Exploring the Potential of Sulfonamide-Dihydropyridine Hybrids as Multitargeted Ligands for Alzheimer’s Disease Treatment

Alzheimer’s disease (AD) is a multifactorial neurodegenerative disease that has a heavy social and economic impact on all societies and for which there is still no cure. Multitarget-directed ligands (MTDLs) seem to be a promising therapeutic strategy for finding an effective treatment for this disease. For this purpose, new MTDLs were designed and synthesized in three steps by simple and cost-efficient procedures targeting calcium channel blockade, cholinesterase inhibition, and antioxidant activity. The biological and physicochemical results collected in this study allowed us the identification two sulfonamide-dihydropyridine hybrids showing simultaneous cholinesterase inhibition, calcium channel blockade, antioxidant capacity and Nrf2-ARE activating effect, that deserve to be further investigated for AD therapy.


Introduction
Alzheimer's disease (AD) is the most common cause of memory impairment and dementia in elderly people [1]. AD is characterized by a series of highly interconnected pathological processes whose main features are: the accumulation and aggregation of abnormal extracellular deposits of amyloid-beta peptide (Aβ), and the intracellular deposits named neurofibrillary tangles (NFTs), composed by aggregates of hyperphosphorylated tau protein [2].
According to the amyloidogenic pathway, two main isoforms of Aβ in AD brains, Aβ 1-40 and Aβ 1-42 , are produced as a result of sequential proteolysis of amyloid precursor protein by β and γ-secretase [3,4]. An inadequate clearance of Aβ peptides induces their accumulation and, subsequently, the formation of senile plaques, associated with AD pathogenesis [5].
Our group has contributed to this field by using multicomponent reactions (MCR) for their facility of execution, time gain, flexibility and the variety of the obtained structures [40][41][42][43].
In the present paper, we describe the design, synthesis and biological assessment of the first novel MTDLs, which simultaneously target ChE inhibition, blockade of calcium channels, associated with antioxidant activity, and Nrf2 activation. These new MTDLs result from the association of sulfonamide moieties into a 1,4-dihydropyridine (1,4-DHP) core ( Figure 1). structures [40][41][42][43].
In the present paper, we describe the design, synthesis and biological assessment of the first novel MTDLs, which simultaneously target ChE inhibition, blockade of calcium channels, associated with antioxidant activity, and Nrf2 activation. These new MTDLs result from the association of sulfonamide moieties into a 1,4-dihydropyridine (1,4-DHP) core ( Figure 1). 1,4-DHPs, such as nilvadipine, are privileged scaffolds in medicinal chemistry, wellknown calcium channel blockers, which have been the subject of phase III clinical trials targeting mild to moderate AD [44]. Indeed, calcium channel blockers may have a neuroprotective effect by preventing Aβ peptide aggregation and NFT formation. It is, therefore, plausible that 1,4-DHPs may prevent or slow the progression of AD [45].
Sulfonamides have a wide range of biological applications for the treatment of diseases, including central nervous system disorders, such as schizophrenia, depression, dementia or AD [46], being particularly able to act as ChE inhibitors [11,47,48] and Nrf2 activators [49][50][51]. Thus, sulfonamide and its derivatives have been used in designing MTDLs against various diseases [14].

Synthesis
The new MTDLs 4a-i were prepared as shown in Scheme 1. The synthetic scheme started from the Hantzsch MCR of commercially available 4-nitrobenzaldehyde (1) with alkyl acetoacetate, and ammonium acetate, in a mixture of EtOH/H2O, under microwave irradiation (MWI), to afford dialkyl 2,6-dimethyl-4-(4-nitrophenyl)-1,4-dihydropyridine- 1,4-DHPs, such as nilvadipine, are privileged scaffolds in medicinal chemistry, wellknown calcium channel blockers, which have been the subject of phase III clinical trials targeting mild to moderate AD [44]. Indeed, calcium channel blockers may have a neuroprotective effect by preventing Aβ peptide aggregation and NFT formation. It is, therefore, plausible that 1,4-DHPs may prevent or slow the progression of AD [45].
Sulfonamides have a wide range of biological applications for the treatment of diseases, including central nervous system disorders, such as schizophrenia, depression, dementia or AD [46], being particularly able to act as ChE inhibitors [11,47,48] and Nrf2 activators [49][50][51]. Thus, sulfonamide and its derivatives have been used in designing MTDLs against various diseases [14].

