Tetrahydrobenzo[ h][1,6]naphthyridine–6-chlorotacrine hybrids as a new family of anti-Alzheimer agents targeting β-amyloid, tau, and cholinesterase pathologies

a Laboratori de Química Farmacèutica (Unitat Associa da al CSIC), Facultat de Farmàcia, and Institut de Biomedicina (IBUB), Unive rsitat de Barcelona, Av. Joan XXIII, 27-31, E-08028, Barcelona, Spain b Departament de Fisicoquímica, Facultat de Farmàcia (C mpus Torribera), and IBUB, Universitat de Barcelona, Prat de la Riba 171 , E-08921, Santa Coloma de Gramenet, Spain c Departament de Fisicoquímica, Facultat de Farmàcia , and Institut de Nanociència i Nanotecnologia (INUB), Universitat de Barcelona, Av. Joan XXIII, 27-3 1, E-08028, Barcelona, Spain d Departament de Farmacologia, de Terapèutica i de T oxicologia, Institut de Neurociències, Universitat Autònoma de Barcelona, E -08193, Bellaterra, Barcelona, Spain e Laboratori de Química Orgànica, Facultat de Farmàc ia, Universitat de Barcelona, and Barcelona Science Park, Baldiri Reixac 10-12, E -08028, Barcelona, Spain


