Tacrine-Based Hybrids: Past, Present, and Future

Alzheimer’s disease (AD) is a neurodegenerative disorder which is characterized by β-amyloid (Aβ) aggregation, τ-hyperphosphorylation, and loss of cholinergic neurons. The other important hallmarks of AD are oxidative stress, metal dyshomeostasis, inflammation, and cell cycle dysregulation. Multiple therapeutic targets may be proposed for the development of anti-AD drugs, and the “one drug–multiple targets” strategy is of current interest. Tacrine (THA) was the first clinically approved cholinesterase (ChE) inhibitor, which was withdrawn due to high hepatotoxicity. However, its high potency in ChE inhibition, low molecular weight, and simple structure make THA a promising scaffold for developing multi-target agents. In this review, we summarized THA-based hybrids published from 2006 to 2022, thus providing an overview of strategies that have been used in drug design and approaches that have resulted in significant cognitive improvements and reduced hepatotoxicity.


Introduction
AD is a progressive multifarious neurodegenerative disorder which is described by a progressive loss of cognitive abilities, such as memory, language skills, and attention, as well as by spatial disorientation and depression. The pathological hallmarks of AD are extracellular accumulation of Aβ plaques composed of Aβ peptides, neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau protein, brain inflammation, and atrophy [1]. Aβ is formed from amyloid precursor protein (APP), which is cleaved by β-secretase (BACE-1) and γ-secretase. Thus, interfering with fibril formation, including metal cation chelation, the disruption of amyloid aggregation, and BACE-1 inhibition, are well-established approaches to the development of anti-AD drugs [2].
Oxidative stress and inflammation are also some of the hallmarks of AD [3]. An increase in reactive oxygen species (ROS) levels is caused by mitochondrial dysfunction, violation of the homeostasis of metal cations, the formation of Aβ fibrils, inflammatory processes, etc. [4].
Calcium regulation is important in learning and memory. The disruption of Ca 2+ level homeostasis caused by the formation of Aβ leads to cell death [5,6]. Blocking Ca 2+ channels is also one of the important strategies in the treatment of AD. Calcium channel blockers (CCBs), one of the more commonly used treatments for hypertension, are also considered as potential drug candidates for anti-AD therapy [7].
Glycogen synthase kinase-3 (GSK-3) is a serine-threonine kinase involved in neurodegeneration. GSK-3β isoform is found to be hyperactive in the brains of AD patients; GSK-3β inhibition is also one of the therapeutic strategies during anti-AD drug development [8,9].
Neurotransmitters such as noradrenaline, dopamine, serotonin (5-HT), and GABA are involved in the pathogenesis of AD; imbalances between neurotransmitters in the temporal cortex and hippocampus have been reported [10]. In addition, in the latter studies of AD, a deficiency of monoamines is found in the brainstem and hippocampus [11]. NMDA antagonists slow the decline in cognitive function in AD patients [3,12].
Different components of the cholinergic system are therapeutic targets in AD treatment. ACh is synthesized from acetyl coenzyme A and choline in the presence of choline acetyltransferase (ChAT). Then, ACh is released into the synapse and binds to either the G-protein coupled muscarinic receptors or the ionotropic nicotinic receptors to transmit signals from one neuron to the other [13]. ACh can be degraded by AChE or BuChE [14]. The activation of the muscarinic M1 receptor exerts a pro-cognitive effect, and an activation of the alpha7 nACh receptor might inhibit the formation of Aβ [15]. Thus, drugs capable of acting on muscarinic and nicotinic receptors are of interest [16,17]. Moreover, the mAChR antagonist scopolamine is used for inducing cognitive and behavioral deficits in animals [18].
Current clinical therapy for AD patients is based on the cholinergic hypothesis, which suggests that the decline of acetylcholine (ACh) levels causes cognitive and memory deficits [19]. An increase in the ACh concentration in a synaptic cleft by various ways, such as the inhibition of both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), is the key approach in the treatment of AD now. AChE inhibitors (AChEIs), including donepezil, galantamine, and rivastigmine, are FDA-approved drugs for AD treatment [20].
Tacrine (9-amine-1,2,3,4-tetrahydroacridine) (THA) was the first FDA-approved ChE inhibitor for the treatment of AD. THA was produced under the brand name Cognex ® and the recommended dose was 40 mg per day. THA acts by inhibiting the metabolism of acetylcholine and thus prolonging its activity and raising levels in the cerebral cortex. Therapy with THA improves mental functioning in patients with mild-to-moderate dementia of Alzheimer disease [21,22]. THA undergoes first pass metabolism by the liver and is extensively metabolized by the cytochrome P450 system, which is supposedly the reason for its high hepatotoxicity. Thus, THA therapy is accompanied by increased serum alanine aminotransferase (ALT) and aspartate aminotransferase (ASAT) levels, which are indicative of liver damage and which were the reason for its withdrawal from use in 2013 [22].
Intensive research resulted in the design of more potent 6-chlorotacrine (6-Cl-THA) and less toxic 7-methoxytacrine (7-MEOTA) drug candidates [23][24][25] (Figure 1). acetyltransferase (ChAT). Then, ACh is released into the synapse and binds to either the G-protein coupled muscarinic receptors or the ionotropic nicotinic receptors to transmit signals from one neuron to the other [13]. ACh can be degraded by AChE or BuChE [14]. The activation of the muscarinic M1 receptor exerts a pro-cognitive effect, and an activation of the alpha7 nACh receptor might inhibit the formation of Aβ [15]. Thus, drugs capable of acting on muscarinic and nicotinic receptors are of interest [16,17]. Moreover, the mAChR antagonist scopolamine is used for inducing cognitive and behavioral deficits in animals [18].
Current clinical therapy for AD patients is based on the cholinergic hypothesis, which suggests that the decline of acetylcholine (ACh) levels causes cognitive and memory deficits [19]. An increase in the ACh concentration in a synaptic cleft by various ways, such as the inhibition of both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), is the key approach in the treatment of AD now. AChE inhibitors (AChEIs), including donepezil, galantamine, and rivastigmine, are FDA-approved drugs for AD treatment [20].
Tacrine (9-amine-1,2,3,4-tetrahydroacridine) (THA) was the first FDA-approved ChE inhibitor for the treatment of AD. THA was produced under the brand name Cognex ® and the recommended dose was 40 mg per day. THA acts by inhibiting the metabolism of acetylcholine and thus prolonging its activity and raising levels in the cerebral cortex. Therapy with THA improves mental functioning in patients with mild-to-moderate dementia of Alzheimer disease [21,22]. THA undergoes first pass metabolism by the liver and is extensively metabolized by the cytochrome P450 system, which is supposedly the reason for its high hepatotoxicity. Thus, THA therapy is accompanied by increased serum alanine aminotransferase (ALT) and aspartate aminotransferase (ASAT) levels, which are indicative of liver damage and which were the reason for its withdrawal from use in 2013 [22].
Intensive research resulted in the design of more potent 6-chlorotacrine (6-Cl-THA) and less toxic 7-methoxytacrine (7-MEOTA) drug candidates [23][24][25] (Figure 1). Despite the hepatotoxicity of THA, its suitability for chemical modification makes it a widely used scaffold for drug development [26]. Chemical modification of amino groups in the THA molecule leads to a decrease in hepatotoxicity [27]. Thus, a conjugation of THA with a second pharmacophoric moiety resulting in THA-based hybrids, pioneered by Pang et al. [28], is still an area of active research and development. Despite the hepatotoxicity of THA, its suitability for chemical modification makes it a widely used scaffold for drug development [26]. Chemical modification of amino groups in the THA molecule leads to a decrease in hepatotoxicity [27]. Thus, a conjugation of THA with a second pharmacophoric moiety resulting in THA-based hybrids, pioneered by Pang et al. [28], is still an area of active research and development.
Since the strategy of ChE inhibition was successful, much attention is paid to the development of drugs that effectively interact with this enzyme. The crystallographic structure of AChE reveals that it has a narrow 20A gorge with two binding sites, the catalytic active site (CAS) at the bottom and the peripheral anionic site (PAS) near the entrance [29,30]. AChE inhibitors can bind to either one or two sites. Importantly, AChE could also promote Aβ formation by interaction through the PAS of AChE, yielding the toxic AChE-Aβ complex [31]. Therefore, the dual binding inhibitors, which target both PAS and CAS, are of interest in AD treatment, and THA-linker-residue hybrids with appropriate linker length are being designed [32].
Based on the above-mentioned multiple cellular and pathological hallmarks of AD, several therapeutic strategies should be used in developing effective anti-AD therapy, and a potential drug candidate should affect several therapeutic targets at once to be effective. Thus, multi-target-directed THA-based hybrids have been of interest for years, and novel potential anti-AD THA-based drugs are still reported. Several reports have considered designing THA-like compounds by replacing or annulating the benzene ring in THA with different heterocyclic systems [33,34] and THA dimers [35]. Additionally, several reviews have reported THA-based hybrids. In 2017, Sameem et al. reported a short review of THA-based scaffolds as multi-target drugs (MTDLs) [36] and Wu et al. reported a review of several THA-based hybrids [27]. In 2019, Girek et al. summarized phyto-THA hybrids [37]. In addition, in 2020, Eckroat et al. summarized structural analogues of THA developed in 2015-2020 [38].
The development of new multi-target drugs based on THA and its analogues is a relevant task, and novel high-quality research works continue to be published. A quick search of the database of articles on THA and its analogues shows that the development of drugs against AD based on THA is of current interest ( Figure 2).
Since the strategy of ChE inhibition was successful, much attention is paid to the velopment of drugs that effectively interact with this enzyme. The crystallographic str ture of AChE reveals that it has a narrow 20A gorge with two binding sites, the cataly active site (CAS) at the bottom and the peripheral anionic site (PAS) near the entra [29,30]. AChE inhibitors can bind to either one or two sites. Importantly, AChE could a promote Aβ formation by interaction through the PAS of AChE, yielding the toxic AC Aβ complex [31]. Therefore, the dual binding inhibitors, which target both PAS and C are of interest in AD treatment, and THA-linker-residue hybrids with appropriate lin length are being designed [32].
Based on the above-mentioned multiple cellular and pathological hallmarks of A several therapeutic strategies should be used in developing effective anti-AD therapy, a a potential drug candidate should affect several therapeutic targets at once to be effect Thus, multi-target-directed THA-based hybrids have been of interest for years, and no potential anti-AD THA-based drugs are still reported. Several reports have conside designing THA-like compounds by replacing or annulating the benzene ring in THA w different heterocyclic systems [33,34] and THA dimers [35]. Additionally, several revie have reported THA-based hybrids. In 2017, Sameem et al. reported a short review of TH based scaffolds as multi-target drugs (MTDLs) [36] and Wu et al. reported a review several THA-based hybrids [27]. In 2019, Girek et al. summarized phyto-THA hybr [37]. In addition, in 2020, Eckroat et al. summarized structural analogues of THA dev oped in 2015-2020 [38].
The development of new multi-target drugs based on THA and its analogues relevant task, and novel high-quality research works continue to be published. A qu search of the database of articles on THA and its analogues shows that the developm of drugs against AD based on THA is of current interest ( Figure 2). In this review, we sum up THA-linker-residue hybrids published from 2006 to 20 with many of them being superior to currently clinically used drugs in terms of their m tiplicity of biological action, low toxicity, and drug efficacy. Tacrine hybrids with antioxidant activity (Section 3), NO-donors (Section 4), with biologically active molecules (Section 5), and with drugs that affect the cholinergic/serotonergic systems (Section 6) are summarized in various chapters. Additionally, two chapters are devoted to tacrine hybrids with natural products (Section 7) and organic ligands (Section 8). Below, the plan of this review is presented in the form of a diagram ( Figure 4). In addition, in Table S1, we summarize all hybrids described in this review, present the best result of inhibitory activity among the series, and the spectrum of biological actions confirmed for hybrids.
Additionally, we summarize the in vivo therapeutic efficacy of hybrids data presented in this review in Table S2.

