Multitarget Drug Design Strategy: Quinone ... - ACS Publications

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Multitarget Drug Design Strategy: Quinone−Tacrine Hybrids Designed To Block Amyloid‑β Aggregation and To Exert Anticholinesterase and Antioxidant Effects Eugenie Nepovimova,†,‡,§,∥ Elisa Uliassi,† Jan Korabecny,‡,§ Luis Emiliano Peña-Altamira,† Sarah Samez,⊥,# Alessandro Pesaresi,⊥ Gregory E. Garcia,⊗ Manuela Bartolini,† Vincenza Andrisano,∇ Christian Bergamini,† Romana Fato,† Doriano Lamba,⊥ Marinella Roberti,† Kamil Kuca,§ Barbara Monti,† and Maria Laura Bolognesi*,† †

Department of Pharmacy and Biotechnology, Alma Mater StudiorumUniversity of Bologna, Via Belmeloro 6, I-40126 Bologna, Italy ‡ Department of Toxicology, Department of Public Health, Centre for Advanced Studies, Faculty of Military Health Sciences, University of Defence, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic § Biomedical Research Centre, University Hospital Hradec Kralove, Sokolska 581, 500 05 Hradec Kralove, Czech Republic ∥ Department of Pharmaceutical Chemistry and Drug Control, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic ⊥ Istituto di Crystallografia, Consiglio Nazionale delle Ricerche, Area Science Park-Basovizza, S.S. 14-Km 163.5, I-34149 Trieste, Italy # Dipartimento di Scienze Chimiche e Farmaceutiche, Università di Trieste, Via L. Giorgieri 1, I-34127 Trieste, Italy ⊗ Research Division, U.S. Army Medical Research Institute of Chemical Defense, 3100 Ricketts, Point Road, Aberdeen Proving Ground, Maryland 21010-5400, United States ∇ Department for Life Quality Studies, Alma Mater StudiorumUniversity of Bologna, Corso d’Augusto 237, I-47921 Rimini, Italy S Supporting Information *

ABSTRACT: We report the identification of multitarget anti-Alzheimer compounds designed by combining a naphthoquinone function and a tacrine fragment. In vitro, 15 compounds displayed excellent acetylcholinesterase (AChE) inhibitory potencies and interesting capabilities to block amyloid-β (Aβ) aggregation. The X-ray analysis of one of those compounds in complex with AChE allowed rationalizing the outstanding activity data (IC50 = 0.72 nM). Two of the compounds showed negligible toxicity in immortalized mouse cortical neurons Neuro2A and primary rat cerebellar granule neurons. However, only one of them was less hepatotoxic than tacrine in HepG2 cells. In T67 cells, both compounds showed antioxidant activity, following NQO1 induction. Furthermore, in Neuro2A, they were able to completely revert the decrease in viability induced by Aβ. Importantly, they crossed the blood-brain barrier, as demonstrated in ex vivo experiments with rats. When ex vivo results were combined with in vitro studies, these two compounds emerged to be promising multitarget lead candidates worthy of further pursuit.

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the AD field, with multitarget drugs and drug candidates currently entering the fray.9 Some of us pioneered this approach, and in 2007 we reported on memoquin (1 in Chart 1), one of the first small chemical entities rationally designed following a multitarget strategy.10 Its outstanding in vitro and in vivo biological profile strengthened the significance of such a strategy for obtaining valuable drug candidates against AD.11 Indeed, in vitro 1 effectively inhibited amyloid-β (Aβ) aggregation and acetylcholinesterase (AChE) and demonstrated potent free-radical

INTRODUCTION

The multitarget approach has been proposed as particularly suitable to combat the heterogeneity and the multifactorial nature of Alzheimer’s disease (AD).1 Characterized by amyloid plaques, neurofibrillary tangles, inflammatory intermediates, and reactive oxygen species (ROS), AD imposes neuronal death via a complex array of networked pathways.2 As a consequence, to be successful the therapeutic tools should be similarly complex, i.e., able to target multiple components of the diseased network.3−8 Building on this strong foundation, an ever-increasing number of multitarget drug discovery efforts have appeared in © XXXX American Chemical Society

Received: July 17, 2014

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dx.doi.org/10.1021/jm5010804 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Chart 1. Design Strategy toward Hybrids 12−29

scavenger activity.10 In vivo, 1 acted as a cognitive enhancer in several AD mouse models:12,13 the remarkable ability of memoquin to rescue both amyloid- and tau-related neurodegeneration further indicates the critical role of the multitarget mechanisms of action.12 Thanks to the unique profile, 1 inspired the development of several series of multitarget-directed ligands (MTDLs) with a therapeutic potential against AD. In an effort to identify derivatives with a lower molecular weight, we were interested in developing monovalent analogues, devoid of the dimeric features of 1.14 The positive results obtained for 2 (Chart 1) illustrated that a naphthoquinone linked to a tertiary benzylamine through a proper spacer were optimal anti-cholinesterase and anti-aggregating features.14 In fact, the protonated amino function allows recognition of Trp86 of the human acetylcholinesterase (hAChE) catalytic site, whereas the naphthoquinone, protruding toward the gorge entrance, allows recognition of Trp286 of the peripheral anionic site (PAS).14 At the same time 2 retained a promising anti-amyloid profile.14 Regarding the anti-aggregating activity, through these and several series of ad hoc designed molecules, we proposed quinone as an anti-amyloid privileged motif.15 Indeed, we demonstrated that it might interfere with several amyloid proteins, due to its possibility to form favorable hydrogen bond and π-stacking interactions.16 The results obtained from our investigations are in agreement with the work of Scherzer-Attali et al., who described naphthoquinone−tryptophan hybrids capable of effectively inhibiting Aβ oligomerization in vitro as well as in vivo.17 In addition, based on recent investigations indicating its capability to prevent cell death in cultured neurons,18 naphthoquinone vitamin K has been suggested as structural

basis for the design of novel neuroprotective agents.19 Collectively, all these data support the potential of a naphthoquinone substructure in chemical space relevant to neurodegenerative diseases. Motivated by these considerations and as a result of our interest in exploring original framework combinations, we propose herein a novel series of hybrids, rationally designed by linking the naphthoquinone moiety to a tacrine scaffold via a methylene spacer. Tacrine (3) was the first drug approved for the treatment of AD. Though its clinical use has been restricted by hepatotoxicity, it remains an important starting point for AD drug discovery endeavors, mainly due to its synthetic accessibility.20 In particular, 6-chlorotacrine 4, which showed an improved AChE inhibitory profile with respect to 3, has been widely exploited.21 The 7-methoxy derivative 5, was found to be still an active AChE inhibitor (AChEI) with significantly lower side effects compared to 3, probably due to a different metabolic fate.22 Thus, several tacrine hybrids, i.e., bifunctional molecules where a tacrine moiety has been chemically linked to another fragment with beneficial anti-AD properties, have been developed as MTDLs.23 For example, tacrine−lipoic acid (6),24 tacrine−melatonin,25 tacrine−ferulic acid,26 tacrine−8hydroxyquinoline hybrids,27 have all shown promising preclinical profiles, but many others have been reported (recently reviewed in23,28). Despite the abundance of tacrine hybrids in the literature, there remains no precedent for its linking to a naphthoquinone moiety. However, it should be mentioned that hybrid compounds bearing a tacrine unit or the closely related huprine B



