New MelatoninâN,N-Dibenzyl(N-methyl)amine Hybrids: Potent

Article pubs.acs.org/jmc

New Melatonin−N,N‑Dibenzyl(N‑methyl)amine Hybrids: Potent Neurogenic Agents with Antioxidant, Cholinergic, and Neuroprotective Properties as Innovative Drugs for Alzheimer’s Disease Beatriz López-Iglesias,† Concepción Pérez,† José A. Morales-García,‡,§ Sandra Alonso-Gil,‡,§ Ana Pérez-Castillo,‡,§ Alejandro Romero,∥,⊥ Manuela G. López,∥ Mercedes Villarroya,∥ Santiago Conde,† and María Isabel Rodríguez-Franco*,† †

Instituto de Química Médica, Consejo Superior de Investigaciones Científicas (IQM-CSIC), C/Juan de la Cierva 3, 28006 Madrid, Spain ‡ Instituto de Investigaciones Biomédicas “Alberto Sols”, Consejo Superior de Investigaciones Científicas (IIB-CSIC), C/Arturo Duperier 4, 28029 Madrid, Spain § Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), C/ Valderrebollo 5, 28031 Madrid, Spain ∥ Instituto Teófilo Hernando and Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, C/Arzobispo Morcillo 4, 28029 Madrid, Spain S Supporting Information *

ABSTRACT: Here, we describe a new family of melatonin−N,N-dibenzyl(Nmethyl)amine hybrids that show a balanced multifunctional profile covering neurogenic, antioxidant, cholinergic, and neuroprotective properties at lowmicromolar concentrations. They promote maturation of neural stem cells into a neuronal phenotype and thus they could contribute to CNS repair. They also protect neural cells against mitochondrial oxidative stress, show antioxidant properties, and inhibit human acetylcholinesterase (AChE). Moreover, they displace propidium from the peripheral anionic site of AChE, preventing the βamyloid aggregation promoted by AChE. In addition, they show low cell toxicity and can penetrate into the CNS. This multifunctional profile highlights these melatonin−N,N-dibenzyl(N-methyl)amine hybrids as useful prototypes in the research of innovative drugs for Alzheimer’s disease.

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INTRODUCTION Alzheimer’s disease (AD) is a dreadful neurological illness and is the most frequent of the degenerative dementias. The slow but progressive impairment of the physical and neurological conditions of AD patients produces devastating effects on the patients themselves and on their caregivers as well as a very high economic burden for families and public health systems.1 AD shows a highly complex network of interconnected pathological processes that result in the accumulation of abnormal deposits of β-amyloid peptide (Aβ) and hyperphosphorylated tau protein as well as massive cell death and the loss of synapses, especially in the cholinergic system.2 Currently, the therapeutic options for the treatment of AD are limited to three acetylcholinesterase (AChE) inhibitors, namely, donepezil, rivastigmine, and galantamine,3 and one Nmethyl-D-aspartate receptor antagonist, memantine.4 The main role of AChE is to hydrolyze the neurotransmitter acetylcholine (ACh) after the occurrence of a nerve impulse. Thus, inhibition of this enzyme has been largely used as a rational approach to palliate memory deficits by increasing ACh in the synaptic cleft. However, AChE also plays other © 2014 American Chemical Society

noncholinergic functions, including an important role in Aβ processing. Several studies have indicated that AChE increases the formation of Aβ fibrils in vitro5 and Aβ plaques in the cerebral cortex of transgenic mouse models of AD.6 These effects seem to be mediated by interactions between Aβ and the peripheral anionic site (PAS) of the enzyme,7 which is supported by the fact that propidium, a noncompetitive AChE inhibitor that binds purely to PAS, significantly decreases AChE-induced Aβ aggregation; however, edrophonium, a pure competitive inhibitor that binds the center active site (CAS), does not show any effect on AChE-induced Aβ fibrillogenesis.8 These findings have led to the design and synthesis of inhibitors of both the CAS and PAS for use as promising diseasemodifying AD drug candidates because they can simultaneously improve cognition and slow the rate of Aβ degeneration.9 Recently, this hypothesis has been validated in murine models of AD that showed an improvement in cognition and a Received: January 13, 2014 Published: April 16, 2014 3773

dx.doi.org/10.1021/jm5000613 | J. Med. Chem. 2014, 57, 3773−3785

Journal of Medicinal Chemistry

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no peripheral MAO inhibitory effects in in vivo experiments.35 Huprine X is a subnanomolar AChE inhibitor that possesses additional effects, such as inhibition of BACE-1, activation of αsecretases, and stimulation of protein kinase C/mitogenactivated protein kinase pathway signaling, and was shown to improve learning and memory in a triple transgenic mouse model of AD.36,37 Our group has also reported several families of MTLs that combined neuroprotective, cholinergic, and antioxidant properties,38−40 including melatonin-based hybrids that have proved their effectiveness in a murine model of AD.11,41,42 Continuing with our interest in the design, synthesis, and biological evaluation of multifunctional molecules, our work is currently focused on taking advantage of the potential neurogenic profile of melatonin-based hybrids, which are endowed with additional anticholinergic properties. Thus, in this work, we have designed new melatonin− N,N-dibenzyl(Nmethyl)amine hybrids (1−14) by binding two fragments with interesting and complementary properties. The melatonin framework, in addition to its above-mentioned neurogenic profile, could demonstrate antioxidant and neuroprotective features and could also interact with the AChE-PAS because of its aromatic character, as we observed in the tacrine−melatonin series. 42 The second selected fragment was the protonable N,N-dibenzyl(N-methyl)amine, which is present in the wellknown AChE inhibitor AP2238, because its interaction with the AChE-CAS has been probed43−45 (Figure 1).

reduction of brain amyloid burden when mice were treated with dual-binding-site AChE inhibitors.10−13 Because of the progressive failure of the endogenous antioxidant systems, the oxidative damage in cellular structures is augmented during aging, and recent findings have shown the involvement of oxidative imbalance in different pathologies. For instance, a post-mortem study of the frontal cortex of AD patients demonstrated a significant disease-dependent increase in oxidative markers that correlated with mini-mental status examination scores.14 Additionally, in a murine AD model, oxidative damage was found to be an event that precedes the appearance of other pathological hallmarks of AD.15 These findings pointed out the early involvement of mitochondrial oxidative stress in the pathogenesis and progression of this disease.16 Thus, drugs that protect neurons from mitochondrial oxidative stress could be useful either for the prevention or treatment of AD.17 Neurogenesis is a dynamic process occurring in the brain of adult vertebrates whereby new nervous cells are generated along the lifespan of an individual. Although it was thought for many years that the creation of nervous tissue was restricted to the first stages of embryonic development, this concept changed in 1962 when Altman demonstrated the formation of new neurons in the brain of adult rats.18 In the following years, neurogenesis was also found in different species of vertebrates, including humans.19,20 The whole process comprises several stages, from the proliferation of progenitor cells to the integration of these new cells into cerebral circuits. Because renewal rates for neuronal cells decrease with aging, drugs capable of helping the replacement of damaged cells with healthy ones would represent an exciting new therapeutic treatment for AD and other neurodegenerative diseases.21 Melatonin (N-acetyl-5-methoxytryptamine) is a ubiquitous molecule produced by a variety of organs and tissues. It is involved in many physiological processes, such as circadian rhythm, endogenous antioxidant regulation, and the immune system. The natural decline in melatonin levels with aging has been associated with insomnia and depression as well as the development of neurodegenerative disorders such as AD.22 Among its extensive pharmacological profile, melatonin shows a potent antioxidant activity by the direct capture of free radicals,23 stimulates the synthesis of several endogenous antioxidant enzymes,24 improves mitochondrial energy metabolism,25 decreases neurofilament hyperphosphorylation, and plays a neuroprotective role against Aβ.26 Moreover, melatonin potentiates the proliferation and differentiation of neural stem cells in the hippocampus of adult mice.27−29 Thus, melatoninbased compounds could show an interesting neurogenic profile of paramount importance in the search of new therapeutics for AD and other neurodegenerative diseases. Furthermore, the complexity of AD pathology has encouraged the design of molecules that are able to interact with two or more complementary targets, with the expectation that these multitarget ligands (MTL) may represent important advantages in the treatment of the disease.30−32 In recent years, many interesting MTLs have been developed, such as memoquin, ladostigil, and huprine X, among others. Memoquin is an AChE and β-secretase-1 (BACE-1) inhibitor with additional antioxidant and Aβ anti-aggregating properties, which has recently been proven to have in vivo efficacy using different mouse models of induced amnesia.33,34 Ladostigil is an inhibitor of cholinesterases (ChEs) and is a brain-selective inhibitor of monoaminoxidases (MAO-A and -B), with little or

Figure 1. Structures of melatonin, AP2238, and new melatonin− N,Ndibenzyl(N-methyl)amine hybrids (1−14).

In this article, we describe the synthesis of new melatonin− N,N-dibenzyl(N-methyl)amine hybrids (1−14) and their biological evaluation, which comprises inhibition of human AChE and BuChE, displacement of propidium from AChEPAS, in vitro CNS permeation, and oxygen radical absorbance capacity (ORAC). Using a human neuroblastoma cell line, we also studied the cell viability and the neuroprotective effects of these hybrids against death induced by mitochondrial oxidative stress. Lastly, we explored the neurogenic effects of a selection of these compounds using primary hippocampal neural stem cells from adult rats.

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RESULTS AND DISCUSSION Synthesis of Melatonin−N, N-Dibenzyl(N-methyl)amine Hybrids . New melatonin−N,N-dibenzyl(N-methyl)amine hybrids 1−14 were obtained as depicted in Scheme 1. Reaction of commercially available 4-(bromomethyl)benzonitrile and differently substituted N-methylbenzylamines gave 4-{[benzyl(methyl)amino]methyl}benzonitriles 15 46−19 (77−93% yield), which were hydrolyzed to corresponding acid 3774



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Scheme 1. Synthesis of Melatonin−N,N-Dibenzyl(N-methyl)amine Hybrids (1−14)

Table 1. Yield (%), Inhibition of h-AChE and h-BuChE [IC50 (μM) or Percentage of Inhibition at 10 μM (%)], and Displacement of Propidium Iodide from the Peripheral Anionic Site of AChE (%) by New Melatonin−N,N-Dibenzyl(Nmethyl)amine Hybrids 1−14 IC50 (μM)b or (inhibition % at 10 μM) R 1 H 2 H 3 H 4 H 5 H 6 2-Cl 7 2-Cl 8 3-Cl 9 3-Cl 10 3-Cl 11 2-OCH3 12 2-OCH3 13 3-OCH3 14 3-OCH3 tacrine BW284c51 a

displacement of propidium iodide (%)b

R′

yield (%)a

h-AChE

h-BuChE

0.3 μM

1.0 μM

H 5-OH 5-OCH3 6-OCH3 6-F H 5-OCH3 H 5-OCH3 6-OCH3 H 5-OCH3 H 5-OCH3

46 30 72 65 65 49 57 55 55 82 61 76 78 63

1.9 ± 0.1 4.2 ± 0.2 6.1 ± 0.4 4.1 ± 0.3 2.5 ± 0.1 3.8 ± 0.2 2.6 ± 0.1 5.1 ± 0.2 5.6 ± 0.4 2.1 ± 0.1 4.7 ± 0.2 5.4 ± 0.2 3.8 ± 0.1 6.8 ± 0.2 0.23 ± 0.07 n.d.

