Design, Synthesis, and Evaluation of Multitarget-Directed Resveratrol

Article pubs.acs.org/jmc

Design, Synthesis, and Evaluation of Multitarget-Directed Resveratrol Derivatives for the Treatment of Alzheimer’s Disease Chuanjun Lu, Yueyan Guo, Jun Yan, Zonghua Luo, Hai-Bin Luo, Ming Yan, Ling Huang,* and Xingshu Li* School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, China S Supporting Information *

ABSTRACT: A series of multitarget-directed resveratrol derivatives was designed and synthesized for the treatment of Alzheimer’s disease (AD). In vitro studies indicated that most of the target compounds exhibit significant inhibition of self-induced β-amyloid (Aβ) aggregation and Cu(II)-induced Aβ1−42 aggregation and acted as potential antioxidants and biometal chelators. In particular, compounds 5d and 10d are potential lead compounds for AD therapy (5d, IC50 = 7.56 μM and 10d, IC50 = 6.51 μM for self-induced Aβ aggregation; the oxygen radical absorbance capacity assay using fluorescein (ORAC-FL) values are 4.72 and 4.70, respectively). Moreover, these compounds are capable of disassembling the highly structured Aβ fibrils generated by self- and Cu(II)-induced Aβ aggregation. Furthermore, 5d crossed the blood−brain barrier (BBB) in vitro and did not exhibit any acute toxicity in mice at doses of up to 2000 mg/kg. Taken together, the data indicate that 5d is a very promising lead compound for AD.

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Fe, ∼340 μM),8 which can promote the aggregation of Aβ. In addition, the interaction of Aβ with Cu2+ contributes to the production of reactive oxygen species (ROS).9 Resveratrol, a natural product with a stilbene structure, has been extensively studied for its wide range of biological effects. Recent evidence has suggested that resveratrol functions as an anti-AD agent through the inhibition of Aβ aggregation by scavenging oxidants and exerting anti-inflammatory activities.10,11 In tests of laboratory animals, resveratrol is also effective in prevention of neurodegenerative diseases,12,13 and its phase II clinical trials for AD patients is ongoing at the present.14 These results indicate that resveratrol could be used as a starting compound in the design of multifunctional drugs for the treatment of AD. We previously reported a series of stilbene derivatives based on the structure of resveratrol that had strong Aβ aggregation inhibitory and antioxidant activity.15 Considering that biometal (Fe, Cu, and Zn)16−19 ions may be crucial participants in the pathological processes of AD,20 we combined resveratrol and the pharmacophore moiety of the well-known metal chelator clioquinol (CQ), which significantly decreased the rate of cognitive decline in moderately severe AD patients in a phase II clinical trial,21 to obtain a novel series of derivatives that are expected to be biometal chelators, antioxidants, and inhibitors of Aβ aggregation (Figure 1).

INTRODUCTION Alzheimer’s disease (AD), the most common fatal neurodegenerative disorder, affects more than 24 million people worldwide.1 Although 100 years have passed since its discovery, effective treatments are lacking.2 Current therapeutic options for the treatment of AD, which include cholinesterase inhibitors (donepezil, rivastigmine, and galantamine)3 and an NMDA receptor antagonist (memantine),4 have resulted in a modest improvement in memory and cognitive function, but they do not prevent progressive neurodegeneration.3 The etiology of AD remains elusive, but multiple factors, such as β-amyloid (Aβ), τ-protein, oxidative stress, dyshomeostasis of biometals, and low levels of acetylcholine, likely play important roles in the development of AD. Therefore, from both a fundamental and practical perspective, multitarget-directed ligands for the treatment of this fatal neurodegenerative disorder are desirable. Among the multiple factors that induce AD, Aβ plays a primary role.5 The histopathological hallmarks of AD are neurofibrillary tangles and senile plaques, which are massive extracellular deposits of aggregated amyloid-β peptide.3 Candidate drugs that are intended to reduce Aβ production, prevent Aβ aggregation, and promote Aβ clearance are currently in clinical trials.6 Oxidative stress is one of the earliest events in AD pathogenesis.7 The free radical and oxidative stress theory of aging also suggests that oxidative damage is an important player in neuronal degeneration. Therefore, the successful protection of neuronal cells from oxidative damage could potentially prevent AD. Recent studies have indicated that excessive biometals, such as iron, zinc, and copper, exist in the brains of AD patients (Cu, ∼400 μM; Zn and Fe, ∼1 mM),3 and it is several-fold to the normal age-matched neuropil (Cu, ∼70 μM; Zn, ∼350 μM; © XXXX American Chemical Society

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RESULTS AND DISCUSSION Chemistry. The synthetic route for the new resveratrol derivatives is shown in Schemes 1 and 2. Commercially available Received: April 18, 2013

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dx.doi.org/10.1021/jm400567s | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Similarly, target compounds 6a,b and 7a,b were obtained by the same reaction of intermediate 4 with other aldehydes, for example, 2-(dimethylamino)benzaldehyde. Target compounds 9a−c, which have a substituted amino group at the 5-position of the 2-hydroxybenzaldehyde moiety, were synthesized with 5c as the intermediate. The reaction of 5c with SnCl2 provided amine intermediate 8, and alkylation with the corresponding aldehydes and sodium cyanoborohydride produced 9 in moderate yields. Compounds with 3,5-dihydroxy at the A ring of target compounds 10a−e were obtained by demethylation of the corresponding dimethoxy compounds 5a−d and 6a in the presence of boron tribromide at −20 °C. Compound 2 was first reacted with 4-(dimethylamino)benzaldehyde or 4-methoxybenzaldehyde, followed by the same method used to prepare 4 to give resveratrol derivatives 11 and 13 (Scheme 2). Finally, the target compounds 12 and 14 were generated by treating 11 and 13 with 5-(dimethylamino)-2hydroxybenzaldehyde and sodium borohydride. Inhibition of Self-Mediated Aβ1−42 Aggregation. The inhibitory activities of the resveratrol derivatives against selfmediated Aβ1−42 aggregation22,23 were evaluated using a thioflavin T (ThT) fluorescence assay.24 Resveratrol and curcumin were used as reference compounds (none of the tested compounds exhibited fluorescence signals under the experimental conditions). The results shown in Table 1 indicate that more than half of the target compounds exhibited more potent inhibition of Aβ aggregation (50.23−79.50%) than curcumin.

Figure 1. Design strategy for resveratrol derivatives.

4-nitrobenzaldehyde was first reacted with NaBH4, and subsequent reaction with PBr3 provided 1-(bromomethyl)-4nitrobenzene, which was refluxed in triethyl phosphite to give Wittig reagent 2. Compound 2 was then reacted with 3,5dimethoxybenzaldehyde in the presence of CH3ONa at 0 °C to give 3. The reduction of 3 by SnCl2 gave amine 4. Compound 4 was reacted with different 2-hydroxybenzaldehyde derivatives in the presence of NaBH4 to provide the target compounds 5a−f. Scheme 1. Synthesis of 5−7, 9, and 10a

Reagents and conditions: (a) NaBH4, MeOH, 0 °C; (b) PBr3, pyridine, 0 °C; (c) triethyl phosphite, 120 °C; (d) 3,5-dimethoxybenzaldehyde, CH3ONa, 0 °C, 1 h, rt, 12 h; (e) SnCl2, EtOAc, 90 °C, 4 h; (f) different 2-hydroxybenzaldehyde derivatives, rt, 4 h; NaBH4, 0 °C, 1 h; (g) different aldehydes, rt, 4 h; NaBH4, 0 °C,1 h; (h) different aldehydes, NaBH3CN, rt; (i) BBr3, CH2Cl2, −20 °C, 2 h, rt, 4 h.

a

B



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Scheme 2. Synthesis of Compounds 12 and 14a

Reagents and conditions: (a) 4-methoxybenzaldehyde (for 11) or 4-(dimethylamino)benzaldehyde (for 13), CH3ONa, 0 °C, 1 h, rt, 12 h; (b) SnCl2, EtOAc, 90 °C, 4 h; (c) 5-(dimethylamino)-2-hydroxybenzaldehyde, rt, 4 h; NaBH4, 0 °C, 1 h.