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Scheme 1. General Method for Synthesis of Tacrine−Quinone Hybrids 12−29a

a

Reagents and conditions: (a) diaminoalkane, phenol, MW irradiation, 150 °C, 40 min; (b) CH2Cl2, RT, 12h

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CHEMISTRY Compounds 12−29 were synthesized following the convergent synthetic approach depicted in Scheme 1. The synthesis of 9chlorotacrines 10a-c has been previously described.36,37 All these chlorides were utilized as key starting materials for the production of intermediates 11a-f, by reaction with the appropriate alkylendiamines in phenol under microwave irradiation.38 Optimization by microwave irradiation enabled not only curtailment of reaction time, but also the reduction of equivalents of phenol from 9 (commonly used for the same reactions under the reflux conditions)39 to 2. The exploitation of this greener procedure afforded 11a−f in good to excellent yields (63−95%). Subsequent reaction of intermediates 11a−f with commercially available 1,4-naphthoquinone (7) and juglone (9), or 2,3-dichloro-1,4-naphthoquinone (8),17 provided the target compounds (12−29) in poor to moderate yields (6−61%). These molecules have been characterized by elemental analysis, NMR spectroscopy, and ESI mass spectrometry.

unit linked to an anthraquinone moiety have been recently published.29,30 In this paper, we report on the design, synthesis, and biological studies of tacrine−naphthoquinone hybrids 12−29 aimed to confront AD on a triple front: (i) by inhibiting AChE, (ii) by contrasting Aβ aggregation, (iii) by reducing oxidative stress. Furthermore, the X-ray crystal structure for complex with Torpedo californica AChE (TcAChE) of the best AChEI (20) was solved.

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DESIGN

In our previous investigations, we demonstrated that 2 had good anti-amyloid and anti-cholinesterase properties.14 The importance of an ethyl and a 2-methoxybenzyl group as substituents on the terminal protonable nitrogen was also confirmed.14 However, exploration of alternative groups with respect to the tertiary benzylamine that could interact with AChE and at the same time have a positive role against amyloid aggregation was never attempted. With this aim, we turned our attention to the scaffolds 3−5, widely exploited by others and us to obtain powerful multitarget lead candidates.31,32 Thus, we sought to combine the anti-cholinesterase activity of 3-5 with the documented inhibitory capability of quinones toward Aβ assembly and oxidative stress, by making the tacrine−quinone hybrids 12−29 depicted in Chart 1. Among the possible naphthoquinone moieties, the following ones were chosen (Scheme 1): (i) 1,4-naphthoquinone (7), recently shown to prevent also β-synuclein fibrillization;33 (ii) 2,3-dichloro-1,4naphthoquinone (dichlone, 8), successfully exploited for the synthesis of naphthoquinone-Trp hybrids;34 (iii) 5-hydroxy-1,4naphthoquinone (juglone, 9), which was reported possessing an optimal anti-AD profile.35 Docking simulations were preliminary carried out to set the optimal length of the linker connecting the two frameworks. Two to three methylenes were the distance leading to best positioning of the binding fragments within the AChE gorge, without excessive linker strain (Figure S1 in Supporting Information).

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RESULTS AND DISCUSSION We have fostered the development of MTDLs as the appropriate option for AD, a multifactorial malady that conventional single-target drugs cannot cure. The MTDLs developed in this report were directed toward three AD-related targets: amyloid toxicity, cholinergic deficit, and oxidative stress. A potential cross-talk among all the three targets has been proposed.40 Oligomeric Aβ is toxic in a variety of neuronal cell models, and the overexpression of Aβ in transgenic mice leads to increased neuronal oxidative stress.41 Oxidative stress, in turn, has been implicated as a contributing factor to neurodegeneration in AD and has a significant connection with the deficits of cholinergic system in the central nervous system (CNS).42 In a vicious cycle, impaired cortical cholinergic neurotransmission may also contribute to β-amyloid plaque pathology.42 Thus, it is conceivable that the proposed molecules, thanks to the simultaneous modulation of three inextricably intertwined pathways, should be more effective in contrasting AD, with a potential disease-modifying effect. C



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Table 1. Inhibition of Aβ Self-Aggregation and AChE and BChE Activities by 12−29 and Reference Compounds 1 and 3−5 inhibition of Aβ1−42 self-aggregation (%) ± SEM, [I] = 10 μMa 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 3 4 5 1

IC50 ± SEM (nM)b for hAChE

22.4 ± 3.1 32.9 ± 2.0 28.0 ± 2.8 31.1 ± 3.5 22.6 ± 5.5 41.2 ± 0.9 39.9 ± 0.9 37.9 ± 3.8 37.5 ± 4.9 not soluble 41.3 ± 2.9 41.6 ± 4.0 29.3 ± 0.4 40.6 ± 2.3 33.2 ± 0.9 52.8 ± 1.8 26.6 ± 1.3 34.8 ± 0.4 0.05) and 73.5 ± 5.13 (p < 0.05) for compound 20 at 12.5 μM, as compared to untreated cells. Considering both in vitro activity profile and neurotoxicity assays as a primary screening of the series, we have selected 16 and 20, which are endowed with good inhibitory activity and are the less neurotoxic derivatives, and investigated their antiAD properties in two cell models. Protective Effects of 16 and 20 on Aβ1−42-Induced Neurotoxicity and tert-Butyl Hydroperoxide-Induced Oxidative Stress. To determine whether 16 and 20 may exert neuroprotective effects, we evaluated their ability to protect neurons against two different harmful stimuli: (i) Aβ and (ii) oxidative stress. We tested whether 16 and 20 could prevent or reduce mitochondrial damage in differentiated N2A cells incubated with Aβ1−42. Significantly decreased levels of cell viability were found in N2A cells (p < 0.001; Figure 3) treated with 10 and 20

Figure 4. Effect of 16 and 20 on ROS formation in T67 cells. The antioxidant activity was evaluated against ROS induced by 100 μM tert-butyl hydroperoxide (TBH) and detected following 2′,7′dichlorodihydrofluorescein diacetate (DCFDA) oxidation. Experiments were performed with T67 cells treated or not with 2.5 μM sulforaphane (S) for 24 h. Data are expressed by mean ± standard deviation. Significance was determined by ANOVA and Tukey posttest between [+TBH] vs [20+S+TBH] (**, p ≤ 0.05) and [+TBH] vs [16+S+TBH] (***, p ≤ 0.001).

several other quinone derivatives.13,56,57 Quinones, chemically speaking, cannot act as antioxidants, rather they could be prooxidants. In fact, we have previously demonstrated that the antioxidant property of 1, in analogy with endogenous ubiquinones,58 pertains mainly to its hydroquinone form. NQO1, an inducible enzyme that catalyzes the two-electron reduction of a variety of natural quinones to hydroquinones, was shown to be involved in the conversion of 1 into the moreantioxidant hydroquinone form.56 The same might apply to 16 and 20, which share the quinone nucleus. Remarkably, NQO1 levels are increased in AD patients, in response to the oxidative stress typical of the pathology.59,60 To mimic the pathological increase of NQO1 in TBH-stressed T67 cells, they were treated with sulforaphane, one of the most potent NQO1 inducer known to date. Figure 4 illustrates that 16 and 20 (at 10 μM) in their oxidized quinone form show a negligible antioxidant activity. This activity was significantly increased in cells pretreated with sulforaphane, confirming that NQO1 is involved also in the bioactivation of these compounds. In particular, treatment with 20 completely suppressed TBHinduced intracellular ROS production, indicating the putative antioxidant properties of this compound. Ex Vivo AChE Inhibition. To preliminary evaluate the CNS penetration of the synthesized compounds, 16 and 20 were subjected to an ex vivo determination of their central AChE inhibitory activity. In this assay, 16 and 20 were administered intraperitoneally (ip) to Wistar rats at two doses of 10 and 50 μmol/kg, 1 h before the animals were sacrificed, and the percentage of brain AChE inhibition versus untreated controls was measured. The results depicted in Figure 5A showed that both 16 and 20 provide dose-dependent inhibition of cholinesterase activity in telencephalon at 1 h postdosing, beginning at 10 mg/kg. As expected, in the cerebellum (Figure

