Marco Catto
email: marco.catto@uniba.it

Leonardo Pisani

Eugenio De

Benny La Mora

Danilo Belviso

Giuseppe Felice Mangiatordi

Andrea Pinto

Annalisa De Palma

Nunzio Denora

Rocco Caliandro

Jacques-Philippe Colletier

Eugenio De La Mora

Benny Danilo Belviso

Jacques- Philippe Colletier

Israel Silman

Orazio Nicolotti

Cosimo Damiano

Jacques- Philippe Colletier

Chiral Separation, X-ray Structure, and Biological Evaluation of a Potent and Reversible Dual Binding Site AChE Inhibitor

Keywords: Dual binding site acetylcholinesterase inhibitors, Alzheimer's disease, coumarin derivatives, X-ray diffraction, molecular docking, chiral separation

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Introduction

The increased life expectancy in developed countries represents an unprecedented challenge for health systems and caregivers, due to the widening incidence of age-related pathologies. Among these, neurodegenerative diseases (NDs), most notably, Alzheimer's disease (AD), are considered as a true emergency, because of their increasing incidence and accompanying social and economic costs. 1 AD is a progressive and fatal neurological disease, involving degeneration of brain areas of the frontal cortex and basal forebrain nuclei, which evolves from memory disorders in its early stages, through progressive behavioral alterations that culminate in a total inability in the later stages of the disease.

Despite decades of intensive research, no disease-modifying therapy is yet available, and therapeutic approaches include solely symptomatic treatments based on acetylcholinesterase (AChE) inhibitors (AChEIs) and memantine, an N-methyl-D-aspartate (NMDA) receptor antagonist. AChE (EC 3.1. 1.7) is the enzyme principally responsible for the termination of nerve impulse transmission at cholinergic synapses, by rapid hydrolysis of the neurotransmitter acetylcholine (ACh). Cholinergic innervation abounds in regions most affected by neurodegeneration in AD, e.g., the hypothalamus and entorhinal neo-cortex, and accordingly selective inhibition of AChE may help slowing the progress of cognitive alteration in the early stages of the disease. Furthermore, it is commonly accepted that impairment of cholinergic innervation from the nucleus basalis and septal diagonal band to the cerebral cortex and hippocampus are involved in producing the cognitive changes. 2 While effectiveness decreases along with the progression of AD, this approach is to date the only option for alleviating symptoms. 3 In the central nervous system (CNS), AChE activity is complemented by that of butyrylcholinesterase (BChE), a related enzyme characterized by a larger active site, and thus capable of accommodating larger substrates. BChE is present in the serum and in the CNS, where its concentration increases with the progression of AD, colocalising with neuritic plaques. 4 These observations have supported the hypothesis that BChE might serve as a target for the symptomatic treatment of late-stage AD. [START_REF] Greig | A New Therapeutic Target in AD Treatment: Attention to Butyrylcholinesterase[END_REF]6 The observation that cymserine analogues, selectively targeting BChE, can partially restore AChE brain levels and cognitive functions in aged rats 7 provides support for this hypothesis.

