Exploiting the Chalcone Scaffold to Develop Multifunctional Agents for Alzheimer’s Disease

Alzheimer’s disease still represents an untreated multifaceted pathology, and drugs able to stop or reverse its progression are urgently needed. In this paper, a series of naturally inspired chalcone-based derivatives was designed as structural simplification of our previously reported benzofuran lead compound, aiming at targeting both acetyl (AChE)- and butyryl (BuChE) cholinesterases that, despite having been studied for years, still deserve considerable attention. In addition, the new compounds could also modulate different pathways involved in disease progression, due to the peculiar trans-α,β-unsaturated ketone in the chalcone framework. All molecules presented in this study were evaluated for cholinesterase inhibition on the human enzymes and for antioxidant and neuroprotective activities on a SH-SY5Y cell line. The results proved that almost all the new compounds were low micromolar inhibitors, showing different selectivity depending on the appended substituent; some of them were also effective antioxidant and neuroprotective agents. In particular, compound 4, endowed with dual AChE/BuChE inhibitory activity, was able to decrease ROS formation and increase GSH levels, resulting in enhanced antioxidant endogenous defense. Moreover, this compound also proved to counteract the neurotoxicity elicited by Aβ1–42 oligomers, showing a promising neuroprotective potential.


Introduction
Alzheimer's disease (AD) is the most common cause of dementia and is becoming a global health concern. Indeed, the continuous rise in the aging population and an exponential increase in AD cases over the next few decades in Europe are expected to result in immense pressure on the social and health-care systems of the region [1,2]. Therefore, there is an urgent need for developing curative or disease-modifying therapies to offset the upcoming AD epidemic. However, despite millions of dollars of investment in drug discovery and clinical trials, no single molecule has yet been approved for its treatment since the advent of cholinesterase inhibitors (ChEIs) and memantine, an N-methyl-d-aspartate (NMDA) receptor antagonist [3].
In AD, atrophy of cells' nuclei in the key areas of the brain, such as the basal forebrain, leads to a noticeable decrease in cholinergic function; cholinergic transmission in the brain is an essential core in a variety of important bioactive compounds [11], belong to the flavonoid family, the largest class of secondary metabolites in plants, where they serve as a defense mechanism to counteract ROS in order to survive and prevent molecular damage. These natural products can be defined as "privileged structures" and they can be properly exploited to develop effective strategies in different fields of drug discovery [12]. Indeed, chalcones possess a peculiar trans-α,β-unsaturated ketone, a functional group perceived as a potential Michael acceptor, that allows the molecule to interact with cysteine residues of different biological targets to obtain the Michael adduct [12][13][14].

Chemistry
According to Scheme 1, compound 1 was synthesized starting from 4-hydroxybenzonitrile, which was alkylated, in the presence of K 2 CO 3 , with 1,7-dibromoheptane obtaining 19, that was then subjected to nucleophilic substitution reaction by N-methylbenzylamine to afford 20. Afterwards, the cyano group was reduced using the Ni/Raney alloy in formic acid [15] to give the aldehyde 21. Benzylic bromination with N-bromosuccinimide (NBS) of 4-methylacetophenone afforded 22, which was subsequently reacted with diethylamine to give 23. Chalcone 1 was obtained via Claisen-Schmidt condensation reaction of 23 with the previously prepared aldehyde 21.
Molecules 2018, 23,1902 3 of 22 The simpler and more flexible framework of the new chalcone-based derivatives allowed the obtaining of less constrained molecules, which could better interact with the narrow gorge of the ChE enzymes. In this new series, the A ring was decorated with a methoxy group or an additional tertiary amino function, charged at physiological pH and linked through spacers of different lengths, to evaluate its possible binding to the peripheral anionic site (PAS) located at the top of the acetylcholinesterase (AChE) gorge. To better address this point, the amino side chain was introduced in meta or para positions on the benzene ring, as in compounds 1-15. Finally, to assess the role of the chalcone α,β-double bond and to evaluate the impact of a further increase in flexibility, the saturated analogs 16-18 were also designed and synthesized. The new compounds were reported in Tables 1 and 2.