Figure 3. Neuroprotection by 12.5 μM of 16 (A) and 20 (B) on differentiated N2A exposed for 24 h to increasing concentrations (0, 5, 10, and 20 μM) of previously aggregated Aβ. Results are expressed as MTT absorbance at 570 nm and are the mean ± SE of three different experiments run in quadruplicate. *p < 0.05 and ***p < 0.001 compared to controls; #p < 0.05, ###p < 0.001 compared to the same concentration of Aβ only, Bonferroni’s posthoc test after two-way ANOVA.

μM of previously aggregated Aβ. Cell viability was significantly increased in N2A cells treated with 16 and 20 at 12.5 μM and then incubated with Aβ, compared to N2A cells only incubated with Aβ. This marked protective effect indicates that 16 and 20 are able to completely revert the decrease in cell viability that Aβ causes (at an almost equimolar ratio). The antioxidant properties of 16 and 20 were estimated through their ROS scavenging effects against human glioma T67 exposed to high levels of tert-butyl hydroperoxide (TBH) (100 μM), and in the absence or presence of pretreatment with sulforaphane (Figure 4). We developed this protocol as a suitable model for unveiling the antioxidant activity of 1 and G



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disease-relevant targets offer a promising approach to the treatment of a multifactorial disease such as AD. MTDLs reported herein have been designed to act on a carefully selected set of primary AD targets. Based on the available biological information, the concomitant inhibition of these targets should produce, in vivo, improved response. The novel hybrids have been created by combining the core components of two leading series of MTDLs, namely naphthoquinone- and tacrine-based ones. Several resulting derivatives showed a very high level of inhibition against AChE in vitro, with 20 showing a remarkable IC50 value of 0.72 nM. The crystal structure showed that the tight binding of 20 with respect to the other derivatives may arise from the presence of (i) the chlorine atom on the tacrine ring interacting through hydrophobic interactions at the CAS; (ii) the linker mostly involved in water-mediated hydrogen-bonding interactions; and (iii) the juglone fragment that nicely accommodates, via van der Waals interactions, within the narrow bottleneck of the AChE gorge. The molecules were then tested against amyloid aggregation. All compounds were active at a concentration of 10 μM, with 27 being the most effective. Finally, in light of the in vitro profiles, nontoxic derivatives 16 and 20 were tested for their neuroprotective activity in two brain cell models. The effectiveness of these molecules as neuroprotective agents might lie in their ability to counteract the ROS-producing neurotoxic effect of Aβ. Importantly, through ex-vivo experiments, we could advance for 16 and 20 BBB permeation and their potential to access their multiple biological targets in the CNS. As a final remark, a comment is deserved on the in vivo relevance of the developed MTDLs 16 and 20. Indeed, their active concentrations in the in vitro and in cell culture amyloidrelated assays are much higher (10 and 12.5 μM) than those necessary to inhibit hAChE (in the nanomolar/subnanomolar range). The same applies to the antioxidant activity (micromolar range). Nevertheless, these data need to be “normalized” on the basis of (i) the amount of target used in the assays and (ii) the physiological/pathological levels of the target itself. As an example, a micromolar concentration of Aβ (10−50 μM) is required to have both amyloid aggregation in a suitable time frame and toxic effects in N2A cells, while in AD-afflicted brain, the Aβ concentration is estimated to be in the nanomolar/ subnanomolar range.62 Therefore, on the basis of the data obtained, it seems plausible that, provided a suitable pharmacokinetic and proper BBB crossing, 16 and 20 may access their inhibitory activity at nanomolar concentration. However, only proof-of-concept by means of in vivo experiments can provide a definite answer to these questions, which are critical in the development of efficacious MTDLs. In conclusion, this work clearly validates the multitarget strategy and demonstrates that structurally novel and distinctive tacrine hybrids with improved AD-profiles can be designed. It is now warranted to further develop these hybrid compounds and explore their full potential for studying and treating AD. In particular, relevant issues such as activity balancing and toxicity should be carefully addressed in the subsequent hit to lead optimization studies.

Figure 5. Ex vivo determination of AChE inhibitory activity of 16 and 20 in telencephalon (A) and, as a negative control, cerebellum (B), after 1 h from i.p. injection (10 and 50 μmol/kg). AChE activity is expressed as mmol/gr prot/h and is the mean ± SE of values obtained from tissues of the two hemispheres from three different animals. *p < 0.05 and ***p < 0.001 compared to controls, Student’s t test.

5B), which produces catalytic inactive AChE,61 activity was not affected. The present experiments suggest that 16 and 20 can cross diffusion barriers associated with the brain and the cell wall to get to the AChE active site. Hepatotoxicity of 16 and 20 on HepG2 Cells. In addition to activity, neurotoxicity, and blood-brain barrier (BBB) permeation, hepatotoxicity would be of critical importance for the drug-likeness of 16 and 20. Indeed, as highlighted before, 3 has been withdrawn from the market for its hepatotoxicity. To this end, preliminary experiments were performed on 16 and 20 in a human hepatoma cell line (HepG2) in comparison with 3 (Figure 6). After 24 h

Figure 6. Toxicity of 3, 16, and 20 to HepG2 cells after 24 h treatment. Results are expressed as percentage of controls and are the mean ± SE of three different experiments run at least in quadruplicate. *p < 0.05 and ***p < 0.001 compared to controls; # p < 0.05 compared to 3, Student’s t test.

incubation at 0−50 μM, a concentration-dependent decrease in the cell viability was observed for 20 and 3, whereas no dramatic variation was found for 16. Encouragingly, 16 showed a superior hepatotoxicity profile with respect to 3.

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CONCLUSIONS By recognizing the heterogeneity and the multifactorial nature of the disease, single chemical entities directed to multiple

EXPERIMENTAL SECTION

Chemistry. General Chemical Methods. Chemical reagents were purchased from Sigma-Aldrich, Fluka and Lancaster (Italy). Chromatographic separations were performed using silica gel columns