Tacrine-Melatonin Hybrids
In 2006, Rodríguez-Franco et al. [39] reported hybrids of THA with melatonin, a pineal neurohormone with strong antioxidant action [134] (Figure 4). Tacrine hybrids with antioxidant activity (Section 3), NO-donors (Section 4), with biologically active molecules (Section 5), and with drugs that affect the cholinergic/serotonergic systems (Section 6) are summarized in various chapters. Additionally, two chapters are devoted to tacrine hybrids with natural products (Section 7) and organic ligands (Section 8). Below, the plan of this review is presented in the form of a diagram ( Figure 4). In addition, in Table S1, we summarize all hybrids described in this review, present the best result of inhibitory activity among the series, and the spectrum of biological actions confirmed for hybrids.
Hybrids were potent inhibitors of cholinesterases at the low nanomolar level. Additionally, hybrid 7 is still one of the most potent inhibitors of human AChE described with IC50 0.008 nM. An antioxidant activity of hybrids was determined by the oxygen radical absorbance capacity assay using fluorescein (ORAC-FL); hybrids showed potent peroxyl radical absorbance capacities ranging from 1.7-to 4-fold the value of trolox, a Vitamin E analogue which was used as a standard. Hybrids 1-12 proved the ability to cross the blood-brain barrier (BBB) in the PAMPA-BBB test.
In 2009, the same scientific group reported an extended series of THA-melatonin hybrids 14-23 [40]. All hybrids were potent inhibitors of mammalian ChEs at the low-nanomolar range. 6-Chloro-and 6,8-dichlorotacrine-melatonin hybrids 3 and 6 showed remarkable selectivity, being from 200-to 1000-fold more active toward hAChE than hBuChE and showing potent peroxyl radical absorbance capacities ranging from 1.5-to 4fold the trolox value. Molecular modeling studies showed that hybrids target both the CAS and the PAS of AChE. A displacement of the binding of propidium iodide (PI) from the PAS at sub-micromolar concentrations was confirmed. In addition, an inhibition of Aβ self-aggregation and neuroprotective properties in a human neuroblastoma line were reported.
Hybrids were potent inhibitors of cholinesterases at the low nanomolar level. Additionally, hybrid 7 is still one of the most potent inhibitors of human AChE described with IC 50 0.008 nM. An antioxidant activity of hybrids was determined by the oxygen radical absorbance capacity assay using fluorescein (ORAC-FL); hybrids showed potent peroxyl radical absorbance capacities ranging from 1.7-to 4-fold the value of trolox, a Vitamin E analogue which was used as a standard. Hybrids 1-12 proved the ability to cross the blood-brain barrier (BBB) in the PAMPA-BBB test.
In 2009, the same scientific group reported an extended series of THA-melatonin hybrids 14-23 [40]. All hybrids were potent inhibitors of mammalian ChEs at the lownanomolar range. 6-Chloro-and 6,8-dichlorotacrine-melatonin hybrids 3 and 6 showed remarkable selectivity, being from 200-to 1000-fold more active toward hAChE than hBuChE and showing potent peroxyl radical absorbance capacities ranging from 1.5-to 4-fold the trolox value. Molecular modeling studies showed that hybrids target both the CAS and the PAS of AChE. A displacement of the binding of propidium iodide (PI) from the PAS at sub-micromolar concentrations was confirmed. In addition, an inhibition of Aβ self-aggregation and neuroprotective properties in a human neuroblastoma line were reported.
In 2005, racemic lipocrine, a lipoic acid with a derivative of THA, was reported [135]. This hybrid inhibited AChE effectively (IC 50 0.253 nM) and reduced AChE-induced Aβ aggregation from ROS formation. Inspired by this, in 2016 Benchekroun et al. designed THA-melatonin hybrids 24-31 with ferulic acid (FA) or lipoic acid [41]. Hybrid 28 was the most effective inhibitor of BuChE with IC 50 1.25 nM and AChE with IC 50 3.62 nM. Additionally, 24-27 were potent antioxidants, showing values from 9.11 trolox equivalents. A neuroprotective effect for hybrids 25-27 was shown on SH-SY5Y neuroblastoma cells, with 26 being the best of the series. Additionally, 26 showed a neuroprotective effect against toxic insults mediated by hydrogen peroxide, Aβ 1−40 , and Aβ 1−42 . Hybrids 24-27 successfully induced the Nrf2 pathway in the AREc32 reporter cell line.

Other Hybrids with Antioxidant Activity
In 2010, Fernández-Bachiller et al. reported THA-based hybrids 32-48 with 8hydroxyquinoline [42] (Figure 5). An inhibition of human cholinesterase showed IC 50 values for all tested hybrids in the nano-and subnanomolar range (0.5-5.5 nM). Hybrid 40 was the best hBuChE inhibitor with IC 50 2 nM. Hybrids conjugated with an unsubstituted 8-hydroxyquinoline fragment and a methylene tether of 7-10 carbons showed the best AChE inhibitory activities, with 35 showing IC 50
The affinity of selected compounds for the PAS was confirmed by the displacement of PI. All hybrids showed permeability values over the above limit in the PAMPA-BBB test. The metal-chelating properties of 35 were confirmed by UV-Vis spectrometry in the presence of Cu 2+ . Finally, 35 showed negligible cell toxicity on human neuroblastoma cell line SHSY5Y.  [42], THA hybrids with benzene/pyridine moieties 49-66 [43,44], and THA-4-oxo-4H-chromene hybrids 67-94 [44].
The affinity of selected compounds for the PAS was confirmed by the displacement of PI. All hybrids showed permeability values over the above limit in the PAMPA-BBB test. The metal-chelating properties of 35 were confirmed by UV-Vis spectrometry in the presence of Cu 2+ . Finally, 35 showed negligible cell toxicity on human neuroblastoma cell line SHSY5Y.
In 2011, Luo et al. reported THA hybrids with substituted benzene or pyridine moieties 49-66 [43,44]. Most compounds showed selectivity for BuChE over AChE, and 58 was found to be the best inhibitor for both ChEs with its IC 50 4.55 nM and 3.41 nM. Kinetic studies of the inhibition of AChE by 58 revealed a mixed-type inhibition, which was confirmed by a molecular modeling study. The antioxidant activity of 58-61 with hydroxyl group was proven via ORAC test; compounds showed potent peroxyl radical absorbance capacities ranging from 1.2-to 2.7-fold of the trolox value. Finally, 58 proved to inhibit self-mediated Aβ aggregation.
In 2012, Fernández-Bachiller et al. reported hybrids of THA with flavonoid scaffold 67-94 derived from 4-oxo-4H-chromene with possible antioxidant and BACE-1 inhibitory activities [45]. Hybrids 67-94 showed a selectivity for BuChE, with 70 as the most active inhibitor. Hybrid 88 was the best hAChE inhibitor with IC 50 35 pM, and 74 was the most active with IC 50 38 pM. Expectedly, hybrids with hydroxyl groups exhibited antioxidant capacities. Hybrid 83 was 1.3-fold more potent than the vitamin E analogue and was the best antioxidant. All hybrids were found to be potent inhibitors of human BACE-1 with IC 50 s from 2 to 22 µM, better than that of apigenin (IC 50 38.5 µM), with the most active as 77 (IC 50 2.1 µM). All hybrids (except for 92 and 93) showed the potential to cross the BBB in the PAMPA-BBB test. Finally, 83 showed potent combined inhibition of human BACE-1 and ChEs.
In 2012, Chao et al. reported hybrids of THA with caffeic acid 95-100 [46] (Figure 6). All hybrids inhibited ChEs, with the most potent AChE inhibitor being 99 (IC 50 0.3 µM). Expectedly, 95-100 showed a radical scavenging activity in a DPPH test due to the presence of a hydroxyl group. Hybrid 99 showed the best antioxidant activity in 4.8 ± 0.9 µM. The inhibition activity of 99 besides Aβ self-or AChE-induced aggregation was proven, as well as its antioxidant properties. Finally, the Cu 2+ -chelating properties of 99 were proven by UV-Vis spectra.
In 2012, Chen et al. reported a hybrid of THA 101 with a flavonolignan silibinin, a natural antioxidant [47,48,136]. Hybrid 101 loses quite a bit of inhibitory activity at BuChE (16-fold lower) and moderately at AChE (3.5-fold lower) when compared with THA. A lower hepatotoxicity of 101 in comparison with THA was revealed on hepatocellular carcinoma HePG2 cells. No histomorphological changes in liver tissue were observed after administration of 101 in vivo, in contrast to the THA administration. The antioxidant effect of 101 was confirmed by an evaluation of the lipid peroxidation products level in vivo after drug administration. In in vivo behavior tests on scopolamine-injected mice, 101 showed the same pro-cognitive effect as THA.
In 2013, Mao et al. reported THA hybrids with Ebselen 102-110, an organoselenium with antioxidant activity, anti-inflammatory, and neuroprotective activities [49,137,138]. Hybrids 102-110 inhibited both AChE and BuChE with nanomolar activity. Hybrid 106 was the best AChE inhibitor with IC50 6.32 nM among the derivatives with unsubstituted Ebselen moiety, whereas 110 with OMe-substituent showed a promising result in 2.55 nM.
Lineweaver-Burk plots of 110 against AChE revealed a mixed-type inhibition, which was also confirmed by molecular modelling. Expectedly, 106 and 110 proved to be antioxidants with eroxynitrite scavenging activity 1.17 and 1.26 times greater than that of ebselen, respectively. Unfortunately, 110 showed high toxicity on human hepatic stellate cells (HSC).
Lineweaver-Burk plots of 110 against AChE revealed a mixed-type inhibition, which was also confirmed by molecular modelling. Expectedly, 106 and 110 proved to be antioxidants with eroxynitrite scavenging activity 1.17 and 1.26 times greater than that of ebselen, respectively. Unfortunately, 110 showed high toxicity on human hepatic stellate cells (HSC).
In 2014, Lan et al. reported THA-(b-carbolines (pyrido [3,4-b]indoles) hybrids 111-127 [50]. All hybrids inhibited both ChEs with IC 50 values from sub-micromolar to nanomolar. Hybrid 122 was the best AChE inhibitor with IC 50 21.6 nM and 125 was the best BuChE inhibitor with IC 50 4.3 nM. In addition, 122 was the best hAChE inhibitor with IC 50 63.2 nM. Kinetic study revealed 122 as a mixed-type inhibitor of AChE, which was also confirmed by molecular modeling studies. Additionally, 122 displayed the most potent antioxidant activity in 1.57 trolox equivalents, and 126 and 122 showed a neuroprotective effect on the rat pheochromocytoma cell line PC12 from H 2 O 2 -induced oxidative stress. Thioflavin T (ThT)-based fluorometric assay with curcumin as a control showed moderate to good antiaggregating potencies of hybrids (22.4-66.5% at 20 µM) with the most effective compounds being 126 and 122; 122 could also inhibit a Cu 2+ -induced Aβ aggregation. UV-Vis spectrometry revealed the ability of 122 to chelate Cu 2+ ions. Finally, the PAMPA-BBB assay revealed the ability of hybrids to cross the BBB.
In 2015, Nepovimova et al. reported hybrids of THA with Trolox 128-148, a watersoluble analogue of vitamin E (±6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) and a "gold standard" antioxidant [51]. Hybrids 128-148 showed moderate inhibition activity toward hAChE with an IC 50 from 13.29 to 0.08 µM. Hybrid 148 was the best hAChE inhibitor with IC 50 80 nM, four-fold weaker than 6-Cl-THA. Overall, 128-148 displayed moderate to good antioxidant capacities, with 148 as the best antioxidant with IC 50 44.09 µM. Mixed-type inhibition of AChE was established for 148 by kinetic assay. The low hepatotoxicity of 148 was shown on HepG2 cells, and a metabolic assay in human liver microsomes showed no potentially metabolic products emerging under experiment. Unfortunately, the limitations in the solubility of 148 did not allow the determining of LD 50 in vivo.
In 2016, Luo et al. reported THA hybrids with N,N-dimethylated flavonoids 149-153 [52]. All hybrids inhibited ChEs in the nanomolar range. Hybrid 152 was the best AChE inhibitor with IC 50 59.61 nM and 153 was the best BuChE inhibitor with IC 50 24.67 nM. The antioxidant activity of hybrids was confirmed using oxygen radical absorbance capacity (ORAC) assay, in which hybrid 153 showed 3.2 trolox equivalents. The antiaggregating activity of hybrids was confirmed by ThT assay. A neuroprotective effect of 152 against H 2 O 2 -induced oxidative stress was shown on PC12 cells, as well as a registered reduction of intracellular ROS levels after treatment with 152.
In 2020, the same scientific group reported significant improvements in cognitive function in APP/PS1 transgenic mice treated with 165 [55]. After 4 weeks of intragastric administration of 165 (1.27 mg/100 g), cognitive function and synaptic plasticity were improved. In addition, the level of Aβ plaques in the DG region in the APP/PS1 mice was reduced.
In 2020, Pérez-Areales et al. reported THA-based hybrids with antioxidant CR-6 192-210 [56]. The most potent hAChE inhibitors were amines 207 (IC 50 442 pM), 208 (IC 50 121 pM), and 209 (IC 50 272 pM), which were 33-, 120-, and 53-fold more potent than the parent Cl-THA. The order of potencies was as follows: amines 207-210 > amides 192-195 > reverse amides 204-206 > O-benzylated amides 196-199. Expectedly, CR-6-chlorotacrine hybrids were found to be less potent inhibitors toward hBuChE than hAChE. However, amines 207 (IC 50 17.4 nM), 208 (IC 50 13.4 nM), and 209 (IC 50 18.3 nM) were the most potent inhibitors of hBuChE, being 29-, 38-, and 28-fold more potent than the parent 6-Cl-THA. Because 6-Cl-THA is known to interact with CAS [139], the lead compound was expected to interact with AChE in the same way, which was confirmed using molecular dynamics simulations. Kinetic studies showed that the hybrid acts as a mixed-type inhibitor of hAChE. The in vitro antioxidant activity of all hybrids featuring a free hydroxyl group was revealed using DPPH assay, with IC 50 values in the 6.9-22.9 µM range. Most of the hybrids were found to be inactive as BACE-1 inhibitors, with only the O-benzylated hybrids 196-199 showing weak inhibition. Favorable brain permeability of hybrids was confirmed using PAMPA-BBB assay. Chronic in vivo efficacy studies with 193 and 197 in double-transgenic APP/PS1 mice have shown positive tendencies in improving cognition and amyloid pathology.
In 2021, Rani et al. reported hybrids of THA with chalcones 211-240, a scaffold with AChE-inhibitory, antioxidant, antiaggregating, anti-inflammatory, neuroprotective, and vasodilator activities [57,140] (Figure 8). Hybrids 216, 225, and 226 showed moderate activity against AChE, and only 225 showed above 50% inhibition at 10 µM against the BuChE. Molecular docking studies showed that 225 and 226 interact with residues of AchE. In vivo behavior studies showed that 216, 225, and 226 attenuated the effect of scopolamine treatment. Moreover, a recovery of scopolamine-induced glutathione depletion in the mice brain was confirmed in group treated with 216, 225 and 226. Additionally, a significant reduction in in-brain malondialdehyde level was detected.  [57,140] (Figure 8). Hybrids 216, 225, and 226 showed moderate activity against AChE, and only 225 showed above 50% inhibition at 10 μM against the BuChE. Molecular docking studies showed that 225 and 226 interact with residues of AchE. In vivo behavior studies showed that 216, 225, and 226 attenuated the effect of scopolamine treatment. Moreover, a recovery of scopolamine-induced glutathione depletion in the mice brain was confirmed in group treated with 216, 225 and 226. Additionally, a significant reduction in in-brain malondialdehyde level was detected.  [58]. Huprines represent a family of potent and selective AChE inhibitors based on THA and (−)-huperzine A (HA) [141]. HA is a Chinese herb extract from Huperzia serrata, a reversible AChE inhibitor [142]. Despite the approval of HA by the FDA of China for AD therapy in 1994, the supply of this drug is still limited due to expensive synthesis [143,144]. Thus, design of potent HA analogs not requiring great synthetic effort is of interest ( Figure 9).  [58]. Huprines represent a family of potent and selective AChE inhibitors based on THA and (−)-huperzine A (HA) [141]. HA is a Chinese herb extract from Huperzia serrata, a reversible AChE inhibitor [142]. Despite the approval of HA by the FDA of China for AD therapy in 1994, the supply of this drug is still limited due to expensive synthesis [143,144]. Thus, design of potent HA analogs not requiring great synthetic effort is of interest ( Figure 9).

