50]. Compared with piperidine, piperazine possesses two basic nitrogen groups, leading us to assume that such combination might enhance ChE inhibition. Therefore, we replace the piperidine in the tail of donepezil with piperazine to design and synthesize four series of novel donepezil-tacrine hybrids (Fig. 4).