a

structure were a benefit for the inhibition of the product of amyloid fibrils. Over the past decade, several small molecules and peptides have been designed to inhibit the conformation transition from the initial α-helices to β-sheets in combination with the C-terminus of Aβ1−42.31−33 To investigate the possible binding modes of the compounds with Aβ1−42, 5d and 10d were selected for molecular docking studies using the CDOCKER program embedded in Accelrys Discovery Studio 2.5.5 (San Diego, CA, U.S.). The structure of Aβ1−42 used in the docking study was obtained from the Protein Data Bank (1IYT).34 All docked conformations are ranked based on docking scores. As shown in Figure 3, both of the ligands are located around the C-terminus of Aβ1−42 and mainly exhibit hydrophobic and hydrogen-bonding interactions. However, 5d and 10d interacted with residues in a different manner. The dimethylamino group of 5d appeared to act as an anchor through hydrophobic interactions with Ile41 and Ala42, whereas the dimethylamino group of 10d was located near Lys28 and Gly29. Hydrogen bonds were formed between the OH group of the C ring and the backbone NH group of Lys28 in both models, with average O−N distances of 2.66 and 2.77 Å for 5d and 10d, respectively. On the basis of the molecular modeling results, we suggest that the resveratrol derivatives inhibit Aβ1−42 aggregation by targeting the C-terminus and inhibiting the formation of β-sheets. Metal-Chelating Properties of 5d and 10d. The ability of 5d and 10d to chelate biometals such as Cu(II), Fe(II), Fe(III), and Zn(II) was studied by UV−vis spectroscopy (Figure 4).35−39 After CuSO4 was added to a solution of 5d, the maximum absorption wavelength shifted from 339 to 348 nm, and the absorbance dropped dramatically, indicating the formation of a 5d−Cu(II) complex (Figure 4A). The maximum absorption at 230 nm exhibited a slight shift when FeSO4, FeCl3, or ZnCl2 was added, suggesting that 5d binds Fe2+, Fe3+, and Zn2+. UV−vis spectroscopic analysis of compound 10d revealed absorption maxima at 232 and 337 nm. As with 5d, the spectrum of the 10d−Cu(II) complex was significantly different from that of 10d alone. When FeSO4, FeCl3, or ZnCl2 was added, the maximum absorption at 232 nm shifted to 222, 223, or 221 nm, respectively. The stoichiometry of the 5d−Cu(II) complex was determined using Job’s method40,41 by preparing a series of solutions in which the total concentration of compound 5d and CuSO4 remained constant but their proportions varied. UV spectroscopy was used to determine the absorbance of the complex of CuSO4 and 5d at different concentrations. As indicated in Figure 4C, when the absorbance changes at 333 nm were plotted, the two straight lines intersected at a mole fraction of 0.758, which implied a 3:1 5d−Cu(II) complex. The Job plot for compound 10d and CuSO4 revealed a break at 0.49, indicating a 1:1 stoichiometry for the 10d−Cu(II) complex.

The complete dose−response curves were obtained by assaying those compounds with inhibition rates greater than 50% (data listed in Table 1). The results indicated that 5d (IC50 = 7.56 μM) was the most potent inhibitor of Aβ1−42 aggregation among the series of compounds featuring a 3,5dimethoxyl group on the A ring. Modifications of 5d with A ring substituted with 3,5-dihydroxy, 4-methoxy, or 4dimethylamino generated 10d, 12, and 14, which all provided good inhibitory activities. In addition, 9a−c, with substituted amino groups on C ring, gave slightly weaker inhibitory activities than 5d. Compounds with 3,5-dihydroxy on A ring, with groups such as hydroxyl, nitro, and substituted amino group on C ring, exhibited good inhibitory activities (10a−e, IC50 of 6.51−14.16 μM). Among them, 10c (IC50 = 7.06 μM and 10d (IC50 = 6.51 μM) provided the best results in this series. The results also indicated that 5d, 10c, and 10d were more potent inhibitors of Aβ1−42 aggregation than resveratrol (IC50 = 15.11 μM). Antioxidant Activity in Vitro. The antioxidant activities of the resveratrol derivatives were evaluated by the oxygen radical absorbance capacity assay method using fluorescein (ORACFL);25,26 the vitamin E analogue Trolox was used as a standard. All compounds exhibited good ORAC-FL values of 2.37−6.27 Trolox equivalents (Table 1). Among these compounds, compound 10a, which features two hydroxy groups on the A ring and one on the C ring, displayed the highest activity (6.27). Because the inhibition of Aβ aggregation is important for candidate AD drugs, we further investigated the multitarget profiles of compounds 5d (3,5-dimethoxyl group on A ring) and 10d (3,5-dihydroxy group on A ring), which exhibited significant inhibition of Aβ aggregation and strong antioxidant activity (4.72 and 4.70). Inhibition of Aβ1−42 Fibril Formation Monitored by Transmission Electron Microscopy (TEM). To further confirm the ability of the resveratrol derivatives to inhibit Aβ1−42 aggregation, the inhibitory activity of compounds 5d and 10d was monitored by TEM (Figure 2b and Figure 2c); resveratrol and clioquinol (CQ) were used as controls (Figure 2d and Figure 2e). After 24 h of incubation at 37 °C, Aβ1−42 alone aggregated into well-defined Aβ fibrils (Figure 2f). By contrast, few Aβ fibrils were observed in the presence of compounds 5d and 10d (Figure 2b and Figure 2c) under identical conditions. Therefore, based on the TEM and ThT measurement results, we can conclude that compounds 5d and 10d effectively inhibit Aβ1−42 fibril formation. Docking Study of Possible Conformations of 5d and 10d with Aβ. The neurotoxicity of the amyloid peptide is associated with the amyloid fibrils.27−29 The formation of a β-sheet structure may promote the aggregation of Aβ1−42.30 Thus, molecules that could inhibit the formation of a β-sheet C



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Table 1. Inhibition of Aβ1−42 Aggregation and Oxygen Radical Absorbance Capacity (ORAC, Trolox Equivalents) of Resveratrol Derivatives 5a−f, 6a,b, 7a,b, 9a−c, 10a−e, 12, and 14

D



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Table 1. continued

The thioflavin-T fluorescence method was used. The values are expressed as the mean ± SD of at least three independent measurements. All values were obtained at a compound concentration of 20 μM. bThe mean ± SD of the three independent experiments. The data are expressed as μmol of Trolox equiv/μmol tested compound. cn.t. means not tested. dThe IC50 (μM) values shown are the mean ± SD of three experiments. a

resveratrol, 78.92% inhibition). These results suggest that 5d and 10d inhibit Cu(II)-induced Aβ aggregation effectively by chelating Cu(II). The TEM images in Figure 5B are consistent with the ThT binding assay results. More well-defined Aβ fibrils (Figure 5Bf) were observed in the presence of Cu(II) than for Aβ alone (Figure 5Bg), and fewer Aβ fibrils were observed when 5d or 10d was added to the samples. Disaggregation of Self- and Metal-Induced Aβ1−42 Aggregation Fibrils by 5d and 10d. The ability of 5d and 10d to disaggregate self-induced Aβ aggregation fibrils was investigated. Aβ fibrils were generated by incubating fresh Aβ for 24 h at 37 °C. The test compound was then added to the sample and incubated for another 24 h at 37 °C (Figure 6). The ThT binding assay demonstrated that both 5d and 10d can disaggregate Aβ fibrils (5d, 67% disaggregation; 10d, 82% disaggregation), as does resveratrol (61% disaggregation at 25 μM). CQ showed a slight effect (16.52% disaggregation). As Figure 6B shows, the TEM assay supported the results of the ThT binding assay.

In addition, the binding affinities of compounds 5d and 10d for Cu2+ were estimated by the software. The association constants obtained by nonlinear least-squares analysis were (4.46 ± 1.20) × 105 M−1 for 5d and (9.23 ± 2.10) × 104 M−1 (see the Supporting Information. The analogous data of CQ are also described in the Supporting Information). Effects of 5d and 10d on Cu(II)-Induced Aβ1−42 Aggregation. To investigate the ability of the resveratrol derivatives to inhibit Cu(II)-induced Aβ1−42 aggregation, we studied compounds 5d and 10d by ThT fluorescence and TEM (Figure 5). For the ThT fluorescence assay, resveratrol and the known metal chelator clioquinol (CQ) were used as the reference compounds.21 The Aβ peptide was first treated with 1 equiv of Cu(II) for 2 min at room temperature and then incubated with or without the testing compounds for 24 h at 37 °C. As shown in Figure 5A, the fluorescence of Aβ treated with Cu(II) is 155.8% that of Aβ alone, which indicates that Cu(II) accelerates Aβ aggregation. By contrast, the fluorescence of Aβ treated with Cu(II) and the tested compounds decreased dramatically (5d, 87.51% inhibition of Cu(II)-induced Aβ1−42 aggregation; 10d, 94.23% inhibition; CQ, 70.94% inhibition; E



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Figure 2. TEM image analysis of Aβ1−42 aggregation in the presence of 5d and 10d. (a) Aβ1−42 (25 μM), 0 h. (b) Aβ1−42 (25 μM) and 5d (20 μM) were incubated at 37 °C for 24 h. (c) Aβ1−42 (25 μM) and 10d (20 μM) were incubated at 37 °C for 24 h. (d) Aβ1−42 (25 μM) and resveratrol (20 μM) were incubated at 37 °C for 24 h. (e) Aβ1−42 (25 μM) and CQ (20 μM) were incubated at 37 °C for 24 h. (f) Aβ1−42 alone (25 μM) was incubated at 37 °C for 24 h.

cellular antioxidant assay based on dichlorofluorescein diacetate (DCFH-DA),43,45 and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), an analogue of vitamin E, was used as the reference compound. First, the colorimetric MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay was performed to examine the potential cytotoxic effects of 5d and 10d. As indicated in Figure 8A, 5d and 10d exhibited no toxicity at 10 μM after incubation for 24 h. Thus, we chose 10 μM as the highest concentration for the subsequent assay. Treatment of SH-SY5Y cells with tert-butyl hydroperoxide significantly increased intracellular oxidative stress. As shown in Figure 8B, both Trolox and the tested compounds exhibited antioxidant activity, but the activities of 5d and 10d were far more potent than that of Trolox. These observations further indicate that resveratrol derivatives have the potential to be efficient multifunctional agents, including antioxidant activity, for the treatment of AD. In Vitro Blood−Brain Barrier Permeation Assay. Blood− brain barrier (BBB) permeability is essential for successful CNS drugs. To measure the possible in vivo BBB permeabilities of 5d and 10d, the parallel artificial membrane permeation assay of the blood−brain barrier (PAMPA-BBB) described by Di et al. was performed.46−48 First, we compared the permeability of 13 commercial drugs with reported values to validate the assay (see Supporting Information, Table S1). A plot of the experimental data versus the reported values produced a good linear correlation, Pe(exp) = 1.4574Pe(lit.) − 1.0773 (R2 = 0.9427) (see the Supporting Information, Figure S4). From this equation and considering the limit established by Di et al. for blood−brain barrier permeation, we concluded that compounds with permeabilities greater than 4.7 × 10−6 cm/s could cross the blood-brain barrier. On the basis of the measured permeability (Table 3), 5d could cross the BBB, whereas the status of 10d was uncertain. On the basis of the biological results, 5d was chosen for further evaluation. Acute Toxicity. To investigate the safety profile of the resveratrol derivatives, the acute toxicity of 5d was determined

