# Analysis of skeletal diversity of multi-target directed ligands (MTDLs) targeting Alzheimer's disease

Uzma Azam, Muhammad Moazzam Naseer, Christophe Rochais

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Uzma Azam, Muhammad Moazzam Naseer, Christophe Rochais. Analysis of skeletal diversity of multi-target directed ligands (MTDLs) targeting Alzheimer's disease. European Journal of Medicinal Chemistry, 2025, 286, pp.117277. 10.1016/j.ejmech.2025.117277. hal-04912969

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Review article

# Analysis of skeletal diversity of multi-target directed ligands (MTDLs) targeting Alzheimer's disease

Uzma Azam<sup>a</sup>, Muhammad Moazzam Naseer<sup>a,b,**</sup>, Christophe Rochais<sup>b,*</sup>

<figure><img src="image_3.png" /></figure>

## ARTICLE INFO

Keywords:

MTDLs

Skeletal diversity

Hybridization approach

Alzheimer's disease

Multifactorial

## ABSTRACT

Alzheimer's disease (AD) remains a significant healthcare challenge, necessitating innovative therapeutic approaches to address its complex and multifactorial nature. Traditional drug discovery strategies targeting single molecular targets are not sufficient for the effective treatment of AD. In recent years, MTDLs have emerged as promising candidates for AD therapy, aiming to simultaneously modulate multiple pathological targets. Among the various strategies employed in MTDL design, pharmacophore hybridization offers a versatile approach to integrate diverse pharmacophoric features within a single molecular scaffold. This strategy provides access to a wide array of chemical space for the design and development of novel therapeutic agents. This review, therefore, provides a comprehensive overview of skeletal diversity exhibited by MTDLs designed recently for AD therapy based on pharmacophore hybridization approach. A diverse range of pharmacophoric elements and core scaffolds hybridized to construct MTDLs that has the potential to target multiple pathological features of AD including amyloid-beta aggregation, tau protein hyperphosphorylation, cholinergic dysfunction, oxidative stress, and neuroinflammation are discussed. Through the comprehensive analysis and integration of structural insights of key biomolecular targets, this review aims to enhance optimization efforts in MTDL design, ultimately striving towards a comprehensive cure for the multifaceted pathophysiology of the disease.

## 1. Introduction

Alzheimer's disease (AD) is a real challenge in modern medicine, characterized by its complex pathophysiology and devastating impact on cognitive function and quality of life [1]. This disease is the leading cause of dementia in the world, accounting for approximately 60–80 % of cases [2,3]. Globally, over 47 million people currently live with dementia, and the total cost of dementia care worldwide is estimated to be around USD 818 billion [4]. Alarmingly, projections suggest that the global prevalence of dementia will increase to 131.5 million by 2050 [4].

In recent years, various hypotheses (Fig. 1) have been proposed to elucidate the complex pathogenesis of AD [5]. Among these hypotheses, the cholinergic hypothesis explains that deficits in cholinergic neurotransmission, specifically due to degeneration of basal forebrain cholinergic neurons, contribute to cognitive decline observed in AD [6–9]. The proteopathy hypothesis suggests that aberrant processing and aggregation of proteins, including beta-amyloid (A/β) and tau, result

in the formation of insoluble plaques and neurofibrillary tangles, thereby disrupting neuronal function and promoting neurodegeneration [10–13]. Additionally, metal ion dyshomeostasis, oxidative stress, and mitochondrial dysfunction are implicated in AD pathogenesis, where dysregulation of metal ions such as copper, zinc and iron, increased production of reactive oxygen species, and impaired energy metabolism, respectively contribute to neuronal damage and death [11,14–18]. Aberrant axonal transport, characterized by disruption of cellular trafficking processes essential for neuronal function and maintenance, has also been proposed as a contributing factor [5]. Moreover, neuroinflammation, involving activation of microglia and astrocytes and release of pro-inflammatory cytokines, exacerbates neuronal injury and contributes to disease progression (Fig. 1) [19–22]. These hypotheses collectively underscore the multifactorial nature of AD and offer valuable insights into the potential targets for therapeutic intervention. Beyond these hypotheses, genetic factors also play a substantial role in AD susceptibility [23–26]. Mutations in genes such as amyloid precursor protein (APP), presenilin 1 (PSEN1), and presenilin 2 (PSEN2) are

<footnote><sup>a</sup> Department of Chemistry, Quaid-i-Azam University, Islamabad, 45320, Pakistan</footnote>

<footnote><sup>b</sup> Université de Caen Normandie, Normandie Univ., CERMN, 14000, Caen, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>** Corresponding author. Department of Chemistry, Quaid-i-Azam University, Islamabad, 45320, Pakistan.</footnote>

<footnote>E-mail addresses: moazzam@qau.edu.pk (M.M. Naseer), christophe.rochais@unicaen.fr (C. Rochais).</footnote>
associated with early-onset familial forms of AD, while the apolipo-
protein E (ApoE) gene particularly the ApoE4 allele, is the strongest
genetic risk factor for late-onset AD [27,28]. Environmental and lifestyle
factors, diet, physical activity, and social engagement are also among
the factors (vide supra) that increase AD risk and progression [29–31].
Therefore, it is quite essential to understand the interplay between these
hypotheses and factors for unraveling the complex etiology of AD and
developing effective therapeutic interventions.

Current FDA-approved drugs for AD (Fig. 2) primarily provides symptomatic relief without halting or reversing the underlying neurodegenerative process [32,33]. Consequently, there is an urgent need for novel therapeutic agents capable of modifying the course of AD and slowing its progression. In recent years, the researchers have increasingly turned their attention to MTDLs as a promising strategy for AD treatment [34]. By simultaneously targeting multiple pathological pathways implicated in AD pathogenesis, MTDLs offer the potential for enhanced efficacy and disease-modifying effects [35]. In the development of MTDLs, the hybridization of various pharmacophores offers a versatile approach to target multiple disease pathways simultaneously [35]. While a wide range of pharmacophores can be employed in MTDL design, the focus has predominantly been on heterocyclic scaffolds [36, 37] due to their inherent structural diversity and functional versatility (vide infra). Indeed, numerous preclinical and clinical studies have demonstrated the promise of MTDLs in modifying AD-related pathology and improving cognitive function [38].

In this review, we aim to highlight the skeletal diversity of MTDLs by
comprehensively summarizing the recent efforts focused on them for
AD. Subsequently, through the analysis of this data, we seek to identify
the most promising compounds against various targets and correlate the
structural characteristics of MTDLs with the potential binding pockets of
biomolecular targets. Accordingly, this review is organized into distinct
sections to carefully explore the landscape of MTDLs based on different
pharmacophores. We begin by presenting MTDLs comprising a single
heterocyclic scaffold, highlighting the diverse array of pharmacological

activities exhibited by these compounds. Subsequently, we report
MTDLs constructed from two or three heterocyclic scaffolds, elucidating
the synergistic effects and enhanced potency achieved through scaffold
hybridization. Additionally, we examine MTDLs that do not incorporate
heterocyclic scaffolds, offering insights into alternative structural
frameworks employed in MTDL design. Finally, we explore MTDLs
derived from pharmacophores present in FDA-approved drugs for AD
treatment, demonstrating the potential of utilizing existing therapeutic
scaffolds to enhance treatment strategies for AD. Through this structured
approach and analysis, we aim to provide a comprehensive overview of
the diverse strategies and molecular frameworks utilized in the devel-
opment of MTDLs that will contribute to better understanding and ul-
timately leading to more effective next-generation therapies for AD.

2. MTDLs based on one heterocyclic scaffold

2.1. Quinoline/quinone/quinazoline/quinazolinone/phthalazinone
derivatives

Quinoline scaffolds are prevalent in both natural and synthetic
compounds, demonstrating numerous biological activities [39]
including anti-Alzheimer activity [40]. Wang et al. reported quinoline
based compound 1 (Table 1) featuring an N, N-dimethylaminoalkyla-
mino moiety at the 4-position of quinoline, demonstrated notable inhi-
bition of Aβ₁.₄₂ aggregation, achieving 95.3 % inhibition at a
concentration of 20 µM [41]. Additionally, this compound exhibited
selective copper (II) chelation, effectively hindered Cu²⁺-induced Aβ
aggregation, and disrupted self-induced Aβ₁.₄₂ aggregation fibrils with
inhibition potential of 4.5 µM. Notably, this compound also displayed
antioxidant properties (IC₅₀ = 6.54 µM), low toxicity (IC₅₀ = 253.7 µM)
and demonstrated a significant neuroprotective effect against
Aβ₁.₄₂-induced toxicity in SH-SY5Y neuroblastoma cells. Moreover, in
vivo studies revealed that this compound effectively reversed
scopolamine-induced memory deficits in mice in the step-down passive

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Proposed hypotheses of AD; (a) cholinergic dysfunction; (b) proteopathy; (c) metal ion dyshomeostasis; (d) oxidative stress; (e) aberrant axonal transport; (f) mitochondrial dysfunction; (g) neuroinflammation.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** FDA approved anti-Alzheimer drugs: (a) AChE inhibitor which was later revoked due to unacceptably high hepatotoxicity; (b & c) AChE inhibitors; (d) AChE and BChE inhibitor; (e) an N-methyl-D-aspartate receptor (NMDAR) antagonist.</figcaption></figure>

avoidance test [41]. Following a similar approach, Knez et al. developed quinoloylnitrone 2 (Table 1), a simple, low molecular weight nitrone having a bidentate ligating site in the form of 8-hydroxy quinoline, exhibited potent inhibition of human butyrylcholinesterase (hBChE) (IC₅₀ = 0.0011 nM vs donepezil 4.15 µM) and human monoamine oxidase (hMAO)-B (IC₅₀ = 4.46 µM) [42]. Additionally, this compound acted as a bio-metal chelator, a free radical scavenger (EC₅₀ = 126.0 µM) in 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, crossed the blood brain barrier (BBB), and showed no cytotoxicity in SH-SY5Y cells. Furthermore, in vivo studies demonstrated the anti-amnesic effect of this compound in a scopolamine-induced mouse model of AD, without adversely affecting motor function or coordination, rather similar to compound 1 [41]. Importantly, chronic treatment of double transgenic APPSwe-PS1δE9 mice with compound 2 resulted in reduced amyloid plaque loads in the hippocampus and cortex of female mice, highlighting its disease-modifying potential [42].

Recognizing the importance of phosphodiesterase 4D (PDE4D) inhibition and the modulation of bio metals in AD therapeutics, Hu et al. synthesized clioquinol-rolipram/roflumilast hybrids 3 and 4 (Table 1) which importantly showed superior activities against PDE4D having an IC₅₀ values of 0.40 µM and 0.49 µM, respectively compared to rolipram (IC₅₀ = 0.62 µM) [43]. Additionally, compounds 3 (IC₅₀ = 0.60 µM) and 4 (IC₅₀ = 0.63 µM) exhibited comparable ORAC values to clioquinol (CQ) (IC₅₀ = 0.60 µM). Moreover, both of these compounds showed enhanced potency compared to clioquinol in inhibiting Aβ aggregation induced by Cu²⁺ (Aβ + Cu²⁺ + CQ, 66%; Aβ + Cu²⁺ + 3, 70.2%; Aβ + Cu²⁺ + 4, 82% at a concentration of 50 µM for each compound) [43]. Similarly, Mo et al. investigated quinoline derivatives 5–7 (Table 1) where quinoline was hybridized with ferulic acid/cinnamic acids as potential anti-Alzheimer’s agents [44]. Interestingly, the derivative 5 i.e. quinoline/ferulic acid hybrid demonstrated the potent inhibition against electric eel acetylcholinesterase (eeAChE) with an IC₅₀ values of 0.62 µM, albeit less potent than tacrine (IC₅₀ = 0.02 µM), whereas the derivative 6, which is a quinoline/cinnamic acid hybrid, exhibited the potent inhibition against equine serum BChE with an IC₅₀ values of 0.10 µM compared to tacrine (IC₅₀ = 8 nM). Notably, the quinoline/cinnamic acid hybrid 7 having CF₃ and a relatively short linker was found to be less potent than 6 against both eeAChE (IC₅₀ = 3.28 µM) and eqBChE

(IC₅₀ = 0.93 µM). Additionally, all these derivatives demonstrated modest neuroprotective properties against H₂O₂-induced oxidative damage in PC12 cells. The derivative 5 showed better cytoprotective effects (cell viability of 74% at 20 µM) than compound 7. Notably, MTT assays on HepG2 cell revealed that compound 7 (93% viability at 10 µM) exhibits lower hepatotoxicity than tacrine (87.9%) [44].

Makhaeva et al. synthesized quinoline derivative 8 (Table 1) showing AChE inhibition with an IC₅₀ values of 1.90 µM compared to donepezil (IC₅₀ = 40 nM) and BChE, with an IC₅₀ value of 84 nM compared to donepezil (IC₅₀ = 19.2 µM) [45]. This compound also exhibited propidium displacement of 13.6% at 20 µM compared to donepezil (10.1%). Furthermore, it showed low activity against carboxylesterase, indicating the absence of clinically unwanted drug-drug interactions. Notably, this compound also demonstrated high antioxidant activity in ABTS assay (IC₅₀ = 14.1 µM) and FRAP assay (IC₅₀ = 0.80 µM), as well as inhibition of luminol chemiluminescence (91% at 20 µM) and lipid peroxidation (IC₅₀ = 9.38 µM) in mouse brain homogenates [45]. Quinolone based compounds 9 and 10 (Table 1) having side chain containing various hydrogen bond donor and acceptors, showed good AChE inhibition with the IC₅₀ values of 0.48 mg/mL and 0.52 mg/mL, respectively as compared to donepezil (0.03 mg/mL) at concentration of 100 µg/mL [46]. The 1-ethylquinolin-4-(1H)-one-3-yl moiety of compounds 9 and 10 that fills the choline-binding pocket by making strong π-π interactions, was supposed to be the main reason of good AChE inhibition. Additionally, good antioxidant capacity of these compounds (SC₅₀ = 70 nM for 9 and 90 nM for 10, respectively) was observed in DPPH assay [46].

Like quinoline, quinazoline also has unique place in heterocyclic and medicinal chemistry with diverse biological applications, making it ideal scaffold to be included drug design and development [47,48]. Mohamed et al. synthesized quinazoline derivatives 11 and 12 (Table 1). Compound 11 was observed to be a dual inhibitor of cholinesterases (ChEs) (AChE IC₅₀ = 7.2 µM; BChE IC₅₀ = 1.6 µM), also prevent Aβ₄₀ aggregation (IC₅₀ = 3.8 µM). While compound 12 could selectively inhibit AChE (IC₅₀ = 9.8 µM), it was the most potent inhibitor of Aβ₄₀ aggregation (IC₅₀ = 0.27 µM) and was ~4 and 1.4 times more potent than the reference agent’s curcumin (IC₅₀ = 3.3 µM) and resveratrol (IC₅₀ = 1.1 µM). Additionally, it also exhibited the good antioxidant
<table><caption>**Table 1 (continued)**</caption><thead><tr><th>Hybrid compounds</th><th>AChE inhibition IC<sub>50</sub> (μM)</th><th>BChE inhibition IC<sub>50</sub> (μM)</th><th>β-Amyloid inhibition (%)</th><th>Antioxidant IC<sub>50</sub> (μM)</th><th>Other activities</th><th>Ref.</th></tr></thead><tbody><tr><td>12</td><td>9.8</td><td>-</td><td>0.27<sup>h</sup></td><td>47</td><td></td><td></td></tr><tr><td>13</td><td>5.31<sup>c</sup></td><td>4.35<sup>c</sup></td><td>39.6<sup>i</sup></td><td>n.t.<sup>a</sup></td><td>Metal chelation</td><td>[51]</td></tr><tr><td>14</td><td>4.09<sup>c</sup></td><td>20.7<sup>c</sup></td><td>43.3<sup>i</sup></td><td></td><td></td><td></td></tr><tr><td>15</td><td>7.61<sup>c</sup></td><td>2.35<sup>c</sup></td><td>63.9<sup>i</sup></td><td></td><td></td><td></td></tr><tr><td>16</td><td>0.39</td><td>10.55</td><td>-</td><td>28.80</td><td>Cell viabilities in neuroblastoma SK-N-SH cell lines, BBB permeability, DNA protection</td><td>[52]</td></tr><tr><td>17</td><td>0.29</td><td>20.24</td><td></td><td>30.09</td><td></td><td></td></tr><tr><td>18</td><td>0.30</td><td>23.26</td><td></td><td>29.32</td><td></td><td></td></tr><tr><td>19</td><td>8.2</td><td>8.9<sup>e</sup></td><td>31.7<sup>j</sup></td><td>0.49</td><td>MAO-B and MAO-A inhibition IC<sub>50</sub> = 0.7 and 6.4 μM, neuroprotection, anti-neuroinflammation, platelet aggregation (14 % at 33 μM), and BBB permeability</td><td>[53]</td></tr></tbody></table>

properties (DPPH scavenging = 47 μM) [49].

Deoxyvasicinone a natural product derived from plants, specifically from the genus Phyllanthus, possesses a quinazolinone ring system as part of its structure [50]. Ma et al. installed structurally diverse aminoacetamide groups at deoxyvasicinone in derivatives **13–15** (Table 1) [51]. These compounds exhibited promising inhibitory activities against hAChE with an IC<sub>50</sub> values of 5.31 nM, 4.09 nM and 7.61 nM, respectively. In addition, compounds **13** and **15** showed the greatest ability to inhibit hBChE, with IC<sub>50</sub> values of 4.35 nM and 2.35 nM, respectively.

Enzyme kinetic studies of compound **15** having a 3,5-difluorobenzyl group at the aminoacetamide pendant demonstrated mixed inhibition mechanism, binding to both peripheral anionic site (PAS) and catalytic anionic site (CAS) of AChE. Most importantly, this compound had the highest inhibitory potential for Aβ<sub>1-42</sub> self-aggregation (63.9 % at 10 μM) as compared to standard curcumin (51.9 %) at same concentration, as well as being an excellent metal chelator [51].

Similarly, quinazolinone derivatives **16–18** (Table 1) containing vanillin, and α-amino phosphonate frameworks were reported by

<footnote>a Not tested.</footnote>

<footnote>b Tested but not active.</footnote>

<footnote>c nM.</footnote>

<footnote>d mg/mL.</footnote>

<footnote>e Percent inhibition.</footnote>

<footnote>f 20 μM.</footnote>

<footnote>g 50 μM.</footnote>

<footnote>h μM.</footnote>

<footnote>i 10 μM.</footnote>

<footnote>j 25 μM.</footnote>
Yelamanda et al. as an effective, selective, and mixed type inhibitors of eeAChE, with an IC₅₀ values of 0.30 µM, 0.29 µM, and 0.30 µM, respectively [52]. The biophysical strategies revealed the high preference of these compounds for AChE. These compounds also had a significant impact on ABTS radical scavenging abilities and maximal DNA protection action in addition to being an effective Aβ₁₄₂ fibril regulators at the molecular scale *via* the formation of micro size co-assembled competent structures, effectively eliminating Aβ₁₄₂ cytotoxicity in SK-N-SH cell lines. Interestingly, they also fulfilled most of the properties of CNS acting drugs during their *in silico* ADMET prediction studies [52]. Similarly, *in vitro* biological assay of phthalazinone derivative **19** (Table 1) demonstrated potent inhibitory activities against MAO-B and

MAO-A with the IC₅₀ values of 0.7 µM and 6.4 µM, respectively in comparison to reference donepezil (IC₅₀ = 3 nM for MAO-A and 2.4 µM for MAO-B) and moderate inhibition against AChE (IC₅₀ = 8.2 µM) compared to donepezil (IC₅₀ = 0.08 µM) [53]. Furthermore, it has been found to inhibit self-induced Aβ₁₄₂ aggregation (31.7 % at 25 µM), platelet aggregation (14 % at 33 µM), antioxidant capacity with the ORAC value of 0.49 µM, neuroprotective potency, anti-neuroinflammation and BBB permeability [53].

## 2.2. Triazole/pyrazole/imidazole/indole derivatives

Several multifunctional molecules containing the triazole moiety

<table><caption>**Table 2**
Anti-Alzheimer's potential of MTDLs based on triazole/pyrazole and indole scaffolds.</caption><thead><tr><th>Hybrid compounds</th><th>AChE inhibition IC<sub>50</sub> (µM)</th><th>BChE inhibition IC<sub>50</sub> (µM)</th><th>β-Amyloid inhibition (%)</th><th>Antioxidant IC<sub>50</sub> (µM)</th><th>Other activities</th><th>Ref.</th></tr></thead><tbody><tr><td>20<br>EtO<sub>2</sub>C-NHAc<br>EtO<sub>2</sub>C-NH</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>78.02<sup>d</sup></td><td>n.t.<sup>a</sup></td><td>Metal chelating ability, neuroprotective potency</td><td>[58]</td></tr><tr><td>21<br>MeO-NH-C<sub>6</sub>H<sub>4</sub>-N-C<sub>6</sub>H<sub>4</sub>-O-C<sub>6</sub>H<sub>5</sub>-O-Me</td><td>6.23<sup>c</sup></td><td>n.a.<sup>b</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>inhibited hCA I and hCA II: IC<sub>50</sub> for 21 = 53.8 and 51.4 nM<br>22 = 11.0 and 78.0 nM, respectively</td><td>[59]</td></tr><tr><td>22<br>CH<sub>3</sub>-N-C<sub>6</sub>H<sub>4</sub>-N-C<sub>6</sub>H<sub>5</sub>-O-C<sub>6</sub>H<sub>5</sub>-N-C<sub>6</sub>H<sub>5</sub>-N-H</td><td>4.65<sup>c</sup></td><td>70.2<sup>c</sup></td><td></td><td></td><td></td><td></td></tr><tr><td>23<br>Br-C<sub>6</sub>H<sub>4</sub>-N-C<sub>6</sub>H<sub>5</sub>-N-C<sub>6</sub>H<sub>5</sub>-N-H</td><td>86<sup>c</sup></td><td>2.54</td><td>-</td><td>n.t.<sup>a</sup></td><td>NMDA (GluN-1a/GluN2B) inhibition IC<sub>50</sub> = 3.88 and 5.05 µM, respectively, BBB permeability, PI displacement potential, neuroprotection</td><td>[61]</td></tr><tr><td>24<br>Cl-C<sub>6</sub>H<sub>4</sub>-N-C<sub>6</sub>H<sub>5</sub>-N-C<sub>6</sub>H<sub>5</sub>-N-H</td><td>135<sup>c</sup></td><td>3.79</td><td></td><td></td><td></td><td></td></tr><tr><td>25<br>Cl-C<sub>6</sub>H<sub>4</sub>-N-C<sub>6</sub>H<sub>5</sub>-N-C<sub>6</sub>H<sub>5</sub>-N-C<sub>6</sub>H<sub>5</sub>-N-O-C<sub>6</sub>H<sub>5</sub></td><td>1.93</td><td>1.16</td><td>50<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>PI displacement, neuroprotective potency, BBB permeability</td><td>[63]</td></tr><tr><td>26<br>Cl-C<sub>6</sub>H<sub>4</sub>-N-C<sub>6</sub>H<sub>5</sub>-N-C<sub>6</sub>H<sub>5</sub>-N-C<sub>6</sub>H<sub>5</sub>-N-C<sub>6</sub>H<sub>5</sub>-N-O-C<sub>6</sub>H<sub>5</sub></td><td>0.46</td><td>0.75</td><td>>50<sup>b</sup></td><td></td><td></td><td></td></tr><tr><td>27<br>Cl-C<sub>6</sub>H<sub>4</sub>-N-C<sub>6</sub>H<sub>5</sub>-N-C<sub>6</sub>H<sub>5</sub>-N-C<sub>6</sub>H<sub>5</sub>-N-C<sub>6</sub>H<sub>5</sub>-N-C<sub>6</sub>H<sub>5</sub>-N-C<sub>6</sub>H<sub>5</sub>-N-O-C<sub>6</sub>H<sub>5</sub></td><td>0.27</td><td>n.a.<sup>b</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>-</td><td>[67]</td></tr><tr><td>28<br>F-C<sub>6</sub>H<sub>4</sub>-N-C<sub>6</sub>H<sub>5</sub>-N-C<sub>6</sub>H<sub>5</sub>-N-C<sub>6</sub>H<sub>5</sub>-N-C<sub>6</sub>H<sub>5</sub>-N-C<sub>6</sub>H<sub>5</sub>-N-O-C<sub>6</sub>H<sub>5</sub></td><td>1.37</td><td>1.85</td><td></td><td></td><td></td><td></td></tr></tbody></table>

<footnote>a Not tested.</footnote>

<footnote>b Tested but not active.</footnote>

<footnote>c nM.</footnote>

<footnote>d 25 µM.</footnote>

<footnote>e 20 µM.</footnote>
have been presented as promising candidates for AD [54–56]. Although the exact function of the triazole in these molecules remains uncertain, their structural and electronic properties may facilitate various interactions with targets relevant to AD [57]. The triazole derivative 20 (Table 2) reported by Kaur et al. not only significantly inhibits the self-induced Aβ42 aggregation (78.02% inhibition at 20 µM, IC50 = 4.57 µM) when compared to the standard curcumin (95% inhibition at 20 µM, IC50 = 1.48 µM), but also potentially disassembled the preformed Aβ42 aggregates [58]. Furthermore, this compound exhibited superior metal chelating ability due to its bidentate behavior involving triazole ring nitrogen electrons to coordinate with the metal ion and retaining copper in a redox-dormant state to prevent reactive oxygen species (ROS) generation in copper-ascorbate redox cycling. Importantly, this compounds has been found non cytotoxic and could inhibit the toxicity induced by Aβ42 aggregates in SH-SY5Y cells [58]. Similarly, Ozil et al. presented triazole derivatives 21 and 22 (Table 2) having dual N-iminoamide-aryl conjugates which has been found as a good candidates against AChE (IC50 = 62.3 nM and 46.5 nM, respectively) and carbonic anhydrases (CA) [59]. Importantly, both these compounds showed superior AChE activity than the standard neostigmine (IC50 = 0.13 µM) [59]. Based on the same strategy, Jones et al. developed a series of triazole-based MTDLs that showed antioxidant capacity, Cu-binding affinity, interaction with the Aβ peptide and modulation of Aβ peptide aggregation, and ability to limit Aβ1.42-induced neurotoxicity in human neuronal culture [60]. Gutti et al. developed triazole-bridged aryl adamantane analogues 23 and 24 (Table 2), exhibiting potent inhibition of AChE with IC50 values of 86 nM and 135 nM, respectively, along with significant inhibition of Aβ aggregation [61]. Additionally, when expressed heterologously in Xenopus laevis oocytes, these compounds demonstrated IC50 values of 3.88 µM and 5.05 µM, respectively, for inhibiting the NMDA receptor (GluN1-1b/GluN2B subunit combination). Permeation across the BBB was confirmed through the PAMPA assay, with these compounds exhibiting Pe of 8.75 × 10⁻⁶ cm/s and 8.24 × 10⁻⁶ cm/s, respectively. Furthermore, both compounds demonstrated safety profiles in SH-SY5Y and HEK-293 cell lines. *In vivo* behavioral studies conducted in rats at an oral dose of 10 mg/kg revealed that these compounds effectively ameliorated cognitive and spatial memory impairment [61].