3D structures of AChE from several species, e.g., Torpedo, electric eel, mouse, and human, 8 in the presence or absence of inhibitors, have been solved by X-ray crystallography, demonstrating the existence of two binding sites at the top and bottom of the active-site gorge, termed the peripheral anionic site (PAS), and the catalytic anionic site (CAS), respectively. 9 Among AChEIs, donepezil (Figure 1) is considered as a reference drug because of its potency and high therapeutic index. Its peculiar inhibitory mechanism involves a dual binding site (DBS) reversible interaction with both the CAS and the PAS of the enzyme, thus resulting in mixed, i.e. neither completely competitive nor noncompetitive, inhibition kinetics. 10,11 Many DBS inhibitors of AChE have been described in the literature, 12 often displaying potent, reversible, and selective inhibition. [START_REF] Pisani | Discovery of Potent Dual Binding Site Acetylcholinesterase Inhibitors via Homo-and Heterodimerization of Coumarin-Based Moieties[END_REF] Many of them exhibit the archetypal structural features of donepezil, i.e., a protonatable N-benzylamine moiety able to interact with the aromatic amino acids of the CAS, and a planar, aromatic, lipophilic terminal portion making hydrophobic interactions (mainly π-π stacking) within the PAS. These structural features were indeed displayed by the coumarin-based racemic compound (±)-cis-1 (Figure 1), which has been studied by some of the authors of the current study. [START_REF] Catto | Synthesis and Biological Evaluation of Coumarin Alkylamines as Potent and Selective Dual Binding Site Inhibitors of Acetylcholinesterase[END_REF] To assess the stereochemical contribution to AChE inhibition of the cis-3-amino-1-ciclohexanecarboxylic acid used as the spacer in (±)-cis-1, we performed its chiral separation by HPLC, and tested the two enantiomers. In parallel, we undertook the design of two new achiral analogues 4 and 6, also shown in Figure 1. Compound 4 includes a 3-atom linear open chain, and 6 encloses a piperidine ring, both joined to the donepezil-like N-benzyl moiety. By employing docking-assisted crystallographic studies, we determined the crystal structure of compound (-)-cis-1 (coded as MC1420), established as the eutomer after chiral separation, complexed with Torpedo californica AChE (TcAChE). Scheme 1 shows the synthetic and experimental procedure for obtaining 4, 6, and the two chiral forms of (±)-cis-1. Permeability and cytotoxicity of the coumarin derivative (±)-cis-1 were also evaluated in vitro as an early assessment of its potential as an AChE inhibitor for alleviating symptoms in AD-associated cognitive impairments. Previous data on coumarin-donepezil hybrid (±)-cis-1 highlighted a good inhibitory potency on electric eel AChE (eeAChE) with very high selectivity over BChE. [START_REF] Catto | Synthesis and Biological Evaluation of Coumarin Alkylamines as Potent and Selective Dual Binding Site Inhibitors of Acetylcholinesterase[END_REF] In order to obtain data comparable with published works in which the inhibition of human isoforms (hAChE, hBChE) were studied, we first investigated the inhibition of hAChE and hBChE by (±)-cis-1. The IC50 value for (±)-cis-1 against hAChE in Table 1 (36.5 nM) is in fair agreement with that previously determined for eeAChE (7.6 nM). [START_REF] Catto | Synthesis and Biological Evaluation of Coumarin Alkylamines as Potent and Selective Dual Binding Site Inhibitors of Acetylcholinesterase[END_REF] The hAChE kinetic inhibition constant (Ki) of (±)-cis-1 was four-fold higher than that measured for donepezil (46.6 vs. 12.7 nM, Table 1). The kinetic profile correlated well with a mixed-mode inhibition, typical for putative DBS inhibitors. A preliminary assessment of safety profile was obtained from a MTT-based cellular test 15 of (±)-cis-1, which was incubated with HepG2 human liver cancer cells in the 20-100 µM concentration range (Figure 2). Cell viability after 2 h was always 80-90% even at the higher concentrations, thus revealing a low intrinsic cytotoxicity. The potential of (±)-cis-1 as a hit compound for pharmacological profiling was further explored by assessing its permeability in a well-validated cell membrane model, which utilizes the MDCK-MDR1 cell line expressing the efflux system P-gp. This cell line is widely considered to reliably mimic blood-brain barrier permeability, accounting for both transcellular and paracellular pathways. 16 The MTT assay of cell viability, performed after 24 and 72 h of coincubation with 100 µM (±)-cis-1, showed lower cell survival compared with the control HepG2 samples, with cell viability dropping to 60% and 37%, respectively (Supporting Information, Table S1). However, the IC50 at this last time point was 30 µM, a value three orders of magnitude higher than the IC50 measured for AChE.

Scheme 1 a

Following a previously reported approach, [START_REF] Pisani | Searching for Multitargeting Neurotherapeutics against Alzheimer's: Discovery of Potent AChE-MAO B Inhibitors through the Decoration of 2H-Chromen-2-one Structural Motif[END_REF] the apparent permeabilities Papp were measured both from the apical to basolateral (Papp AP) and from the basolateral to apical (Papp BL) compartments.

Diazepam and FD-4 were used as markers of transcellular and paracellular pathways, respectively.

The permeability values shown in Table 2 are comparable to those of reference compounds, while the efflux ratio lower than 2 that was found indicates that the compound is not a substrate for P-gp. With this information in hand, we proceeded with the enantiomeric separation of (±)-cis-1. Due to the presence of a secondary N-benzylamine group, we performed a preliminary N-Boc protection, leading to the lipophilic derivative 7 (Scheme 1). Resolution of racemate (±)-cis-7 into the single enantiomers was achieved by semi-preparative chiral HPLC (Figure 3), using a Kromasil 5-AmyCoat chiral stationary phase with isopropanol/n-hexane 1:1 v/v as the mobile phase, followed by Boc deprotection. Inhibition data reported in Table 1 show a eudismic ratio of ca. 20 for the (-) enantiomer (coded as MC1420), with IC50 for hAChE very close to that of donepezil. Interestingly, an opposite eudismic ratio was found for hBChE inhibition, leading to high (730-fold) AChE/BChE selectivity of MC1420.