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Article

by flash (silica gel 40, 0.0400.063 mm; Merck) chromatography. CEM Discover SP focused microwave reactor was used for microwavemediated reactions. The course of the reactions was observed by thin layer chromatography (TLC) on Merck (0.25 mm) glasspacked precoated silica gel plates (60 F254), then visualized with an UV lamp. Melting points were determined on a Gallenkamp melting point apparatus and are uncorrected. NMR spectra were recorded on Varian V XR 200 and MR 400 instruments, respectively. Chemical shifts are reported in parts per millions (ppm) relative to tetramethylsilane (TMS), and spin multiplicities are given as s (singlet), br s (broad singlet), d (doublet), t (triplet), q (quartet), or m (multiplet). Lowresolution mass spectra ESI-MS were recorded on a Waters ZQ 4000 apparatus. From all new compounds satisfactory elemental analyses were obtained, confirming >95% purity. General Procedure for Synthesis of Intermediates (11a−f). 9Chlorotacrine (10a,b, 2.30 mmol), alkylenediamine (9.19 mmol), and phenol (0.43 g, 4.59 mmol) were charged in a pressure-tight microwave tube containing a stirring bar. The reaction mixture was submitted to microwave irradiation for 40 min at 150 °C, with an irradiation power of 150 W. To the reaction mixture was added 8 mL of 10% water solution KOH, and the mixture was extracted with 30 mL of CH2Cl2. The organic layer was washed with brine and water and dried over Na2SO4. Excess solvent was evaporated under reduced pressure. A crude residue was purified by flash chromatography (ethyl acetate/methanol/33% water solution of NH3; 9.5:0.5:0.3) to afford 9aminoalkyleneamino-1,2,3,4-tetrahydroacridine as yellow oil. Analytical data of intermediates 11a−f are in good agreement with the literature data.37,39,63 General Procedure for Synthesis of Tacrine−Naphthoquinone Hybrids (12−29). Intermediate (11a−f, 2.49 mmol) was dissolved in 2 mL of CH2Cl2. Also appropriate naphthoquinone (2.98 mmol) was dissolved in 2 mL of CH2Cl2. The solution containing intermediate (11a−f) was added dropwise to the solution of naphthoquinone and was allowed to stir for 12 h at room temperature. The excess solvent was evaporated. A crude residue was purified by flash-chromatography (ethyl acetate/methanol; 9.5:0.5). The residue was recrystallized with CHCl3/hexane to give final compound as orange solid. 2-[2-(7-Methoxy-1,2,3,4-tetrahydroacridin-9-yl-amino)ethyl]amino-1,4-naphthoquinone (12). Yield 28%; mp 177−179 °C. 1H NMR (CDCl3): δ (ppm) 8.05 (d, 1H, J = 7.2 Hz), 8.00 (d, 1H, J = 8.0 Hz), 7.79 (d, 1H, J = 9.2 Hz), 7.71 (q, 1H, J = 12.2 Hz) 7.58 (q, 1H, J = 10.6 Hz), 7.19−7.15 (m, 2H), 6.20 (bs, 1H), 5.73 (s, 1H), 3.83 (s, 3H), 3.66 (t, 2H, J = 6.0 Hz), 3.41 (q, 2H, J = 11.8 Hz), 3.00 (t, 2H, J = 5.8 Hz), 2.72 (t, 2H, J = 5.8 Hz), 1.86 (t, 4H, J = 3.0 Hz). 13C NMR (CDCl3): δ (ppm) 183.11, 181.68, 156.81, 156.21, 147.91, 135.00, 133.53, 132.32, 130.55, 130.03, 126.48, 126.36, 121.71, 120.99, 101.60, 100.95, 55.67, 46.27, 43.29, 33.44, 25.20, 23.04, 22.81. ESI-MS: m/z 428 [M+]. 2-[3-(7-Methoxy-1,2,3,4-tetrahydroacridin-9-yl-amino)propyl]amino-1,4-naphthoquinone (13). Yield 27%; mp 160−162 °C. 1H NMR (CDCl3): δ (ppm) 8.09 (q, 1H, J = 7.6 Hz), 8.02 (q, 1H, J = 7.6 Hz), 7.82 (d, 1H, J = 9.2 Hz), 7.75−7.71 (m, 1H) 7.63−7.59 (m, 1H), 7.23 (q, 1H, J = 8.8 Hz), 7.15 (d, 1H, J = 2.8 Hz), 5.73 (s, 1H), 3.85 (s, 3H), 3.48 (t, 2H, J = 6.8 Hz), 3.34 (d, 2H, J = 6.0 Hz), 3.04 (s, 2H), 2.75 (s, 2H), 2.04 (q, 2H, J = 14.8 Hz), 1.92−1.89 (m, 4H). 13C NMR (CDCl3): δ (ppm) 183.07, 181.87, 156.57, 156.53, 149.09, 147.94, 143.46, 134.99, 133.67, 132.23, 130.58, 126.46, 126.37, 121.81, 120.53, 118.80, 101.30, 101.23, 55.63, 46.66, 40.71, 33.87, 30.10, 25.05, 23.15, 22.93. ESI-MS: m/z 442 [M+]. 2-[2-(1,2,3,4-Tetrahydroacridin-9-yl-amino)ethyl]amino-1,4naphthoquinone (14). Yield 13%; mp 185−187 °C. 1H NMR (CDCl3): δ (ppm) 8.01 (d, 1H, J = 3.8 Hz), 7.94−7.85 (m, 3H), 7.67−7.63 (m, 1H), 7.56−7.46 (m, 2H), 7.31−7.27 (m, 1H), 5.69 (s, 1H), 4.50 (bs, 1H), 3.72 (d, 2H, J = 5.2 Hz), 3.40 (q, 2H, J = 11.8 Hz), 2.99 (t, 2H, J = 6.0 Hz), 2.67 (t, 2H, J = 5.8 Hz), 1.83−1.78 (m, 4H). 13 C NMR (CDCl3): δ (ppm) 183.13, 181.69, 149.33, 147.86, 135.03, 133.54, 132.35, 130.55, 129.29, 128.71, 126.52, 126.40, 124.71, 122.06, 118.35, 101.70, 46.78, 43.47, 34.17, 25.15, 23.08, 22.87. ESI-MS: m/z 397 [M+].