3.7 ± 0.1 9.5 ± 0.5 7.8 ± 0.5 50.0 ± 2.1 7.0 ± 0.1 >10 (38%) >10 (37%) >10 (38%) >10 (38%) >10 (22%) 8.2 ± 0.4 >10 (47%) 9.6 ± 0.2 >10 (45%) 0.040 ± 0.002 n.d.

37.6 ± 0.22 8.0 ± 0.54 45.2 ± 1.80 24.1 ± 1.01 8.1 ± 0.87 7.7 ± 0.08 n.d. 10.9 ± 0.30 4.1 ± 0.70 16.8 ± 0.06 11.5 ± 0.32 n.d. 21.8 ± 0.56 n.d. n.d. n.d.

5.6 ± 0.11 22.4 ± 0.37 25.3 ± 0.96 24.9 ± 0.30 n.d. 25.8 ± 0.90 n.d. 15.6 ± 0.32 11.3 ± 0.48 11.4 ± 0.01 16.9 ± 1.21 n.d. 21.4 ± 1.18 n.d. n.d. n.d.

3.0 μM 51.4 25.3 67.3 17.8 34.5 34.7 n.d. 29.9 31.8 33.4 37.9 30.4 43.7 n.d. n.d. 16.0

± ± ± ± ± ±

1.80 0.70 0.08 0.31 0.29 0.61

± ± ± ± ± ±

0.60 0.37 0.38 1.23 0.76 1.10

± 2.4

Percentage of isolated product (%). bResults are the mean ± SEM (n = 3). n.d., not determined.

(N-methyl)amine hybrids 1−14 were assayed as inhibitors of human AChE (h-AChE) and BuChE (h-BuChE) by following the method of Ellman et al.,47 using tacrine as a reference (Table 1). All tested compounds displayed inhibition of hAChE with IC50’s in the low-micromolar range and showed little fluctuation after the introduction of different substituents in the benzene or indole ring. However, h-BuChE inhibition showed a higher dependency on the presence of substituents: only unsubstituted hybrid 1 displayed low-micromolar inhibition of h-BuChE (IC50 = 3.7 μM), whereas the introduction of any substituent in 2−14 penalized the enzyme−hybrid interactions by 1 order of magnitude and showed IC50’s around 10 μM or higher.

derivatives 20−24 in good yields (69−97%). Then, these intermediate acids were activated with benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) and subsequently coupled with commercially available tryptamines in the presence of triethylamine in dimethylformamide solutions at room temperature overnight to afford the desired melatonin− N,N-dibenzyl(N-methyl)amine derivatives (1−14) in moderate to good yields (30−82%). All new hybrids (1−14) were purified by chromatographic techniques and structurally characterized by their analytical (HPLC and combustion analysis) and spectroscopic data (1H NMR, 13C NMR, and MS). Inhibition of Human Cholinesterases and Propidium Displacement from AChE. New melatonin−N, N-dibenzyl3775



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As previously mentioned, the enzyme AChE interacts with Aβ oligomers and stimulates conformational changes that contribute to their aggregation and precipitation into amyloid plaques.7 The enzymatic region that promotes these abnormal modifications is placed in the PAS of AChE48 because the selective PAS ligand propidium inhibits AChE-induced Aβ aggregation, whereas the competitive CAS ligand edrophonium does not.8 Therefore, the propidium displacement assay is a well-accepted method and is usually used as a first approach to study the inhibition of Aβ aggregation by PAS ligands.49 Propidium iodide undergoes a 10-fold increase in fluorescence when it is bound to AChE-PAS. In the presence of another inhibitor, the decay in the fluorescence signal is related to the displacement percentage of propidium from this enzymatic site and thus it is accepted as a measure of the affinity of such a compound for AChE-PAS.50 Thus, the experimental affinity of new compounds 1−14 for AChE-PAS was studied by competitive enzymatic assays in the presence of propidium iodide as a first and indirect probe of the inhibition of Aβ aggregation. New hybrids 1−14 were evaluated at three concentrations, 0.3, 1.0, and 3.0 μM, and the results are gathered in Table 1. All compounds displaced the propidium cation from the AChEPAS better than 4,4′-(3-oxopentane-1,5-diyl)bis(N,N-dimethylN-prop-2-en-1-ylanilinium) dibromide (BW248c51), a PAS ligand used as a reference in this type of experiment.40 These results suggested that the new hybrids were able to bind to AChE-PAS and thus they could prevent Aβ aggregation stimulated by this enzyme. For the majority of compounds, a concentration−response relationship was obtained, yielding a maximum at 3.0 μM, which is a concentration that is very close to the IC50 values observed for h-AChE inhibition (1.9−6.8 μM). However, some compounds (1, 3, and 10) displayed a Ushaped line in the concentration−response data, as observed in other MTLs in a previous work.40 This could be explained by the fact that these multifunctional derivatives could interact with other points of the enzymatic gorge, depending on their concentration. At 3.0 μM, hybrid 3, which is derived from 5-methoxyindole and an unsubstituted dibenzylamine fragment, showed the best result with 67% propidium displacement. Other outstanding compounds were 1 (51%), 11 (38%), and 13 (44%). In Vitro Blood−Brain Barrier Permeation Assay. To explore whether new hybrids 1−14 could enter into the brain, we used a parallel artificial membrane permeation assay for the blood−brain barrier (PAMPA-BBB), described by Di et al.,51 and then optimized these for molecules with limited water solubility.38,41 The in vitro permeabilities (Pe) of new compounds 1−14 and 10 commercial drugs through a lipid extract of porcine brain were determined using a mixture of PBS/EtOH (70:30). Assay validation was performed by comparing the experimental permeability with the reported values of these commercial drugs, which gave a good linear correlation, Pe (exptl) = 0.57 Pe (bibl) + 2.36 (r2 = 0.919) (see the Supporting Information). From this equation and taking into account the described limits for BBB permeation,51 we found that compounds with permeability values above 4.6 × 10−6 cm s−1 could penetrate into the CNS by passive diffusion. All melatonin− N,N-dibenzyl(N-methyl)amine hybrids 1−14 showed permeability values over this limit, pointing out that these compounds could cross the BBB (Table 2). Evaluation of the Oxygen Radical Absorbance Capacity. The oxygen radical absorbance capacity (ORAC)

Table 2. Permeability Values from the PAMPA-BBB Assay (Pe, 10−6 cm s−1) and Oxygen Radical Absorbance Capacity by Fluorescence (ORAC-FL, Trolox Equiv) of Hybrids 1−14 PAMPA-BBB assay compd

R

1 H 2 H 3 H 4 H 5 H 6 2-Cl 7 2-Cl 8 3-Cl 9 3-Cl 10 3-Cl 11 2-OCH3 12 2-OCH3 13 3-OCH3 14 3-OCH3 melatonin a

R′

Pe (10

H 5-OH 5-OCH3 6-OCH3 6-F H 5-OCH3 H 5-OCH3 6-OCH3 H 5-OCH3 H 5-OCH3

−6

−1 a

cm s )

7.4 ± 5.0 ± 7.1 ± 7.2 ± 6.6 ± 5.8 ± 5.9 ± 7.1 ± 7.9 ± 7.9 ± 8.8 ± 9.9 ± 8.8 ± 8.7 ± n.d.

0.2 0.5 0.4 0.1 0.2 0.1 0.4 0.1 0.2 0.1 0.3 0.3 0.1 0.4

ORAC-FL (trolox equiv)a 3.2 4.3 2.5 1.5 2.1 2.8 2.5 2.9 2.6 1.8 2.9 2.4 2.7 2.5 2.4

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.03 0.1 0.04 0.01 0.04 0.02 0.2 0.1 0.1 0.04 0.1 0.1 0.04 0.04 0.1

Results are the mean ± SD (n = 3).

of new melatonin− N,N-dibenzyl(N-methyl)amine hybrids 1− 14 was determined as a measure of their antioxidant properties (Table 2) following a well-established protocol.52 Trolox, the aromatic part of vitamin E and the part that is responsible for radical capturing, was used as an internal standard to which the unit value is arbitrarily given, ORAC (trolox) = 1. Thus, results were expressed as trolox equivalents (micromoles of trolox/ micromoles of tested compound) on a comparative scale that shows if the products are better (ORAC > 1) or worse than trolox (ORAC < 1). Melatonin was used as a positive standard for comparative purposes, giving an ORAC value of 2.4 that fully agreed with the value previously described by Sofic et al. (2.0 trolox equiv). 53 Tested compounds showed potent peroxyl radical absorbance capacities ranging from 1.5- to 4.3fold of the trolox value and thus they could be considered as potent antioxidant agents. As expected, the ORAC of these compounds is mainly located in the melatonin-like fragment, where the presence of a phenolic group clearly yielded the best ORAC value (2, R′ = 5OH, ORAC = 4.3 trolox equiv). However, the introduction of a halogen or a methoxy group in the indole and/or the N,Ndibenzyl(N-methyl)amine fragment penalized the ORAC ability to between 0.3 and 1.7 trolox equiv compared to unsubstituted hybrid 1 (ORAC = 3.2 trolox equiv). It is worth mentioning that 5-methoxyindole derivatives 3 and 9 showed better ORAC values (2.5 and 2.6 trolox equiv, respectively) than their 6-methoxy counterparts 4 and 10 (1.5 and 1.8 trolox equiv, respectively), suggesting the involvement of the indolic position 5 in the trapping mechanism of these hybrids. Cell Viability and Neuroprotection Studies. To explore the therapeutic potential of new melatonin−dibenzylamine hybrids 1−14, cell viability and neuroprotective capacity against mitochondrial oxidative stress were evaluated using the human neuroblastoma cell line SH-SY5Y with a mixture of rotenone and oligomycin A as the toxic insult. The combination of such toxic agents induces mitochondrial ROS as a consequence of the blockade of complexes I and V of the mitochondrial electronic chain; thus, it is a good model of endogenous oxidative stress.54 In these experiments, compounds were tested at 1.0 μM, a concentration close to their IC50 in h-AChE 3776



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which were induced to proliferate following described protocols.56−58 Then, we analyzed whether addition of different compounds could promote early stages of neurogenesis and neuronal maturation by staining the cells for β-tubulin (antibody clone TuJ1) and MAP-2 (microtubule-associated protein 2), respectively. All tested compounds, including references, were used at a concentration of 10 μM, which was determined on the basis of our experience with neurosphere-based neurogenic experiments.57,58 Moreover, we performed additional viability studies with compounds 3, 11, and 14 at 10 μM using the human neuroblastoma cell line. None of tested compounds caused a significant reduction of cell viability (measured as MTT reduction) or cell death (measured as LDH release) after exposing the SH-SY5Y cells for 48 h to compounds at 10 μM (see Figure S1 and Table S4 in the Supporting Information). These results indicated that compounds 3, 11, and 14 were not toxic at the concentration used in the neurogenic studies. Figures 3 and 4 show the neurogenic effects of compounds 3, 11, and 14 at 10 μM on neural stem cell cultures, which are compared with the effect of vehicle (basal), melatonin (endogenous substrate of melatonin receptors), and luzindole (antagonist of melatonin receptors). In each bar chart, quantifications of TuJ+ or MAP-2+ cells are shown using five neurospheres per condition (Figures 3 and 4). As expected, melatonin induced both early neurogenesis and cell maturation, whereas luzindole was not able to promote either of these processes. All tested melatonin− N,N-dibenzyl(N-methyl)amine hybrids, 3, 11, and 14, were able to stimulate early neurogenesis and cell maturation into a neuronal phenotype and were more effective than melatonin itself. By using TuJ1 fluorescence immunodetection, compound 11 was found to be the most effective hybrid for early neurogenesis, being twice as potent as melatonin (Figure 3). However, in the cell maturation experiments, the most efficient agent was hybrid 3, derived from 5-methoxyindole and an unsubstituted N,N-dibenzyl(Nmethyl)amine, which was almost twice as efficient as melatonin. Moreover, hybrid 3 showed a dense crown of both β-tubulinand MAP-2-positive cells around the neurosphere core and additional positive cells outside of it, suggesting a migration out of the neurosphere (Figure 4).