241-249
retained the high potency of the parent huprine hBuChE. The nine-atoms linker was found to be optimal for the i and hBuChE, yielding 243, which surpassed the nanomolar poten studies of AChE inhibition showed a dual site binding, and the PAS was confirmed by a PI displacement. All compounds showe the DPPH assay, with the best antioxidant being 247 (IC50 = 31.7 BACE-1 inhibitors, with the most active being 241. The ability of was proven in PAMPA-BBB assay. Additionally, biodistribution revealed an ability of 243 and 249 to accumulate in the brain. Finally, the therapeutic efficacy of 243 and 249 was investig and old (10 month) APP/PS1 mice. Mice treated with 249 enhance in old APP/PS1 mice in all the performed tests, while neither 249 young transgenic mice. A decrease in the Aβ42/Aβ40 ratio in the br 249 was revealed. In addition, 249 significantly increased the str mission, and reduced hippocampal levels of the oxidative stress neuroinflammation (astrogliosis) marker GFAP.

Tacrine-Ferulic Acid Hybrids
FA is a well-known antioxidant with multiple biological acti tiple activity and ease of chemical modifications, the design of F terest ( Figure 10) [146].
In 2008, Fang et al. reported THA-FA hybrids 250-254 [59]. A ChEs, with the most potent hybrids being 252 and 253 toward bo optimal length of linker in 6-7 atoms. Kinetic study of AChE in reversible and noncompetitive inhibition. All hybrids showed m dant activity confirmed by ORAC-fluorescein assay and hybrid equivalents.
In 2010, an investigation of the in vivo anti-AD effectiveness

241-249
retained the high potency of the parent huprine Y against hAChE and hBuChE. The nine-atoms linker was found to be optimal for the inhibition of both hAChE and hBuChE, yielding 243, which surpassed the nanomolar potency of huprine Y. Kinetic studies of AChE inhibition showed a dual site binding, and the interaction of 243 with PAS was confirmed by a PI displacement. All compounds showed antioxidant activity in the DPPH assay, with the best antioxidant being 247 (IC 50 = 31.7 µM), and proved to be BACE-1 inhibitors, with the most active being 241. The ability of hybrids to cross the BBB was proven in PAMPA-BBB assay. Additionally, biodistribution studies in C57BL6 mice revealed an ability of 243 and 249 to accumulate in the brain.
Finally, the therapeutic efficacy of 243 and 249 was investigated in young (5 month) and old (10 month) APP/PS1 mice. Mice treated with 249 enhanced learning and memory in old APP/PS1 mice in all the performed tests, while neither 249 nor 243 were effective in young transgenic mice. A decrease in the Aβ 42 /Aβ 40 ratio in the brains of mice treated with 249 was revealed. In addition, 249 significantly increased the strength of synaptic transmission, and reduced hippocampal levels of the oxidative stress marker 4-HNE and the neuroinflammation (astrogliosis) marker GFAP.

Tacrine-Ferulic Acid Hybrids
FA is a well-known antioxidant with multiple biological actions [145]. Due to its multiple activity and ease of chemical modifications, the design of FA-THA hybrids is of interest ( Figure 10) [146].
In 2008, Fang et al. reported THA-FA hybrids 250-254 [59]. All compounds inhibited ChEs, with the most potent hybrids being 252 and 253 toward both ChEs, suggesting the optimal length of linker in 6-7 atoms. Kinetic study of AChE inhibition by 253 showed reversible and noncompetitive inhibition. All hybrids showed moderate to good antioxidant activity confirmed by ORAC-fluorescein assay and hybrid 253 showed 1.5 trolox equivalents.
In 2010, an investigation of the in vivo anti-AD effectiveness of 254 was reported [60]. Unfortunately, no beneficial effect on scopolamine-induced cognition impairment was detected.
In 2012, Chen et al. designed THA-FA-NO donor trihybrids 257-276 [30]. NO-do nating hybrids showed better or comparable inhibition activity compared to parent 250-254 and a decrease in antioxidant activity. The in vitro reactivity of 257-276 as NO-dona tors was confirmed using the Griess reaction [147], in which 262 and 273 showed the height levels of nitrite. Additionally, ex vivo organ bath tests (coronary arteries from rats vascular relaxation assay for 251, 257, 262, 273, and 256 revealed a high activity for al hybrids. Hybrid 262 showed a comparable EC50 with positive control isosorbide dinitrate (ISDN). Hybrids 251, 262, and 256 were active in improving memory impairment in sco polamine-induced mice in a transfer latency time (TLT) test. Importantly, 262 possessed better performance than the non-nitrate hybrid 256. Finally, the levels of ASAT and ALT in serum of drug-treated mice were determined; 251 and 262 possessed higher safety than THA, and 262 showed the lowest hepatotoxicity.  [30]. NOdonating hybrids showed better or comparable inhibition activity compared to parent 250-254 and a decrease in antioxidant activity. The in vitro reactivity of 257-276 as NOdonators was confirmed using the Griess reaction [147], in which 262 and 273 showed the height levels of nitrite. Additionally, ex vivo organ bath tests (coronary arteries from rats) vascular relaxation assay for 251, 257, 262, 273, and 256 revealed a high activity for all hybrids. Hybrid 262 showed a comparable EC 50 with positive control isosorbide dinitrate (ISDN). Hybrids 251, 262, and 256 were active in improving memory impairment in scopolamine-induced mice in a transfer latency time (TLT) test. Importantly, 262 possessed better performance than the non-nitrate hybrid 256. Finally, the levels of ASAT and ALT in serum of drug-treated mice were determined; 251 and 262 possessed higher safety than THA, and 262 showed the lowest hepatotoxicity.
Fu et al. designed THA-FA hybrids with piperazine linker [62]. All hybrids presented inhibitory activity for both ChEs and selectivity for AChE. The best AChE inhibitors were 279 with IC 50 52.7 nM and 280 with IC 50 61.7 nM. Low antiaggregating properties of hybrids were revealed, and the Cu 2+ -chelating properties of 280 were confirmed by UV-Vis spectroscopy. Finally, the protective effects of 280 against Aβ-induced neurotoxicity were shown on Neuro-2A cells.
In 2018, Zhu et al. reported THA-FA hybrids 282-294 with different substituents in the benzene ring [63]. Hybrid 288 was the most potent AChE inhibitor with IC 50 37.02 nM. The presence of electron-withdrawing substituents contributed to the inhibition of BuChE; hybrid 292 with CF 3 substituent showed IC 50 52 nM. Molecular docking showed binding of 288 with both the CAS and PAS of AChE. Further, 285, 288, and 291 displayed inhibition on the aggregation of Aβ. When hybrids 285 and 288 were studied for in vivo behavioral analysis in scopolamine-induced cognition-impairment, treatment with 288 led to a remarkable improvement of memory in the scopolamine-induced cognitive impairment in the Morris water maze test. Finally, ALT and ASAT levels were measured after the treatment of animals with 285 and 288, and the hybrids proved to be safe, which was also confirmed by morphologic results.

Tacrine Hybrids with NO-Donating Molecules
Nitric oxide (NO) is a key signaling molecule involved in the regulation of many physiological processes [148]. NO plays roles in regulating synaptic plasticity, neurosecretion, and the sleep-wake cycle, and is considered as a molecule for the treatment of AD, which can cause therapeutic effects by increasing blood supply and regulating cerebral circulation [149][150][151][152][153][154]. In recent years, NO-donating and NO mimetic strategies in AD treatment proved to be effective, and several THA-based hybrids with NO-donating properties were reported ( Figure 11).

Tacrine Hybrids with NO-Donating Molecules
Nitric oxide (NO) is a key signaling molecule involved in the regulation of many physiological processes [148]. NO plays roles in regulating synaptic plasticity, neurosecretion, and the sleep-wake cycle, and is considered as a molecule for the treatment of AD, which can cause therapeutic effects by increasing blood supply and regulating cerebral circulation [149][150][151][152][153][154]. In recent years, NO-donating and NO mimetic strategies in AD treatment proved to be effective, and several THA-based hybrids with NO-donating properties were reported ( Figure 11).
In 2008, Fang et al. designed and synthesized a series of THA hybrids with NO-donating nitrato-and diazeniumdiolate moieties [64]. With the exception of 308 (226.0 nM), all compounds inhibited AChE with IC50 from 5.2 to 93 nM. Inhibition of BuChE was also similar to THA, with IC50 values from 5.2 to 41.0 nM. Hybrid 308 showеd selectivity for the inhibition of BuChE over AChE, with IC50 7 and 226 nM.
When the vasorelaxation effects of hybrids were accessed via a test with PGF2R-precontracted porcine pulmonary artery, 295, 303, and 308 showed moderate effect. When ASAT, lactate dehydogenase (LDH), albumin levels in serum, and concentration of protein in liver tissue after injection of 303 were measured, the hybrid did not show any hepatotoxicity. Figure 11. THA hybrids with NO-donating moieties 295-308 and model compound 309 [64], hybrids 310-316, and model 317 [65].
In 2008, nitrate-THA hybrids 310-317 with shorter and longer diamine side chains were reported [65]. All hybrids retain the ChE inhibitory effect of THA, with the most interesting being 310 (IC50 9.1 nM) and 314 (IC50 7.7 nM). In in vivo tests on the scopolamine-induced cognition impairment animal model, hybrids 295, 303, and 310 showed improving recognition activity whereas the analogue 317 did not. This result indicates that the nitrate group of 303 may not only contribute to the vessel relaxant activity, but also is Figure 11. THA hybrids with NO-donating moieties 295-308 and model compound 309 [64], hybrids 310-316, and model 317 [65].
In 2008, Fang et al. designed and synthesized a series of THA hybrids with NOdonating nitrato-and diazeniumdiolate moieties [64]. With the exception of 308 (226.0 nM), all compounds inhibited AChE with IC 50 from 5.2 to 93 nM. Inhibition of BuChE was also similar to THA, with IC 50 values from 5.2 to 41.0 nM. Hybrid 308 showed selectivity for the inhibition of BuChE over AChE, with IC 50 7 and 226 nM.
When the vasorelaxation effects of hybrids were accessed via a test with PGF2Rprecontracted porcine pulmonary artery, 295, 303, and 308 showed moderate effect. When ASAT, lactate dehydogenase (LDH), albumin levels in serum, and concentration of protein in liver tissue after injection of 303 were measured, the hybrid did not show any hepatotoxicity.
In 2008, nitrate-THA hybrids 310-317 with shorter and longer diamine side chains were reported [65]. All hybrids retain the ChE inhibitory effect of THA, with the most interesting being 310 (IC 50 9.1 nM) and 314 (IC 50 7.7 nM). In in vivo tests on the scopolamineinduced cognition impairment animal model, hybrids 295, 303, and 310 showed improving recognition activity whereas the analogue 317 did not. This result indicates that the nitrate group of 303 may not only contribute to the vessel relaxant activity, but also is essential for the ChE inhibitory effect. Finally, 295 and 303 did not show obvious signs of hepatotoxicity.