The Ki for MC1420 was 19.1 nM, close to the value of 12.7 nM found for donepezil, and the kinetic data fitted a Michaelis-Menten model of a mixed-type inhibition, with very low variance (residuals < ± 1%; r 2 = 0.996; Figure 4). As shown in Table 1, the newly synthesized derivatives 4 and 6 (Scheme 1) were unable to reproduce the good inhibitory capacity of racemate 1. As far as compound 4 is concerned, we replaced the conformationally constrained cyclohexyl spacer with a linear open chain. This structural variation turned out to be detrimental, resulting in a 20-fold activity drop in inhibition of hAChE. The second structural variation was designed to keep the six-atom ring spacer, while incorporating the basic nitrogen into an N-benzylpiperidine fragment, which is a typical pharmacophore motif of donepezil and related structures. The isonipecotamide derivative 6 showed a 6-fold drop in hAChE inhibition compared to rac-1 while retaining fair AChE inhibition and very high AChE/BChE selectivity. It is noteworthy that the butylamide 4 showed strong inhibition of hBChE, thus resulting as a good and fairly selective inhibitor of this isozyme. The decrease in activity returned by these achiral analogues, irrespective of the limited variation in distance between the basic nitrogen and the coumarin moiety, interacting at the CAS and PAS, respectively, prompted us to elucidate the interactions of MC1420

with its target protein at the molecular level.

We thus determined the crystal structure of the complex of eutomer MC1420 with TcAChE. MC1420

was soaked into trigonal crystals of TcAChE, obtained as described earlier, [START_REF] Dighe | Rivastigmine and Metabolite Analogues with Putative Alzheimer's Disease-modifying Properties in a Caenorhabditis elegans Model[END_REF] and the structure of the TcAChE/MC1420 complex (PDB ID: 6TT0) was solved at 2.8 Å resolution from data collected at 100 K at a synchrotron beamline, following cryoprotection and flash-cooling of crystals. At this stage, the absolute configuration of MC1420 at the 1,3-cis-cyclohexyl ring spacer, namely, either (1R,3S)

or (1S,3R), was unknown. Therefore, the structural refinement was performed assuming both the configurations of the spacer. In both cases, it was observed that the ligand molecule binds to the CAS through its N-benzyl moiety, with the coumarin group anchored at the PAS (Figure 5). This binding The experimental electron density map revealed the presence of a water molecule (W1 in Figure 5) at 2.8 Å from the oxygen atom of the amide group of MC1420, and 2.7 Å from the nitrogen atom of the Phe288 backbone, distances both compatible with H-bond interactions. This water-mediated interaction, along with the two stacking interactions described above, comprise the whole set of significant protein-ligand interactions. The oxygen of W1 has a thermal factor (59.4 Å 2 ) that agrees very well with the average B-factor of atoms within 5 Å of W1 (60 Å 2 ). Importantly, the B-factor is obtained by using W1 at full crystallographic occupancy, suggesting that this water molecule and related water-mediated interactions are present in each unit of the crystal. By performing hydration analysis of the ligand by wet script, two water molecules were identified close to the one observed experimentally (Figure S1 in Supporting Information). Interestingly, W1 corresponds to a conserved water as identified by Koellner et al., [START_REF] Koellner | Active-site Gorge and Buried Water Molecules in Crystal Structures of Acetylcholinesterase from Torpedo californica[END_REF] and it therefore preexists at this position before binding of the compound. The structure offers an illustration that this structural water is indeed fully part of the gorge, where it helps to accommodate (and determine the binding affinity) of MC1420 in the gorge.

Despite the ligand being clearly visible in the experimental Fo-Fc electron density map, the absolute configuration of the (-)-cis-1 ligand used in the crystal preparation could not be unambiguously determined at the achieved resolution. We additionally performed molecular docking simulations, followed by binding free energy calculations for both configurations. Given the importance of W1

for ligand binding, this water molecule was included in the docking calculations performed.

(1R,3S)-cis-1 returned not only a better docking score (-13.56 kcal/mol) and a better binding free energy (-99.28 kcal/mol) than (1S,3R)-cis-1 (-11.74 kcal/mol and -81.00 kcal/mol respectively), but also a more plausible pose, as shown by comparing Figures 5 and6. We may postulate that this difference can be accounted for by an additional interaction with the PAS, established only by the (1R,3S) configuration (Figure 6). The S configuration of the carbon atom at position 3 of the cyclohexane should more likely permit an orientation of the charged amine adjacent to the benzyl ring, prone to form a salt bridge with Asp72. Taken together, these data suggest that MC1420 in the (1R,3S)-cis configuration should be more favored in binding compared to (1S,3R)-cis configuration.

