Chromaffin cells as a model to evaluate mechanisms of cell death and neuroprotective compounds.

In this review, we show how chromaffin cells have contributed to evaluate neuroprotective compounds with diverse mechanisms of action. Chromaffin cells are considered paraneurons, as they share many common features with neurons: (i) they synthesize, store, and release neurotransmitters upon stimulation and (ii) they express voltage-dependent calcium, sodium, and potassium channels, in addition to a wide variety of receptors. All these characteristics, together with the fact that primary cultures from bovine adrenal glands or chromaffin cells from the tumor pheochromocytoma cell line PC12 are easy to culture, make them an ideal model to study neurotoxic mechanisms and neuroprotective drugs. In the first part of this review, we will analyze the different cytotoxicity models related to calcium dyshomeostasis and neurodegenerative disorders like Alzheimer's or Parkinson's. Along the second part of the review, we describe how different classes of drugs have been evaluated in chromaffin cells to determine their neuroprotective profile in different neurodegenerative-related models.

Synthesis, structure, theoretical and experimental in vitro antioxidant/pharmacological properties of α-aryl, N-alkyl nitrones, as potential agents for the treatment of cerebral ischemia.

The synthesis, structure, theoretical and experimental in vitro antioxidant properties using the DPPH, ORAC, and benzoic acid, as well as preliminary in vitro pharmacological activities of (Z)-α-aryl and heteroaryl N-alkyl-nitrones 6-15, 18, 19, 21, and 23, is reported. In the in vitro antioxidant activity, for the DPPH radical test, only nitrones bearing free phenol groups gave the best RSA (%) values, nitrones 13 and 14 showing the highest values in this assay. In the ORAC analysis, the most potent radical scavenger was nitrone indole 21, followed by the N-benzyl benzene-type nitrones 10 and 15. Interestingly enough, the archetypal nitrone 7 (PBN) gave a low RSA value (1.4%) in the DPPH test, or was inactive in the ORAC assay. Concerning the ability to scavenge the hydroxyl radical, all the nitrones studied proved active in this experiment, showing high values in the 94-97% range, the most potent being nitrone 14. The theoretical calculations for the prediction of the antioxidant power, and the potential of ionization confirm that nitrones 9 and 10 are among the best compounds in electron transfer processes, a result that is also in good agreement with the experimental values in the DPPH assay. The calculated energy values for the reaction of ROS (hydroxyl, peroxyl) with the nitrones predict that the most favourable adduct-spin will take place between nitrones 9, 10, and 21, a fact that would be in agreement with their experimentally observed scavenger ability. The in vitro pharmacological analysis showed that the neuroprotective profile of the target molecules was in general low, with values ranging from 0% to 18.7%, in human neuroblastoma cells stressed with a mixture of rotenone/oligomycin-A, being nitrones 18, and 6-8 the most potent, as they show values in the range 24-18.4%.

Cholinergic and neuroprotective drugs for the treatment of Alzheimer and neuronal vascular diseases. II. Synthesis, biological assessment, and molecular modelling of new tacrine analogues from highly substituted 2-aminopyridine-3-carbonitriles.