2-[3-(1,2,3,4-Tetrahydroacridin-9-yl-amino)propyl)amino-1,4naphthoquinone (15). Yield 28%; mp 154−156 °C. 1H NMR (CDCl3): δ (ppm) 8.10 (q, 1H, J = 7.4 Hz), 8.03 (q, 1H, J = 7.8 Hz), 7.92 (q, 2H, J = 12.4 Hz), 7.75−7.71 (m, 1H), 7.64−7.60 (m, 1H), 7.56 (s, 1H), 7.36 (s, 1H), 5.74 (s, 1H), 3.60 (t, 2H, J = 6.8 Hz), 3.35 (d, 2H, J = 6.0 Hz), 3.08 (s, 2H), 2.72 (s, 2H), 2.08 (q, 2H, J = 14.6 Hz), 1.91 (s, 4H). 13C NMR (CDCl3): δ (ppm) 183.07, 181.86, 147.94, 134.98, 133.66, 132.24, 130.60, 129.32, 126.46, 126.38, 124.59, 122.66, 101.31, 46.66, 40.39, 30.07, 29.84, 25.01, 22.91, 22.45. ESIMS: m/z 412 [M+]. 2-[2-(6-Chloro-1,2,3,4-tetrahydroacridin-9-yl-amino)ethyl]amino-1,4-naphthoquinone (16). Yield 14%; mp 197−199 °C. 1H NMR (CDCl3): δ (ppm) 8.05 (q, 2H, J = 21.4 Hz), 7.87 (d, 1H, J = 2.0 Hz), 7.81 (d, 1H, J = 8.8 Hz), 7.73 (t, 1H, J = 7.4 Hz) 7.62 (t, 1H, J = 7.4 Hz), 7.29 (q, 1H, J = 9.2 Hz), 5.57 (bs, 1H), 5.74 (s, 1H), 3.99 (bs, 1H), 3.72 (d, 2H, J = 5.2 Hz), 3.41 (q, 2H, J = 11.6 Hz), 3.01 (d, 2H, J = 5.6 Hz), 2.70 (d, 2H, J = 5.6 Hz), 1.88 (t, 4H, J = 3.2 Hz). 13C NMR (CDCl3): δ (ppm) 183.11, 181.66, 160.44, 149.46, 147.80, 135.06, 134.40, 133.50, 132.41, 130.51, 128.24, 126.54, 126.42, 125.41, 123.71, 119.23, 118.59, 118.38, 101.80, 46.92, 43.48, 34.21, 29.84, 25.01, 22.96. ESI-MS: m/z 432 [M+], 434 [M+ + 2], 454 [M+ + Na]. 2-[3-(6-Chloro-1,2,3,4-tetrahydroacridin-9-yl-amino)propyl]amino-1,4-naphthoquinone (17). Yield 13%; mp 121−123 °C. 1H NMR (CDCl3): δ (ppm) 8.07 (d, 1H, J = 7.6 Hz), 8.01 (d, 1H, J = 7.6 Hz), 7.83 (q, 2H, J = 17.0 Hz), 7.71 (t, 1H, J = 7.4 Hz) 7.60 (t, 1H, J = 7.6 Hz), 7.25 (q, 1H, J = 9.2 Hz), 5.96 (bs, 1H), 5.70 (s, 1H), 3.53 (t, 2H, J = 6.6 Hz), 3.30 (q, 2H, J = 12.8 Hz), 3.01 (s, 2H), 2.67 (s, 2H), 2.07−2.00 (m, 2H), 1.89 (t, 4H, J = 3.0 Hz). 13C NMR (CDCl3): δ (ppm) 183.07, 181.84, 160.16, 150.15, 147.83, 135.01, 134.29, 133.63, 132.26, 130.56, 128.05, 126.48, 126.39, 125.03, 124.02, 118.98, 117.46, 101.35, 47.04, 40.39, 34.19, 30.20, 24.93, 22.99, 22.74. ESI-MS: m/z 446 [M+], 448 [M+ + 2]. 2-[2-(1,2,3,4-Tetrahydroacridin-9-yl-amino)ethyl]aminojuglone (18). Yield 10%; mp 171−173 °C. 1H NMR (CDCl3): δ (ppm) 12.90 (s, 1H), 7.89 (q, 2H, J = 16.6 Hz), 7.56 (q, 2H, J = 12.6 Hz), 7.47 (t, 1H, J = 7.8 Hz), 7.37 (t, 1H, J = 7.6 Hz), 7.25 (s, 1H), 6.20 (bs, 1H), 5.63 (s, 1H), 3.73 (t, 2H, J = 5.6 Hz), 3.43 (q, 2H, J = 11.4 Hz), 3.05 (d, 2H, J = 6.0 Hz), 2.73 (d, 2H, J = 6.0 Hz), 1.90 (s, 4H). 13C NMR (CDCl3): δ (ppm) 188.92, 180.71, 161.08, 158.63, 149.39, 148.41, 146.80, 134.11, 130.30, 128.75, 126.02, 124.62, 121.93, 120.50, 119.18, 117.88, 114.69, 100.37, 46.44, 43.32, 33.63, 25.00, 22.83, 22.55. ESIMS: m/z 414 [M+]. 2-[3-(1,2,3,4-Tetrahydroacridin-9-yl-amino)propyl]aminojuglone (19). Yield 6%; mp 162−164 °C. 1H NMR (CDCl3): δ (ppm) 12.97 (s, 1H), 7.88 (q, 2H, J = 7.4 Hz), 7.56−7.51 (m, 2H), 7.44 (q, 1H, J = 8.4 Hz), 7.36−7.32 (m, 1H), 7.24−7.21 (m, 1H), 6.26 (bs, 1H), 5.59 (s, 1H), 3.57 (t, 2H, J = 7.0 Hz), 3.33 (q, 2H, J = 12.4 Hz), 3.04 (d, 2H, J = 5.6 Hz), 2.70 (s, 2H), 2.08−2.01 (m, 2H), 1.88 (t, 4H, J = 3.2 Hz). 13C NMR (CDCl3): δ (ppm) 189.06, 181.06, 161.26, 158.34, 150.47, 148.61, 134.16, 130.52, 128.94, 128.36, 126.17, 124.51, 122.41, 120.34, 119.27, 117.05, 114.95, 100.14, 46.69, 40.55, 33.61, 30.04, 25.06, 23.01, 22.67. ESI-MS: m/z 428 [M+]. 2-[2-(6-Chloro-1,2,3,4-tetrahydroacridin-9-yl-amino)ethyl]aminojuglone (20). Yield 23%; mp 169−171 °C. 1H NMR (CDCl3): δ (ppm) 12.87 (s, 1H), 7.85 (d, 1H, J = 2.0 Hz), 7.80 (t, 1H, J = 6.4 Hz), 7.53 (t, 1H, J = 3.6 Hz), 7.44 (q, 1H, J = 10.8 Hz), 7.28−7.21 (m, 2H), 6.23 (bs, 1H), 5.59 (s, 1H), 3.72 (q, 2H, J = 11.8 Hz), 3.41 (q, 2H, J = 11.6 Hz), 2.99 (d, 2H, J = 5.6 Hz), 2.66 (q, 2H, J = 12.4 Hz), 1.87 (t, 4H, J = 3.2 Hz). 13C NMR (CDCl3): δ (ppm) 189.06, 180.82, 161.25, 148.50, 134.31, 130.41, 126.23, 125.46, 123.74, 119.37, 114.79, 100.61, 46.72, 43.47, 33.81, 25.03, 22.87, 22.56. ESI-MS: m/z 448 [M+], 450 [M+ + 2]. 2-[3-(6-Chloro-1,2,3,4-tetrahydroacridin-9-yl-amino)propyl]aminojuglone (21). Yield 17%; mp 180−182 °C. 1H NMR (CDCl3): δ (ppm) 12.94 (s, 1H), 7.83 (q, 2H, J = 21.0 Hz), 7.55 (q, 1H, J = 7.2 Hz), 7.45 (t, 1H, J = 7.8 Hz), 7.27−7.22 (m, 2H), 6.17 (bs, 1H), 5.58 (s, 1H), 3.54 (t, 2H, J = 7.0 Hz), 3.32 (q, 2H, J = 13.0 Hz), 3.01 (s, 2H), 2.67 (s, 2H), 2.07−2.00 (m, 2H), 1.89 (t, 4H, J = 3.0 Hz). 13C NMR (CDCl3): δ (ppm) 189.05, 181.03, 161.27, 150.34, 148.53, 134.53, 134.19, 130.48, 127.55, 126.21, 125.12, 124.04, 119.30, 118.75, I