Tacrine-Phenothiazine Hybrids
Phenothiazine is a first-generation heterocyclic anti-psychotic medication that can also prevent tau filament formation [155]. In 2014, Hui et al. reported THA-phenothiazine heterodimers 318-320, which were designed based on molecular docking simulation [66] ( Figure 12). Hybrid 318 was the most potent AChE inhibitor with IC 50 89 nM. Hybrid 318 proved the ability to reduce P-Tau accumulation in N2a cells, and the ability of 318 to bind Aβ fibrils was confirmed using surface plasmon resonance (SPR).
, 24, x FOR PEER REVIEW 15 of 54 essential for the ChE inhibitory effect. Finally, 295 and 303 did not show obvious signs of hepatotoxicity.

Tacrine-Phenothiazine Hybrids
Phenothiazine is a first-generation heterocyclic anti-psychotic medication that can also prevent tau filament formation [155]. In 2014, Hui et al. reported THA-phenothiazine heterodimers 318-320, which were designed based on molecular docking simulation [66] ( Figure 12). Hybrid 318 was the most potent AChE inhibitor with IC50 89 nM. Hybrid 318 proved the ability to reduce P-Tau accumulation in N2a cells, and the ability of 318 to bind Aβ fibrils was confirmed using surface plasmon resonance (SPR).
In 2021, Gorecki et al. reported THA-phenothiazine heterodimers 321-356 [67]. All hybrids were potent hChEs inhibitors. 6-Cl-THA-based derivatives were more potent on hAChE (IC50 8-1500 nM) than THA analogues. The most selective compounds were 332 with IC50 8/190 nM and 321 with IC50 2040/15 nM. All compounds showed a toxicity on HePG2 cells in the micromolar range. Hybrids 330, 332, 336, and 344 proved their potential ability to cross the BBB. 6-Cl-THA-based hybrids showed the ability to inhibit τ (306−336) aggregation; the chain length was found to influence the inhibitory potency, with optimal 2-3 methylene units. Length of the linker also proved to be crucial in self-induced Aβ aggregation, with hybrids 332 and 335 showing the best inhibitory potency. Finally, in vivo safety studies revealed a good tolerance of hybrid 332.

Tacrine-Benzotiazole/Benzofuran Derivatives
Hybrids with Aβ-affinic benzofuran/benzotiazole moieties were repeatedly designed as dual action drugs capable of both Aβ binding and cholinesterase inhibition ( Figure 13). Both benzotiazole and benzofuran are well-known scaffolds for Aβ binding; thus, 11 C-Pittsburgh Compound-B (PiB) is a non-invasive tool for amyloid imaging in humans [156,157] Pioneer THA-benzofuran/benzotiazole conjugates were reported by Huang et al.  In 2021, Gorecki et al. reported THA-phenothiazine heterodimers 321-356 [67]. All hybrids were potent hChEs inhibitors. 6-Cl-THA-based derivatives were more potent on hAChE (IC 50 8-1500 nM) than THA analogues. The most selective compounds were 332 with IC 50 8/190 nM and 321 with IC 50 2040/15 nM. All compounds showed a toxicity on HePG2 cells in the micromolar range. Hybrids 330, 332, 336, and 344 proved their potential ability to cross the BBB. 6-Cl-THA-based hybrids showed the ability to inhibit τ (306-336) aggregation; the chain length was found to influence the inhibitory potency, with optimal 2-3 methylene units. Length of the linker also proved to be crucial in self-induced Aβ aggregation, with hybrids 332 and 335 showing the best inhibitory potency. Finally, in vivo safety studies revealed a good tolerance of hybrid 332.
Pioneer THA-benzofuran/benzotiazole conjugates were reported by Huang et al. [68]. Hybrids 357-364 inhibited ChEs with IC 50 values in the micromolar range. Hybrid 359 exhibited the most potent inhibition of AChE with IC 50 0.017 µM. Kinetics study of AChE inhibition showed 359 to bind both the CAS and PAS of AChE. The ability of hybrids to inhibit Aβ aggregation was assessed by the ThT assay, in which 359 demonstrated similar Aβ aggregation inhibitory activity with curcumin.
In 2013, Keri et al. also designed THA-benzotiazole hybrids 365-369 [69]. All hybrids displayed high inhibitory activities the against AChE enzyme, and with IC 50 values in the micromolar range 365 was chosen as the lead compound with IC 50 0.34 µM. All the compounds showed some ability to inhibit the Aβ 42 self-aggregation, which was confirmed by ThT assay.
In 2016, Zha et al. reported hybrids 370-395 based on THA, as well as its analogues with different side cycle size, with benzofuran scaffolds [70]. The inhibitory activities against hAChE ranged from 7.49 µM to 0.86 nM. The most potent hybrid, 386, showed a subnanomolar inhibitory potency, 493 times more potent than THA. The most selective hBuChE inhibitors were hybrids with hexamethyl chain 388-391. Hybrid 386 was also the only derivative slightly selective for hAChE (2.5-fold). hBuChE inhibitory activity was associated with the presence of a 7-methoxy substituent on the benzofuran nucleus. The highest hBuChE inhibition was achieved with hybrids 384 and 390 (0.49 nM and 0.48 nM). Kinetic study of hAChE inhibition by 386 revealed mixed-type inhibition. Further, the ability of 386 to inhibit AChE-induced Aβ fibril formation was confirmed by a ThT assay. An inhibition of Aβ self-aggregation and an inhibitory potency against hBACE-1 were confirmed. Finally, treatment of scopolamine-induced ICR mice with 386 led to considerable amelioration in cognition impairment. In ASAT and ALT levels measurements in serum after treatment with similar doses of 386, THA and bis-THA were measured and revealed low hepatotoxicity of 386.
In 2019, THA-benzothiazole hybrids 396-401 were reported by Rajeshwari et al. [71]. The docking study revealed favorable interactions of hybrids with THA moiety binding CAS. All hybrids inhibited AChE (IC 50 0.06-0.27 µM), but 397 exhibited the best inhibitory activity (IC 50 0.06 µM). In addition, all hybrids inhibited Aβ self-aggregation. A neuroprotective effect of hybrids was confirmed on SH-SY5Y cells treated with Aβ peptide or ascorbate/iron. Hybrids 397, 398, and 401 prevented Aβ-induced cell toxicity; 396, 398, 399, and 401 also showed the ability to inhibit Aβ-self-aggregation. However, the log BB assessment showed that hybrids are not drug candidates for oral administration.
In 2021, Nepovimova et al. reported THA-benzotiazole hybrids 414-438 [73]. All hybrids were potent hAChE inhibitors with IC 50 values in the micromolar to nanomolar range. Hybrids based on 7-MEOTA (414-420) displayed the poorest inhibition of hAChE; THA-based hybrids (421-427) showed moderate results, whereas hybrids based on 6-Cl-THA (428-434) were the best hAChE inhibitors. Based on a set of test results, hybrid 436 was chosen as lead, and its interactions with AChE were simulated by molecular docking. 6-Cl-THA moiety was found to occupy the PAS of hAChES, in contrast to its previously reported CAS binding [139]. The antiaggregating potential of 414-434 was confirmed using ThT assay. Additionally, the inhibition effects of 416 and 429 were confirmed using steady-state fluorescence and microscopy techniques. In addition, 436 showed the lowest hepatotoxicity, which was confirmed by the MTT test. The BBB penetration ability of hybrids was confirmed by a PAMPA-BBB test. Finally, an insignificant therapeutic effect of 436 was observed in scopolamine-treated mice.

Tacrine Hybrids with NSAIDS
Inflammation is an important therapeutic target, and is one of the important factors in clinical symptoms of AD [158]. Thus, nonsteroidal anti-inflammatory drugs (NSAIDs) are of interest in AD therapy [159]. NSAIDs were reported to reduce inflammatory markers and reverse spatial memory deficits in APPsw transgenic mice or improve memory and learning, and decrease stress-related behaviors in FAD5X/Ppara-null mice [160][161][162].
In  Similar hybrids 444-455 fortified with NO-donating ability were designed [75]. All hybrids showed comparable or better BuChE inhibitory activity (IC50s 3.9 -13.9 nM) than parent hybrids 439 -441. The best results were obtained for 447 and 455, with IC50 4309.5 and 1456.4 nM against AChE. Kinetic study revealed a mixed-type inhibition of 447. All hybrids showed promising levels of nitrite generated in Griess reactions. Vasorelaxation activity of 447 and 455 was confirmed in an ex vivo organ bath (coronary arteries from rats).
The same scientific group reported hybrids 456-463 [76]. All hybrids (ex. 463) exhibited similar or higher inhibitory activities compared with THA. The most potent hybrids Behavior studies in vivo were performed using a scopolamine-induced impairment in passive avoidance test. An improving memory impairment in the group treated parent with 442 (hybrid without NO-donating group), and the group treated with 456 was observed compared to the scopolamine group. Hybrid 456 showed no difference in comparison with THA and hybrid 442 (p > 0.05). ASAT and ALT levels were determined after mice were treated with THA, hybrids 442, and 456 at equimolar doses. Hybrids 442 and  [75], 456-463 [76], THA-indometacine hybrids 464-471 [77], ROS-responsive ibuprofen-THA hybrids 472-475 [78].
Similar hybrids 444-455 fortified with NO-donating ability were designed [75]. All hybrids showed comparable or better BuChE inhibitory activity (IC 50 s 3.9-13.9 nM) than parent hybrids 439-441. The best results were obtained for 447 and 455, with IC 50 4309.5 and 1456.4 nM against AChE. Kinetic study revealed a mixed-type inhibition of 447. All hybrids showed promising levels of nitrite generated in Griess reactions. Vasorelaxation activity of 447 and 455 was confirmed in an ex vivo organ bath (coronary arteries from rats).
The same scientific group reported hybrids 456-463 [76]. All hybrids (ex. 463) exhibited similar or higher inhibitory activities compared with THA. The most potent hybrids were 456 and 460 with IC 50  Behavior studies in vivo were performed using a scopolamine-induced impairment in passive avoidance test. An improving memory impairment in the group treated parent with 442 (hybrid without NO-donating group), and the group treated with 456 was observed compared to the scopolamine group. Hybrid 456 showed no difference in comparison with THA and hybrid 442 (p > 0.05). ASAT and ALT levels were determined after mice were treated with THA, hybrids 442, and 456 at equimolar doses. Hybrids 442 and 456 displayed higher safety than THA, and NO-donating hybrid 456 showed the lowest hepatotoxicity.
In 2021, Zawada et al. reported THA-indometacine hybrids 464-471 [77]. The IC 50 values for hybrids range from 10 to 260 nM. The most active compound against AChE was 471 (IC 50  Hybrids 472-475 showed moderate or no obvious AChE inhibitory activity in the absence of H 2 O 2 . An ability of 475 to inhibit proinflammatory cytokines TNF-α and IL-1β in endotoxin lipopolysaccharide (LPS)-treated microglial cells (BV-2) was revealed, as well as the regulating of apoptosis-related proteins. In addition, 475 showed neglected hepatotoxicity in HepG2 cells. Finally, the therapeutic effect of 475 and the improving spatial memory of Aβ-induced AD model rats were confirmed.
In 2021, Liu et al. designed ROS-responsive ibupro Low neurotoxicity of hybrids was proven on SH-SY5Y ce 475 against H2O2, and H2O2-scavenging capability were grade into counterparts in the presence of H2O2 was confi Hybrids 472-475 showed moderate or no obvious absence of H2O2. An ability of 475 to inhibit proinflamma in endotoxin lipopolysaccharide (LPS)-treated microglia well as the regulating of apoptosis-related proteins. In a hepatotoxicity in HepG2 cells. Finally, the therapeutic e spatial memory of Aβ-induced AD model rats were confi