Chemistry
According to Scheme 1, compound 1 was synthesized starting from 4-hydroxybenzonitrile, which was alkylated, in the presence of K2CO3, with 1,7-dibromoheptane obtaining 19, that was then subjected to nucleophilic substitution reaction by N-methylbenzylamine to afford 20. Afterwards, the cyano group was reduced using the Ni/Raney alloy in formic acid [15] to give the aldehyde 21.
The synthesis of compounds 2-18 were accomplished as shown in Scheme 2. The 3-or 4hydroxyacetophenone was alkylated, in the presence of K2CO3, with the selected bromochloroalkane to afford the chloroalkyloxy derivatives 24-29, which were then subjected to nucleophilic substitution Scheme 1. Reagents and conditions: (i) Br(CH 2 ) 7 Br, K 2 CO 3 , acetone, reflux; (ii) N-methylbenzylamine, Et 3 N, toluene, reflux; (iii) Ni/Raney, HCOOH, reflux; (iv) NBS, (PhCOO) 2 , CCl 4 , reflux; (v) Et 2 NH, toluene, reflux; (vi) KOH, EtOH, r.t.          In order to explain this unexpected result, docking studies were performed (see below). Finally, the α,β unsaturation of derivatives 3-5 was selectively hydrogenated, aiming at evaluating its role in the orientation of the molecules. This modification, leading to the more flexible compounds 16-18 (Table 2), allowed maintaining a micromolar inhibitory potency, even if lower with respect to 3-5. Regarding hBuChE, the introduction of a methoxy group on the A ring, both in para and in meta positions, as in compounds 2 and 12, led to a consistent increase in potency with respect to AChE (3.42 µM vs. 98.5 µM for 2 and 8.58 µM vs. 105 µM for 12, Table 1). A similar trend was observed for the benzofuran-based lead compound (3.42 and 8.58 µM, respectively, and 38.1 µM [9]).
Differently from what was noticed for hAChE inhibition, the substitution of the methoxy with a diethylamino group, as in compounds 1, 4 and 5, allowed maintaining of the activity in the micromolar range, although with different selectivity depending on the position of the chain. Indeed, the subset bearing the diethylamino group in para position showed a greater affinity for hAChE, while the meta substituted one proved to be more potent on hBuChE. In this small subset of metaderivatives, compound 13, with 2-methylene units, was the most active and the most potent of the whole series on this enzyme.
As expected, the larger size of the active site gorge in BuChE, due to the presence of the smaller valine and leucine instead of the aromatic phenylanine residues [17], enabled a better fit of molecules with bulky substituents, with the exception of compounds 10 and 11, which proved to be inactive on both ChEs.
To complete the picture, the hydrogenated compounds 16-18 (Table 2) were three/four-fold less potent on BuChE with respect to AChE.
In a healthy brain, AChE is the major player in the breakdown of the neurotransmitter acetylcholine (ACh). Conversely, in an AD affected brain, AChE levels progressively decrease, while BuChE increases and becomes prevailing [18]. As a result, the regulation of ACh central levels in AD patients likely involves both enzymes, which thus represents validated therapeutic targets to counteract the cholinergic deficit [19,20]. Moreover, recent studies indicate that BuChE can be associated with Aβ plaques, due to its accumulation in these structures, and inhibition of this enzyme could be responsible for a reduced Aβ deposition in subcortical regions of the brain [21]. In this overall framework, compound 4 emerges as the most promising, being an effective low micromolar inhibitor of both enzymes.

Docking Studies
To explain the binding mode and the affinities of newly synthesized compounds to target enzymes AChE and BuChE, docking studies were performed and interactions of compounds 4 and 6 with AChE and BuChE were evaluated. Three-dimensional (3D) and two-dimensional (2D) The synthesis of compounds 2-18 were accomplished as shown in Scheme 2. The 3-or 4-hydroxyacetophenone was alkylated, in the presence of K 2 CO 3 , with the selected bromochloroalkane to afford the chloroalkyloxy derivatives 24-29, which were then subjected to nucleophilic substitution by diethylamine in refluxing toluene to give compounds 30-35. The chalcones 3-5 and 13-15 were obtained via Claisen-Schmidt condensation of 30-35 with the previously described aldehyde 21 (see Scheme 1). With the same procedure (Scheme 2, right side), compounds 2 and 12 were obtained starting from the selected methoxyacetophenone and 21. Finally, compounds 3-5 were hydrogenated with Pd/CaCO 3 to obtain the final derivatives 16-18.
The derivatives bearing amines other than diethylamine were prepared according to Scheme 3: 24 was first subjected to Claisen-Schmidt condensation reaction with 21 to give chalcone 36 that, via a parallel synthesis procedure, was then subjected to nucleophilic substitution reaction by the selected amine (6-11).