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2.73 (s, 2H), 1.88 (t, 4H, J = 3.0 Hz). 13C NMR (CDCl3): δ (ppm) 180.12, 176.81, 158.92, 149.65, 147.25, 144.23, 135.10, 132.79, 132.54, 129.82, 128.95, 128.76, 127.04, 126.96, 124.63, 122.09, 120.69, 118.00, 49.27, 45.79, 33.98, 25.22, 23.05, 22.81. ESI-MS: m/z 432 [M+], 434 [M+ + 2]. 2-Chloro-3-[3-(1,2,3,4-tetrahydroacridin-9-yl-amino)propyl]amino-1,4-naphthoquinone (29). Yield 16%; mp 122−124 °C. 1H NMR (CDCl3): δ (ppm) 8.11 (q, 1H, J = 7.8 Hz), 7.97 (q, 1H, J = 7.8 Hz), 7.90−7.83 (m, 2H), 7.69 (q, 1H, J = 7.8 Hz), 7.60 (q, 1H, J = 7.6 Hz), 7.50 (s, 1H), 7.32 (s, 1H), 6.10 (bs, 1H), 3.94 (d, 2H, J = 6.8 Hz), 3.57 (t, 2H, J = 6.8 Hz), 3.03 (s, 2H), 2.71 (s, 2H), 2.03 (t, 2H, J = 7.0 Hz), 1.89 (q, 4H, J = 6.4 Hz). 13C NMR (CDCl3): δ (ppm) 180.47, 176.86, 158.79, 150.26, 147.38, 144.19, 135.14, 132.72, 132.66, 129.84, 128.90, 128.61, 127.01, 126.98, 124.25, 122.43, 120.51, 117.08, 46.46, 42.71, 34.01, 33.06, 25.10, 23.11, 22.84. ESI-MS: m/z 446 [M+], 448 [M+ + 2]. Inhibition of Human AChE and BChE. The method of Ellman et al. was followed.45 A Sunrise multichannel spectrophotometer (Tecan, Salzburg, Austria) was used for all measurements of cholinesterase activity. Photometric 96-well microplates made from polystyrene (Nunc, Rockilde, Denmark) were used for measurement purposes. Human recombinant AChE (hAChE) and human plasmatic BChE (hBChE) (Aldrich; commercially purified by affinity chromatography) were suspended into phosphate buffer (pH 7.4) up to final activity 0.002 U/μL. Cholinesterase (5 μL), solution of 0.4 mg/mL 5,5′dithio-bis(2-nitrobenzoic) acid (40 μL), 1 mM acetylthiocholine chloride in phosphate buffer (20 μL), and an appropriate concentration of inhibitor (1 mM−0.1 nM; 5 μL) were injected per well. Absorbance was measured at 412 nm after 5 min incubation using automatic shaking of the microplate. Percentage of inhibition (I) was calculated from the measured data as follows:

117.19, 114.91, 100.21, 46.85, 40.47, 33.81, 30.13, 24.93, 22.93, 22.60. ESI-MS: m/z 462 [M+], 464 [M+ + 2]. 2-[2-(7-Methoxy-1,2,3,4-tetrahydroacridin-9-yl-amino)ethyl]aminojuglone (22). Yield 15%; mp 184−186 °C. 1H NMR (CDCl3): δ (ppm) 12.90 (s, 1H), 7.88 (d, 1H, J = 9.6 Hz), 7.55 (q, 1H, J = 7.4 Hz), 7.45 (q, 1H, J = 8.2 Hz), 7.24−7.18 (m, 2H), 7.13 (d, 1H, J = 2.4 Hz), 6.38 (bs, 1H), 5.60 (s, 1H), 3.84 (s, 3H), 3.66 (t, 2H, J = 5.8 Hz), 3.41 (q, 2H, J = 11.2 Hz), 3.01 (t, 2H, J = 6.2 Hz), 2.72 (t, 2H, J = 5.6 Hz), 1.87 (q, 4H, J = 10.0 Hz). 13C NMR (CDCl3): δ (ppm) 189.08, 180.90, 161.26, 156.85, 156.30, 148.60, 134.25, 130.49, 130.11, 126.18, 121.75, 120.93, 119.30, 114.88, 100.93, 100.51, 55.65, 46.21, 43.37, 33.44, 25.23, 23.03, 22.84. ESI-MS: m/z 444 [M+]. 2-[3-(7-Methoxy-1,2,3,4-tetrahydroacridin-9-yl-amino)propyl]aminojuglone (23). Yield 26%; mp 170−172 °C. 1H NMR (CDCl3): δ (ppm) 12.97 (s, 1H), 7.78 (d, 1H, J = 9.2 Hz), 7.51 (d, 1H, J = 7.6 Hz), 7.43 (q, 1H, J = 11.6 Hz), 7.24−7.18 (m, 3H), 6.43 (bs, 1H), 5.56 (s, 1H), 3.86−3.80 (m, 3H), 3.49 (t, 2H, J = 6.8 Hz), 3.32 (q, 2H, J = 12.4 Hz), 2.98 (d, 2H, J = 6.0 Hz), 2.70 (d, 2H, J = 5.6 Hz), 2.05− 1.98 (m, 2H), 1.85 (d, 4H, J = 2.8 Hz). 13C NMR (CDCl3): δ (ppm) 189.00, 181.01, 161.22, 156.61, 155.67, 149.81, 148.68, 134.10, 130.51, 129.25, 126.08, 121.28, 120.87, 119.21, 117.72, 114.93, 101.52, 99.98, 55.67, 46.21, 40.67, 32.99, 29.96, 25.15, 23.00, 22.76. ESI-MS: m/z 458 [M+]. 2-Chloro-3-[2-(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl-amino)ethyl]amino-1,4-naphthoquinone (24). Yield 61%; mp 130−132 °C. 1 H NMR (CDCl3): δ (ppm) 8.08 (d, 1H, J = 7.2 Hz), 7.92 (d, 1H, J = 6.8 Hz), 7.73−7.66 (m, 2H), 7.61−7.57 (m, 1H), 7.13 (t, 2H, J = 5.2 Hz), 6.25 (bs, 1H), 4.04 (q, 2H, J = 11.6 Hz), 3.83 (s, 3H), 3.68 (t, 2H, J = 5.6 Hz), 2.97 (s, 2H), 2.74 (s, 2H), 1.86 (t, 4H, J = 3.2 Hz). 13 C NMR (CDCl3): δ (ppm) 180.06, 176.75, 156.67, 156.41, 148.61, 144.20, 143.03, 135.06, 132.73, 132.52, 130.29, 129.76, 126.97, 126.90, 121.65, 120.78, 118.97, 111.49, 100.94, 55.64, 48.77, 45.69, 33.63, 25.24, 23.07, 22.82. ESI-MS: m/z 462 [M+], 464 [M+ + 2]. 2-Chloro-3-[3-(7-methoxy1,2,3,4-tetrahydroacridin-9-yl-amino)propyl]amino-1,4-naphthoquinone (25). Yield 24%; mp 150−152 °C. 1H NMR (CDCl3): δ (ppm) 8.09 (d, 1H, J = 8.0 Hz), 7.95 (d, 1H, J = 7.2 Hz), 7.79 (d, 1H, J = 8.8 Hz), 7.68 (t, 1H, J = 7.4 Hz), 7.58 (t, 1H, J = 7.4 Hz), 7.17 (t, 2H, J = 6.4 Hz), 6.24 (bs, 1H), 3.96 (q, 2H, J = 13.4 Hz), 3.85 (s, 3H), 3.53 (t, 2H, J = 6.6 Hz), 2.99 (s, 2H), 2.71 (s, 2H), 2.06−1.99 (m, 2H), 1.85 (s, 4H). 13C NMR (CDCl3): δ (ppm) 180.45, 176.82, 156.49, 155.71, 149.90, 144.26, 142.02, 135.10, 132.69, 129.83, 128.98, 126.95, 121.19, 120.86, 117.49, 101.52, 55.67, 45.92, 42.83, 33.02, 25.15, 23.03, 22.73, 22.62. ESI-MS: m/z 476 [M+], 478 [M+ + 2]. 2-Chloro-3-[2-(6-chloro-1,2,3,4-tetrahydroacridin-9-yl-amino)ethyl]amino-1,4-naphthoquinone (26). Yield 32%; mp 165−167 °C. 1 H NMR (CDCl3): δ (ppm) 8.07 (d, 1H, J = 7.2 Hz), 7.92 (d, 1H, J = 7.2 Hz), 7.80 (d, 1H, J = 9.2 Hz), 7.70 (q, 2H, J = 14.8 Hz), 7.61 (d, 1H, J = 7.6 Hz), 7.22 (t, 1H, J = 4.4 Hz), 6.08 (bs, 1H), 4.03 (q, 2H, J = 11.6 Hz), 3.73 (s, 2H), 2.96 (s, 2H), 2.69 (s, 2H), 1.87 (s, 4H). 13C NMR (CDCl3): δ (ppm) 179.82, 176.57, 160.15, 149.47, 147.85, 143.96, 134.96, 134.19, 132.65, 132.26, 129.53, 127.82, 126.84, 126.77, 125.08, 123.56, 118.84, 117.90, 49.19, 45.68, 33.94, 24.93, 22.77, 22.52. ESI-MS: m/z 466 [M+], 468 [M+ + 2]. 2-Chloro-3-[3-(6-chloro-1,2,3,4-tetrahydroacridin-9-yl-amino)propyl]amino-1,4-naphthoquinone (27). Yield 19%; mp 120−122 °C. 1H NMR (CDCl3): δ (ppm) 8.10 (q, 1H, J = 7.8 Hz), 7.95 (q, 1H, J = 7.8 Hz), 7.79 (q, 2H, J = 16.6 Hz), 7.70 (q, 1H, J = 9.0 Hz), 7.60 (q, 1H, J = 7.8 Hz), 7.24−7.21 (m, 1H), 3.92 (d, 2H, J = 7.2 Hz), 3.57 (s, 2H), 2.99 (s, 2H), 2.67 (s, 2H), 2.03 (t, 2H, J = 7.2 Hz), 1.88 (q, 4H, J = 6.4 Hz). 13C NMR (CDCl3): δ (ppm) 180.40, 176.82, 159.77, 150.46, 147.80, 144.13, 135.16, 134.35, 132.70, 132.63, 129.77, 127.58, 127.00, 126.97, 124.85, 124.23, 118.58, 116.78, 46.45, 42.54, 33.90, 33.10, 24.92, 22.96, 22.64. ESI-MS: m/z 480 [M+], 482 [M+ + 2]. 2-Chloro-3-[2-(1,2,3,4-tetrahydroacridin-9-yl-amino)ethyl]amino-1,4-naphthoquinone (28). Yield 30%; mp 183−185 °C. 1H NMR (CDCl3): δ (ppm) 8.09 (q, 1H, J = 7.8 Hz), 7.95 (q, 1H, J = 7.4 Hz), 7.88 (d, 1H, J = 8.4 Hz), 7.82 (d, 1H, J = 8.4 Hz), 7.70 (d, 1H, J = 1.2 Hz), 7.61 (t, 1H, J = 4.0 Hz), 7.49 (s, 1H), 7.33 (s, 1H), 6.13 (bs, 1H), 4.04 (t, 2H, J = 6.0 Hz), 3.74 (t, 2H, J = 5.8 Hz), 3.00 (s, 2H),