Tacrine-Hupyridone Hybrids
Pyridones have been utilized as privileged scaffolds hupyridone hybrids were first described in 1999 [164,165 study of therapeutic efficacy of several THA dimers, inclu [166]. In addition, in 2021 Mak et al. summarized multifun hupyridone [167]. Herein, we will provide the most poten described by Carlier et al. [164] (Figure 15).  476 possessed a nanomolar AChE inhibition (IC 50 8.8 nM). Since both THA and huperzine A can increase the expression of brain-derived neurotrophic factor (BDNF) in the brain, 476 was also suggested to elevate BDNF expression concurrently [168,169].
In 2018, Chen et al. reported 476 to prevent the surgery-induced decrease in BDNF in the hippocampus of aged mice [79]. Hybrid 476 might act on BDNF to enhance cognitive performance. Additionally, 476 proved to increase the expression of pAkt and pERK, and ChAT-positive area in the hippocampal regions of surgery-treated mice proved to effectively attenuate scopolamine-induced cognitive impairments in vivo and be less toxic than THA [80].
Recently, Xuan et al. reported that 476 produces cognitive-enhancing effects in APP/PS1 and Aβ oligomers-treated mice [81]. Neuroprotective effects of 476 were proved, including the inhibition of Aβ aggregation, the activation of the BDNF/TrkB pathway, the alleviation of neuroinflammation, and the decrease in AChE activity.

Tacrine-Donepezil Hybrids
Donepezil is a specific and reversible inhibitor of AChE, and is an FDA-approved drug for the symptomatic treatment of AD [170]. THA-donepezil hybrids were designed by different scientific groups. The first donepezil-THA hybrid was reported in 2004 by Shao et al. [82].
Camps et al. reported [83] donepezil-THA hybrids in order to obtain more effective AChE inhibitors than previously reported by Shao  All hybrids were hAChE inhibitors, exhibiting IC50 values in the subnanomolar range. Hybrids 477-484 were more potent AChE inhibitors than were 485 (IC50 6.0 nM) and 486 (IC50 25 nM). Hybrid 480 was the most effective inhibitor (IC50 90 pM). Hybrids 477, 478, 481, and 482 were more potent BuChE inhibitors than was 485 (IC50 76 nM), though none of them was as potent as 486 (IC50 0.6 nM). Hybrids 479 and 480 proved their ability to bind with PAS via displacement of ThT. In addition, six out of the eight hybrids exhibited an Aβ antiaggregating activity.

Tacrine-Huprine Hybrids
Huprine is a potent AChE inhibitor based on THA scaffold [174].   Most of the hybrids displayed well-balanced potencies in the low nanomolar range when tested in vitro on the two recombinant human enzymes, hsEH and hAChE. All hybrids retained the hsEH inhibitory activity of TPPU (IC 50 3.7 nM) with IC 50 s in the subnanomolar to low nanomolar range, with hybrids 487 and (−)-490 displaying an even higher potency. Regarding hAChE inhibition, 487 and 489 retained the potency of 6-Cl-THA, and 488 was five-fold more potent. (−)-(7S,11S)-490 proved to be 850-fold more potent than its enantiomer, in line with the eudismic ratio of huprine Y. Hybrid 489 was chosen as the lead, with IC 50 12.9 nM against hAChE and IC 50 179 nM against hBuChE. Molecular dynamics simulations revealed 489 to interact with both sites of AChE. Hybrid 489 was the most stable compound in human microsomes. In vivo investigation in senescenceaccelerated mouse-prone 8 (SAMP8) revealed a significant amelioration in short-term and long-term working memory after oral administration of 489 (2 mg kg/day).

Tacrine-Bifendate Hybrids
In 2018, Cen et al. reported hybrids of THA with Bifendate (503-507), which is used for the treatment of chronic viral hepatitis B in China, and which was also reported to protect the liver mitochondria in mice from THA-induced injury [86,175,176] (Figure 19).

Tacrine-Bifendate Hybrids
In 2018, Cen et al. reported hybrids of THA with Bifendate (503-507), which for the treatment of chronic viral hepatitis B in China, and which was also rep protect the liver mitochondria in mice from THA-induced injury [86,175,176] (Fig   Figure 19. THA -Bifendate hybrids 503-507 [86]. Hybrids 503-507 showed potent inhibitory activities at the nanomolar concen and good selectivity for BuChE. Hybrid 506 was the most potent AChE inhibi 27.32 nM). Hybrid 504 was the most potent inhibitor of BuChE (IC50 4.02 nM). In a 506 showed a high inhibition of hAChE. Hybrids 503-507 prevented the self-med aggregation, and the antiaggregating potential of 506 was confirmed by TEM stu Figure 19. THA -Bifendate hybrids 503-507 [86].
Hybrids 503-507 showed potent inhibitory activities at the nanomolar concentrations and good selectivity for BuChE. Hybrid 506 was the most potent AChE inhibitor (IC 50 27.32 nM). Hybrid 504 was the most potent inhibitor of BuChE (IC 50 4.02 nM). In addition, 506 showed a high inhibition of hAChE. Hybrids 503-507 prevented the self-mediated Aβ aggregation, and the antiaggregating potential of 506 was confirmed by TEM study. Low hepatotoxicity of 506 was confirmed on HepG2 and HL-7702 cells. No increases in ALT and ASAT levels were observed after the administration of 506 in mice, but amelioration of the cognition functions in the scopolamine treated ICR mice was proven.

Tacrine hybrids with HDAC Inhibitors
Histone deacetylases (HDACs) are generally considered as therapeutic targets in the treatment of AD. The roles of histone deacetylases HDACs on cognitive impairments have been demonstrated in studies of AD animal models [177]. Furthermore, different types of HDACs may have distinct roles in the cognitive changes of AD. In

Tacrine Hybrids with Thio Derivatives
Modulation of synaptic plasticity, especially the long-term potentiation (LTP), has been proposed as a potential therapeutic strategy for improving cognitive function of AD patients [179]. Compounds with mercapto group, such as dithiothreitol (DTT), glutathione (GSH), and N-acetyl cysteine (NAC), can facilitate the induction of LTP in normal rats and even reverse the LTP impairment in aged rats [180]. Inspired by this, several THA hybrids with thio derivatives were reported ( Figure 21). All hybrids inhibited ChEs, with improved inhibition on AChE compared to THA. Hybrids 517 and 535 were the most potent inhibitors of AChE (IC 50 0.12 and 0.26 nM). The inhibitory potency on HDACs of hybrids 508-535 was determined against HeLa nuclear extract. Hybrids 511, 517-520, 523-524, 528, and 535 showed superior or comparable inhibitory potency when compared with reference compounds SAHA or PXD101; hybrids 517, 520, and 524 were the most potent, with IC 50 0.23, 0.32, and 0.28 nM. An antioxidant activity of all hybrids (except 520) was confirmed using ABTS assay. Hybrids 508-535 also exhibited an inhibition of Aβ self-aggregation. An ability of hybrids to chelate copper ions was confirmed. Kinetic study of AChE inhibition by 517 revealed mixed-type inhibition. Additionally, hybrids 512, 517, 519, and 524 were predicted as a BBB penetrant.

Tacrine Hybrids with Thio Derivatives
Modulation of synaptic plasticity, especially the long-term potentiation (LTP), has been proposed as a potential therapeutic strategy for improving cognitive function of AD patients [179]. Compounds with mercapto group, such as dithiothreitol (DTT), glutathione (GSH), and N-acetyl cysteine (NAC), can facilitate the induction of LTP in normal rats and even reverse the LTP impairment in aged rats [180]. Inspired by this, several THA hybrids with thio derivatives were reported ( Figure 21). been proposed as a potential therapeutic strategy for improving cognitive func patients [179]. Compounds with mercapto group, such as dithiothreitol (DTT) one (GSH), and N-acetyl cysteine (NAC), can facilitate the induction of LTP in n and even reverse the LTP impairment in aged rats [180]. Inspired by this, sev hybrids with thio derivatives were reported ( Figure 21).  [88]. Hybrids generally retained the ChE inhibitory effect, and 540 disp most potent inhibitory activity against AchE with pIC50 7.37 ± 0.02. Mercaptot rivatives were more potent inhibitors of BuchE than AchE, similar to that of TH for 540. Effects of hybrids on high-frequency stimulation (HF)-induced LTP i  [88]. Hybrids generally retained the ChE inhibitory effect, and 540 displayed the most potent inhibitory activity against AchE with pIC 50 7.37 ± 0.02. Mercaptotacrine derivatives were more potent inhibitors of BuchE than AchE, similar to that of THA, except for 540. Effects of hybrids on high-frequency stimulation (HF)-induced LTP in the CA1 region of Sprague−Dawley rat hippocampal slices were evaluated and an increase in the magnitude of LTP during the incubation of hippocampal slices with 537 and 541 was detected. Additionally, an enhanced hippocampal LTP after intracerebroventricular (icv) injection of 541 was detected in vivo. A neuroprotective action of 536, 538, and 540 against H 2 O 2 -induced oxidative stress was proven on human neuroblastoma cell line SH-SY5Y. All hybrids showed neuroprotective effects in a concentration-dependent manner, whereas hybrids 537, 539, and 541 presented a U-shaped dose-protection dependency. Finally, AST and ALT activities in serum samples were measured after administration of the drugs in vivo; hybrids 536 and 537 showed little hepatotoxicity.
Levels of H 2 S and activity of its synthesized enzyme cystathione β-synthase (CBS) are severely reduced in the brains of AD patients [181,182]. Treatment with H 2 S or a H 2 S donor improves cognitive function in AD patients and rat models [183].
In 2019, Cheng et al. reported THA-based hybrid 554 with H 2 S-releasing moieties (ACS81) [90]. Hybrid 554 improved cognitive and locomotor activity in AD mice, while also reducing inflammation and increasing synaptic plasticity in the hippocampus. Furthermore, hepatotoxicity studies confirmed that 554 was much safer than THA. Treatment with 554 was able to inhibit the AChE levels in the serum and hippocampus of AlCl 3 -treated AD mice with comparable effects to THA. Additionally, 554 inhibited hippocampal inflammation, as evidenced by the decreased mRNA expression of proinflammatory cytokines (TNF-α, IL-6, and IL-1β). Hybrid 554 also increased hippocampal H 2 S levels, decreased inflammation, and improved synaptic plasticity in the hippocampus. Importantly, 554 did not show evidence of hepatotoxicity or liver inflammation as measured by hepatic transaminases and proinflammatory cytokines.