inhibition. The percentage of cell death was determined by measuring the amount of lactic dehydrogenase (LDH), an enzyme that is released to the extracellular medium when neurons die.55 First, possible cytotoxic effects of 1−14 were studied by exposing cells to compounds at 1.0 μM for 24 h. In all cases, the LDH percentage was lower or equal to the basal value, suggesting that cell viability reached 100% (data not shown). Then, cells were incubated with hybrids 1−14 at 1.0 μM for 24 h before the addition of a mixture of rotenone (30 μM) and oligomycin A (10 μM) and then were maintained for another 24 h. Melatonin was used as a positive reference, and results are shown in Figure 2 (and Table S3 in the Supporting

Figure 2. Effect of compounds 1−14 and melatonin on cell death induced by the combination of rotenone and oligomycin A. Human neuroblastoma SH-SY5Y cells were exposed for 24 h to compounds (1.0 μM) and then exposed for another 24 h to the combination of rotenone (30 μM) and oligomycin A (10 μM) in the presence of compounds. Cell viability was assessed by measuring LDH release. Data are expressed as the mean ± SEM of triplicates of seven different batches of cells. ***p < 0.001 with respect to toxic agents alone (ANOVA followed by Bonferroni post-test).

Information). All new compounds displayed good levels of protection ranging from 12 to 36%. Among hybrids with an unsubstituted N,N-dibenzyl(N-methyl)amine fragment (1−5), the introduction of a 5- or a 6-substituent in the indole ring increased the protection percentage, especially in the case of the 5-methoxy group. In fact, hybrid 3, derived from 5methoxyindole and an unsubstituted dibenzylamine fragment, was the best neuroprotective agent, saving 36% of the cells from the damage induced by mitochondrial ROS. Regarding 5-methoxyindole derivatives (R′ = 5-OCH3), the introduction of a chlorine atom in the N,N-dibenzyl(Nmethyl)amine fragment had a detrimental effect on neuroprotection, reducing values from the highest value of 36% (3, R = H) to 27% (7, R = 2-Cl) and 12% (9, R = 3-Cl). However, the introduction of a second methoxy group in position 3 maintained an approximately equal degree of neuroprotection, 30% (14, R = 3-OCH3). Neurogenic Studies. To evaluate the potential ability of the new hybrids to repair brain damage, we performed in vitro neurogenesis studies. Considering the structure of the neurogenic melatonin, which is a 5-methoxyindole derivative, hybrids bearing one or two methoxy groups in different positions were selected for these experiments, namely, 3 (R = H, R′ = 5OCH3), 11 (R = 2-OCH3, R′ = H), and 14 (R = 3-OCH3, R′ = 5-OCH3) (see Scheme 1 for R and R′ definitions). Primary neural stem cells were obtained from the subgranular zone of the dentate gyrus of the hippocampus of adult Wistar rats,

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CONCLUSIONS The results presented here clearly show that the new melatonin− N,N-dibenzyl(N-methyl)amine hybrids display a balanced biological profile. At low-micromolar concentrations, they stimulate early stages of neurogenesis from neural stem cells and neuronal maturation into a neuronal phenotype, inhibit h-AChE, displace propidium from AChE-PAS, and protect a human neuroblastoma cell line against damage caused by mitochondrial free radicals. In addition, they are CNSpermeable compounds with low cell toxicity that show good antioxidant properties. Thus, it is expected that these new melatonin−N,Ndibenzyl(N-methyl)amine hybrids could promote autorepair processes from neural stem cells in the CNS, protect neurons from mitochondrial oxidative stress, increase patient memory, and reduce the formation of amyloid plaques. These biological properties, along with their ability to penetrate into the CNS, highlight these new hybrids as exciting new multifunctional prototypes in the search for new regenerative drugs for the treatment of AD and other neurodegenerative pathologies. 3777



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Figure 3. Neural stem cell cultures (neurospheres), derived from the subgranular zone of the dentate gyrus of the hippocampus of adult Wistar rats, treated with vehicle (basal), melatonin, luzindole (MT antagonist), 3, 11, and 14 at 10 μM for 48 h. Neuronal cells were detected using an anti-β-tubulin antibody (TuJ1 clone, green) as an early neurogenesis marker. DAPI (blue) was used for nuclear staining. Scale bar, 200 μm. Quantification of TuJ+ cells in neurospheres is shown. Values are the mean ± SD from five neurospheres per condition. *** p ≤ 0.001.

Figure 4. Neural stem cell cultures (neurospheres), derived from the subgranular zone of the dentate gyrus of the hippocampus of adult Wistar rats, treated with vehicle (basal), melatonin, luzindole (MT antagonist), 3, 11, and 14 at 10 μM for 48 h. MAP-2 (red) was used as a marker that is associated with neuronal maturation, and DAPI (blue) was used for nuclear staining. Scale bar, 200 μm. Quantification of MAP-2+ cells in neurospheres is shown. Values are the mean ± SD from five neurospheres per condition. *** p ≤ 0.001.

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detector (λ = 214−274 nm) and using a Delta Pak C18 5 μm, 300 Å column. Melting points (uncorrected) were determined with a Reichert-Jung Thermovar apparatus. 1H and 13C NMR spectra were recorded in CDCl3 or CD3OD solutions using a Varian XL-300 spectrometer. Chemical shifts are reported in δ (ppm) relative to internal Me4Si. J values are given in hertz, and spin multiplicities are expressed as s (singlet), d (doublet), t (triplet), q (quartet), or m (multiplet). Mass spectra (MS) were obtained by electron spray ionization (ESI) in positive mode using a Hewlett-Packard MSD 1100 spectrometer.

EXPERIMENTAL SECTION

Chemistry. General Methods. Reagents and solvents were purchased from common commercial suppliers and were used without further purification. Chromatographic separations were performed on silica gel (Kielgel 60 Merck of 230−400 mesh), and compounds were detected with UV light (λ = 254 nm). HPLC analyses were used to confirm the purity of all compounds (>95%) and were performed on Waters 6000 equipment at a flow rate of 1.0 mL/min with a UV 3778



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N-Benzyl-1-(4-carboxyphenyl)-N-methylmethanaminium Chloride (20). From 4-{[benzyl(methyl)amino]methyl}benzonitrile (15) (898 mg, 3.8 mmol), 727 mg (75%) of 20 was obtained as a pure white solid. mp 230−231 °C. ESI-MS m/z 256 [MH]+. 1H NMR (CD3OD) δ 8.00 (d, 2H, J = 8.2 Hz), 7.49 (d, 2H, J = 8.2 Hz), 7.39 (m, 5H), 3.98 (s, 2H), 3.95 (s, 2H), 2.43 (s, 3H). 13C NMR (CD3OD) δ 172.0 (CO), 139.2 (C), 135.7 (C), 135.4 (C), 131.3 (2CH), 131.0 (2CH), 130.9 (2CH), 129.8 (2CH), 129.7 (CH), 61.8 (CH2), 61.3 (CH2), 41.0 (CH3). Purity: 99% (by HPLC). Anal. (C16H18ClNO2) C, H, N. N-(4-Carboxybenzyl)-1-(2-chlorophenyl)-N-methylmethanaminium Chloride (21). From 4-{[(2-chlorobenzyl)(methyl)amino]methyl}benzonitrile (16) (1.03 g, 3.8 mmol), 967 mg (88%) of 21 was obtained as a white solid. mp 144−146 °C. ESI-MS m/z 290 [MH]+, 292 [MH + 2]+. 1H NMR (CD3OD) δ 8.00 (d, 2H, J = 8.4 Hz), 7.57 (dd, 1H, J = 7.0 Hz, J = 2.4 Hz) 7.51 (d, 2H, J = 8.4 Hz), 7.41 (dd, 1H, J = 7.3 Hz, J = 2.0 Hz), 7.33 (dd, 1H, J = 7.3 Hz, J = 2.0 Hz), 7.30 (dd, 1H, J = 7.3 Hz, J = 2.4 Hz), 3.91 (s, 4H,), 2.38 (s, 3H). 13C NMR (CD3OD) δ 170.3 (C), 142.2 (C), 135.8 (C), 135.0 (C), 133.0 (CH), 132.8 (CH), 130.9 (2CH), 130.8 (2CH), 130.7 (2CH), 128.3 (CH), 62.2 (CH2), 58.9 (CH2), 41.9 (CH3). Purity: 99% (by HPLC). Anal. (C16H17Cl2NO2) C, H, N. N-(4-Carboxybenzyl)-1-(3-chlorophenyl)-N-methylmethanaminium Chloride (22). From 4-{[(3-chlorobenzyl)(methyl)amino]methyl}benzonitrile (17) (1.03 g, 3.8 mmol), 1.07 g (97%) of 22 was obtained as a white solid. mp 140−142 °C. ESI-MS m/z 290 [MH]+, 292 [MH + 2]+. 1H NMR (CD3OD) δ 8.13 (d, 2H, J = 8.4 Hz), 7.64 (d, 2H, J = 8.4 Hz), 7.61 (s, 1H), 7.51 (m, 3H), 4.48 (s, 2H), 4.42 (s, 2H), 2.73 (s, 3H). 13C NMR (CD3OD) δ 167.5 (C), 135.0 (C), 134.1 (C), 132.6 (C), 131.6 (C), 131.3 (2CH), 131.1 (CH), 130.8 (CH), 130.4 (2CH), 130.3 (CH), 129.6 (CH), 59.3 (CH2), 59.1 (CH2), 38.6 (CH3). Purity: 100% (by HPLC). Anal. (C16H17Cl2NO2) C, H, N. N-(4-Carboxybenzyl)-1-(2-methoxyphenyl)-N-methylmethanaminium Chloride (23). From 4-{[(2-methoxybenzyl)(methyl)amino]methyl}benzonitrile (18) (1.01 g, 3.8 mmol), 1.05 g (97%) of 23 was obtained as a white solid. mp 164−166 °C. ESI-MS m/z 286 [MH]+. 1H NMR (CD3OD) δ 8.11 (d, 2H, J = 8.4 Hz), 7.69 (d, 2H, J = 8.4 Hz), 7.45 (td, 1H, J = 7.7 Hz, J = 1.7 Hz), 7.40 (dd, 1H, J = 7.7 Hz, J = 1.7 Hz), 7.05 (dd, 1H, J = 7.7 Hz, J = 0.9 Hz), 7.00 (td, 1H, J = 7.7, J = 0.9 Hz), 3.80 (s, 3H), 3.29 (s, 2H), 3.23 (s, 2H), 2.81 (s, 3H). 13 C NMR (CD3OD) δ 168.8 (C), 159.4 (C), 135.8 (C), 133.6 (C), 133.5 (CH), 132.4 (2CH), 131.4 (2CH), 129.5 (CH), 122.1 (CH), 118.9 (C) 62.2 (CH2), 60.7 (CH3), 56.1 (CH2), 55.9 (CH2) 41.2 (CH3). Purity: 99% (by HPLC). Anal. (C17H20ClNO3) C, H, N. N-(4-Carboxybenzyl)-1-(3-methoxyphenyl)-N-methylmethanaminium Chloride (24). From 4-{[(3-methoxybenzyl)(methyl)amino]methyl}benzonitrile (19) (1.01 g, 3.8 mmol), 748 mg (69%) of 24 was obtained as a white solid. mp 100−102 °C. ESI-MS m/z 286 [MH]+. 1H NMR (CD3OD) δ 8.00 (d, 2H, J = 8.3 Hz), 7.47 (d, 2H, J = 8.3 Hz), 7.31 (t, 1H, J = 7.9 Hz), 7.00 (s, 1H), 6.95 (m, 2H), 3.92 (s, 2H), 3.87 (s, 2H), 3.80 (s, 3H), 2.41 (s, 3H). 13C NMR (CD3OD) δ 161.5 (CO), 139.8 (C), 137.3 (C), 135.4 (C), 130.9 (2CH), 130.8 (2CH), 130.7 (CH), 123.2 (CH), 122.9 (C), 116.5 (CH), 115.0 (CH), 62.0 (CH2), 61.5 (CH2), 55.7 (CH3), 41.3 (CH3). Purity: 99% (by HPLC). Anal. (C17H20ClNO3) C, H, N. General Procedure for the Synthesis of Melatonin− N,NDibenzyl(N-methyl)amino Hybrids (1−14). To a solution of the corresponding 4-{[benzyl(methyl)amino]methyl}benzoic acid 20−24 (0.5 mmol), triethylamine (1.2 mmol), and PyBOP (0.6 mmol) in DMF (8 mL) was added the appropriated 2-(1H-indol-3-yl)ethanamine (0.5 mmol). The reaction mixture was stirred at room temperature overnight, and DMF was then evaporated to dryness under reduced pressure. The residue was dissolved in CH2Cl2 (10 mL) and consecutively washed with a 10% aqueous NaHCO3 solution (3 × 10 mL) and H2O (10 mL). The organic phase was dried over sodium sulfate and evaporated to dryness under reduced pressure. The residue was purified on a silica gel column using mixtures of hexane/EtOAc as eluent, obtaining the corresponding dibenzylamino−melatonin derivative as a pure solid.