Like triazole, pyrazole which is also a five membered ring containing two nitrogen atoms [62], has also attracted considerable attention for the development of MTDLs for AD. For example, 3,5-diarylpyrazole derivatives 25 and 26 (Table 2) were evaluated for their inhibitory activities against AChE, BChE, and PAMPA by Gutti et al. [63]. The derivative 25 showed good AChE (IC50 = 1.93 µM), BChE (IC50 = 1.16 µM), hAChE (IC50 = 1.75 µM) and BBB permeation (Pe = 9.49 × 10⁻⁶ cm/s), as compared to standard donepezil (IC50 = 19 nM, against AChE, 0.94 µM for BChE and 22 nM against hAChE) and BBB permeation (Pe = 13.92 × 10⁻⁶ cm/s). In comparison, the derivative 26 was found to be more active than 25 against all the enzymes, showing AChE with an IC50 values of 0.46 µM, BChE with an IC50 values of 0.75 µM and hAChE with an IC50 values of 0.47 µM. Interestingly, propidium iodide (PI) was significantly displaced from AChE's PAS by both derivatives. Moreover, they were discovered to be less harmful to MC65 cells with the reduction in metal-induced Aβ1.42 aggregation [63]. Similarly, imidazole is another important five membered heterocyclic scaffold having two nitrogen at 1,3-positions of the ring with diverse biological applications [64]. Because COX and 5-LOX-derived eicosanoids are thought to participate to Aβ plaque deposition, Correc et al. explored a series of 1, 5-diarylimidazoles as MT-stabilizing agents and/or inhibitors of the cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) processes [65].

Indole is a well-known pharmacophore in medicinal chemistry [66]. Parveen et al. investigated biaryl indole derivatives 27 and 28 (Table 2) against ChEs [67]. Interestingly, the derivative 27 was found to be highly selective, showing IC50 values of 0.27 µM against AChE with no activity against BChE. In contrast, its fluoro analog 28 was however discovered as dual inhibitor of AChE and BChE showing IC50 values of

1.37 µM and 1.85 µM, respectively. This difference in behavior can be attributed to the increased lipophilicity of the compound 28, affecting its distribution, metabolism, membrane permeability and tissue distribution [67].

## 2.3. Pyridine/piperidine/piperazine derivatives

Mounting evidence suggests that Aβ aggregation, glycogen synthase kinase 3β (GSK-3β), and oxidative stress are inextricably linked, and their simultaneous modulation may have a positive and synergistic impact in addressing impairments associated with AD [68]. The pyridine derivative 29 (Table 3) with 3,4-dihydroxy substitution reported by De Simone et al. to have the ability to inhibit GSK-3β (IC50 = 24.36 µM) and Aβ42 self-aggregation (IC50 = 9.0 µM), and to counteract with ROS (kinh = 6.8 × 10⁵ M⁻¹s⁻¹) even in phosphate buffer at pH = 7.4 (kinh = 3.2 × 10⁵ M⁻¹s⁻¹) [69]. Most importantly, this compound demonstrated high BBB permeability (Pe = 5.5 × 10⁻⁶ cm s⁻¹), no significant cytotoxic effects in immature cortical neurons up to 50 µM, and neuroprotective properties against glutamate-induced toxic insult at micromolar concentrations [69]. A similar compound 30 (Table 3) having N-benzyl pyridinium moiety demonstrated not only potent AChE activity (IC50 = 12.1 nM for eeAChE, 8.6 nM for hAChE compared to donepezil, IC50 = 40.2 nM) but also high selectivity towards AChE over BChE [70]. It also inhibited Aβ1.42 aggregation (64.7 % at 20 µM), provided neuroprotection against amyloid-induced cell toxicity in PC12 cells and showed good BBB permeability in the PAMPA-BBB assay [70].

Similarly, pyridine derivatives 31 and 32 (Table 3) decorated with azo-stilbene moiety demonstrated the significant AChE inhibition (IC50 = 4.18 µM and 3.86 µM, respectively) [71]. They possessed strong antioxidant properties, inviolable binding affinity with Cu²⁺ and Zn²⁺ metal ions, as indicated by their high stability constants and pM values. Additionally, both compounds effectively chelated copper ions from Aβ42 and exhibited the capability to modulate the aggregation of the Aβ42 peptide, both in the presence and absence of metal ions. Remarkably, these compounds displayed a low nanomolar Ki value in the thioflavin (ThT) competition assay, underscoring their potent interaction with Aβ fibrils. Additionally, a low neurotoxicity has also been observed especially for compound 31 in cellular toxicity studies [71]. Similarly, the dihydropyridine analog 33 (Table 3) has been reported to as an appealing antioxidant (IC50 = 1.75 TE), Ca²⁺ channel antagonist (47 % at 10 µM) and a good neuroprotectant (22.49 % against H₂O₂ at the concentration of 10 µM) [72].

MAO-B inhibition has emerged as a potential therapeutic strategy for AD [73,74]. Additionally, targeting BChE presents a feasible approach for alleviating symptoms arising from cholinergic dysregulation [73]. Kosak et al. investigated two structurally similar piperidine derivatives 34 and 35 (Table 3), namely N-propargylpiperidine sulfonamides, wherein 34 exhibited dual inhibition of MAO IC50 (hMAO-B) = 53.9 µM) and BChE (IC50 (hBChE) = 2.6 µM), while derivative 35 selectively inhibited hBChE (IC50 = 0.127 µM) [75]. Interestingly, both derivatives demonstrated favorable selectivity for hBChE over AChE, penetrated the BBB, exhibited neuroprotective effects against Aβ1.42 and were non-cytotoxic. These findings suggest that substituting N-piperidine with the propargyl functional group may lead to the development of selective hBChE and MAO-B inhibitors with neuroprotective properties in comparison to the unsubstituted piperidine derivatives [75]. Another piperidine based compound 36 (Table 3) that has the potential of influencing both Aβ protein accumulation and the toxicity of ROS in neuronal cells, was synthesized by Lanthier et al. by joining two scaffolds of interest, a typical aminochlorobenzophenone found in many 5-HT₄R agonists and an antioxidant chembotype, on piperidine nucleus [76]. This compound demonstrated potent 5-HT₄ receptor (Ki = 12.7 nM) and antioxidant activity (IC50 = 3.40 µM) [76].

The piperidine derivative 37 (Table 3) having two aryl rings conjugated through N-iminoamide moiety exhibited selective and non-competitive inhibition of AChE (IC50 = 5.9 µM) with a selectivity
<table><caption>**Table 3**
Anti-Alzheimer's potential of MTDLs based on pyridine/piperidine and piperazine scaffolds.</caption><thead><tr><th>Hybrid compounds</th><th>AChE inhibition IC<sub>50</sub> (µM)</th><th>BChE inhibition IC<sub>50</sub> (µM)</th><th>β-Amyloid inhibition (%)</th><th>Antioxidant IC<sub>50</sub> (µM)</th><th>Other activities</th><th>Ref.</th></tr></thead><tbody><tr><td>29</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>9.0<sup>d</sup></td><td>n.t.<sup>a</sup></td><td>GSK-3β (IC<sub>50</sub> = 24.36 µM, chelated Cu<sup>2+</sup>, radical scavenger, BBB permeation, neuroprotective properties)</td><td>[69]</td></tr><tr><td>30</td><td>12.1<sup>c</sup></td><td>2.6</td><td>64.7<sup>a</sup></td><td>n.a.<sup>b</sup></td><td>BBB permeation, metal chelation, neuroprotective properties</td><td>[70]</td></tr><tr><td>31</td><td>4.18</td><td>n.t.<sup>a</sup></td><td>30<sup>f</sup></td><td>2.26</td><td>Metal chelation, low neurotoxicity</td><td>[71]</td></tr><tr><td>32</td><td>3.86</td><td></td><td>40<sup>f</sup></td><td>1.86</td><td></td><td></td></tr><tr><td>33</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>1.75</td><td>Ca<sup>2+</sup> channel antagonist (47 % inhibition), substantial neuroprotection (22.49 %) against H<sub>2</sub>O<sub>2</sub></td><td>[72]</td></tr><tr><td>34</td><td>n.a.<sup>b</sup></td><td>2.6</td><td>n.a.<sup>b</sup></td><td>n.t.<sup>a</sup></td><td>34 exhibited hMAO-B (IC<sub>50</sub> = 53.9 µM), neuroprotective activity</td><td>[75]</td></tr><tr><td>35</td><td></td><td>0.12</td><td></td><td></td><td></td><td></td></tr><tr><td>36</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>3.40</td><td>5-HT<sub>4</sub>R inhibition IC<sub>50</sub> = 7.5 nM</td><td>[76]</td></tr><tr><td>37</td><td>5.9</td><td>&gt;30</td><td>30<sup>f</sup></td><td>7.45</td><td>Neuroprotective activity, low cytotoxicity</td><td>[77]</td></tr><tr><td>38</td><td>33.7<sup>c</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>(h)5-HT<sub>6</sub>R inhibition K<sub>i</sub> = 219, (h)5-HT<sub>4</sub>R K<sub>i</sub> = 210 and an anti-amnesic effect</td><td>[78]</td></tr><tr><td>39</td><td>7.9</td><td>31.7</td><td>n.t.<sup>a</sup></td><td>2.5</td><td>Inhibited MAO-B IC<sub>50</sub> = 9.9 for BACE-1 = 8.3 µM, neuroprotective activity, metal chelation, BBB permeation</td><td>[79]</td></tr></tbody></table>

<footnote>a Not tested.</footnote>

<footnote>b Tested but not active.</footnote>

<footnote>c nM.</footnote>

<footnote>d µM.</footnote>

<footnote>e 20 µM.</footnote>

<footnote>f 75 µM.</footnote>
index (SI > 5.1), indicating a preference for AChE over BChE inhibition. It also demonstrated antioxidant activity with an IC₅₀ value of 7.45 µM against neuronal *t*-BOOH-induced oxidative stress [77]. These compounds displayed neuroprotective potential against *t*-BOOH and Aβ₁.₄₂-induced neurotoxicity with low cytotoxicity in SH-SY5Y cells and favorable druggability properties [77]. Hatat et al. reported amino-chlorobenzophenone linked piperidine derivative **38** (Table 3) structural analog of **36**, aiming to exert an integrative anti-amnesic effect [78]. This derivative has the capability of activating 5-HT₄ receptors (Kᵢ = 210), blocking 5-HT₆ receptors (Kᵢ = 219), inhibiting AChE activity (IC₅₀ = 33.7 nM) and demonstrated an anti-amnesic effects in vivo in a scopolamine-induced cognitive deficit model at a dose of 0.3 mg/kg [78].

Like piperidine, piperazine derivatives also have tremendous biological significance. Zhu *et al.* reported piperazine derivative **39** (Table 3) having 2-acetylphenol-O-alkylhydroxyethyl amine moiety that exhibited notable *in vitro* inhibitory potency with IC₅₀ values of 7.9 µM against *ee*AChE, 9.9 µM against MAO-B, and 8.3 µM against BACE-1 [79]. Its mechanism of inhibition against AChE was determined to be of mixed type, capable of binding to both CAS and PAS of AChE. Furthermore, this compound demonstrated significant antioxidant capacity (ORAC = 2.5 equiv.) and exhibited neuroprotective activity. Additionally, it displayed selective chelation of metal ions and demonstrated permeability across the BBB [79].

## 2.4. Coumarin/chromone/benzofuran derivatives

Coumarin, a well-known natural product having a benzopyrone structure, is another attractive heterocyclic scaffold possessing diverse applications [80]. Yang et al. reported 4-hydroxycoumarin derivative **40** (Table 4) with a dimethylaminopropyl pendant that displayed significant inhibitory activity against AChE with an IC₅₀ values of 4.28 µM [81]. Additionally, this compound effectively hindered self-induced and Cu²⁺-induced aggregation of Aβ₁.₄₂, exhibiting superior inhibition activity at 10 µM (64.27 %) compared to curcumin (49.8 %), and demonstrated robust antioxidant properties (IC₅₀ = 2.90 µM). Moreover, it showed notable metal-chelating ability, selectively chelate Cu²⁺, Al³⁺, Fe³⁺, Fe²⁺, and Zn²⁺ ions. Most importantly, *in silico* studies indicated its favorable BBB permeability and ADMET properties. Interestingly, the introduction of another hydroxy group at this derivative led to decrease in AChE, BChE and Aβ inhibition with considerable increase in antioxidant properties [81]. Wang et al. reported structurally similar compound **41** (Table 4) with additional hydroxy group at 7-position of coumarin that showed inhibition of AChE with an IC₅₀ value of 11.15 µM, BChE with an IC₅₀ value > 100 µM and antioxidant properties with an IC₅₀ value of 1.44 µM [82]. Molecular docking and ADMET studies predict that this compound could effectively interact with target proteins and cross the BBB [82].

Yang et al. investigated coumarin derivative **42** (Table 4) having pargyline pharmacophore as dual inhibitor of Aβ aggregation and MAOs [83]. This derivative displayed remarkable inhibitory activity against MAOs, with IC₅₀ values of 27 nM for MAO-B and 3.27 µM for MAO-A compared to pargyline (IC₅₀ = 0.19 µM against MAO-B and 3.52 µM against MAO-A) and exhibited significant inhibition of Aβ₁.₄₂ aggregation. Moreover, it demonstrated low toxicity and exhibited effective BBB permeability [83]. Rasagiline is a selective and an irreversible inhibitor of MAO-B [84]. Xie et al. investigated a chromone derivative **43** (Table 4) having N-propargyl moiety from rasagiline for their ability to inhibit MAO and Aβ aggregation [85]. It displayed superior *h*MAO-B potency (IC₅₀ = 4.0 nM) and selectivity (SI > 25000) compared to rasagiline (IC₅₀ = 141.70 nM, SI > 355) and effectively inhibited Aβ₁.₄₂ aggregation (40.78 % at 25 µM). Molecular dynamics studies of this compound confirmed competitive reversible modulation of *h*MAO-B, and it demonstrated low toxicity and favorable neuroprotective effects. Importantly, this compound also showed promising pharmacokinetic profiles following its oral and intravenous administration [85].

Agbo et al. reported furan fused coumarin derivative **44** (Table 4) that displayed substantial anti-AChE activity (IC₅₀ = 2.8 µM) and inhibited BACE-1 (IC₅₀ = 15.3 µM), also showed COX-2 (IC₅₀ = 8.6 µM) and LOX-5 (IC₅₀ = 13.9 µM) activities [86]. Furthermore, it demonstrated potent DPPH radical scavenging activity (IC₅₀ = 6.8 µM), highlighting its antioxidant properties. The 2-arylbenzofuran derivative **45** (Table 4) reported by Montanari et al. for its inhibitory activity against ChEs and cannabinoid receptors [87]. This compound exhibited inhibitory activity against BChE (IC₅₀ = 13.4 µM), displayed neuroprotective effects against Aβ₁.₄₂ oligomers, and acted as a potent and selective CB2 ligand, modulating microglial phenotype from pro-inflammatory M1 to neuroprotective M2 [87].

## 2.5. Carbazole/indolinone/oxazole/oxadiazole derivatives

Carbazole which is considered as indole derivatives, manifest diverse applications across various domains owing to its special structural features [88]. Mishra et al. conducted an extensive investigation into the multifaceted attributes of carbazole derivative **46** (Table 4), elucidating its efficacy as inhibitor of AChE (IC₅₀ = 7.72 µM) [89]. Furthermore, this compound showed inhibition of Aβ aggregation (67.12 % at 10 µM), antioxidant activity (81.64 % at 50 µM), and metal chelation capabilities. Evaluation of this compound against H₂O₂-induced toxicity in SH-SY5Y cells corroborated its neuroprotective attributes without exhibiting cytotoxic effects. In addition, it also demonstrated efficacy in ameliorating memory imbalance and cognition impairment in a scopolamine-induced dementia mouse model [89].

The isoindoline-1,3-dione derivative **47** (Table 4) exhibited substantial inhibitory activities against *ee*AChE (IC₅₀ = 3.33 µM) and *h*BACE-1 (43.7 % at 50 µM), along with inhibition of Aβ aggregation (IC₅₀ = 24.9 % at 10 µM) [90]. Molecular modeling studies also confirmed its potential interactions with the binding sites of AChE and BACE-1 [90]. Similarly, Mehreen et al. reported a series of phenoxy pendant 2,3-indolinones with remarkable inhibitory potential against AChE comparable to the standard drug, donepezil [91]. Likewise, N-phenyl thiosemicarbazones of 2,3-indolinones inhibited Aβ-induced neurotoxicity and ROS production at concentrations as low as 1 µM [92].

The distinctive structural features of oxazole derivatives offer promising avenues for exploration across diverse scientific realms [93]. Luo et al. reported a polyfunctional oxazole derivative **48** (Table 4) exhibiting potent inhibitory activity against GSK-3β (IC₅₀ = 0.25 µM) and also demonstrated good neuroprotective properties [94]. Mechanical studies unveiled the inhibition of GSK-3β and downstream p-Tau expression, reduction of H₂O₂-induced ROS damage, restoration of mitochondrial membrane potential imbalance, suppression of Ca²⁺ influx, and mitigation of apoptosis. Moreover, this compound activated the Keap1-Nrf2-ARE signaling pathway, upregulating downstream oxidative stress proteins, thereby exhibiting cytoprotective effects [94].

Two structurally similar 1,3,4-oxadiazole derivatives **49** and **50** (Table 4) having extensive *π*-conjugation were evaluated for inhibition against AChE, BChE, and BACE-1 [95]. Interestingly, derivative **49** with *tri*-fluormethyl substitution demonstrated slightly superior AChE inhibition (IC₅₀ = 68 nM) in comparison to the derivative **50** with tri-fluoromethyloxy substitution (IC₅₀ = 9.2 nM). In contrast, derivative **50** was found more effective and comparable, against BChE and BACE-1 with IC₅₀ values of 0.16 µM and 0.21 µM respectively as compared to **49** (IC₅₀ = 0.22 µM and IC₅₀ = 0.25 µM). Both derivatives also displayed significant anti-Aβ aggregatory activity, neuroprotective effects against neuroblastoma SH-SY5Y cells, and improved cognitive function *in vivo*. Pharmacokinetic studies suggested enhanced oral absorptivity for compound **49** [95].

## 2.6. Natural product derivatives

Herbal remedies have garnered significant attention for their therapeutic potential in managing and attenuating various diseases, including
<table><caption>**Table 4**
Anti-Alzheimer's potential of MTDLs based on coumarin/benzofuran/carbazole/isoindolinone and oxadiazole scaffolds.</caption><thead><tr><th>Hybrid compounds</th><th>AChE<br>inhibition IC<sub>50</sub><br>(µM)</th><th>BChE<br>inhibition IC<sub>50</sub><br>(µM)</th><th>β-Amyloid<br>inhibition (%)</th><th>Antioxidant<br>IC<sub>50</sub> (µM)</th><th>Other activities</th><th>Ref.</th></tr></thead><tbody><tr><td>40</td><td>4.28</td><td>65.48</td><td>64.27<sup>d</sup></td><td>2.90</td><td>Metal chelating ability, BBB permeability</td><td>[81]</td></tr><tr><td>41</td><td>11.15</td><td>&gt;100</td><td>29.08<sup>d</sup></td><td>1.44</td><td>Metal chelation, BBB permeability</td><td>[82]</td></tr><tr><td>42</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>54.0<sup>e</sup></td><td>n.t.<sup>a</sup></td><td>IC<sub>50</sub> = 2.7 nM for MAO-B and 3.27 µM for MAO-A, low toxicity, BBB permeability</td><td>[83]</td></tr><tr><td>43</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>40.78<sup>e</sup></td><td>n.t.<sup>a</sup></td><td>hMAO-B potency with IC<sub>50</sub> = 4.0 nM, low toxicity, good neuroprotective potential, BBB permeability</td><td>[85]</td></tr><tr><td>44</td><td>2.8</td><td>6.3</td><td>n.t.<sup>a</sup></td><td>6.8</td><td>BACE-1 IC<sub>50</sub> = 15.3 µM, COX-2 = 8.6 µM, and LOX-5 = 13.9 µM</td><td>[86]</td></tr><tr><td>45</td><td>n.a.<sup>b</sup></td><td>13.4</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>Powerful and selective CB<sub>2</sub> ligand, and had cytotoxic effect</td><td>[87]</td></tr><tr><td>46</td><td>7.72</td><td>&gt;100</td><td>67.12<sup>d</sup></td><td>81.64<sup>g</sup></td><td>Metal chelation, enhanced memory imbalance and cognition impairment, neuroprotective potency</td><td>[89]</td></tr><tr><td>47</td><td>3.33</td><td>14.1</td><td>24.9<sup>d</sup></td><td>n.t.<sup>a</sup></td><td>hBACE-1 inhibition 43.7 % at 50 µM</td><td>[90]</td></tr><tr><td>48</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>GSK-3β inhibition IC<sub>50</sub> = 0.25 µM, neuroprotective capabilities</td><td>[94]</td></tr><tr><td>49</td><td>6.8<sup>c</sup></td><td>0.22</td><td>24–66<sup>f</sup></td><td>n.t.<sup>b</sup></td><td>Inhibited BACE-1: 49 IC<sub>50</sub> = 0.25 µM, 50 = 0.21 µM, exhibited PI deflection, improved cognitive impairments, BBB permeability</td><td>[95]</td></tr><tr><td>50</td><td>9.2<sup>c</sup></td><td>0.16</td><td>22–49<sup>f</sup></td><td></td><td></td><td></td></tr></tbody></table>

<footnote>a Not tested.</footnote>

<footnote>b Tested but not active.</footnote>

<footnote>c nM.</footnote>

<footnote>d 10 µM.</footnote>

<footnote>e 25 µM.</footnote>

<footnote>f 5-20 µM.</footnote>

<footnote>g Percent inhibition.</footnote>
neurological disorders like AD [96,97]. Sang et al. reported scutellarein derivative 51 (Table 5) having O-acetamidoalkylbenzylamine moiety that has the ability to inhibit AChE (IC₅₀ = 5.1 nM), self-induced Aβ₁₄₂ aggregation (57.1 % at 25 µM), Cu²⁺-induced Aβ₁₄₂ aggregation (43.4 % at 25 µM), hAChE-induced Aβ₁₄₀ aggregation (IC₅₀ = 3.9 µM), and Cu²⁺-induced aggregation of the tightly packed Aβ₁₄₂ fibrils (66.3 % at 25 µM), besides acting as a promising antioxidant (1.3 Trolox equiv.) and bio metals chelator [98]. Moreover, this natural product derivative demonstrated neuroprotective effects against H₂O₂-induced cellular damage and low cytotoxicity in neuronal cell line SH-SY5Y cells in addition to ameliorating scopolamine-induced memory deficits in mice. Molecular modeling studies suggested its interactions with both the CAS and PAS of AChE [98].

Deferiprone, an orally administered iron chelator [99] and resveratrol, a natural antioxidant and Aβ peptide aggregation inhibitor were merged together by Xu et al. to obtain derivatives 52 and 53 (Table 5) that has the ability to act as promising triple-function MTDLs, exhibiting substantial inhibition of Aβ₁₄₂ aggregation (58.43 % and 65.30 % at 20 µM, respectively [100]. They showed stronger ABTS⁺ scavenging activity (IC₅₀ = 1.73 µM and 4.02 µM, respectively) than Trolox, and pFE (III) values (19.60 and 19.0, respectively) comparable to deferiprone [100]. In another study, Piemontese et al. identified and evaluated secondary metabolites of plants or fungi with suitable physical properties, and found that the compound 54 (Table 5) has significant antioxidant activity (EC₅₀ = 2.6 µM/µM of DPPH), has the ability to inhibit AChE (IC₅₀ = 8.13 µM), Aβ₁₄₀ aggregation (IC₅₀ = 50 µM), BChE (IC₅₀ = 7 nM), and has metal chelation potential [101]. Similarly, Li et al. reported the hesperetin derivative 55 (Table 5) that displayed potent inhibitory activity against AChE (IC₅₀ = 9.37 nM), high selectivity over BChE (selectivity index value of 305), and significant inhibition of self-induced Aβ aggregation (76.3 % at 20 µM) [102]. It also exhibited antioxidant properties and neuroprotective effect in vitro, with low cytotoxicity towards the PC12 cells [102].

## 2.7. *Bridged polycyclic/chalcone derivatives*

Bridged polycyclic molecules with predominantly planar and/or achiral conjugates represent a novel class of compounds with potential therapeutic applications. Titov et al. conducted a biological evaluation of N-bridged cyclopenta[a]indene derivatives 56 (Table 5), revealing its selective inhibition of potent BChE (IC₅₀ = 3.4 nM) [103]. This compound also exhibit low intrinsic cytotoxicity in SH-SY5Y cells (IC₅₀ = 53 µM) and favorable brain permeation (P<sub>e</sub> = 3.43 × 10⁻⁶ cm/s), thereby demonstrating neuroprotective effects against glutamatergic neurotoxicity [103]. Similarly, novel polycyclic amine cage derivatives, with or without a propagylamine function, were designed to possess innate multifunctional neuroprotective effects [104]. Calcium regulation tests on SH-SY5Y human neuroblastoma cells revealed significant VGCC blocking activity and substantial NMDA antagonism, with compound 57 (Table 5) displaying the highest activity (88.3 %) at 10 µM. Moreover, this compound exhibited potent activity against hMAO-B (IC₅₀ = 1.70 µM) as compared to selegiline (IC₅₀ = <10 nM), indicating selectivity over MAO-A isoenzyme and classification as a reversible MAO inhibitor based on time-dependency studies [104]. Pont et al. investigated polycyclic derivatives 58 and 59 (Table 5) targeting BACE-1 secondary and catalytic sites, demonstrating effective *in vitro* activity against hBACE-1 and ChEs [105]. Both compounds exhibited moderate anti-aggregating activity against Aβ₄₂ and tau, with favorable brain permeation and significant cognitive benefits in an AD mouse model. These compounds showed potential as disease-modifying, cognition-enhancing anti-AD leads by repairing long- and short-term memory, modulating APP processing, minimizing tau phosphorylation, and increasing synaptic protein levels [105].