Noteworthy, the eutomer in both configurations should form a water-bridged H-bond with Phe288 in the acyl pocket. In agreement with the experimental findings, even the best solutions for achiral compounds 4 and 6

returned poorer docking scores (-11.44 kcal/mol and -12.34 kcal/mol, respectively) as well as poorer binding free energies (-79.49 kcal/mol and -78.70 kcal/mol, respectively) relative to (1R,3S)-cis-1.

The obtained top-scored docking poses are reported in the Supporting Information (Figure S2).

In conclusion, our efforts to design selective and reversible AChE/BChE inhibitors led us to the synthesis of the AChE-selective hit compound (±)-cis-1 (Figure 1), [START_REF] Catto | Synthesis and Biological Evaluation of Coumarin Alkylamines as Potent and Selective Dual Binding Site Inhibitors of Acetylcholinesterase[END_REF] which showed in vitro good safety and capacity to cross the BBB as assessed by HepG2 and MDCKII-MDR cell-based assays, respectively. After chiral resolution, the (-)-cis-1 enantiomer MC1420 resulted the eutomer in hAChE inhibition, thus justifying the use of X-ray crystallography to resolve its binding mode in complex with TcAChE. The structure confirmed the dual binding mode of interaction predicted for MC1420, whereas docking calculations suggested that MC1420 should more favorably bind the enzyme in (1R,3S) absolute configuration than in (1S,3R).

Furthermore, in an attempt to overcome stereoisomeric limitations in a possible pharmacological evaluation, two achiral congeners of (±)-cis-1, i.e. 4 and 6, were synthesized and tested. These compounds did not replicate the strong inhibitory potency of 1. However, the butylamide 4 displayed good inhibition of BChE (IC50 = 181 nM) and the piperidine derivative 6 a high (about two orders of magnitude) AChE/BChE selectivity, suggesting that both of them could deserve further consideration.

Materials and methods

Structure determination of the MC1420/TcAChE complex

After chiral separation of (±)-cis-1 racemate, the (-)-cis-1 enantiomer MC1420 was soaked for 2 h at 1 mM concentration in hanging drops containing trigonal crystals of TcAChE, obtained as described previously. [START_REF] Dighe | Rivastigmine and Metabolite Analogues with Putative Alzheimer's Disease-modifying Properties in a Caenorhabditis elegans Model[END_REF] Crystals were then flash-cooled, in situ, at 100 K under the gaseous nitrogen stream of a cryo-cooler (Oxford Cryosystems, Oxford, United Kingdom). Data collection was carried out on beamline ID29, at the European Synchrotron Facility (ESRF) at a wavelength of 1.074 Å. Initial phases were determined by rigid-body refinement using as a model the native TcAChE structure (PDB ID 2VT7). The Fo-Fc difference map showed continuous positive electron density at s > 3.5 within the active site gorge of the enzyme (CAS and PAS). Both enantiomers compatible with MC1420 in cis conformation (1R,3S and 1S,3R of the cyclohexane ring) were tested in the refinement procedure. The restrain description file required for the refinement procedure was generated by using the PRODRG server [START_REF] Schüttelkopf | A Tool for High-Throughput Crystallography of Protein-Ligand Complexes[END_REF] and modified to ensure planarity of the coumarin and amide groups. Molecular geometry was optimized by eLBOW, [START_REF] Moriarty | Electronic Ligand Builder and Optimization Workbench (eLBOW): a tool for ligand coordinate and restraint generation[END_REF] under the crystallographic suite PHENIX. [START_REF] Adams | A Comprehensive Python-based System for Macromolecular Structure Solution[END_REF] The ligand was fitted into the positive Fo-Fc Fourier difference map by using COOT. [START_REF] Emsley | Features and Development of Coot[END_REF] Water molecules were then added to the protein-ligand complex and the resulting crystal structure was refined using phenix.refine, [START_REF] Afonine | Towards Automated Crystallographic Structure Refinement with phenix.refine[END_REF] included in the Phenix crystallographic software suite. [START_REF] Adams | A Comprehensive Python-based System for Macromolecular Structure Solution[END_REF] The structural model was validated using the Phenix implementation of MolProbity. [START_REF] Chen | MolProbity: All-atom Structure Validation for Macromolecular Crystallography[END_REF]

Docking simulations

Both enantiomers of (±)-cis-1 were docked into the refined X-ray structure of the complex 6TT0. The protein structure was prepared using Protein Preparation Wizard [START_REF]Schrödinger Suite 2018-4 Protein Preparation Wizard[END_REF] for adding missing hydrogen atoms, reconstructing incomplete side chains and loops, and assigning ambiguous protonation states.