Elemental analyses were carried out in a PerkinElmer 240C in the ́ Centro de Quimica Orgánica “Manuel Lora-Tamayo” (CSIC), and the results are within ±0.4% of the theoretical values. General Procedure for the Synthesis of 4-{[Benzyl(methyl)amino]methyl}benzonitriles (15−19). A mixture of 4(bromomethyl)benzonitrile (5 mmol) and the corresponding amine (15 mmol) in ethyl ether (50 mL) was refluxed for 10 h. After cooling to room temperature, a white solid was removed by filtration, and the solvent was evaporated to dryness under reduced pressure. The residue was purified on a silica gel column using mixtures of hexane/ EtOAc as eluent, obtaining the corresponding 4-{[benzyl(methyl)amino]methyl}benzonitrile as a pure solid. Analytical and spectroscopic data of 15 were consistent with those previously described.46 4-{[(2-Chlorobenzyl)(methyl)amino]methyl}benzonitrile (16). Reagents used were 4-(bromomethyl)benzonitrile (941 mg, 4.8 mmol) and 1-(2-chlorophenyl)-N-methylmethanamine (2.23 g, 14.3 mmol). Purification involved the use of hexane/EtOAc (15:1) as eluent. 16: colorless syrup (1.15 g, 88%). ESI-MS m/z 271 [MH]+, 273 [MH + 2]+. 1H NMR (CDCl3) δ 7.60 (d, 2H, J = 8.4 Hz), 7.51 (dd, 1H, J = 7.6 Hz, J = 1.5 Hz), 7.49 (d, 2H, J = 8.4 Hz), 7.36 (dd, 1H, J = 7.6 Hz, J = 1.2 Hz) 7.26 (td, 1H, J = 7.6 Hz, J = 1.2 Hz), 7.20 (td, 1H, J = 7.6 Hz, J = 1.5 Hz), 3.67 (s, 2H), 3.63 (s, 2H), 2.22 (s, 3H). 13C NMR (CDCl3) δ 145.1 (C), 136.3 (C), 134.3 (C), 132.1 (2CH), 130.6 (CH), 129.6 (CH), 129.3 (2CH), 128.3 (2CH), 126.6 (CH), 119.0 (C), 110.7 (C), 61.5 (CH2), 58.7 (CH2), 42.3 (CH3). Purity: 100% (by HPLC). Anal. (C16H15ClN2) C, H, N. 4-{[(3-Chlorobenzyl)(methyl)amino]methyl}benzonitrile (17). Reagents used were 4-(bromomethyl)benzonitrile (941 mg, 4.8 mmol) and 1-(3-chlorophenyl)-N-methylmethanamine (2.23 g, 14.3 mmol). Purification involved the use of hexane/EtOAc (15:1) as eluent. 17: colorless syrup (1.29 g, 83%). ESI-MS m/z 271 [MH]+, 273 [MH + 2]+. 1H NMR (CDCl3) δ 7.60 (d, 2H, J = 8.4 Hz), 7.47 (d, 2H, J = 8.4 Hz), 7.36 (s, 1H), 7.26 (m, 3H), 3.55 (s, 2H), 3.50 (s, 2H), 2.17 (s, 3H). 13C NMR (CDCl3) δ 145.2(C), 141.3 (C), 134.5 (C), 132.4 (2CH), 129.9 (CH), 129.5 (2CH), 129.0 (CH), 127.6 (CH), 127.1 (CH), 119.2 (C), 111.1 (C), 61.7 (CH2), 61.6 (CH2), 42.6 (CH3). Purity: 100% (by HPLC). Anal. (C16H15ClN2) C, H, N. 4-{[(2-Methoxybenzyl)(methyl)amino]methyl}benzonitrile (18). Reagents used were 4-(bromomethyl)benzonitrile (941 mg, 4.8 mmol) and 1-(2-methoxyphenyl)-N-methylmethanamine (1.96 g, 14.3 mmol). Purification involved the use of hexane/EtOAc (12:1) as eluent. 18: colorless syrup (1.58 g, 93%). ESI-MS m/z 267 [MH]+. 1H NMR (CDCl3) δ 7.60 (d, 2H, J = 8.2 Hz), 7.50 (d, 2H, J = 8.2 Hz), 7.40 (dd, 1H, J = 7.9 Hz, J = 1.6 Hz), 7.25 (td, 1H, J = 7.9 Hz, J = 1.6 Hz), 6.96 (td, 1H, J = 7.9 Hz, J = 0.7 Hz), 6.87 (dd, 1H, J = 7.9 Hz, J = 0.7 Hz), 3.81 (s, 3H), 3.60 (s, 2H), 3.57 (s, 2H), 2.24 (s, 3H). 13C NMR (CDCl3) δ 158.0 (C), 145.8 (C), 132.3 (2CH), 130.5 (CH), 129.6 (2CH), 128.4 (CH), 126.9 (C), 120.6 (CH), 119.4 (C), 110.8 (C), 110.6 (CH), 61.9 (CH2), 55.6 (CH3), 55.5 (CH2), 42.8 (CH3). Purity: 100% (by HPLC). Anal. (C17H18N2O) C, H, N. 4-{[(3-Methoxybenzyl)(methyl)amino]methyl}benzonitrile (19). Reagents used were 4-(bromomethyl)benzonitrile (941 mg, 4.8 mmol) and 1-(3-methoxyphenyl)-N-methylmethanamine (1.96 g, 14.3 mmol). Purification involved the use of hexane/EtOAc (12:1) as eluent. 19: colorless syrup (1.33 g, 77%). ESI-MS m/z 267 [MH]+. 1H NMR (CDCl3) δ 7.52 (d, 2H, J = 8.4 Hz), 7.41 (d, 2H, J = 8.4 Hz), 7.18 (t, 1H, J = 8.1 Hz), 6.88 (s, 1H), 6.87 (dd, 1H, J = 8.1 Hz, J = 1.7 Hz), 6.74 (dd, 1H, J = 8.1 Hz, J = 1.7 Hz), 3.74 (s, 3H), 3.47 (s, 2H), 3.45 (s, 2H), 2.12 (s, 3H). 13C NMR (CDCl3) δ 159.5 (C), 145.2 (C), 140.4 (C), 131.9 (2CH), 129.2 (CH), 129.1 (2CH), 120.9 (CH), 118.9 (C), 114.2 (CH), 112.2 (CH), 110.5 (C), 61.8 (CH2), 61.0 (CH2), 55.1 (CH2), 42.3 (CH3). Purity: 100% (by HPLC). Anal. (C17H18N2O) C, H, N. General Procedure for the Synthesis of 4-{[Benzyl(methyl)amino]methyl}benzoic Acids (20−24). To a solution of the corresponding nitrile (3.8 mmol) in dioxane (6 mL) was added a solution of NaOH (2 M aq., 29 mL), and the mixture was refluxed for 4 h. After cooling to room temperature, the solution was made acidic with HCl (2 M aqueous), and the resulting acid was isolated by filtration. 3779



