Multifunctional Small Molecules as Potential Anti-Alzheimer’s Disease Agents

Alzheimer’s disease (AD) is a severe multifactorial neurodegenerative disorder characterized by a progressive loss of neurons in the brain. Despite research efforts, the pathogenesis and mechanism of AD progression are not yet completely understood. There are only a few symptomatic drugs approved for the treatment of AD. The multifactorial character of AD suggests that it is important to develop molecules able to target the numerous pathological mechanisms associated with the disease. Thus, in the context of the worldwide recognized interest of multifunctional ligand therapy, we report herein the synthesis, characterization, physicochemical and biological evaluation of a set of five (1a–e) new ferulic acid-based hybrid compounds, namely feroyl-benzyloxyamidic derivatives enclosing different substituent groups, as potential anti-Alzheimer’s disease agents. These hybrids can keep both the radical scavenging activity and metal chelation capacity of the naturally occurring ferulic acid scaffold, presenting also good/mild capacity for inhibition of self-Aβ aggregation and fairly good inhibition of Cu-induced Aβ aggregation. The predicted pharmacokinetic properties point towards good absorption, comparable to known oral drugs.


Introduction
Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder characterized by a progressive loss of neurons in the brain. Early symptoms are memory decline and language problems, followed by other cognitive serious dysfunctions related to brain atrophy [1]. AD is the most common cause of dementia and, in 2019, it was estimated that 50 million individuals were affected by dementia worldwide. This number is projected to reach 152 million cases by 2050 [2]. Despite research efforts, the pathogenesis and mechanism of AD progression are not yet completely understood. However, it is wellknown that a common feature in AD patients is the presence of extracellular amyloid-β (Aβ) plaques and intracellular neurofibrillary tangles (NFT) of hyperphosphorylated tau protein, the two major hallmarks in AD [3].
There are only a few symptomatic drugs approved for the treatment of AD. Four of them hamper the pathway that downregulates the neurotransmitter acetylcholine (ACh) acting as acetylcholinesterase inhibitors (AChE)-tacrine, donepezil, rivastigmine and galantamine-and the fifth is a N-methyl-D-aspartate (NMDA) receptor antagonist (memantine) [4]. Moreover, on 7 June 2021, the Food and Drug Administration (FDA) approved Herein, we report the synthesis and characterization of a new set of hybrid compounds enclosing the ferulic acid scaffold, followed by the evaluation of their physicochemical and biological properties, envisaging their potential role as anti-AD agents.
The FA derivatives 1a-e were synthesized coupling the commercially available ferulic acid (6) and hydrochloride benzylhydroxylamines 5a-e variously substituted. The Oarylmethylhydroxylamine hydrochloride 5a-e were synthetized according to the procedure previously described [34][35][36]. Briefly, the O-arylmethylhydroxylamine hydrochloride 5a-e were obtained by reaction between the suitably substituted benzyl bromide 2ae and the N-hydroxyphthalimide (3) by Mitsunobu reaction, and successive deprotection

Chemistry
The (E)-N-(benzyloxy)-3-(4-hydroxy-3-methoxyphenyl)acrylamide compounds, 1a-e, were obtained following the synthetic procedure reported in Scheme 1. Herein, we report the synthesis and characterization of a new set of hybrid compounds enclosing the ferulic acid scaffold, followed by the evaluation of their physicochemical and biological properties, envisaging their potential role as anti-AD agents.
The FA derivatives 1a-e were synthesized coupling the commercially available ferulic acid (6) and hydrochloride benzylhydroxylamines 5a-e variously substituted. The Oarylmethylhydroxylamine hydrochloride 5a-e were synthetized according to the procedure previously described [34][35][36]. Briefly, the O-arylmethylhydroxylamine hydrochloride 5a-e were obtained by reaction between the suitably substituted benzyl bromide 2a-e and the N-hydroxyphthalimide (3) by Mitsunobu reaction, and successive deprotection of the phthalimido group with ammonia solution 7N in MeOH. Compounds 5a-e were purified by crystallization and isolated as their hydrochloride salts.
Finally, the coupling reaction of the free amino group of these benzylhydroxylamines with the carboxylic group of FA was carried out in anhydrous DMF and inert argon atmosphere, in the presence of the carboxyl activating agent N-(3-dimethylaminopropyl)-Nethylcarbodiimide hydrochloride (EDCI), hydroxybenzotriazole (HOBt) and N-methylmorfoline. The reaction mixture was stirred at r.t. for 24 h, followed by the corresponding workup and purification to afford the final compounds 1a-e as pure solids under good yields (57-69%).