Inhibition of Cholinesterase Activity
The inhibitory potencies of compounds 1-18 toward human recombinant AChE (hAChE) and BuChE from human serum (hBuChE) were determined using Ellman's assay [16] and the IC 50 values were reported in Tables 1 and 2. Regarding hAChE, the introduction of a methoxy group on the chalcone A ring, independently of its para or meta position, as in compounds 2 and 12, respectively, led to a consistent drop in potency with respect to the benzofuran-based lead compound (about 100 µM vs 40.7 µM [9], Table 1). The substitution of the methoxy with a methylendiethylamino group, in compound 1, induced a 35-fold increase in inhibitory activity (2.81 µM), leading us to speculate on a positive contribution of the diethylamino group. An additional structural modification, namely the lengthening of the side chain from 2 to 4 methylene units, combined with the introduction of an oxygen atom, allowed an increase in activity of one order of magnitude, as in the para-alkoxymethylendiethylamino derivatives 3-5, showing potency in the sub-micromolar range. In this context, the meta-substitution seemed to negatively affect activity, compounds 13-15 being less effective as AChE inhibitors than the corresponding para-substituted compounds. In this small metasubset, compound 15, with 4-methylene units, was the most active.
One of the aims of this paper was the design of structurally simpler and more flexible derivatives with respect to our previously reported benzofuran-based lead compound, in order to exploit this increased adaptability in a better interaction with AChE. The results indicate that the introduction of a para-diethylaminoethoxy group (compound 3) led to a 10-fold increase in potency. Moreover, a positive contribution of the oxygen atom in the side chain can be recognized, maybe due to its H-bond acceptor feature, since compound 1, bearing a simple diethylaminomethyl group, proved to be less active. The derivatives bearing amines other than diethylamine were prepared according to Scheme 3: 24 was first subjected to Claisen-Schmidt condensation reaction with 21 to give chalcone 36 that, via a parallel synthesis procedure, was then subjected to nucleophilic substitution reaction by the selected amine (6-11). Scheme 2. Reagents and conditions: (i) Cl(CH 2 ) n Br, K 2 CO 3 , acetone, reflux; (ii) selected amine, toluene, reflux; (iii) 21, KOH, EtOH, r.t.; (iv) H 2 , Pd/CaCO 3 , THF, r.t.
Taking this into account, for a deeper evaluation of the contribution of the terminal amino group in the interaction with PAS, the ethoxy spacer was maintained and diethylamine was replaced with different bulkier amines (Table 1).
In order to explain this unexpected result, docking studies were performed (see below).
Finally, the α,β unsaturation of derivatives 3-5 was selectively hydrogenated, aiming at evaluating its role in the orientation of the molecules. This modification, leading to the more flexible compounds 16-18 (Table 2), allowed maintaining a micromolar inhibitory potency, even if lower with respect to 3-5.
Regarding hBuChE, the introduction of a methoxy group on the A ring, both in para and in meta positions, as in compounds 2 and 12, led to a consistent increase in potency with respect to AChE (3.42 µM vs. 98.5 µM for 2 and 8.58 µM vs. 105 µM for 12, Table 1). A similar trend was observed for the benzofuran-based lead compound (3.42 and 8.58 µM, respectively, and 38.1 µM [9]).

Inhibition of Cholinesterase Activity
The inhibitory potencies of compounds 1-18 toward human recombinant AChE (hAChE) and BuChE from human serum (hBuChE) were determined using Ellman's assay [16] and the IC50 values were reported in Tables 1 and 2. Regarding hAChE, the introduction of a methoxy group on the chalcone A ring, independently of its para or meta position, as in compounds 2 and 12, respectively, led to a consistent drop in potency with respect to the benzofuran-based lead compound (about 100 µM vs 40.7 µM [9], Table 1). The substitution of the methoxy with a methylendiethylamino group, in compound 1, induced a 35-fold increase in inhibitory activity (2.81 µM), leading us to speculate on a positive contribution of the diethylamino group. An additional structural modification, namely the lengthening of the side chain from 2 to 4 methylene units, combined with the introduction of an oxygen atom, allowed an increase in activity of one order of magnitude, as in the para-alkoxymethylendiethylamino derivatives 3-5, showing potency in the sub-micromolar range. In this context, the meta-substitution seemed to negatively affect activity, compounds 13-15 being less effective as AChE inhibitors than the corresponding para-substituted compounds. In this small meta-subset, compound 15, with 4methylene units, was the most active.
One of the aims of this paper was the design of structurally simpler and more flexible derivatives with respect to our previously reported benzofuran-based lead compound, in order to exploit this increased adaptability in a better interaction with AChE. The results indicate that the introduction of a para-diethylaminoethoxy group (compound 3) led to a 10-fold increase in potency. Moreover, a positive contribution of the oxygen atom in the side chain can be recognized, maybe due to its Hbond acceptor feature, since compound 1, bearing a simple diethylaminomethyl group, proved to be less active.
Taking this into account, for a deeper evaluation of the contribution of the terminal amino group in the interaction with PAS, the ethoxy spacer was maintained and diethylamine was replaced with different bulkier amines (Table 1). Differently from what was noticed for hAChE inhibition, the substitution of the methoxy with a diethylamino group, as in compounds 1, 4 and 5, allowed maintaining of the activity in the micromolar range, although with different selectivity depending on the position of the chain. Indeed, the subset bearing the diethylamino group in para position showed a greater affinity for hAChE, while the meta substituted one proved to be more potent on hBuChE. In this small subset of meta-derivatives, compound 13, with 2-methylene units, was the most active and the most potent of the whole series on this enzyme.
As expected, the larger size of the active site gorge in BuChE, due to the presence of the smaller valine and leucine instead of the aromatic phenylanine residues [17], enabled a better fit of molecules with bulky substituents, with the exception of compounds 10 and 11, which proved to be inactive on both ChEs.
To complete the picture, the hydrogenated compounds 16-18 (Table 2) were three/four-fold less potent on BuChE with respect to AChE.
In a healthy brain, AChE is the major player in the breakdown of the neurotransmitter acetylcholine (ACh). Conversely, in an AD affected brain, AChE levels progressively decrease, while BuChE increases and becomes prevailing [18]. As a result, the regulation of ACh central levels in AD patients likely involves both enzymes, which thus represent validated therapeutic targets to counteract the cholinergic deficit [19,20]. Moreover, recent studies indicate that BuChE can be associated with Aβ plaques, due to its accumulation in these structures, and inhibition of this enzyme could be responsible for a reduced Aβ deposition in subcortical regions of the brain [21]. In this overall framework, compound 4 emerges as the most promising, being an effective low micromolar inhibitor of both enzymes.