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N-[2-(1H-Indol-3-yl)ethyl]-4-{[benzyl(methyl)amino]methyl}benzamide (1). Reagents used were 4-{[benzyl(methyl)amino]methyl}benzoic acid (20) (127.6 mg, 0.5 mmol), 2-(1H-indol-3yl)ethanamine (80.1 mg, 0.5 mmol), PyBOP (312.2 mg, 0.6 mmol), and Et3N (156 μL, 1.2 mmol). Purification involved the use of hexane/ EtOAc (1:3) as eluent. Hybrid 1: white solid. mp 94−96 °C (91.4 mg, 46%). ESI-MS m/z 398 [MH]+. 1H NMR (CDCl3) δ 8.20 (s, NH), 7.65 (s, 1H), 7.62 (d, 2H, J = 8.2 Hz), 7.37 (d, 2H, J = 8.2 Hz), 7.33 (m, 5H), 7.25 (m, 1H), 7.21 (td, 1H, J = 7.1 Hz, J = 1.0 Hz), 7.12 (td, 1H, J = 7.9 Hz, J = 1.0 Hz), 7.05 (m, 1H), 6.22 (t, NH, J = 5.4 Hz), 3.79 (q, 2H, J = 6.6 Hz), 3.53 (s, 2H), 3.51 (s, 2H), 3.09 (t, 2H, J = 6.6 Hz), 2.16 (s, 3H). 13C NMR (CDCl3) δ 167.4 (CONH), 142.9 (C), 138.8 (C), 136.4 (C), 133.4 (C), 128.9 (2CH), 128.9 (2CH), 128.3 (2CH), 127.3 (C), 127.1 (CH), 126.8 (2CH), 122.2 (CH), 122.1 (CH), 119.5 (CH), 118.7 (CH), 113.0 (C), 111.3 (CH), 61.8 (CH2), 61.3 (CH2), 42.2 (CH3), 40.2 (CH2), 25.3 (CH2). Purity: 99% (by HPLC). Anal. (C26H27N3O) C, H, N. 4-{[Benzyl(methyl)amino]methyl}-N-[2-(5-hydroxy-1H-indol3-yl)ethyl]benzamide (2). Reagents used were 4-{[benzyl(methyl)amino]methyl}benzoic acid (20) (127.6 mg, 0.5 mmol), 3-(2aminoethyl)-1H-indol-5-ol (88.0 mg, 0.5 mmol), PyBOP (312.2 mg, 0.6 mmol), and Et3N (156 μL, 1.2 mmol). Purification involved the use of hexane/EtOAc (2:1) as eluent. Compound 2: white solid. mp 75−77 °C (62.0 mg, 30%). ESI-MS m/z 414 [MH]+. 1H NMR (CDCl3) δ 7.74 (d, 2H, J = 8.2 Hz), 7.41 (d, 2H, J = 8.2 Hz), 7.32 (m, 4H), 7.24 (t, 1H, J = 7.1 Hz), 7.15 (d, 1H, J = 8.5 Hz), 7.03 (s, 1H), 6.99 (d, 1H, J = 2.2 Hz), 6.66 (dd, 1H, J = 8.5 Hz, J = 2.2 Hz), 3.63 (d, 2H, J = 7.4 Hz), 3.54 (s, 2H), 3.51 (s, 2H), 2.98 (t, 2H, J = 7.4 Hz), 2.16 (s, 3H). 13C NMR (CDCl3) δ 170.6 (CONH), 151.7 (C), 144.2 (C), 140.1 (C), 135.3 (C), 133.6 (C), 130.8 (2CH), 130.7 (2CH), 130.0 (C), 129.8 (2CH), 128.8 (CH), 128.7 (2CH), 124.7 (CH), 113.2 (CH), 113.1 (C), 112.9 (CH), 104.1 (CH), 63.3 (CH2), 62.7 (CH2), 42.9 (CH3), 42.5 (CH2), 26.8 (CH2). Purity: 99% (by HPLC). Anal. (C26H27N3O2) C, H, N. 4-{[Benzyl(methyl)amino]methyl}-N-[2-(5-methoxy-1Hindol-3-yl)ethyl]benzamide (3). Reagents used were 4-{[benzyl(methyl)amino]methyl}benzoic acid (20) (127.6 mg, 0.5 mmol), 2(5-methoxy-1H-indol-3-yl)ethanamine (95.1 mg, 0.5 mmol), PyBOP (312.2 mg, 0.6 mmol), and Et3N (156 μL, 1.2 mmol). Purification involved the use of hexane/EtOAc (1:1) as eluent. Hybrid 3: yellow solid. mp 102−103 °C (154 mg, 72%). ESI-MS m/z 428 [MH]+. 1H NMR (CDCl3) δ 8.68 (s, NH), 7.66 (d, 2H, J = 8.2 Hz), 7.36 (d, 2H, J = 8.2 Hz), 7.32 (m, 4H), 7.20 (d, 1H, J = 8.8 Hz), 7.04 (d, 1H, J = 2.3 Hz), 6.95 (d, 1H, J = 2.3 Hz), 6.84 (dd, 1H, J = 8.8 Hz, J = 2.3 Hz), 6.52 (t, NH, J = 5.5 Hz), 3.75 (q, 2H, J = 6.7 Hz), 3.74 (s, 3H), 3.51 (s, 4H), 3.03 (t, 2H, J = 6.7 Hz), 2.16 (s, 3H). 13C NMR (CDCl3) δ 167.4 (CONH), 153.8 (C), 143.0 (C), 138.8 (C), 133.1 (C), 131.5 (C), 128.8 (2CH), 128.8 (2CH), 128.2 (2CH), 127.6 (C), 127.0 (CH), 126.8 (2CH), 123.0 (CH), 112.3 (C), 112.2 (CH), 112.1 (CH), 100.2 (CH), 61.7 (CH2), 61.1 (CH2), 55.6 (CH3), 42.1 (CH3), 40.4 (CH 2 ), 25.2 (CH 2 ). Purity: 98% (by HPLC). Anal. (C27H29N3O2) C, H, N. 4-{[Benzyl(methyl)amino]methyl}-N-[2-(6-methoxy-1Hindol-3-yl)ethyl]benzamide (4). Reagents used were 4-{[benzyl(methyl)amino]methyl}benzoic acid (20) (127.6 mg, 0.5 mmol), 2(6-methoxy-1H-indol-3-yl)ethanamine (95.1 mg, 0.5 mmol), PyBOP (312.2 mg, 0.6 mmol), and Et3N (156 μL, 1.2 mmol). Purification involved the use of hexane/EtOAc (1:1) as eluent. Derivative 4: yellow solid. mp 119−121 °C (139 mg, 65%). ESI-MS m/z 428 [MH]+. 1H NMR (acetone-d6) δ 7.87 (d, 2H, J = 8.2 Hz), 7.49 (d, 1H, J = 8.6 Hz), 7.44 (d, 2H, J = 8.2 Hz), 7.37 (m, 2H), 7.31 (m, 2H), 7.23 (m, 1H), 7.05 (s, 1H), 6.90 (d, 1H, J = 2.2 Hz), 6.68 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz), 3.76 (s, 3H), 3.69 (t, 2H, J = 7.4 Hz), 3.54 (s, 2H), 3.51 (s, 2H), 3.02 (t, 2H, J = 7.4 Hz), 2.12 (s, 3H). 13C NMR (acetone-d6) δ 167.2 (CONH), 157.2 (C), 143.7 (C), 140.2 (C), 138.2 (C), 134.7 (C), 129.5 (2CH), 129.3 (2CH), 129.0 (2CH), 127.9 (2CH), 127.7 (CH), 122.9 (C), 121.7 (CH), 119.9 (CH), 113.4 (C), 109.6 (CH), 95.2 (CH), 62.3 (CH2), 61.9 (CH2), 55.6 (CH3), 42.3 (CH3), 41.2 (CH2), 26.3 (CH2). Purity: 99% (by HPLC). Anal. (C27H29N3O2) C, H, N.

4-{[Benzyl(methyl)amino]methyl}-N-[2-(6-fluoro-1H-indol-3yl)ethyl]benzamide (5). Reagents used were 4-{[benzyl(methyl)amino]methyl}benzoic acid (20) (127.63 mg, 0.5 mmol), 2-(6-fluoro1H-indol-3-yl)ethanamine (89.0 mg, 0.5 mmol), PyBOP (312.2 mg, 0.6 mmol), and Et3N (156 μL, 1.2 mmol). Purification involved the use of hexane/EtOAc (2:3) as eluent. Hybrid 5: yellow solid. mp 83− 85 °C (135 mg, 65%). ESI-MS m/z 416 [MH]+. 1H NMR (CDCl3) δ 8.63 (s, NH), 7.63 (d, 2H, J = 8.2 Hz), 7.49 (dd, 1H, J = 9.0 Hz, J = 5.2 Hz), 7.36 (d, 2H, J = 8.2 Hz), 7.32 (m, 4H), 7.24 (m, 1H), 6.99 (dd, 1H, J = 9.7 Hz, J = 2.3 Hz), 6.96 (d, 1H, J = 2.3 Hz), 6.84 (td, 1H, J = 9.0 Hz, J = 2.3 Hz), 6.40 (t, 1H, J = 5.7 Hz), 3.74 (q, 2H, J = 6.7 Hz), 3.24 (s, 2H), 3.51 (s, 2H), 3.02 (t, 2H, J = 6.7 Hz), 2.16 (s, 3H). 13 C NMR (CDCl3) δ 167.5 (CONH), 161.1 (C), 158.8 (C), 143.0 (C), 138.7 (C), 136.3 (C, J=12.2), 128.9 (2CH), 128.8 (2CH), 128.3 (2CH), 127.1 (CH), 126.8 (2CH), 123.9 (C), 122.4 (CH), 122.3 (CH), 119.4 (C), 119.3 (CH), 112.8 (CH), 108.0 (CH, JC,F = 24.4 Hz), 97.6 (CH, JC,F = 25.9 Hz), 61.8 (CH2), 61.2 (CH2), 42.1 (CH3), 40.2 (CH 3 ), 25.2 (CH 2 ). Purity: 98% (by HPLC). Anal. (C26H26FN3O) C, H, N. N-[2-(1H-Indol-3-yl)ethyl]-4-{[(2-chlorobenzyl)(methyl)amino]methyl} benzamide (6). Reagents used were 4-{[(2chlorobenzyl)(methyl)amino]methyl}benzoic acid (21) (135.4 mg, 0.5 mmol), 2-(1H-indol-3-yl)ethanamine (80.1 mg, 0.5 mmol), PyBOP (312.2 mg, 0.6 mmol), and Et3N (156 μL, 1.2 mmol). Purification involved the use of hexane/EtOAc (1:1) as eluent. Compound 6: white solid. mp 97−99 °C (106 mg, 49%). ESI-MS m/z 432 [MH]+, 434 [MH + 2]+. 1H NMR (CDCl3) δ 8.20 (s, NH), 7.65 (d, 1H, J = 8.1 Hz), 7.62 (d, 2H, J = 8.3 Hz), 7.52 (dd, 1H, J = 7.6 Hz, J = 1.2 Hz), 7.38 (d, 2H, J = 8.3 Hz), 7.37 (d, 1H, J = 8.1 Hz), 7.34 (dd, 1H, J = 7.8 Hz, J = 1.0 Hz).7.21 (m, 4H), 7.13 (t, 1H, J = 7.2 Hz), 7.06 (d, J = 2.0 Hz), 6.23 (t, NH, J = 5.4 Hz), 3.80 (q, 2H, J = 6.6 Hz), 3.63 (s, 2H), 3.60 (s, 2H), 3.09 (t, 2H, J = 6.6 Hz), 2.19 (s, 3H). 13C NMR (CDCl3) δ 167.3 (CONH), 143.0 (C), 136.5 (C), 136.4 (C), 134.2 (C), 133.4 (C), 130.6 (CH), 129.4 (CH), 128.9 (2CH), 128.1 (CH), 127.2 (CH), 126.8 (2CH), 126.6 (CH), 122.2 (CH), 122.1 (CH), 119.5 (CH), 118.7 (CH), 113.0 (C), 111.3 (CH), 61.7 (CH2), 58.5 (CH2), 42.2 (CH3), 40.2 (CH2), 25.3 (CH2). Purity: 100% (by HPLC). Anal. (C26H26ClN3O) C, H, N. 4-{[(2-Chlorobenzyl)(methyl)amino]methyl}-N-[2-(5-methoxy-1H-indol-3-yl)ethyl]benzamide (7). Reagents used were 4{[(2-chlorobenzyl)(methyl)amino]methyl}benzoic acid (21) (135.4 mg, 0.5 mmol), 2-(5-methoxy-1H-indol-3-yl)ethanamine (95.1 mg, 0.5 mmol), PyBOP (312.2 mg, 0.6 mmol), and Et3N (156 μL, 1.2 mmol). Purification involved the use of hexane/EtOAc (1:1) as eluent. Hybrid 7: white solid. mp 120−122 °C (132 mg, 57%). ESI-MS m/z 462 [MH]+, 464 [MH + 2]+. 1H NMR (CD3OD) δ 7.73 (d, 2H, J = 8.3 Hz), 7.54 (d, 1H, J = 8.3 Hz), 7.46 (d, 1H, J = 8.3 Hz), 7.44 (d, 2H, J = 8.6 Hz), 7.32 (d, 1H, J = 8.3 Hz), 7.36 (m, 1H), 7.21 (d, 1H, J = 8.8 Hz), 6.72 (dd, 1H, J = 8.8 Hz, J = 2.4 Hz), 3.72 (s, 3H), 3.66 (s, 2H), 3.63 (s, 2H), 3.34 (t, 2H, J = 7.3 Hz), 3.03 (t, 2H, J = 7.3 Hz), 2.20 (s, 3H). 13C NMR (CD3OD) δ 170.1 (CONH), 154.9 (C), 137.5 (C), 135.5 (C), 134.8 (C), 133.3 (C), 132.3 (C), 132.3 (CH), 130.5 (CH), 130.4 (CH), 130.3 (C), 130.2 (2CH), 129.7 (CH), 129.2 (C), 128.3 (2CH), 113.3 (C), 112.9 (CH), 112.7 (CH), 101.2 (CH), 62.6 (CH2), 59.4 (CH2), 56.1 (CH3), 42.5 (CH2), 42.3 (CH3), 26.3 (CH2). Purity: 99% (by HPLC). Anal. (C27H28ClN3O2) C, H, N. N-[2-(1H-Indol-3-yl)ethyl]-4-{[(3-chlorobenzyl)(methyl)amino]methyl}benzamide (8). Reagents used were 4-{[(3chlorobenzyl)(methyl)amino]methyl}benzoic acid (22) (135.4 mg, 0.5 mmol), 2-(1H-indol-3-yl)ethanamine (80.1 mg, 0.5 mmol), PyBOP (312.2 mg, 0.6 mmol), and Et3N (156 μL, 1.2 mmol). Purification involved the use of hexane/EtOAc (2:1) as eluent. Derivative 8: white solid. mp 110−112 °C (119 mg, 55%). ESI-MS m/ z 432 [MH]+, 434 [MH + 2]+. 1H NMR (CDCl3) δ 8.21(s, NH), 7.64 (d, 2H, J = 8.3 Hz), 7.64 (m, 1H), 7.37 (d, 2H, J = 8.3 Hz), 7.37 (m, 2H), 7.23 (m, 4H), 7.13 (td, 1H, J = 7.5 Hz, J = 1.0 Hz), 7.06 (d, 1H, J = 2.5 Hz), 6.23 (t, NH, J = 5.5 Hz), 3.80 (q, 2H, J = 6.7 Hz), 3.52 (s, 2H), 3.46 (s, 2H), 3.09 (t, 2H, J = 6.7 Hz), 2.16 (s, 3H). 13C NMR (CDCl3) δ 167.4 (CONH), 142.8 (C), 141.3 (C), 136.4 (C), 134.2 (C), 133.5 (C), 129.5 (CH), 128.8 (2CH), 128.7 (2CH), 127.3 (C), 3780