Antioxidant Activity
Cinnamic acid derivatives, such as FA, have been shown to avoid chain-breaking in the oxidation of low density lipoproteins, related with their hydrogen or electron-donating capacity and to the stability of the formed phenoxyl radicals [37].
Commercial ferulic acid (FA) and all the newly synthesized compounds 1a-e were studied for their radical scavenging activity, following the protocol previously reported [38,39]. The activity of each compound is expressed as EC 50 and is related to its interaction with the free stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH•). Analysis of the results contained in Table 1 shows that benzyloxyamidic derivatives 1a-e have good antioxidant activity, similar to the one of ferulic acid and slightly lower than the one of ascorbic acid [40], with an EC 50 value in the order of low µM. The transformation of the carboxylic portion of ferulic acid and the increasing size of the molecule do not affect their radical scavenging capacity, which must be related to the proton donor phenolic group, both in the precursor and in the hybrids.

Metal Chelation Studies
Besides antioxidant capacity, FA can also play other roles such as chelation of transition metal ions (e.g., copper, iron), which are catalysts of oxidative stress, and interference in metal-induced Aβ aggregation.
In order to evaluate the chelation capacity of the herein developed FA derivatives, compounds 1a and 1d were selected to be investigated on their acid-base behavior and metal chelating ability towards Cu 2+ and Fe 3+ ions, by using UV-Vis spectrophotometric titrations. These results were further compared to those of FA, studied by pH-potentiometric titrations.

Acid-base Properties
To evaluate the metal complexation capacity of the selected compounds, their protonation constants were determined.
Compounds were obtained in their neutral form, H2L (FA) and HL (1a, 1d), respectively. Due to some water-solubility limitations, especially for the benzyloxyamidic derivatives 1a and d, a mixed (20%, w/w) DMSO/water medium was chosen. The values obtained for the protonation constants are reported in Table 2.

Metal Chelation Studies
Besides antioxidant capacity, FA can also play other roles such as chelation of transition metal ions (e.g., copper, iron), which are catalysts of oxidative stress, and interference in metal-induced Aβ aggregation.
In order to evaluate the chelation capacity of the herein developed FA derivatives, compounds 1a and 1d were selected to be investigated on their acid-base behavior and metal chelating ability towards Cu 2+ and Fe 3+ ions, by using UV-Vis spectrophotometric titrations. These results were further compared to those of FA, studied by pH-potentiometric titrations.

Acid-Base Properties
To evaluate the metal complexation capacity of the selected compounds, their protonation constants were determined.
Compounds were obtained in their neutral form, H 2 L (FA) and HL (1a, 1d), respectively. Due to some water-solubility limitations, especially for the benzyloxyamidic derivatives 1a and d, a mixed (20%, w/w) DMSO/water medium was chosen. The values obtained for the protonation constants are reported in Table 2.

Metal Chelation Studies
Besides antioxidant capacity, FA can also play other roles such as chelation of transition metal ions (e.g., copper, iron), which are catalysts of oxidative stress, and interference in metal-induced Aβ aggregation.
In order to evaluate the chelation capacity of the herein developed FA derivatives, compounds 1a and 1d were selected to be investigated on their acid-base behavior and metal chelating ability towards Cu 2+ and Fe 3+ ions, by using UV-Vis spectrophotometric titrations. These results were further compared to those of FA, studied by pH-potentiometric titrations.