Docking Studies
To explain the binding mode and the affinities of newly synthesized compounds to target enzymes AChE and BuChE, docking studies were performed and interactions of compounds 4 and 6 with AChE and BuChE were evaluated. Three-dimensional (3D) and two-dimensional (2D) representations of the most energetically profitable poses of these compounds docked in the active site of hAChE are presented in Figure 2.
Molecules 2018, 23, 1902 8 of 22 representations of the most energetically profitable poses of these compounds docked in the active site of hAChE are presented in Figure 2. The most energetically favorable binding mode of compound 4 revealed that the Nbenzylmethylamine moiety binds to the CAS while the diethylamino group is located in the pocket forming PAS and the linker occupied the center of both cavities (Figure 2a). The AChE-4 complex is stabilized by π-cation and π-π stacking interactions (Figure 2b).
Analysis of the optimal binding mode for compound 6 places the ligand within the active site of The most energetically favorable binding mode of compound 4 revealed that the N-benzylmethylamine moiety binds to the CAS while the diethylamino group is located in the pocket forming PAS and the linker occupied the center of both cavities (Figure 2a). The AChE-4 complex is stabilized by π-cation and π-π stacking interactions (Figure 2b).
Analysis of the optimal binding mode for compound 6 places the ligand within the active site of AChE. The simulations showed a clear preference to accommodate the N-benzylmethylamine moiety within the CAS while the morpholine moiety is turned away from the PAS. The key factors to stabilize the enzyme-ligand complex were found to be π-cation, π-π T-shaped, π-π stacking and hydrogen bonding interactions (Figure 2c). Figure 3 illustrates the docked pose of compound 4 superimposed with the docked pose of compound 6, pointing out to the fact that these two molecules do not share a similar binding mode. Both compounds 4 and 6 could interact with the catalytic active site with Trp86 and Glu202 residues; however, compound 4 also interacted in the peripheral anionic site. The higher binding of compound 4 within AChE binding pocket can be attributed to the binding orientation and geometry permitted by this ligand, spanning both cavities of AChE (CAS and PAS).
Molecules 2018, 23, 1902 9 of 22 within the CAS while the morpholine moiety is turned away from the PAS. The key factors to stabilize the enzyme-ligand complex were found to be π-cation, π-π T-shaped, π-π stacking and hydrogen bonding interactions (Figure 2c). Figure 3 illustrates the docked pose of compound 4 superimposed with the docked pose of compound 6, pointing out to the fact that these two molecules do not share a similar binding mode. Both compounds 4 and 6 could interact with the catalytic active site with Trp86 and Glu202 residues; however, compound 4 also interacted in the peripheral anionic site. The higher binding of compound 4 within AChE binding pocket can be attributed to the binding orientation and geometry permitted by this ligand, spanning both cavities of AChE (CAS and PAS). Compounds 4 and 6 were modeled into the structure of hBuChE (PDB: 4BDS, Figure 4). Both ligands fit into the active site of hBuChE mainly through π-π and hydrogen bonds interactions, which are found to be essential for binding. Thus, compound 4 interacts inside the hBuChE binding cavity in the same region occupied by compound 6. These compounds find interactions in the middle of the active-site gorge and the N-benzylmethylamine moiety is pointed toward the catalytic triad residues, His438, Ser198 and Glu325.
As shown in Figure 5, it was found that the N-benzylmethylamine moiety of both ligands occupied the catalytic site of hBuChE through π-π stacking interactions with Trp231 and Phe329. Furthermore, the binding in compound 6 is also supported by the formation of a hydrogen bond between a morpholine oxygen atom of the ligand and Asn289. In addition, the other ammonium groups are located at the beginning of the gorge. These ligands bind to hAChE with extended conformations, whereas they bind to hBuChE with folded conformations  Compounds 4 and 6 were modeled into the structure of hBuChE (PDB: 4BDS, Figure 4). Both ligands fit into the active site of hBuChE mainly through π-π and hydrogen bonds interactions, which are found to be essential for binding. Thus, compound 4 interacts inside the hBuChE binding cavity in the same region occupied by compound 6. These compounds find interactions in the middle of the active-site gorge and the N-benzylmethylamine moiety is pointed toward the catalytic triad residues, His438, Ser198 and Glu325.
Molecules 2018, 23, 1902 9 of 22 within the CAS while the morpholine moiety is turned away from the PAS. The key factors to stabilize the enzyme-ligand complex were found to be π-cation, π-π T-shaped, π-π stacking and hydrogen bonding interactions (Figure 2c). Figure 3 illustrates the docked pose of compound 4 superimposed with the docked pose of compound 6, pointing out to the fact that these two molecules do not share a similar binding mode. Both compounds 4 and 6 could interact with the catalytic active site with Trp86 and Glu202 residues; however, compound 4 also interacted in the peripheral anionic site. The higher binding of compound 4 within AChE binding pocket can be attributed to the binding orientation and geometry permitted by this ligand, spanning both cavities of AChE (CAS and PAS). Compounds 4 and 6 were modeled into the structure of hBuChE (PDB: 4BDS, Figure 4). Both ligands fit into the active site of hBuChE mainly through π-π and hydrogen bonds interactions, which are found to be essential for binding. Thus, compound 4 interacts inside the hBuChE binding cavity in the same region occupied by compound 6. These compounds find interactions in the middle of the active-site gorge and the N-benzylmethylamine moiety is pointed toward the catalytic triad residues, His438, Ser198 and Glu325.
As shown in Figure 5, it was found that the N-benzylmethylamine moiety of both ligands occupied the catalytic site of hBuChE through π-π stacking interactions with Trp231 and Phe329. Furthermore, the binding in compound 6 is also supported by the formation of a hydrogen bond between a morpholine oxygen atom of the ligand and Asn289. In addition, the other ammonium groups are located at the beginning of the gorge. These ligands bind to hAChE with extended conformations, whereas they bind to hBuChE with folded conformations  As shown in Figure 5, it was found that the N-benzylmethylamine moiety of both ligands occupied the catalytic site of hBuChE through π-π stacking interactions with Trp231 and Phe329. Furthermore, the binding in compound 6 is also supported by the formation of a hydrogen bond between a morpholine oxygen atom of the ligand and Asn289. In addition, the other ammonium groups are located at the beginning of the gorge. These ligands bind to hAChE with extended conformations, whereas they bind to hBuChE with folded conformations