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J = 7.7 Hz, J = 1.0 Hz), 6.85 (m, 2H), 6.27 (t, NH, J = 5.6 Hz), 3.79 (s, 3H), 3.77 (s, 3H), 3.75 (q, 2H, J = 6.6 Hz), 3.58 (s, 2H) 3.55 (s, 2H), 3.05 (t, 2H, J = 6.6 Hz), 2.20 (s, 3H). 13C NMR (CDCl3) δ 167.4 (CONH), 157.7 (C), 154.0 (C), 143.3 (C), 133.2 (C), 131.5 (C), 130.3 (CH), 128.9 (2CH), 128.0 (CH), 127.7 (C), 126.8 (C), 126.7 (2CH), 122.9 (CH), 120.3 (CH), 112.8 (C), 112.5 (CH), 112.0 (CH), 110.3 (CH), 100.3 (CH), 61.7 (CH2), 55.8 (CH2), 55.3 (CH3), 55.2 (CH3), 42.4 (CH3), 40.3 (CH2), 25.3 (CH2). Purity: 100% (by HPLC). Anal. (C28H31N3O3) C, H, N. N-[2-(1H-Indol-3-yl)ethyl]-4-{[(3-methoxybenzyl)(methyl)amino]methyl}benzamide (13). Reagents used were 4-{[(3methoxybenzyl)(methyl)amino]methyl}benzoic acid (24) (142.7 mg, 0.5 mmol), 2-(1H-indol-3-yl)ethanamine (80.1 mg, 0.5 mmol), PyBOP (312.2 mg, 0.6 mmol), and Et3N (156 μL, 1.2 mmol). Purification involved the use of EtOAc as eluent. Compound 13: yellow solid. mp 81−83 °C (166 mg, 78%). ESI-MS m/z 428 [MH]+. 1 H NMR (CD3OD) δ 7.74 (d, 2H, J = 8.4 Hz), 7.60 (dd, 1H, J = 8.1 Hz, J = 1.0 Hz), 7.42 (d, 2H, J = 8.4 Hz), 7.32 (dd, 1H, J = 8.1 Hz, J = 1.0 Hz), 7.22 (t, 1H, J = 8.1 Hz), 7.09 (s, IH), 7.06 (td, 1H, J = 8.1 Hz, J = 1.0 Hz), 6.98 (td, 1H, J = 8.1 Hz, J = 1.0 Hz), 6.93 (d, 1H, J = 2.0 Hz), 6.91 (d, 1H, J = 8.1 Hz), 6.80 (dd, 1H, J = 8.1 Hz, J = 2.0 Hz), 3.77 (s, 3H), 3.66 (t, 2H, J = 7.4 Hz), 3.53 (s, 2H) 3.48 (s, 2H), 3.05 (t, 2H, J = 7.4 Hz), 2.16 (s, 3H). 13C NMR (CD3OD) δ 170.1 (CONH), 161.2 (C), 143.8 (C), 141.3 (C), 138.1 (C), 134.8 (C), 130.3 (CH), 130.3 (2CH), 128.8 (C), 128.3 (2CH), 123.4 (CH), 122.5 (CH), 122.3 (CH), 119.6 (CH), 119.4 (CH), 115.7 (CH), 113.7 (CH), 113.4 (C), 112.2(CH), 62.7 (CH2), 62.1 (CH2), 55.6 (CH3), 42.5 (CH3), 42.2 (CH2), 26.3 (CH2). Purity: 99% (by HPLC). Anal. (C27H29N3O2) C, H, N. N-[2-(5-Methoxy-1H-indol-3-yl)ethyl]-4-{[(3methoxybenzyl)(methyl)amino]methyl}benzamide (14). Reagents used were 4-{[(3-methoxybenzyl)(methyl)amino]methyl}benzoic acid (24) (142.7 mg, 0.5 mmol), 2-(5-methoxy-1H-indol-3yl)ethanamine (95.1 mg, 0.5 mmol), PyBOP (312.2 mg, 0.6 mmol), and Et3N (156 μL, 1.2 mmol). Purification involved the use of hexane/ EtOAc (2:1) as eluent. Hybrid 14: white solid. mp 82−84 °C (144 mg, 63%). ESI-MS m/z 458 [MH]+. 1H NMR (CD3OD) δ 7.71 (d, 2H, J = 8.3 Hz), 7.38 (d, 2H, J = 8.3 Hz), 7.21 (d, 1H, J = 8.7 Hz), 7.20 (t, 1H, J = 7.6 Hz), 7.06 (d, 1H, J = 2.4 Hz), 7.05 (s, 1H), 6.90 (d, 1H, J = 1.2 Hz), 6.88 (d, 1H, J = 7.6 Hz), 6.78 (dd, 1H, J = 7.6 Hz, J = 1.2 Hz), 6.72 (dd, 1H, J = 8.7 Hz, J = 2.4 Hz), 3.75 (s, 3H), 3.70 (s, 3H), 3.63 (t, 2H, J = 7.3 Hz), 3.49 (s, 2H) 3.44 (s, 2H), 3.02 (t, 2H, J = 7.3 Hz), 2.13 (s, 3H). 13C NMR (CD3OD) δ 170.0 (CONH), 161.2 (C), 154.9 (C), 143.8 (C), 141.3 (C), 134.7 (C), 133.3 (C), 130.3 (CH), 130.2 (2CH), 129.2 (C), 128.3 (2CH), 124.2 (CH), 122.5 (CH), 115.6 (CH), 113.7 (CH), 113.3 (C), 112.9 (CH), 112.7 (CH), 101.2 (CH), 62.7 (CH2), 62.1 (CH2), 56.1 (CH3), 55.6 (CH3), 42.5 (CH2), 42.3 (CH3), 26.3 (CH2). Purity: 100% (by HPLC). Anal. (C28H31N3O3) C, H, N. Biochemical Studies. Inhibition of Human AChE and BuChE. The method of Ellman et al. was followed.47 The assay solution consisted of 0.1 M phosphate buffer, pH 8.0, 400 μM 5,5′-dithiobis(2nitrobenzoic acid) (DTNB, Ellman’s reagent), 0.05 U mL−1 h-AChE (human recombinant acetylcholinesterase, Sigma Chemical Co.) or 0.024 U mL−1 h-BuChE (butyrylcholinesterase from human serum, Sigma Chemical Co.), and 800 μM acetylthiocholine iodide or 500 μM butyrylthiocholine as the substrate of the enzymatic reaction, respectively. The compounds tested were added to the assay solution and preincubated with the enzyme for 5 min at 30 °C. After that period, the substrate was added. The absorbance changes at 412 nm were recorded for 5 min with a UV/vis microplate spectrophotometer (Multiskan Spectrum, Thermo Electron Co.). The reaction rates were compared, and the inhibition percentage resulting from the presence of test compound was calculated. The IC50 is defined as the concentration of each compound that reduces 50% of the enzymatic activity without any inhibitor. Measurement of Propidium Iodide Displacement from the Peripheral Anionic Site (PAS) of AChE. A soltion of AChE from bovine erythrocytes at a concentration of 5 μM in 0.1 mM Tris buffer, pH 8.0, was used. Aliquots of compounds were added to obtain final