I=1−

ΔA i ΔA 0

ΔAi indicates absorbance change provided by cholinesterase exposed to anticholinerase compound. ΔA0 indicates absorbance change caused by intact cholinesterase, where phosphate buffer was applied in the same way as the anticholinerase compound. IC50 was determined using GrapPad Prism 6. (La Jolla, CA, USA). Percentage of inhibition was overlaid by proper curve chosen according to optimal correlation coefficient. All results are determined as the mean of three independent measurements. Subsequently, IC50 was computed, and standard error of the mean was established. Crystallization of TcAChE−20 Complex. TcAChE was isolated, purified, and crystallized essentially as previously described,64 with the only exception being the affinity chromatography ligand, mono(aminocaproyl)-p-aminophenyltrimethylammonium. Owing to its relatively limited solubility in water, 20 was dissolved in DMSO (100 mM). The crystals of the complex were obtained by soaking the native crystals at 4 °C for 12 h, in 2 mM 20, 30% PEG [poly(ethylene glycol)] 200, 8% DMSO, 100 mM MES at pH 6.0. Structure Determination of TcAChE−20 Complex. X-ray diffraction data were collected at the XRD-1 beamline of the Italian Synchrotron Facility ELETTRA (Trieste, Italy). A PILATUS 2 M detector (Dectris Ltd.) and focusing optics were employed for the measurements. The crystals were flash-cooled in a nitrogen stream at 100 K, using an Oxford Cryosystems cooling device (Oxford, UK). Data processing was done with MOSFLM version 7.0.765,66 and the CCP4 package version 6.3.0.67 The enzyme−inhibitor structure was determined by Patterson search methods with the PHASER package 2.3.068 using the refined coordinates of the native TcAChE (PDB ID 1EA5)47 after removal of the water molecules. Crystallographic refinement of the complex was performed with REFMAC5 version 5.7.69 All data within the resolution range were included with no σcutoff. Bulk solvent correction and anisotropic scaling were applied. The Fourier maps were computed with σ-A weighted (2|F0| − |Fc|, Φc) and (|F0| − |Fc|, Φc) coefficients after initial native protein rigid body refinement, followed by maximum likelihood positional and individual J