Acid-base Properties
To evaluate the metal complexation capacity of the selected compounds, their protonation constants were determined.
Compounds were obtained in their neutral form, H2L (FA) and HL (1a, 1d), respectively. Due to some water-solubility limitations, especially for the benzyloxyamidic derivatives 1a and d, a mixed (20%, w/w) DMSO/water medium was chosen. The values obtained for the protonation constants are reported in Table 2. In order to evaluate the chelation capacity of the herein developed FA derivatives, compounds 1a and 1d were selected to be investigated on their acid-base behavior and metal chelating ability towards Cu 2+ and Fe 3+ ions, by using UV-Vis spectrophotometric titrations. These results were further compared to those of FA, studied by pH-potentiometric titrations.

Acid-base Properties
To evaluate the metal complexation capacity of the selected compounds, their protonation constants were determined.
Compounds were obtained in their neutral form, H2L (FA) and HL (1a, 1d), respectively. Due to some water-solubility limitations, especially for the benzyloxyamidic derivatives 1a and d, a mixed (20%, w/w) DMSO/water medium was chosen. The values obtained for the protonation constants are reported in Table 2.  The values were obtained by fitting analysis of the experimental pH-potentiometric (FA) and spectrophotometric data (1a, 1d) with an equilibrium model using Hyperquad 2008 [41] and Psequad [42] programs, respectively. Figure 2 includes the potentiometric titration curves obtained for FA, as an example. The values were obtained by fitting analysis of the experimental pH-potentiometric (FA) and spectrophotometric data (1a, 1d) with an equilibrium model using Hyperquad 2008 [41] and Psequad [42] programs, respectively. Figure 2 includes the potentiometric titration curves obtained for FA, as an example.  The values were obtained by fitting analysis of the experimental pH-potentiometric (FA) and spectrophotometric data (1a, 1d) with an equilibrium model using Hyperquad 2008 [41] and Psequad [42] programs, respectively. Figure 2 includes the potentiometric titration curves obtained for FA, as an example. The protonation constants calculated for ferulic acid (FA) and depicted in Table 2 are in accordance with values previously reported (log K1 = 8.77-8.94, log K2 = 4.46-4.56) [43][44][45], taking into consideration that the literature values were determined in aqueous medium and/or different ionic strength conditions. These constants correspond to the protonation of the phenolic and carboxylic groups of FA, respectively. Table 2 also encloses the protonation constants corresponding to the phenolic oxygen atom of 1a (8.75) and 1d (8.93), determined by spectrophotometric titration, which agree with log K1 of FA, as expected. The protonation constants corresponding to the NH amidic group of compounds 1a and 1d could not be determined because the NH2 + group is extremely acidic (typically The protonation constants calculated for ferulic acid (FA) and depicted in Table 2 are in accordance with values previously reported (log K 1 = 8.77-8.94, log K 2 = 4.46-4.56) [43][44][45], taking into consideration that the literature values were determined in aqueous medium and/or different ionic strength conditions. These constants correspond to the protonation of the phenolic and carboxylic groups of FA, respectively. Table 2 also encloses the pro- tonation constants corresponding to the phenolic oxygen atom of 1a (8.75) and 1d (8.93), determined by spectrophotometric titration, which agree with log K 1 of FA, as expected. The protonation constants corresponding to the NH amidic group of compounds 1a and 1d could not be determined because the NH 2 + group is extremely acidic (typically pK a < 0). In fact, due to the presence of the neighbor carbonyl group, the lone pair of electrons is no longer localized on the nitrogen atom and so the basicity of this center is quite reduced.
Examples of species distribution curves, determined at the experimental conditions used in the pH-potentiometric and spectrophotometric titrations, are shown in Figure 3. The predominant species at assumed physiological conditions, pH 7.4 and concentration C L = 10 −5 M, are HL − for FA (ca. 99%) and the neutral HL for 1a (96%) and 1d (97%). The existence of the neutral HL species in extremely high concentrations (96-97%) explains the need to use a mixed 20% w/w DMSO/water medium in the solution studies, as the lipo-hydrophilic character is not only determined by the molecular charge but also by solute-solvent interactions.
Molecules 2021, 26, x FOR PEER REVIEW 7 of 17 pKa < 0). In fact, due to the presence of the neighbor carbonyl group, the lone pair of electrons is no longer localized on the nitrogen atom and so the basicity of this center is quite reduced. An example of species distribution curves, determined at the experimental conditions used in the pH-potentiometric and spectrophotometric titrations, is shown in Figure  3. The predominant species at assumed physiological conditions, pH 7.4 and concentration CL = 10 −5 M, are HLfor FA (ca. 99%) and the neutral HL for 1a (96%) and 1d (97%). The existence of the neutral HL species in extremely high concentrations (96-97%) explains the need to use a mixed 20% w/w DMSO/water medium in the solution studies, as the lipo-hydrophilic character is not only determined by the molecular charge but also by solute-solvent interactions.