Kolaj et al. reported chalcone derivative 60 (Table 5) for its anti-oligomeric potency (IC₅₀ = 1.8 µM) and anti-fibrillary potential [106]. However, it exhibited only moderate anti-fibrillary activity, highlighting

the need for distinct therapeutic strategies targeting Aβ oligomerization and fibrillization in AD [106]. Another chalcone based compound 61 (Table 5) displayed good inhibitory potential against AChE, BChE, and MAO-B, along with antioxidant and metal-chelating properties [107]. Additionally, it showed selective MAO-B inhibition (IC₅₀ = 0.57 µM) and potential neuroprotective effects, crossing the BBB *in vitro* and enhancing cognitive function *in vivo* [107]. Similarly, Mannich base derivative 62 (Table 5) demonstrated potent activities against self- and Cu²⁺-induced Aβ₁₄₂ aggregation, selective downregulation of AChE and MAO-B, antioxidant capacity, and metal-chelating properties [108]. It also exhibited excellent anti-neuroinflammatory activity and adequate BBB permeation [108]. Likewise, compound 63 (Table 5), a N-methyl-piperazinylalkyl-O-chalcone derivative, displayed high AChE inhibitory activity (IC₅₀ = 11.6 nM) compared to donepezil (IC₅₀ = 15.68 nM), while also exhibiting effective anti-AGEs activity and suppression of free radical generation [109]. *In vivo* testing against STZ-induced dementia in rats demonstrated improved memory skills and reduced brain AChE activity and oxidative stress [109].

# 3. MTDLs based on two/three heterocyclic scaffolds

## 3.1. *Quinoline/quinazolinone hybrids*

### 3.1.1. *Quinoline-carbazole hybrids*

Zhang et al. synthesized an aminoquinoline-carbazole hybrid 64 (Table 6) that exhibited preferential copper chelation, and effectively suppressed self-induce and Cu²⁺-induced Aβ₁₄₂ aggregation (22.9 and 22.3 %, respectively at 20 µM concentration of 64) [110]. Notably, it demonstrated the ability to penetrate the BBB with P<sub>e</sub> = 16.3 × 10⁻⁶ cm s⁻¹. Furthermore, this derivative provided significant neuroprotection against glutamate induced HT22 cell death [110]. Another, quinolone-carbazole hybrid 65 (Table 6) exhibited good activity against AChE with an IC₅₀ value of 0.11 µM and BChE with an IC₅₀ value of 20 nM [111]. This compound also displayed inhibitory activity against self-induced Aβ₁₄₂ aggregation (27.4 %) and AChE-induced Aβ₁₄₂ aggregation (24.9 %) at 100 µM concentration. Ligand-protein docking simulation results and kinetic studies demonstrated that it could efficiently bind to the enzyme's PAS and CAS with mixed-type inhibitory activity. Furthermore, this compound significantly protected PC12 cells from H₂O₂-induced toxicity [111].

### 3.1.2. *Quinoline-pyridine hybrids*

Czarnecka et al. reported quinoline-pyridine hybrid 66 (Table 6) that exhibited the potent AChE activity with an IC₅₀ values of 52 nM, surpassing the reference (donepezil IC₅₀ = 0.103 µM) [112]. The high level of AChE inhibition was attributed to the perfect size of the linker, facilitating interactions with PAS. Furthermore, this hybrid molecule inhibited BChE, suppressed Aβ₁₄₂ aggregation by 25.7 % at 5 µM to 92.8 % at 100 µM concentration, and demonstrated significant anti-inflammatory effects [112]. In another study by the same group, quinoline-pyridine hybrid 67 (Table 6), having similar structure to 66 but different linker size and substitution pattern at the pyridine ring, was also identified as the promising compound, exhibiting inhibitory therapeutic potential against eeAChE and eqBChE with IC₅₀ values of 67 nM and 0.153 µM, respectively [113]. Additionally, this compound demonstrated non-hepatotoxicity, capability to inhibit Aβ-aggregation (79 % at 100 µM concentration), and inhibited ChE activity in a mixed manner. Using yeast three-hybrid (Y3H) technology, the renowned ligand-receptor associations were confirmed. However, this derivative did not exhibit antioxidant potential, as confirmed by two different tests [113].

### 3.1.3. *Quinoline-piperidine/pyrrolidine hybrids*

Chen et al. reported quinoline-piperidine hybrid 68 (Table 6) based on oxoisoaporphine and benzopiperidine scaffolds, that exhibited strong inhibition of nitric oxide (NO) and inducible nitric oxide synthase
<table><caption>**Table 5**
Anti-Alzheimer's potential of MTDLs based on natural products/bridged polycyclic and chalcone scaffolds.</caption><thead><tr><th>Hybrid compounds</th><th>AChE inhibition IC<sub>50</sub> (μM)</th><th>BChEinhibition IC<sub>50</sub> (μM)</th><th>β-Amyloid inhibition (%)</th><th>Antioxidant IC<sub>50</sub> (μM)</th><th>Other activities</th><th>Ref.</th></tr></thead><tbody><tr><td>51</td><td>5.1<sup>c</sup></td><td>&gt;500</td><td>57.1<sup>d</sup></td><td>1.13</td><td>Bio metals chelation, neuroprotection, low cytotoxicity, scopolamine-induced memory loss</td><td>[98]</td></tr><tr><td>52</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>58.4<sup>e</sup></td><td>1.73</td><td>Iron chelating activity of 52 with pFe(III) = 19.60<br>53 = 19.0</td><td>[100]</td></tr><tr><td>53</td><td></td><td></td><td>65.30<sup>e</sup></td><td>4.02</td><td></td><td></td></tr><tr><td>54</td><td>8.13</td><td>7<sup>c</sup></td><td>50<sup>f</sup></td><td>2.6</td><td>Metal chelation</td><td>[101]</td></tr><tr><td>55</td><td>9.37<sup>c</sup></td><td>2862<sup>c</sup></td><td>76.3<sup>e</sup></td><td>3.0</td><td>Protected PC12 neurons, neuroprotective activity</td><td>[102]</td></tr><tr><td>56</td><td>20.1</td><td>3.4<sup>c</sup></td><td>n.a.<sup>b</sup></td><td>n.t.<sup>a</sup></td><td>Inhibited hMAO-A (IC<sub>50</sub> = 24 μM, hMAO-B (IC<sub>50</sub> = 1.70 μM), neuroprotection, BBB permeability</td><td>[103]</td></tr><tr><td>57</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>NMDA inhibition 88.3 %, neuroprotection, less neurotoxicity, MAO-B inhibition IC<sub>50</sub> = 1.70</td><td>[104]</td></tr><tr><td>58</td><td>3.60<sup>c</sup></td><td>620<sup>c</sup></td><td>47.9<sup>e</sup></td><td>-</td><td>Inhibited hBACE-1 IC<sub>50</sub> = 1.50 and 2.71 μM, respectively, inhibited tau aggregation 29.6 and 65.8 % at 10 μM respectively, metal binding, neuroprotection, BBB permeation.</td><td>[105]</td></tr><tr><td>59</td><td>2.57<sup>c</sup></td><td>43.0<sup>c</sup></td><td>49.5<sup>e</sup></td><td>39.4</td><td></td><td></td></tr><tr><td>60</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>20.57<sup>b</sup></td><td>n.t.<sup>a</sup></td><td>Anti A/β oligomeric activity, anti-fibrillary potential</td><td>[106]</td></tr><tr><td>61</td><td>1.3</td><td>1.2</td><td>81.2<sup>d</sup></td><td>1.30</td><td>MAO-B inhibition with IC<sub>50</sub> value of 0.57 μM, metal chelation, BBB permeation. enhanced scopolamine-induced cognitive deficits</td><td>[107]</td></tr></tbody></table>

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<table><caption>**Table 5 (continued)**</caption><thead><tr><th>Hybrid compounds</th><th>AChE<br>inhibition<br>IC<sub>50</sub> (μM)</th><th>BChEinhibition<br>IC<sub>50</sub> (μM)</th><th>β-Amyloid<br>inhibition (%)</th><th>Antioxidant<br>IC<sub>50</sub> (μM)</th><th>Other activities</th><th>Ref.</th></tr></thead><tbody><tr><td><img src="image_62.png" alt="62"></td><td>7.15</td><td>n.a.<sup>b</sup></td><td>70.65<sup>d</sup></td><td>0.97</td><td>MAO-B IC<sub>50</sub> = 0.43 μM, metal chelating property, BBB permeability</td><td>[108]</td></tr><tr><td><img src="image_63.png" alt="63"></td><td>11.6</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>18.51</td><td>AGEs inhibitory activity (IC<sub>50</sub> = 38.47 μM)</td><td>[109]</td></tr></tbody></table>

a Not tested.

b Tested but not active.

c nM.

d 25 μM.

e 20 μM.

f μM.

g 10 μM.

h 100 μM.

(iNOS) production and modulated cytokine production by reducing tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1/β) formation in lipopolysaccharide-activated BV-2 microglia and RAW 264.7 macrophages [114]. Additionally, when incubated with SH-SY5Y cells transfected with human APP containing Swedish abnormalities, this hybrid significantly reduced Aβ<sub>42</sub> aggregation. Furthermore, it significantly suppressed the activity of AChE (IC<sub>50</sub> = 31 nM) and BChE (IC<sub>50</sub> = 0.51 μM), and showed significant delayed dysfunction caused by Aβ<sub>1-42</sub> toxicity (0.54 % at 5 μM) in GMC101 [114]. In another study, quinoline-piperidine hybrids 69 and 70 (Table 6) displayed AChE and BChE inhibition potentials comparable to the reference drug galantamine [115]. Particularly, compound 69 exhibited the most potent inhibition of eeAChE and eqBChE, with IC<sub>50</sub> values of 1.20 μM and 18.52 μM, respectively (galantamine IC<sub>50</sub> = 1.28 μM for eeAChE and 24.41 μM for eqBChE). Kinetic and molecular docking studies revealed that it acted as a mixed-type antagonist, binding to both CAS and PAS of AChE. Compound 70 displaced PI from the PAS of eeAChE by 25.80 %. Additionally, both compounds exhibited remarkable scavenging of DPPH radicals and demonstrated potent biometal chelating abilities towards Al<sup>3+</sup>, Fe<sup>2+</sup>, Cu<sup>2+</sup>, and Zn<sup>2+</sup> ions [115].

Wang et al. reported a series of quinolone-piperidine/pyrrolidine hybrids 71-74 (Table 6) each having benzo[g]quinoline-5,10-dione and piperidine or pyrrolidine scaffolds, as MTDLs for AD [116]. Hybrids 71 and 72 significantly inhibited self-induced Aβ aggregation in vitro, exhibiting 57.4 % and 61.7 % inhibition at 100 μM, respectively compared to curcumin (48.1 %) [116]. In addition, derivatives 73 and 74 demonstrated moderate inhibitory potency toward AChE, with IC<sub>50</sub> values of 1.08 μM and 1.12 μM, respectively, compared to tacrine (0.14 μM). However, the tested compounds showed less inhibitory potential against BChE. Further investigation revealed that compound 74 could reduce hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>)-induced neurotoxicity in SH-SY5Y cells (cell viability = 83 %). Additionally, a PAMPA assay for the BBB predicted that these compounds have the ability to penetrate the BBB and reach their targets in the central nervous system (CNS) [116].

### 3.1.4. Quinoline-morpholine hybrids

Zaib et al. reported quinoline-morpholine hybrid 75 (Table 6) having N-iminothiourea linkage [117]. Interestingly, this hybrid showed promising AChE inhibition with an IC<sub>50</sub> values of 0.12 μM, which is five times higher than the standard galantamine (IC<sub>50</sub> = 0.62 μM). The high level of inhibition of this compound was attributed to the synergistic effect of electron-rich methoxy group and the ethyl morpholine moiety [117].

### 3.1.5. Quinoline-thiazole hybrids

Barré et al. investigated dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) as a promising target for the development of new drugs to target both ChEs and DYRK1A [118]. The oxidized form, quinolinium salt 76 (Table 6) exhibited potent inhibition of hAChE with IC<sub>50</sub> value of 81.4 nM. Notably, this compound also inhibited BChE (IC<sub>50</sub> = 482 nM). Moreover, it displaced PI more effectively (26 %) than donepezil (18 %). However, this compound did not demonstrate the ability to penetrate the BBB and showed relatively low activity against DYRK1A (5 % inhibition) [118]. Similarly, new tetrahydro-1H-pyrazolo[3,4-b]quinoline derivatives 77 and 78 (Table 6) acted as dual inhibitors of AChE and COX-2 [119]. In vitro testing revealed that both compounds exhibited nearly twice the inhibition effect of tacrine on AChE, with inhibition values of 22.9 % and 18.43 %, respectively, compared to tacrine (17.43 %). Additionally, the anti-inflammatory activity of these hybrids surpassed that of celecoxib in an in vitro COX-1/COX-2 inhibition assay, with higher selectivity indices (SI = 13.13 and 8.99, respectively). Moreover, these candidates demonstrated enhanced anti-inflammatory activity by inhibiting COX-2 protein induction [119].

### 3.1.6. Quinoline-oxadiazole hybrids

New S-alkyl phthalimide analog of 5-(2-phenylquinolin-4-yl)-1,3,4-oxadiazole-2-thiol 79 (Table 6) was evaluated for its inhibitory activity against MAO-A, MAO-B, and AChE enzymes [120]. This analog demonstrated the most efficient inhibition of AChE, with an IC<sub>50</sub> value of 1.02 μM. Additionally, this quinoline-oxadiazole hybrid exhibited inhibitory activity against MAO-A and MAO-B, with IC<sub>50</sub> values of 3.31 μM and 3.39 μM, respectively [120].

### 3.1.7. Quinazolinone-piperidine hybrids

In screening assays, deoxyvasicinone analogue 80 (Table 6) containing an electron-donating group (methoxy) at the meta-position exhibited significant suppression of both AChE activity and cellular tau oligomerization [121]. It demonstrated an IC<sub>50</sub> of 0.91 μM for AChE inhibition and an EC<sub>50</sub> of 3.83 μM for downregulating cellular tau oligomerization. Additionally, the neuroprotective effects of this compound were evaluated in tau-expressing SH-SY5Y cells treated with Aβ oligomers [121].

## 3.2. Triazole/triazine/imidazole/indole/pyrazole hybrids

### 3.2.1. Triazole-chromone hybrids

Triazole-containing 3-phenylcoumarin-lipoic acid hybrids 81 and 82 (Table 7), featuring four carbon spacers, were explored as MTDLs [122].
<table><caption>**Table 6**
Anti-Alzheimer's potential of MTDLs based on quinoline/quinazolinone hybrids.</caption><thead><tr><th>Hybrid compounds</th><th>AChE inhibition IC<sub>50</sub> (µM)</th><th>BChE inhibition IC<sub>50</sub> (µM)</th><th>β-Amyloid inhibition (%)</th><th>Antioxidant IC<sub>50</sub> (µM)</th><th>Other activities</th><th>Ref.</th></tr></thead><tbody><tr><td>64</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>22.3<sup>e</sup></td><td>n.a.<sup>b</sup></td><td>BBB permeation metal chelation, neuroprotection</td><td>[110]</td></tr><tr><td>65</td><td>0.11</td><td>20<sup>c</sup></td><td>27.4<sup>f</sup></td><td>n.t.<sup>a</sup></td><td>Neuroprotection, AChE-induced Aβ<sub>1-42</sub> aggregation 24.9 %</td><td>[111]</td></tr><tr><td>66</td><td>52<sup>c</sup></td><td>0.158</td><td>92.8<sup>f</sup></td><td>n.t.<sup>a</sup></td><td>Good anti-inflammatory effect, non-cytotoxic properties</td><td>[112]</td></tr><tr><td>67</td><td>67<sup>c</sup></td><td>0.153</td><td>79<sup>f</sup></td><td>n.a.<sup>b</sup></td><td>Non-hepatotoxicity</td><td>[113]</td></tr><tr><td>68</td><td>31<sup>c</sup></td><td>0.51</td><td>0.54<sup>g</sup></td><td>n.t.<sup>a</sup></td><td>Anti-inflammatory activity, Inhibition of LPS-induced TNF-α and IL-1β</td><td>[114]</td></tr><tr><td>69</td><td>1.20</td><td>18.52</td><td>n.t.<sup>a</sup></td><td>n.a.<sup>b</sup></td><td>Dislocation of PI, metal chelation</td><td>[115]</td></tr><tr><td>70</td><td>1.23</td><td>22.11</td><td></td><td></td><td></td><td></td></tr><tr><td>71</td><td>14.18</td><td>36.23</td><td>57.4<sup>f</sup></td><td>n.t.<sup>a</sup></td><td>BBB permeability</td><td>[116]</td></tr><tr><td>72</td><td>1.12</td><td>15.38</td><td>61.7<sup>f</sup></td><td></td><td></td><td></td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 6 (continued)**</caption><thead><tr><th>Hybrid compounds</th><th>AChE inhibition IC<sub>50</sub> (µM)</th><th>BChE inhibition IC<sub>50</sub> (µM)</th><th>β-Amyloid inhibition (%)</th><th>Antioxidant IC<sub>50</sub> (µM)</th><th>Other activities</th><th>Ref.</th></tr></thead><tbody><tr><td>73</td><td>1.08</td><td>13.17</td><td>52.8<sup>f</sup></td><td></td><td></td><td></td></tr><tr><td>74</td><td>10.94</td><td>59.06</td><td>53.6<sup>f</sup></td><td></td><td></td><td></td></tr><tr><td>75</td><td>0.12</td><td>8.12<sup>c</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>-</td><td>[117]</td></tr><tr><td>76</td><td>81.4<sup>c</sup></td><td>482<sup>c</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>Inhibited PI displacement</td><td>[118]</td></tr><tr><td>77</td><td>22.9<sup>d</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>77 showed SGPT = 96.16; 78: 61.16, 77 inhibited COX-1 and COX-2 IC<sub>50</sub> = 9.98 and 0.76 µM<br>78 with IC<sub>50</sub> = 7.55 and 0.84 µM, respectively</td><td>[119]</td></tr><tr><td>78</td><td>18.43<sup>d</sup></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>79</td><td>1.02</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>Inhibited MAO-A and MAO-B IC<sub>50</sub> = 3.31 and 3.39 µM, respectively</td><td>[120]</td></tr><tr><td>80</td><td>0.91</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>Neuroprotection, BBB permeation, tau oligomerization (IC<sub>50</sub> = 3.83 mM)</td><td>[121]</td></tr></tbody></table>

Hybrid **81**, a benzocoumarin derivative, exhibited potent AChE inhibition with an IC₅₀ value of 7.1 µM, while also significantly reducing intracellular ROS levels. Moreover, it acted as an antioxidant and metal chelator, shielding SH-SY5Y cells from Aβ₁₄₂-induced injury. Conversely, the 8-methoxycoumarin analogue **82**, though slightly less effective against AChE than **81**, displayed reasonable protection against H₂O₂-induced cell death in PC12 cells (29.4 %) and substantial

inhibition of self-induced Aβ aggregation (62.4 %) at 100 µM concentration [122]. Similarly, a cycloaddition Click reaction led to the discovery of triazole linked coumarin-lipoic acid hybrid **83** (Table 7) [123]. This compound, exhibiting an IC₅₀ value of 16.4 µM, demonstrated notable AChE inhibition and promising inhibition of Aβ aggregation. Additionally, it displayed protective effects against H₂O₂-induced cytotoxicity and showed preferential bio-metal chelation, along with

<footnote>a Not tested.</footnote>

<footnote>b Tested but not active.</footnote>

<footnote>c nM.</footnote>

<footnote>d Percent inhibition.</footnote>

<footnote>e 20 µM.</footnote>

<footnote>f 100 µM.</footnote>

<footnote>g µM.</footnote>
<table><caption>**Table 7**
Anti-Alzheimer's potential of MTDLs based on triazole/triazine hybrids.</caption><thead><tr><th>Hybrid compounds</th><th>AChE inhibition IC<sub>50</sub> (µM)</th><th>BChE inhibition IC<sub>50</sub> (µM)</th><th>β-Amyloid inhibition (%)</th><th>Antioxidant IC<sub>50</sub> (µM)</th><th>Other activities</th><th>Ref.</th></tr></thead><tbody><tr><td>81</td><td>7.1</td><td>68.6</td><td>n.a.<sup>b</sup></td><td>85.46</td><td>Neuroprotection, metal chelation</td><td>[122]</td></tr><tr><td>82</td><td>12.6</td><td>86.7</td><td>62.4<sup>d</sup></td><td>n.a.<sup>b</sup></td><td></td><td></td></tr><tr><td>83</td><td>16.4</td><td>49.37</td><td>51.2<sup>d</sup></td><td>87.5</td><td>Bio-metal chelation, protection of SH-SY5Y cells against H<sub>2</sub>O<sub>2</sub>-induced cytotoxicity and neuroprotection</td><td>[123]</td></tr><tr><td>84</td><td>&gt;100</td><td>7.55</td><td>13.1<sup>d</sup></td><td>85.57</td><td>Neuroprotection, metal chelation</td><td>[124]</td></tr><tr><td>85</td><td>21.71</td><td>&gt;100</td><td>32.6<sup>e</sup></td><td>n.t.<sup>a</sup></td><td>Neuroprotective ability, metal chelation</td><td>[125]</td></tr><tr><td>86</td><td>n.t.<sup>a</sup></td><td>18.0</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>Inhibited 5-LOX IC<sub>50</sub> = 21.0 µM, Anti-tyrosinase 72%, low cytotoxicity</td><td>[126]</td></tr><tr><td>87</td><td>59<sup>e</sup></td><td>≥10</td><td>34.26<sup>f</sup></td><td>n.t.<sup>a</sup></td><td>Neuroprotection, low cytotoxicity</td><td>[127]</td></tr><tr><td>88</td><td>14.05</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>92.80<sup>b</sup></td><td>Inhibited hCA-I with IC<sub>50</sub> = 2.72, hCA-II = 3.93 nM</td><td>[128]</td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 7 (continued)**</caption><thead><tr><th>Hybrid compounds</th><th>AChE inhibition IC<sub>50</sub> (µM)</th><th>BChE inhibition IC<sub>50</sub> (µM)</th><th>β-Amyloid inhibition (%)</th><th>Antioxidant IC<sub>50</sub> (µM)</th><th>Other activities</th><th>Ref.</th></tr></thead><tbody><tr><td>89</td><td>84<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>–</td><td>[129]</td></tr><tr><td>90</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>2.43<sup>g</sup></td><td>n.t.<sup>a</sup></td><td>Chelates metal ions, inhibits metal mediated Aβ aggregation, controls ROS production, and reduces oxidative stress</td><td>[130]</td></tr><tr><td>91</td><td>1.06<sup>c</sup></td><td>7.3<sup>c</sup></td><td>7.68<sup>g</sup></td><td>92.0</td><td>BBB permeation, enhanced basal synaptic efficiency, substantially lowered hippocampal oxidative stress and neurological inflammation</td><td>[131]</td></tr><tr><td>92</td><td>55<sup>c</sup></td><td>11.09</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>Inhibited BACE-1 IC<sub>50</sub> = 11.09 µM</td><td>[135]</td></tr><tr><td>93</td><td>0.2</td><td>4.6</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>Exhibited remarkable displacement of PI, a significant improvement in mental problems and issues, BBB permeation</td><td>[136]</td></tr><tr><td>94</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>>50</td><td>94 Inhibited BACE-1 IC<sub>50</sub> = 8.55 µM, 95 IC<sub>50</sub> = 11.42 µM, neuroprotective activities, prevention against amyloid peptide toxicity metal chelation</td><td>[137]</td></tr><tr><td>95</td><td></td><td></td><td></td><td>44.42</td><td></td><td></td></tr></tbody></table>

neuroprotection against H<sub>2</sub>O<sub>2</sub>- and Aβ<sub>1-42</sub>-induced cytotoxicity in SH-SY5Y cells [123]. Similarly, Jalili-Baleh et al. reported a triazole based novel chromone-lipoic acid hybrid **84** (Table 7) [124]. This hybrid, with a chain length of four carbon atoms, emerged as the most efficacious BChE inhibitor, with an IC<sub>50</sub> value of 7.55 µM, demonstrating substantial neuroprotective activity and antioxidant properties [124].

*In vitro* biological assessments revealed triazole tethered 2H-chromen-2-one hybrid **85** (Table 7), featuring 3,4-diF on the aryl ring, as the most efficacious BChE inhibitor, with an IC<sub>50</sub> value of 21.71 µM [125]. This compound exhibited inhibition of self-induced Aβ<sub>1-42</sub> aggregation and AChE-induced Aβ aggregation, along with chelation of bio-metals

[125]. Additionally, a novel 1,2,3-triazole linked coumarinopyrazole ligand **86** (Table 7) demonstrated significant BChE inhibition, with an IC<sub>50</sub> value of 18.0 µM, surpassing its regioisomer [126]. This compound also exhibited inhibitory effects on the 5-LOX enzyme and anti-tyrosinase activity, with cytotoxicity against HCT-116 cells, indicating its potential for further investigation [126]. Likewise, a new benzotriazole-coumarin hybrid **87** (Table 7) was identified as a multifunctional agent, showing potent AChE inhibition (IC<sub>50</sub> = 59 nM) with a mixed-type inhibition scenario [127]. This compound also displayed inhibition of Cu<sup>2+</sup>-induced Aβ<sub>1-42</sub> aggregation and chelating properties for metal ions, along with DNA protective capacity against degenerative

<footnote>a Not tested.</footnote>

<footnote>b Tested but not active.</footnote>

<footnote>c nM.</footnote>

<footnote>d 100 µM.</footnote>

<footnote>e 10 µM.</footnote>

<footnote>f 50 µM.</footnote>

<footnote>g µM.</footnote>

<footnote>h Percent inhibiton.</footnote>
properties of OH radicals. Molecular modeling studies confirmed the ability of this hybrid to block both the PAS and the CAS of AChE [127].