Antioxidant Activity
To deeply investigate the biological profile of the new derivatives, a small subset of compounds was evaluated to establish their antioxidant potential, which could be associated to the chalcone scaffold. To this aim, derivatives 2, 4 and 17 were selected, due to their different structural features: 2 possesses a simple methoxy group, 4 is a dual AChE/BuChE inhibitor bearing an amino terminal group and 17 is devoid of the chalcone peculiar double bond.
To establish the range of concentrations not associated with neurotoxicity, SH-SY5Y cells viability was evaluated after a 24 h treatment with compounds 2, 4 and 17 at different concentrations (2.5-80 µM) using the MTT assay. The obtained results highlighted that concentrations up to 2.5 µM did not affect neuronal viability (data not shown). Therefore, to perform the subsequent antioxidant and neuroprotective assays, a concentration of 1.25 µM was selected for all the studied compounds.
To determine the antioxidant activity, the intracellular ROS formation induced by t-BuOOH was evaluated after a 24 h treatment with compounds 2, 4 and 17 (1.25 µM) using the fluorescent probe H2DCF-DA. Data showed that the studied compounds prevented ROS formation in the order of strength 4 > 17 > 2, with inhibition percentages of 27%, 23% and 16%, respectively ( Figure 6).
In parallel, glutathione (GSH) levels in SH-SY5Y cells were evaluated after a 24 h treatment with compounds 2, 4 and 17 (1.25 µM) using the fluorescent probe monochlorobimane (MCB). This evaluation was based on experimental evidence indicating that oxidative stress and aging reduced GSH levels. Data showed that compounds 4 and 17 significantly increased GSH levels, while compound 2 decreased GSH basal levels (Figure 7).

Antioxidant Activity
To deeply investigate the biological profile of the new derivatives, a small subset of compounds was evaluated to establish their antioxidant potential, which could be associated to the chalcone scaffold. To this aim, derivatives 2, 4 and 17 were selected, due to their different structural features: 2 possesses a simple methoxy group, 4 is a dual AChE/BuChE inhibitor bearing an amino terminal group and 17 is devoid of the chalcone peculiar double bond.
To establish the range of concentrations not associated with neurotoxicity, SH-SY5Y cells viability was evaluated after a 24 h treatment with compounds 2, 4 and 17 at different concentrations (2.5-80 µM) using the MTT assay. The obtained results highlighted that concentrations up to 2.5 µM did not affect neuronal viability (data not shown). Therefore, to perform the subsequent antioxidant and neuroprotective assays, a concentration of 1.25 µM was selected for all the studied compounds.
To determine the antioxidant activity, the intracellular ROS formation induced by t-BuOOH was evaluated after a 24 h treatment with compounds 2, 4 and 17 (1.25 µM) using the fluorescent probe H 2 DCF-DA. Data showed that the studied compounds prevented ROS formation in the order of strength 4 > 17 > 2, with inhibition percentages of 27%, 23% and 16%, respectively ( Figure 6).
In parallel, glutathione (GSH) levels in SH-SY5Y cells were evaluated after a 24 h treatment with compounds 2, 4 and 17 (1.25 µM) using the fluorescent probe monochlorobimane (MCB). This evaluation was based on experimental evidence indicating that oxidative stress and aging reduced GSH levels. Data showed that compounds 4 and 17 significantly increased GSH levels, while compound 2 decreased GSH basal levels (Figure 7).

Neuroprotective Activity
It is well documented that soluble Aβ oligomers are neurotoxic species, able to trigger cognitive deficits also in the absence of plaques. Thus, they can be considered critical factors in the pathogenesis of AD by causing synaptic dysfunction and neuronal death [22].

Neuroprotective Activity
It is well documented that soluble Aβ oligomers are neurotoxic species, able to trigger cognitive deficits also in the absence of plaques. Thus, they can be considered critical factors in the pathogenesis of AD by causing synaptic dysfunction and neuronal death [22].