The synthesis, biological assessment, and molecular modelling of new tacrine analogues 11-22 is described. Compounds 11-22 have been obtained by Friedländer-type reaction of 2-aminopyridine-3-carbonitriles 1-10 with cyclohexanone or 1-benzyl-4-piperidone. The biological evaluation showed that some of these molecules were good AChE inhibitors, in the nanomolar range, and quite selective regarding the inhibition of BuChE, the most potent being 5-amino-2-(dimethylamino)-6,7,8,9-tetrahydrobenzo[1,8-b]-naphthyridine-3-carbonitrile (11) [IC(50) (EeAChE: 14nM); IC(50) (eqBuChE: 5.2µM]. Kinetic studies on the easily available and potent anticholinesterasic compound 5-amino-2-(methoxy)-6,7,8,9-tetrahydrobenzo[1,8-b]-naphthyridine-3-carbonitrile (16) [IC(50) (EeAChE: 64nM); IC(50) (eqBuChE: 9.6µM] showed that this compound is a mixed-type inhibitor (K(i)=69.2nM) of EeAChE. Molecular modelling on inhibitor 16 confirms that this compound, as expected and similarly to tacrine, binds at the catalytic active site of EeAChE. The neuroprotective profile of molecules 11-22 has been investigated in SH-SY5Y neuroblastoma cells stressed with a mixture of oligomycin-A/rotenone. Compound 16 was also able to rescue by 50% cell death induced by okadaic acid in SH-SY5Y cells. From these results we conclude that the neuroprotective profile of these molecules is moderate, the most potent being compounds 12 and 17 which reduced cell death by 29%. Compound 16 does not affect ACh- nor K(+)-induced calcium signals in bovine chromaffin cells. Consequently, tacrine analogues 11-22 can be considered attractive therapeutic molecules on two key pharmacological targets playing key roles in the progression of Alzheimer, that is, cholinergic dysfunction and oxidative stress, as well as in neuronal cerebrovascular diseases.

Multipotent drugs with cholinergic and neuroprotective properties for the treatment of Alzheimer and neuronal vascular diseases. I. Synthesis, biological assessment, and molecular modeling of simple and readily available 2-aminopyridine-, and 2-chloropyridine-3,5-dicarbonitriles.

The synthesis, molecular modeling, and pharmacological analysis of new multipotent simple, and readily available 2-aminopyridine-3,5-dicarbonitriles (3-20), and 2-chloropyridine-3,5-dicarbonitriles (21-28), prepared from 2-amino-6-chloropyridine-3,5-dicarbonitrile (1) and 2-amino-6-chloro-4-phenylpyridine-3,5-dicarbonitrile (2) is described. The biological evaluation showed that some of these molecules were modest inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), in the micromolar range. The 2-amino (3, 4), and 2-chloro derivatives 21-23, 25, 26 were AChE selective inhibitors, whereas 2-amino derivatives 5, 14 proved to be selective for BuChE. Only inhibitor 24 was equipotent for both cholinesterases. Kinetic studies on compound 23 showed that this compound is a mixed-type inhibitor of AChE showing a K(i) of 6.33 microM. No clear SAR can be obtained form these data, but apparently, compounds bearing small groups such as the N,N'-dimethylamino or the pyrrolidino, regardless of the presence of a 2-amino, or 6-chloro substituent in the pyridine ring, preferentially inhibit AChE. Molecular modeling on inhibitors 4, 5, 22, and 23 has been carried out to give a better insight into the binding mode on the catalytic active site (CAS), and peripheral anionic site (PAS) of AChE. The most important differences in the observed binding relay on the modifications of the group at C2, as the amino group forms two hydrogen bonds that direct the binding mode, while in the case of compounds with a chlorine atom, this is not possible. The neuroprotective profile of these molecules has been investigated. In the LDH test, only compounds 26, 3, 22, and 24 showed neuroprotection with values in the range 37.8-31.6% in SH-SY5Y neuroblastoma cells stressed with a mixture of oligomycin-A/rotenone, but in the MTT test only compound 17 (32.9%) showed a similar profile. Consequently, these compounds can be considered as attractive multipotent therapeutic molecules on two key pharmacological receptors playing key roles in the progress of Alzheimer, that is, cholinergic dysfunction and oxidative stress, and neuronal vascular diseases.

New tacrine-dihydropyridine hybrids that inhibit acetylcholinesterase, calcium entry, and exhibit neuroprotection properties.

In this communication, we describe the synthesis and biological evaluation of tacripyrimedones 1-5, a series of new tacrine-1,4-dihydropyridine hybrids bearing the general structure of 11-amino-12-aryl-3,3-dimethyl-3,4,5,7,8,9,10,12-octahydrodibenzo[b,g][1,8]naphthyridine-1(2H)-one. These multifunctional compounds are moderately potent and selective AChEIs, with no activity toward BuChE. Kinetic analysis and molecular modeling studies point out that the new compounds preferentially bind the peripheral anionic site of AChE. In addition, compounds 1-5 show an excellent neuroprotective profile, and a moderate blocking effect of L-type voltage-dependent calcium channels due to the mitigation of [Ca(2+)] elevation elicited by K(+) depolarization. Therefore, they represent a new family of molecules with potential therapeutic application for the treatment of Alzheimer's disease.