Article

isotropic temperature factor refinements. A prominent electron density feature in the catalytic gorge of TcAChE and along the gorge itself allowed unambiguous fitting of the inhibitor. Carbohydrates (N-acetyl β-D-glucosamine units linked at Asn59, Asn416, and Asn453) were built in by inspecting the electron density maps. Peaks in the difference Fourier maps, above the contour level of 1.8 rmsd, were automatically added as water molecules to the atomic model and retained if they met stereochemical requirements, and their B factors, after refinement, were below 80 Å2. Maps inspection and model corrections during refinement were based on the graphics program COOT version 0.7.70 Atomic coordinates and structure factor amplitudes of the TcAChE− 20 complex have been deposited in the Brookhaven Protein Data Bank under the PDB ID code 4TVK. The crystal parameters, data collection, and refinement statistics are summarized in Table S2. Figures were created using LIGPLOT+ version 1.4.5 (see Supporting Information) and PyMOL version 1.7.1.3. Inhibitory Potency on Aβ1−42 Self-Aggregation. 1,1,1,3,3,3Hexafloro-2-propanol (HFIP)-pretreated Aβ1−42 samples (Bachem AG, Switzerland) were solubilized with a CH3CN/0.3 mM Na2CO3/ 250 mM NaOH (48.4/48.4/3.2) mixture to obtain a stable stock solution (Aβ1−42 = 500 μM).16,71 Experiments were performed by incubating the peptide in 10 mM phosphate buffer (pH = 8.0) containing 10 mM NaCl, at 30 °C for 24 h (final Aβ concentration 50 μM) with and without inhibitors at 10.0 μM (Aβ/inhib. = 5/1). Blanks containing the tested inhibitors were also prepared and tested. To quantify amyloid fibril formation, the thioflavin T fluorescence method was used.71 After incubation, samples were diluted to a final volume of 2.0 mL with 50 mM glycine−NaOH buffer (pH 8.5) containing 1.5 μM thioflavin T. A 300-s time scan of fluorescence intensity was carried out (λexc = 446 nm; λem = 490 nm), and values at plateau were averaged after subtracting the background fluorescence of 1.5 μM thioflavin T solution. The fluorescence intensities were compared, and the percent inhibition due to the presence of the tested inhibitor was calculated. Cell Cultures. N2A cells (American Type Culture Collection, ATCC, through LGC Standards S.r.l., Sesto San Giovanni, MI, Italy) were grown in 10% fetal bovine serum (FBS)/ Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2 mM L-glutamine and 50 units/mL of penicillin/streptomycin (Life Technologies Italia, Monza, MB, Italy) in a 37 °C incubator containing 5% CO2/95% humidified air. For experiments, cells were plated on 96-well plates at a density of 0.1 × 106 cells/well and subjected to differentiation through dibutyryl cAMP treatment (1 mM dbcAMP, Sigma-Aldrich, Milan, Italy) in complete medium, which was renewed every other day for a week. Primary cerebellar granule neurons (CGNs) were prepared from 7day-old Wistar rats, as previously described.72 Briefly, cells were dissociated from cerebella and plated on 96-well plates, previously coated with 10 mg/L poly-L-lysine at the density of 0.12 × 106 cells/ well in BME with 10% heat-inactivated FBS (Life Technologies), 2 mM glutamine, 100 μM gentamicin sulfate, and 25 mM KCl (SigmaAldrich). Sixteen hours later, 10 μM cytosine arabino-furanoside (Sigma-Aldrich) was added to avoid glial proliferation. After 7 days in vitro (DIV), differentiated neurons were shifted to serum-free medium for compound testing. The T67 human glioma cell line was derived by Lauro et al.73 from a World Health Organization (WHO) grade III gemistocytic astrocytoma. T67 cells were cultured in DMEM (BioWhittaker, Cambrex BioScience, Belgium), supplemented with 10% FBS (BioWhittaker, Cambrex BioScience, Belgium), 100 UI/mL penicillin, 100 μg/mL streptomycin, and 40 μg/mL gentamycin, in a 5% CO2 atmosphere at 37 °C, with saturating humidity. Cell stocks were cryopreserved by standard methods and stored in liquid nitrogen. Cell viability was measured by trypan blue exclusion. Determination of Neurotoxic and Neuroprotective Activities in N2A Cells or CGNs. To test the toxicity of compounds, differentiated N2A cells or CGNs were exposed to increasing concentration of the compounds, from 0 (controls) to 50 μM, for 24 h. To test neuroprotection from Aβ toxicity of selected compounds 16 and 20 on differentiated N2A, aggregated synthetic Aβ1−42 from

Biopeptide Co., Inc. (San Diego, CA) was prepared by incubating the synthetic peptide in 95% PBS/5% DMSO (5 mM) at 37 °C for 72 h, sonicating, and further centrifugating at 15000g at RT for 10 min. Increasing concentrations of aggregated Aβ1−42 (0, 5, 10, and 20 μM) were used to treat N2A differentiated for 7 days with dbcAMP, in the absence (control) or presence of 0, 12.5, or 25 μM of selected compounds for 24 h, in serum-free medium. Neuronal survival was analyzed by using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay.74 For MTT assay, thiazolyl blue was added to culture medium at final concentration of 0.1 mg/mL. Following 20 min at 37 °C in the dark, MTT precipitate was dissolved in 0.1 M Tris-HCl buffer containing 5% Triton X-100 (all from Sigma-Aldrich), and the absorbance was read at 570 nm in a multiplate spectophotometric reader (Bio-Rad Laboratories S.r.l., Segrate, MI, Italy). Determination of Antioxidant Activity in T67 Cells. To evaluate the antioxidant activity of the compounds, T67 cells were seeded in 24-well plates at 1 × 105 cells/well. Experiments were performed after 24 h of incubation at 37 °C in 5% CO2 with 4methylsulfinylbutyl isothiocyanate, sulphoraphane (2.5 μM). After this time cells were washed and treated for 24 h with 10 μM of compounds 16 and 20. The antioxidant activity of 16 and 20 compounds was evaluated after 30 min of incubation with 10 μM fluorescent probe (2′,7′dichlorofluorescein diacetate, DCFH-DA) in DMEM, by measuring the intracellular ROS formation evoked by 30 min exposure of T67 cells to 100 μM tert-butyl hydroperoxide (TBH) in PBS. The fluorescence increase of the cells from each well was measured (λexc = 485 nm; λem = 535 nm) with a spectrofluorometer (Wallac Victor multilabel S9 counter, PerkinElmer Inc., Boston, MA).57 Data are reported as the mean ± standard deviation of at least three independent experiments. Ex Vivo AChE Inhibition. To test the brain permeability, two selected compound were subjected to an ex vivo determination of its AChE inhibitory activity by using a similar methodology previously adopted by Munoz-Torrero.21 Briefly, each compound was administered intraperitoneally (ip) to young adult Wistar rats at doses of either 10 or 50 μmol/kg. Experiments were performed in accordance with the Italian and European Community law for the use of experimental animals and were approved by a local bioethical committee. Animals were killed by decapitation at 1 h after injection, brain was immediately dissected, and both telencephalon and cerebellum were collected. Tissues were homogenized in ice-cold 50 mM Tris-HCl buffer at pH 7.4 and added with Triton X-100 to a final 0.5% concentration (all chemicals were from Sigma-Aldrich). Homogenates were used to assay the AChE activity according to the standard colorimetric method45 and to measure the total protein content for normalization.75 Determination of Hepatotoxicity of 16 and 20 on HepG2 Cells. HepG2 cells (human hepatocellular liver carcinoma cell line from American Type Culture Collection, ATCC), were grown in DMEM supplemented with 10% FBS and 50 units/mL of penicillin/ streptomycin (Life Technologies Italia, Monza, MB, Italy) at 37 °C in a humidified atmosphere containing 5% CO2. For the experiments, cells (0.5 × 105 cells/well) were seeded in 96-well plate in complete medium; after 24 h, the medium was removed, and cells were exposed to the increasing concentrations of compounds 3 or 16 or 20 (0, 12.5, 25, and 50 μM) in DMEM with no serum for further 24 h. Cell survival was measured through MTT assay.74

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ASSOCIATED CONTENT

S Supporting Information *

Elemental analysis of derivatives 12−29; computational studies; crystallographic data collection and refinement statistics for 20; figures displaying additional data from X-ray studies. This material is available free of charge via the Internet at http:// pubs.acs.org. K



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Accession Codes

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The PDB accession code for 20-bound TcAChE X-ray structure is 4TVK.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +39 0512099717. E-mail: marialaura.bolognesi@ unibo.it. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the University of Bologna, Grant Agency of the Czech Republic (No. P303/11/1907), and MH CZ-DRO (University Hospital Hradec Kralove, No. 00179906). We are grateful to the ELETTRA XRD1 beamline staff for their assistance during the data collection. Thanks are also due to UniRimini S.p.A. and CIRI (PORFESR project).

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ABBREVIATIONS AChE, acetylcholinesterase; Aβ, amyloid-β; TcAChE, Torpedo californica acetylcholinesterase; N2A, immortalized mouse cortical neurons Neuro2A; AD, Alzheimer’s disease; ROS, reactive oxygen species; MTDLs, multitarget-directed ligands; hAChE, human acetylcholinesterase; PAS, peripheral anionic site; AChEI, acetylcholinesterase inhibitor; CAS, catalytic anionic site; CNS, central nervous system; ACh, acetylcholine; BChE, butyrylcholinesterase; hBChE, human butyrylcholinesterase; CGNs, cerebellar granule neurons; TBH, tert-butyl hydroperoxide; BBB, blood-brain barrier

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