### 3.2.2. Triazole-pyridine hybrids

The inhibitory effects of the bis-1,2,4-triazole-3-thiol-pyridine hybrid **88** (Table 7) on AChE and *h*CA I and II were examined [128]. This hybrid exhibited potent inhibition against AChE, with an IC₅₀ value of 14.05 nM and a Ki value of 8.47 nM. Additionally, it demonstrated inhibitory activity against cytosolic *h*CA I and II, with IC₅₀ values of 2.72 nM for *h*CA I and 3.93 nM for *h*CA II. Moreover, the radical scavenging activity of the bis-1,2,4-triazole-pyridine hybrid was verified using the ABTS cation radical assay. Specifically, this hybrid, featuring a 3-methoxyphenyl substituent, exhibited the highest activity, with a value of 92.80 % [128].

### 3.2.3. Triazole-imidazole hybrids

Faraji et al. evaluated a 1,2,3-triazole linked benzimidazole **89** (Table 7) for its *in vitro* inhibition potential against AChE [129]. The study employed the compound **89**, featuring a 2-methylenethio linker and a 1-(2-fluorobenzyl)-1,2,3-triazole linked to a benzimidazole group, exhibited significant inhibition of AChE, reaching 84 % inhibition at a concentration of 100 µM. Docking simulations further elucidated that it predominantly bound to the CAS of the AChE enzyme [129].

### 3.2.4. Triazole-peptide hybrids

The peptidomimetic hybrid **90** (Table 7), a triazole-peptide hybrid, exhibited remarkable inhibitory effects on Aβ aggregation, with an IC₅₀ value of 2.43 µM, and demonstrated efficacy in disassembling preformed Aβ₄₂ fibrils [130]. Additionally, this hybrid displayed metal ion chelation properties, effectively inhibiting metal mediated Aβ aggregation, reducing ROS production, and mitigating oxidative stress. Notably, this compound exhibited low cytotoxicity and conferred protection to differentiated SH-SY5Y against Aβ-induced toxicity. Furthermore, it destabilized the Aβ₄₂ protofibril structure by significantly diminishing the binding affinity between chains D-E, thereby disrupting and deforming the protofibril architecture [130].

### 3.2.5. Triazole-huprine Y hybrids

Viayna et al. devised hybrid **91** (Table 7) with nanomolar efficacy against *h*AChE (IC₅₀ = 1.06 nM) and *h*BChE (IC₅₀ = 7.3 nM), while preserving or enhancing the antioxidant properties of capsaicin through the fusion of the scaffolds of the cholinesterase inhibitor huprine Y and the antioxidant capsaicin [131]. Biodistribution studies conducted in C57BL6 mice demonstrated brain permeation (Pₑ = 8.8 × 10⁻⁶ cm/s), with this compound exhibiting a superior brain/plasma ratio. Chronic administration of this hybrid to 10-month-old APP/PS1 mice ameliorated memory and cognition deficits, as evidenced by three distinct behavioral tests, and delayed the progression of Alzheimer-like disease, as indicated by a substantially reduced Aβ₄₂/Aβ₄₀ ratio in the hippocampus. Furthermore, it augmented basal synaptic efficiency and significantly attenuated hippocampal oxidative stress and neuroinflammation [131].

### 3.2.6. Triazine-piperazine hybrids

Triazine stands out as a versatile scaffold [132] for crafting multi-target hybrids, owing to its structural planarity and capacity for intercalating between β-amyloid sheets [133,134]. Leveraging the triazine scaffold with diverse functional groups, the presented compounds hold promise for significant anti-aggregation effects, potentially leading to Aβ disaggregation [133,134]. Maliszewski et al. investigated a novel MTDL analogue of nitrogen mustard featuring the 1,3,5-triazine ring substituted with dipeptide residue **92** (Table 7) for inhibitory activity against both AChE and BACE-1 enzymes [135]. This hybrid bearing a 1,3,5-triazine ring substituted with Lys-Ala-OMe, exhibited the most potent inhibition against both AChE (IC₅₀ = 55 nM) and BACE-1 (IC₅₀ = 11.09 µM) enzymes [135]. Similarly, Tripathi et al.

developed a biphenyl-3-oxo-1,2,4-triazine-piperazine-based MTDL **93** (Table 7) featuring a three-carbon atom linker linked with a benzylpiperazine terminal group, demonstrated significant non-competitive inhibition against AChE (IC₅₀ = 0.2 µM) compared to the standard donepezil (IC₅₀ = 0.1 µM) [136]. Additionally, this compound exhibited notable displacement of PI from the PAS of AChE (22.22 %) and demonstrated favorable BBB permeation in the PAMPA-BBB assay (Pₑ = 6.93 × 10⁻⁶ cm/s). *In vivo* behavioral studies of the representative compound revealed significant improvement in cognitive impairments in scopolamine-induced amnesia mouse models. Moreover, *ex vivo* studies indicated that it effectively inhibited AChE and reversed scopolamine-induced oxidative stress. Molecular docking and dynamics simulation analyses unveiled its capacity for mutual binding and active site interactions with AChE's PAS and CAS residues [136].

### 3.2.7. Triazine-triazole hybrids

The biological assessment of 1,2,4-triazines appended with 1,2,3-triazoles, exemplified by hybrids **94** and **95** (Table 7), revealed promising MTDLs with notable BACE-1 inhibitory activity [137]. Specifically, derivatives bearing Cl and NO₂ groups at the 4-position of the phenyl ring, namely compounds **94** (IC₅₀ = 8.55 µM) and **95** (IC₅₀ = 11.42 µM), exhibited significant inhibition against BACE-1. Additionally, examination of the neuroprotective properties of these compounds in the PC12 neuronal cell line demonstrated modest mitigation of amyloid peptide toxicity, with 10 % protection for **94** and 14 % protection for **95** at a concentration of 5 µM. Moreover, it demonstrated metal chelating activity along with reasonable antioxidant potential (IC₅₀ = 44.42 µM). Molecular docking studies of these molecules revealed enhanced binding to several amino acids of BACE-1, critical for effective inhibition [137].

### 3.2.8. Imidazole-piperazine hybrids

Ramrao et al. employed bio-isosteric replacement and hybrid pharmacophore linking strategies to synthesize a novel 4,5-diphenyl-1H-imidazole linked piperazine MTDL designated as hybrid **96** (Table 8) [138]. This hybrid, featuring two carbon atoms connected to the terminal group of benzylpiperazine, exhibited significant inhibitory potential against *ee*AChE (IC₅₀ = 0.42 µM), *eq*BChE (IC₅₀ = 0.47 µM), and BACE-1 (IC₅₀ = 0.39 µM) targets. PI displacement assays indicated that it effectively interacted with the PAS of AChE, showing 29.09 % displacement at 10 µM concentration and 41.43 % at 50 µM, respectively. Furthermore, it demonstrated inhibition of both self-induced (21.28–30.85 %) and AChE-induced (25.70–51.07 %) Aβ aggregation at 5, 10 & 20 µM concentration in the ThT assay. *In vitro* and *in vivo* studies using molecular docking and molecular modeling simulations elucidated the active site interactions of this compound with AChE and BACE-1 [138]. Similarly, alkyl-substituted benzimidazole-piperazine hybrids **97** and **98** (Table 8) exhibited varying degrees of effectiveness as inhibitors of BChE [139]. These hybrids emerged as the most potent BChE inhibitors, with IC₅₀ values of 5.18 µM and 5.22 µM, respectively. Kinetic studies revealed that hybrid **97** acts as a selective non-competitive BChE inhibitor, and molecular modeling studies suggested their access to the CAS of BChE. Additionally, both these hybrids demonstrated neuroprotective effects against H₂O₂-induced cytotoxicity (cell viability = 244 % and 115 %, respectively at 10 µM concentration) and Aβ-induced cytotoxicity in SH-SY5Y cells (94.93 % and >100, respectively) [139].

### 3.2.9. Indole-piperazine hybrids

The indole-piperazine, methylene-linked hybrid **99** (Table 8) has been identified as an irreversible MAO-B inhibitor (IC₅₀ = 154 nM with 91 % inhibition) and an inverse agonist of 5-HT6 receptor (5-HT6R) at Gs signaling (Ki = 38 nM) during the development of dual-acting 5-HT6R antagonists/MAO-B inhibitors [140]. The incorporation of propargyl and N-methylpropargylamine (derived from rasagiline and pargyline, respectively) led to an enhancement in inhibition activity,
<table><caption>**Table 8**
Anti-Alzheimer's potential of MTDLs based on imidazole/indole/pyrazole hybrids.</caption><thead><tr><th>Hybrid compounds</th><th>AChE inhibition IC<sub>50</sub> (µM)</th><th>BChE inhibition IC<sub>50</sub> (µM)</th><th>β-Amyloid inhibition (%)</th><th>Antioxidant IC<sub>50</sub> (µM)</th><th>Other activities</th><th>Ref.</th></tr></thead><tbody><tr><td>96</td><td>0.42</td><td>0.47</td><td>21.28-30.85<sup>c</sup></td><td>n.t.<sup>a</sup></td><td>Inhibited BACE-1 (IC<sub>50</sub> = 0.39 µM), displacement of PI</td><td>[138]</td></tr><tr><td>97</td><td>34.85</td><td>5.18</td><td>94.93<sup>d</sup></td><td>n.t.<sup>a</sup></td><td>No cytotoxicity, neuroprotective effect</td><td>[139]</td></tr><tr><td>98</td><td>97.2</td><td>5.22</td><td>&gt;100<sup>d</sup></td><td>n.t.<sup>a</sup></td><td></td><td></td></tr><tr><td>99</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>MAO-B inhibition (IC<sub>50</sub> = 154 nM, no hepatotoxicity, moderate BBB permeability, reversed scopolamine-induced cognitive dysfunction, Glioprotective properties)</td><td>[140]</td></tr><tr><td>100</td><td>28.8<sup>b</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>Inhibited 5-HT<sub>4</sub>R with K<sub>i</sub> = 37.5 nM and affinity for σ<sub>-1</sub>R with K<sub>i</sub> = 5.1 nM</td><td>[141]</td></tr><tr><td>101</td><td>11.7<sup>b</sup></td><td>45.7<sup>b</sup></td><td>85.04<sup>d</sup></td><td>n.t.<sup>a</sup></td><td>BBB permeation</td><td>[142]</td></tr><tr><td>102</td><td>&gt;100</td><td>&gt;100</td><td>81.65<sup>e</sup></td><td>0.27</td><td>Metal chelation, low toxicity</td><td>[143]</td></tr><tr><td>103</td><td>45<sup>b</sup></td><td>&gt;100</td><td>79.47<sup>e</sup></td><td>0.28</td><td></td><td></td></tr><tr><td>104</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>81.91<sup>e</sup></td><td>n.t.<sup>a</sup></td><td>MAO-B antagonist (IC<sub>50</sub> = 0.41 µM, metal-ion chelating potential, anti-neuroinflammation (NO, TNF-α), neuroprotective activity, and BBB permeation</td><td>[144]</td></tr><tr><td>105</td><td>0.35</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>Tau aggregation (EC<sub>50</sub> = 0.24 µM), neuroprotection, cell viability (75.1 %), BBB permeation</td><td>[145]</td></tr></tbody></table>

<footnote>a Not tested.</footnote>

<footnote>b nM.</footnote>

<footnote>c 5-20 µM.</footnote>

<footnote>d 100 µM.</footnote>

<footnote>e 25 µM.</footnote>
underscoring the pivotal role of this hybrid in forming a covalent bond with the flavin adenine dinucleotide (FAD) cofactor in the enzymatic target. In rat microsomal tests, this compound demonstrated moderate metabolic stability, artificial membrane permeability (P<sub>e</sub> = 4.84 × 10<sup>-6</sup> cm/s), absence of hepatotoxicity (IC<sub>50</sub> = 52.7 µM), and good distribution to the brain. Additionally, it exhibited glioprotective properties in a cultivated astrocyte model using 6-hydroxydopamine (6-OHDA) as the cytotoxic agent. Ultimately, this hybrid completely reversed scopolamine-induced cognitive dysfunction in rats in the novel object recognition (NOR) task [140].

### 3.2.10. Indole-piperidine hybrids

Lalut et al. conducted pharmacomodulation to develop a novel indole-piperidine hybrid **100** (Table 8) starting from donecopride, a dual AChE inhibitor and 5-HT4R agonist [141]. This derivative exhibited nanomolar-level inhibition of human AChE, and its affinity for the σ-1 receptor (σ-1R) was inversely correlated with its affinity for the 5-HT4R. In *in vivo* experiments demonstrated that this hybrid conferred protective effects against dizocilpine-induced cognitive impairment in the passive avoidance test in mice, consistent with its potent *in vitro* affinity for the σ-1R. Furthermore, it inhibited hAChE with an IC<sub>50</sub> value of 28.8 nM, inhibited the 5-HT4R with a Ki value of 37.5 nM, and exhibited an affinity for the σ-1R with a Ki value of 5.1 nM [141].

### 3.2.11. Indole-huprine Y-hybrids

From the biological evaluation, heterodimer **101** (Table 8) (with a six-carbon chain between HupY and L-Trp) was identified as a MTDL with the most balanced properties [142]. It exhibited good inhibitory potential against AChE with an IC<sub>50</sub> value of 11.7 nM and for BChE with an IC<sub>50</sub> value of 45.7 nM, respectively. Furthermore, it blocked Aβ<sub>1-42</sub> self-aggregation (85.04 %) at 100 µM and demonstrated the ability to cross the BBB with a P<sub>e</sub> value of 10.0 × 10<sup>-6</sup> cm/s. Kinetic analysis of this compound's AChE inhibition affirmed its non-competitive mode of inhibitory activity against human AChE and suggested interaction with the PAS [142].

### 3.2.12. Pyrazole-piperazine hybrids

Umar et al. reported selective and effective AChE and Aβ aggregation inhibitors, benzopyrazole-piperazine hybrids **102** and **103** (Table 8) [143]. These hybrids displayed the highest inhibition percentage (81.65 % and 79.47 %, respectively) of self-induced Aβ<sub>1-42</sub> aggregation at 25 µM concentration, compared to curcumin (52.70 % at 25 µM). Furthermore, hybrid **103** featuring bis(N-(1H-pyrazolo[3,4-b]pyridin-3-yl) exhibited excellent anti-AChE activity (IC<sub>50</sub> = 45 nM), with kinetic analysis revealing its nature as a mixed-type blocker. TEM analysis suggested that the planar structure of pyrazolopyridine may facilitate intercalation between Aβ fibrils while inhibiting aggregation. Additionally, this hybrid exhibited Aβ-disaggregation (53.80 % at 25 µM), antioxidation (IC<sub>50</sub> = 0.28 µM), and metal-chelation activity [143].

### 3.2.13. Pyrazole-phthalide hybrids

Cong et al. discovered a novel 5-(2-hydroxyphenyl)-2-phthalide-3-(3H)-pyrazolone hybrid **104** (Table 8) as the most potent MAO-B antagonist (IC<sub>50</sub> = 0.41 µM, SI > 24.4) and antioxidant (1.16 Trolox equiv.) [144]. It also exhibited anti-Aβ aggregation effects, with 56.03 % and 57.51 % suppression of self- and Cu<sup>2+</sup>-induced Aβ<sub>1-42</sub> aggregation, and 81.91 % and 82.40 % disintegration of self- and Cu<sup>2+</sup>-induced Aβ<sub>1-42</sub> fibrils at 25 µM, respectively. Furthermore, this hybrid demonstrated significant metal-ion chelating potential, anti-neuroinflammation (NO, TNF-α), neuroprotective activity, and BBB permeation. More importantly, *in vivo* behavioral testing revealed that it significantly improved memory and cognitive deficits in Aβ<sub>1-42</sub> induced AD mouse models [144].

### 3.2.14. Bis-pyrazole hybrids

The biphenyl pyrazole scaffold **105** (Table 8) demonstrated binding

to monomeric tau with sub-micromolar affinity and hinders tau oligomer establishment during initial phases [145]. This hybrid also displayed inhibition of AChE activity with an IC<sub>50</sub> value of 0.35 µM, compared to the positive control donepezil with an IC<sub>50</sub> value of 7.56 µM. Further investigation into the effect of this hybrid on tau oligomerization in a FRET-based cellular experiment, employing a dose-dependent assay, revealed an EC<sub>50</sub> of 0.24 µM. Therefore, within a cellular context, this hybrid effectively inhibits premature tau oligomerization. Remarkably, this lead compound surpasses single-targeted AChE and tau-aggregation antagonists in an induced cytotoxicity model of SH-SY5Y cells (cell viability = 75.1 %). The PAMPA-BBB indicated a high potential for penetration of the BBB and lipid membrane with a P<sub>e</sub> value of 33.5 × 10<sup>-6</sup> cm/s, while donepezil exhibited a value of 23.4 × 10<sup>-6</sup> cm/s [145].

## 3.3. Pyridine/piperidine/pyrimidine/piperazine hybrids

### 3.3.1. Pyridine-resveratrol hybrids

The pyridoxine-resveratrol hybrids **106** and **107** (Table 9) were identified as MTDLs with inhibitory effects on AChE (IC<sub>50</sub> = 2.11 µM and 1.56 µM, respectively) and MAO-B (IC<sub>50</sub> = 12.4 µM and 14.01 µM, respectively) [146]. These hybrids also inhibited MAO-A (12.8 % and 21.3 %, respectively) at 10 µM. An inhibition kinetic study suggested that hybrid **106** operates as a mixed-type inhibitor of AChE, binding to both the CAS and PAS. Additionally, a molecular modeling study elucidated the binding mode of these hybrids with MAO-B. Furthermore, these compounds demonstrated BChE inhibition, robust antioxidant properties and metal-chelating abilities [146].

### 3.3.2. Pyridine-thiophene hybrids

*In vitro* evaluation of pyridine-thiophene hybrid **108** (Table 9) displayed a significant inhibitory activity against eeAChE, with an IC<sub>50</sub> value of 3.08 µM, and demonstrated higher affinity for BChE (IC<sub>50</sub> = 1.65 µM, SI = 0.5) [147]. Moreover, this hybrid exhibited BBB permeability and anti-convulsive properties in BALB-C mice using the pentylenetetrazole (PTZ) seizure model [147].

### 3.3.3. Pyridine-piperidine hybrids

Malek et al. introduced a 1,4-dihydropyridine-piperidine MTDL designated as compound **109** (Table 9), developed through a linkage strategy [148]. This compound exhibited notable blockade activity against Ca<sup>2+</sup> channels (IC<sub>50</sub> = 21 µM) and displayed effective affinity towards hH3R (Ki = 565 nM). Additionally, it demonstrated specific inhibition of hBChE (IC<sub>50</sub> = 7.83 µM), possessed robust antioxidant properties (3.6 Trolox eq.), exhibited high permeability across the BBB (P<sub>e</sub> = 8.6 × 10<sup>-6</sup> cm/s), and showed potential for ameliorating cognitive deficits induced by lipopolysaccharide [148].

### 3.3.4. Pyridine-chromone hybrids

Yuanyuan et al. introduced pyridine-chromone hybrid **110** (Table 9) by merging hydroxypyridinone and chromone elements, owing to its significant iron-chelating ability (pFe<sup>3+</sup> = 18.52) and its selective inhibition of human MAO-B with an IC<sub>50</sub> value of 67.02 nM [149]. Furthermore, this hybrid possessed the capability to penetrate the BBB and can notably ameliorate cognitive impairment induced by scopolamine in AD mice [149].

### 3.3.5. Piperidine-pyrrolidine hybrids

Wichur et al. investigated novel piperidine-pyrrolidine hybrids **111** and **112** (Table 9) for their inhibitory potential against BChE, BACE-1, Aβ, and tau protein aggregation, alongside assessing antioxidant and metal-chelating properties [150]. These hybrids effectively inhibited the aggregation of Aβ and tau proteins at 10 µM (**111**: 45 % for Aβ, 53 % for tau; **112**: 49 % for Aβ, 54 % for tau). Furthermore, both hybrids exhibited free radical scavenging potential in the ABTS assay and antioxidant properties in the FRAP assay, in addition to preferentially
<table><caption>**Table 9**
Anti-Alzheimer's potential of MTDLs based on pyridine/piperidine/pyrimidine/piperazine hybrids.</caption><thead><tr><th>Hybrid compounds</th><th>AChE<br>inhibition IC<sub>50</sub><br>(µM)</th><th>BChE<br>inhibition IC<sub>50</sub><br>(µM)</th><th>β-Amyloid<br>inhibition (%)</th><th>Antioxidant<br>IC<sub>50</sub> (µ/M)</th><th>Other activities</th><th>Ref.</th></tr></thead><tbody><tr><td>106</td><td>2.11</td><td>16.7</td><td>n.t.<sup>a</sup></td><td>2.56</td><td>Inhibited MAO B, IC<sub>50</sub> = 12.4 µM and 14.01 µM, respectively also inhibited MAO-A (12.8 % and 21.3 %, respectively) metal chelation</td><td>[146]</td></tr><tr><td>107</td><td>1.56</td><td>8.9</td><td></td><td>1.76</td><td></td><td></td></tr><tr><td>108</td><td>3.08</td><td>1.65</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>Anti-convulsive potential, BBB permeation</td><td>[147]</td></tr><tr><td>109</td><td>22.5</td><td>7.83</td><td>n.t.<sup>a</sup></td><td>3.6</td><td>Good Ca<sup>2+</sup> channels blockade activity, efficacious affinity against hH<sub>3</sub>R, BBB permeability</td><td>[148]</td></tr><tr><td>110</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>hMAO-B inhibition IC<sub>50</sub> = 67.02 nM, BBB permeability, iron-chelating potential (pFe<sup>3+</sup> = 18.52)</td><td>[149]</td></tr><tr><td>111</td><td>40.8<sup>b</sup></td><td>2.39</td><td>45<sup>c</sup></td><td>96<sup>c</sup></td><td>Chelate copper ions, inhibit tau protein aggregation (53 % and 54 % at 10 µM, respectively), inhibited BACE-1 (25 % and &lt;10 %, respectively)</td><td>[150]</td></tr><tr><td>112</td><td>29.9<sup>b</sup></td><td>1.94</td><td>49<sup>c</sup></td><td>94<sup>c</sup></td><td></td><td></td></tr><tr><td>113</td><td>6.47</td><td>20.01<sup>b</sup></td><td>39<sup>c</sup></td><td>n.t.<sup>a</sup></td><td>BACE-1 inhibitory activity (IC<sub>50</sub> = 26.3 % at 50 µM), BBB permeation</td><td>[151]</td></tr><tr><td>114</td><td>63.5<sup>b</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>K<sub>i</sub> = 59 nM for 5-HT<sub>4</sub>R</td><td>[155]</td></tr><tr><td>115</td><td>-</td><td>-</td><td>1.3<sup>d</sup></td><td>n.t.<sup>a</sup></td><td>Suppressed tau protein IC<sub>50</sub> = 1.8 µM, neuroprotective effect against tau toxicity</td><td>[156]</td></tr><tr><td>116</td><td>32<sup>b</sup></td><td>21.83</td><td>n.t.<sup>a</sup></td><td>1.520</td><td>Inhibited MAO-B with the IC<sub>50</sub> = 2.11 µM, BBB permeation, and good copper chelation</td><td>[158]</td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 9 (continued)**</caption><thead><tr><th>Hybrid compounds</th><th>AChE inhibition IC<sub>50</sub> (μM)</th><th>BChE inhibition IC<sub>50</sub> (μM)</th><th>β-Amyloid inhibition (%)</th><th>Antioxidant IC<sub>50</sub> (μ/M)</th><th>Other activities</th><th>Ref.</th></tr></thead><tbody><tr><td>117</td><td>40<sup>b</sup></td><td>25.03</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>Lowered spatial memory impairment and upgraded cognitive deficits, BBB permeation, inhibited MAO B, metal chelation, neuroprotection</td><td>[159]</td></tr><tr><td>118</td><td>10.36</td><td>17.21</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>Inhibited MAO-B with the IC<sub>50</sub> = 0.13 and 0.10 μM, respectively, no neurotoxicity, BBB permeation</td><td>[160]</td></tr><tr><td>119</td><td>69<sup>b</sup></td><td>0.98</td><td></td><td></td><td></td><td></td></tr><tr><td>120</td><td>1.83</td><td>10.1</td><td>n.t.<sup>a</sup></td><td>30<sup>b</sup></td><td>Displaced PI, BBB permeation</td><td>[161]</td></tr><tr><td>121</td><td>127<sup>b</sup></td><td>91<sup>b</sup></td><td>3.16<sup>d</sup></td><td>6.40</td><td>BBB permeation, cytotoxicity</td><td>[162]</td></tr><tr><td>122</td><td>70<sup>b</sup></td><td>59<sup>b</sup></td><td>2.08<sup>d</sup></td><td>8.09</td><td></td><td></td></tr><tr><td>123</td><td>99<sup>b</sup></td><td>185<sup>b</sup></td><td>2.14<sup>d</sup></td><td>7.20</td><td></td><td></td></tr><tr><td>124</td><td>0.71</td><td>n.t.<sup>a</sup></td><td></td><td></td><td>Exhibited EC<sub>50</sub> values of 2.21 μM and 2.71 μM for cellular tau-oligomerization, respectively, neuroprotection</td><td>[163]</td></tr><tr><td>125</td><td>1.09</td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

<footnote>a Not tested.</footnote>

<footnote>b nM.</footnote>

<footnote>c 10 μM.</footnote>

<footnote>d μM.</footnote>

<footnote>e Percent inhibition.</footnote>
chelating copper ions. Both these compounds also emerged as the most potent BChE inhibitors, with IC₅₀ values of 2.39 µM and 1.94 µM, respectively [150]. Similarly, phthalimide and saccharin hybrids linked by different alicyclic fragments were evaluated against AChE, BACE-1, and Aβ inhibition [151]. *In vitro* findings revealed that the phthalimide derivative containing a 3-aminopiperidine fragment, compound **113** (Table 9), exhibited preferential AChE inhibiting activity (IC₅₀ = 6.47 µM), along with BACE-1 inhibition (IC₅₀ = 26.3 % at 50 µM), and demonstrated significant Aβ anti-aggregating properties (39 % at 10 µM) [151].

### 3.3.6. Piperidine-benzisoxazole hybrids

The preclinical candidate donecopride, known for inhibiting AChE while acting as a 5-HT4R agonist [152–154]. In a pharmacomodulation study a novel piperidine-benzisoxazole hybrid **114** (Table 9) was developed using rigidification approach as a potent AChE inhibitor (IC₅₀ = 63.5 nM) and Ki = 59 nM for 5-HT4R [155]. The chemical synthesis of this ligand was optimized, and further subjected to *in vitro* and *in cellulo* testing against each target. Docking assessments in the *h*AChE binding site provided further insights into the SAR [155].