In the second disaggregation study (Figure 7A), 5d, 10d, CQ, and resveratrol (50 μM) were added individually to Aβ fibrils generated by reacting Aβ with 1 equiv of Cu(II) for 24 h at 37 °C with constant agitation. The ThT binding assay indicated that 5d and 10d noticeably disaggregated Aβ fibrils compared to CQ at 50 μM (5d, 88.81% disaggregation; 10d, 85.87% disaggregation; CQ, 40.95% disaggregation). Resveratrol disaggregated 86.43% Cu(II)-induced Aβ fibrils as well. Figure 7B shows the TEM images of the Aβ species, which indicate that 5d and 10d were capable of disassembling the Aβ fibrils from Cu(II)-induced aggregation. Inhibition of Monoamine Oxidase (MAO) and Acetylcholinesterase (AChE) Activities. MAO is also an important target for the treatment of multifactorial diseases. Selective inhibitors for MAO-A have been used as effective antidepressants,42 and MAO-B inhibitors are associated with the treatment of neurodegenerative disorders.43 AD patients commonly present depressive symptoms, which suggests that dual inhibition of MAO-A and MAO-B could be beneficial to AD therapy.44 To further study the multipotent biological profile of the target compounds, the ability of compounds 5d and 10d to inhibit recombinant human MAO-A and MAO-B was evaluated. Ladostigil, an MAO-B inhibitor approved by the FDA for use in phase II clinical trials, and clorgyline, an irreversible and selective inhibitor of MAO-A, were used as reference compounds. Compounds 5d and 10d exhibited a strong, balanced MAO inhibitory activity (Table 2), as expected. A docking study of the compounds to MAO-B revealed that 5d and 10d have similar conformations in the binding site of MAO-B. Both inhibitors formed two hydrogen bonds with protein residues (see Supporting Information, Figures S1 and S2). Furthermore, 5d and 10d exhibited moderate ChE inhibitory activity (Table 2; 5d, IC50 = 36.04 μM for AChE; 10d, IC50 = 6.27 μM for AChE and IC50 = 21.25 μM for BuChE). The docking models are shown in the Supporting Information (Figures S3 and S4). Intracellular Antioxidant Activity. The antioxidant activity of 5d and 10d in SH-SY5Y cells was evaluated in a F



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Figure 3. Docking studies of 5d (colored blue) and 10d (colored red) with Aβ1−42 (PDB code 1IYT). (a) Surface representations of 5d and 10d interacting with Aβ1−42 in one possible binding conformation near the C-terminus (residues that hydrophobically interact with inhibitors are shown as yellow sticks. The dashed lines indicate possible hydrogen-bond contacts). (b) Cartoon representations of 5d and 10d interacting with Aβ1−42. (c) Association of 5d (colored blue) and the C-terminus of Aβ1−42 obtained from docking calculations. The hydrogen-bonding interaction between the ligand and residue Lys28 is indicated by the green line. (d) Association of 10d (colored red) and the C-terminus of Aβ1−42 obtained from docking calculations. The hydrogen-bonding interaction between the ligand and residue Lys28 is indicated by the green line.

in KM mice at doses of 0, 677, 1333, and 2000 mg/kg (n = 5 per group) by oral administration. From the first 4 h through 14 days after administration, all mice remained alive and appeared healthy based on criteria related to fur sleekness, water and food consumption, and body weight. All animals

were sacrificed on the 14th day after drug administration and macroscopically examined for possible damage to the heart, liver, and kidneys. The results showed that the animals treated with compound 5d did not develop any acute toxicity or mortality immediately or during the post-treatment period. G



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Figure 4. (A) UV spectrum of compound 5d (20 μM) alone and in the presence of CuSO4 (40 μM), FeSO4 (40 μM), FeCl3 (40 μM), or ZnCl2 (40 μM) in 20% (v/v) ethanol/buffer (20 mM HEPES, 150 mM NaCl, pH 7.4). (B) UV spectrum of compound 10d (20 μM) alone and in the presence of CuSO4 (40 μM), FeSO4 (40 μM), FeCl3 (40 μM), or ZnCl2 (40 μM) in 20% (v/v) ethanol/buffer (20 mM HEPES, 150 mM NaCl, pH 7.4). (C) Determination of the stoichiometry of complex 5d−Cu(II) by Job’s method. (D) Determination of the stoichiometry of complex 10d−Cu(II) by Job’s method.

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General Procedure II. p-Toluenesulfonic acid (0.2 mmol) was added to a solution of amine (2 mmol) and aldehyde (2 mmol) in ethanol, and the resulting mixture was stirred at room temperature. When the substrates disappeared (as detected by TLC), NaBH4 (6 mmol) was added at 0 °C. After the mixture was stirred for 1 h, water (10 mL) was added, and the mixture was extracted with ethyl acetate. The solvents were removed under reduced pressure to obtain the crude product, which was purified by recrystallization from ethyl acetate. General Procedure III. NaBH3CN (6 mmol) was added to a solution of 8 (2 mmol) and aldehyde (2 mmol) in acetonitrile, and the resulting mixture was stirred at room temperature in a nitrogen atmosphere. When the substrates disappeared (as detected by TLC), the solvents were removed under reduced pressure. Water (10 mL) was then added, and the mixture was extracted with ethyl acetate. The organic solvent phase was concentrated under vacuum. The crude product was purified by flash chromatography on silica gel. General Procedure IV. BBr3 (6 equiv) was added dropwise at −20 °C under nitrogen to a solution of compound 5 or 6a (1 equiv) in dried CH2Cl2. The resulting solution was slowly warmed to room temperature and stirred overnight. Ice−water was added slowly, and the mixture was filtered to provide the crude product, which was purified by recrystallization from methanol. Diethyl 4-Nitrobenzylphosphonate (2). Compound 2 was synthesized by following a previously reported procedure.49 Yellow oil, yield: 90%. 1H NMR (400 MHz, CDCl3) δ 8.18 (dd, J = 8.7, 0.7 Hz, 2H), 7.49 (dd, J = 8.8, 2.5 Hz, 2H), 4.16−3.94 (m, 4H), 3.26 (d, J = 22.4 Hz, 2H), 1.27 (t, J = 7.1 Hz, 6H). (E)-1,3-Dimethoxy-5-(4-nitrostyryl)benzene (3). To a solution of CH3ONa (0.972 g, 18 mmol) in DMF (15 mL) was added 2 (3.279 g, 12 mmol). After the mixture was stirred at 0 °C under nitrogen for 1 h, 3,5-dimethoxybenzaldehyde (1.66 g, 10 mmol) in DMF (10 mL) was added dropwise to the solution. The reaction mixture was slowly warmed to room temperature and stirred overnight. Ice−water was added, and the precipitate was filtered to give the crude product. Purification of the crude product by recrystallization from ethyl acetate provided the target product. Yellow solid, yield: 70%. 1H NMR (400 MHz, CDCl3) δ 8.22 (d, J = 8.7 Hz, 2H), 7.63 (d, J = 8.7 Hz, 2H),

CONCLUSION On the basis of the knowledge that resveratrol possesses a variety of bioactivities, we have developed a novel series of resveratrol derivatives as multitarget agents for the treatment of AD. Among the synthesized compounds, 5d and 10d exhibited significant inhibition of Aβ aggregation, metal-chelating ability, disaggregation of Aβ fibrils generated by self- and Cu(II)induced Aβ aggregation, antioxidant activity, acceptable MAO-A and MAO-B inhibition, moderate AChE inhibition, and low neurotoxicity. Furthermore, 5d could cross the blood− brain barrier (BBB) in vitro and did not exhibit acute toxicity in mice at doses up to 2000 mg/kg. These properties highlight the potential of these new compounds to be developed as new multifunctional drugs in the treatment of Alzheimer’s disease.