Synthesis
The new MTDLs 4a-i were prepared as shown in Scheme 1. The synthetic scheme started from the Hantzsch MCR of commercially available 4-nitrobenzaldehyde (1) with alkyl acetoacetate, and ammonium acetate, in a mixture of EtOH/H 2 O, under microwave irradiation (MWI), to afford dialkyl 2,6-dimethyl-4-(4-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate 2a-c. The catalytic hydrogenation of compounds 2a-c, in the presence of Pd/C, in ethyl acetate, at room temperature (rt), for 24 h, afforded the corresponding amino compounds 3a-c. Finally, compounds 4a-i were prepared by reacting compounds 3a-c with (benzene, p-toluene or methane)-sulfonyl chlorides in pyridine at rt (2-3 h). The resulting mixture was then purified by column chromatography using n-hexane/ethyl acetate as eluent to obtain the target compounds with yields ranging from 53% to 76%. All synthesized compounds were characterized by 1 H and 13 C NMR and elemental analysis. These data are reported in the experimental section and the Supporting Information.
of Pd/C, in ethyl acetate, at room temperature (rt), for 24 h, afforded the corresponding amino compounds 3a-c. Finally, compounds 4a-i were prepared by reacting compounds 3a-c with (benzene, p-toluene or methane)-sulfonyl chlorides in pyridine at rt (2-3 h). The resulting mixture was then purified by column chromatography using n-hexane/ethyl acetate as eluent to obtain the target compounds with yields ranging from 53% to 76%. All synthesized compounds were characterized by 1 H and 13 C NMR and elemental analysis. These data are reported in the experimental section and the Supporting Information. Scheme 1. Synthesis of Multi-Target-Directed Ligands 4a-i.

Biological Assesment
To verify the efficacy of our design, the compounds were subjected to biological (ChE inhibition, calcium channel blockade, transcriptional activation of Nrf2) and physicochemical evaluation (antioxidant activity).

Cholinesterases Inhibition
The ChE inhibition of compounds 4a-i was evaluated using EeAChE and eqBChE and donepezil and tacrine as references.
As indicated in Table 1, only compound 4f exhibited EeAChE inhibition with an IC50 equal to 12.6 µM compared to donepezil which showed an IC50 equal to 20.8 nM. All other compounds showed less than 50% inhibition at 10 µM. However, their IC50s could not be determined due to the solubility limit of these compounds in the buffer solution at very high concentrations. Regarding eqBChE inhibition, compounds 4a (R1 = Me, R2 = Ph), 4d (R1 = Et, R2 = pMePh), and 4f (R1 = Et, R2 = Me) exhibited IC50s equal to 5.0, 0.30 and 8.7 µM, respectively, compared to tacrine which showed an IC50 equal to 2.2 nM. The best compound, 4d, is only 135-fold less active than tacrine, one of the known strongest BChE inhibitors.

Biological Assesment
To verify the efficacy of our design, the compounds were subjected to biological (ChE inhibition, calcium channel blockade, transcriptional activation of Nrf2) and physicochemical evaluation (antioxidant activity).