### 3.3.7. Piperidine-thioxanthenone hybrids

The study investigated thioxanthene-9-one and xanthene-9-one hybrids that had previously shown promise in suppressing ChEs and Aβ₄₀ aggregation [156] now focusing on their effect on tau protein aggregation [157]. The piperidine-thioxanthenone hybrid **115** (Table 9) stood out with exceptional biological activity, demonstrating potent suppression of both tau protein and Aβ₄₀ aggregation, with IC₅₀ values of 1.8 µM and 1.3 µM, respectively. Additionally, this compound exhibited a neuroprotective effect against tau toxicity induced by okadoic acid in human SH-SY5Y cells, showing efficacy comparable to that of estradiol and PD38 at equivalent concentrations [156].

### 3.3.8. Pyrimidine-imidazole hybrids

Xu et al. introduced propargylamine-modified pyrimidinylthiourea hybrids as dual inhibitors of ChEs and MAOs, along with other functions relevant to AD [158]. The pyrimidine-benzimidazole hybrid **116** (Table 9) emerged as a potent dual inhibitor, effectively targeting both AChE and MAO-B. It exhibited an IC₅₀ value of 32 nM for AChE inhibition, outperforming donepezil (IC₅₀ = 6.39 µM), and an IC₅₀ value of 2.11 µM for MAO-B inhibition, comparable to pargyline (IC₅₀ = 0.22 µM). Additionally, this hybrid demonstrated enhanced antioxidant capacity, BBB permeation, and effective copper chelation *in vitro*. Animal studies further validated the efficacy of this compound, as its hydrochloric salt formulation inhibited AChE/MAO-B activities in the brain and ameliorated scopolamine-induced memory loss in mice [158].

### 3.3.9. Pyrimidine-piperidine hybrids

The N-propargyl-substituted diphenylpyrimidine-piperidine hybrid **117** (Table 9) exhibited remarkable potency as an AChE inhibitor, with an IC₅₀ value of 40 nM, compared to pargyline with an IC₅₀ value of 15 nM [159]. Additionally, it demonstrated a high selectivity index of 626 over BChE and inhibited MAO-B with an IC₅₀ value of 0.37 µM. In ROS inhibition studies, this hybrid reduced intracellular ROS levels in SH-SY5Y cells by 68 %. Moreover, *in vivo* investigations involving oral administration of the HCl salt of hybrid **117** to rats revealed its ability to cross the BBB and reach the target site. In mice, this salt significantly attenuated spatial memory impairment and improved cognitive deficits [159].

### 3.3.10. Pyrimidine-indole hybrids

The 2-arylidine hybrids **118** and **119** (Table 9) of thiazolopyrimidine, emerged as the potent inhibitors of MAO-B with the IC₅₀ values of 0.13 µM and 0.10 µM, respectively as compared to safanamide IC₅₀ = 30 nM [160]. In addition, these hybrids also showed reasonable inhibition for ChEs, kinetic studies revealed a mixed inhibition mode of

these inhibitors. The impact of functionalities and linker length on effectiveness was revealed by SAR analysis. Acute toxicity testing revealed that both hybrids were safe up to a dose of 2000 mg/kg. PAMPA-BBB analysis revealed the BBB permeation of these compounds (Pₑ = 6.20 × 10⁻⁶ cm/s and 7.45 × 10⁻⁶ cm/s, respectively). Whereas MTT assays on neuroblastoma SH-SY5Y cells revealed no neurotoxicity for these compounds at the tested concentrations [160].

#### 3.3.11. Piperazine-amiridine hybrids

Piperazine-amiridine hybrid **120** (Table 9), featuring a 4-nitrophenyl substituent, demonstrated an AChE IC₅₀ of 1.83 µM (Ki = 1.50 µM and αKi = 2.58 µM), while exhibiting a lower BChE IC₅₀ of 10.1 µM compared to the standards amiridine (IC₅₀ = 0.18 µM) and tacrine (IC₅₀ = 21 nM) [161]. Furthermore, this hybrid displayed low activity against carboxylesterase, indicating a reduced likelihood of unwanted drug-drug interactions in clinical use. Additionally, the hybrid molecule exhibited significant antioxidant capability, with Trolox equiv. antioxidant capacity of 30 nM [161].

#### 3.3.12. Piperazine-thienopyrimidine hybrids

The AChE inhibition exhibited by the piperazine-tetrahydrodrobenzothienopyrimidine, hybrids **121–123** (Table 9), was comparable to that of donepezil [162]. Among these hybrids, **122** demonstrated the most potent inhibition of both AChE and BChE, with IC₅₀ values of 70 nM and 59 nM, respectively. Additionally, all three hybrids exhibited significant antioxidant properties. Compounds **122** and **123** were found to inhibit Aβ aggregation (IC₅₀ = 2.08 µM, 2.14 µM) more effectively than donepezil (IC₅₀ = 2.93 µM). Moreover, cytotoxicity studies revealed that both these compounds were less cytotoxic (IC₅₀ = 218.7 µM and 229.3 µM) than donepezil, while compound **121** exhibited marginal cytotoxicity (IC₅₀ = 134.2 µM) towards PC12 cells compared to donepezil (IC₅₀ = 140.9 µM). Based on the findings from molecular docking and molecular dynamics simulations, compound **122**, which consists of a phenylpiperazine molecule linked to a thienopyrimidine scaffold *via* a two-atom spacer, emerged as a promising multifunctional therapeutic candidate for AD treatment. It functions as an effective dual AChE/BChE inhibitor and also demonstrates superior inhibition of Aβ aggregation compared to donepezil [162].

#### 3.3.13. Piperazine-pyrazine hybrids

Madhav et al. outlined the development of piperazine-pyrazine hybrids **124** and **125** (Table 9) using a multicomponent Petasis reaction [163]. Notably, these hybrids exhibited promising activity, with IC₅₀ values of 0.71 µM and 1.09 µM against *h*AChE, respectively, while demonstrating EC₅₀ values of 2.21 µM and 2.71 µM for cellular tau oligomerization, respectively. Furthermore, when evaluated on tau-expressing SH-SY5Y cells using an MTT assay, hybrid **124** displayed superior neuroprotective effects compared to donepezil [163].

### 3.4. Oxadiazole/thiadiazole/thiazole/thiazolidinedione hybrids

#### 3.4.1. Oxadiazole-pyridine hybrids

The effects of 1,2,4-oxadiazole-pyridine hybrids on glucose uptake in HepG2 cells were investigated due to the significance of abnormal glucose metabolism in AD [164]. Hybrids **126** and **127** (Table 10) demonstrated excellent dual potency in GSK-3β inhibition, with IC₅₀ values of 1.02 µM and 0.19 µM, respectively, and anti-neuroinflammatory potency, with IC₅₀ values of 0.47 µM and 6.94 µM, respectively. Compound **127** exhibited a greater effect on glucose consumption than the positive drug metformin and displayed a neuroprotective effect. It also significantly reduced Aβ-induced tau hyperphosphorylation by inhibiting GSK-3β at the cellular level. Both compounds inhibited the formation of intracellular ROS. Additionally, these hybrids exhibited normal BBB permeability and were neurotoxic up to a concentration of 50 µM. *In vivo* studies demonstrated that hybrid **127** alleviated memory loss in scopolamine-induced mouse models
<table><caption>**Table 10**
Anti-Alzheimer's potential of MTDLs based on oxadiazole/thiazole/thiazolidinedione hybrids.</caption><thead><tr><th>Hybrid compounds</th><th>AChE inhibition IC<sub>50</sub> (µM)</th><th>BChE inhibition IC<sub>50</sub> (µM)</th><th>β-Amyloid inhibition (%)</th><th>Antioxidant IC<sub>50</sub> (µM)</th><th>Other activities</th><th>Ref.</th></tr></thead><tbody><tr><td><img src="image_126.png" alt="Chemical structure of 126"></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>n.a.<sup>b</sup></td><td>GSK-3β inhibition with the IC<sub>50</sub> = 1.02 and 0.19 µM, respectively, anti-neuroinflammatory potency with IC<sub>50</sub> values of 0.47 µM and 6.94 µM, respectively, BBB permeability</td><td>[164]</td></tr><tr><td><img src="image_127.png" alt="Chemical structure of 127"></td><td>1.1</td><td>7.80</td><td>38.2-65.9<sup>e</sup></td><td>n.a.<sup>b</sup></td><td>Improved learning and memory, BBB permeability</td><td>[165]</td></tr><tr><td><img src="image_128.png" alt="Chemical structure of 128"></td><td>54<sup>c</sup></td><td>0.79</td><td>n.a.<sup>b</sup></td><td>n.t.<sup>a</sup></td><td>Inhibited hBACE-1, IC<sub>50</sub> = 98 nM, displacement of PI, improved learning and cognition, BBB permeability, neuroprotection</td><td>[166]</td></tr><tr><td><img src="image_129.png" alt="Chemical structure of 129"></td><td>91<sup>c</sup></td><td>106<sup>c</sup></td><td>33.33-65.66<sup>e</sup></td><td>n.t.<sup>a</sup></td><td>Inhibited (hBACE-1) (IC<sub>50</sub> = 207 and 143 nM, respectively, BBB permeation, potential dismantling of A/β aggregates, and neuroprotection</td><td>[167]</td></tr><tr><td><img src="image_130.png" alt="Chemical structure of 130"></td><td>64<sup>c</sup></td><td>74<sup>c</sup></td><td>35.66-73.33<sup>e</sup></td><td></td><td></td><td></td></tr><tr><td><img src="image_131.png" alt="Chemical structure of 131"></td><td>1.08</td><td>1.65</td><td>20-61<sup>e</sup></td><td>n.t.<sup>a</sup></td><td>Inhibited hBACE-1 (IC<sub>50</sub> = 0.65 and 0.75 µM, respectively), displaced PI</td><td>[168]</td></tr><tr><td><img src="image_132.png" alt="Chemical structure of 132"></td><td>0.91</td><td>1.58</td><td>22-69<sup>e</sup></td><td></td><td></td><td></td></tr><tr><td><img src="image_133.png" alt="Chemical structure of 133"></td><td>6.40</td><td>7.50</td><td>n.t.<sup>a</sup></td><td></td><td></td><td>[169]</td></tr><tr><td><img src="image_134.png" alt="Chemical structure of 134"></td><td>5.80</td><td>7.20</td><td></td><td></td><td></td><td></td></tr><tr><td><img src="image_135.png" alt="Chemical structure of 135"></td><td>6.90</td><td>7.60</td><td></td><td></td><td></td><td></td></tr><tr><td><img src="image_136.png" alt="Chemical structure of 136"></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 10 (continued)**</caption><thead><tr><th>Hybrid compounds</th><th>AChE inhibition IC<sub>50</sub> (µM)</th><th>BChE inhibition IC<sub>50</sub> (µM)</th><th>β-Amyloid inhibition (%)</th><th>Antioxidant IC<sub>50</sub> (µM)</th><th>Other activities</th><th>Ref.</th></tr></thead><tbody><tr><td>137</td><td>13.96</td><td>14.62</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>Anti-amnestic properties, BBB permeability</td><td>[170]</td></tr><tr><td>138</td><td>2.32</td><td>&gt;100</td><td>53.30<sup>f</sup></td><td>n.t.<sup>a</sup></td><td>BBB permeability, neuroprotection, low toxicity, metal chelation</td><td>[171]</td></tr><tr><td>139</td><td>20.8<sup>d</sup></td><td>43.3<sup>d</sup></td><td>60.6<sup>g</sup></td><td>n.t.<sup>a</sup></td><td>–</td><td>[172]</td></tr><tr><td>140</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>BACE-1 (IC<sub>50</sub> = 9 µM), anti-inflammation (70 %) antioxidant, anti-amyloid, neuroprotective, and anti- amnesic properties</td><td>[173]</td></tr><tr><td>141</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>Inhibited BACE-1 IC<sub>50</sub> = 3 µM, rat paw edema, anti- amnesic, anti-amyloid, and neuroprotective properties</td><td>[174]</td></tr><tr><td>142</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>Inhibited GSK-3/β IC<sub>50</sub> = 4.93 and 0.89 µM respectively, nontoxic, BBB permeability, also inhibited tau aggregations (60 % and 80 % at 10 µM, respectively)</td><td>[176]</td></tr><tr><td>143</td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

[164]. Similarly, a novel 1,3,4-oxadiazole-pyridine hybrid, **128** (Table 10), with 4-hydroxyl substituents, was reported by Mishra et al. for its potential AChE inhibition along with remarkable antioxidant properties [165]. This hybrid demonstrated the most potent inhibition of hAChE with a non-competitive enzyme inhibition (IC<sub>50</sub> = 1.1 µM; Ki = 0.96 µM). Furthermore, the PAMPA-BBB assay revealed significant BBB permeability of this hybrid (P<sub>e</sub> = 4.65 × 10<sup>-6</sup> cm/s). In addition, it inhibited AChE-induced A/β aggregation (38.2–65.9 % at 5–20 µM) in the ThT assay. *In vivo* behavioral studies showed that this hybrid improved learning and memory in a dose-dependent manner. *Ex vivo* studies confirmed substantial AChE inhibition and antioxidant potential of the lead compound in brain homogenates [165].

The hybrid **129** (Table 10), 1,3,4-oxadiazole-pyridylpiperazine with 2,4-difluoro substituents at the terminal phenyl ring, was identified as the most promising multifunctional ligand [166]. It exhibited inhibitory activity against hAChE (IC<sub>50</sub> = 54 nM), hBChE (IC<sub>50</sub> = 0.79 µM vs donepezil 0.36 µM), and hBACE-1 (IC<sub>50</sub> = 98 nM). Enzyme kinetics of

the hybrid against hAChE revealed a mixed type of inhibition (Ki = 30 nM). Moreover, it demonstrated significant displacement of PI from hAChE's PAS (22.4 % at 10 µM, compared to donepezil 16.9 % at the same concentration). It exhibited excellent BBB permeability in the PAMPA assay (P<sub>e</sub> = 6.1 × 10<sup>-6</sup> cm/s) and neuroprotective ability against SH-SY5Y neuroblastoma cell lines (69–89 %). This hybrid also showed self- and AChE-induced anti-A/β aggregation activity in ThT assays. Additionally, *in vivo* behavioral studies demonstrated that novel hybrid improved learning and cognition in scopolamine- and A/β-induced cognitive dysregulation, assessed using the Y-maze and Morris water maze. *Ex vivo* studies indicated the reduced AChE activity and exhibited antioxidant potential, with adequate oral absorptivity as determined by pharmacokinetic studies [166].

### 3.4.2. Oxadiazole-pyrrolidine hybrids

Oxadiazole-pyrrolidine hybrids **130** and **131** (Table 10) with -CF3 as a substituent were identified as the potent inhibitor, inhibiting hBACE-1

<footnote>a Not tested.</footnote>

<footnote>b Tested but not active.</footnote>

<footnote>c nM.</footnote>

<footnote>d Percent inhibition.</footnote>

<footnote>e 5-20 µM.</footnote>

<footnote>f 50 µM.</footnote>

<footnote>g 100 µM.</footnote>
with IC₅₀ values of 207 nM and 143 nM, respectively as compared to donepezil (IC₅₀ = 362 nM) [167]. They also demonstrated inhibition of hAChE with IC₅₀ values of 91 nM and 64 nM, respectively, and hBChE with IC₅₀ values of 106 nM and 74 nM, respectively. Furthermore, these hybrids were also found to possess significant AChE-PAS binding capacity, BBB permeation, potential dismantling of Aβ aggregates, and neuroprotection capabilities on SHSY-5Y cell lines. The Y-Maze and Morris water maze tests revealed that both hybrids reduced cognitive impairment caused by scopolamine and Aβ. In addition, ex-vivo activity on rat brain homogenate revealed a decline in AChE level and oxidative stress. Pharmacokinetic studies determined that hybrid 131 has a significant oral absorption profile [167].

### 3.4.3. Oxadiazole-piperidine hybrids

The oxadiazole-piperidine hybrids 132 and 133 (Table 10) were evaluated to assess their potential to inhibit hAChE, hBChE, hBACE-1, and Aβ aggregation, aiming to enhance cognitive and memory functions associated with AD [168]. These hybrids emerged as standout candidates, displaying multifaceted inhibitory profiles against hAChE (IC₅₀ = 1.08 µM and 0.91 µM, respectively), hBChE (IC₅₀ = 1.65 µM and 1.58 µM, respectively vs donepezil 1.41 µM), and hBACE-1 (IC₅₀ = 0.65 µM and 0.75 µM, respectively) enzymes in in vitro experiments [168]. Additionally, both hybrids exhibited slightly higher anti-Aβ aggregation properties in ThT assays conducted in the presence of self- and hAChE-induced aggregation (132: 20–61 %, and 38–79 %, respectively and 133: 22–69 % and 41–81 %, respectively) compared to donepezil (self-induced: 21–37 %, hAChE-induced 28–72 %) at 5, 10 and 20 µM concentrations. Importantly, these hybrids effectively displaced PI from the cholinesterase PAS region, demonstrating excellent permeability across the BBB and no associated neurotoxicity. Notably, hybrid 133 was found to ameliorate cognitive and memory functions in behavioral rat models of AD induced by scopolamine and Aβ [168].

### 3.4.4. Oxadiazole-benzoxazole hybrids

In vitro experiments demonstrated substantial inhibitory activities of oxadiazole-benzoxazole hybrids 134–136 (Table 10) against AChE and BChE [169]. Notably, these compounds exhibited significant activity, even surpassing the effectiveness of standard inhibitors, with IC₅₀ values of 6.40 µM, 5.80 µM, and 6.90 µM, respectively, against AChE, compared to donepezil (IC₅₀ = 33.65 µM). Furthermore, these hybrids also inhibited BChE with IC₅₀ values of 7.50 µM, 7.20 µM, and 7.60 µM, respectively, compared to donepezil with IC₅₀ = 35.80 µM [169].

### 3.4.5. Thiazole-piperazine hybrids

Jończyk et al. conducted in vitro assays on thiazol-piperazine hybrid 137 (Table 10) to assess its potential against AChE and BChE, while molecular dynamics simulation investigations revealed potential interactions between this active hybrid and AChE, BChE, and the human H3 histamine receptor [170]. Computational findings indicated a very good physicochemical profile and high drug-likeness of this hybrid. In test conditions, the PAMPA assay demonstrated excellent BBB permeability of this hybrid (P<sub>e</sub> = 9.85 × 10<sup>-6</sup> cm/s). This hybrid exhibited a well-balanced multifunctional profile with therapeutic potential toward the studied targets: H3 antagonist activity (pA2 = 8.27), inhibitory activity against both AChE (IC₅₀ = 13.96 µM) and BChE with IC₅₀ = 14.62 µM. In the passive avoidance test on mice, in vivo pharmacological studies revealed that it possesses anti-amnestic properties. Similarly, benzothiazole-piperazine hybrid 138 (Table 10), with pyridine as a substituent, demonstrated balanced functions, such as effective, noncompetitive, and selective inhibition of AChE (IC₅₀ = 2.32 µM), good copper chelation, and Aβ₁₄₂ aggregation inhibition (53.30 % at 50 µM) [171]. The ability of the hybrid to inhibit Aβ fibrils was also validated by confocal laser scanning microscopy and TEM analysis. Besides this, it exhibited low toxicity and could prevent cell viability loss caused by H₂O₂ neurotoxicity in SH-SY5Y cells. Noticeably, this hybrid dramatically improved cognitive processing and memory tasks in a mouse model

of scopolamine-induced memory deficit [171].

### 3.4.6. Thiazole-imidazole hybrids

The new 2-phenylbenzo[d]thiazole substituted hybrid 139 (Table 10) showed potential as a dual inhibitor of Aβ aggregation and ChE inhibition [172]. In vitro studies revealed that this hybrid, containing an imidazole ring, strongly inhibited Aβ₁₄₀ aggregation (49.2 %) as well as Aβ₁₄₂ aggregation (60.6 %) at 100 µM. However, it exhibited weak inhibitory potential against both AChE and BChE [172].

### 3.4.7. Thiazole-thiadiazine hybrids

The novel thiazolyl-thiadiazine MTDL 140 (Table 10) exhibited inhibition of BACE-1, and in vivo studies showcased effective anti-inflammatory activity with inherent gastrointestinal safety [173]. This hybrid demonstrated significant BACE-1 inhibition (IC₅₀ = 9 µM) along with properties such as anti-inflammatory activity (70 %), antioxidant effects, anti-amyloid activity, neuroprotection, and anti-amnesic effects [173].

### 3.4.8. Thiazole-quinoxaline hybrids

The bisthiazole-quinoxaline hybrid 141 (Table 10) inhibited BACE-1 (IC₅₀ of 3 µM), owing to its -NH₂ functionality that forms a strong hydrogen bond with the catalytic site [174]. Additionally, it demonstrated significant inhibition of rat paw edema in acute and chronic inflammation models, with a reduction of 69 %. In the AlCl₃-induced AD model, it showed remarkable anti-amnesic, anti-amyloid, antioxidant, and neuroprotective properties in treated rats. Moreover, behavioral assessments indicated improved cognitive functions, further supporting the gastrointestinal safety of this hybrid [174].

### 3.4.9. Thiazolidinedione-indole hybrids

The modified tau protein system stands out as a significant target for AD, with therapeutic strategies focusing on inhibiting the phosphorylating tau kinase GSK-3β and the tau aggregation process [175]. Recognizing this, Gandini et al. reported 2,4-thiazolidinedione-indole hybrids 142 and 143 (Table 10) [176]. These hybrids exhibited micromolar IC₅₀ values for GSK-3β (4.93 and 0.89 µM, respectively) and demonstrated 60 % and 80 % inhibition of AcPHF6 aggregation at 10 µM, respectively. Moreover, they showed PAMPA-BBB permeation and cellular tolerability. Hybrid 143 also inhibited both K18 and full-length tau aggregations. Remarkably, in an okadaic acid-induced neurodegeneration cell model, both derivatives enhanced cell viability, they represent the first stable, nontoxic, dual-acting derivatives capable of targeting tau aggregation at two distinct sites [176].

## 3.5. Coumarin/benzofuran hybrids

### 3.5.1. Coumarin-piperidine hybrids

Coumarin, recognized for its multifunctional nature, serves as a promising scaffold for the development of MTDLs with therapeutic implications in neurodegenerative diseases, particularly AD [177]. In a study by Rullo et al. 7-benzyloxy-2H-chromen-2-one-piperidine hybrid 144 (Table 11) was evaluated as MTDLs targeting AChE, BChE and MAO-B, key enzymes in AD pathology [178]. This hybrid demonstrated sub-micromolar inhibition of MAO-A (inhibition = 32 % at 10 µM), MAO-B (IC₅₀ = 0.27 µM), AChE (IC₅₀ = 3.1 µM), and BChE (IC₅₀ = 2.3 µM). Conversely, an aliphatic 6-membered nitrogen-containing cycle in this hybrid resulted in decreased MAO-B inhibitory potency [178]. Similarly, a novel coumarin-dithiocarbamate hybrid, designated as 145 (Table 11), exhibited potent inhibition of AChE (IC₅₀ = 27 nM) and downregulation of Aβ aggregation (inhibition = 40.19 % at 25 µM), comparable to the inhibition of curcumin (39.62 %) at same concentration [179]. The kinetic and molecular modeling studies of this hybrid revealed it as a mixed-type inhibitor interacting with both CAS and PAS of AChE. Moreover, this hybrid displayed specific metal-chelating ability, good BBB permeation (P<sub>e</sub> = 5.75 × 10<sup>-6</sup> cm/s), and low toxicity on
<table><caption>**Table 11**
Anti-Alzheimer's potential of MTDLs based on coumarin/benzofuran/carbazole/isatin hybrids.</caption><thead><tr><th>Hybrid compounds</th><th>AChE inhibition IC<sub>50</sub> (µM)</th><th>BChE inhibition IC<sub>50</sub> (µ/M)</th><th>β-Amyloid inhibition (%)</th><th>Antioxidant IC<sub>50</sub> (µM)</th><th>Other activities</th><th>Ref.</th></tr></thead><tbody><tr><td>144</td><td>3.1</td><td>2.3</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>MAO-A inhibition = 32 % at 10 µM, MAO-B inhibition IC<sub>50</sub> = 0.27 µM</td><td>[178]</td></tr><tr><td>145</td><td>27<sup>c</sup></td><td>15.29</td><td>40.19<sup>d</sup></td><td>n.t.<sup>a</sup></td><td>Metal chelating ability, good BBB permeation, overturn the cognitive dysfunction and low toxicity</td><td>[179]</td></tr><tr><td>146</td><td>44<sup>c</sup></td><td>23.28<sup>c</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>hMAO-B inhibition IC<sub>50</sub> = 0.10 µM, BBB permeability, no toxicity, overturn the cognitive impairments</td><td>[180]</td></tr><tr><td>147</td><td>0.10</td><td>14.38</td><td>38.25<sup>d</sup></td><td>n.t.<sup>a</sup></td><td>BBB permeability, low toxicity, improve memory</td><td>[181]</td></tr><tr><td>148</td><td>29.56</td><td>45.87</td><td>n.t.<sup>a</sup></td><td>67.07</td><td>Possess anti-inflammatory activity, BBB permeability.</td><td>[182]</td></tr><tr><td>149</td><td>28.68</td><td>47.39</td><td>n.t.<sup>a</sup></td><td>65.57</td><td></td><td></td></tr><tr><td>150</td><td>0.27</td><td>n.t.<sup>a</sup></td><td>64.5<sup>e</sup></td><td>347</td><td>-</td><td>[183]</td></tr><tr><td>151</td><td>34.96<sup>c</sup></td><td>133.15<sup>c</sup></td><td>337.77<sup>f</sup></td><td>n.t.<sup>a</sup></td><td>Good ability for MMP-2 inhibition (IC<sub>50</sub> = 441.33 nM), chelates metal and BBB permeability</td><td>[184]</td></tr><tr><td>152</td><td>0.64</td><td>0.55</td><td>29.8<sup>g</sup></td><td>n.t.<sup>a</sup></td><td>Neuroprotection, BBB permeability</td><td>[185]</td></tr><tr><td>153</td><td>3.75</td><td>n.a.<sup>b</sup></td><td>n.t.<sup>a</sup></td><td>0.81</td><td>-</td><td>[186]</td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 11 (continued)**</caption><thead><tr><th>Hybrid compounds</th><th>AChE inhibition IC<sub>50</sub> (µM)</th><th>BChE inhibition IC<sub>50</sub> (µ/M)</th><th>β-Amyloid inhibition (%)</th><th>Antioxidant IC<sub>50</sub> (µM)</th><th>Other activities</th><th>Ref.</th></tr></thead><tbody><tr><td>154</td><td>8.72</td><td>0.50</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>–</td><td>[187]</td></tr><tr><td>155</td><td>6.72</td><td>>100</td><td></td><td></td><td></td><td></td></tr><tr><td>156</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>74<sup>g</sup></td><td>n.t.<sup>a</sup></td><td>Tau aggregation 66.1 %, neurotoxicity survival 94.3 % at 10 µM, BBB permeation</td><td>[3]</td></tr><tr><td>157: n=3<br>158: n=4</td><td>18.2<br>27.5</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>BBB permeability, no hepatotoxicity, immunotoxicity, and cytotoxicity</td><td>[189]</td></tr><tr><td>159</td><td>>100</td><td>4.78</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>Neuroprotective properties, no BACE-1 inhibition</td><td>[190]</td></tr></tbody></table>

SH-SY5Y cells. Furthermore, at doses of up to 1000 mg/kg, it showed no cytotoxic effect in mice and improved cognitive dysfunction in a scopolamine-induced AD mouse model [179].