The ligand was prepared using LigPrep 26 in order to properly generate all the possible tautomers and ionization states at a pH value of 7.0 ± 2.0. The files thus obtained were used for docking simulations performed by Grid-based ligand docking with energetics (GLIDE). [START_REF]Schrödinger Suite 2018-4 Protein Preparation Wizard[END_REF][START_REF] Friesner | Extra Precision GLIDE: Docking and Scoring Incorporating a Model of Hydrophobic Enclosure for Protein-Ligand Complexes[END_REF] During the docking process, the protein was held fixed, whereas full conformational flexibility was allowed for the ligand. The default Force Field OPLS_2005, [START_REF] Robertson | Improved Peptide and Protein Torsional Energetics with the OPLSAA Force Field[END_REF] and all the default settings of the extra precision (XP) protocol were used. A cubic grid was used that was centered on the refined structure of the cognate ligand, having an edge of 10 Å for the inner box and 30 Å for the outer box. Finally, a water molecule (referred to as W1) was kept in the binding site during docking simulations. Indeed, W1 arises from experimental electron density indicating a water-mediated H-bond involving the carbonyl group of the ligand and the backbone of Phe288.

MM-GBSA calculations

The binding free energies (DG) between protein and ligands were computed by applying the Molecular Mechanics/Generalized Born Surface Area on the obtained top-scored docking poses. [START_REF] Genheden | The MM/PBSA and MM/GBSA Methods to Estimate Ligand-Binding Affinities[END_REF] More specifically, Prime 30 was the software used, and the following Equation 1 was applied:

ΔEbind = ΔEMM + ΔGsolv + ΔGSA (1)
where ΔEMM, ΔGsolv and ΔGSA represent the difference between the contribution made by the ligandprotein complex and the sum of those made by the ligand and the protein taken alone, in terms of minimized energy, solvation energy and surface area energy, respectively. Flexibility was allowed for all residues having at least one atom within a distance of 3 Å from the ligand.

Associated Content

Supporting Information

Syntheses of compounds 4-6, procedures for chiral separation of (±)-cis-1, cell viability assays (Table S1), hydration calculations for MC1420 (Figure S1), docking of compounds 4 and 6 (Figure S2), and crystallographic data (Table S2) are reported as Supporting Information. This material is available free of charge via the internet at http://pubs.acs.org.

Lay summary

Enantiomerically pure MC1420, a brain-permeant, potent dual binding site AChE inhibitor, has been crystallized in complex with TcAChE and its binding mode investigated by comparing X-ray data and docking studies.

Materials and methods

Chemistry

Starting materials, reagents and analytical grade solvents were commercially available and purchased from Merck Sigma-Aldrich, Milan, Italy. All reactions were routinely monitored with thin layer chromatography (TLC) on aluminum sheets (Merck Kieselgel 60 F254); spots were displayed through UV lamp. For reactions requiring anhydrous environment, glassware was preliminary dried by heating on flame burner and then by cooling under argon stream.

, N-Boc protected isonipecotic acid, HOBt, DIC,

) semi-preparative chiral HPLC.

Synthesis of 4-(benzylamino)-N-(6,7-dimethoxy-2H-2-oxochromen-3-yl)butyramide hydrochloride (4)

121 mg (0.4 mmol) of 3 1 (Scheme 1) dissolved in 3 mL of anhydrous acetone were added portionwise to a solution of 436 µL (0.4 mmol) of benzylamine in 3 mL of anhydrous acetone held at reflux. After 24 h the reaction was warmed to room temperature and the solvent was evaporated under vacuum. The crude oil was purified by column chromatography (eluent CH2Cl2/MeOH 9:1).

The oil obtained by purification was treated with 1 mL of HCl

Synthesis of N-(6,7-dimethoxy-2H-2-oxochromen-3-yl)piperidine-4-carboxamide (5)

136 mg (0.54 mmol) of commercial N-Boc-piperidine-4-carboxylic acid and 90 mg (0.54 mmol) of 1-hydroxybenzotriazole were dissolved in 10 mL of anhydrous dichloromethane (DCM). After 5 min. the reaction was cooled to 0 °C trough an external ice bath and 92 µL (0.54 mmol) of N,Ndiisopropylcarbodiimide were added dropwise. After 5 min 60 mg (0.27 mmol) of 2 1 were added (Scheme 1) and the reaction was slowly warmed to room temperature and stirred for 36h. The The resulting acid solution was neutralized with NaHCO3, the inorganic precipitate filtered off and washed with DCM. The solution was extracted with DCM (3×20 mL) and the organic layers were collected and dried over anhydrous Na2SO4. The solvent was removed by rotatory evaporation yielding an orange solid: yield 84%.