Neuroprotective Activity
It is well documented that soluble Aβ oligomers are neurotoxic species, able to trigger cognitive deficits also in the absence of plaques. Thus, they can be considered critical factors in the pathogenesis of AD by causing synaptic dysfunction and neuronal death [22].
The neuroprotective activity toward Aβ 1-42 oligomers (OAβ 1-42 ) (10 µM) induced toxicity in SH-SY5Y cells was evaluated after 4 h treatment with compounds 2, 4, 17 (1.25 µM) using the MTT formazan exocytosis assay. As shown in Figure 8, compound 4 partially counteracted the neurotoxic effects induced by OAβ 1-42 increasing SH-SY5Y cells viability, while compound 2 reinforced the neurotoxic effects induced by OAβ  . No neuroprotective effect was observed for compound 17. Taken together, the data for antioxidant and neuroprotective activities point at compound 4 as the most promising one, being endowed with a better profile than compounds 2 and 17, suggesting that both the amino terminal group and the chalcone peculiar double bond were crucial structural features for inducing these effects. Undoubtedly, the α,β-unsaturated carbonyl moiety in compound 4, acting as a Michael acceptor, could interfere with Keap1-Nrf2 binding, causing the subsequent activation of Nrf2 signaling pathway [23]. Indeed, several studies postulate electrophylic compounds as a possible Cys-based modification of Keap1 allowing its dissociation from Nrf2 and leading to the transcription of cytoprotective genes [24,25]. In this respect, a fine-tuning of the chalcone electrophilicity, due to the substituents introduced, can be considered an important feature to minimize the risk of off-target effects. On the other hand, compound 17, only devoid of the α,β double bond with respect to 4, could still increase the GSH levels, probably acting with a different mechanism.
In summary, the increased flexibility of these newly synthesized chalcone-based derivatives led to an improved cholinesterase inhibitory activity with respect to the benzofuran lead compound, possibly due to a better fit into the catalytic ChEs gorges. On the other hand, the presence of the distinctive α,β-unsaturated carbonyl moiety also allowed introducing an appreciable antioxidant and neuroprotective potential. In this series, compound 4, endowed with a dual AChE/BuChE low micromolar inhibitory activity and a relevant antioxidant and neuroprotective profile, emerged as an effective multipotent molecule, suitable to be further developed in view of the multifaceted character of AD.

Chemistry
General Methods. Melting points were measured in glass capillary tubes on a Büchi SMP-20 apparatus and are uncorrected. 1 H-NMR and 13 C-NMR spectra were recorded in CDCl3, unless otherwise indicated, on a Varian Gemini spectrometer 400 MHz and 101 MHz, respectively. Chemical shifts are reported in parts per million (ppm) relative to tetramethylsilane (TMS), and spin multiplicities are given as s (singlet), d (doublet), t (triplet), m (multiplet) or br (broad). Direct infusion Taken together, the data for antioxidant and neuroprotective activities point at compound 4 as the most promising one, being endowed with a better profile than compounds 2 and 17, suggesting that both the amino terminal group and the chalcone peculiar double bond were crucial structural features for inducing these effects. Undoubtedly, the α,β-unsaturated carbonyl moiety in compound 4, acting as a Michael acceptor, could interfere with Keap1-Nrf2 binding, causing the subsequent activation of Nrf2 signaling pathway [23]. Indeed, several studies postulate for electrophylic compounds a possible Cys-based modification of Keap1 allowing its dissociation from Nrf2 and leading to the transcription of cytoprotective genes [24,25]. In this respect, a fine-tuning of the chalcone electrophilicity, due to the substituents introduced, can be considered an important feature to minimize the risk of off-target effects. On the other hand, compound 17, only devoid of the α,β double bond with respect to 4, could still increase the GSH levels, probably acting with a different mechanism.
In summary, the increased flexibility of these newly synthesized chalcone-based derivatives led to an improved cholinesterase inhibitory activity with respect to the benzofuran lead compound, possibly due to a better fit into the catalytic ChEs gorges. On the other hand, the presence of the distinctive α,β-unsaturated carbonyl moiety also allowed introducing an appreciable antioxidant and neuroprotective potential. In this series, compound 4, endowed with a dual AChE/BuChE low micromolar inhibitory activity and a relevant antioxidant and neuroprotective profile, emerged as an effective multipotent molecule, suitable to be further developed in view of the multifaceted character of AD.

General method for the synthesis of compounds 24-29:
A stirred mixture of 4-hydroxyacetophenone (0.022 mol), selected bromochloroalkane (0.044 mol) and K 2 CO 3 (6 g) was refluxed in acetone (150 mL) for 20 h. The suspension was filtered while hot, and the solvent was removed under reduced pressure. After adding petroleum ether, the residue was kept in the freezer overnight and the white solid that formed was filtered off and purified by flash chromatography on silica gel (petroleum ether/ethyl acetate 9:1).

1-(3-(4-Chlorobutoxy)phenyl)ethan-1-one
General method for the synthesis of compounds 30-35. A stirred solution of selected chloroderivative (6.5 mmol) and diethylamine (13 mmol) in toluene (100 mL) was refluxed for 24 h. The mixture was washed with water (3 × 25 mL) and the organic layer was dried. The solvent was removed under reduced pressure and the residue was purified by flash chromatography on silica gel (toluene/acetone 4:1).