Synthesis of 6-amino-1,4-dihydropyridines that prevent calcium overload and neuronal death.

The synthesis and pharmacology of 6-amino-1,4-dihydropyridines, such as ethyl 6-amino-4-aryl-5-cyano-1,4-dihydro-2-methyl-3-pyridinecarboxylic acids (3-16) and 2-amino-4-aryl-7,7-dimethyl-5-oxo-1,4,5,6,7,8-hexahydro-3-quinolinenitriles (17-21) are described. Compounds 18 and 21, at the concentration of 0.3 microM, proved to be the best blockers of the [Ca(2+)] overload induced by depolarization with high [K(+)] of SH-SY5Y neuroblastoma cells, with values of 63.8% and 50.4%, respectively. Most of the compounds induced a remarkable neuroprotective effect against toxicity caused by high [K(+)]-elicited [Ca(2+)] overload, and against H(2)O(2)-generated free radicals, in SH-SY5Y cells.

Chondroitin sulfate protects SH-SY5Y cells from oxidative stress by inducing heme oxygenase-1 via phosphatidylinositol 3-kinase/Akt.

We investigated the mechanism of the neuroprotective properties of chondroitin sulfate (CS), an endogenous perineuronal net glycosaminoglycan, in human neuroblastoma SH-SY5Y cells subjected to oxidative stress. Preincubation with CS for 24 h afforded concentration-dependent protection against H2O2-induced toxicity (50 microM for 24 h) measured as lactic dehydrogenase released to the incubation media; cell death was prevented at the concentrations of 600 and 1000 microM. Cell death caused by a combination of 10 microM rotenone plus 1 microM oligomycin-A (Rot/oligo) was also reduced by CS at concentrations ranging from 0.3 to 100 microM; in this toxicity model, maximum protection was achieved at 3 microM (48%). No significant protection was observed in a cell death model of Ca2+ overload (70 mM K+, for 24 h). H2O2 and Rot/oligo generated reactive oxygen species (ROS) measured as an increase in the fluorescence of dichlorofluorescein diacetate-loaded cells. CS drastically reduced ROS generation induced by both H2O2 (extracellular ROS) and Rot/oligo (intracellular ROS). CS also increased the expression of phosphorylated Akt and heme oxygenase-1 by 2-fold. The protective effects of CS were prevented by chelerythrine, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), cycloheximide, and Sn(IV)-protoporphyrin IX. Taken together, these results show that CS can protect SH-SY5Y cells under oxidative stress conditions by activating protein kinase C, which phosphorylates Akt that, via the phosphatidylinositol 3-kinase/Akt pathway, induces the synthesis of the antioxidant protein heme oxygenase-1.

Enzymatic and solid-phase synthesis of new donepezil-based L- and d-glutamic acid derivatives and their pharmacological evaluation in models related to Alzheimer's disease and cerebral ischemia.

Previously, we have described N-Bz-L-Glu[NH-2-(1-benzylpiperidin-4-yl)ethyl]-O-nHex (IQM9.21, L-1) as an interesting multifunctional neuroprotective compound for the potential treatment of neurodegenerative diseases. Here, we describe new derivatives and different synthetic routes, such as chemoenzymatic and solid-phase synthesis, aiming to improve the previously described route in solution. The lipase-catalysed amidation of L- and D-Glu diesters with N-benzyl-4-(2-aminoethyl)piperidine has been studied, using Candida antarctica and Mucor miehei lipases. In all cases, the α-amidated compound was obtained as the main product, pointing out that regioselectivity was independent of the reacting Glu enantiomer and the nature of the lipase. An efficient solid-phase route has been used to produce new donepezil-based L- and D-Glu derivatives, resulting in good yield. At micromolar concentrations, the new compounds inhibited human cholinesterases and protected neurons against toxic insults related to Alzheimer's disease and cerebral ischemia. The CNS-permeable compounds N-Cbz-L-Glu(OEt)-[NH-2-(1-benzylpiperidin-4-yl)ethyl] (L-3) and L-1 blocked the voltage-dependent calcium channels and L-3 protected rat hippocampal slices against oxygen-glucose deprivation, becoming promising anti-Alzheimer and anti-stroke lead compounds.