Another coumarin-dithiocarbamate hybrid, labeled as **146** (Table 11), exhibited potent inhibition of hMAO-B (IC<sub>50</sub> = 0.10 µM as compared to rasagiline IC<sub>50</sub> = 0.14 µM) and balanced inhibition of eeAChE (IC<sub>50</sub> = 44 nM) [180]. Kinetic and molecular modeling studies revealed it as a dual binding site inhibitor for AChE and a competitor blocker for MAO-B. Additionally, it displayed good BBB penetration (P<sub>e</sub> = 5.75 × 10<sup>-6</sup> cm/s), no toxicity to SH-SY5Y cells, and improved cognitive impairments in a scopolamine-induced AD mouse model [180]. Likewise, a chromanone-dithiocarbamate hybrid, designated as **147** (Table 11), was assessed and exhibited robust inhibitory potential against eeAChE (IC<sub>50</sub> = 0.10 µM) and demonstrated efficacy in inhibiting both AChE-induced and self-induced Aβ aggregation as [181]. The kinetic analysis and docking studies revealed that this hybrid targeted both the CAS and PAS of AChE, positioning it as a dual binding site inhibitor. Moreover, it displayed good BBB penetration, reduced neurotoxicity, and improved memory in a scopolamine-induced mouse model of AD [181].

### 3.5.2. Coumarin-oxadiazole hybrids

The anti-ChE activity of coumarin-oxadiazole hybrids **148** and **149** (Table 11) was assessed, revealing them as potent AChE inhibitors with IC<sub>50</sub> values of 29.56 µM and 28.68 µM, respectively [182]. Additionally, these hybrids exhibited notable antioxidant properties as determined by DPPH assay, with IC<sub>50</sub> values of 67.07 µM and 65.57 µM, respectively.

Moreover, these hybrids demonstrated higher SI values of 1.65 and 1.55, respectively, compared to the standard galantamine (SI = 1.13). Molecular docking studies further elucidated the binding interactions of these potent AChE inhibitors with binding energy scores of 9.7 and 10.1 kcal/mol for both compounds, respectively, indicating favorable binding affinity to the AChE active site. Additionally, these hybrid molecules exhibited promising *in vitro* antioxidant and anti-inflammatory properties [182].

### 3.5.3. Coumarin-benzofuran hybrids

Hiremathad et al. conducted an evaluation of the coumarin-benzofuran hybrid **150** (Table 11) featuring a methoxy functional group and a shorter linker (n = 0) [183]. Molecular modeling simulations indicated that the combination of coumarin and benzofuran could mimic the structure of donepezil when employing linkers of sizes ranging from 0 to 2 atoms. Notably, this hybrid exhibited the highest DPPH radical scavenging capacity with a CE<sub>50</sub> value of 347 µM. Furthermore, it demonstrated significant AChE inhibition potential with an IC<sub>50</sub> value of 0.27 µM, along with inhibiting Aβ self-aggregation by 64.5 % at 40 µM. Overall, the presence of the methoxy substitution and the appropriate linker length were identified as contributing factors to the observed inhibitory effects [183].

### 3.5.4. Bis-coumarin hybrids

Phosphazine and phosphazide hybrids incorporating coumarin moieties were investigated for their potential as selective and potent inhibitors of AChE and Aβ aggregation [184]. The phosphazide linked

<footnote>a Not tested.</footnote>

<footnote>b Tested but not active.</footnote>

<footnote>c nM.</footnote>

<footnote>d 25 µM.</footnote>

<footnote>e 40 µM.</footnote>

<footnote>f µM.</footnote>

<footnote>g 10 µM.</footnote>
bis-coumarin hybrid **151** (Table 11), featuring a coumarin ring with a
formyl group, demonstrated remarkable AChE inhibition, with an IC₅₀
value of 34.96 nM, comparable to that of donepezil (IC₅₀ = 34.42 nM),
and exhibited a high SI (AChE/BChE; 3.81). Additionally, this hybrid
displayed significant inhibition of MMP-2 (IC₅₀ = 441.33 nM) and
self-induced Aβ₁.₄₂ aggregation (IC₅₀ = 337.77 nM). The impact of this
hybrid on Zn²⁺-induced Aβ₁.₄₂ aggregation was also evaluated. Kinetic
analysis revealed that it acts as a competitive AChE blocker. Moreover, it
demonstrated strong metal-chelating properties and was predicted to
permeate the BBB. Importantly, this hybrid exhibited no toxicity to
SH-SY5Y cells (CC₅₀ = 537.3 nM), yielding a safety index of 15.37.
Furthermore, in mouse models of scopolamine-induced memory loss, it
significantly ameliorated memory impairment (% alteration = 74.81)
and exhibited transfer latency time comparable to that of donepezil
[184].

**3.5.5. Benzofuran-pyridine hybrids**

A newly synthesized multitargeted 3-aminobenzofuran-pyridine
hybrid, designated as compound **152** (Table 11), has been identified
as a potent inhibitor of AChE and BChE [185]. This hybrid molecule,
featuring a 2-fluorobenzyl substituent, exhibited the most promising
anti-ChE inhibitory activities, with IC₅₀ values of 0.64 µM for AChE and
0.55 µM for BChE as compared to donepezil (IC₅₀ = 16 nM and 3.99 µM
for AChE and BChE, respectively). Additionally, it demonstrated inhi-
bition of self- and AChE-induced Aβ-aggregation (29.8 % at 10 µM and
30.1 % at 100 µM, respectively) and exhibited notable inhibition in MTT
assay against PC12 cells (86.4 %). Kinetic analysis revealed that it acts as
a mixed-type inhibitor of AChE [185].

3.6. Carbazole/isatin hybrids

3.6.1. Carbazole-chromone hybrids

Shi et al. synthesized carbazole-chromone hybrid **153** (Table 11) featuring a linker length of 5 carbon atoms, by linking two distinct pharmacophores, carbazole and coumarin [186]. This hybrid molecule demonstrated the most potent inhibitory activity against eeAChE, with an IC₅₀ of 3.75 µM, and 70.51 µM for hAChE. Additionally, this hybrid exhibited modest antioxidant activity, with an IC₅₀ value of 0.81 µM. Molecular docking studies revealed that it could interact with both the CAS and PAS of AChE [186]. Similarly, carbazole-coumarin hybrids **154** and **155** (Table 11) were identified as dual-binding site AChE inhibitors [187]. Hybrid **155**, with a linker length of 5 carbons, demonstrated good AChE inhibitory activity, with an IC₅₀ value of 6.72 µM, and showed high selectivity over BChE, consistent with hybrid **153** [186]. Meanwhile, compound **154**, featuring a linker length of 4 carbons, exhibited the most potent BChE inhibitory activity, with an IC₅₀ of 0.50 µM. The SAR analysis indicated the significance of linker length in assessing AChE inhibitory activity, while the structure of the coumarin moieties influenced the hybrids' BChE inhibition activities. Molecular docking analysis of hybrid **155** further elucidated its interaction with critical amino acids at AChE's CAS and PAS [187].

**3.6.2. Carbazole-thiazolidinedione hybrids**

Gandini et al. conducted *in vitro* experiments using intact *E. coli* cells expressing Aβ₄₂ and tau proteins to assess the aggregating behavior of the carbazole-thiazolidinedione hybrid compound **156** (Table 11) [3]. Subsequently, this hybrid underwent *in vivo* testing in a *Drosophila melanogaster* AD model, where it exhibited the most promising characteristics, displaying high activity, non-toxicity, and BBB permeability. This carbazole based hybrid significantly enhanced the lifespan and locomotor abilities of Aβ₄₂-expressing flies, surpassing the effects of doxycycline. Moreover, it demonstrated the capacity to mitigate Aβ₄₂ aggregates in the brain of flies [3].

3.6.3. Isatin-thiazoline hybrids

Isatin derivatives are known for their diverse pharmacological

properties owing to their ability to inhibit various enzymes and re-
ceptors [188]. Isatin-linked thiazoline AChE inhibitors **157** and **158**
(Table 11), capable of interacting with both the CAS and PAS of the
enzyme concurrently, have been identified as potential agents for AD
[189]. These hybrid molecules demonstrated significant AChE potency,
with IC₅₀ values of 18.2 µM and 27.5 µM, respectively, surpassing
rivastigmine (IC₅₀ = 275 µM) and comparable to galantamine (IC₅₀ =
8.7 µM), both clinically used AChE inhibitors. Moreover, kinetic studies
revealed that hybrid **157** exhibited a non-competitive inhibition pattern,
while molecular modeling predicted simultaneous interaction with both
the CAS and PAS. The SAR analysis indicated that the optimal carbon
number between the isatin and 2-thiazoline moieties is three to four
[189].

3.6.4. Isatin-triazole hybrids

Saeedi et al. investigated isatin-triazole hybrid **159** (Table 11), featuring an ortho methyl substituent on the benzyl ring for their potential inhibition of ChEs and BACE-1 in *in vitro* [190]. This lead compound, exhibited an IC₅₀ value of 4.78 µM for anti-BChE activity, surpassing that of the reference drug donepezil (IC₅₀ = 5.19 µM). Notably, *in vitro* assessments suggested that the presence of an ortho substitution on the benzyl moiety enhances the anti-BChE activity of the hybrid. Molecular docking analysis indicated that this hybrid could engage with both the PAS and CAS of BChE. Furthermore, this optimized hybrid was evaluated for its inhibition of BACE-1, but it did not exhibit inhibitory activity within the range of 10–50 µM. However, it demonstrated neuroprotective effects, with a percentage of 23.39 % at 5 µM concentration [190].

3.7. Natural product hybrids

3.7.1. Homoisoflavonoid-piperidine hybrids

Homoisoflavonoid Mannich base hybrid **160** (Table 12) was identified as promising MTDL, exhibiting preferential inhibition of AChE and MAO-B with IC₅₀ values of 2.49 nM and 1.74 µM, respectively [191]. Additionally, this hybrid exhibited notable inhibition of self- and Cu²⁺-induced Aβ₁.₄₂ aggregation (39.3 % and 47 % at 25 µM, respectively), antioxidant activity (IC₅₀ = 2.25 µM), and bio-metal chelating potential. Moreover, it displayed high permeability across the BBB. The SAR analysis highlighted the significant impact of the amine functional group and its substituted location on the therapeutic potential of these compounds [191].

3.7.2. Flavonoid-pyrrolidine hybrids

Flavonoids, a class of polyphenolic compounds commonly found in plant-based foods, are frequently consumed by humans through dietary intake. Given their abundance and potential health benefits, various flavonoids are under investigation in clinical trials for their potential to target several key features of AD [192]. Faraji et al. developed multifunctional flavonoid-pyrrolidine hybrid **161** (Table 12) using a linkage approach and assessed their abilities to inhibit acetylcholinesterase, attenuate Aβ aggregation, and provide neuroprotection [193]. This hybrid exhibited the most potent anti-AChE activity, with an IC₅₀ value of 10 nM, and demonstrated reasonable inhibition of BChE, with an IC₅₀ of 2.4 µM. Additionally, this hybrid significantly reduced H₂O₂-induced cell death in PC12 neurons, with cell viability reaching 64.45 %. Moreover, it effectively inhibited both AChE-induced and self-induced Aβ peptide aggregation by 51.3 % at 100 µM and 49.2 % at 10 µM, respectively [193].

3.7.3. Naringenin-piperidine hybrids

Naringenin, a naturally occurring flavanone abundantly found in
citrus fruits, tomatoes, and grapefruits, exhibits a diverse range of bio-
logical activities, notably strong neuroprotective effects [194]. In-
vestigations into naringenin hybrids have revealed that
naringenin-piperidine hybrid **162** (Table 12) possessed good
<table><caption>**Table 12**
Anti-Alzheimer's potential of MTDLs based on hybrids containing a natural product.</caption><thead><tr><th>Hybrid compounds</th><th>ACHE inhibition IC<sub>50</sub> (µM)</th><th>BChE inhibition IC<sub>50</sub> (µM)</th><th>β-Amyloid inhibition (%)</th><th>Antioxidant IC<sub>50</sub> (µM)</th><th>Other activities</th><th>Ref.</th></tr></thead><tbody><tr><td>160</td><td>2.49<sup>b</sup></td><td>19.8<sup>b</sup></td><td>39.3<sup>c</sup></td><td>2.25</td><td>MAO-B inhibition IC<sub>50</sub> = 1.74 µM, bio metal chelating potential, and high BBB permeation.</td><td>[191]</td></tr><tr><td>161</td><td>10<sup>b</sup></td><td>2.4</td><td>49.2<sup>d</sup></td><td>n.t.<sup>a</sup></td><td>Neuroprotective properties</td><td>[193]</td></tr><tr><td>162</td><td>62.52</td><td>94<sup>b</sup></td><td>69.2<sup>c</sup></td><td>1.13</td><td>Good metal chelating properties</td><td>[195]</td></tr><tr><td>163</td><td>37.1<sup>b</sup></td><td>12.9</td><td>58.1<sup>c</sup></td><td>1.66</td><td>Significant neuroprotection, bio metal chelation, BBB permeation</td><td>[197]</td></tr><tr><td>164</td><td>n.t.<sup>a</sup></td><td></td><td>84.74<sup>e</sup></td><td>71.80<sup>g</sup></td><td>Improved memory problems in behavioral tests, protected hippocampal neurons.</td><td>[199]</td></tr><tr><td>165</td><td></td><td></td><td>75.06<sup>e</sup></td><td>71.63<sup>g</sup></td><td></td><td></td></tr><tr><td>166</td><td>n.t.<sup>a</sup></td><td></td><td>54.4<sup>f</sup></td><td>n.t.<sup>a</sup></td><td>Neuroprotective effects against H<sub>2</sub>O<sub>2</sub>, 6-OHDA, BBB permeation</td><td>[200]</td></tr><tr><td>167</td><td>n.t.<sup>a</sup></td><td></td><td></td><td></td><td>Activity against neuroblastoma cell line with the IC<sub>50</sub> value of 23.98 and 12.99 µM, respectively</td><td>[201]</td></tr><tr><td>168</td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

inhibitory potential against AChE (IC<sub>50</sub> = 62.52 µM) and potent inhibitory effect against BChE, with an IC<sub>50</sub> value of 94 nM [195]. These observations suggest that the presence of oxygen atoms in the morpholine unit exerts electron-withdrawing effects, thereby reducing the

electronic density of amines. Molecular docking studies further revealed that it interacts with both CAS and PAS of BChE. Additionally, this hybrid exhibited superior hydroxyl free radical (*OH) scavenging activity compared to vitamin C, and cyclic voltammetry analyses

<footnote>a Not tested.</footnote>

<footnote>b nM.</footnote>

<footnote>c 25 µM.</footnote>

<footnote>d 100 µM.</footnote>

<footnote>e 20 µM.</footnote>

<footnote>f 10 µM.</footnote>

<footnote>g Percent inhibition.</footnote>
demonstrated its effective scavenging of superoxide anion radical (O<sup>2-</sup>) with an IC<sub>50</sub> value of 1.13 µM. Moreover, it demonstrated favorable metal chelating properties and exhibited anti-Aβ aggregation capabilities, particularly in inhibiting Cu<sup>2+</sup>-induced Aβ<sub>1-42</sub> aggregation by 69.2 % at 25 µM [195].

### 3.7.4. Aurone-piperidine hybrids

Aurones, a subgroup of flavonoids, have garnered significant interest in medicinal chemistry due to their diverse biological activities and potential as MTDLs [196]. The aurone Mannich base hybrid **163** (Table 12), featuring an amine substitution at the 4'-position, exhibited exceptional inhibitory potency against ratAChE, eeAChE, and hAChE, with IC<sub>50</sub> values of 87.8 nM, 21.2 nM, and 37.1 nM, respectively [197]. Moreover, this hybrid demonstrated potent antioxidant activity, with an IC<sub>50</sub> value of 1.66 µM, and conferred significant neuroprotection against H<sub>2</sub>O<sub>2</sub>-induced injury in PC12 cells. Additionally, it exhibited bio-metal chelating properties and displayed noteworthy inhibition of both self-induced and Cu<sup>2+</sup>-induced Aβ<sub>1-42</sub> aggregation, with inhibition potentials of 58.1 % at 25 µM for both scenarios. Importantly, this hybrid demonstrated high BBB permeation with a P<sub>e</sub> value of 13.48 × 10<sup>-6</sup> cm/s [197].

### 3.7.5. Sarsasapogenin-triazole hybrids

Sarsasapogenin, an active constituent found in Rhizoma anemarrhenae, has emerged as a promising bioactive lead compound for the treatment of AD [198]. In a study by Wang et al. sarsasapogenin-triazolyl hybrids **164** and **165** (Table 12) were assessed for their potential neuroprotective effects against H<sub>2</sub>O<sub>2</sub>-induced neurotoxicity in SH-SY5Y cells and their ability to inhibit Aβ<sub>1-42</sub> aggregation [199]. These results demonstrated that both hybrids exhibited significant inhibition of Aβ<sub>1-42</sub> fibril formation, with inhibition percentages of 84.74 % and 75.06 % at 20 µM, respectively, surpassing the inhibition achieved by curcumin (55.87 %) at same concentration. This potent inhibitory activity observed in compounds containing a benzyl group was attributed to their steric hindrance and the potential of benzyl groups to engage in π-π stacking interactions with the aromatic residues of Aβ. Additionally, these hybrids demonstrated moderate neuroprotective effects in SH-SY5Y cells against H<sub>2</sub>O<sub>2</sub>-induced neurotoxicity, with cell viabilities of 71.80 % and 71.63 %, respectively, at a concentration of 12.5 µM, compared to Trolox (78.38 % at 12.5 µM). Furthermore, oral administration of both hybrids significantly ameliorated memory deficits in behavioral tests, and TUNEL staining revealed a reduction in neurodegeneration in the brain tissues treated with these compounds [199].

### 3.7.6. Diosgenin-indole hybrids

Zhou et al. investigated the neuroprotective effects of diosgenin-indole hybrid **166** (Table 12) against damage induced by H<sub>2</sub>O<sub>2</sub>, 6-hydroxydopamine (6-OHDA), and Aβ toxicity [200]. Through initial SAR studies, it was determined that the incorporation of an indole fragment and an electron-donating group at the C-5 position of the indole ring contributed to enhanced neuroprotective activities. This compound exhibited significant protection against cellular damage induced by H<sub>2</sub>O<sub>2</sub> (52.9 %), 6-OHDA (38.4 %), and Aβ<sub>1-42</sub> (54.4 %) at 10 µM. Molecular docking studies further revealed that it exhibited strong binding affinity for Aβ<sub>1-42</sub>, suggesting its potential as an Aβ-targeting agent. Additionally, this hybrid demonstrated a significant reduction in H<sub>2</sub>O<sub>2</sub>-induced ROS production. In *in vivo* experiments, administration of this hybrid effectively ameliorated cognitive and memory deficits in Aβ-injected mice [200].

### 3.7.7. Flavone-nipecotic acid hybrids

Dhunmati et al. assessed flavone linked nipecotic acid hybrids **167** and **168** (Table 12) for their potential using the SH-SY5Y neuroblastoma cell line [201]. These evaluated hybrid compounds exhibited notable *in vitro* activity against SH-SY5Y cell line, displaying IC<sub>50</sub> values of 23.98

µM and 12.99 µM, respectively. Docking predictions for these compounds were consistent with the *in vitro* findings, further supporting their potential therapeutic relevance [201].

## 4. MTDLs without heterocyclic scaffold

### 4.1. Chalcone derivatives

Sang et al. introduced dimethylamino chalcone-O-alkylamine MTDL **169** (Table 13), exhibiting significant inhibition and disaggregation of Aβ, along with promising selective AChE inhibition, bio metal chelation, and selective MAO-B inhibition [202]. This hybrid displayed notable inhibitory potential against self-induced Aβ aggregation (IC<sub>50</sub> = 0.88 µM) and effective disaggregation towards self-induced Aβ aggregation (95.1 % at 25 µM). Moreover, it demonstrated remarkable antioxidant activity (IC<sub>50</sub> = 2.1 µM), potent AChE inhibition (IC<sub>50</sub> = 0.13 µM), and MAO-B inhibition (IC<sub>50</sub> = 1.0 µM), along with significant neuroprotection. This hybrid acted as an efficient bio metal chelator, hindering Cu<sup>2+</sup>-induced Aβ aggregation (95.3 %, 25 µM), and could disassemble well-structured Aβ fibrils (88.1 %, 25 µM). Additionally, it exhibited *in vitro* BBB permeability. Intriguingly, at doses up to 1000 mg/kg, it showed no toxicity in mice and ameliorated scopolamine-induced memory impairment [202]. Similarly, 4'-OH-flurbiprofen-chalcone hybrids **170** and **171** (Table 13), demonstrated superior inhibitory effects on self-induced Aβ<sub>1-42</sub> aggregation (60 % and 78.2 %, respectively) and Cu<sup>2+</sup>-induced Aβ<sub>1-42</sub> aggregation (52.4 % and 95 %, respectively) at 25 µM concentration [203]. Furthermore, these representative hybrids demonstrated good antioxidant (IC<sub>50</sub> = 3.10 µM and 3.30 µM, respectively), MAO inhibition, bio metal chelating, and *in vitro* anti-neuroinflammatory activities. Hybrid **171** also demonstrated suitable BBB permeation (P<sub>e</sub> = 6.66 × 10<sup>-6</sup> cm/s). These findings underscored the significance of incorporating the chalcone unit and free 4'-OH into the flurbiprofen nucleus for radical scavenging potential. Moreover, hybrids with two phenolic hydroxyl groups exhibited higher antioxidant potential than those with only one phenolic hydroxyl group [203].

### 4.2. Miscellaneous

Although quinones may have toxicological effects but they are also known for their good antioxidant properties [204]. This may contribute to overall health benefits. Campora et al. reported naphthoquinone derivative **172** (Table 13) with an aromatic side chain that showed significant inhibition of Aβ<sub>40</sub> aggregation (IC<sub>50</sub> = 3.2 µM), AChE (IC<sub>50</sub> = 9.2 µM), MAO-A (IC<sub>50</sub> = 3.6 µM) and remarkable MAO-B inhibition (IC<sub>50</sub> = 7.7 nM) [205]. This compound also found to be a potent inhibitor of Aβ<sub>42</sub> aggregation comparable to quercetin [205]. Sağlık et al. investigated indanone derivatives **173** and **174** (Table 13) that showed remarkable inhibition of Aβ aggregation (173: 82.74 and 75.62 %; 174: 80.26 and 71.89 % at the concentrations of 10<sup>-3</sup> and 10<sup>-4</sup> M), AChE (IC<sub>50</sub> = 24.8 nM and 22.4 nM), moderate BChE inhibition (61.28 and 61.12 %), good MAO-A (IC<sub>50</sub> = 0.11 µM and 0.12 µM) and MAO-B inhibition (IC<sub>50</sub> = 40.9 nM and 41.2 nM), and noteworthy antioxidant capacity (IC<sub>50</sub> = 0.19 and 0.25 µM), respectively [206]. This compound displayed mixed inhibition kinetics and superior efficacy against MAO-A. Evaluation of cytotoxicity in NIH/3T3 mouse fibroblast healthy cell line using the MTT method revealed acceptable safety profiles for both compounds [206].