Introduction
Alzheimer's disease (AD) currently represents one of the most important unmet medical needs worldwide. Worryingly, because prevalence and mortality figures associated with AD will keep increasing, this condition will be even more pronounced in the upcoming decades, unless efficient disease-modifying drugs become available [1].
Current treatments for AD involve the use of acetylcholinesterase (AChE) inhibitors (donepezil, rivastigmine and galantamine) or NMDA receptor antagonists (memantine), which restore neurotransmitter deficits that are responsible for the symptomatic phase of the disease (cognitive, functional, and neuropsychiatric deficits) that appears a decade or more after the onset of the neurodegenerative process.
It is becoming increasingly apparent that the simultaneous modulation of several crucial targets that play early roles in the neuropathogenic process is a promising approach to derive effective drugs that can modify the course of AD [3,4]. Obviously, administration of these multitarget drugs depends on a precise knowledge of the timing of the critical neuropathologies to be hit and on the development of suitable biomarkers that enable timely therapeutic interventions and the assessment of their impact on AD progression. Whereas these important issues are addressed [2,5,6], the parallel development of multitarget drugs hitting different combinations of key biological targets should be actively pursued.
Neuropathologies related to the β-amyloid peptide (Aβ) and tau protein are thought to be at the root of the neurodegenerative process [2]. Also, AChE seems to play a role in the early phases of the disease, inasmuch as it can bind to Aβ, thereby accelerating its aggregation into oligomers and fibrils and increasing the neurotoxicity of Aβ aggregates [7]. The Aβ proaggregating action of AChE has been reported to reside in its peripheral anionic site (PAS) [7c], which is located at the mouth of a 20 Å deep gorge that leads to the catalytic anionic site (CAS) of the enzyme [8].
We have recently developed a new family of potent AChE inhibitors able to simultaneously bind at the CAS and PAS of the enzyme, i.e. dual binding site inhibitors, which were designed by molecular hybridization of the novel 3,4-dihydro-2Hpyrano [3,2-c]quinoline scaffold of ester 1 and the potent AChE CAS inhibitor 6chlorotacrine, 2 ( Fig. 1) [9]. The most potent hybrid of the series, compound 3 ( Fig. 1) retained the high human AChE inhibitory activity of the parent 6-chlorotacrine (3: IC 50 = 7 nM in human erythrocyte AChE; IC 50 = 19 nM in human recombinant AChE (hAChE)), and additionally exhibited a potent inhibitory activity of human butyrylcholinesterase (hBChE) and a weak Aβ antiaggregating activity (29% inhibition of AChE-induced Aβ40 aggregation at 100 µM and 21% inhibition of self-induced Aβ42 aggregation at 50 µM) [9].
Even though the 5-(4-chlorophenyl)-3,4-dihydro-2H-pyrano [3,2-c]quinoline moiety of 3 might establish π-π stacking interactions with the aromatic PAS residues Trp286 and Tyr72 (hAChE numbering), concomitant to the interactions of the 6-chlorotacrine unit at the CAS, the 5-(4-chlorophenyl)-3,4-dihydro-2H-pyrano [3,2-c]quinoline ester 1, used as synthetic precursor of 3, was found to be essentially inactive as AChE inhibitor. With the aim of optimizing this tricyclic scaffold as a PAS-binding moiety, we recently designed a family of tetrahydrobenzo[h] [1,6]naphthyridines, which mainly resulted from the substitution of the oxygen atom at position 1 of the 3,4-dihydro-2Hpyrano [3,2-c]quinoline system of ester 1 by an amine nitrogen atom [10]. The rationale behind this structural modification was that the increased basicity of the pyridine nitrogen atom would enable i) its protonation at physiological pH, and hence, ii) the establishement of cation-π interactions, apart from π-π stacking, with the aromatic PAS residues. Indeed, the most potent compound of the series, 4 ( Fig. 1), resulting from a double O → NH bioisosteric replacement from ester 1 at position 1 and at the side chain in position 9, exhibited a nanomolar hAChE inhibitory activity (IC 50 = 65 nM) [10].