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EXPERIMENTAL SECTION

Chemistry. Mass spectra were generated on an Agilent LC−MS 6120 instrument with an ESI mass selective detector in positive ion mode. Melting points were determined on an SRS-Opti Melt automated melting point instrument. NMR spectra were acquired on a Bruker Avance III spectrometer with TMS as the internal standard. The purity of the synthesized compounds was determined by high-performance liquid chromatography (HPLC) with a TC-C18 column (4.6 × 250 mm, 5 μm), an acetonitrile/water or acetonitrile/water (0.1% TFA) mobile phase, and a flow rate of 1 mL/min. General Procedure I. Compounds 4, 8, 11, and 13 were synthesized according to a previously reported procedure.49 Briefly, a mixture of a nitro compound (1 equiv) and stannous chloride (4 equiv) in ethyl acetate was refluxed for 4 h. Saturated sodium bicarbonate was added to achieve alkalinity. The mixture was filtered, and the filtrate was extracted by ethyl acetate to obtain the crude product, which was purified by silica gel column chromatography (EtOAC/petroleum ether as eluent) to provide the target product. H



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Figure 5. (Top) Scheme of the inhibition experiments. (A) Results of the ThT binding assay. Statistical significance was analyzed using Student’s t test and ANOVA: (∗∗∗) p < 0.001, versus Aβ1−42 + Cu2+; (##) p < 0.01, versus Aβ1−42 alone. (B) TEM images (the inhibition of Cu(II)-induced Aβ1−42 aggregation ([Aβ1−42] = 25 μM, [5d] = 50 μM, [10d] = 50 μM, [CQ] = 50 μM, [resveratrol] = 50 μM, [Cu2+] = 25 μM, 37 °C, 24 h): (a) Aβ1−42, 0 h; (b) Aβ1−42 + Cu2+ + 5d; (c) Aβ1−42 + Cu2+ + 10d; (d) Aβ1−42 + Cu2+ + CQ; (e) Aβ1−42 + Cu2+ + resveratrol; (f) Aβ1−42 + Cu2+; (g) Aβ1−42 alone. 7.16 (q, J = 16.3 Hz, 2H), 6.70 (d, J = 1.7 Hz, 2H), 6.46 (s, 1H), 3.85 (s, 6H). (E)-4-(3,5-Dimethoxystyryl)aniline (4). Compound 4 was prepared according to general procedure I. White solid, yield: 70%. 1 H NMR (400 MHz, CDCl3) δ 7.33 (d, J = 8.3 Hz, 2H), 7.00 (d, J = 16.2 Hz, 1H), 6.85 (d, J = 16.2 Hz, 1H), 6.66 (dd, J = 14.4, 5.0 Hz, 4H), 6.36 (s, 1H), 3.83 (s, 6H). (E)-2-((4-(3,5-Dimethoxystyryl)phenylamino)methyl)phenol (5a). Compound 5a was prepared according to general procedure II. White solid, yield: 60%. Mp: 131.1−131.9 °C. 1H NMR (400 MHz, CDCl3) δ 8.00 (br, 1H), 7.39 (d, J = 8.1 Hz, 2H), 7.19 (m, 2H), 7.01 (d, J = 16.2 Hz, 1H), 6.92−6.85 (m, 3H), 6.81 (d, J = 8.2 Hz, 2H), 6.64 (s, 2H), 6.37 (s, 1H), 4.42 (s, 2H), 4.01 (br, 1H), 3.82 (s, 6H). 13 C NMR (101 MHz, CDCl3) δ 160.98, 156.46, 146.95, 139.74, 129.79, 129.27, 128.81, 127.73, 126.26, 122.89, 120.19, 116.60, 115.79, 104.34, 99.61, 55.35, 48.26. LC−MS (ESI): 362.2 [M + H]+. HRMS calcd for C23H23NO3 [M + H]+: 362.1751. Found: 362.1734. HPLC purity: 96.3%. (E)-2-((4-(3,5-Dimethoxystyryl)phenylamino)methyl)-6-methoxyphenol (5b). Compound 5b was prepared according to general procedure II. Yellow solid, yield: 75%. Mp: 138.5−140.2 °C. 1H NMR (400 MHz, CDCl3) δ 7.34 (d, J = 8.2 Hz, 2H), 6.99 (d, J = 16.4 Hz, 1H), 6.89 (d, J = 4.2 Hz, 1H), 6.84−6.81 (m, 3H), 6.67 (d, J = 8.3 Hz,

2H), 6.62 (s, 2H), 6.34 (s, 1H), 6.14 (br, 1H), 4.40 (s, 2H), 4.28 (br, 1H), 3.90 (s, 3H), 3.82 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 160.96, 148.04, 146.67, 143.89, 140.21, 129.41, 127.82, 127.00, 124.62, 124.58, 121.16, 119.65, 113.52, 109.95, 104.17, 99.30, 56.06, 55.35, 43.63. LC−MS (ESI): 392.2 [M + H]+. HRMS calcd for C24H25NO4 [M + H]+: 392.1856. Found: 392.1844. HPLC purity: 96.8%. (E)-2-((4-(3,5-Dimethoxystyryl)phenylamino)methyl)-4-nitrophenol (5c). Compound 5c was prepared according to general procedure II. Yellow solid, yield: 80%. Mp: 134.2−135.0 °C. 1H NMR (400 MHz, CDCl3) δ 8.17−8.08 (m, 2H), 7.41 (d, J = 8.4 Hz, 2H), 7.04−6.79 (m, 5H), 6.64 (d, J = 2.1 Hz, 2H), 6.38 (t, J = 2.0 Hz, 1H), 4.53 (s, 2H), 3.82 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 162.95, 160.98, 145.67, 140.88, 139.47, 131.09, 128.41, 127.83, 127.06, 125.46, 124.60, 122.94, 117.10, 116.52, 104.42, 99.73, 55.37, 48.69. LC−MS (ESI): 407.1 [M + H]+. HRMS calcd for C23H22N2O5 [M + H]+: 407.1601. Found: 407.1602. HPLC purity: 97.4%. (E)-2-((4-(3,5-Dimethoxystyryl)phenylamino)methyl)-4(dimethylamino)phenol (5d). Compound 5d was prepared according to general procedure II. Yellow solid, yield: 58%. Mp: 106.7−107.2 °C. 1H NMR (400 MHz, CDCl3) δ 7.39 (d, J = 8.4 Hz, 2H), 7.01 (d, J = 16.2 Hz, 1H), 6.88 (d, J = 16.2 Hz, 1H), 6.81 (dd, J = 8.3, 4.4 Hz, 3H), 6.70 (d, J = 8.7 Hz, 1H), 6.64 (d, J = 1.9 Hz, 3H), 6.37 (s, 1H), 4.38 (s, 2H), 4.00 (br, 1H), 3.83 (s, 6H), 2.87 (s, 6H). 13 C NMR (101 MHz, CDCl3) δ 160.98, 147.31, 139.84, 129.35, I



Article

Figure 6. (Top) Schematic of the disaggregation experiments. (A) Results of the ThT binding assay. Statistical significance was analyzed by ANOVA: (∗∗) p < 0.01, (∗∗∗) p < 0.001, versus control. (B) TEM images ([Aβ1−42] = 25 μM, [5d] = 25 μM, [10d] = 25 μM, [CQ] = 25 μM, [resveratrol] = 25 μM, 37 °C, 24 h): (a) Aβ1−42, 0 h; (b) Aβ1−42 + 5d; (c) Aβ1−42 + 10d; (d) Aβ1−42 + CQ; (e) Aβ1−42 + resveratrol; (f) Aβ1−42 alone. for C24H25NO4 [M + H]+: 392.1856. Found: 392.1855. HPLC purity: 96.3%. (E)-2-((4-(3,5-Dimethoxystyryl)phenylamino)methyl)-N,N-dimethylaniline (6a). Compound 6a was prepared according to general procedure II. Yellow solid, yield: 52%. Mp: 87.6−89.4 °C. 1 H NMR (400 MHz, CDCl3) δ 7.34 (t, J = 7.6 Hz, 3H), 7.23 (dd, J = 9.7, 5.3 Hz, 1H), 7.13 (d, J = 8.0 Hz, 1H), 7.01 (d, J = 11.7 Hz, 1H), 6.97 (d, J = 16.9 Hz, 1H), 6.81 (d, J = 16.2 Hz, 1H), 6.62 (d, J = 6.5 Hz, 4H), 6.34 (s, 1H), 4.52 (br, 1H), 4.40 (s, 2H), 3.80 (s, 6H), 2.72 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 160.96, 152.54, 148.39, 140.26, 133.04, 129.48, 129.16, 128.04, 127.86, 126.51, 124.32, 123.41, 119.36, 113.01, 104.13, 99.23, 55.34, 44.91, 44.50. LC−MS (ESI): 389.2 [M + H]+. HRMS calcd for C25H28N2O2 [M + H]+: 389.2224. Found: 389.2220. HPLC purity: 96.3%. (E)-4-(3,5-Dimethoxystyryl)-N-(2-methoxybenzyl)aniline (6b). Compound 6b was prepared according to general procedure II. Yellow solid, yield: 50%. Mp: 84.2−85.5 °C. 1H NMR (400 MHz, CDCl3) δ 7.39−7.26 (m, 4H), 7.01 (d, J = 16.2 Hz, 1H), 6.93−6.91 (m, 2H), 6.84 (d, J = 16.1 Hz, 1H), 6.66−6.65 (m, 4H), 6.37 (s, 1H), 4.38 (s, 2H), 4.26 (br, 1H), 3.89 (s, 3H), 3.84 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 161.03, 157.45, 148.33, 140.33, 129.54, 128.85, 128.43, 127.88, 127.19, 126.55, 124.37, 120.60, 113.12, 110.39, 104.21, 99.31, 55.34, 43.35. LC−MS (ESI): 376.2 [M + H]+. HRMS calcd