The fluoren-9-amine MTDL **175** (Table 13) displayed selective BChE inhibition (IC<sub>50</sub> = 0.47 µM), antagonistic activity on NMDARs (GluN1/GluN2A = 41.02 %, GluN1/GluN2B = 17.24 %), and low cytotoxicity in the CHO-K1 cell line (IC<sub>50</sub> = 111 µM) [207]. Docking studies revealed interaction of its tricyclic core with Trp82 within the enzyme's anionic site, akin to tacrine. Kinetic analysis indicated selective inhibition of BChE by this hybrid [207]. Similarly, flurbiprofen-clioginol MTDLs **176** and **177** (Table 13) effectively inhibited Aβ-aggregation, both
<table><caption>**Table 13**
Anti-Alzheimer's potential of MTDLs based on chalcones/miscellaneous compounds.</caption><thead><tr><th>Hybrid compounds</th><th>AChE<br>inhibition<br>IC<sub>50</sub> (µM)</th><th>BChE<br>inhibition<br>IC<sub>50</sub> (µM)</th><th>β-Amyloid<br>inhibition (%)</th><th>Antioxidant<br>IC<sub>50</sub> (µM)</th><th>Other activities</th><th>Ref.</th></tr></thead><tbody><tr><td>H<sub>3</sub>C<sup>N</sup>-CH<sub>3</sub><br>169</td><td>0.13</td><td>236</td><td>0.88<sup>e</sup></td><td>2.1</td><td>MAO-B inhibition IC<sub>50</sub> = 1.0 µM, bio metal chelator, decompile the well-structured Aβ fibrils (88.1 %, 25 µM). BBB permeation</td><td>[202]</td></tr><tr><td>COOCH<sub>3</sub><br>170</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>60<sup>f</sup></td><td>3.10</td><td>MAO inhibition, bio metal chelation, and anti-neuroinflammatory activities, BBB permeation</td><td>[203]</td></tr><tr><td>COOCH<sub>3</sub><br>171</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>78.2<sup>f</sup></td><td>3.30</td><td></td><td></td></tr><tr><td>Cl-172</td><td>9.2</td><td>26</td><td>3.2<sup>e</sup></td><td>n.t.<sup>a</sup></td><td>Inhibited hMAO-A (IC<sub>50</sub> = 3.6 µM) and hMAO-B (IC<sub>50</sub> = 7.7 nM), cell viability 89 %</td><td>[205]</td></tr><tr><td>OMe-173</td><td>24.8<sup>e</sup></td><td>61.28<sup>d</sup></td><td>82.74<sup>g</sup></td><td>0.188</td><td>173 and 174 inhibited MAO-A (IC<sub>50</sub> = 0.11 µM and 0.12 µM, respectively), MAO-B (IC<sub>50</sub> = 40.9 nM and 41.2 nM, respectively), low cytotoxicity.</td><td>[206]</td></tr><tr><td>OMe-174</td><td>22.4<sup>e</sup></td><td>61.12<sup>d</sup></td><td>80.26<sup>g</sup></td><td>0.246</td><td></td><td></td></tr><tr><td>H-175</td><td>13.98</td><td>0.47</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>Antagonistic activity on the NMDARs (GluN1/GluN2A = 41.02 µM, GluN1/GluN2B = 17.24 µM) and a low cell viability in the CHO-K1 cell line (IC<sub>50</sub> = 111 µM)</td><td>[207]</td></tr><tr><td>CH<sub>3</sub>-176</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>96.33<sup>e</sup></td><td>2.44</td><td>Bio metal chelating abilities, anti-neuroinflammatory behavior, and adequate BBB permeation</td><td>[208]</td></tr><tr><td>CH<sub>3</sub>-OCH<sub>3</sub>-177</td><td></td><td></td><td>96.09<sup>g</sup></td><td>1.91</td><td></td><td></td></tr><tr><td>CH<sub>3</sub>C<sup>N</sup>-CH<sub>3</sub>-178</td><td>10.4</td><td>106</td><td>42.5<sup>f</sup></td><td>2.46</td><td>Disaggregation of self- and Cu<sup>2+</sup>-induced Aβ/1.42 aggregation fibrils, bio metal chelating, anti-neuroinflammatory, and BBB permeation</td><td>[209]</td></tr><tr><td>O<sub>2</sub>N-179</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>Inhibited MAO-B more effectively (IC<sub>50</sub> value = 9.2 and 1.6 nM, BACE-1 IC<sub>50</sub> = 8.02 and 8.21 µM respectively, BBB permeation</td><td>[210]</td></tr></tbody></table>

(continued on next page)
Nitro group-bearing enamides **179** and **180** (Table 13) showed potent inhibition of MAO-B ($IC_{50}$ values of 9.2 nM and 16 nM, respectively) compared to standard drugs like lazabemide ($IC_{50}$ = 0.11 µM) and pargyline ($IC_{50}$ = 0.14 µM) [210]. They exhibited higher selectivity for MAO-B over MAO-A, with SI values of >1652.2 and > 2500.0, respectively. Kinetic studies revealed that they are reversible and competitive inhibitors of MAO-B, with Ki values of 13 nM and 4.89 nM, respectively. Additionally, these hybrids inhibited BACE-1 with $IC_{50}$ values 8.02 µM and 8.21 µM, lower than the standard quercetin 13.40 µM, and they demonstrated successful BBB permeation in PAMPA assay [210].

*p*-aminobenzoic acid hybrid **181** (Table 13) exhibited promising inhibitory activity against both AChE ($IC_{50}$ = 46 nM) and BChE ($IC_{50}$ = 16.96 µM), with significant cognitive improvement observed in amnesia animal models compared to standard donepezil with $IC_{50}$ = 40 nM for AChE and 15.24 µM for BChE [211]. Similarly, palladium (II) complexes of thiosemicarbazone-based molecules were identified as effective antiaggregating agents in *in cellulo* investigations with *E. coli* [212]. Compounds **182** and **183** (Table 13), incorporating propargylamine substituted derivatives with salicylic and cinnamic scaffolds, displayed ChE and MAO inhibition properties [213]. Compound **182** was the most potent inhibitor of AChE ($IC_{50}$ = 8.05 µM), while carbamate **183** showed the highest activity and selectivity for BChE ($IC_{50}$ = 26.09 µM), indicating its potential as a true MTDL with balanced inhibitory activity against both ChEs. These compounds also exhibited antioxidant properties and were predicted to be active after oral administration, with the ability to reach brain tissue [213].

Pasieka et al. evaluated the hydroxyethylamine derivative **184** (Table 13) with a 3-*t*-butylbenzylamine substitution, and found that it exhibited potent inhibition against several enzymes, including *ee*AChE ($IC_{50}$ = 2.86 µM), *eq*BChE ($IC_{50}$ = 60 nM), *h*BChE ($IC_{50}$ = 20 nM), and *h*BACE-1 ($IC_{50}$ = 5.9 µM) [214]. Moreover, it showed inhibition of *A*β aggregation (57.9 % at 10 µM) as well as inhibition of mGAT1 ($IC_{50}$ = 10.96 µM) and mGAT2 ($IC_{50}$ = 19.05 µM). The substitution of benzyl-amine led to increased activity, as observed in the SAR analysis [214]. Similarly, in *in vitro* assays the pterostilbene *β*-amino alcohol-based hybrid **185** (Table 13) exhibited a potent inhibition of *ee*AChE ($IC_{50}$ = 24.04 µM), outperforming pterostilbene itself [215]. Furthermore, it demonstrated significant antioxidant activity ($IC_{50}$ = 1.20 µM) and provided substantial neuroprotection against $H_2O_2$-induced PC12 cell injury (74.90 %). Additionally, it inhibited self-induced $A\beta_{1-42}$ aggregation (40.23 % at 25 µM) and exhibited high BBB permeability *in vitro* ($P_e$ = 8.12 × 10⁻⁶ cm/s) [215]. The biological evaluation of the 2-hydroxyl-4-benzyloxybenzyl aniline derivative **186** (Table 13) revealed its remarkable inhibition of MAO-B, with an $IC_{50}$ value of 14 nM as compared to rasagiline ($IC_{50}$ = 94.6 nM) [216]. Additionally, it demonstrated high antioxidant capacity ($IC_{50}$ = 2.14 µM), adequate BBB permeation ($P_e$ = 8.62 × 10⁻⁶ cm/s), good metal chelating ability, and a significant neuroprotective effect [216]. Shi et al. investigated the Schiff base derivative **187** (Table 13), identified it as a highly selective MAO-B inhibitor, with an $IC_{50}$ of 8.4 nM as compared to rasagiline $IC_{50}$ = 29 nM, supported by docking studies [217]. Furthermore, it exhibited antioxidant potential ($IC_{50}$ = 2.3 µM) and significantly inhibited self-induced $A\beta_{1-42}$ aggregation (31.8 % at 25 µM). It also acted as a selective metal chelator, inhibiting Cu²⁺-induced $A\beta_{1-42}$ aggregation (62.3 % at 25 µM). Moreover, this hybrid demonstrated a significant neuro-protective effect against $H_2O_2$-induced PC12 cell injury. Importantly, this compound exhibited good BBB permeation ($P_e$ = 13.29 × 10⁻⁶ cm/s) along with favorable drug-like attributes [217].

## 5. MTDLs based on FDA approved drugs

Numerous instances of MTDLs have been documented in scientific literature, synthesized through the amalgamation of FDA-approved drugs with heterocyclic moieties. These heterocycles are coupled to the pharmacophores of the FDA-approved drugs employing three

principal strategies: linkage, fusion, and incorporation [218]. This approach aims to capitalize on the synergistic effects arising from the integration of diverse molecular frameworks, thereby enhancing the therapeutic potential of the resulting compounds.

### 5.1. Linkage of tacrine pharmacophore with one or more heterocyclic scaffolds

Despite its hepatotoxicity, tacrine remains a cornerstone for designing multifunctional molecules targeting ChEs, metal ions and *A*β aggregations, with ongoing efforts to reduce its adverse effects. Through the linkage approach, tacrine's pharmacophore is tethered to various heterocyclic scaffolds like pyridines, pyrimidines, quinolines, imidazoles, and thiazoles. This strategy enables modulation of tacrine's interactions with molecular targets, potentially reducing hepatotoxicity while enhancing therapeutic efficacy. By combining tacrine's pharmacophore with diverse heterocyclic motifs, researchers aim to develop multifunctional agents with improved pharmacological properties, underscoring tacrine's versatility in drug design for complex diseases [219].

#### 5.1.1. Tacrine-quinoline/acridine/pyrazoline/pyridine/pyrrolidine hybrids

##### 5.1.1.1. Tacrine-quinoline hybrids.
Hepnarova et al. synthesized tacrine-quinolone hybrid compound **188** (Table 14) by linking tacrine to benzyl quinolone carboxylic acid [220]. This hybrid exhibited potent inhibition of *h*AChE with an $IC_{50}$ value of 419 nM and *h*BChE with an $IC_{50}$ value of 3.72 µM, comparable to the control drug THA (320 nM and 881 nM, respectively). Additionally, this hybrid demonstrated inhibition of muscarinic acetylcholine receptor M1 ($IC_{50}$ = 4.01 µM). Kinetic analysis revealed a mixed-type inhibition profile against *h*AChE, consistent with docking studies. Furthermore, it exhibited potential inhibition against hepatotoxicity in HepG2 cell lines ($IC_{50}$ = 4.99 µM) and demonstrated promising penetrability through the BBB with $P_e$ = 3.08 × 10⁻⁶ cm/s [220]. Similarly, Uliassi et al. reported that the tacrine-quinolinetrione hybrid **189** (Table 14) impeded ChEs with an $IC_{50}$ values of 58.3 nM for *h*AChE and 109 nM for *h*BChE compared to tacrine ($IC_{50}$ = 415 nM and 35 nM, respectively) [221]. Furthermore, it showed antioxidant activity in menadione-induced oxidative stress of SH-SY5Y cells. Importantly, this hybrid demonstrated low hepatotoxicity and effective anti-amyloid aggregation inhibition (33.4 % at 10 µM concentration) [221].

##### 5.1.1.2. Tacrine-acridine hybrids.
The tacrine-acridine hybrid **190** (Table 14) reported by Chufarova et al., stood out as a significantly more active inhibitor than tacrine itself, with an $IC_{50}$ of 7.6 pM for AChE and 1.7 pM for BChE, compared to $IC_{50}$ values of 89914.1 pM and 14982.4 pM, respectively, for tacrine [222]. This hybrid also demonstrated potent inhibition of *A*β aggregation, with 57.77 % inhibition at the highest concentration of 100 µM, maintaining substantial inhibition as concentrations decreased. Moreover, molecular docking revealed a dual-binding mode for this hybrid, targeting both the CAS and PAS of AChE simultaneously [222].

##### 5.1.1.3. Tacrine-piperazine hybrids.
A 6-chlorotacrine (huprine)-TPPU hybrid **191** (Table 14) acted as dual inhibitors of the enzymes soluble epoxide hydrolase (sEH) ($IC_{50}$ = 4.6 nM) and *h*AChE ($IC_{50}$ = 12.9 nM), and provide cumulative effects against neuroinflammation and memory impairment [223]. Computational studies confirmed that both enzymes occupied the entire gorge, from the main site to a secondary site, including an AChE cryptic pocket. In *in vitro*, the lead compound demonstrated dual nanomolar potencies, adequate brain permeability ($P_e$ = 8.4 × 10⁻⁶ cm/s), aqueous solubility (7.5 µM), human microsomal stability and a lack of neurotoxicity. Additionally, it rescued memory, synaptic resilience, and neuroinflammation in an AD mouse model after
<table><caption>**Table 14**
Anti-Alzheimer's potential of MTDLs based on tacrine and quinoline/acridine/pyrazoline/pyridine/pyrrolidine hybrids.</caption><thead><tr><th>Hybrid compounds</th><th>AChE<br>inhibition IC<sub>50</sub><br>(µM)</th><th>BChE<br>inhibition IC<sub>50</sub><br>(µM)</th><th>β-Amyloid<br>inhibition (%)</th><th>Antioxidant<br>IC<sub>50</sub> (µM)</th><th>Other activities</th><th>Ref.</th></tr></thead><tbody><tr><td>188</td><td>419<sup>b</sup></td><td>3.72</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>Inhibit muscarinic M1 receptor, cross BBB, neuroprotective properties</td><td>[220]</td></tr><tr><td>189</td><td>58.3<sup>b</sup></td><td>109<sup>b</sup></td><td>33.4<sup>d</sup></td><td>—</td><td>—</td><td>[221]</td></tr><tr><td>190</td><td>7.6<sup>c</sup></td><td>1.7<sup>c</sup></td><td>57.77<sup>e</sup></td><td>n.t.<sup>a</sup></td><td>No hepatotoxicity, neuroprotection, BBB permeation</td><td>[222]</td></tr><tr><td>191</td><td>12.9<sup>b</sup><br>4.12<sup>b</sup></td><td>179<sup>b</sup></td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>BBB permeability, microsomal stability, enzymes soluble epoxide hydrolase inhibition IC<sub>50</sub> = 4.6 nM against <i>hsEH</i> and 22,5 nM against <i>msEH</i></td><td>[223]</td></tr><tr><td>192</td><td>14<sup>b</sup></td><td>22<sup>b</sup></td><td>94.4<sup>d</sup></td><td>n.t.<sup>a</sup></td><td>5-HT<sub>6</sub> receptor antagonist (K<sub>i</sub> = 18 nM) BBB permeability</td><td>[224]</td></tr><tr><td>193</td><td>0.13</td><td>0.45</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>PDE4D inhibitory activity with IC<sub>50</sub> = 0.27 µM</td><td>[225]</td></tr><tr><td>194</td><td>6.4<sup>b</sup></td><td>260<sup>b</sup></td><td>46<sup>f</sup></td><td>n.t.<sup>a</sup></td><td><i>h</i>GSK-3β kinase activity (IC<sub>50</sub> = 66 nM), anti-proliferative activity, inhibited tau protein hyperphosphorylation</td><td>[34]</td></tr><tr><td>195</td><td>3.05</td><td>3.19</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>Modest CCB (30.40 at 1 µM) with no substantial hepatotoxicity, BBB permeation</td><td>[226]</td></tr><tr><td>196</td><td>2.0<sup>b</sup></td><td>69</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>Inhibited COX-2; (IC<sub>50</sub> = 29 nM), 5-LOX; (IC<sub>50</sub> = 540 nM), devoid of hepatotoxicity and neurotoxicity</td><td>[227]</td></tr></tbody></table>

<footnote>a Not tested.</footnote>

<footnote>b nM.</footnote>

<footnote>c pM.</footnote>

<footnote>d 10 µM.</footnote>

<footnote>e 200 µM.</footnote>

<footnote>f 20 µM.</footnote>
low dose chronic oral administration [223].

5.1.1.4. *Tacrine-pyrazoline hybrids.* Wieckowska et al. developed MTDL 192 (Table 14) by combining pharmacophore fragment targeting serotonin 5-HT6 receptors, AChE/BChE inhibition, and Aβ aggregation inhibition [224]. This hybrid displayed balanced activity as a 5-HT6R antagonist (Ki = 18 nM) and served as a noncompetitive inhibitor of ChEs (hAChE; IC₅₀ = 14 nM vs donepezil IC₅₀ = 10 nM, eqBChE; IC₅₀ = 22 nM vs donepezil IC₅₀ = 6 nM). Moreover, *in vitro* studies revealed that this hybrid exhibited superior Aβ anti-aggregation activity at 10 µM concentration compared to resveratrol (94.4 % vs 79 %) and demonstrated the ability to cross the BBB (Pₑ = 5.7 x 10⁻⁶ cm/s) [224].

5.1.1.5. *Tacrine-pyridine hybrids.* Pan et al. designed tacrine-pyrazolo[3,4-b]pyridine hybrid 193 (Table 14), featuring a six-carbon linker between tacrine and the pyrazolo[3,4-b] pyridine moiety, exhibiting potent AChE inhibition with an IC₅₀ value of 0.13 µM, comparable to tacrine (IC₅₀ = 0.11 µM) [225]. Additionally, it exhibited good compromise in inhibiting AChE and BChE, along with PDE4D with IC₅₀ values of 0.45 µM and 0.27 µM, respectively, [225]. Similarly, Jiang et al. developed a new GSK-3/β/AChE dual-target inhibitor 194 (Table 14) by linking tacrine with pyridothiazole ester [34]. This hybrid demonstrated nanomolar inhibition of hAChE (IC₅₀ = 6.4 nM) and hGSK-3/β kinase activity (IC₅₀ = 66 nM), outperforming tacrine (IC₅₀ = 230 nM against hAChE). Moreover, at 20 µM concentration, it showed significant inhibition of Aβ self-aggregation (inhibitory rate = 46 %) and reduced tau protein hyperphosphorylation in mouse neuroblastoma N2α-Tau cells. *In vivo* studies further demonstrated that this hybrid notably ameliorated neurological dysfunction in ICR mice treated with scopolamine while exhibiting lower hepatotoxicity compared to tacrine [34].

On a different front, Chioua et al. synthesized quinoline-thione, 195 (Table 14) by incorporating tacrine, a ChE inhibitor, and 3,4-dihydropyrimidin-2(1H)-thione, efficient calcium channel blockers (CCBs). This hybrid displayed reasonable to intense CCB activity, comparable to or exceeding the reference CCB drug nimodipine, while also exhibiting moderate to good ChEI properties [226]. Notably, this methoxy derivative emerged as a well-balanced ChEI/CCB agent, functioning as a low micromolar *h*ChEI and displaying modest CCB activity, with no significant hepatotoxicity towards HepG2 cells, and effective anticipated oral absorption and BBB permeation [226].

5.1.1.6. *Tacrine-pyrrolidine hybrids.* Using a linkage approach, a multi-potent tacrine-pyrrolidine hybrid, designated as compound 196 (Table 14), was developed to simultaneously target COX-2, 5-LOX, AChE, and BChE [227]. This hybrid demonstrated excellent and balanced inhibitory activity against all four tested targets, with IC₅₀ values as follows: COX-2 (29 nM vs celecoxib 50 nM), 5-LOX (540 nM vs zileuton 580 nM), *ee*AChE (2.0 nM vs tacrine 410 nM), and *eq*BChE (69 nM vs tacrine 60 nM), positioning it as an optimal multipotent hybrid compound. Notably, this tacrine derivative exhibited no hepatotoxicity or neurotoxicity against SH-SY5Y cell lines, with a cell viability of 99.74 µM compared to donepezil's 97.40 µM at a 1 µM concentration, as confirmed by biochemical analysis and histopathological examination of mice liver. Moreover, in an *in vivo* acute toxicity study, it demonstrated safety up to a dose of 1000 mg/kg [227].

5.1.2. Tacrine-triazole/benzimidazole/thiadizole/phenothiazine hybrids

5.1.2.1. *Tacrine-triazole hybrids.* AlFadly et al. introduced two families of MTDLs based on tacrine-triazole and tacrine-thiosemicarbazide, designed to possess anti-inflammatory and anticholinesterase activity through triple targeting properties [228]. Among these, triazoles hybrids 197 and 198, and thiosemicarbazide hybrid 199 (Table 15) emerged as promising hits, demonstrating effective inhibition of *h*BChE

(IC₅₀ = 678 nM, 757 nM, and 326 nM, respectively) and COX-2 (IC₅₀ = 43 nM, 61 nM, and 110 nM, respectively). Additionally, these hybrids showed potent inhibition of 15-LOX (IC₅₀ = 6.14 µM, 9.11 µM, and 12.31 µM, respectively) surpassing reference inhibitor zileuton (IC₅₀ = 13.84 µM). Cell-based assays confirmed their anti-neuroinflammatory activity by reducing IL-1β and TNF-α expression levels in PC12 neuron cells. Importantly, these hybrids exhibited excellent safety profiles in hepatic and murine brain cell lines and were safe up to 100 µM in PC12 cells. Furthermore, they demonstrated enhanced BBB permeation compared to tacrine, along with favorable physicochemical characteristics, drug-likeness, and ligand effectiveness indices [228]. Similarly, Oukoloff et al. developed a novel class of MTDLs by combining tacrine and valmerin scaffolds to target AChE and GSK-3α/β simultaneously [229]. The most potent hybrid, 200 (Table 15), exhibited inhibitory potential with IC₅₀ values of 9.5 nM for *h*AChE and 7 nM for GSK-3α/β. Additionally, this hybrid demonstrated low cytotoxicity across a panel of human cell lines, including SH-SY5Y cells, and effectively crossed the BBB without interfering with efflux pumps like P-gp [229].

5.1.2.2. *Tacrine-benzimidazole hybrids.* Tacrine-hydroxyphenylbenzimi-dazole hybrids 201–203 (Table 15) offer a promising approach to combat the multifactorial nature of AD [230]. Hybrid 201 displayed significantly improved AChE inhibitory activity (IC₅₀ = 6.3 nM) compared to tacrine alone (IC₅₀ = 350 µM), while both hybrids exhibited high inhibition of self-induced Aβ aggregation (74.6 % and 70.9 %, respectively) and Cu⁺²-induced Aβ aggregation (77.3 % and 59.5 %, respectively) at 40 µM. Additionally, these compounds demonstrated moderate antioxidant potential, with hybrid 203 showing the highest activity (EC₅₀ = 564 µM) likely due to the presence of a hydroxyl group in the linker. Moreover, neuroprotective effects were observed with hybrid 203, suggesting its potential to mitigate neurotoxicity induced by Aβ and Fe/AscH(−) in neuronal cells [230].

5.1.2.3. *Tacrine-thiadiazole hybrids.* Makhaeva et al. employed tacrine linkage with 1,2,4-thiadiazole derivatives using two distinct spacers, pentylaminopropene, and pentylaminopropane [231]. The resulting conjugates, 204 and 205 (Table 15), displayed predominant inhibition of BChE with notable potency (IC₅₀ = 725 nM and 380 nM, respectively). Additionally, both hybrids effectively displaced PI from the AChE PAS (25.2 µM and 26.8 µM, respectively, compared to donepezil 10.1 µM), suggesting their potential to inhibit AChE-induced Aβ aggregation. Moreover, these compounds exhibited significant radical-scavenging capacity, with conjugate 205 demonstrating higher anti-BChE activity, enhanced anti-aggregate potential, and reduced carboxylesterase potency compared to hybrid 204. Conjugate 204 showed superior radical scavenging (IC₅₀ = 1.28 µM), likely attributed to the HOMO localization in the enamine-thiadiazole system. Predictions indicated medium and high BBB permeation for both conjugates, respectively, with a medium likelihood of causing cardiac toxicity [231].

5.1.2.4. *Tacrine-phenothiazine hybrids.* Phenothiazine serves as the principal pharmacophore of methylene blue (MB), recognized for its ability to impede the accumulation of soluble tau and the formation of tau filaments, along with its neuroprotective attributes [232–234]. Gorecki et al. synthesized tacrine-phenothiazine hybrids 206 and 207 (Table 15) with the aim of targeting ChE and τ-peptide aggregation [235]. Compound 206 emerged as a potent and selective AChE inhibitor, displaying an IC₅₀ of 8 nM, compared to tacrine's IC₅₀ of 320 nM, while hybrid 207 acted as a potent BChE inhibitor, with an IC₅₀ of 19 nM versus tacrine's 80 nM. Notably, both hybrids exhibited significant inhibition of τ(306–336) peptide aggregation, with percent inhibition values of 50.5 and 55.6 %, respectively at 50 µM. Moreover, hybrid 206 demonstrated remarkable efficacy in inhibiting self-induced Aβ₁-42 aggregation (74.1 % at 50 µM). Although *in vitro* studies indicated
<table><caption>**Table 15**
Anti-Alzheimer's potential of MTDLs based on tacrine and triazole/benzimidazole/thiadiazole/phenothiazine hybrids.</caption><thead><tr><th>Hybrid compounds</th><th>AChE<br>inhibition<br>IC<sub>50</sub> (nM)</th><th>BChE<br>inhibition<br>IC<sub>50</sub> (nM)</th><th>β-Amyloid<br>inhibition (%)</th><th>Antioxidant<br>IC<sub>50</sub> (μM)</th><th>Other activities</th><th>Ref.</th></tr></thead><tbody><tr><td>197</td><td>7.03<sup>a</sup></td><td>678</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>COX-2 (IC<sub>50</sub> = 43, 61 and 110 nM respectively, excellent safety profile in hepatic and murine brain cell lines, BBB permeation</td><td>[228]</td></tr><tr><td>198</td><td>3.75<sup>a</sup></td><td>757</td><td></td><td></td><td></td><td></td></tr><tr><td>199</td><td>218</td><td>326</td><td></td><td></td><td></td><td></td></tr><tr><td>200</td><td>9.5</td><td>395</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>Inhibitory potential with IC<sub>50</sub> = 7 nM for GSK-3α/β, exhibited low cytotoxicity, BBB permeation</td><td>[229]</td></tr><tr><td>201</td><td>30.9</td><td>n.t.<sup>a</sup></td><td>74.6<sup>d</sup></td><td>&gt;1000</td><td>Radical scavenging and metal chelating capacity and prevent neurotoxicity.</td><td>[230]</td></tr><tr><td>202</td><td>6.3</td><td></td><td>39.4<sup>d</sup></td><td>n.a.<sup>b</sup></td><td></td><td></td></tr><tr><td>203</td><td>16.8</td><td></td><td>70.9<sup>d</sup></td><td>564</td><td></td><td></td></tr><tr><td>204</td><td>1.24</td><td>725</td><td>n.t.<sup>a</sup></td><td>1.28</td><td>Greater anti-aggregate potential, and a lower carboxylesterase potency, BBB permeability, no cardiac toxicity, displaced PI</td><td>[231]</td></tr><tr><td>205</td><td>0.43<sup>a</sup></td><td>380</td><td>n.t.<sup>a</sup></td><td>0.38</td><td>Radical scavenging, BBB permeability, no cardiac toxicity, displaced PI</td><td>[231]</td></tr></tbody></table>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

(continued on next page)
<table><caption>**Table 15 (continued)**</caption><thead><tr><th>Hybrid compounds</th><th>AChE<br>inhibition<br>IC<sub>50</sub> (nM)</th><th>BChE<br>inhibition<br>IC<sub>50</sub> (nM)</th><th>β-Amyloid<br>inhibition (%)</th><th>Antioxidant<br>IC<sub>50</sub> (μM)</th><th>Other activities</th><th>Ref.</th></tr></thead><tbody><tr><td>206</td><td>2040</td><td>19</td><td>n.a.<sup>b</sup></td><td>n.t.<sup>a</sup></td><td>Inhibited τ<sub>(306-336)</sub> peptide aggregation with percent inhibition 55.6 and 50.5 % at 50 μM respectively, showed cytotoxicity towards HepG2 cells and cerebellar granule neurons, BBB permeability</td><td>[235]</td></tr><tr><td>207</td><td>8.0</td><td>190</td><td>74.1<sup>e</sup></td><td></td><td></td><td></td></tr></tbody></table>

cytotoxicity towards HepG2 cells and cerebellar granule neurons, administration of this hybrid to mice at 14 mg/kg (i. p.) did not lead to any pathophysiological abnormalities. Both *in vitro* and *in vivo* models confirmed the ability of it to penetrate the CNS, with a relatively slow elimination half-time and a maximum brain concentration near the IC₅₀ value for AChE inhibition [235].