Cholinesterases Inhibition
The ChE inhibition of compounds 4a-i was evaluated using EeAChE and eqBChE and donepezil and tacrine as references.
As indicated in Table 1, only compound 4f exhibited EeAChE inhibition with an IC 50 equal to 12.6 µM compared to donepezil which showed an IC 50 equal to 20.8 nM. All other compounds showed less than 50% inhibition at 10 µM. However, their IC 50 s could not be determined due to the solubility limit of these compounds in the buffer solution at very high concentrations. Regarding eqBChE inhibition, compounds 4a (R 1 = Me, R 2 = Ph), 4d (R 1 = Et, R 2 = pMePh), and 4f (R 1 = Et, R 2 = Me) exhibited IC 50s equal to 5.0, 0.30 and 8.7 µM, respectively, compared to tacrine which showed an IC 50 equal to 2.2 nM. The best compound, 4d, is only 135-fold less active than tacrine, one of the known strongest BChE inhibitors. Regarding the selectivity, most of the active compounds were preferential inhibitors of BChE. Compounds 4a (R 1 = Me, R 2 = Ph) and 4d (R 1 = Et, R 2 = pMePh) were totally selective to BChE, while compound 4f, bearing a methylsulfonamide group and an ethyl ester, was active on both AChE and BChE enzymes with a selectivity value equal to 1.5.
Due to the small number of molecules active on the ChEs, it is difficult to discuss structure-activity relationships (SAR) in sufficient depth.

Calcium Channel Inhibition
The calcium channel blockade of compounds 4a-i, and nimodipine as a reference, was evaluated at 10 µM concentration and is reported in Table 1. Eight of the nine compounds showed calcium channel inhibition with values ranging from 22% for 4d (R 1 = Et, R 2 = pMePh) to 51% for 4h (R 1 = iPr, R 2 = pMePh). The most potent compounds corresponded, in decreasing order, to 4h with 51% and 4g (R 1 = iPr, R 2 = Ph) with 50% comparing thus very favourably with nimodipine (52%).
According to the SAR and for the same sulfonamide group, the best results were always obtained for compounds bearing the isopropyl ester group, followed by those bearing the methyl ester group, except for compound 4i. For the same ester group, the nature of the sulfonamide scaffold does not play a significant role in the activity.

Antioxidant Assay
The antioxidant capacity of compounds 4a-i was evaluated by the oxygen radical absorbance capacity (ORAC) method [52], using melatonin as a reference. Radical scavenging activities are reported as Trolox Equivalents (TE). As shown in Table 1, all compounds exhibited antioxidant capacity with values ranging from 0.86 TE for 4g (R 1 = iPr, R 2 = Ph) to 3.01 TE for 4c (R 1 , R 2 = Me). The best compound, 4c, is more active than melatonin which showed a 2.45 TE [53].
Therefore, these three compounds were chosen to evaluate their Nrf2 transcriptional activation potencies.

Nrf2 Transcriptional Activation Potencies of MTDLs 4a, 4d and 4f
The Nrf2-ARE activating effect of selected 4a, 4d and 4f was evaluated in vitro using a cell-based luciferase assay in the AREc32 cell line [29], tert-butylhydroquinone (TBHQ) being used as positive control.
Prior to this, the cytotoxicity of the compounds against AREc32 cells was assessed by measuring cell viability. All three compounds showed no toxicity up to 50 µM.
As shown in Figure 2, no significant activity was observed for compound 4d. Interestingly, compounds 4a and 4f induced the Nrf2 transcriptional pathway significantly and successfully as early as 25 µM for 4a and 50 µM for 4f.
Prior to this, the cytotoxicity of the compounds against AREc32 cells was assessed by measuring cell viability. All three compounds showed no toxicity up to 50 µM.
As shown in Figure 2, no significant activity was observed for compound 4d. Interestingly, compounds 4a and 4f induced the Nrf2 transcriptional pathway significantly and successfully as early as 25 µM for 4a and 50 µM for 4f. The concentrations required to double the specific activity (CD) of the luciferase reporter were then calculated to compare relative potencies. ( Table 2). As expected, compounds 4a and 4f were the best compounds showing a CD value equal to 19.3 and 44.3 µM, respectively, compared to TBHQ, which showed 1.2 µM. Interestingly, compound 4a is only 16-fold less active than TBHQ, this one being one of the most potent activators of Nrf2. Nevertheless, compound 4f, with a CD value equal to 44.3 µM, i.e., 36-fold less active than TBHQ, shows, however, a 1.4-fold higher activity than melatonin (CD = 60 µM) [54], which is known for its ability to induce the transcriptional pathway [55]. Table 2. Induction potencies of compounds 4a, 4d, 4f or TBHQ in Nrf2/ARE-luciferase reporter cells.