Metal Complexation
The chelating ability of FA and of selected compounds 1a and 1d was studied by using the same experimental techniques (potentiometry, UV-Vis spectrophotometry) and experimental medium used for the titration of the ligand alone (20% w/w DMSO/water). In the following calculations for the 1:1, 1:2 and 1:3 metal/ligand (M/L) systems (M = Fe, Cu), the log Ki values previously obtained by each experimental method were used in the equilibrium model of the complexation studies performed by the same methodology.
Alterations in the deprotonation profiles of the ligand titration curves due to the presence of the metal ions are evident in Figure 2, since the curves for the M/L systems appear well below that of FA for a > −1 (M = Fe) or a > 0 (M = Cu). This evidence supports the formation of metal complexes with the deprotonated (L − ) and mono-protonated (HL) forms of the ligand, whose stability order follows the expected trend Fe > Cu. Equilibrium models obtained from the fitting analysis of the potentiometric curves (M = Fe, Cu) and the UV-Vis spectral data (M = Fe, Cu) are reported in Table 2.
The potentiometric curves and the equilibrium models obtained for FA seem to point toward the formation of metal complexes involving the carboxylic and the phenolic group for acidic lower pH values, as pointed out in the literature [45,46]. The co-existence of a bidentate O-phenol, O-methoxy coordination is also believed to be involved, with predominance at higher pH values. In fact, this bidentate O-phenol, O-methoxy coordination core occurs in the complexation of compounds 1a and 1d (see Figure 4) and so it must also compete with the carboxylate group in the M/L systems of FA.