Synthesis of 1-benzyl-N-(6,7-dimethoxy-2H-2-oxochromen-3-yl)piperidine-4-carboxamide (6)

67 mg (0.15 mmol) of 5 (Scheme 1) and 10 mg (0.7 mmol) of K2CO3 were suspended in 2 mL of anhydrous acetone. 0.018 mL (0.15 mmol) of benzyl bromide were added and the mixture stirred for 24 h at room temperature. The salt was filtered and washed with acetone and THF and the solvent was concentrated under vacuum. A brown solid was obtained and purified by column chromatography (eluent DCM/MeOH 95:5) yielding a yellow solid: yield 65%. MP 151-4 °C (dec).

Chromatographic resolution of (±)-cis-1.

125 mg of chromatographically pure (±)-cis-1 (0.28 mmol) were dissolved in 5 mL of anhydrous THF; 87 mg of di-t-butyldicarbonate (0.40 mmol) were added and the solution stirred at room temperature for 6 h (Scheme 1). The solvent was evaporated to dryness and the crude residue purified on column chromatography (eluent ethyl acetate/n-hexane 1:1), giving (±)-cis-7 in quantitative yield.

The separation of the two enantiomers (+)-cis-7 and (-)-cis-7 was carried out with a Kromasil 5-

Enzyme inhibition assays

Inhibition of human acetylcholinesterase (hAChE) in phosphate buffer pH 8.0 was assessed by means of the classical Ellman's assay, 2 implemented on a 96-well plate procedure. 3 Butyrylcholinesterase (hBChE) was also assayed to assess AChE/BChE selectivity. Inhibition kinetics of (±)-cis-1 and its enantiomers were determined for hAChE (Table 1). Acetyl-or butyrylthiocholine iodide were used as substrate and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) as the chromophoric reagent. All enzymes and reagents were purchased from Sigma Aldrich Italy.

Solutions of tested compounds were prepared starting from 10 mM stock solutions in DMSO, that were diluted with aqueous assay medium to a final content of organic solvent always lower than 1%. ChE-catalyzed hydrolysis was followed by measuring the increase of absorbance at 412 nm every 30 sec for 5 min at 25 °C using a 96-well microplate reader Infinite M1000 Pro (Tecan, Milan, Italy). The concentration of compound which yielded 50% inhibition of the ChE activity (IC50) was calculated by non-linear regression of the response-log(concentration) curve, using GraphPad Prism (GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego, CA, USA). Kinetic studies were performed in the same incubation conditions, using six concentrations of substrate (from 0.033 to 0.200 mM) and four concentrations of inhibitor in a range comprising the IC50 value. Inhibition constants and kinetic parameters were calculated within the "Enzyme kinetics" module of Prism.

MTT assays.

Cytotoxicity of compound (±)-cis-1 was assayed on HepG2 human liver cancer cell line and on Madin-Darby canine kidney cells (MDCK-MDR1), by a MTT-based assay previously described. 4 Briefly, viable cells were seeded in a sterile 96-well plate Cell Culture Cluster (Corning, NY, USA)

and incubated with different concentrations of tested compounds for 2 h (on HepG2 cells), 24 and 72 h (on MDCK-MDR1 cells) at 37 °C in 5% CO2. At the end of incubation, the culture medium was replaced by a solution of MTT 0.5 mg/mL in PBS. After 4 h incubation at 37 °C in 5% CO2 the supernatants were aspirated and 100 µL of DMSO were added to each well. The absorbance at 570 nm was measured using a plate reader Victor V3 (Perkin-Elmer, Milan, Italy). Results are expressed as the percentage of MTT reduction respect to control cells. All experiments were carried out in sextuplicate and were repeated twice.

Permeability assays.

Blood-brain barrier permeability was estimated with a cellular model using transfected Madin-Darby canine kidney cells (MDCK-MDR1) expressing P-glycoprotein (P-gp), 5 as previously described. 6 Briefly, MDCK-MDR1 cells were seeded at a concentration of 1×10 5 cells per well on the apical side of Transwell inserts, in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin and 2 mM L-glutamine (EuroClone, Milan, Italy). Cells were maintained at 37 °C, 5% CO2, and saturated humidity.

Figure 1 .

 1 Figure 1. Ligand-based design of dual binding site inhibitors of AChE.

  ( a ) Reagents and conditions: (a), 4-chlorobutyryl chloride, THF, triethylamine, rt; (b), benzylamine, KI, acetone, rt; (c), N-Boc protected isonipecotic acid, HOBt, DIC, CH2Cl2, rt; (d), TFA, CH2Cl2, 0 °C. (e), benzyl bromide, K2CO3, acetone, rt; (f) Boc2O, THF, rt; (g) semi-preparative chiral HPLC.

Figure 2 .

 2 Figure 2. HepG2 cells viability, measured by the MTT assay, in the absence (black bar) and presence (grey

Figure 3 .