### 5.1.3. Tacrine-isatin/indole/coumarin/chromone/benzofuran hybrids

#### 5.1.3.1. Tacrine-isatin hybrids.
Riazimontazer et al. developed tacrine-isatin Schiff base hybrids **208–210** (Table 16), inhibitors of AChE-induced Aβ aggregation [236]. The hybrids **208** and **209** exhibited significant AChE inhibition, with IC₅₀ values of 0.42 nM and 0.62 nM, respectively, surpassing tacrine's potency (IC₅₀ = 38.72 nM). Meanwhile, compound **210** demonstrated potent inhibition of BChE with an IC₅₀ value of 0.11 nM, which was 56-fold more potent than tacrine's inhibition (IC₅₀ = 6.21 nM). Additionally, these compounds exhibited remarkable metal-chelating properties. Kinetic and molecular modeling studies revealed that hybrid **208** acts as a mixed-type blocker, capable of binding to both the CAS and PAS of AChE [236].

#### 5.1.3.2. Tacrine-indole hybrids.
Wichur et al. utilized the tacrine backbone to develop a promising tacrine-indole based MTDL **211** (Table 16) [237]. It exhibited 5-HT6 receptor antagonist activity with a Ki value of 13 nM, showing comparable potency to methiothepine (Ki = 0.30 nM). Furthermore, this hybrid demonstrated potent inhibition of both AChE and BChE, with IC₅₀ values of 8.42 nM and 24.39 nM, respectively. Additionally, it effectively impeded Aβ aggregation *in vitro*, with 74.8 % inhibition at 10 μM, and inhibited tau aggregation *in cellulo* by 79 % at same concentration. Importantly, this hybrid exhibited acceptable metabolic stability in mouse liver microsomes [237].

#### 5.1.3.3. Tacrine-coumarin hybrids.
Najafi et al. synthesized a class of tacrine-coumarin linked triazole hybrids, represented by compounds **212** and **213** (Table 16), which were identified as potent dual binding site ChEIs [238]. Hybrid **212** exhibited remarkable anti-AChE activity with an IC₅₀ value of 27 nM, while compound **213** demonstrated the most potent anti-BChE activity with an IC₅₀ value of 6 nM. Both hybrids showed significantly greater activity than the reference drugs tacrine (AChE; 48 nM, BChE; 10 nM) and donepezil (AChE; 39 nM, BChE; 8.416 μM). Additionally, compound **212** displayed a low BACE-1 inhibition effect and demonstrated neuroprotection against PC12 cells exposed to Aβ<sub>25-35</sub>. Moreover, *in vivo* studies using the Morris water maze task revealed that hybrid **212** significantly reversed scopolamine-induced memory loss in rats [238].

#### 5.1.3.4. Tacrine-chromone hybrids.
Pérez-Areales et al. combined the scaffold of the polar antioxidant lead 7-methoxy-2,2-dimethylchroman-6-ol with that of the lipophilic cholinesterase inhibitor 6-chlorotacrine to develop compounds with favorable brain permeation and multiple biological activities *in vitro* [239]. The lead compounds **214** and **215** (Table 16) demonstrated potent inhibition of AChE with IC₅₀ values of 0.27 nM and 0.12 nM, respectively, and BChE with IC₅₀ values of 18.3 nM and 13.4 nM, respectively. Additionally, these hybrids exhibited inhibition of β-site APP cleaving BACE-1 enzyme with inhibition percentages of 28.1 and 24.8 % at 5 μM, respectively. Moreover, both compounds showed inhibition of Aβ<sub>42</sub> (34 % and 27.9 % at 10 μM) and tau aggregation (48.2 % and 35.5 % at 10 μM) and BBB permeation. *In vivo* studies on wild-type and APP/PS1 mice, both hybrids were well-tolerated and demonstrated beneficial effects on memory retention, amyloid abnormalities, and oxidative stress [239].

#### 5.1.3.5. Tacrine-chromene hybrids.
Fares et al. investigated the potential of MTDLs **216** and **217** (Table 16), synthesized by fusion of tacrine and chromene (natural products exhibiting a wide range of biological activities [240]). These hybrids demonstrated potent inhibition of hAChE, with IC₅₀ values of 0.44 μM and 0.25 μM, respectively [241]. Notably, their efficacy in inhibiting BChE surpassed that of rivastigmine by 10- and 5-fold, with IC₅₀ values of 80 nM and 14 nM, respectively (rivastigmine IC₅₀ = 81 nM). Additionally, these compounds showed binding to the PAS, influencing Aβ aggregation and reducing Aβ-related neurodegeneration. Compound **217** displayed superior effectiveness compared to curcumin, with an IC₅₀ of 74 nM and 76 % inhibition at 10 μM. Moreover, both hybrids exhibited potent BACE-1 inhibition with IC₅₀ values of 38 nM and 44 nM, respectively, nearly doubling the activity of curcumin (IC₅₀ = 67 nM). Furthermore, these hybrids displayed inhibitory activity against MAO-B, with IC₅₀ values of 5.15 μM and 2.42 μM, respectively. They also possessed antioxidant properties and demonstrated effective metal-chelating capabilities for Fe<sup>2+</sup>, Zn<sup>2+</sup>, and Cu<sup>2+</sup>, mitigating oxidative stress in AD-affected brains. Importantly,

<footnote>a Not tested.</footnote>

<footnote>b Tested but not active.</footnote>

<footnote>c μM.</footnote>

<footnote>d 40 μM.</footnote>

<footnote>e 50 μM.</footnote>
<table><caption>**Table 16**
Anti-Alzheimer's potential of MTDLs based on tacrine and isatin/indole/coumarin/benzofuran hybrids.</caption><thead><tr><th>Hybrid compounds</th><th>AChE<br>inhibition<br>IC<sub>50</sub> (nM)</th><th>BChE<br>inhibition<br>IC<sub>50</sub> (nM)</th><th>β-Amyloid<br>inhibition (%)</th><th>Antioxidant<br>IC<sub>50</sub> (μM)</th><th>Other activities</th><th>Ref.</th></tr></thead><tbody><tr><td>208</td><td>0.42</td><td>0.57</td><td>n.a.<sup>b</sup></td><td></td><td>Metal chelating property</td><td>[236]</td></tr><tr><td>209</td><td>0.62</td><td>3.52</td><td>84.6<sup>c</sup></td><td></td><td></td><td></td></tr><tr><td>210</td><td>57.85</td><td>0.11</td><td>n.a.<sup>b</sup></td><td></td><td></td><td></td></tr><tr><td>211</td><td>8.42</td><td>24.39</td><td>74.8<sup>c</sup></td><td>n.t.<sup>a</sup></td><td>Inhibited 5-HT<sub>6</sub>R with K<sub>i</sub> = 13.0 nM, inhibited tau aggregation 79 % at 10 μM, demonstrated acceptable metabolic stability on mouse liver microsomes</td><td>[237]</td></tr><tr><td>212</td><td>27</td><td>104</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>Inhibited BACE-1 13.97 %, neuroprotection against PC12 cells, overturned scopolamine-induced memory loss</td><td>[238]</td></tr><tr><td>213</td><td>95</td><td>6.0</td><td></td><td></td><td></td><td></td></tr><tr><td>214</td><td>0.27</td><td>18.3</td><td>34<sup>c</sup></td><td>18.5</td><td>BACE-1 (inhibition = 28.1 and 24.8 %), tau aggregation inhibition (48.2 and 35.5 % at 10 μM, respectively) and BBB permeation</td><td>[239]</td></tr><tr><td>215</td><td>0.12</td><td>13.4</td><td>27.9<sup>c</sup></td><td>6.9</td><td></td><td></td></tr><tr><td>216</td><td>44</td><td>80</td><td>2.85<sup>d</sup></td><td>0.88</td><td>Exhibited BACE-1 inhibition with IC<sub>50</sub> values of 38 and 44 nM, respectively, inhibitory activity against MAO-B, with IC<sub>50</sub> values of 5.15 and 2.42 μM, respectively, metal chelation, BBB permeability</td><td>[241]</td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 16 (continued)**</caption><thead><tr><th>Hybrid compounds</th><th>AChE inhibition IC<sub>50</sub> (nM)</th><th>BChE inhibition IC<sub>50</sub> (nM)</th><th>β-Amyloid inhibition (%)</th><th>Antioxidant IC<sub>50</sub> (μM)</th><th>Other activities</th><th>Ref.</th></tr></thead><tbody><tr><td>217</td><td>25</td><td>14</td><td>0.74<sup>d</sup></td><td>1.10</td><td></td><td></td></tr><tr><td>218</td><td>13</td><td>n.t.<sup>a</sup></td><td>57.8<sup>e</sup></td><td>n.t.</td><td>Neuroprotective effects, metal (Fe, Cu) chelating ability</td><td>[242]</td></tr><tr><td>219</td><td>13</td><td></td><td>31.5<sup>e</sup></td><td></td><td></td><td></td></tr></tbody></table>

these hybrids exhibited acceptable safety profiles when tested on normal cells SH-SY5Y and HepG2 and could penetrate the BBB [241].

5.1.3.6. *Tacrine-benzofuran hybrids.* Fancellu et al. explored the reposi- tioning of the well-known AChE inhibitor, tacrine, by coupling it to benzofuran derivatives [242]. The conjugates **218** and **219** (Table 16) exhibited very good AChE inhibition, with IC₅₀ values of 13 nM for both, compared to tacrine with an IC₅₀ of 35 nM. These hybrids demonstrated good potential for inhibiting self-induced Aβ aggregation, with per- centages of 57.8 % and 31.5 %, respectively, compared to tacrine (20 %) at 25 µM). Additionally, compound **219** showed significant inhibition of Cu⁺-mediated Aβ aggregation (53.4 % at 25 µM). The effectiveness of inhibition depended on the size of the linker and substituents attached to the main moiety. Furthermore, both hybrids exhibited neuroprotection in neuroblastoma cell viability tests following Aβ₁.₄₂-induced toxicity [242].

## 5.2. Linkage of tacrine pharmacophore with non-heterocyclic scaffolds

Elkina et al. combined tacrine with an antioxidant, 2-tolylhydraziny- lidene-1,3-diketone, using an aminoalkylene linker to create hybrid compound **220** (Table 17) [243]. This new conjugate effectively inhibited AChE (IC₅₀ = 0.241 µM) and BChE (IC₅₀ = 98.5 nM), with only weak inhibition of carboxylesterase (12.2 % at 20 µM). In the ABTS test, this conjugate molecule illustrated metal chelating ability for Cu²⁺, Fe²⁺, and Zn²⁺, as well as high antiradical activity. In the FRAP test, non-fluorinated hybrid **220** also demonstrated Fe³⁺ reducing activity [243]. Cashew nutshell liquid (CNSL) is an inexpensive food waste that possesses anti-inflammatory properties [244,245]. Rossi et al. reported the first sustainable MTDLs, derived from CNSL by combining func- tionalized CNSL components and tacrine templates [246]. Based on hepatic, neuronal, and microglial cell toxicity, MTDLs were selected. Enzymatic studies disclosed compounds **221** and **222** (Table 17) as potent and selective BChE inhibitors, with IC₅₀ values of 0.035 nM and 0.26 nM, respectively, compared to tetrahydroacridine 45.8 nM. Both these hybrids also showed anti-neuroinflammatory and neuroprotective activities in BV-2 microglial cells at 0.01 µM, verifying the design

rationale. Furthermore, they may also reach the brain at their active nanomolar concentration based on PAMPA-BBB data [246]. Pérez-Areales et al. utilized the fluorobenzohomoadamantanamine scaffold, a known NMDA receptor antagonist, to hybridize with tacrine *via* the linkage approach. The resulting hybrids, **223–225** (Table 17), exhibited greater potencies than their parent compounds against AChE and BChE, with up to 44-fold increased potency over 6-chlorotacrine. Additionally, these hybrids showed potency against NMDA receptors, with up to 2-fold increased potency over the parent compounds ben- zohomoadamantanamine and memantine as well as predicted to be brain-permeable [247].

### 5.2.1. Tacrine-cinnamic/squaric/benzoic acid/phenol/salicylamide/vanillin/O-phosphorylated/resveratrol hybrids

5.2.1.1. *Tacrine-cinnamic acid hybrids.* Chen et al. introduced small molecules, tacrine-cinnamic acid hybrids **226–228** (Table 17), as an effective ChE inhibitors [248]. These compounds exhibited strong in- hibition of hAChE, with IC₅₀ values of 2.7 nM, 6.9 nM, and 3.7 nM, respectively. Moreover, these representatives displayed suppression of Aβ self-aggregation by 31.82 %, 42.22 %, and 34.57 %, respectively, compared to resveratrol (30.36 %) at 25 µM, in an *in vivo* assay. The optimal compounds also exhibited promising results in ameliorating scopolamine-induced cognition deficits and showed initial safety in hepatotoxicity assessment [248].

5.2.1.2. *Tacrine-squaric acid hybrids.* Svobodova et al. capitalized on the versatile nature of squaric acid as a structural framework [249], which can be readily converted into amide-bearing compounds with hydrogen bond donor and acceptor groups, allowing interaction with multiple complementary sites [250]. By combining this framework with various tacrine-based derivatives, they synthesized hybrids **229** and **230** (Table 17), which exhibited potent inhibition against hAChE and hBChE, respectively, with IC₅₀ values of 2.0 nM and 21 nM, compared to THA (320 nM for hAChE and 80 nM for hBChE). Kinetic analysis of these hybrids demonstrated mixed-type inhibition, suggesting simultaneous interaction with both anionic sites of the enzymes [250].

<footnote>a Not tested.</footnote>

<footnote>b Tested but not active.</footnote>

<footnote>c 10 µM.</footnote>

<footnote>d µM.</footnote>

<footnote>e 25 µM.</footnote>
<table><caption>**Table 17**
Anti-Alzheimer's potential of MTDLs based on tacrine and non-heterocyclic scaffold hybrids.</caption><thead><tr><th>Hybrid compounds</th><th>AChE<br>inhibition IC<sub>50</sub><br>(nM)</th><th>BChE<br>inhibition IC<sub>50</sub><br>(nM)</th><th>β-Amyloid<br>inhibition (%)</th><th>Antioxidant<br>IC<sub>50</sub> (μM)</th><th>Other activities</th><th>Ref.</th></tr></thead><tbody><tr><td>220</td><td>0.241<sup>c</sup></td><td>98.5</td><td>n.t.<sup>a</sup></td><td>19.4</td><td>Weak inhibition of carboxylesterase (12.2 %), metal chelating ability, as well as high antiradical activity</td><td>[243]</td></tr><tr><td>221</td><td>2.54</td><td>0.0352</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>Anti-neuroinflammatory and neuroprotective activities, BBB permeability.</td><td>[246]</td></tr><tr><td>222</td><td>1260</td><td>0.26</td><td></td><td></td><td></td><td></td></tr><tr><td>223</td><td>1.4<sup>c</sup></td><td>0.2</td><td>12.3<sup>d</sup></td><td>n.t.<sup>a</sup></td><td>NMDA receptors inhibition IC<sub>50</sub> = 0.9, 1.2 and 3.4 μM, respectively, BBB permeation, Tau aggregation in E. coli (% inhibition = 20.7, 6.9 and 22.9 % at 10 μM</td><td>[247]</td></tr><tr><td>224: n = 3<br>225: n = 4</td><td>0.3<sup>c</sup><br>2.5<sup>c</sup></td><td>0.5<br>0.02</td><td>&lt;5<sup>d</sup></td><td></td><td></td><td></td></tr><tr><td>226</td><td>2.7</td><td>6.5</td><td>31.82<sup>e</sup></td><td>n.t.<sup>a</sup></td><td>No remarkable hepatotoxicity, good performance in ameliorating scopolamine-induced cognition deficits.</td><td>[248]</td></tr><tr><td>227</td><td>6.9</td><td>12.9</td><td>42.22<sup>e</sup></td><td></td><td></td><td></td></tr><tr><td>228</td><td>3.7</td><td>3.7</td><td>34.57<sup>e</sup></td><td></td><td></td><td></td></tr><tr><td>229</td><td>2.0</td><td>110</td><td>n.t.<sup>a</sup></td><td></td><td>Low cytotoxicity, BBB permeation</td><td>[250]</td></tr><tr><td>230</td><td>13</td><td>21</td><td></td><td></td><td></td><td></td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 17 (continued)**</caption><thead><tr><th>Hybrid compounds</th><th>AChE inhibition IC<sub>50</sub> (nM)</th><th>BChE inhibition IC<sub>50</sub> (nM)</th><th>β-Amyloid inhibition (%)</th><th>Antioxidant IC<sub>50</sub> (μM)</th><th>Other activities</th><th>Ref.</th></tr></thead><tbody><tr><td>231</td><td>25</td><td>0.12<sup>c</sup></td><td>32<sup>d</sup></td><td>n.t.<sup>a</sup></td><td>Neuroprotective properties, a free-radical scavenger, be less toxic, anti-inflammatory properties</td><td>[251]</td></tr><tr><td>232</td><td>31.2</td><td>8.0</td><td>84.92<sup>d</sup></td><td>n.t.<sup>a</sup></td><td>—</td><td>[252]</td></tr><tr><td>233</td><td>41.3</td><td>1.39</td><td>80.43<sup>d</sup></td><td>n.t.<sup>a</sup></td><td>No significant cytotoxic effect</td><td>[253]</td></tr><tr><td>234</td><td>168</td><td>0.51</td><td>63.8<sup>e</sup></td><td>n.t.<sup>a</sup></td><td>Neuroprotective activity, low hepatotoxicity and complete stability under the physiological conditions</td><td>[254]</td></tr><tr><td>235</td><td>82.6</td><td>15.6</td><td>80.1<sup>e</sup></td><td>n.t.<sup>b</sup></td><td>PI displacement from eeAChE (19.8 %), Porcine(pppp) Liver(pppp)CES (23 %), effective chelator of Cu<sup>2+</sup>, Fe<sup>2+</sup>, and Zn<sup>2+</sup></td><td>[255]</td></tr><tr><td>236</td><td>0.22<sup>e</sup></td><td>10.4</td><td>n.t.<sup>a</sup></td><td>0.73</td><td>Porcine Liver CES (29.5 %), PI displacement (8.2 %)</td><td>[256]</td></tr><tr><td>237</td><td>2.13<sup>e</sup></td><td>n.t.<sup>a</sup></td><td>77<sup>e</sup></td><td>6.4</td><td>Exhibited protection of SH-SY5Y cell line against H<sub>2</sub>O<sub>2</sub></td><td>[258]</td></tr><tr><td>238</td><td>0.12</td><td>361.52</td><td>34.2<sup>h</sup></td><td>9.05</td><td>Inhibited HDAC1 with the IC<sub>50</sub> = 0.23 nM, metal chelating attributes, BBB penetrant</td><td>[259]</td></tr><tr><td>239</td><td>6.11</td><td>12.86</td><td>n.t.<sup>a</sup></td><td>n.t.<sup>a</sup></td><td>Lack hepatocytotoxicity, neuroprotection activity</td><td>[260]</td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 17 (continued)**</caption><thead><tr><th>Hybrid compounds</th><th>AChE inhibition IC<sub>50</sub> (nM)</th><th>BChE inhibition IC<sub>50</sub> (nM)</th><th>β-Amyloid inhibition (%)</th><th>Antioxidant IC<sub>50</sub> (μM)</th><th>Other activities</th><th>Ref.</th></tr></thead><tbody><tr><td>Ph<sub>2</sub>Ph<sub>2</sub>HN<sub>2</sub>Ph<sub>2</sub>HN<sub>2</sub>β<sub>12</sub>HN<sub>2</sub>Cl<sub>2</sub></td><td>9.95</td><td>1.96</td><td></td><td></td><td>Cytotoxic effect against SH-SY5Y cells, reduction of hepatotoxicity</td><td></td></tr><tr><td>Et<sub>2</sub>Et<sub>2</sub>HN<sub>2</sub>Ph<sub>2</sub>HN<sub>2</sub>Cl<sub>2</sub></td><td>676.7</td><td>111.3</td><td></td><td></td><td></td><td></td></tr><tr><td>MeO<sub>2</sub>OMe<sub>2</sub>HN<sub>2</sub>Cl<sub>2</sub></td><td>0.80<sup>a</sup></td><td>n.a.<sup>b</sup></td><td>17.6<sup>f</sup></td><td>&gt;5000</td><td>Demonstrated anti-inflammatory, immunomodulatory characteristics, high BBB permeability as well as low cytotoxicity</td><td>[262]</td></tr></tbody></table>

a Not tested.

b Tested but not active.

c μM.

d 100 μM.

e 25 μM.

f 50 μM.

g 10 μM.

h 20 μM.

### 5.2.1.3. Tacrine-benzoic acids hybrids.
Czarnecka et al. explored the synthesis of tacrine and 3,5-dichlorobenzoic acid hybrid **231** (Table 17), it emerged as the most potent inhibitor of ChEs (IC<sub>50</sub> for eeAChE = 25 nM and IC<sub>50</sub> for eqBChE = 0.123 µM), outperforming tacrine in inhibiting both enzymes (IC<sub>50</sub> = 78 nM and 15 nM, for both targets respectively) [251]. This hybrid also exhibited the ability to inhibit Aβ aggregation (32 % at 100 µM), targeting both the CAS and PAS of AChE, with additional neuroprotective and free-radical scavenging properties and minimal toxicity compared to tacrine after oral administration. Moreover, it displayed anti-inflammatory properties by showing little inhibitory activity against hyaluronidase (HYAL) [251]. Similarly, Ski-biński et al. designed iodobenzoic acid derivative linked to tacrine **232** (Table 17) that stood out as a potent AChE inhibitor (IC<sub>50</sub> = 31.2 nM) and BChE inhibitor (IC<sub>50</sub> = 8.0 nM), exhibiting stronger inhibition compared to tacrine. This hybrid displayed a mixed inhibition pattern against AChE and acted as a PAS inhibitor. Additionally, it effectively inhibited amyloid aggregation at various concentrations [252].

Furthermore, the development of 2-fluorobenzoic acid or 3-fluorobenzoic acid derivatives linked to tacrine by Czarnecka et al. yielded hybrid **233** (Table 17) as a potential inhibitor of ChEs and Aβ aggregation [253]. It exhibited higher inhibitory potency against both AChE (IC<sub>50</sub> = 41.37 nM) and BChE (IC<sub>50</sub> = 1.39 nM) compared to tacrine (IC<sub>50</sub> = 80.82 nM for AChE and 19.55 nM for BChE). Kinetic assays revealed it as the most effective inhibitor against AChE, while aggregation experiments demonstrated significant inhibition of Aβ aggregation at various concentrations [253].

### 5.2.1.4. Tacrine-phenol hybrids.
Roldan-Pena et al. developed tacrine-phenolic heterodimers by hybridizing both pharmacophores, modifying the nature of imino, amino, ether, and the length of the linker, along with the type of substituents such as hydroxy, methoxy, and benzyloxy, their number, and position on the aromatic part [254]. The lead compound **234** (Table 17), with an ether-type linker, exhibited an 85-fold increase in activity compared to parent tacrine, with an IC<sub>50</sub> value of 0.515 nM against hBChE, compared to tacrine's 44.2 nM.

Additionally, this hybrid inhibited the self-aggregation of Aβ<sub>42</sub> (63.8 % at 50 µM) and demonstrated neuroprotective activity. It shows promise for treating neuronal injury and senescence. Moreover, the lead compound exhibited low hepatotoxicity and complete stability under physiological conditions [254].

### 5.2.1.5. Tacrine-salicylamide hybrids.
Makhaeva et al. developed conjugates of tacrine-salicylimine hybrid **235** (Table 17) with broad-spectrum biological action. This conjugate displayed IC<sub>50</sub> values of 82.6 nM for AChE and 15.6 nM for BChE, with modest inhibition of off-target carboxylesterase [255]. In the ThT test, this conjugate inhibited Aβ<sub>42</sub>-self-aggregation (80.1 % at 100 µM), with enhanced inhibition observed with spacer expansion. Notably, the salicylamide hybrid exhibited strong ABTS<sup>•+</sup>-scavenging activity. Additionally, it demonstrated potent Cu<sup>2+</sup>, Fe<sup>2+</sup>, and Zn<sup>2+</sup> chelation and exhibited less cytotoxicity on mouse hepatocytes compared to tacrine, along with expected intestinal absorption and BBB permeability [255]. Similarly, Grishchenko et al. investigated new conjugate of tacrine and salicylamide **236** (Table 17) with alkylene spacers as potential multifunctional medications [256]. This hybrid molecule showed the strongest inhibitory activity against AChE (IC<sub>50</sub> = 0.224 µM) and BChE (IC<sub>50</sub> = 10.4 nM). It also displayed weak inhibition of carboxylesterase, suggesting limited potential for undesirable drug-drug interactions in clinical trials. Moreover, this conjugate acted as a mixed-type reversible suppressor of both ChEs and demonstrated dual binding to the CAS and PAS of AChE in molecular docking studies, indicating its ability to block AChE-induced Aβ aggregation. Importantly, the lead compound exhibited significant ABTS<sup>•+</sup>-scavenging activity and metal chelating activity towards Cu<sup>2+</sup>, Fe<sup>2+</sup>, and Zn<sup>2+</sup> [256].

### 5.2.1.6. Tacrine-vanillin hybrids.
Vanillin, a natural antioxidant, serves as a promising starting point for synthesizing new compounds with enhanced antioxidant properties, as well as Aβ peptide aggregating and AChE inhibiting properties [257]. Scipioni et al. reported the synthesis of vanillin derivatives containing either a tacrine or a naphthalimido