Novel multipotent tacrine-dihydropyridine hybrids with improved acetylcholinesterase inhibitory and neuroprotective activities as potential drugs for the treatment of Alzheimer's disease.

In this work we describe the synthesis and biological evaluation of the tacrine-1,4-dihydropyridine (DHP) hybrids (3-11). These multipotent molecules are the result of the juxtaposition of an acetylcholinesterase inhibitor (AChEI) such as tacrine (1) and a 1,4-DHP such as nimodipine (2). Compounds 3-11 are very selective and potent AChEIs and show an excellent neuroprotective profile and a moderate Ca2+ channel blockade effect. Consequently, these molecules are new potential drugs for the treatment of Alzheimer's disease.

Depolarization preconditioning produces cytoprotection against veratridine-induced chromaffin cell death.

The hypothesis that K(+) channels and cell depolarization are involved in neuronal death and neuroprotection was tested in bovine chromaffin cells subjected to two treatment periods: the first period (preconditioning period) lasted 6 to 48 h and consisted of treatment with high K(+) solutions or with tetraethylammonium (TEA), a K(+) channel blocker; the second period consisted of incubation with veratridine for 24 h, to cause cell damage. Preconditioning with high K(+) (20-80 mM) or TEA (10-30 mM) for 24 h caused 20-60% cytoprotection against veratridine-induced cell death in bovine chromaffin cells. The absence of Ca(2+) ions during the first 9 h of an 18-h preconditioning period abolished the cytoprotection. Preconditioning with K(+) or TEA increased by 2.5-fold the expression of brain-derived neurotrophic factor and by nearly 2-fold the expression of the antiapoptotic protein Bcl-2. However, preconditioning did not modify the veratridine-evoked Ca(2+) signal. High K(+) shifted the Em by about 10 mV and TEA evoked a transient burst of action potentials superimposed on a sustained depolarization. We conclude that preconditioning may protect chromaffin cells from death by blocking K(+) channels that depolarize the cell and cause a cytosolic Ca(2+) signal, leading to enhanced expression of BDNF and Bcl-2.

Synthesis and biological evaluation of new 4H-pyrano[2,3-b]quinoline derivatives that block acetylcholinesterase and cell calcium signals, and cause neuroprotection against calcium overload and free radicals.

The synthesis and biological evaluation of ethyl 5-amino-4-(3-pyridyl)-2-methyl-6,7,8,9-tetrahydro-4H-pyrano[2,3-b]quinoline-3-carboxylates (9-11) is described. We have found that these compounds inhibit AChE with a mild potency, mitigates the [Ca(2+)](c) triggered by high K(+), and cause neuroprotection against Ca(2+) overloading and free radical-induced neuronal death.

Captan and fenitrothion dissipation in field-treated cauliflowers and effect of household processing.

Field trial studies have been performed with captan and fenitrothion on cauliflower to propose maximum residue limits and to study the dissipation of the pesticides. Residue levels have been determined at different times following good laboratory practice using gas chromatography with mass spectrometric detection. The behaviour of residue levels of these compounds after household processing has been analysed using gas chromatography with electron capture detection. Seven days after treatment, residue levels of captan could be detected, but not of fenitrothion. The half-lives of dissipation for captan and fenitrothion were calculated as 0.9 and 1.8 days respectively. Washing did not significantly affect the residual amounts of captan and fenitrothion observed in raw vegetables; however, after cooking, captan had degraded completely, whereas residue levels of fenitrothion were not modified significantly.