Metal Complexation
The chelating ability of FA and of selected compounds 1a and 1d was studied by using the same experimental techniques (potentiometry, UV-Vis spectrophotometry) and experimental medium used for the titration of the ligand alone (20% w/w DMSO/water). In the following calculations for the 1:1, 1:2 and 1:3 metal/ligand (M/L) systems (M = Fe, Cu), the log K i values previously obtained by each experimental method were used in the equilibrium model of the complexation studies performed by the same methodology.
Alterations in the deprotonation profiles of the ligand titration curves due to the presence of the metal ions are evident in Figure 2, since the curves for the M/L systems appear well below that of FA for a > −1 (M = Fe) or a > 0 (M = Cu). This evidence supports the formation of metal complexes with the deprotonated (L − ) and mono-protonated (HL) forms of the ligand, whose stability order (Fe > Cu) follows the expected trend. Equilibrium models obtained from the fitting analysis of the potentiometric curves (M = Fe, Cu) and the UV-Vis spectral data (M = Fe, Cu) are reported in Table 2.
The potentiometric curves and the equilibrium models obtained for FA seem to point toward the formation of metal complexes involving the carboxylic and the phenolic group for acidic lower pH values, as pointed out in the literature [45,46]. The co-existence of a bidentate O-phenol, O-methoxy coordination is also believed to be involved, with predominance at higher pH values. In fact, this bidentate O-phenol, O-methoxy coordination core occurs in the complexation of compounds 1a and 1d (see Figure 4) and so it must also compete with the carboxylate group in the M/L systems of FA.  In the obtained metal complex models for FA, MHL corresponds to the species with the ligand phenolic oxygen atom protonated. ML, ML2 and ML3 species correspond to complexes involving the completely deprotonated form of the ligand, while MH-1L, MH-2L, MH-1L2 and MH-2L2 are mixed ligand-hydroxy metal complexes.
The stability constants found for the metal complex systems Fe 3+ /FA and Cu 2+ /FA are similar to those already published [45,46] and small differences between the respective values can be attributed to the different working media and ionic strength. Figure 5 presents an illustrative example of the spectral data obtained along the spectrophotometric titration of the Fe 3+ /1d system (1:3). Species distribution curves for some of the 1:2 and 1:3 M/L systems herein studied, at the used experimental conditions, are reported in Figure 6. In the obtained metal complex models for FA, MHL corresponds to the species with the ligand phenolic oxygen atom protonated. ML, ML 2 and ML 3 species correspond to complexes involving the completely deprotonated form of the ligand, while MH -1 L, MH -2 L, MH -1 L 2 and MH -2 L 2 are mixed ligand-hydroxide metal complexes.
The stability constants found for the metal complex systems Fe 3+ /FA and Cu 2+ /FA are similar to those already published [45,46] and small differences between the respective values can be attributed to the different working media and ionic strength. Figure 5 presents an illustrative example of the spectral data obtained along the spectrophotometric titration of the Fe 3+ /1d system (1:3).  In the obtained metal complex models for FA, MHL corresponds to the species with the ligand phenolic oxygen atom protonated. ML, ML2 and ML3 species correspond to complexes involving the completely deprotonated form of the ligand, while MH-1L, MH-2L, MH-1L2 and MH-2L2 are mixed ligand-hydroxy metal complexes.
The stability constants found for the metal complex systems Fe 3+ /FA and Cu 2+ /FA are similar to those already published [45,46] and small differences between the respective values can be attributed to the different working media and ionic strength. Figure 5 presents an illustrative example of the spectral data obtained along the spectrophotometric titration of the Fe 3+ /1d system (1:3). Species distribution curves for some of the 1:2 and 1:3 M/L systems herein studied, at the used experimental conditions, are reported in Figure 6. Species distribution curves for some of the 1:2 and 1:3 M/L systems herein studied, at the used experimental conditions, are reported in Figure 6. From the analysis of Table 2 and Figure 6, FA appears as a somewhat weake chelator than 1a at low pH values, since at pH ca. 2, there is free iron in the Fe 3+ /FA s (ca. 90%), while in the presence of 1a, there is 100% FeL. In fact, as already stated, pH, the metal complexation with FA seems to occur between the carboxylic group a phenolic oxygen [28,29]. Moreover, evidence for the formation of FeL3 complexes in ing 1a or 1d was not found from the treatment of the experimental data.
Concerning the Cu 2+ /L systems, the coordination with this metal ion typically o at higher pH values than with Fe 3+ , which indicates that FA, 1a and 1d are stronger tors for iron than for copper ions.
Comparison of the metal chelating capacity of the studied compounds can b formed by analysis of the respective pM values (at pH 7.4, CL/CM = 10, CM = 10 −6 M Both compounds 1a and 1d have similar chelating capacities towards iron and co which are also analogous to those of FA. Thus, these results suggest that in both cas metal coordination should involve mostly feroyl phenolic groups, even though t carboxylic groups may also have some role at the lowest pH values.

Inhibition of Aβ1-42 Aggregation
The studied compounds (1a-e) were tested in vitro to evaluate their activity as itors of Aβ1-42 peptide aggregation, through the Thioflavin T (ThT) fluorescence [48,49]. This method is based on the strong ability of the dye ThT to bind β-amyloid and oligomers through ionic and hydrophobic interactions, while its interaction w amyloid monomers is extremely weak. The intercalation of compounds in the β structure of amyloid protein may prevent fibrils aggregation and ThT binding, an evaluated by fluorimetry. The fluorescence emission measurements were performed overnight incubation of the self-mediated or Cu 2+ -induced Aβ fibril aggregates wi studied compounds. From the analysis of Table 2 and Figure 6, FA appears as a somewhat weaker iron chelator than 1a at low pH values, since at pH ca. 2, there is free iron in the Fe 3+ /FA system (ca. 90%), while in the presence of 1a, there is 100% FeL. In fact, as already stated, at low pH, the metal complexation with FA seems to occur between the carboxylic group and the phenolic oxygen [28,29]. Moreover, evidence for the formation of FeL 3 complexes involving 1a or 1d was not found from the treatment of the experimental data.
Concerning the Cu 2+ /L systems, the coordination with this metal ion typically occurs at higher pH values than with Fe 3+ , which indicates that FA, 1a and 1d are stronger chelators for iron than for copper ions.
Comparison of the metal chelating capacity of the studied compounds can be performed by analysis of the respective pM values (at pH 7.4, C L /C M = 10, C M = 10 −6 M) [47]. Both compounds 1a and 1d have similar chelating capacities towards iron and copper, which are also analogous to those of FA. Thus, these results suggest that in both cases, the metal coordination should involve mostly feroyl phenolic groups, even though the FA carboxylic groups may also have some role at the lowest pH values.