Molecular dynamics (MD) simulations confirmed the expected binding of the tricyclic moiety of 4 at the AChE PAS (cation-π / π-π interactions with Trp286) and hydrogen bond between the protonated pyridine nitrogen atom and the hydroxyl group of the PAS residue Tyr72) as well as additional interactions of the amide function at position 9 with midgorge residues (hydrogen bond between the amide NH group and the hydroxyl group of Tyr124) [10].
Once the PAS-binding moiety had been optimized, we inferred that molecular hybridization with the CAS binder 6-chlorotacrine should provide further improvement of the AChE inhibitory activity. The disposition of the amide chain at position 9 of the tetrahydrobenzo[h] [1,6]naphthyridine 4 along the midgorge, with its nitrogen atom at a distance of ~6 Å from the position occupied by the exocyclic amino group of tacrine in its complex with Torpedo californica AChE (Fig. 2) [11], suggested that a linker of 3 methylenes should be optimal to connect both moieties retaining all their interactions with AChE residues all along the enzyme gorge. Thus, the tetrahydrobenzo[h] [1,6]naphthyridine-6-chlorotacrine hybrid 5a ( Fig. 1) was rationally designed as a novel multiple-site AChE inhibitor, bearing optimized PAS-binding moiety and tether length.
Herein, we describe the synthesis of the tetrahydrobenzo[h] [1,6]naphthyridine-6chlorotacrine hybrid 5a and its longer tetra-, penta-, and octa-methylene-linked homologues 5b-d, the evaluation of their inhibitory activities against hAChE and hBChE, and the study of their binding mode to hAChE by MD simulations. To further expand the potential multitarget profile of hybrids 5a-d, their inhibitory activities against the aggregation of Aβ42 and tau protein in intact Escherichia coli cells, as a simplified in vivo model of aggregation of amyloidogenic proteins, were also evaluated.
Moreover, the brain penetration of these hybrids, and therefore the ability to reach their targets at the central nervous system (CNS), was assessed by a parallel artificial membrane permeation assay (PAMPA-BBB). [1,6]naphthyridine-6-chlorotacrine hybrids Apart from the rationally designed trimethylene-linked hybrid 5a, we planned the synthesis of the longer tetra-and penta-methylene homologues 5b and 5c, still bearing relatively short linkers. We also envisioned the synthesis of the octamethylene-linked analogue 5d, mainly for comparison with the octamethylene-linked 3,4-dihydro-2Hpyrano [3,2-c]quinoline-based hybrid 3, to gain further insight into the effect of the O → NH bioisosteric replacement at position 1 of the tricyclic system, while keeping the same tether length.
Coupling of the carboxylic acid with the known aminoalkyltacrine derivatives 7a-d [14], using HOBt and EDC in the presence of Et 3 N in a 10:1 mixture EtOAc/DMF, followed by silica gel column chromatography purification afforded the expected N-Boc-protected amides, in some cases together with directly deprotected amides (5a and 5c). Treatment of the N-Boc-protected amides with 4 M HCl in dioxane at room temperature afforded the target amides 5a-d in 34-80% total overall yields from ester 6 (Scheme 1).
The novel tetrahydrobenzonaphthyridine-6-chlorotacrine hybrids 5a-d were fully characterized in the form of dihydrochloride salts through their spectroscopic data (IR, 1 H and 13 C NMR) and HRMS and their purity was assessed by elemental analysis. The biological characterization was also performed with the dihydrochloride salts.
Please, insert here Scheme 1.