 3 Figure 3. Chromatograms of enantiopure samples obtained by chiral resolution of (±)-cis-7.

Figure 4 .

 4 Figure 4. Michaelis-Menten plot for inhibition of hAChE by MC1420 at various inhibitor concentrations. The

  mode is driven mainly by two π-π stacking interactions, that of the aromatic ring of the N-benzyl group of the ligand with the indole of Trp84 (Trp86 in hAChE) in the CAS, and of the coumarin ring with the indole moiety of Trp279 (Trp286 in hAChE) in the PAS. Regardless of the ligand's absolute configuration, the rings involved in these stacking interactions are almost parallel (interplanar angle < 15°) and display the typical parallel-displaced geometry. Interestingly, the interplanar distance between the coumarin group and Trp279 is about 0.5 Å shorter than that between Trp84 in the CAS and the N-benzyl group of the ligand.

Figure 5 .

 5 Figure 5. X-ray structure of the MC1420/TcAChE complex (PDB ID: 6TT0). Data refined for the 1R,3S-cis-

Figure 6 .

 6 Figure 6. Top-scored docking poses for the (1R,3S)-cis-(A) and (1S,3R)-cis-(B) configurational isomers of

  Scheme 1 a

  mixture was filtered and the solvent was removed by rotatory evaporation. The resulting oil was concentrated to dryness yielding a brown solid. Purification by column chromatography (eluent ethyl acetate/n-hexane 2:1) afforded 1-Boc-N-(6,7-dimethoxy-2H-2-oxochromen-3-yl)piperidine-4carboxamide as yellow solid: yield 98%. ESI-MS m/z: 455 [M +Na] + , 431 [M-H] -. 146 mg (0.27 mmol) of this intermediate were dissolved in 3 mL of a solution of TFA 50% in DCM at 0°C with stirring for 15 min. The reaction mixture was then warmed to room temperature and stirred for 5h.

AmyCoat ( 21 . 2 × 1 ( 5

 21215 250 mm) chiral stationary phase with isopropanol/n-hexane 1:1 v/v as the eluent (flow rate 15 mL/min; λ = 220 nm). (+)-7, tR = 9.8 min; [α]D 25 = + 34 deg•mL•dm -1 •g -1 (5 mg/mL; DCM). (-)-7, tR = 14.5 min; [α]D 25 = -34 deg•mL•dm -1 •g -1 (5 mg/mL; DCM).After separation, compounds (+)-7 and (-)-7 (48 mg each, 0.09 mmol) were deprotected by dissolving them in 3 mL of trifluoroacetic acid 10% in DCM at 0 °C. After the same workup described above for 5, pure final compounds were obtained in quantitative yield.1 H-NMR and ESI-MS spectra were fully consistent with those previously described for (±)-cis-1.1 Both free bases were crystallized as hydrochloride from a solution of HCl 1.25N in ethanol: MP 193-5 °C (dec). Analytical calculated % for C25H28N2O5•HCl•1.5 H2O C 60.06; H 6.45; N 5.60; found (+)-cis-1 C 60.04; H 6.19; N 5.51; (-)-cis-1 C 59.77; H 6.26; N 5.61. (+)-cis-1, [α]D 25 = + 50 deg•mL•dm -1 •g -mg/mL; DCM); (-)-cis-1, [α]D 25 = -50 deg•mL•dm -1 •g -1 (5 mg/mL; DCM).

Monolayer formation (approximately 8

 8 days after seeding) was monitored by microscopy, and the TEER (Trans Epithelial Electrical Resistance) was measured every day using an EVOM electrode epithelial volt-ohm meter (World Precision Instruments, Friedberg, Germany). Formation of tight junctions was assessed by measuring the flux of the transcellular standard diazepam (75 µM) and of paracellular standard fluorescein isothiocyanate-dextran (FD4, Sigma) (200 µg/mL) from the apical to the basolateral compartment. These probes were quantified with a PE double-beam UV-visible spectrophotometer Lambda Bio 20 (PerkinElmer, Milan, Italy) and a Victor3 fluorimeter (Wallac Victor3, 1420 Multilabel Counter, Perkin-Elmer) at excitation and emission wavelengths of 485 and

Figure S2 .

 S2 Figure S2. Top-scored docking poses of 4 (A) and 6 (B) within the binding site of TcAChE. The ligand

Table 1 .

 1 Inhibition data of title compounds. a

	Entry	IC50 (nM)	Ki hAChE (nM)
		hAChE	hBChE
	(±)-cis-1	36.5 ± 5.4	6250 ± 906	46.6 ± 3.6 (mixed)
	(+)-cis-1	380 ± 45	2330 ± 219	93.4 ± 3.2 (mixed)
	(-)-cis-1 (MC1420)	19.2 ± 3.0	14100 ± 205	19.1 ± 1.4 (mixed)
	4	748 ± 24	181 ± 7	n.d.
	6	223 ± 5	21 ± 2% b	n.d.
	Donepezil	16.1 ± 2.7	2900 ± 500	12.7 ± 1.0 (mixed)
	( a ) Values are mean ± SEM of three independent experiments; n.d.: not determined. ( b ) % inhibition at 10 µM.