128.94, 127.73, 125.98, 115.46, 104.34, 99.59, 55.35, 48.32, 41.93. LC−MS (ESI): 405.2 [M + H]+. HRMS calcd for C25H28N2O3 [M + H]+: 405.2173. Found: 405.2160. HPLC purity: 98.6%. (E)-4-Chloro-2-((4-(3,5-dimethoxystyryl)phenylamino)methyl)phenol (5e). Compound 5e was prepared according to general procedure II. Yellow solid, yield: 80%. Mp: 100.2−101.4 °C. 1 H NMR (400 MHz, CDCl3) δ 8.21 (br, 1H), 7.41 (d, J = 8.1 Hz, 2H), 7.18 (d, J = 10.0 Hz, 2H), 7.02 (d, J = 16.2 Hz, 1H), 6.91 (d, J = 16.2 Hz, 1H), 6.82 (d, J = 8.0 Hz, 3H), 6.66 (s, 2H), 6.39 (s, 1H), 4.40 (s, 2H), 4.05 (br, 1H), 3.84 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 161.00, 155.20, 146.50, 139.67, 130.22, 128.95, 128.68, 128.32, 127.77, 126.55, 124.76, 124.40, 117.94, 115.95, 104.42, 99.68, 55.35, 48.11. LC−MS (ESI): 396.1 [M + H]+. HRMS calcd for C23H22NO3Cl [M + H]+: 396.1361. Found: 396.1348. HPLC purity: 98.0%. (E)-2-((4-(3,5-Dimethoxystyryl)phenylamino)methyl)-5-methoxyphenol (5f). Compound 5f was prepared according to general procedure II. Yellow solid, yield: 60%. Mp: 132.5−133.7 °C. 1H NMR (400 MHz, CDCl3) δ 8.15 (br, 1H), 7.41 (d, J = 8.2 Hz, 2H), 7.07 (d, J = 8.2 Hz, 1H), 7.03 (d, J = 16.4 Hz, 1H), 6.90 (d, J = 16.2 Hz, 1H), 6.84 (d, J = 8.2 Hz, 2H), 6.66 (s, 2H), 6.53−6.44 (m, 2H), 6.39 (s, 1H), 4.38 (s, 2H), 3.84 (s, 6H), 3.80 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 160.97, 160.79, 157.62, 147.00, 139.73, 129.82, 129.41, 128.80, 127.71, 126.26, 115.91, 115.07, 106.04, 104.33, 102.26, 99.60, 55.36, 47.89. LC−MS (ESI): 392.2 [M + H]+. HRMS calcd J



Article

Figure 7. (Top) Schematic of the disaggregation experiments. (A) Results of the ThT binding assay. Statistical significance was analyzed by ANOVA: (∗∗∗) p < 0.001, versus control. (B) TEM images ([Aβ1−42] = 25 μM, [5d] = 50 μM, [10d] = 50 μM, [CQ] = 50 μM, [resveratrol] = 50 μM, [Cu2+] = 25 μM, 37 °C, 24 h): (a) Aβ1−42, 0 h; (b) Aβ1−42 + Cu2+ + 5d; (c) Aβ1−42 + Cu2+ + 10d; (d) Aβ1−42 + Cu2+ + CQ; (e) Aβ1−42 + Cu2+ + resveratrol; (f) Aβ1−42 + Cu2+.

Table 2. ChE and MAO Inhibitory Activities of 5d and 10d IC50 (μM)a compd

AChEb

BuChEc

MAO-A

MAO-B

5d 10d clorgyline ladostigil

36.04 ± 1.18 6.27 ± 0.43 nt 50 ± 4.8

>50 21.25 ± 3.32 nt nt

8.19 ± 0.81 7.08 ± 0.67 4.1 ± 0.2 nM nt

12.16 ± 1.04 14.09 ± 1.06 nt 37.1 ± 3.1

The values represent the mean ± standard error of at least three different experiments. nt: not tested. bAChE from electric eel was used. cBuChE from equine serum was used.

a

for C24H25NO3 [M + H]+: 376.1907. Found: 376.1902. HPLC purity: 98.1%. (E)-N-((1H-Pyrrol-3-yl)methyl)-4-(3,5-dimethoxystyryl)aniline (7a). Compound 7a was prepared according to general procedure II. Gray solid, yield: 56%. Mp: 112.9−114.1 °C. 1H NMR (400 MHz, CDCl3) δ 8.30 (br, 1H), 7.35 (d, J = 8.0 Hz, 2H), 7.00 (d, J = 16.2 Hz, 1H), 6.84 (d, J = 16.2 Hz, 1H), 6.73 (s, 1H), 6.70−6.59 (m, 4H), 6.35 (s, 1H), 6.21−6.11 (m, 2H), 4.34 (s, 2H), 4.03 (br, 1H), 3.82 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 160.96, 147.86,

140.07, 129.28, 129.22, 127.94, 127.31, 124.88, 117.60, 113.20, 108.60, 106.42, 104.20, 99.35, 55.36, 41.67. LC−MS (ESI): 335.2 [M + H]+. HRMS calcd for C21H22N2O2 [M + H]+: 335.1754. Found: 335.1754. HPLC purity: 98.9%. (E)-N-((1H-Indol-3-yl)methyl)-4-(3,5-dimethoxystyryl)aniline (7b). Compound 7b was prepared according to general procedure II. White solid, yield: 40%. Mp: 151.4−153.5 °C. 1H NMR (400 MHz, CDCl3) δ 8.05 (br, 1H), 7.67 (d, J = 7.8 Hz, 1H), 7.38 (t, J = 8.4 Hz, 3H), 7.24−7.11 (m, 3H), 7.01 (d, J = 16.2 Hz, 1H), 6.83 (d, J = 16.2 K



Article

Figure 8. (A) Effects of 5d and 10d on cell viability in SH-SY5Y cells. (B) Percentage increase in intracellular ROS induced by exposure to t-BuOOH, as determined by DCFH-DA. The results are reported as the mean of three independent experiments performed in sextuplicate. Statistical significance was analyzed using ANOVA: (∗) p < 0.5, (∗∗) p < 0.01, (∗∗∗) p < 0.001, versus control.

Table 3. Permeability Pe (×10−6 cm/s) in the PAMPA-BBB Assay of the Selected Compounds and Their Predictive Penetration in the CNS compda

Pe (×10−6 cm/s)b

prediction

5d·HCl 10d·HCl

7.13 ± 0.64 2.55 ± 0.31

CNS+ CNS±

[M + H]+. HRMS calcd for C27H32N2O3 [M + H]+: 433.2486. Found: 433.2468. HPLC purity: 96.0% (E)-2-((4-(3,5-Dimethoxystyryl)phenylamino)methyl)-4(isopropylamino)phenol (9c). Compound 9c was prepared according to general procedure III. Yellow oil, yield: 21%. Mp: 91.1−93.2 °C. 1H NMR (400 MHz, CDCl3) δ 7.38 (d, J = 8.5 Hz, 2H), 7.01 (d, J = 16.2 Hz, 1H), 6.87 (d, J = 16.2 Hz, 1H), 6.79 (d, J = 8.5 Hz, 2H), 6.75 (d, J = 8.5 Hz, 1H), 6.64 (d, J = 2.2 Hz, 2H), 6.52 (dd, J = 8.5, 2.8 Hz, 1H), 6.48 (d, J = 2.6 Hz, 1H), 6.37 (t, J = 2.1 Hz, 1H), 4.33 (s, 2H), 3.82 (s, 6H), 3.54 (dt, J = 12.5, 6.3 Hz, 1H), 1.19 (d, J = 6.3 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 160.98, 148.14, 147.41, 141.00, 139.88, 129.05, 129.00, 127.74, 125.80, 124.15, 117.24, 115.30, 115.0, 115.02, 104.33, 99.55, 55.36, 47.76, 45.59, 23.02. LC−MS (ESI): 419.2 [M + H]+. HRMS calcd for C26H30N2O3 [M + H]+: 419.2329. Found: 419.2310. HPLC purity: 95.7%. (E)-5-(4-(2-Hydroxybenzylamino)styryl)benzene-1,3-diol (10a). Compound 10a was prepared according to general procedure IV. Green solid, yield: 70%. Mp: 152.7−156.5 °C. 1H NMR (400 MHz, MeOD) δ 7.68 (d, J = 8.5 Hz, 2H), 7.40 (d, J = 8.5 Hz, 2H), 7.33−7.26 (m, 1H), 7.21 (d, J = 7.5 Hz, 1H), 7.14−7.03 (m, 2H), 6.95 (d, J = 8.1 Hz, 1H), 6.86 (d, J = 7.5 Hz, 1H), 6.52 (d, J = 2.0 Hz, 2H), 6.25 (s, 1H), 4.56 (s, 2H). 13C NMR (101 MHz, MeOD) δ 159.77, 157.69, 140.73, 135.12, 133.00, 132.68, 128.96, 127.46, 124.47, 120.93, 118.28, 116.29, 106.44, 103.84, 53.69. LC−MS (ESI): 334.1 [M + H]+. HRMS calcd for C21H19NO3 [M + H]+: 334.1438. Found: 334.1436. HPLC purity: 95.6%. (E)-3-((4-(3,5-Dihydroxystyryl)phenylamino)methyl)benzene-1,2-diol (10b). Compound 10b was prepared according to general procedure IV. Yellow solid, yield: 78%. Decomposed ∼210 °C. 1 H NMR (400 MHz, MeOD) δ 7.68 (d, J = 8.5 Hz, 2H), 7.41 (d, J = 8.4 Hz, 2H), 7.16−7.00 (m, 2H), 6.88 (dd, J = 6.3, 3.1 Hz, 1H), 6.76− 6.63 (m, 2H), 6.52 (d, J = 1.9 Hz, 2H), 6.24 (s, 1H), 4.54 (s, 2H). 13C NMR (101 MHz, MeOD) δ 159.81, 146.48, 146.10, 140.61, 140.15, 135.30, 132.49, 128.97, 127.50, 124.41, 123.12, 120.83, 118.62, 117.96, 106.45, 103.81, 53.52. LC−MS (ESI): 350.2 [M + H]+. HRMS calcd for C21H19NO4 [M + H]+: 350.1387. Found: 350.1390. HPLC purity: 96.1%. (E)-5-(4-(2-Hydroxy-5-nitrobenzylamino)styryl)benzene-1,3diol (10c). Compound 10c was prepared according to general procedure IV. Red solid, yield: 81%. Mp: 160.9−161.3 °C. 1H NMR (400 MHz, MeOD) δ 8.15 (s, 1H), 7.99 (dd, J = 6.8, 2.1 Hz, 1H), 7.27 (d, J = 7.0 Hz, 2H), 6.91 (dd, J = 8.8, 7.0 Hz, 2H), 6.77−6.69 (m, 1H), 6.61 (d, J = 7.0 Hz, 2H), 6.44 (d, J = 1.8 Hz, 2H), 6.17 (d, J = 2.0 Hz, 1H), 4.35 (s, 2H). 13C NMR (101 MHz, MeOD) δ 162.78, 159.56, 149.48, 141.89, 141.72, 129.96, 128.72, 128.08, 125.48, 125.35, 125.29, 115.82, 114.20, 105.76, 102.45, 43.22. LC−MS (ESI): 379.1 [M + H]+. HRMS calcd for C21H18N2O5 [M + H]+: 379.1288. Found: 379.1287. HPLC purity: 98.8%. (E)-5-(4-(5-(Dimethylamino)-2-hydroxybenzylamino)styryl)benzene-1,3-diol (10d). Compound 10d was prepared according to general procedure IV. Yellow solid, yield: 60%. Mp: 180.7−181.8 °C.