Tacrine Hybrids with Fluorescent Probes
PI is well-known to bind the AChE [184]. Inspired by PI structure, in 2009 Camps et al. designed 6-Cl-THA-based hybrids 555-564 with 5-phenylpyrano [3,2-c]quinoline [91] ( Figure 22). All hybrids showed nanomolar activity toward ChEs. The most potent hAChE inhibitor was 557. Hybrids 560-564 turned out to be two-to three-fold more potent toward the human enzyme, with no significant dependency on the length of the linker. Hybrids 560-564 expectedly proved to be more potent BuChE inhibitors that chloro-substituted 555-559, with IC 50 values in the nanomolar range, up to three-to four-fold more potent than 6-Cl-THA. Molecular modeling and kinetic studies confirmed the dual site binding to hAChE. Hybrids 560, 563, and 564 can be considered as moderate inhibitors of Aβ self-aggregation. In addition, 564 showed as a potent BACE-1 inhibitor. Finally, these hybrids are able to cross BBB according to PAMPA-BBB assay.  [1,6]naphthyridine 565-568 [92], THA-lophine hybrids 569-591 [93].
All the 6-Cl-THA-based hybrids turned out to be potent inhibitors of hAChE, with 565 being the most potent (IC50 6.27 pM). Additionally, 565-568 exhibited an inhibition of Aβ42 and tau aggregation. All hybrids were predicted to cross the BBB.
In 2013, Costa et al. reported THA-based hybrids 569-591 with 2,4,5-triphenyl-1Himidazole (lophine), which can be used as a fluorescent-labeling reagent and was reported as a ChE inhibitor [93,185]. Hybrids 569-591 were found to be potent inhibitors, with IC50 in the nanomolar range. The most active AChE inhibitor was 570 (IC50 5.87 nM) and the most active BuChE inhibitor was 581 (IC50 7.10 nM), which was inactive toward AChE.
The most potent inhibitor of AChE was 600 (IC50 45 nM). Hybrids 593 and 599 were also of high potency and excellent selectivity for AChE. A Ca 2+ influx induced by K + depolarization in SH-SY5Y cells was evaluated. Most of the hybrids demonstrated a significant Ca 2+ blockade, with the most potent being 598 with a blockade similar to that obtained for nimodipine. Finally, 593, 599, and 600 showed neuroprotective properties against Ca 2+ overload and H2O2-induced oxidative stress on SH-SY5Y cells.  [1,6]naphthyridine 565-568 [92], THA-lophine hybrids 569-591 [93].
All the 6-Cl-THA-based hybrids turned out to be potent inhibitors of hAChE, with 565 being the most potent (IC 50 6.27 pM). Additionally, 565-568 exhibited an inhibition of Aβ 42 and tau aggregation. All hybrids were predicted to cross the BBB.
In 2013, Costa et al. reported THA-based hybrids 569-591 with 2,4,5-triphenyl-1Himidazole (lophine), which can be used as a fluorescent-labeling reagent and was reported as a ChE inhibitor [93,185]. Hybrids 569-591 were found to be potent inhibitors, with IC 50 in the nanomolar range. The most active AChE inhibitor was 570 (IC 50 5.87 nM) and the most active BuChE inhibitor was 581 (IC 50 7.10 nM), which was inactive toward AChE.
The most potent inhibitor of AChE was 600 (IC 50 45 nM). Hybrids 593 and 599 were also of high potency and excellent selectivity for AChE. A Ca 2+ influx induced by K + depolarization in SH-SY5Y cells was evaluated. Most of the hybrids demonstrated a significant Ca 2+ blockade, with the most potent being 598 with a blockade similar to that obtained for nimodipine. Finally, 593, 599, and 600 showed neuroprotective properties against Ca 2+ overload and H 2 O 2 -induced oxidative stress on SH-SY5Y cells.
In 2009, an expanded series of THA-nimodipine hybrids (601-605) was reported [95]. Most of the tacripyrines were more potent inhibitors of AChE than THA. Hybrid 600 was again chosen as the lead compound (IC 50 45 nM). Molecular modeling results showed binding of (R)-600 with PAS. Hybrid 600 proved to be an inhibitor of AChE-induced Aβ 40 aggregation and Aβ 42 self-aggregation. Most hybrids promoted significant Ca 2+ blockade, with the most potent being 604, whose activity was similar with nimodipine. PAMPA-BBB assay showed that almost all tacripyrines could cross the BBB and reach their biological targets.
In 2011, the same scientific group provided the pharmacological analysis of both enantiomers of 600 [96]. Both enantiomers showed similar results in inhibiting cholinesterase activity, AChE-induced Aβ aggregation, and Aβ self-aggregation in vitro. (S)-600 afforded significant protection against Aβ 25-35 -induced toxicity when tested on SH-SY5Y cells.
In 2015, Xiu-Lian et al. reported a similar THA-based hybrid 606 [97]. Hybrid 606 in low concentrations proved its ability to reduce tau phosphorylation levels, which was confirmed on HEK293/tau cells. In addition, its ability to inhibit the generation and release of Aβ was confirmed on mouse neuroblastoma N2a/APP cells.
In 2018, hybrids of THA with dihydropyirimidine-thiones 607-618 were reported [98]. Most tacripyrimidines showed selectivity for hBuChE with IC 50 from 0.372 mM (616) to 154 mM (614). Additionally, most tacripyrimidines 607-618 inhibited hAChE with IC 50 from 3.05 mM (611) to 31.0 mM (615). The most selective and potent hAChEI was 617 (hAChE: IC 50 0.0373 mM). All tacripyrimidines except 616 significantly inhibited Ca 2+ influx induced by K-depolarization in SH-SY5Y cells. A hepatotoxicity study revealed most tacripyrimidines to be similarly or slightly less toxic than THA. Finally, 611 was chosen as a well-balanced inhibitor of ChEs and a calcium channel blocker, with no toxicity toward HepG2 cells up to 300 mM and excellent predicted oral absorption and BBB permeability.
In 2015, Sola et al. reported hybrids 619-622 based on a THA, 6-Cl-THA, or huprine Y with 2-(2-oxopyrrolidin-1-yl)butyramide moiety of levetiracetam, an antiepileptic drug [99] that was reported to improve memory performance in mouse models of AD [187] (Figure 24). tacripyrimidines to be similarly or slightly less toxic than THA. Finally as a well-balanced inhibitor of ChEs and a calcium channel blocker, wi ward HepG2 cells up to 300 mM and excellent predicted oral absorption ability.
In 2015, Sola et al. reported hybrids 619-622 based on a THA, 6-Cl-Y with 2-(2-oxopyrrolidin-1-yl)butyramide moiety of levetiracetam, an a [99] that was reported to improve memory performance in mouse mo ( Figure 24).  All hybrids were potent inhibitors of hAChE, with IC 50 in the low nanomolar range. Huprine is a stereoactive drug, with (7R,11R)-huprine derivatives being more potent hBuChE inhibitors than (7S,11S)-counterparts [188]. Expectedly, 621 and 622 showed different inhibition activity. However, no differences in inhibitory activity of (7S,11S)huprine Y hybrids were detected. Hybrids 621 and 622 exhibited a moderately potent Aβ 42 and tau antiaggregating activity. The inhibition of mouse brain AChE after i.p. administration of the levetiracetam-based hybrids was also confirmed. A significant reduction in the frequency of spontaneous convulsions in APP/PS1 mice treated with the levetiracetam-huprine hybrid 621 was revealed. APP/PS1 mice treated with hybrids 619 and 621 exhibited significantly increased recognition indices when compared to vehicletreated animals. In addition, immunohistochemical determination revealed a reduction of the Aβ burden in the cortex of APP/PS1 mice after chronic treatment with 621. Finally, chronic treatment with 621 led to a significant reduction of GFAP positive astrocytes around Aβ plaques and Iba1 positive microglial cells in APP/PS1 mice.

Tacrine Hybrids with Modulators of Serotonin Receptors
In 2016, Wigockowska et al. designed hybrids 623-628 as potential ChE inhibitors and 5-HT 6 antagonists. As a 5-HT 6 antagonist, 1-(phenylsulfonyl)-4-(piperazin-1-yl)-1H-indole was chosen (Figure 25) [100]. exhibited significantly increased recognition indices when compared to vehicle-trea animals. In addition, immunohistochemical determination revealed a reduction of the burden in the cortex of APP/PS1 mice after chronic treatment with 621. Finally, chr treatment with 621 led to a significant reduction of GFAP positive astrocytes around plaques and Iba1 positive microglial cells in APP/PS1 mice.

Tacrine Hybrids with Modulators of Serotonin Receptors
In 2016, Wigockowska et al. designed hybrids 623-628 as potential ChE inhibi and 5-HT6 antagonists. As a 5-HT6 antagonist, 1-(phenylsulfonyl)-4-(piperazin-1-yl)indole was chosen (Figure 25) [100]. All hybrids displayed high affinities for 5-HT6 receptor in the low nanomolar ra IC50 values for hybrids were in the range from 7.1 to 57.0 nM for AChE and from 8. 21.3 nM for BuChE. Hybrid 626 was chosen for its balanced activity as lead compo based on a set of test results. Hybrid 626 significantly diminished serotonin-induced cium mobilization, confirming its 5-HT6 antagonistic properties (Kb = 27.0 nM). A kin study revealed a non-competitive type of inhibition of AChE/BuChE. Hybrid 626 creased the rats' locomotion activity and reduced scopolamine-induced hyperlocomo in rats.
In 2018, an expanded library of similar hybrids 629-638 was synthesized [101]. affinity of hybrids for recombinant human 5-HT6 receptor was found to increase with linker length and reached a Ki value of 18 nM for 632, which was confirmed by a radio and binding assay. All hybrids were potent ChEs inhibitors, with the most active be 634 (IC50 50 nM against AChE) and 632 (IC50 14 nM against hAChE). A kinetic study vealed a non-competitive mode of action for 632. Further, the inhibitory effect on In 2018, an expanded library of similar hybrids 629-638 was synthesized [101]. The affinity of hybrids for recombinant human 5-HT 6 receptor was found to increase with the linker length and reached a Ki value of 18 nM for 632, which was confirmed by a radioligand binding assay. All hybrids were potent ChEs inhibitors, with the most active being 634 (IC 50 50 nM against AChE) and 632 (IC 50 14 nM against hAChE). A kinetic study revealed a non-competitive mode of action for 632. Further, the inhibitory effect on Aβ aggregation was determined by ThT assay, and the most active hybrids were 632, 633, 635-638. A PAMPA-BBB test showed a possible effective CNS permeability of hybrids. An in vitro metabolic stability study on human liver microsomes did not detect any hepatotoxic metabolites.
In 2017, Li et al. reported hybrids of THA with Vilazodone 639-668, an inhibitor of serotonin reuptake and partial agonist of 5-HT 1 A receptor [102,189] (Figure 26). aggregation was determined by ThT assay, and the most active hybrids were 632, 633, 635-638. A PAMPA-BBB test showed a possible effective CNS permeability of hybrids. An in vitro metabolic stability study on human liver microsomes did not detect any hepatotoxic metabolites. In 2017, Li et al. reported hybrids of THA with Vilazodone 639-668, an inhibitor of serotonin reuptake and partial agonist of 5-HT1A receptor [102,189] (Figure 26).
Activities of hybrids such as 5-HT1A agonists and 5-HT reuptake inhibitors were evaluated; hybrid 643 showed relatively balanced activities against the three targets. Low hepatotoxicity of 643 was confirmed on HepG2 cells. Low cardiotoxicity of 643 was confirmed by hERG activity inhibition. The antidepressive effect of 643 was confirmed by the Activities of hybrids such as 5-HT 1 A agonists and 5-HT reuptake inhibitors were evaluated; hybrid 643 showed relatively balanced activities against the three targets. Low hepatotoxicity of 643 was confirmed on HepG2 cells. Low cardiotoxicity of 643 was confirmed by hERG activity inhibition. The antidepressive effect of 643 was confirmed by the tail suspension test in vivo, and cognitive improvements were detected in scopolaminetreated mice.

Tacrine Hybrids with Modulator of Muscarinic Receptors
In 2007, Elsinghorst et al. reported THA-based hybrids 669-674 with gallamine, an archetypal muscarinic allosteric agent [103] (Figure 27). An increasing substitution of the gallamine-derived moiety tends to reduce the inhibitory potency. Hybrid 669 was found to be a potent inhibitor of AChE (IC 50 500 pM). The interaction of the building blocks and hybrids 669-674 with M2 receptors was measured in receptors whose acetylcholine binding site was blocked by the radioligand

Tacrine Hybrids with Cannabinoid CB1 Receptor Antagonists
Cannabinoid signaling systems are involved in a variety of physiological processes. The selective CB1 antagonist/inverse agonist drug Rimonabant is an FDA-approved drug to treat obesity and metabolic-related disorders [194]. In 2007, Wise et al. reported [195] a combination of rimonabant and donezepil, a CB1 antagonist and an AChE inhibitor, as an effective memory-enhanced therapy.

H]Nmethylscopolamine ([ 3 H]NMS). All hybrids induced an allosteric inhibition of [ 3 H]NMS
dissociation. The most potent log K Xdiss of 680 was more than three log units higher com-pared to xanomeline. In vivo studies in rats revealed the ability of 687 to significantly enhance scopolamine action.
In 2020, Maspero et al. reported THA hybrids 712-717 with xanomeline, a selective muscarinic acetylcholine receptor agonist and M1/M4 preferring muscarinic acetylcholine receptor activator [106,193]. Hybrids 712-717 were able to inhibit AChE; eight methylene units were optimal for the highest AChE inhibition. The most active inhibitor was 715 with pIC 50 9.55. However, hybrids 712-717 were unable to activate the M1 receptor subtype.