127.2 (CH), 126.9 (2CH), 122.2 (CH), 122.1 (CH), 119.5 (CH), 118.7 (CH), 113.0 (C), 111.3 (CH), 61.4 (CH2), 61.2 (CH2), 42.3 (CH3), 40.2 (CH2), 25.3 (CH2). Purity: 100% (by HPLC). Anal. (C26H26ClN3O) C, H, N. 4-{[(3-Chlorobenzyl)(methyl)amino]methyl}-N-[2-(5-methoxy-1H-indol-3-yl)ethyl]benzamide (9). Reagents used were 4{[(3-chlorobenzyl)(methyl)amino]methyl}benzoic acid (22) (135.4 mg, 0.5 mmol), 2-(5-methoxy-1H-indol-3-yl)ethanamine (95.1 mg, 0.5 mmol), PyBOP (312.2 mg, 0.6 mmol), and Et3N (156 μL, 1.2 mmol). Purification involved the use of hexane/EtOAc (2:1) as eluent. Hybrid 9: yellow solid. mp 108−110 °C (119 mg, 55%). ESI-MS m/z 462 [MH]+, 464 [MH + 2]+. 1H NMR (CDCl3) δ 8.15 (s, NH), 7.62 (d, 2H, J = 8.3 Hz), 7.35 (d, 2H, J = 8.3 Hz), 7.34 (s, 2H), 7.25 (d, 1H, J = 8.6 Hz), 7.22 (m, 3H), 7.03 (s. 2H), 6.85 (dd, 1H, J = 8.6 Hz, J = 2.3 Hz), 6.27 (t, NH, J = 5.6 Hz), 3.77 (q, 2H, J = 6.5 Hz), 3.86 (s, 3H), 3.51 (s, 2H) 3.45 (s, 2H), 3.04 (t, 2H, J = 6.5 Hz), 2.14 (s, 3H). 13C NMR (CDCl3) δ 167.6 (CONH), 154.3 (C), 143.1 (C), 141.5 (C), 134.4 (C), 133.6 (C), 131.7 (C), 129.8 (CH), 129.1 (2CH), 129.0 (2CH), 127.9 (C), 127.5 (CH), 127.2 (2CH), 123.2 (CH), 113.0 (C), 112.8 (CH), 112.3 (CH), 100.5 (CH), 61.7 (CH2), 61.4 (CH2), 56.0 (CH3), 42.5 (CH3), 40.5 (CH2), 25.5 (CH2). Purity: 100% (by HPLC). Anal. (C27H28ClN3O2) C, H, N. 4-{[(3-Chlorobenzyl)(methyl)amino]methyl}-N-[2-(6-methoxy-1H-indol-3-yl)ethyl]benzamide (10). Reagents used were 4{[(3-chlorobenzyl)(methyl)amino]methyl}benzoic acid (22) (135.4 mg, 0.5 mmol), 2-(6-methoxy-1H-indol-3-yl)ethanamine (95.1 mg, 0.5 mmol), PyBOP (312.2 mg, 0.6 mmol), and Et3N (156 μL, 1.2 mmol). Purification involved the use of hexane/EtOAc (1:1) as eluent. Hybrid 10: yellow solid. mp 58−60 °C (189 mg, 82%). ESI-MS m/z 462 [MH]+, 464 [MH + 2]+. 1H NMR (CDCl3) δ 8.31 (s, NH), 7.65 (d, 2H, J = 8.4 Hz), 7.48 (d, 1H, J = 8.6 Hz), 7.36 (d, 2H, J = 8.4 Hz), 7.35 (s, 1H), 7.23 (m, 3H), 6.91 (d, 1H, J = 2.2 Hz), 6.84 (d, 1H, J = 2.2 Hz), 6.78 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz), 6.35 (t, NH, J = 5.6 Hz), 3.81 (s, 3H), 3.75 (q, 2H, J = 6.6 Hz), 3.52 (s, 2H) 3.46 (s, 2H), 3.03 (t, 2H, J = 6.6 Hz), 2.15 (s, 3H). 13C NMR (CDCl3) δ 167.4 (CONH), 156.5 (C), 142.7 (C), 141.2 (C), 137.2 (C), 134.1 (C), 133.4 (C), 129.85 (CH), 128.8 (2CH), 128.7 (2CH), 127.2 (CH), 126.9 (2CH), 121.6 (C), 120.9 (CH), 119.3 (C), 112.7 (C), 109.4 (CH), 94.7 (CH), 61.4 (CH2), 61.2 (CH2), 55.6 (CH3), 42.2 (CH3), 40.2 (CH 2 ), 25.3 (CH 2 ). Purity: 100% (by HPLC). Anal. (C27H28ClN3O2) C, H, N. N-[2-(1H-Indol-3-yl)ethyl]-4-{[(2-methoxybenzyl)(methyl)amino]methyl}benzamide (11). Reagents used were 4-{[(2methoxybenzyl)(methyl)amino]methyl}benzoic acid (23) (142.7 mg, 0.5 mmol), 2-(1H-indol-3-yl)ethanamine (80.1 mg, 0.5 mmol), PyBOP (312.2 mg, 0.6 mmol), and Et3N (156 μL, 1.2 mmol). Purification involved the use of hexane/EtOAc (1:1) as eluent. Compound 11: white solid. mp 80−82 °C (130 mg, 61%). ESI-MS m/ z 428 [MH]+. 1H NMR (CD3OD) δ 7.74 (d, 2H, J = 8.3 Hz), 7.60 (d, 1H, J = 8.1 Hz), 7.42 (d, 2H, J = 8.3 Hz), 7.31 (m, 2H), 7.24 (td, 1H, J = 7.8 Hz, J = 1.7 Hz), 7.09 (s, 1H), 7.06 (m, 1H), 6.97 (td, 1H, J = 8.1 Hz, J = 1.0 Hz), 6.93 (m, 1H), 6.91 (td, 1H, J = 7.6 Hz, J = 1.0 Hz), 3.78 (s, 3H), 3.66 (t, 2H, J = 7.4 Hz), 3.54 (s, 2H) 3.34 (s, 2H), 3.06 (t, 2H, J = 7.4 Hz), 2.18 (s, 3H). 13C NMR (CD3OD) δ 170.1 (CONH), 159.4 (C), 143.7 (C), 138.1 (C), 134.7 (C), 132.1 (CH), 130.4 (2CH), 129.7 (CH), 128.8 (C), 128.2 (2CH), 127.0 (C), 123.4 (CH), 122.3 (CH), 121.2 (CH), 119.6 (CH), 119.4 (CH), 113.4 (C), 112.2 (CH), 111.6 (CH), 62.5 (CH2), 56.0 (CH3), 55.7 (CH2), 42.6 (CH3), 42.2 (CH2), 26.3 (CH2). Purity: 100% (by HPLC). Anal. (C27H29N3O3) C, H, N. N-[2-(5-Methoxy-1H-indol-3-yl)ethyl]-4-{[(2methoxybenzyl)(methyl)amino]methyl}benzamide (12). Reagents used were 4-{[(2-methoxybenzyl)(methyl)amino]methyl}benzoic acid (23) (142.7 mg, 0.5 mmol), 2-(5-methoxy-1H-indol-3yl)ethanamine (95.1 mg, 0.5 mmol), PyBOP (312.2 mg, 0.6 mmol), and Et3N (156 μL, 1.2 mmol). Purification involved the use of EtOAc as eluent. Hybrid 12: yellow solid. mp 58−60 °C (174 mg, 76%). ESIMS m/z 458 [MH]+. 1H NMR (CDCl3) δ 8.18 (s, NH), 7.62 (d, 2H, J = 8.6 Hz), 7.40 (m, 1H), 7.39 (d, 2H, J = 8.6 Hz), 7.25 (d, 1H, J = 9.0 Hz), 7.22 (td, 1H, J = 7.7 Hz, J = 2.0 Hz), 7.03 (m, 2H), 6.94 (td, 1H, 3781



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penicillin, and 50 μg mL−1 streptomycin (reagents from GIBCO, Madrid, Spain). Cultures were seeded into flasks containing supplemented medium and were maintained at 37 °C in 5% CO2/ humidified air. Stock cultures were passaged 1:4 twice weekly. For assays, SH-SY5Y cells were subcultured in 48-well plates at a seeding density of 105 cells per well. For the cytotoxicity experiments, cells were treated with drugs before they reached confluence in DMEM free of serum. Cell Viability Experiments. To study the cytotoxic effects of compounds alone, cells were plated at a density of 105 cells per well at least 48 h before the toxicity measurements. Cells were exposed for 24 h to the compound at 1 μM, and the quantitative assessment of cell death was made by measurement of the percent of the intracellular enzyme lactate dehydrogenase (LDH) released to the extracellular medium (cytotoxicity detection kit, Roche). The quantity of LDH was evaluated in a microplate reader (Anthos 2010 or Labsystems iMES Reader MS) at 492 nm (λ excitation) and 620 nm (λ emission). Neuroprotection Studies. To study the cytoprotective action of the compounds against cell death induced by the mixture of rotenone (30 μM) and oligomycin A (10 μM), drugs were given at time zero and maintained for 24 h. Then, the media were replaced by fresh media that still contained the drug plus the cytotoxic insult, which was left for an additional 24 h period. Thereafter, cell survival was assessed by measuring LDH activity. Measurement of Lactic Dehydrogenase (LDH) Activity. Extracellular and intracellular LDH activity was measured by UV/vis using a cytotoxicity cell death kit (Roche-Boehringer Mannheim, Germany) according to the manufacturer’s indications. Total LDH activity was defined as the sum of intracellular and extracellular LDH activity, and released LDH was defined as the percentage of extracellular compared with total LDH activity. Data were expressed as the mean (±SEM) of at least three different cultures in quadruplicate. LDH released was calculated for each individual experiment relative to 100% of the extracellular LDH released by the vehicle with respect to the total. To determine percent protection, LDH release was normalized as follows: in each individual triplicate experiment, LDH release obtained in nontreated cells (basal) was subtracted from the LDH released upon the toxic treatment and normalized to 100%, and that value was subtracted from 100. Measurement of Cell Viability by MTT Assay. Cell viability was also measured by quantitative colorimetric assay with 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) (Sigma-Aldrich), as described previously.60 Briefly, 50 μL of the MTT labeling reagent, at a final concentration of 0.5 mg mL−1, was added to each well at the end of the incubation period, and the plate was placed in a humidified incubator at 37 °C with 5% CO2 and 95% air (v/v) for an additional 2 h. Then, the insoluble formazan was dissolved with dimethyl sulfoxide; colorimetric determination of MTT reduction was measured at 540 nm. Control cells treated with 0.1% DMSO were taken as 100% viability. Neurogenesis Assays. Animals. Adult (8−12 weeks old) male Wistar rats (n = 6 per group), housed in a 12 h light−dark cycle animal facility, were used in this study. All procedures with animals were specifically approved by the Ethics Committee for Animal Experimentation of the CSIC and carried out in accordance with National (normative 1201/2005) and International recommendations (Directive 2010/63 from the European Communities Council). Special care was taken to minimize animal suffering. Neurosphere Cultures. Neurospheres (NS) were derived from the subgranular zone of the dentate gyrus of the hippocampus of adult Wistar rats, which were induced to proliferate using established passaging methods to achieve optimal cellular expansion according to published protocols.56 Rats were decapitated, and brains were dissected, obtaining the hippocampus as described.57 Briefly, cells were seeded into 12-well dishes and cultured in DMEM/F12 (1:1, Invitrogen) containing 10 ng mL−1 epidermal growth factor (EGF, Peprotech, London, UK), 10 ng mL−1 fibroblast growth factor (FGF, Peprotech), and B27 medium (Gibco). After 3 days in culture, primary NS cultures were treated with different compounds. To determine the ability of compounds to induce differentiation, NS from 10 day old