a

Compounds were dissolved in DMSO at 5 mg/mL and diluted with PBS/EtOH (70:30). The final concentration of compounds was 100 μg/mL. bValues are expressed as the mean ± SD of three independent experiments.

Hz, 1H), 6.68 (d, J = 8.3 Hz, 2H), 6.64 (s, 2H), 6.35 (s, 1H), 4.51 (s, 2H), 4.02 (br, 1H), 3.82 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 160.98, 148.31, 140.25, 136.46, 129.47, 127.87, 126.67, 126.59, 124.41, 122.71, 122.48, 119.84, 118.95, 113.78, 112.90, 111.29, 104.16, 99.30, 55.34, 39.96. LC−MS (ESI): 385.2 [M + H]+. HRMS calcd for C25H24N2O2 [M + H]+: 385.1911. Found: 385.1911. HPLC purity: 97.8%. (E)-4-Amino-2-((4-(3,5-dimethoxystyryl)phenylamino)methyl)phenol (8). Compound 8 was prepared according to general procedure I. Yellow solid, yield: 45%. 1H NMR (400 MHz, DMSO-d6) δ 7.29 (d, J = 8.5 Hz, 2H), 7.07 (d, J = 16.3 Hz, 1H), 6.81 (d, J = 16.3 Hz, 1H), 6.66 (d, J = 2.0 Hz, 2H), 6.59−6.51 (m, 3H), 6.48 (d, J = 2.6 Hz, 1H), 6.32 (s, 1H), 6.30 (d, J = 2.7 Hz, 1H), 4.11 (s, 2H), 3.76 (s, 6H). (E)-2-((4-(3,5-Dimethoxystyryl)phenylamino)methyl)-4(propylamino)phenol (9a). Compound 9a was prepared according to general procedure III. Yellow solid, yield: 38%. Mp: 120.3−123.4 °C. 1H NMR (400 MHz, CDCl3) δ 7.39 (d, J = 8.4 Hz, 2H), 7.01 (d, J = 16.4 Hz, 1H), 6.88 (d, J = 16.3 Hz, 1H), 6.80 (d, J = 8.3 Hz, 2H), 6.76 (d, J = 8.6 Hz, 1H), 6.64 (s, 2H), 6.56−6.47 (m, 2H), 6.37 (s, 1H), 4.35 (s, 2H), 3.83 (s, 6H), 3.04 (t, J = 7.1 Hz, 2H), 1.66−1.60 (m, 2H), 1.00 (t, J = 7.4 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 160.97, 147.95, 147.48, 142.28, 139.90, 129.03, 128.92, 127.76, 125.71, 124.21, 117.17, 115.26, 114.05, 113.89, 104.33, 99.52, 55.37, 47.63, 47.04, 22.82, 11.70. LC−MS (ESI): 419.2 [M + H]+. HRMS calcd for C26H30N2O3 [M + H]+: 419.2329. Found: 419.2321. HPLC purity: 95.5%. (E)-2-((4-(3,5-Dimethoxystyryl)phenylamino)methyl)-4(isobutylamino)phenol (9b). Compound 9b was prepared according to general procedure III. Yellow solid, yield: 26%. Mp: 86.7− 87.1 °C. 1H NMR (400 MHz, CDCl3) δ 7.40 (d, J = 8.5 Hz, 2H), 7.03 (d, J = 16.2 Hz, 1H), 6.89 (d, J = 16.2 Hz, 1H), 6.81 (d, J = 8.5 Hz, 2H), 6.77 (d, J = 8.5 Hz, 1H), 6.66 (d, J = 2.2 Hz, 2H), 6.57−6.47 (m, 2H), 6.38 (t, J = 2.1 Hz, 1H), 4.36 (s, 2H), 3.84 (s, 6H), 2.90 (d, J = 6.7 Hz, 2H), 1.88 (dt, J = 13.3, 6.7 Hz, 1H), 1.00 (d, J = 6.6 Hz, 6H). 13 C NMR (101 MHz, CDCl3) δ 160.98, 147.85, 147.37, 142.48, 139.86, 129.18, 128.97, 127.74, 125.88, 123.94, 117.24, 115.38, 113.76, 113.67, 104.32, 99.56, 55.35, 52.98, 47.98, 28.09, 20.53. LC−MS (ESI): 433.2 L



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H NMR (400 MHz, MeOD) δ 7.27 (d, J = 8.4 Hz, 2H), 6.91 (d, J = 17.3 Hz, 2H), 6.76−6.61 (m, 5H), 6.43 (s, 2H), 6.15 (s, 1H), 4.31 (s, 2H), 2.72 (s, 6H). 13C NMR (101 MHz, MeOD) δ 158.13, 148.58, 148.35, 144.88, 140.28, 128.63, 127.20, 126.55, 125.95, 123.87, 116.41, 115.31, 115.01, 113.21, 104.31, 101.00, 43.15, 41.82. LC−MS (ESI): 377.2 [M + H]+. HRMS calcd for C23H24N2O3 [M + H]+: 377.1860. Found: 377.1851. HPLC purity: 98.2%. (E)-5-(4-(2-(Dimethylamino)benzylamino)styryl)benzene1,3-diol (10e). Compound 10e was prepared according to general procedure IV. Yellow semisolid, yield: 56%. 1H NMR (400 MHz, MeOD) δ 7.25 (d, J = 7.5 Hz, 1H), 7.13 (d, J = 8.4 Hz, 2H), 7.09− 7.03 (m, 2H), 6.89 (d, J = 7.4 Hz, 1H), 6.79 (d, J = 16.2 Hz, 1H), 6.59 (d, J = 16.2 Hz, 1H), 6.47 (d, J = 8.3 Hz, 2H), 6.32 (d, J = 1.7 Hz, 2H), 6.04 (s, 1H), 4.28 (s, 2H), 2.61 (s, 6H). 13C NMR (101 MHz, MeOD) δ 159.59, 153.74, 150.07, 141.76, 134.97, 130.05, 129.78, 128.64, 127.53, 125.10, 124.48, 120.15, 114.08, 105.65, 102.35, 45.44, 44.52. LC−MS (ESI): 361.3 [M + H]+. HRMS calcd for C23H24N2O2 [M + H]+: 361.1911. Found: 361.1922. HPLC purity: 96.1%. (E)-4-(4-Methoxystyryl)aniline (11). Compound 11 was prepared according to general procedure I. Gray solid, yield: 65%. 1H NMR (400 MHz, CDCl3) δ 7.40 (d, J = 7.7 Hz, 2H), 7.30 (d, J = 7.4 Hz, 2H), 6.88 (s, 4H), 6.66 (d, J = 7.5 Hz, 2H), 3.82 (s, 3H), 3.70 (br, 2H). (E)-4-(Dimethylamino)-2-((4-(4-methoxystyryl)phenylamino)methyl)phenol (12). Compound 12 was prepared according to general procedure II. Yellow solid, yield: 71%. Mp: 147.2−148.4 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.76 (br, 1H), 7.42 (d, J = 8.0 Hz, 2H), 7.27 (d, J = 7.9 Hz, 2H), 6.89 (dd, J = 18.1, 10.5 Hz, 3H), 6.70 (d, J = 10.5 Hz, 2H), 6.60 (d, J = 8.0 Hz, 2H), 6.51 (d, J = 8.5 Hz, 1H), 6.12 (s, 1H), 4.18 (d, J = 5.2 Hz, 2H), 3.75 (s, 3H), 2.78 (br, 1H), 2.69 (s, 6H). 13C NMR (101 MHz, DMSO-d6) δ 158.11, 148.49, 146.85, 144.32, 130.59, 127.12, 126.86, 126.81, 125.73, 125.03, 122.55, 115.41, 114.45, 114.04, 112.94, 112.30, 55.02, 41.67, 41.40. LC−MS (ESI): 375.2 [M + H]+. HRMS calcd for C24H26N2O2 [M + H]+: 375.2067. Found: 375.2061. HPLC purity: 95.4%. (E)-4-(4-Aminostyryl)-N,N-dimethylaniline (13). Compound 13 was prepared according to general procedure I. Yellow solid, yield: 60%. 1H NMR (400 MHz, CDCl3) δ 7.37 (d, J = 8.8 Hz, 2H), 7.30 (d, J = 8.4 Hz, 2H), 6.85 (d, J = 2.3 Hz, 2H), 6.72 (d, J = 8.6 Hz, 2H), 6.66 (d, J = 8.5 Hz, 2H), 2.97 (s, 6H). (E)-4-(Dimethylamino)-2-((4-(4-(dimethylamino)styryl)phenylamino)methyl)phenol (14). Compound 14 was prepared according to general procedure II. Yellow solid, yield: 52%. Mp: 172.9−175.5 °C. 1H NMR (400 MHz, CDCl3) δ 7.36 (t, J = 8.6 Hz, 4H), 6.84 (dt, J = 16.3, 12.3 Hz, 5H), 6.70 (d, J = 8.5 Hz, 3H), 6.64 (s, 1H), 4.36 (s, 2H), 2.96 (s, 6H), 2.86 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 149.84, 148.54, 146.30, 145.23, 130.80, 127.22, 127.06, 126.41, 126.29, 124.18, 123.43, 117.09, 115.78, 114.83, 114.59, 112.59, 48.95, 41.95, 40.53. LC−MS (ESI): 388.2 [M + H]+. HRMS calcd for C25H29N3O [M + H]+: 388.2383. Found: 388.2376. HPLC purity: 98.6%. Biological Assays. ThT Assay. Aβ1−42 (Millipore; counterion, NaOH) was dissolved in ammonium hydroxide (1% v/v) to give a stock solution (2000 μM), which was aliquoted into small samples and stored at −80 °C. For the inhibition of self-mediated Aβ1−42 aggregation experiment, the Aβ stock solution was diluted with 50 mM phosphate buffer (pH 7.4) to 50 μM before use. A mixture of the peptide (10 μL, 25 μM, final concentration) with or without the tested compound (10 μL, 20 μM, final concentration) was incubated at 37 °C for 48 h. Blanks using 50 mM phosphate buffer (pH 7.4) instead of Aβ with or without inhibitors were also carried out. The sample was diluted to a final volume of 200 μL with 50 mM glycine−NaOH buffer (pH 8.0) containing thioflavin T (5 μM). Then the fluorescence intensities were recorded 5 min later (excitation, 450 nm; emission, 485 nm).24 The percent inhibition of aggregation was calculated by the expression (1 − IFi/IFc) × 100, in which IFi and IFc are the fluorescence intensities obtained for Aβ in the presence and absence of inhibitors after subtracting the background, respectively. 1