Inhibition of Aβ 1-42 Aggregation
The studied compounds (1a-e) were tested in vitro to evaluate their activity as inhibitors of Aβ 1-42 peptide aggregation, through the Thioflavin T (ThT) fluorescence assay [48,49]. This method is based on the strong ability of the dye ThT to bind β-amyloid fibrils and oligomers through ionic and hydrophobic interactions, while its interaction with β-amyloid monomers is extremely weak. The intercalation of compounds in the β-sheet structure of amyloid protein may prevent fibrils aggregation and ThT binding, and it is evaluated by fluorimetry. The fluorescence emission measurements were performed after overnight incubation of the self-mediated or Cu 2+ -induced Aβ fibril aggregates with the studied compounds.
Results are expressed as percentage of aggregation inhibition (Table 3) and tacrine was assayed as a model compound to verify the validity of the method used. All compounds present a good-moderate level of inhibition in both kinds of induced Aβ aggregation. Among the FA benzyloxyamide derivatives 1a-e, the chlorine substituent (1d, 1e) seems to slightly decrease the self-aggregation activity of β-amyloid; otherwise, compounds 1a and 1b substituted with the -OCH 3 group, ortho and meta, and 1c with the CF 3 group in the ortho position, have higher activities toward self-mediated β-amyloid aggregation. All the tested ligands showed an increase in the inhibitory activity for β-amyloid aggregation, when in the presence of the biometal ion Cu 2+ , probably due to their capacity for metal chelation.