Compounds CD (μM) 4a
19.3 ± 6.7 4d >50 4f 44.3 ± 4.7 TBHQ 1.2 ± 0.2 Data was represented as concentration-response curves and adjusted to non-linear equation. The CD value was used to quantify and compare the induction potency and expressed as the concentration required to double the specific luciferase reporter activity and are means ± SEM of at least four different experiments. The concentrations required to double the specific activity (CD) of the luciferase reporter were then calculated to compare relative potencies. ( Table 2). As expected, compounds 4a and 4f were the best compounds showing a CD value equal to 19.3 and 44.3 µM, respectively, compared to TBHQ, which showed 1.2 µM. Interestingly, compound 4a is only 16-fold less active than TBHQ, this one being one of the most potent activators of Nrf2. Nevertheless, compound 4f, with a CD value equal to 44.3 µM, i.e., 36-fold less active than TBHQ, shows, however, a 1.4-fold higher activity than melatonin (CD = 60 µM) [54], which is known for its ability to induce the transcriptional pathway [55]. Table 2. Induction potencies of compounds 4a, 4d, 4f or TBHQ in Nrf2/ARE-luciferase reporter cells.
Data was represented as concentration-response curves and adjusted to non-linear equation. The CD value was used to quantify and compare the induction potency and expressed as the concentration required to double the specific luciferase reporter activity and are means ± SEM of at least four different experiments.