Inhibition of Human Cholinesterases
The capacity of 1-18 to inhibit human cholinesterase activity was assessed using the Ellman's assay [16]. The assays were performed on a Jasco V-530 double beam spectrophotometer connected to a HAAKE DC30 thermostating system (Thermo Haake, Karlsruhe, Germany). Stock solutions of the tested compound (2 mM) were prepared in methanol and diluted in methanol. The assay solution consisted of a 0.1 M phosphate buffer, pH 8.0, with the addition of 340 µM 5,5 -dithiobis(2-nitrobenzoic acid), 0.02 unit/mL human recombinant AChE or BuChE from human serum (Sigma-Aldrich, Milan, Italy), and 550 µM substrate, i.e., acetylthiocholine iodide or butyrylthiocholine iodide, respectively (Sigma-Aldrich). Inhibitors were added to the assay solution at increasing concentrations and preincubated at 37 • C with the enzyme for 20 min before the addition of substrate. The rate of absorbance increase at 412 nm was followed for 3 min. In parallel, blanks containing all components except the enzyme were prepared to account for the non-enzymatic hydrolysis of the substrate. The reaction rates were compared and the percent inhibition due to the presence of the tested compound was calculated. Each concentration was analyzed at least in duplicate. Inhibition plots for each compound were obtained by plotting the % inhibition versus the logarithm of inhibitor concentration in the assay solution. The linear regression parameters were determined for each curve and the IC 50 was extrapolated. For each tested compound two independent assessments of the IC 50 value were carried out.

Determination of Neurotoxicity
To establish the range of concentrations not associated with neurotoxicity, SH-SY5Y cells were seeded in a 96-well plate at 2 × 10 4 cells/well, incubated for 24 h and subsequently treated with different concentrations of compounds 2, 4 and 17 (2.5-80 µM) for 24 h at 37 • C in 5% CO 2 . Neuronal cells viability, in terms of mitochondrial metabolic function, was evaluated by the reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to its insoluble formazan, as previously described [26]. Briefly, the treatment medium was replaced with MTT in Hank's Balanced Salt Solution (HBSS) (0.5 mg/mL) for 2 h at 37 • C in 5% CO 2 . After washing with HBSS, formazan crystals were dissolved in isopropanol. The amount of formazan was measured (570 nm, reference filter 690 nm) using a multilabel plate reader (VICTOR™ X3, PerkinElmer, Waltham, MA, USA). The quantity of formazan was directly proportional to the number of viable cells.

Determination of Antioxidant Activity
The intracellular antioxidant activity of the studied compounds was evaluated in SH-SY5Y cells as previously described [27]. Briefly, SH-SY5Y cells were seeded in a 96-well plate at 2 × 10 4 cells/well and incubated for 24 h at 37 • C in 5% CO 2 . Subsequently, SH-SY5Y cells were incubated for 24 h with compounds 2, 4 and 17 (1.25 µM). At the end of incubation, the treatment medium was removed and 100 µL of a fluorescent probe, 2 -7 dichlorodihydrofluorescein diacetate (H 2 DCF-DA) (10 µg/mL), was added to each well. After 30 min of incubation at room temperature, H 2 DCF-DA solution was replaced with a solution of tert-butyl hydroperoxide (t-BuOOH) (100 µM) for 30 min. The reactive oxygen species (ROS) formation was measured (excitation at 485 nm and emission at 535 nm) using a multilabel plate reader (VICTOR™ X3, PerkinElmer). The antioxidant activity in terms of inhibition percentage in ROS formation induced by t-BuOOH, is calculated using the following formula: where Ptc = % of increase in ROS formation induced by t-BuOOH in the presence of the studied compounds; Pt = % of increase in ROS formation induced by t-BuOOH.

Determination of Glutathione Levels
The GSH levels were evaluated in SH-SY5Y cells as previously described [28]. SH-SY5Y cells were seeded in a black 96-well plate at 2 × 10 4 cells/well and incubated for 24 h at 37 • C in 5% CO 2 . Subsequently, SH-SY5Y cells were incubated for 24 h with compounds 2, 4 and 17 [1.25 µM]. At the end of incubation, the treatment medium was removed, and 100 µL of a fluorescent probe, monochlorobimane (MCB), was added to each well. After 30 min of incubation at 37 • C in 5% CO 2 , the GSH levels were measured (excitation at 355 nm and emission at 460 nm) using a multilabel plate reader (VICTOR™ X3, PerkinElmer). Data are expressed as concentration of GSH (µM) obtained by a GSH standard curve.

Determination of Neuroprotective Activity toward Aβ 1-42 Oligomers
To evaluate the neuroprotective activity of the studied compounds, SH-SY5Y cells were seeded in a 96-well plate at 3 × 10 4 cells/well, incubated for 24 h and subsequently treated with Aβ 1-42 oligomers (OAβ 1-42 ) [10 µM] in the presence of compounds 2, 4 and 17 [1.25 µM] for 4 h. The neuroprotective activity, in terms of increase in intracellular MTT granules, was measured by MTT formazan exocytosis assay, as previously described [30]. Briefly, the treatment medium was replaced with MTT in HBSS (0.5 mg/mL) for 1 h at 37 • C in 5% CO 2 . After the incubation, intracellular MTT granules were completely solubilized in Tween-20 (10% v/v). The absorbance of Tween-20 soluble MTT was measured at 570 nm (reference filter 690 nm) using a multilabel plate reader (VICTOR™ X3, PerkinElmer). Data are expressed as percentage of neuronal viability versus control.