Evaluation of AChE inhibitory activity
The inhibitory activity of the novel hybrids 5a-d against hAChE was evaluated by the method of Ellman et al. [15]. The 3,4-dihydro-2H-pyrano [3,2-c]quinoline derivatives 1 and 3, as well as 6-chlorotacrine, 2, were also evaluated under the same assay conditions as reference compounds. Also, the reported activity of compound 4 [10] was considered for comparison purposes.
All the tetrahydrobenzonaphthyridine-6-chlorotacrine hybrids turned out to be very potent inhibitors of hAChE (Table 1). Indeed, in agreement with the rational design Please, insert here Table 1.
Overall, hybrid 5a constitutes one of the most potent noncovalent inhibitors of hAChE so far reported, even though a few examples of other picomolar or even femtomolar inhibitors of AChE have also been described [14a,16].

Binding mode within AChE: Molecular modelling studies
To shed light on the structural basis of the surprisingly high AChE inhibitory activity determined for compound 5a, the binding mode to hAChE was explored by means of MD simulations, taking advantage of the X-ray crystallographic structure of the hAChE complex with huprine W (PDB entry 4BDT [17]). The initial pose of the ligand was guided by the structural information available for the binding mode of huprine X to Torpedo californica AChE (PDB entry 1E66 [18]), which matches well the structure of huprine W bound to hAChE, and by the recently reported binding mode of compound 4 [10].
The analysis of the 100 ns MD trajectory supported the structural integrity and stability of the ligand bound to the hAChE gorge. Thus, with the sole exception of a slight reorientation of the tetrahydrobenzo[h] [1,6]naphthyridine moiety in the PAS, which was originated from a conformational change in the loop defined by residues 289-292, the RMSD of the ligand remained fully stable during the last 60 ns of the trajectory (Fig.   S1, Supplementary Material). The hybrid 5a establishes a complex network of interactions with the residues of the binding site (Fig. 3). As expected, the 6chlorotacrine moiety was tightly bound in the CAS due to the cation-π interactions with the aromatic rings of Trp86 and Tyr337 (average distances of 3.9 Å between the indole or phenol rings and the aminoacridine unit) and the hydrogen bond of the protonated acridine nitrogen atom with the carbonyl oxygen of His447 (average distance of 2.9 Å).
On the other hand, the tetrahydrobenzo[h] [1,6]naphthyridine moiety was firmly stacked against Trp286, thus enabling the formation of a cation-π interaction between the protonated quinoline nitrogen atom and the indole ring of Trp286. The binding of this moiety was also assisted by transient hydrogen bond interactions between the NH group and the hydroxyl oxygen of Tyr72. The most remarkable finding concerns the interactions formed by the amide group in the tether, as the amide NH group is involved in a hydrogen bond with Asp74 (average distance of 3.6 Å), which is in turn hydrogenbonded to the hydroxyl group of Tyr341 (average distance of 2.8 Å), while the amide carbonyl oxygen forms either direct or water-mediated interactions with the hydroxyl group of Tyr337 (average distance of 4.9 Å). As a further test, the binding free energy of compound 5a was determined with the Solvated Interaction Energy (SIE) method [19], which relies on MM/PBSA calculations in conjunction with weighting scaling factors for the free energy components suitably parameterized to reproduce the experimental binding affinities for a diverse set of protein-ligand complexes. The SIE binding affinity obtained for compound 5a is -12.5 kcal/mol (Table S1, Supplementary Material), which is 4 kcal/mol lower than that determined for compound 4 (-8.5 kcal/mol [10]), which is in agreement with the ~10 4 ratio between the IC 50 values reported in Table 1.
These findings provide a basis to explain the abrupt change in inhibitory activity between compounds 5a and 5b, as the enlargement of the oligomethylene chain between tacrine and the amide group would disrupt the interactions with the midgorge residues. On the other hand, the amide group that is present in the tether of some tacrine-indole heterodimers reported by Muñoz-Ruiz et al. as picomolar AChE inhibitors was also suggested to participate in a complex network of interactions with midgorge residues [14a]. In the most potent tacrine-indole heterodimer the amide group, located at six methylene groups from the tacrine unit, was suggested to interact with Tyr124 and Tyr337. In hybrid 5a the shortening of the chain to three methylenes enables the formation of a distinct interaction pattern with Asp74 and Tyr337. Overall, these findings reinforce the significant contribution played by the midgorge in complementing both PAS and CAS and modulating the affinity of AChE inhibitors.