Tacrine Hybrids with Cannabinoid CB1 Receptor Antagonists
Cannabinoid signaling systems are involved in a variety of physiological processes. The selective CB1 antagonist/inverse agonist drug Rimonabant is an FDA-approved drug to treat obesity and metabolic-related disorders [194]. In 2007, Wise et al. reported [195] a combination of rimonabant and donezepil, a CB1 antagonist and an AChE inhibitor, as an effective memory-enhanced therapy.
In Hybrids exhibited AChE inhibiting activities and significant cannabinoid CB1 receptor antagonistic properties. The most effective AchE inhibitor was 720 (pIC50 6.5). Hybrids 718-721 showed significant CB1 receptor affinities and, in general, acted as CB1 receptor antagonists, while 720 showed significant CB1 receptor affinity with Ki = 48 nM.
When the effects of hybrids on the increase in intracellular calcium evoked by NMDA in neurons loaded with Fura-2 was evaluated [196], the most potent NMDA antagonists were 730 and 731. Unfortunately, low BBB permeation for 731 and 732 was predicted, whereas when substituted at the bridgehead amino group 729 and 730 were predicted to be able to cross the BBB. All hybrids exhibited good inhibitory activity toward ChEs. The most potent cholinesterase inhibitor was 725, with an IC 50 0.47 µM for hAChE and 0.11 µM for hBuChE.
When the effects of hybrids on the increase in intracellular calcium evoked by NMDA in neurons loaded with Fura-2 was evaluated [196], the most potent NMDA antagonists were 730 and 731. Unfortunately, low BBB permeation for 731 and 732 was predicted, whereas when substituted at the bridgehead amino group 729 and 730 were predicted to be able to cross the BBB. THA-tianeptine hybrids were potent inhibitors of cholinesterases, th AChE inhibitor was 736 (IC50 6.79 nM), and 737 was the most active and sele iting BuChE (IC50 3.59 nM). Molecular modeling studies showed that THA CAS, while tianeptine binds to PAS. Additionally, 737 and 739 were able to vitro basal secretion of S100B, a calcium-binding protein which is known to eral processes associated with AD [197]. THA-tianeptine hybrids were potent inhibitors of cholinesterases, the most active AChE inhibitor was 736 (IC 50 6.79 nM), and 737 was the most active and selective in inhibiting BuChE (IC 50 3.59 nM). Molecular modeling studies showed that THA moiety targets CAS, while tianeptine binds to PAS. Additionally, 737 and 739 were able to reduce the in vitro basal secretion of S100B, a calcium-binding protein which is known to regulate several processes associated with AD [197].

Tacrine Hybrids with MAO Inhibitors
MAO inhibitors (namely, two isoforms MAO-A and MAO-B) are considered as promising therapeutic agents for AD [198][199][200]. Ladostigil is a drug with cholinesterase and brain-selective monoamine oxidase inhibitory activities approved for phase IIb clinical trial [189].
In THA-tianeptine hybrids were potent inhibitors of cholinesterases, the most active AChE inhibitor was 736 (IC50 6.79 nM), and 737 was the most active and selective in inhibiting BuChE (IC50 3.59 nM). Molecular modeling studies showed that THA moiety targets CAS, while tianeptine binds to PAS. Additionally, 737 and 739 were able to reduce the in vitro basal secretion of S100B, a calcium-binding protein which is known to regulate several processes associated with AD [197].

Tacrine Hybrids with MAO Inhibitors
MAO inhibitors (namely, two isoforms MAO-A and MAO-B) are considered as promising therapeutic agents for AD [198][199][200]. Ladostigil is a drug with cholinesterase and brain-selective monoamine oxidase inhibitory activities approved for phase IIb clinical trial [189].
In Hybrids 748-751 were inhibitors of AChE, with the most active being 749 (IC50 36.1 nM). In addition, 755 exhibited the best IC50 toward BuChE in 2.03 nM. Hybrid 754 was chosen as lead due to balanced activity based on a set of test results. A kinetic study  [112]. Coumarin moiety was chosen due to its MAO inhibitory activity [202] and AChE inhibitory activity [203]. Hybrid 766 gave the highest AChE inhibitory activity with IC 50 17.70 nM. The substituents in coumarin moiety were found to worsen the inhibitory activity. Hybrids 774 (IC 50 31.88 nM for AChE) and 771 (IC 50 50.76 nM for BuChE) were the most potent inhibitors, with their inhibitory activity 1.8-and 1.3-fold less than those of their no substituted analog 766. All hybrids showed inhibition activity against hMAO-A and hMAO-B, with the most selective toward MAO-B being 773 (IC 50 0.24 mM). An inhibitory activity of 773 as a mixed-type competitive inhibitor was confirmed. Finally, 773 showed negligible toxicity on SH-SY5Y cells.

Tacrine Hybrids with Natural Products
In 2013, Xie et al. reported THA-coumarin hybrids 781-800, structural analogues of hybrids 761-780 ( Figure 32) [112,113]. Hybrids showed moderate activity toward both ChEs, with the best AChE inhibitor being 786 (IC 50 0.092 µM), and the most effective BuChE inhibitor being 790 (IC 50 0.099 µM). Kinetic study revealed a mixed-type inhibition for 786. An ability of hybrids to inhibit self-induced Aβ aggregation was confirmed using a ThT-test, with 786 as the most potent AChE inhibitor that also showed the highest inhibitory potency. The metal-chelating ability of hybrids was confirmed using UV-Vis spectrometry in the presence of Cu 2+ and Fe 2+ . Finally, a low toxicity of 786 was confirmed on SH-SY5Y cells.
lective toward MAO-B being 773 (IC50 0.24 mM). An inhibitory activity of 773 as a mixedtype competitive inhibitor was confirmed. Finally, 773 showed negligible toxicity on SH-SY5Y cells.