Docking Studies
Molecular dockings were performed by using the Autodock Vina [31] software 1, The Scripps Research Institute., La Jolla, CA, USA). Diprotonated compounds 4 and 6 were prepared with Discovery Studio, version 2.1, software package (BIOVIA, San Diego, CA, USA), using standard bond lengths and bond angles. With the CHARMm force field [32] and partial atomic charges, the molecular geometries of the compounds were energy-minimized using the adopted-based Newton-Rapson algorithm until the rms gradient was below 0.01 kcal (mol Å) −1 .
The crystal structure of hAChE complexed with fasciculin-II (PDB: 1B41) was obtained from the Protein Data Bank (PDB). The protein was prepared by removing all water molecules, heteroatoms, any co-crystallized solvent and the ligand. Protein model tool in Discovery Studio, version 2.1, software package was used to assign proper bonds, bond orders, hybridization and charges. CHARMm force field was applied using the receptor-ligand interactions tool in Discovery Studio, version 2.1, software package. AutoDockTools (ADT; version 1.5.4, The Scripps Research Institute, La Jolla, CA, USA) was used to add hydrogens and partial charges for proteins and ligands using Gasteiger charges. The structures of the ligands were then loaded in ADT Tools and flexible torsions were assigned with AutoTors module, and the acyclic dihedral angles were allowed to rotate freely. Some selected side chains into the hAChE macromolecule were also allowed to change their conformations. Using the AutoTors module, the macromolecule side chains chosen to be flexible are: Trp286, Tyr124, Tyr337, Tyr72, Asp74, Thr75, Trp86 and Tyr341. The docking box was displayed using ADT and it is big enough to include the whole protein target ("blind docking"). A grid box of 60 × 60 × 72 with grid point spacing of 1 20 of 22 g the Autodock Vina [31] software 1, The Scripps nated compounds 4 and 6 were prepared with IOVIA, San Diego, CA, USA), using standard bond force field [32] and partial atomic charges, the rgy-minimized using the adopted-based Newtonw 0.01 kcal (mol Å) −1 . ith fasciculin-II (PDB: 1B41) was obtained from the ed by removing all water molecules, heteroatoms, ein model tool in Discovery Studio, version 2.1, bonds, bond orders, hybridization and charges. ptor-ligand interactions tool in Discovery Studio, DT; version 1.5.4, The Scripps Research Institute, nd partial charges for proteins and ligands using ere then loaded in ADT Tools and flexible torsions yclic dihedral angles were allowed to rotate freely. cromolecule were also allowed to change their acromolecule side chains chosen to be flexible are: p86 and Tyr341. The docking box was displayed ole protein target ("blind docking"). A grid box of ositioned at the middle of the protein (x = 116.546; as been used (PDB ID: 4BDS). Docking calculations ribed before for hAChE, not including flexibility at d dockings where a box of 66 × 66 × 70 Ǻ with grid e of the protein (x = 136.0; y = 123.59; z = 38.56). m_modes, which was set to 40. The docked ed on the binding energy and the top ranked t docking-energy conformation was considered as results generated were directly loaded into the d ligands and macromolecule were analyzed. , was positioned at the middle of the protein (x = 116.546; y = 110.33; z = −134.181). The three-dimensional structure of hBuChE has been used (PDB ID: 4BDS). Docking calculations were performed following the same protocol described before for hAChE, not including flexibility at the receptor. All dockings were performed as blind dockings where a box of 66 × 66 × 70 force field [32] and partial atomic charges, the ergy-minimized using the adopted-based Newtonow 0.01 kcal (mol Å) −1 . ith fasciculin-II (PDB: 1B41) was obtained from the red by removing all water molecules, heteroatoms, tein model tool in Discovery Studio, version 2.1, bonds, bond orders, hybridization and charges. eptor-ligand interactions tool in Discovery Studio, ADT; version 1.5.4, The Scripps Research Institute, and partial charges for proteins and ligands using ere then loaded in ADT Tools and flexible torsions cyclic dihedral angles were allowed to rotate freely. acromolecule were also allowed to change their acromolecule side chains chosen to be flexible are: rp86 and Tyr341. The docking box was displayed hole protein target ("blind docking"). A grid box of ositioned at the middle of the protein (x = 116.546; has been used (PDB ID: 4BDS). Docking calculations cribed before for hAChE, not including flexibility at nd dockings where a box of 66 × 66 × 70 Ǻ with grid le of the protein (x = 136.0; y = 123.59; z = 38.56). um_modes, which was set to 40. The docked sed on the binding energy and the top ranked st docking-energy conformation was considered as g results generated were directly loaded into the ed ligands and macromolecule were analyzed. , was positioned at the middle of the protein (x = 136.0; y = 123.59; z = 38.56). Default parameters were used except num_modes, which was set to 40. The docked conformations of each ligand were ranked based on the binding energy and the top ranked conformations were visually analyzed. The lowest docking-energy conformation was considered as the most stable orientation. Finally, the docking results generated were directly loaded into the Discovery Studio and interactions between docked ligands and macromolecule were analyzed.