Molecular Docking Studies of Compounds 4a and 4f
To explore the possible binding modes and the interactions of compounds 4a and 4f with EeAChE and eqBChE, docking studies were carried out using AutoDock Vina [56] software v.1.2.0.
The 3D structure of EeAChE was retrieved from the Protein Data Bank (PDB ID: 1C2B), a single catalytic subunit of the enzyme was used, and the flexibility of eight side chains has been considered by allowing side chain flexibility during the docking.
As shown in Figure 3 the most energetically favoured binding mode places the ligand 4a in the peripheral anionic site (PAS); therefore, no interactions with the catalytic triad residues can be established.
The 3D structure of EeAChE was retrieved from the Protein Data Bank (PDB ID: 1C2B), a single catalytic subunit of the enzyme was used, and the flexibility of eight side chains has been considered by allowing side chain flexibility during the docking.
As shown in Figure 3 the most energetically favoured binding mode places the ligand 4a in the peripheral anionic site (PAS); therefore, no interactions with the catalytic triad residues can be established. In the complex, 4a adopts a folded conformation where the phenyl ring of the sulfonamide moiety is interacting with Tyr341 and Trp286 via π-π T-shaped interactions and with Tyr72 via π-π stacked interactions. The oxygen atoms of sulfonamide and ester groups create a hydrogen bond with the hydroxyl group of Tyr72 and the indole NH of Trp286, respectively. Additionally, π-π T-shaped interactions were observed between the phenyl substituent of the dihydropyridine moiety and Tyr341 (Figure 4a). In the complex, 4a adopts a folded conformation where the phenyl ring of the sulfonamide moiety is interacting with Tyr341 and Trp286 via π-π T-shaped interactions and with Tyr72 via π-π stacked interactions. The oxygen atoms of sulfonamide and ester groups create a hydrogen bond with the hydroxyl group of Tyr72 and the indole NH of Trp286, respectively. Additionally, π-π T-shaped interactions were observed between the phenyl substituent of the dihydropyridine moiety and Tyr341 (Figure 4a).  Molecular docking showed that compound 4f is located in a different way than compound 4a (Figure 3). The docking results for compound 4f suggest that it can fit well in the active site of EeAChE and interact with important amino acid residues. The less buried sulphonamide moiety permits the ligand to reach the bottom of the narrow gorge and to interact with two amino acids of the catalytic triad.
The methylsulfonamide moiety is pointed toward the catalytic triad residues, and it Molecular docking showed that compound 4f is located in a different way than compound 4a (Figure 3). The docking results for compound 4f suggest that it can fit well in the active site of EeAChE and interact with important amino acid residues. The less buried sulphonamide moiety permits the ligand to reach the bottom of the narrow gorge and to interact with two amino acids of the catalytic triad.
The methylsulfonamide moiety is pointed toward the catalytic triad residues, and it interacts via carbon hydrogen interactions with Ser203 and with His447 forming a hydrogen bond. It was also found that this moiety of the ligand is forming π-sulfur interactions with Trp86. The phenyl ring, in the middle of the gorge, interacts with Tyr337 via π-π stacked interactions. One of the ester and methyl groups is in the acyl binding pocket interacting with Phe338 and Phe297 via π-alkyl interactions and Phe295 via van der Waals interactions. These groups can also interact with two amino acids in the PAS (Tyr341 and Tyr124) (Figure 4b).
The other ester and methyl groups are in the PAS, with the oxygen atoms involved in two hydrogen bonds with Tyr124 and Tyr337. In addition, these moieties can form π-alkyl interactions with Tyr341, Tyr337 and Tyr124 (Figure 4b).
To elucidate the interactions between compounds 4a and 4f and eqBChE, we also performed docking studies into the active site of the homology-modelled eqBChE. Without the X-ray structure of horse BChE (hBChE), a homology model has been retrieved from the SWISS-MODEL Repository [57] to rationalize the experimental data.
The ligand 4a is placed into the binding pocket of eqBChE, interacting with the residues involved in catalysis (CAS), with the residues in the oxyanion hole (OH), with the residues in the acyl-binding pocket (ABP) and with the residues in the PAS (Figure 5a). The protein complex with the best-docked pose of inhibitor showed that the phenyl ring in the dihydropyridine moiety is in the middle of the receptor cavity and with both the benzenesulfonamide and dihydropyridine groups located deep inside the gorge.  The dihydropyridine moiety binds in the CAS region of the enzyme. Specifically, the NH and methyl groups form a hydrogen bond and π-alkyl interactions with the catalytic triad residue His438, respectively. Besides, the dihydropyridine moiety is parallel to Trp82, establishing π-sigma and π-alkyl interactions with three methyl groups. One of the methyl groups is engaged in a network of π-alkyl and alkyl interactions with Trp430, Tyr440, Ala328, Met437 and Tyr440. The benzenesulfonamide ring is involved in π-π Tshaped interactions with Trp231 and Phe329 (ABP) and in π-alkyl interactions with Leu286 (ABP). Gly116 (OH) allowed an amide-π stacked interaction with this benzene ring. The ligand displays an additional hydrogen bond between the NH of the sulfonamide moiety and Leu285. An ester group interacts weakly with Asp70 and Tyr332, in the PAS (Figure 6a). The dihydropyridine moiety binds in the CAS region of the enzyme. Specifically, the NH and methyl groups form a hydrogen bond and π-alkyl interactions with the catalytic triad residue His438, respectively. Besides, the dihydropyridine moiety is parallel to Trp82, establishing π-sigma and π-alkyl interactions with three methyl groups. One of the methyl groups is engaged in a network of π-alkyl and alkyl interactions with Trp430, Tyr440, Ala328, Met437 and Tyr440. The benzenesulfonamide ring is involved in π-π T-shaped interactions with Trp231 and Phe329 (ABP) and in π-alkyl interactions with Leu286 (ABP). Gly116 (OH) allowed an amide-π stacked interaction with this benzene ring. The ligand displays an additional hydrogen bond between the NH of the sulfonamide moiety and Leu285. An ester group interacts weakly with Asp70 and Tyr332, in the PAS (Figure 6a). triad residue His438, respectively. Besides, the dihydropyridine moiety is parallel to Trp82, establishing π-sigma and π-alkyl interactions with three methyl groups. One of the methyl groups is engaged in a network of π-alkyl and alkyl interactions with Trp430, Tyr440, Ala328, Met437 and Tyr440. The benzenesulfonamide ring is involved in π-π Tshaped interactions with Trp231 and Phe329 (ABP) and in π-alkyl interactions with Leu286 (ABP). Gly116 (OH) allowed an amide-π stacked interaction with this benzene ring. The ligand displays an additional hydrogen bond between the NH of the sulfonamide moiety and Leu285. An ester group interacts weakly with Asp70 and Tyr332, in the PAS (Figure 6a).  The docking results revealed that BChE could effectively accommodate compound 4f inside the active site gorge (Figure 5b). The dihydropyridine moiety is oriented toward the bottom of the active site and it binds in the CAS region of the enzyme, establishing a key π-alkyl interaction with His438 and alkyl, π-alkyl and carbon-hydrogen interactions with two key amino acids of the ABP (Trp82 and Ala328). Leu285 residue and Gly116 (OH) further stabilized the position of the methylsulfonamide moiety in the active-site gorge via hydrogen bond and carbon-hydrogen interactions (Figure 6b). Besides, the esters groups display additional interactions with Tyr332 and Asp70, in the PAS (Figure 6b).
Based on docking results, compound 4a interacted with the mouth of the active gorge of EeAChE. In contrast, the most potent compound, 4f, can fit well in the active site interacting with important amino acid residues on both pockets, the CAS and PAS. Docking studies also revealed the capability of compounds 4a and 4f to bind to CAS and PAS of eqBChE and induce its inhibitory effect.