For the inhibition of copper-mediated Aβ1−42 aggregation experiment, the Aβ stock solution was diluted in 20 μM HEPES (pH 6.6) with 150 μM NaCl. The mixture of the peptide (10 μL, 25 μM, final concentration) with or without copper (10 μL, 25 μM, final concentration) and the tested compound (10 μL, 50 μM, final concentration) was incubated at 37 °C for 24 h. Then 20 μL of the sample was diluted to a final volume of 200 μL with 50 mM glycine− NaOH buffer (pH 8.0) containing thioflavin T (5 μM). The detection method was the same as that of self-mediated Aβ1−42 aggregation experiment. For the disaggregation of self-induced Aβ fibrils experiment, the Aβ stock solution was diluted with 10 mM phosphate buffer (pH 7.4). The peptide (15 μL, 50 μM) was incubated at 37 °C for 24 h. The tested compound (15 μL, 50 μM) was then added and incubated at 37 °C for another 24 h. Then 20 μL of the sample was diluted to a final volume of 200 μL with 50 mM glycine−NaOH buffer (pH 8.0) containing thioflavin T (5 μM). The detection method was the same as above. For the disaggregation of copper-induced Aβ fibrils experiment, the Aβ stock solution was diluted in 20 μM HEPES (pH 6.6) with 150 μM NaCl. The mixture of the peptide (10 μL, 25 μM, final concentration) with copper (10 μL, 25 μM, final concentration) was incubated at 37 °C for 24 h. The tested compound (10 μL, 50 μM, final concentration) was then added and incubated at 37 °C for another 24 h.50 Then 20 μL of the sample was diluted to a final volume of 200 μL with 50 mM glycine−NaOH buffer (pH 8.0) containing thioflavin T (5 μM). The detection method was the same as above. TEM Assay.50 For the metal-free experiment, the Aβ stock solution was diluted with 10 mM phosphate buffer (pH 7.4). For the copper-induced experiment, the Aβ stock solution was diluted with 20 μM HEPES (pH 6.6) and 150 μM NaCl. The sample preparation was the same as for the ThT assay. Aliquots (10 μL) of the samples were placed on a carbon-coated copper/rhodium grid for 2 min. Each grid was stained with uranyl acetate (1%, 5 μL) for 2 min. After draining off the excess staining solution, the specimen was transferred for imaging by transmission electron microscopy (JEOL JEM-1400). All compounds are solubilized in the buffer which was used for the experiment. Oxygen Radical Absorbance Capacity (ORAC-FL) Assay.51 The tested compound and fluorescein (FL) stock solution were diluted with 75 mM phosphate buffer (pH 7.4) to 10 and 0.117 μM, respectively. The solution of (±)-6-hydroxy-2,5,7,8-tetramethylchroman2-carboxylic acid (Trolox) was diluted with the same buffer to 100, 80, 60, 50, 40, 20, and 10 μM. The solution of 2,2′-azobis(amidinopropane)dihydrochloride (AAPH) was prepared before the experiment by dissolving 108.4 mg of AAPH in 10 mL of 75 mM phosphate buffer (pH 7.4) to a final concentration of 40 mM. The mixture of the tested compound (20 μL) and FL (120 μL, 70 nM, final concentration) was preincubated for 10 min at 37 °C, and then 60 μL of the AAPH solution was added. The fluorescence was recorded every minute for 120 min (excitation, 485 nm; emission, 520 nm). A blank using phosphate buffer instead of the tested compound was also carried out. All reaction mixtures were prepared in triplicate, and at least three independent runs were performed for each sample. The antioxidant curves (fluorescence versus time) were normalized to the curve of the blank. The area under the fluorescence decay curve (AUC) was calculated using following equation: i = 120

AUC = 1 +

∑

(fi /f0 )

i=1

where f 0 is the initial fluorescence reading at 0 min and f i is the fluorescence reading at time i. The net AUC was calculated by the expression AUCsample − AUCblank. Regression equations between net AUC and Trolox concentrations were calculated. ORAC-FL value for each sample was calculated by using the standard curve, which means the ORAC-FL value of tested compound expressed as Trolox equivalents.52 Metal-Chelating Study. The chelating studies were performed with a UV−vis spectrophotometer. The absorption spectra of each M



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compound (20 μM, final concentration) alone or in the presence of CuSO4, FeSO4, or ZnCl2 (40 μM, final concentration) for 30 min in 20% (v/v) ethanol/buffer (20 mM HEPES, 150 mM NaCl, pH 7.4) were recorded at room temperature. The stoichiometry of the compound−Cu 2+ complex was determined by Job’s method. Separate solutions of compound and CuSO4 were used to prepare solutions in which the sum of the concentrations of both species (2.0 × 10−5 M) was constant in all samples, but the proportions of either component varied between 0% and 100%.53 Blanks using 20% (v/v) ethanol/buffer instead of Cu2+ were also carried out. The UV spectra were normalized to the respective blanks. The absorbance differences at 333 nm for 5d and 339 nm for 10d were plotted versus the mole fraction. The minimum revealed the stoichiometry of the compound−Cu2+ complex. In Vitro Inhibition of ChE. Acetylcholinesterase (AChE, EC 3.1.1.7, from the electric eel), butyrylcholinesterase (BuChE, EC 3.1.1.8, from equine serum), 5,5′-dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent, DTNB), acetylthiocholine chloride (ATC), and butylthiocholine chloride (BTC) were purchased from Sigma Aldrich. All in vitro AChE assays were performed in 0.1 M KH2PO4/K2HPO4 buffer, pH 8.0, using a Shimadzu UV-2450 spectrophotometer. AChE solutions were prepared to give 2.0 units/mL in 2 mL aliquots. The assay medium (1 mL) consisted of phosphate buffer (pH 8.0), 50 μL of 0.01 M DTNB, 10 μL of enzyme, and 50 μL of 0.01 M substrate. The test compounds were added to the assay solution and preincubated with the enzyme at 37 °C for 15 min, followed by the addition of substrate. Activity was determined by measuring the increase in absorbance at 412 nm at 1 min intervals at 37 °C. Calculations were performed according to the method of Ellman et al.54 Each concentration was assayed in triplicate. Blanks containing all components except AChE were carried out. The percent inhibition was calculated by the following expression: (1 − Ai/Ac) × 100, where Ai and Ac are the absorbance obtained for AChE in the presence and absence of inhibitors, respectively, after subtracting the respective background. The in vitro BuChE assay (BuChE or ACh as the enzyme substrate) was performed using a method similar to that described above. In Vitro Inhibition of Monoamine Oxidase. Adequate amounts of recombinant hMAO-A or hMAO-B (Sigma-Aldrich) were acquired and adjusted to 12.5 μg/mL for hMAO-A and 75 μg/mL for hMAO-B. The test drugs (20 μL) and MAO (80 μL) were incubated for 15 min at 37 °C in a flat, black-bottomed 96-well microtest plate in the dark. The reaction was initiated by adding 200 μM Amplex Red reagent, 2 U/mL horseradish peroxidase, and 2 mM p-tyramine for hMAO-A or 2 mM benzylamine for hMAO-B and incubated at 37 °C for 20 min. Activity was quantified in a multidetection microplate fluorescence reader based on the fluorescence generated (excitation, 545 nm; emission, 590 nm). The specific fluorescence emission was calculated after subtraction of the background activity. The background activity was determined from wells containing all components except the hMAO isoforms, which were replaced by a sodium phosphate buffer solution. The percent inhibition was calculated by the following expression: (1 − IFi/IFc) × 100 in which IFi and IFc are the fluorescence intensities obtained for hMAO in the presence and absence of inhibitors after subtracting the respective background. Docking Study. The simulation system was built based on the structure obtained from the Protein Data Bank (PDB codes: 1IYT for Aβ1−42, 2V61 for MAO-B, 2CMF for AChE). The heteroatoms and water molecules were removed, and all hydrogen atoms were subsequently added to the protein. Then force field was assigned to the enzyme. The ligand binding site was defined as 13 Å from the original ligand. Prior to the docking calculations, the original ligand was removed. The 3D structures of 5d and 10d were generated and optimized with the Discovery Studio 2.1 package (Accelrys Inc., San Diego, CA). The CDOCKER program of the Discovery Studio 2.1 software, which allows full flexibility of ligands, was used to perform docking simulations. The docking and subsequent scoring were performed using the default parameters of the CDOCKER program. CDOCKER_INTERACTION_ENERGY is used like a score where a lower value indicates a more favorable binding.