Butyrylcholinesterase inhibition
BChE is partly responsible for acetylcholine hydrolysis and hence, for the cholinergic deficit of AD patients, especially in advanced stages of the disease, when the levels of AChE in CNS markedly decrease. Thus, BChE also represents a biological target of interest for AD treatment [20]. The BChE inhibitory activity of hybrids 5a-d against hBChE was evaluated by the method of Ellman et al. [15].
The parent compounds 6-chlorotacrine, 2, and 4 are selective inhibitors of hAChE, albeit still exhibiting a moderately potent hBChE inhibitory activity, with IC 50 values in the submicromolar range. Not unexpectedly, hybrids 5a-d were also more potent against hAChE than hBChE, displaying submicromolar IC 50 values for hBChE inhibition ( Table 1). In this case, molecular hybridization resulted in an increased hBChE inhibitory activity relative to the parent compound 4 (3-8-fold) but in a slightly decreased potency relative to 6-chlorotacrine, 2, with the exception of hybrid 5a, which was equipotent to 6-chlorotacrine.

Aβ42 and tau aggregation inhibition
The aggregation of the amyloidogenic proteins Aβ, especially the most aggregationprone and neurotoxic 42 amino acid form thereof (Aβ42), and tau are widely thought to constitute early pathogenic events in AD, and hence, they are the target of many drug candidates purported to modify the natural course of the disease [21].
We have recently developed a new methodology for the evaluation of the effects of putative inhibitors on the aggregation and subsequent formation of insoluble inclusion bodies of any amyloidogenic protein that can be overexpressed in E. coli cells [22]. The method relies on monitoring the changes in the fluorescence of Thioflavin S (Th-S) that are produced upon binding to amyloid aggregates rich in β-sheet structures. Compounds that are able to cross the membranes of E. coli cells and inhibit the aggregation of overexpressed amyloidogenic proteins will lead to a decrease in the fluorescence of Th-S. This method is fast, simple and inexpensive, as it avoids the use of synthetic peptides.
We have successfully used this method for the evaluation of inhibitors of Aβ42 and tau aggregation. Interestingly, the results obtained in the screening of Aβ42 aggregation inhibitors correlated very well with the results previously reported from in vitro assays using synthetic peptides, thereby validating this methodology [22].
The inhibitory activity of hybrids 5a-d against Aβ42 and tau aggregation was assessed using this methodology. In general, very similar results and SAR trends were found for both activities. At 10 µM, hybrids 5a-d exhibited percentages of inhibition in the ranges 52-77% and 41-69% against Aβ42 and tau aggregation, respectively (Table 1).
Molecular hybridization led to increased Aβ42 and tau anti-aggregating activities, hybrids 5a-d being more potent than 6-chlorotacrine (5-7-fold more potent against Aβ42 aggregation and 30-50-fold more potent against tau aggregation). The parent compound 4 could not be tested in these assays, but in in vitro tests it had been found to be a weak inhibitor of Aβ42 aggregation (15% inhibition at 10 µM) [10], so presumably hybrids 5a-d are also more potent than 4.
The length of the linker seemed to have a subtle effect on Aβ42 and tau anti-aggregating activities. These activities slightly increased with the tether length so that the longer homologue 5d was 1.5-fold more potent than the shorter counterpart 5a for both activities. On the other hand, the O → NH bioisosteric replacement had a similar effect on both activities, the tetrahydrobenzo[h] [1,6]naphthyridine-based hybrid 5d being roughly 1.5-fold more potent than the 3,4-dihydro-2H-pyrano [3,2-c]quinoline-based hybrid 3 for Aβ42 and tau aggregation inhibition.
The parallel results obtained for these hybrids against both Aβ42 and tau aggregation further support the notion that diseases based on the pathological aggregation of one or several amyloidogenic proteins might share common mechanisms and might be confronted with common therapeutic interventions [23].
Overall, hybrids 5a-d can be considered as moderately potent dual Aβ42 and tau antiaggregating compounds, with IC 50 values that must lie around or below 10 µM. Because these anti-aggregating activities have been determined without involving the presence of AChE, the high AChE inhibitory activity of hybrids 5a-d cannot be responsible for their Aβ42 and tau anti-aggregating activities, which might be ascribed, instead, to a direct interaction with Aβ42 and tau. The precise mechanisms through which these hybrids bind Aβ42 and tau and/or exert their anti-aggregating activities are unknown, even though it has been reported that the presence of several aromatic moieties with extended π-conjugated systems (including biphenyls and phenyl-substituted benzoheteroaromatic systems, similar to the phenylquinoline moiety present in hybrids 5a-d) may enable binding to Aβ42 [24].
The dual Aβ42 and tau anti-aggregating profile is of much interest for diseasemodifying anti-Alzheimer agents. However, it must be recognized that the antiaggregating activities of hybrids 5a-d are not well balanced relative to their cholinesterase inhibitory activities, especially AChE and particularly in the case of its picomolar inhibitor 5a, which represents an important issue in multitarget compounds.