concentrations of 0.3, 1.0, and 3.0 μM, and the solutions were kept at room temperature for at least 6 h. Afterward, the samples were incubated for 15 min with propidium iodide at a final concentration of 20 μM, and the fluorescence was measured in a fluorescence microplate reader (Fluostar Optima, BMG, Germany). Wavelengths of excitation and emission were 485 and 620 nm, respectively. In Vitro Blood−Brain Barrier Permeation Assay (PAMPABBB). Prediction of the brain penetration was evaluated using a parallel artificial membrane permeation assay (PAMPA-BBB) in a similar manner as previously described.38,41,51 Pipetting was performed with a semi-automatic robot (CyBi-SELMA), and UV absorbance was read with a microplate spectrophotometer (Multiskan Spectrum, Thermo Electron Co.). Commercial drugs, phosphate buffered saline solution at pH 7.4 (PBS), and dodecane were purchased from Sigma, Aldrich, Acros, and Fluka. Millex filter units (PVDF membrane, diameter 25 mm, pore size 0.45 μm) were acquired from Millipore. The porcine brain lipid (PBL) was obtained from Avanti Polar Lipids. The donor microplate was a 96-well filter plate (PVDF membrane, pore size 0.45 μm), and the acceptor microplate was an indented 96well plate, both from Millipore. The acceptor 96-well microplate was filled with 200 μL of PBS/ethanol (70:30), and the filter surface of the donor microplate was impregnated with 4 μL of PBL in dodecane (20 mg mL−1). Compounds were dissolved in PBS/ethanol (70:30) at 100 μg mL−1, filtered through a Millex filter, and then added to the donor wells (200 μL). The donor filter plate was carefully placed on the acceptor plate to form a sandwich, which was left undisturbed for 240 min at 25 °C. After incubation, the donor plate was carefully removed, and the concentration of compounds in the acceptor wells was determined by UV/vis spectroscopy. Every sample was analyzed at five wavelengths in four wells and in at least three independent runs, and the results are given as the mean ± standard deviation. In each experiment, 11 quality control standards of known BBB permeability were included to validate the analysis set. Oxygen Radical Absorbance Capacity Assay. The ORAC method was followed52,59 using a Polarstar Galaxy plate reader (BMG Labtechnologies GmbH, Offenburg, Germany) with 485-P excitation and 520-P emission filters. The equipment was controlled by Fluorostar Galaxy software (version 4.11-0) for fluorescence measurement. 2,2′-Azobis(amidinopropane) dihydrochloride (AAPH), (±)-6hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (trolox), and fluorescein (FL) were purchased from Sigma-Aldrich. The reaction was carried out in 75 mM phosphate buffer (pH 7.4), and the final reaction mixture was 200 μL. Antioxidant (20 μL) and FL (120 μL; 70 mM, final concentration) solutions were placed in a black 96-well microplate (96F untreat, Nunc). The mixture was preincubated for 15 min at 37 °C, and AAPH solution (60 μL, 12 mM, final concentration) was then added rapidly using a multichannel pipet. The microplate was immediately placed in the reader, and the fluorescence was recorded every minute for 80 min. The microplate was automatically shaken prior each reading. Samples were measured at eight different concentrations (0.1−1 μM). A blank (FL + AAPH in phosphate buffer), instead of the sample solution, and eight calibration solutions using trolox (1−8 μM) were also carried out in each assay. All reaction mixtures were prepared in duplicate, and at least three independent assays were performed for each sample. Raw data were exported from Fluostar Galaxy Software to an Excel sheet for further calculations. Antioxidant curves (fluorescence vs time) were first normalized to the curve of the blank corresponding to the same assay, and the area under the fluorescence decay curve (AUC) was calculated. The net AUC corresponding to a sample was calculated by subtracting the AUC corresponding to the blank. Regression equations between net AUC and antioxidant concentration were calculated for all samples. ORACFL values were expressed as trolox equivalents by using the standard curve calculated for each assay, where the ORAC-FL value of trolox was taken to be 1.0. Studies of Cell Viability and Neuroprotection. Culture of SHSY5Y Cells. SH-SY5Y cells, at passages between 3 and 16 after defreezing, were maintained in a Dulbecco’s modified Eagle’s medium (DMEM) containing 15 nonessential amino acids and supplemented with 10% fetal calf serum (FCS), 1 mM glutamine, 50 units mL−1 3782



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cultures were plated onto 100 μg mL−1 poly-L-lysine-coated coverslips and treated for 48 h in the presence of serum but in the absence of exogenous growth factors. Immunocytochemistry. After treatment, cells were processed for immunocytochemistry with two types of neurogenesis-associated neuronal markers: anti-β-tubulin antibody (clone TuJ1), associated with early stages of neurogenesis, and MAP-2 (microtubule-associated protein 2), a marker of neuronal maturation. DAPI staining (4′,6diamidino-2-phenylindole) was used as a nuclear marker. Basal values were obtained under the same conditions but in the absence of any product. Melatonin (endogenous ligand of the melatoninergic receptors) and luzindole (melatoninergic antagonist) were used as controls. The images were obtained using a Nikon fluorescence microscope 90i that was coupled to a digital camera Qi. The microscope configuration was adjusted to produce the optimum signal-to-noise ratio. For quantifying the number of cells producing a given marker in any experiment, the number of positive cells in the neurosphere was counted as previously described.58 Cell numbers were estimated from a total of five neurospheres per condition over three independent experiments. Statistical Determinations. One-way ANOVA analysis for comparisons between different treatments on neurospheres was performed using the SPSS statistical software package (version 20.0) for Windows (Chicago, IL) followed by Student’s t post hoc test. A p value ≤ 0.05 was considered to be statistically significant.

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ligand; ORAC-FL, oxygen radical absorbance capacity by fluorescence; PAMPA-BBB, parallel artificial membrane permeation assay for blood−brain barrier permeation; PAS, peripheral anionic site; PyBOP, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate

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ASSOCIATED CONTENT

* Supporting Information S

Elemental analyses of new melatonin−N,N-dibenzyl(Nmethyl)amine hybrids 1−14 and intermediates 16−24; permeability in the PAMPA-BBB assay of 10 commercial drugs; neuroprotection of the human neuroblastoma cell line SH-SY5Y against the combination of rotenone and oligomycin by hybrids 1−14; percentages of MTT reduction and LDH release in SH-SY5Y cells exposed to compounds 3, 11, and 14; and molecular modelling overlay of AP2238 and 3. This material is available free of charge via the Internet at http:// pubs.acs.org.

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REFERENCES

(1) Sloane, P. D.; Zimmerman, S.; Suchindran, C.; Reed, P.; Wang, L.; Boustani, M.; Sudha, S. The public health impact of Alzheimer’s disease, 2000−2050: Potential implication of treatment advances. Annu. Rev. Public Health 2002, 23, 213−231. (2) Querfurth, H. W.; LaFerla, F. M. Alzheimer’s disease. N. Engl. J. Med. 2010, 362, 329−344. (3) Bond, M.; Rogers, G.; Peters, J.; Anderson, R.; Hoyle, M.; Miners, A.; Moxham, T.; Davis, S.; Thokala, P.; Wailoo, A.; Jeffreys, M.; Hyde, C. The effectiveness and cost-effectiveness of donepezil, galantamine, rivastigmine and memantine for the treatment of Alzheimer’s disease (review of technology appraisal no. 111): A systematic review and economic model. Health Technol. Assess. 2012, 16, 1−470. (4) Wilkinson, D.; Wirth, Y.; Goebel, C. Memantine in patients with moderate to severe Alzheimer’s disease: Meta-analyses using realistic definitions of response. Dementia Geriatr. Cognit. Disord. 2013, 37, 71− 85. (5) Inestrosa, N. C.; Dinamarca, M. C.; Alvarez, A. Amyloid− cholinesterase interactions. Implications for Alzheimer’s disease. FEBS J. 2008, 275, 625−632. (6) Rees, T.; Hammond, P. I.; Soreq, H.; Younkin, S.; Brimijoin, S. Acetylcholinesterase promotes beta-amyloid plaques in cerebral cortex. Neurobiol. Aging 2003, 24, 777−787. (7) De Ferrari, G. V.; Canales, M. A.; Shin, I.; Weiner, L. M.; Silman, I.; Inestrosa, N. C. A structural motif of acetylcholinesterase that promotes amyloid beta-peptide fibril formation. Biochemistry 2001, 40, 10447−10457. (8) Bartolini, M.; Bertucci, C.; Cavrini, V.; Andrisano, V. betaAmyloid aggregation induced by human acetylcholinesterase: inhibition studies. Biochem. Pharmacol. 2003, 65, 407−416. (9) Viayna, E.; Sabate, R.; Muñoz-Torrero, D. Dual inhibitors of betaamyloid aggregation and acetylcholinesterase as multi-target antiAlzheimer drug candidates. Curr. Top. Med. Chem. 2013, 13, 1820− 1842. (10) García-Palomero, E.; Muñoz, P.; Usán, P.; García, P.; Delgado, E.; De Austria, C.; Valenzuela, R.; Rubio, L.; Medina, M.; Martinez, A. Potent beta-amyloid modulators. Neurodegener. Dis. 2008, 5, 153−156. (11) Spuch, C.; Antequera, D.; Fernández-Bachiller, M. I.; RodríguezFranco, M. I.; Carro, E. A new tacrine−melatonin hybrid reduces amyloid burden and behavioral deficits in a mouse model of Alzheimer’s disease. Neurotoxic. Res. 2010, 17, 421−431. (12) Antequera, D.; Bolos, M.; Spuch, C.; Pascual, C.; Ferrer, I.; Fernández-Bachiller, M. I.; Rodríguez-Franco, M. I.; Carro, E. Effects of a tacrine-8-hydroxyquinoline hybrid (IQM-622) on Abeta accumulation and cell death: Involvement in hippocampal neuronal loss in Alzheimer’s disease. Neurobiol. Dis. 2012, 46, 682−691. (13) Lorrio, S.; Gómez-Rangel, V.; Negredo, P.; Egea, J.; León, R.; Romero, A.; Dal-Cim, T.; Villarroya, M.; Rodríguez-Franco, M. I.; Conde, S.; Arce, M. P.; Roda, J. M.; García, A. G.; López, M. G. Novel multitarget ligand ITH33/IQM9.21 provides neuroprotection in in vitro and in vivo models related to brain ischemia. Neuropharmacology 2013, 67, 403−411. (14) Ansari, M. A.; Scheff, S. W. Oxidative stress in the progression of Alzheimer disease in the frontal cortex. J. Neuropathol. Exp. Neurol. 2010, 69, 155−167. (15) Gu, F.; Zhu, M.; Shi, J.; Hu, Y.; Zhao, Z. Enhanced oxidative stress is an early event during development of Alzheimer-like pathologies in presenilin conditional knock-out mice. Neurosci. Lett. 2008, 440, 44−48.

AUTHOR INFORMATION

Corresponding Author

*Phone: 34-91-5622900. Fax: 34-91-5644853. E-mail: [email protected]. Present Address

(A.R.) Departamento de Toxicologiá y Farmacologia,́ Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain. ⊥

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge the financial support of the Spanish Ministry of Economy and Competitiveness (projects SAF201231035, SAF2009-13015-C02-01, and SAF2012-32223), the Fundación de Investigación Médica Mutua Madrileña Autó movilistica (AP103952012), and the CSIC (PIE-201280E074). B.L.-I. thanks the CSIC for an I3P Training Contract.

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ABBREVIATIONS USED Aβ, β-amyloid peptide; ACh, acetylcholine; AChE, acetylcholinesterase; AD, Alzheimer’s disease; BACE-1 , β-secretase-1; BBB, blood−brain barrier; BuChE, butyrylcholinesterase; CAS, catalytic active site; CNS, central nervous system; hAChE, human AChE; hBuChE, human BuChE; MTL, multitarget 3783



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