Discussion
In this study, our focus was on the design, synthesis, and biological evaluation of a novel class of compounds obtained by combining sulfonamide moieties with a 1,4-dihydropyridine scaffold. These compounds exhibit ChE inhibition, calcium channel blockade, Nrf2 pathway activation, and antioxidant activity, well-established therapeutic targets for AD.
We found that, without exception, all compounds exhibited antioxidant activity as measured by the ORAC test. This fluorescence-based test measures the ability of molecules to trap the radical derived from AAPH, suggesting that these molecules can donate a hydrogen atom, specifically from the DHP, as well as the ability to stabilize a free radical.
At the same time, almost all the prepared compounds showed calcium channel blockade, comparable to nimodipine used as a reference. Further investigation is ongoing in our laboratory to establish the mode of action of such compounds on calcium channel activity, as well as to develop a complete SAR profile with a larger number of analogues.
Conversely, a few compounds are active on ChE. Two compounds, 4a and 4d, showed fully selective inhibition of BChE with IC 50 values in the micromolar range, whereas compound 4f showed dual inhibition of both ChEs.
These three selected compounds can induce the Nrf2 transcriptional pathway significantly and successfully, with compound 4a exhibiting an effect as early as 25 µM and compound 4f at 50 µM. They can therefore activate endogenous antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase. This is likely due to the presence of the sulfonamide moiety and its antioxidant activities.
From this work, compounds 4a and 4f were identified as the first generation of MTDLs combining simultaneous ChE inhibition, calcium channel blockade, antioxidant capacity, and an activation effect of Nrf2-ARE. These compounds could serve as templates for developing more potent multitarget compounds for targets related to AD.
In addition, these molecules show great promise compared to the sulfonamides recently described in the literature [12,49]. Regarding AChE inhibition, these molecules are less active than the sulfonamides described by Yamali et al. [48], which exhibit nanomolar activities between 8 and 15 nM, but they are at least 10-fold more active than those described by the group of Enriz and Imramovsky [11], which show activities between 55 and 150 µM. For BChE, it is worth noting that the sulfonamides prepared in this study are significantly more active than those described in the literature [12]. As an example, compound 4d, the most potent BChE inhibitor, is 173 to 1700 times more active than the sulfonamides described by Enriz and Imramovsky [11], which show IC 50 values between 52 and 112 µM, and 23 to 70 times more active than the sulfonamides reported by Singh's group [47], where the IC 50 values range from 7 to 21 µM.
The binding modes of these compounds towards both ChEs were determined through molecular docking. The results showed that compound 4a interacts with the mouth of the active gorge of AChE, while the most potent compound, 4f, fits well in the active site and interacts with important amino acid residues in both CAS and PAS. Docking studies also revealed the capability of compounds 4a and 4f to bind to both CAS and PAS of BChE, resulting in their inhibitory effect.