In Silico Pharmacokinetic Properties
To predict the drug-likeness of the studied compounds, pharmacokinetic properties were evaluated using in silico tools, namely descriptors provided by the QIKPROP program (v. 2.5) [51]. To estimate compounds' absorption across biological membranes and their possible toxicity, the following parameters (Table 4) have been calculated: the lipohydrophilic character (clog P), the blood-brain barrier (BBB) partition coefficient (log BB), the ability to be absorbed through the intestinal tract (Caco-2 cell permeability) and the CNS (Central Nervous System) activity, along with verification of Lipinski's rule of five. These descriptors are useful to have an idea of the possible formulation for oral use of new compounds as anti-AD drugs. -0 a (Acceptable <500); b Predicted octanol/water partition coefficient log P (acceptable range −2.0 to 6.5); c Predicted BBB permeability (acceptable range −3 to 1.2); d Predicted Caco-2 cell permeability in nm/s (acceptable range: <25 is poor and >500 is great); e Percentage of human oral absorption (acceptable range: <25% is poor and >80% is high); f qualitative CNS activity parameter from (-) inactive, (++) active; g Number of violations of Lipinski's rule of five. The rules are MW < 500, clog P o/w < 5, donor HB ≤ 5, acceptor HB ≤ 10. Compounds that satisfy these rules are considered drug-like (the "five" refers to the limits, which are multiples of 5).
From analysis of the results reported in Table 4, which includes compounds 1a-e as well as the parent FA, all the compounds are small molecules with low molecular weight (MW) and are aligned with the five points of Lipinski's rule.
Log BB is calculated dividing the concentration of drug in the brain by the concentration in the blood and it measures the ability of drugs to pass the BBB. All compounds have a log BB value inside the range accepted to be potentially carried to the brain.
In investigating ADME of novel pharmaceutical molecules, another important step is the prediction of human intestinal permeation by non-active transport, expressed by Caco-2 permeability, as it is usually determined by Caco-2 cell line derived from human colorectal epithelial cancer [52]. The rate of absorption calculated for each compound is high or extremely high as concerns compound 1e, considering 500 nm/s as the value of reference for great permeability, which represents a good improvement relative to FA (87 nm/s). Furthermore, the estimation of CNS activity of studied ligands is not high, although they might pass the BBB. This result can be related to an unlikely cerebral toxicity.
Overall, the pharmacokinetic parameters depicted in Table 4 were found to be within the acceptable range (see Table 4 footnote). A solution of commercially available (E)-4-hydroxy-3-methoxy cinnamic acid (ferulic acid, FA) (6), (1 eq) in anhydrous DMF (2 mL) under inert atmosphere for Argon, was treated with hydroxybenzotriazole (HOBt) (1.2 eq), N-methylmorpholine (3 eq), the opportune benzylhydroxylamine hydrochloride 5a-e (3.1 eq) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDCI) (1.4 eq). The resulting mixture was stirred at room temperature (r.t.) for 24 h. Then, the mixture was extracted with AcOEt and washed with H 2 O. The organic phase was dried, filtered, and evaporated to give the crude derivatives that, after purification by flash chromatography (eluent: CHCl 3 , MeOH and NH 3 in ratio 9:1:0.1, respectively), afforded the corresponding hybrid FA compounds 1a-e. addition method using 0.1M HCl (Titrisol). The 0.1M HCl solution, used in calibration of the glass electrode and in spectrophotometric titrations, was prepared from a Titrisol ampoule. The titrant was prepared from carbonate free commercial concentrate (Titrisol, KOH 0.1 M ampoules). The KOH solution was standardized by titration with a solution of potassium hydrogen phthalate using Gran's method and it was rejected when the percentage of carbonate was greater than 0.5% of the total amount of base. The potentiometric studies were performed with an automated potentiometric apparatus controlled by PASAT program and containing a Crison micropH 2002 millivoltmeter, a Crison microBu 2031 burette and a Haake thermostatic bath (T = 25.0 ± 0.1 • C). The spectrophotometric titrations were carried out with a Perkin-Elmer Lambda 35 spectrophotometer.

Materials and Methods
The inhibition of β-amyloid (Aβ) aggregation was studied through the Th-T fluorescence assay, which was performed using a Varian Cary Eclipse fluorimeter at the following wavelengths: excitation (446 nm) and emission (485 nm). Amyloid-β peptide (1-42) was purchased from GeneCust as lyophilized powder stored at −20 • C.

Antioxidant Activity
Compounds were evaluated for their radical scavenging activity by the Blois method using DPPH (2,2-diphenyl-1-picrylhydrazyl), a stable free-radical. It absorbs in methanol solution at 517-520 nm and it is a scavenger for other radicals converting itself in the reduced form DPPHH. The assay consists of preparing solutions of increasing concentration of each compound starting from a stock solution (2-3 × 10 −4 M), adding to them 2.5 mL DPPH (0.002% in MeOH, 50-100 µM) and the necessary volume of MeOH to reach the final volume 3.5 mL for each sample. The control sample was made up of DPPH and MeOH. Solutions were protected from light at r.t. for 30 min and then absorbance was measured at 517 nm with a Perkin Elmer Lambda 35 UV-Vis spectrophotometer, using methanolic solution as the blank in the other cell. All the studied compounds have good solubility in MeOH, so their solutions could be prepared. The assay was repeated three times for all compounds and the antioxidant activity, expressed as EC 50 , was calculated by Equation (1).
Results correspond to the % of antioxidant activity related to ligand concentration and are expressed by the average EC 50 values of the three assays, that is the concentration of substrate which causes 50% loss of DPPH activity. L] l , were calculated by fitting the pH-potentiometric and spectrophotometric data with, respectively, the Hyperquad 2008 [41] and Psequad [42] programs. The metal hydrolysis model was determined under the defined experimental conditions (I = 0.1 M KCl, 20% w/w DMSO/H 2 O, T = 25.0 ± 0.1 • C) and the following values of stability constants were included in the fitting of experimental data towards the equilibrium models related to the Fe 3+ /L and Cu 2+ /L systems: log β (FeH -2 ) = −6.78, log β (FeH -3 ) = −10.78; log β (Cu 2 H -2 ) = −9.94. The species distribution curves were obtained with the Hyss program [41].