Blood-brain barrier permeation assay
Anti-Alzheimer drug candidates, like any other CNS drug, must be able to efficiently enter into the brain, which requires a good ability to cross the blood-brain barrier (BBB) and a low P-glycoprotein efflux liability [25]. The large molecular weight of hybrids 5a-d (>500) might compromise their ability to cross biological membranes, including BBB [26]. However, a number of distinct anti-Alzheimer hybrid compounds with molecular weights over 500 have shown good oral availability and/or brain permeability in ex vivo and in vivo studies in mice [27]. Indeed, the positive results obtained for the hybrids 5a-d in the aggregation studies in E. coli cells were already indicative of their ability to cross biological membranes, but a more accurate determination of their ability to cross the BBB was necessary. In this light, the brain permeability of the hybrids 5a-d was predicted using an in vitro model of passive transcellular permeation, namely the widely known PAMPA-BBB assay [28]. Thus, the in vitro permeability (P e ) through a lipid extract of porcine brain was determined using a mixture of phosphate-buffered saline (PBS)/EtOH (70:30). Assay validation was made by comparing the experimental and reported permeability values of 14 commercial drugs (Table S2, Supplementary Material), which provided a good linear correlation: P e (exp) = 1.5010 P e (lit) -0.8618 (R 2 = 0.9199). Using this equation and the limits established by Di et al. for BBB permeation [28], the following ranges of permeability were established: P e (10 −6 cm s −1 ) > 5.1 for compounds with high BBB permeation (CNS+); P e (10 −6 cm s −1 ) < 2.1 for compounds with low BBB permeation (CNS−); and 5.1 > P e (10 −6 cm s −1 ) > 2.1 for compounds with uncertain BBB permeation (CNS+/−).
All the tetrahydrobenzo[h] [1,6]naphthyridine-6-chlorotacrine hybrids, 5a-d, were predicted to be able to cross the BBB. The measured P e values for 5a-d were found to slightly increase with the tether length, and hence, with lipophilicity, and were clearly above the threshold for high BBB permeation (Table 1), thereby anticipating their ability to enter the brain and reach their different CNS targets.
Of note, the predicted in vitro ability of the novel hybrids to cross the BBB was also confirmed through the BBB permeation index obtained using a recently reported in silico multiclassification method (Table 2), which was developed utilizing a comprehensive data set containing around 12000 diverse compounds [29]. This method was also used to assess the intestinal absorption of the novel compounds, which was predicted to be positive in all cases. Finally, the predicted rat acute toxicity of the hybrids was clearly lower than that predicted for the anti-Alzheimer AChE inhibitor tacrine, thereby supporting the safety of these compounds ( Table 2).

Conclusion
In this work we have further advanced in the hit-to-lead optimization process that, starting from the 3,4-dihydro-2H-pyrano [3,2-c]quinoline carboxylic ester 1, had led to the potent hAChE inhibitors 3 [9] and 4 [10] by molecular hybridization with 6chlorotacrine and by double O → NH bioisosteric replacement, respectively. Herein, combination of the optimized AChE PAS-binding moiety present in 4, molecular hybridization with 6-chlorotacrine, and a MD-driven optimization of the tether length has led to the discovery of the 1,2,3,4-tetrahydrobenzo[h] [1,6]naphthyridine-6chlorotacrine hybrid 5a as a picomolar inhibitor of hAChE.
Apart from 5a, other three longer homologues, i.e. 5b-d, have been also synthesized and found to be potent inhibitors of hAChE, exhibiting IC 50 values in the low nanomolar range. Like the parent compounds 6-chlorotacrine, 2, and the tetrahydrobenzo[h] [1,6]naphthyridine 4, hybrids 5a-d have been found to be selective for hAChE vs. hBChE inhibition, albeit still keeping a potent hBChE inhibitory activity.
Very interestingly, these hybrids turned out to be moderately potent dual inhibitors of Torr (standard conditions) at least for 2 days and possess a purity ≥95% as evidenced by their elemental analyses and HPLC measurements. Of note, as previously reported for some tacrine-related dimeric compounds [31], the new hybrids herein described have the ability to retain molecules of water, which cannot be removed after drying the analytical samples under the aforementioned standard conditions. Thus, the elemental analyses of these compounds showed the presence of variable amounts of water, which have been indicated in the corresponding compound formulas.

. Determination of AChE and BChE inhibitory activities
The inhibitory activities against human recombinant AChE (Sigma-Aldrich) and human serum BChE (Sigma-Aldrich) were evaluated spectrophotometrically by the method of Ellman et al. [15].