Table 2 .

 2 Permeability assay data. a

Entry Papp AP (×10 -5 cm/s) Papp BL (×10 -5 cm/s) ER (Papp BL/ Papp AP)

	(±)-cis-1	3.7 ±1.2	0.84 ± 0.20	0.22
	Diazepam	2.0 ± 0.2	1.4 ± 0.2	0.70
	FD-4	0.69 ± 0.10	0.65 ± 0.12	0.93
	(

a ) Values are mean ± SEM of three independent experiments.

Table of Contents

 of Graphic (For Table of Contents Use Only)

	Chiral Separation, X-

ray Structure and Biological Evaluation of a Potent and Reversible Dual Binding Site AChE Inhibitor Marco

Catto*, Leonardo Pisani, Eugenio de la Mora, Benny Danilo Belviso, Giuseppe Felice Mangiatordi, Andrea Pinto, Annalisa De Palma, Nunzio Denora, Rocco Caliandro, Jacques-Philippe Colletier, Israel Silman, Orazio Nicolotti, Cosimo Damiano Altomare

Table S2 . Data collection and refinement statistics.

 S2

		MC1420
	PDB ID	6TT0
	Wavelength (Å)	1.074
	Resolution range	39.48 -2.8 (2.9 -2.8)
	Space group	P 31 2 1
	Unit cell	111.42 111.42 137.37 90 90
		120
	Total reflections	44657 (4385)
	Unique reflections	24409 (2405)
	Multiplicity	1.8 (1.8)
	Completeness (%)	98.06 (98.81)
	Mean I/sigma(I)	10.50 (1.57)

Supporting Information

535 nm, respectively. The apparent permeabilities (Papp), in units of cm/sec, in both directions, were calculated using the following equation S1: Papp=(Va/area×time)×([Drug]acceptor/[Drug]initial) (eq. S1)

where "Va" is the volume in the acceptor well, "area" is the surface area of the membrane, "time" is the total transport time, "[Drug] acceptor" is the concentration of the drug measured by UVspectroscopy and "[Drug] initial" is the initial drug concentration in the Apical (AP) or Basolateral (BL) chamber.

To assess the Papp AP or BL, across the BBB, (±)-cis-1 was added to the apical or to basolateral compartment of the Transwell model in 0.5 mL or 1.5 mL of PBS (EuroClone, Milan, Italy), respectively, at a concentration of 75 µM. The receiving compartments contained 1.5 mL or 0.5 mL of PBS, respectively. After 2 h of incubation, the medium in the apical and basolateral compartments were collected separately. (±)-cis-1 was quantified through UV-visible spectroscopy using the UV-visible spectrophotometer Lambda Bio 20 equipped with 10 mm path-length-matched quartz cells. Papp AP (cm/sec) and Papp BL (cm/sec) were calculated as described above. The efflux ratio (ER) was calculated using the following equation S2:

ER=(Papp, BL-AP)/(Papp, AP-BL) (eq. S2)

Papp, BL-AP: apparent permeability of basal-to-apical transport; Papp, AP-BL: apparent permeability of apical-to-basal transport. An efflux ratio greater than 2 indicates that a test compound is likely to be a substrate for P-gp transport.

Table S1. MDCK-MDR1 cell MTT assay. a

Entry % cell viability at [drug]= 100 µM IC50 (µM) 72 h 24 h

72 h (±)-cis-1 60 ± 2 37 ± 2 30.2 ± 1.6 ( a ) Values are mean ± SEM of three independent experiments.

Hydration calculations.

Hydration calculations were performed on MC1420 independently from crystallographic data, to identify protein-ligand interactions mediated by water molecules, by using WET, 7 a python script included in the AUTODOCK v. 4.2 docking suite. 8 The hydration procedure consists of the following steps: i) H-bond donors and acceptors in the ligand are recognized; ii) the ligand is 8 saturated with water molecules along H-bond vectors from the heavy atoms; iii) water molecules positions are checked against experimental occurrences. [9][10][11][12] The procedure provides 12 water molecules, two of them that are involved in H-bonds with oxygen of the amidic group of the ligand and N of the amidic group of Phe288. Such interactions are similar to that of the experimentally obtained W1 water molecule. Figure S1 shows that water molecules obtained by WET (cyan spheres) are close to W1 water molecule (red sphere).