Inhibition of Self and Cu 2+ -Mediated Aβ 1-42 Aggregation
The method used is based on thioflavin T (ThT) fluorescence emission, which depends on the interaction of this dye with Aβ fibrils [49]. The assay is performed with the Aβ 1-42 peptide sample, which was previously prepared by solubilization of its lyophilized powder in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), an organic solvent useful to solubilize and monomerize the β-sheets protein aggregates. After 24 h, the solution was divided into Eppendorf tubes kept in ice, and then, HFIP was left to evaporate overnight (T = 25 • C). The resultant films were stored in the freezer. To perform the assay, each film of Aβ was re-dissolved in a solution of 69.5 µL CH 3 CN/Na 2 CO 3 (300 µM)/NaOH (250 mM) in ratio 48.3:48.3:3.4 µL, and 392.5 µL of phosphate buffer 0.215 M (pH = 8) was added to the resulting alkaline solution to obtain a final concentration of 40 µM. Compounds were dissolved in MeOH (1 mg/mL) and diluted with phosphate buffer to reach a ligand stock solution of 480 µM. Two different types of experiments were prepared in a final volume of 60 µL: control ligand assays (Aβ 1-42 aggregation in the absence of inhibitor) and ligand assays (effect of ligand on Aβ 1-42 aggregation). For each experiment, a blank sample without Aβ 1-42 was used to monitor the effect of the compounds in fluorescence. To study the inhibition of aggregation in the presence of Cu 2+ , which is known to promote it, an intermediate stock solution of CuCl 2 in phosphate buffer (240 µM) was prepared. Then, it was aliquoted to obtain a final concentration of 40 µM in the samples. All samples were incubated in a water bath for 24 h at 37 • C. Then, 180 µL of ThT (5 µM) in glycine-NaOH buffer (50 mM, pH = 8.5) is added to each solution and 200 µL of it is placed in a 96-well plate (BD Falcon) to be read with the fluorimeter. After 5 min of incubation with ThT, fluorescence was measured at 446 nm (L excitation) and 485 nm (L emission).

In Silico ADME Properties
The drug-likeness of all compounds was investigated by in silico calculations using the software QIKPROP (version 2.5) provided by MAESTRO [51]. The following pharmacokinetic descriptors or ADME (absorption, distribution, metabolism and excretion) properties were calculated, namely, to predict: the lipophilicity (clog P), the blood-brain barrier partition coefficient (log BB), the ability to be absorbed through the intestinal tract (Caco-2 cell permeability), the CNS activity, along with the verification of Lipinski's rule of five. The prediction of those parameters gives us an idea of the ADME profile of the new molecules as possible drugs for oral use and of their absorption in the CNS.

Conclusions
The absence of a cure for the severe and complex Alzheimer's disease (AD) is the main rationale for the work described herein, focused on the development and study of a new set of multifunctional ferulic acid (FA) derivatives as potential anti-AD agents. In particular, a set of hybrids enclosing the ferulic acid scaffold and benzyloxyamines with different substituent groups were synthesized and evaluated for their physicochemical and biological properties. These compounds demonstrated good chelating capacity towards redox-active metal ions (Cu 2+ and Fe 3+ ) and good radical scavenging capacity, both these properties being conferred by the FA moiety. They also evidenced moderate/good capacity for inhibition of self-induced beta-amyloid (Aβ) aggregation, which was considerably improved in the case of Cu-induced aggregation, attributable to their Cu-chelation ability. Finally, their in silico predicted ADME properties are in the range of known oral drugs and also satisfied Lipinski's rule of five, indicating good absorption and, hence, good bioavailability. Thus, these compounds appear with promising lead structure for further developments as anti-Alzheimer agents.