Materials and Methods
Monitoring of reaction progress was performed by analytical thin-layer chromatography (TLC) on aluminium sheets precoated with silica gel (Type 60 F254, 0.25 mm; from Merck, Darmstadt, Germany). NMR spectra were measured on a BRUCKER DRX-400 AVANCE spectrometer using dimethylsulfoxide (DMSO-d 6 ) or chloroform (CDCl 3 ) as solvents. Chemical shifts are expressed in parts per million (ppm) and multiplicities of 1 H NMR signals were labelled as follows: s: singlet; d: doublet; t: triplet; q: quartet; and m: multiplet and coupling constants were expressed in hertz (Hz). Elemental analyses were performed on a Carlo-Erba CHNS apparatus. The purity of the compounds 4a-i was checked and confirmed to be >95% by elemental analyses, conducted on a Carlo-Erba EA 1108. These compounds were also found to be ≥95% pure by HPLC analysis using a Hitachi Chromaster instrument equipped with a BDS Hypersil C18 column (4.6 mm × 250 mm, Ø = 5 µm). The mobile phase is a mixture of methanol-and aqueous potassium phosphate 0.1 g% and phosphoric acid at 85% at 0.05 g% (60:40, v/v), and peaks were detected at 230 nm (see chromatograms of 4a and 4f in the Supporting Information)

General Synthesis of Compounds 2a-c
A solution of 1 mmol of 4-nitro-benzaldehyde and 2.5 mmol of the appropriate alkyl acetoacetate in the presence of 1.2 mmol of ammonium acetate was heated under MWI at 150 • C for 5 min. After cooling, an ethanol/water mixture (1:1) was added and the precipitate formed was collected in solid form by filtration to give compounds 2a-c with yields ranging from 75% to 96%.

General Synthesis of Compounds 3a-c
To a solution of compounds 2a-c (1 equiv., 1 mmol) in 75 mL of ethyl acetate, 150 mg of palladium on carbon and sodium sulfate (6 equiv., 6 mmol, 2 g) were added. The reaction mixture was subjected to hydrogenation at 40 psi hydrogen pressure for 24 h. The solution was then filtered on Celite. The filtrate was concentrated and purified using flash column chromatography (EtOAc, n-hexane) to obtain the desired compounds 3a-c in solid form.  13

General Synthesis of Compounds 4a-i
To a solution of compounds 3a-c (1 mmol) in 4 mL pyridine, cooled at 0 • C, 1 mmol of sulfonylchloride derivatives was added at 0 • C and the mixture was stirred at rt for 2~3 h. Then, 5 mL of 3N HCl ethyl acetate was added at 0 • C followed by filtration to remove the pyridine hydrochloride formed. 8 mL of a 1N hydrochloric acid solution was added to the filtrate, followed by three extractions with 3 × 15 mL ethyl acetate. The organic layers were combined, dried over Na 2 SO 4 and evaporated. The residue obtained was purified by flash column chromatography with an n-hexane ethyl acetate mixture to give the expected product in solid form.

Calcium Channel Inhibition
Assessment of calcium channel blockade of compounds 4a-i was performed according to the previously described protocol using the FLIPR calcium indicator 6 [59]. Briefly, FLIPR-loaded SH-SY5Y cells were exposed for 10 min to a 10 µm concentration of the tested compounds and to nimodipine used as a reference. 0.1% DMSO was used as a vehicle. The fluorescence change from calcium flux induced with KCl and CaCl2 (90 and 5 mM, respectively) was then recorded (λEx = 485 nm; λEm = 525 nm). Data were collected in three independent experiments with eight technical replicates per experiment. Outliers detected by the Grubbs test were excluded from further analysis.

Oxygen Radical Absorbance Capacity Assay
Antioxidant activity of 4a-i was performed by ORAC according to the protocol previously described [54]. Briefly, in a black 96-well microplate (Nunc), fluorescein and the tested compound were incubated for 15 min at 37 • C. 2,2 -Azobis(amidinopropane) dihydrochloride was then added and fluorescence was measured every minute for 1 h (λEx = 485 nm; λEm = 535 nm). All reactions were performed in triplicate, and at least three different assays were performed for each sample.