Cell Culture. The human neuron-like cell line SH-SY5Y was obtained from Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences (Shanghai, China). Cells were grown at 37 °C in a humidified incubator with 5% CO2 in Dulbecco’s modified Eagle medium (DMEM, GIBCO) supplemented with 10% fetal calf serum (FCS, GIBCO), 1 mM glutamine, 100 IU/mL penicillin, and 100 μg/mL streptomycin. Determination of Cytotoxicity. Cytotoxicity was evaluated with the colorimetric MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium bromide] assay.55 SH-SY5Y cells were seeded at 5 × 104 cells/well in 96-well plates. After 24 h, the medium was removed and replaced with the tested compounds at different concentrations for 24 h at 37 °C. After replacement of the tested compounds with 80 μL of medium and 20 μL of MTT in PBS (0.5 mg/mL, final concentration), the cells were incubated for another 4 h. After the removal of MTT, the formazan crystals were dissolved in DMSO. The amount of formazan was measured (570 nm). Cell viability was expressed as the percentage of control cells and was calculated using the formula Ft/Fnt × 100, where Ft is the absorbance of the treated neurons after subtracting the absorbance of zero day control and Fnt is the absorbance of the untreated neurons after subtracting the absorbance of zero day control. Antioxidant Activity in SH-SY5Y Cells. SH-SY5Y cells were seeded at 1 × 104 cells/well in 96-well plates. After 24 h, the medium was removed and replaced with the tested compounds (2.5, 5, 10 μM) at 37 °C and incubated for another 24 h. Trolox was used as the control with concentrations of 5, 10, and 20 μM. The cells were washed with PBS and incubated with 5 μM DCFH-DA (a fluorescent probe) in PBS at 37 °C and 5% CO2 for 30 min. After removal of the DCFH-DA and further washing, the cells were exposed to 0.1 mM t-BuOOH (a compound used to induce oxidative stress) in PBS for 30 min. At the end of the incubation, the fluorescence of the cells in each well was measured (excitation, 485 nm; emission, 535 nm) with a multifunctional microplate reader (Molecular Devices, Flex Station 3). The antioxidant activity was expressed as the percentage of control cells and calculated with the formula [(Ft − Fnt)/(Ft′ − Fnt)] × 100, where Ft is the absorbance of neurons treated with the tested compound, Ft′ is the absorbance of neurons not treated with the tested compound, and Fnt is the absorbance of neurons not treated with t-BuOOH. In Vitro Blood−Brain Barrier Permeation Assay. The blood− brain barrier penetration of compounds was evaluated using the parallel artificial membrane permeation assay (PAMPA) described by Di et al.46−48 Commercial drugs were purchased from Sigma and Alfa Aesar. Porcine brain lipid (PBL) was obtained from Avanti Polar Lipids. The donor microplate (PVDF membrane, pore size 0.45 mm) and acceptor microplate were both from Millipore. The 96-well UV plate (COSTAR) was from Corning Incorporated. The acceptor 96-well microplate was filled with 300 μL of PBS/EtOH (7:3), and the filter membrane was impregnated with 4 μL of PBL in dodecane (20 mg/mL). Compounds were dissolved in DMSO at 5 mg/mL and diluted 50-fold in PBS/EtOH (7:3) to a final concentration of 100 μg/mL. Then 200 μL of the solution was added to the donor wells. The acceptor filter plate was carefully placed on the donor plate to form a sandwich, which was left undisturbed for 10 h at 25 °C. After incubation, the donor plate was carefully removed, and the concentration of compounds in the acceptor wells was determined using the UV plate reader (Flexstation 3). Every sample was analyzed at five wavelengths in four wells and in at least three independent runs. Pe was calculated using the following expression: Pe = {−VdVa/[(Vd + Va)At]}ln(1 − drugacceptor/drugequilibrium) where Vd is the volume of donor well, Va is the volume in the acceptor well, A is the filter area, t is the permeation time, drugacceptor is the absorbance obtained in the acceptor well, and drugequilibrium is the theoretical equilibrium absorbance. The results are given as the mean ± standard deviation. In the experiment, 13 quality control standards of known BBB permeability were included to validate the analysis set. A plot of the experimental data versus literature values gave a strong linear correlation, Pe(exp) = 1.4574Pe(lit.) − 1.0773 (R2 = 0.9427) (Figure S5). From this equation and the limit established by Di et al. (Pe(lit.) = 4.0 × 10−6 cm/s) for blood−brain barrier permeation, we concluded that compounds with a N



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permeability greater than 4.7 × 10−6 cm/s could cross the blood−brain barrier (Table S2). Acute Toxicity. A total of 20 KM mice (male, 22 days, 18−20 g) purchased from the laboratory animal center of Sun Yat-sen University (Guangzhou, China) were used to evaluate the acute toxicity of compound 5d. Mice were maintained with a 12 h light/dark cycle (light from 07:00 to 19:00) at 20−22 °C with a relative humidity of 60−70%. Sterile food and water were provided according to institutional guidelines. Prior to each experiment, mice were fasted overnight and allowed free access to water. Compound 5d was suspended in 0.5% carboxymethylcellulose sodium (CMC-Na) salt solution (677, 1333, and 2000 mg/kg) and given via oral administration according to the divided experimental groups. After the administration of the compounds, the mice were observed continuously for the first 4 h for any abnormal behavior and mortality changes, intermittently for the next 24 h, and occasionally thereafter for 14 days for the onset of any delayed effects. All animals were sacrificed on the 14th day after drug administration and were macroscopically examined for possible damage to the heart, liver, and kidneys.56 Statistical Analysis. The results are expressed as the mean ± SD of at least three independent experiments. Data were subjected to Student’s t test or one-way analysis of variance (ANOVA) followed by Dunnett’s test. P values less than 0.05 were accepted to indicate the significance.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Docking study, in vitro blood−brain barrier permeation assay, association constant study, and HPLC chromatograms of target compounds. This material is available free of charge via the Internet at http://pubs.acs.org. Accession Codes

The PDB codes for the structures of Aβ1−42, MAO-B, and AChE are 1IYT, 2V61, and 2CMF, respectively.

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AUTHOR INFORMATION

Corresponding Author

*For L.H.: phone, +086-20-3994-3051; fax, +086-20-39943051; e-mail, [email protected]. For X.L.: phone, +086-20-3994-3050; fax, +086-20-3994-3050; e-mail, lixsh@ mail.sysu.edu.cn. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the China Postdoctoral Science Foundation (Grant 2012M251651), the Ph.D. Programs Foundation of the Ministry of Education of China (Grant 20120171120045), the Guangdong Engineering Research Center of Chiral Drugs, and the Natural Science Foundation of China (Grant 20972198) for financial support of this study.

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ABBREVIATIONS USED AD, Alzheimer’s disease; Aβ, β-amyloid peptide; MAO-A, monoamine oxidase A; MAO-B, monoamine oxidase B; ACh, acetylcholine; ROS, reactive oxygen species; AChE, acetylcholinesterase; ThT, thioflavin T; TEM, transmission electron microscopy; ORAC-FL, oxygen radical absorbance capacity assay method using fluorescein; CQ, clioquinol; DCFH-DA, dichlorofluorescein diacetate; Trolox, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; MTT, 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; HPLC, highperformance liquid chromatography; AAPH, 2,2′-azobis(amidinopropane) dihydrochloride; DTNB, 5,5′-dithiobis(2nitrobenzoic acid); ATC, acetylthiocholine chloride; BTC, butylthiocholine chloride; BBB, blood−brain barrier O



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