PAMPA-BBB assay
To evaluate the brain penetration of hybrids 5, a parallel artificial membrane permeation assay for blood-brain barrier was used, following the method described by Di et al. [28]. The in vitro permeability (P e ) of fourteen commercial drugs through lipid extract of porcine brain membrane together with the test compounds was determined.
Commercial drugs and the synthesized compounds were tested using a mixture

Molecular modelling
4.3.1. Setup of the system. Molecular modelling was performed using the X-ray crystallographic structure of hAChE in complex with huprine W (PDB ID: 4BDT [15]).
The structure was refined by removal of N-acetyl-D-glucosamine and sulphate anions and addition of missing hydrogen atoms. The enzyme was modelled in its physiological active form with neutral His447 and deprotonated Glu334, which together with Ser203 form the catalytic triad. The ionization state for the rest of ionizable residues was assessed from PROPKA3 [33] calculations. Accordingly, the standard ionization state at neutral pH was considered but for residues Glu285, Glu450 and Glu452, which were protonated. Finally, three disulfide bridges were defined between Cys residues 257-272, 529-409, and 69-96, respectively.

Molecular dynamics simulations.
The binding mode of hybrid 5a to hAChE was explored by means of MD simulations. Starting from that proposed initial pose, a 100 ns MD simulation was performed using the PMEMD module of AMBER12 [34] software package, the parm99SB [35] force field for the protein and the GAFF-derived parameters [36] for the ligand, whose geometrical parameters were optimized at the B3LYP/6-31G(d) level [37] and its charge distribution was described by using electrostatic potential charges with the RESP procedure [38]. Na + cations were added to neutralize the negative charge of the system with the XLEAP module of AMBER12.
The system was immersed in an octahedral box of TIP3P [39] water molecules, preserving the crystallographic waters inside the binding cavity. The final system contained around 52000 atoms.
The geometry of the system was minimized in four steps. First, water molecules and counterions were refined through 7000 steps of conjugate gradient and 3000 steps of steepest descent algorithm. Then, the position of hydrogen atoms was optimized using 4500 steps of conjugate gradient and 500 steps of steepest descent algorithm. At the third stage, hydrogen atoms, water molecules and counterions were further optimized using 11500 steps of conjugate gradient and 2500 steps of steepest descent algorithm. Thermalization of the system was performed in five steps of 25 ps, increasing the temperature from 50 to 298 K. Concomitantly, the residues that define the binding site were restrained during thermalization using a variable restraining force. Thus, a force constant of 25 kcal mol -1 Å -2 was used in the first stage of the thermalization and was subsequently decreased by increments of 5 kcal mol -1 Å -2 in the next stages. Then, an additional step of 250 ps was performed in order to equilibrate the system density at constant pressure (1 bar) and temperature (298 K). Finally, a 100 ns trajectory was run using a time step of 2 fs. SHAKE was used for those bonds containing hydrogen atoms in conjunction with periodic boundary conditions at constant volume and temperature, particle mesh Ewald for the treatment of long electrostatic interactions, and a cutoff of 10 Å for nonbonded interactions. Moreover, in the initial 20 ns of the simulation the distance between the protonated nitrogen in the 6-chlorotacrine moiety of the inhibitor and the carbonyl oxygen of His447 was constrained to avoid artefactual rearrangements in the CAS of the enzyme.
The structural analysis was performed using in-house software and standard codes of AMBER12. The solvent interaction energies (SIE) technique developed by Purisima and co-workers [19] was used to estimate the interaction free energies for the AChE technical assistance in the HPLC purity measurements and in predicting safety data, respectively.

Appendix A. Supplementary material
Supplementary data related to this article can be found at http://dx.doi.org/. These data include data of the elemental analyses of hybrids 5a-d, additional results from the molecular modeling studies and PAMPA-BBB assay, as well as copies of the 1 H and 13 C NMR spectra of the tested compounds.  [1,6]naphthyridine-6chlorotacrine hybrids and reference compounds against AChE, BChE, Aβ42 and tau aggregation, and BBB predicted permeabilities.  [1,6]naphthyridine 4 in AChE with the X-ray structure of the complex Torpedo californica AChE-tacrine.  [1,6]naphthyridine-6-chlorotacrine hybrid 5a (magenta) in the average structure obtained from the snapshots sampled in the last 5 ns of the trajectory.
The residues involved in interactions are shown as green-coloured sticks.