Nrf2 Transcriptional Activation Potencies of MTDLs 4a, 4d and 4f
The evaluation of Nrf2 transcriptional activation potencies of the tested compounds was performed using an Nrf2/ARE-luciferase reporter HEK293 stable cell line (Signosis, Santa Clara, CA, USA). First, we determined the cytotoxicity of the tested compounds using the MTT assay. For this purpose, the cells were seeded at a density of 2 × 10 4 cells per well in DMEM high glucose medium supplemented with 10% FBS using transparent 96-well culture plates. After 48 h at 37 • C, in 95% air/5% CO 2 , the culture medium was replaced with fresh DMEM containing only 0.1% FBS. Different concentrations of the tested compounds or DMSO (0.1%) were added to this culture medium. After 24 h of incubation with the tested compounds, the per cent of cell viability was measured. All reactions were performed in duplicate and repeated in at least four cell cultures.
The evaluation of Nrf2 transcriptional activation was then performed using noncytotoxic concentrations of the tested compounds according to the protocol previously described [60]. Briefly, the cells were seeded as described for the MTT assay, except that white 96-well culture plates were used. After 48 h of incubation, the cells were treated with the tested compounds or DMSO (0.1%). After 24 h of treatment, luciferase activity was measured using the Bright-Glo Luciferase Assay System (Promega, Madison, WI, USA) according to the manufacturer's instructions. All reactions were performed in duplicate and repeated in at least four different cultures.

Molecular Docking of Compounds 4a and 4f into EeAChE and eqBChE
Compounds 4a and 4f were assembled within Discovery Studio (DS, version 2022), software package, using standard bond lengths and bond angles. The molecular geometries of 4a and 4f were energy-minimized using the adopted-based Newton-Raphson algorithm with the CHARMm force field [61]. Structures were considered fully optimized when the energy changes between iterations were less than 0.01 kcal/mol [62]. The ligand was set up for docking with the help of AutoDockTools (ADT; version 1.5.7) to define the torsional degrees of freedom to be considered during the docking process. All the acyclic dihedral angles in the ligand were allowed to rotate freely.
The 3D coordinates of EeAChE (PDB ID: 1C2B), were obtained from the PDB. Then, the water molecules, heteroatoms, co-crystallized solvent and ligand were removed. Proper bonds, bond orders, hybridization and charges were assigned using the protein model tool in the DS software package Version 2022. AutoDockTools (ADT; version 1.5.7) was used to add hydrogens and partial charges for proteins and ligands using Gasteiger charges. To give flexibility to the binding site, eight residues (Trp286, Tyr124, Tyr337, Tyr72, Asp74, Thr75, Trp86 and Tyr341) lining the AChE binding site were allowed to move using the AutoTors module.
The search space was defined as a box with the following parameters: size x = 60, size y = 60, size z = 72 with grid points separated 1 Ǻ and centred at the middle of the protein (x = 21.5911; y = 87.752; z = 23.591).
The hBChE model has been retrieved from the SWISS-MODEL Repository [57]. A putative three-dimensional structure of eqBChE has been created based on the crystal structure of hBChE (PDB ID: 2PM8), these two enzymes exhibited 89% sequence identity. Initial protein preparation and docking calculations were performed following the same protocol described for EeAChE. A cube of 75 Ǻ with grid points separated by 1 Ǻ , was positioned in the middle of the protein (x = 29.885; y = −54.992; z = 58.141).
Docking calculations were performed with the program Autodock Vina v.1.2.0. [56] as blinds dockings where the num_modes was set to 40 and the other parameters were left as default parameters Finally, the most favourable conformations based on the binding energy were selected for analyzing the interactions between the enzymes and inhibitors, using DS. Funding: This work was supported by the Regional Council of Franche-Comté (2022Y-13659 and 13660 Accurate Project).