Tacrine Hybrids with Natural Products
In 2013, Xie et al. reported THA-coumarin hybrids 781-800, structural analogues of hybrids 761-780 ( Figure 32) [112,113]. Hybrids showed moderate activity toward both ChEs, with the best AChE inhibitor being 786 (IC50 0.092 μM), and the most effective BuChE inhibitor being 790 (IC50 0.099 μM). Kinetic study revealed a mixed-type inhibition for 786. An ability of hybrids to inhibit self-induced Aβ aggregation was confirmed using a ThT-test, with 786 as the most potent AChE inhibitor that also showed the highest inhibitory potency. The metal-chelating ability of hybrids was confirmed using UV-Vis spectrometry in the presence of Cu 2+ and Fe 2+ . Finally, a low toxicity of 786 was confirmed on SH-SY5Y cells.
In 2014, Thiratmatrakul et al. reported hybrids of THA with phytochemicals carbazoles 835-837 [117]. All hybrids showed potent ABTS radical scavenging capacities with IC 50 in the range of 8.34-11.24 µM, and selectivity against AChE over BuChE. Hybrid 835 displayed the most potent inhibitory activity and inhibition selectivity toward AChE, (IC 50 0.48 µM). A neuroprotective effect of hybrids against H 2 O 2 -induced oxidative stress was shown on NG108-15cells and 835 proved to be most potent in protecting cell damage. Additionally, neuroprotective effect of hybrids against Aβ peptide induced toxicity was shown on C6 astroglioma cells. Hybrid 835 was also the most potent in increasing cell viability. Behavioral studies indicated that 835 could improve scopolamine-induced cognitive deficits in mice.
In 2017, Jeřábek et al. reported THA-resveratrol hybrids 845-852 [119]. The most potent AChE inhibitor was 845 (IC 50 0.8 µM). Some antiaggregating properties of hybrids were revealed by ThT assay. Only 852 showed no neurotoxicity on primary rat cerebellar granule neurons (CGNs). Nitrite production in LPS-treated glial cells was evaluated, which was significantly reduced by treatment of cells with 852. Finally, an ability of 852 to modulate the switch from the M1 to M2 phenotype on glial cells was investigated; a decrease in iNOS and slightly attenuating MRC1 expression was detected. Unfortunately, hepatotoxicity of hybrids on HePG2 cells was shown.
In 2018, Lopes et al. reported THA hybrids with natural-based D-xylose, D-ribose and D-galactose 853-861 [120]. Hybrid 857 showed an IC 50 2.2 nM against AChE and of 4.93 nM against BuChE. Docking studies revealed that sugar moieties are stabilized in the PAS region through cation-π and CH/π interactions with Trp279.
Cell toxicity studies revealed the order of toxicity is as follows: THA derivatives < 7-MEOTA analogues < 6-Cl-THA derivatives. The maximum tolerated dose of (S)-873 was found to be 70 mg/kg, meaning (S)-873 is safer than THA. The therapeutic effect of (S)-873 in a scopolamine-induced cognitive deficit rat model confirmed the pro-cognitive potential of the hybrid.
In 2019, Cheng et al. reported THA-indole hybrids 883-889 [122]. A moderate inhibition activity was shown by all hybrids. Hybrid 887 with IC 50 0.173 µM against AChE was chosen as lead based on a set of test results. Kinetic study that revealed 887 is a mixed type ChE inhibitor. In addition, 887 exhibits a much stronger effect in modulating neural network activity compared to THA, indicating better antidementia and nootropic potentials.
In 2021, Rossi et al. reported THA-based hybrids conjugated with anacardic acid, cardanol, and cardols 890-902 [123]. All hybrids were effective AChE inhibitors, with the most active being 891 (IC 50 2.54 nM). As BuChE inhibitors, 890, 891, and 893 were the top-ranked with IC 50 0.0352 nM, 0.265 nM, and 0.177 nM. A crystal structure of 890 with hBuChE revealed that 890 accommodates the active site gorge of hBuChE. Toxicity studies revealed low hepato-and neurotoxicity in hybrids. Anti-inflammatory activity of 890 and 891 was shown on LPS-treated microglial BV-2 cells; a protective activity against neurotoxic insults was detected, as was the suppression of LPS-induced IL-1β, COX-2, and iNOS expression (TNF-α only for 891). The PAMPA-BBB test predicted that both hybrids have the potential to cross the BBB.
In 2014, Chen et al. reported a first example of photoswitchable THA-based hybrids [125]. Hybrids 918-921 showed reversible light-controlled behavior. The absorbances around 270 nm decreased upon UV-irradiation, whereas the absorbance band at 340 nm increased, and a new absorbance band maximum at 525 nm evolved. However, in all cases, the inhibitory activity against AChE was practically the same before and after irradiation of hybrids, except for the 921 (IC 50 4.3 nM for "open" molecule and 1.8 nM for "closed'). Computational docking studies suggest that 921 might bind to both CAS and the PAS of AChE in both opened and closed forms. Additionally, when AChE-induced Aβ aggregation under the action of both photoforms of 921 were investigated, ring-closed 921 showed lower inhibition than a ring-open form.
In 2019, Cheng et al. reported THA-indole hybrids 883-889 [122]. A moderate inhibition activity was shown by all hybrids. Hybrid 887 with IC50 0.173 μM against AChE was chosen as lead based on a set of test results. Kinetic study that revealed 887 is a mixed type ChE inhibitor. In addition, 887 exhibits a much stronger effect in modulating neural network activity compared to THA, indicating better antidementia and nootropic potentials.
In 2021, Rossi et al. reported THA-based hybrids conjugated with anacardic acid, cardanol, and cardols 890-902 [123]. All hybrids were effective AChE inhibitors, with the most active being 891 (IC50 2.54 nM). As BuChE inhibitors, 890, 891, and 893 were the topranked with IC50 0.0352 nM, 0.265 nM, and 0.177 nM. A crystal structure of 890 with hBuChE revealed that 890 accommodates the active site gorge of hBuChE. Toxicity studies revealed low hepato-and neurotoxicity in hybrids. Anti-inflammatory activity of 890 and 891 was shown on LPS-treated microglial BV-2 cells; a protective activity against neurotoxic insults was detected, as was the suppression of LPS-induced IL-1β, COX-2, and iNOS expression (TNF-α only for 891). The PAMPA-BBB test predicted that both hybrids have the potential to cross the BBB.
In 2015, Mao et al. reported THA-propargylamine hybrids 940-944 [127]. Hybrid 941 showed good inhibition activity for both ChEs (IC 50  In 2017, Najaf et al. reported THA-1,2,3-triazole hybrids 959-973 [129]. All hybrids showed moderate inhibition activity against ChEs, with the best inhibitor being 970 (IC 50 0.521 mM); 968 was the best BuChE inhibitor (IC 50 0.055 mM). Kinetic study confirmed mixed types of inhibition for both AChE and BuChE. In vivo evaluation of 970 confirmed memory improvement in scopolamine-induced impairment.  Br-and Cl-as substituents in THA unit were found to enhance AChE inhibition, fluorine-substituted pyridine groups were found to intensify to GSK-3β target, and alkylamine linkers with a linear chain of seven carbons were chosen as the most beneficial moiety. Hybrid 1035 was chosen as the compound with excellent dual AChE/GSK-3 inhibition (AChE: IC50 51.1 nM; GSK/3β: IC50 89.3 nM). Br-and Cl-as substituents in THA unit were found to enhance AChE inhibition, fluorine-substituted pyridine groups were found to intensify to GSK-3β target, and alkylamine linkers with a linear chain of seven carbons were chosen as the most beneficial moiety. Hybrid 1035 was chosen as the compound with excellent dual AChE/GSK-3 inhibition (AChE: IC 50  The docking studies for 1035 proved both CAS and PAS binding. Hybrid 1035 could fit the binding pockets of AChE and GSK-3β and exhibited good affinity with the interactions of several secondary bonds through the cooperation of the THA unit, alkylamine linker, and pyrimidone moiety, making it an excellent dual AChE/GSK-3β inhibitor. Additionally, 1035 was proven to regulate the tau protein pathway in SH-SY5Y-derived neurons, and to alleviate glyceraldehyde-induced cytotoxicity in DSH-SY5Y cells. A kinase selectivity profiling study showed that 1035 is a pan-GSK-3 inhibitor and possessed good kinase selectivity profiles. The capacity of 1035 to successfully permeate the BBB was confirmed by UPLC-MS/MS. Finally, treatment of scopolamine-induced ICR mice with 1035 led to significant amelioration of memory and spatial behavior.
In 2021, Ozten et al. reported carbamate hybrids of THA 1040-1052 [132] (Figure 38).  [128], THA-1,2,3-triazole hybrids 959-973 [129], Schiff base hybrids 974-989 [130]. Br-and Cl-as substituents in THA unit were found to enhance AChE inhibition, fluorine-substituted pyridine groups were found to intensify to GSK-3β target, and alkylamine linkers with a linear chain of seven carbons were chosen as the most beneficial moiety. Hybrid 1035 was chosen as the compound with excellent dual AChE/GSK-3 inhibition (AChE: IC50 51.1 nM; GSK/3β: IC50 89.3 nM). Hybrids were mostly more neurocytoxic than THA. Only 1064 showed a sig All hybrids inhibited both ChEs, but 1050 was chosen as the best inhibitor of AChE and BuChE (IC 50  The docking studies for 1035 proved both CAS and PAS binding. Hybrid 10 fit the binding pockets of AChE and GSK-3β and exhibited good affinity with the tions of several secondary bonds through the cooperation of the THA unit, alk linker, and pyrimidone moiety, making it an excellent dual AChE/GSK-3β inhib ditionally, 1035 was proven to regulate the tau protein pathway in SH-SY5Y-deriv rons, and to alleviate glyceraldehyde-induced cytotoxicity in DSH-SY5Y cells. A selectivity profiling study showed that 1035 is a pan-GSK-3 inhibitor and possess kinase selectivity profiles. The capacity of 1035 to successfully permeate the BBB w firmed by UPLC-MS/MS. Finally, treatment of scopolamine-induced ICR mice w led to significant amelioration of memory and spatial behavior.
In Hybrids were mostly more neurocytoxic than THA. Only 1064 showed a sig reduction of hepatotoxicity against HepG2 cells when compared with THA. In C Figure 39. THA hybrids with phosphorus moieties 1053-1066 [133].
Hybrids were mostly more neurocytoxic than THA. Only 1064 showed a significant reduction of hepatotoxicity against HepG2 cells when compared with THA. In ChEs test inhibition, 1053 and 1058 showed similar activity with THA. The most active hybrid was 1055 with IC 50 6.11 nM against AChE and 12.86 nM against BuChE. In addition, 1060 and 1053 were potent against BuChE, with IC 50 1.969 nM and 6.753 nM.
THA-melatonin hybrid 7, first described in 2006, is still one of the most potent hybrids described to date. At the same time, THA-melatonin hybrids 3 and 6 showed selectivity for one esterase, which also makes them promising scaffolds for the development of anti-AD drugs. The conjugation of additional lipoic or ferulic acid moieties reduces inhibition activity (24)(25)(26)(27)(28)(29)(30)(31), apparently due to steric hindrances arising in the resulting hybrid when binding to the target ChE [41].
THA-silibinin hybrid 101 demonstrated low hepatotoxicity in vivo, but was not superior to THA in improving cognitive abilities, presumably due to high steric hindrance [47]. Hybrid 165 showed both cognitive improvements and a decrease in hepatotoxicity in vivo, which clearly indicates the effectiveness of antioxidant strategy [54]. In addition, a reduction in Aβ plaque levels in the APP/PS1 mice certainly proves the effectiveness of simultaneous anti-amyloid and anti-AChE approaches in the treatment of AD.
An extremely interesting result was observed for hybrids of THA and FA. Thus, 254 with linker length n = 8 did not show cognitive improvement in vivo, while hydride 255 with linker length n = 6 showed significant cognitive improvements [51,52]. This once again emphasizes the importance of choosing the linker length both for the effectiveness of cholinesterase inhibition and for anti-AD therapy in vivo. It is also important to note that THA-FA hybrids 277-281 were linked through a conformationally rigid piperazine linker [62]. Comparison of the inhibition data of these hybrids with their analogues clearly indicates that the restriction of the conformational mobility of the linker negatively affects the inhibitory effect.
Also, the effectiveness of introducing NO-donors into THA hybrids for anti-AD therapeutic efficacy should be noted. Thus, a comparison of the in vivo efficacy of hybrids 262 (with the NO-donating group) and 256 (without the NO-donating group) clearly indicates that the vasorelaxant effect of NO-donors not only reduces the hepatotoxicity of hybrids, but also improves cognitive functions in vivo better than hybrids that do not possess a vasorelaxant effect [53]. Convincing evidence for the effectiveness of NOgroup donors was also concluded from the results of in vivo studies of hybrids 295, 310, and 317. Hybrid 317 (without the NO-donating group) did not show improvement in cognitive functions in tests in vivo, while 295 and 310 showed improvements in cognitive functions, apparently due to the vasorelaxant effect [64,65]. It is also worth noting the THA-flurbiprofen hybrids 439-443 which were then optimized by conjugation with NOdonor molecules to give 444-463 [74][75][76]. At the same time, 456 did not outperform THA in improving its cognitive functions; however, the reduced hepatoxicity of 456 is attributed by the authors to its ability to donate NO.
Not only the length of the linker is critical in drug design, but also its nature and donor properties. Thus, the amide linker between THA and benzofuran in 370-381 renders a negative effect on inhibitory activity compared to the amine linker in 382-387. Additionally, both aromatic moieties in a hybrid should not be sterically expanded to be able to pack into the enzyme binding site. Thus, 388-395 show selectivity for BuChE, apparently due to the steric impossibility of binding to AChE. Regarding the negative effect of an amide linker on inhibition activity, 781-800 exhibited moderate anti-AD activity, while in subsequent work, similar hybrids 761-780 were designed not with an amide, but with an amine linker [112,113]. Optimization of the molecular structure resulted in an increase in anticholinesterase activity, and in the ability of 761-780 to inhibit MAO.
The exceptionally successful design of hybrids with analogues of PI deserves special attention. The known possibility of the second moiety to bind to the AChE provides a successful design of the anti-AChE drug, in which the optimal length of the linker between the CAS and PAS binding fragment should be selected. Thus, 565 is one of the most potent inhibitors of hAChE reported to date, with an IC 50 6.27 pM [92]. It is interesting to note that the design of 565 is also a result of careful drug development, as originally synthesized 555-564 were not effective inhibitors [91]. However, the successful design made it possible to obtain one of the strongest inhibitors described to date. In vivo studies on the efficacy of 565 would be extremely interesting.
Of obvious interest are hybrids acting on the cholinergic system. Thus, 592-605, which are both cholinesterase inhibitors and calcium channel blockers, were developed and the leader compound (S)-600 was chosen, of which studies of the in vivo therapeutic efficacy would be very interesting [95,96]. In addition, similar hybrids 607-618, representing a weak structural analogue based on dihydropyirimidine-thiones, are also able to block calcium channels [98]. For 611, the study of anti-AD activity for (R) and (S) enantiomers would certainly be interesting.
Hybrids 669-674 and 675-690 are all cholinesterase inhibitors and muscarinic receptor agonists; 687 shows therapeutic efficacy in vivo [103,104]. Both the hybrid series 675-690 and 712-717 are THA-xanomeline hybrids [104,106]. At the same time, the first series are successful activators of M1 receptors, while the second series are not able to affect receptors, which once again confirms the importance of not only the choice of the second moiety, but also the design of the linker. Additionally, THA-BOCA hybrids 691-711 turned out to be antagonists of muscarinic receptors, which confirms the complexity of receptor-targeted drug design [105]. The design of hybrids of THA with a memantine residue is a classic "dual drug" idea, while 722-728 do not show significant anticholinesterase activity [108]. At the same time, the change in the linker and the conjugation of the free amino group to the adamantane core significantly increased the activity of the resulting hybrids, and 729-732 turned out to be promising cholinesterase inhibitors and NMDA receptor antagonists [109]. Unfortunately, these hybrids are presumably unable to cross the BBB, possibly due to steric hindrance. Hybrids 759 and 773 are promising anticholinergic agents and MAO-B inhibitors, and their in vivo efficacies as anti-AD agents are also of interest [111,112].
Of particular note is the therapeutic efficacy of hybrids based on Huprine Y. Thus, Huprine Y-capsaicin hybrid 249 significantly enhanced learning and memory in old APP/PS1 mice [58]. Huprine-THA heterodimers (±)-494 and (±)-500 inhibited mouse brain AChE activity [85]. In addition, Huprine Y-based hybrid (SSS)-621 with 2-(2oxopyrrolidin-1-yl)butyramide moiety of levetiracetam significantly increased recognition indices in vivo, reduced Aβ burden in the cortex, and reduced GFAP positive astrocytes around Aβ plaques [99]. Additionally, both enantiomers of hybrid (+)-and (−)-831 are able to lower the levels of hippocampal Aβ and increase the levels of APP both in initial and advanced stages of AD in vivo [116]. It should also be noted that the right and left hybrids (+) and (−)-831 showed a tremendous difference in the inhibition of hAChE, and both showed anti-AD efficacy in vivo.
THA-carbazoles hydride 835 is not an effective cholinesterase inhibitor compared to the other hybrids described above, but demonstrated an ability to cause cognitive improvements in vivo [117]. As hybrids of THA with natural products, resveratrol 845-852 showed high hepatotoxicity [119]. THA hybrids with sugar moieties 853-861 and THA-anacardic acid hybrids 890-891 showed high efficacy along with low toxicity, and their further studies are promising [120,123]. An interesting result is the activity of the (S)-873 enantiomer toward AChE [121].
The design of photoactive THA hybrids 918-921 is an interesting concept, but its practical applicability is questionable [125]. The spectacular work on the development of quinone-THA hybrids by Nepovimova et al., in which 926 is a selective AChE inhibitor, exhibits multiple activities and inhibits AChE in vivo [126]. Hybrids 940-944 with propargylamine as the second residue are striking in their simplicity and efficiency; further research on hybrids would be interesting [132]. 7-MEOTA-based hybrids with p-anisidine 945-958 are not as active, presumably due to the low activity of the parental 7-MEOTA [128]. Despite the fact that simple THA-triazole hybrids 959-973 are not active compared to other previously described hybrids, 970 showed therapeutic efficacy in vivo [129]. Hybrids of THA with isatin, especially 984, also showed good activity [130]. An extremely interesting THA hybrid is pyrimidone 1035, which exhibits AChE/GSK-3 inhibition activity and also exhibits therapeutic efficacy in vivo [131].
Comparison of structurally similar hybrids 128-148 and 192-210 with antioxidants Trolox and Cp-6 is also of interest [51,56]. Hybrids 128-148 with an amide linker showed moderate inhibitory activity, while hybrids 192-201 showed picomolar activity. The importance of the linker choice is confirmed again, which is confirmed by the order of activities in the second series: amines 207-210 > amides 192-195 > reverse amides 204-206 > Obenzylated amides 196-199. That is, the amine linker between THA and the second residue without an acceptor amide bond plays one of the key roles in the activity of the hybrid.

Conclusions
The design of THA hybrids is a promising alternative to anti-AD drugs used in clinical practice due to their high efficiency and multiple biological actions. In addition, synthesis of THA hybrids results in reducing hepatotoxicity. A wide possibility opens up with the conjugation of THA with a second biological active moiety, which provides an additional biological activity and several mechanisms of anti-AD therapy in one drug. Thus, in this review, many hybrids are able not only to inhibit cholinesterase, but also to affect amyloid aggregation and reduce the number of Aβ plaques in the brain. Additionally, many of the described hybrids showed a high selectivity for one of the cholinesterase, are able to donate NO and cause a vasorelaxant effect, inhibit BACE-1, cause an antidepressant and a neuroprotective effect, and chelate metal cations, thereby counteracting the formation of toxic amyloid aggregates. The impressive results of in vivo studies also confirm that THA derivatives are able to improve cognitive functions by not only cholinesterase inhibition, but also by reducing the number of amyloid plaques in AD brains, while not causing noticeable hepatotoxicity.