Residue determination of captan and folpet in vegetable samples by gas chromatography/negative chemical ionization-mass spectrometry.

A gas chromatography/negative chemical ionization-mass spectrometry (GC/NCI-MS) method has been developed for the simultaneous determination of the fungicides captan and folpet in khaki (persimmon; flesh and peel) and cauliflower. Samples were extracted with acetone in the presence of 0.1 M zinc acetate solution in order to avoid degradation of fungicides and were purified using solid-phase extraction with divinylbenzene polymeric cartridges. Purified extracts were evaporated and dissolved in hexane prior to injection into the GC/NCI-MS system. Isotope-labeled captan and folpet were used as surrogate/internal standards, and quantification was performed using matrix-matched calibration. The method showed linear response in the concentration range tested (50-2500 ng/mL). The method was fully validated with untreated blank samples of khaki (flesh and peel) and cauliflower spiked at 0.05 and 0.5 mg/kg. Satisfactory recoveries between 82 and 106% and relative standard deviations lower than 11% in all cases (n = 5) were obtained. The limit of detection for both compounds were estimated to be 0.01 mg/kg. The developed method has been applied to treated and untreated samples collected from residue trials.

Galantamine prevents apoptosis induced by beta-amyloid and thapsigargin: involvement of nicotinic acetylcholine receptors.

Galantamine is currently used to treat Alzheimer's disease patients; it behaves as a mild blocker of acetylcholinesterase (AChE) and has an allosteric modulating action on nicotinic acetylcholine receptors (nAChRs). In this study, we observed that galantamine prevented cell death induced by the peptide beta-amyloid(1-40) and thapsigargin in the human neuroblastoma cell line SH-SY5Y, as well as in bovine chromaffin cells. The protective effect of galantamine was concentration-dependent in both cell types; maximum protection was produced at 300 nM. The antiapoptotic effect of galantamine at 300 nM, against beta-amyloid(1-40) or thapsigargin-induced toxicity, was reversed by alpha-bungarotoxin. At neuroprotective concentrations, galantamine caused a mild and sustained elevation of the cytosolic concentration of calcium, [Ca2+]c, measured in single cells loaded with Fura-2. Incubation of the cells for 48 h with 300 nM galantamine doubled the density of alpha7 nicotinic receptors and tripled the expression of the antiapoptotic protein Bcl-2. These results strongly suggest that galantamine can prevent apoptotic cell death by inducing neuroprotection through a mechanism related to that described for nicotine, i.e. activation of nAChRs and upregulation of Bcl-2. These findings might explain the long-term beneficial effects of galantamine in patients suffering of Alzheimer's disease.

Differences in the vascular selectivity and tolerance between the NO donor/beta-blocker PF9404C and nitroglycerin.

The vascular selectivity of PF9404C ((2'S),(2S)-3-isopropylamine,1-[4-(2,3-dinitroxy)propoxymethyl]-phenoxy-2'-propanol), a new beta-blocker with nitric oxide (NO)-donor and vasodilator properties, was studied in different rabbit arteries and veins. Phenylephrine (10(-6) M) or 35 mM K+ were used to pre-contract the arteries and veins prior to study the relaxant effects of PF9404C and nitroglycerin. The potency of both drugs to depress the phenylephrine-induced contraction was greater than that shown in the blockade of the K(+)-evoked contraction in most of the vessels studied, with the exception of the central ear artery. PF9404C exhibited about three-fold higher potency than nitroglycerin to relax the majority of the vessels studied, especially when they were contracted with K+, and showed a certain selectivity of action for the renal artery. PF9404C produced autotolerance but this effect was about 20-fold less pronounced than that observed with nitroglycerin. Cross-tolerance in those preparations pre-exposed to PF9404C that were relaxed later on with nitroglycerin was much greater than autotolerance. The tolerance for nitroglycerin was practically abolished in the presence of N-acetylcysteine. However, this was not the case for PF9404C. These results indicate that, although sharing the property of being NO donors, PF9404C and nitroglycerin show a different profile in causing vasodilation; furthermore, the tolerance to this effect is lesser for PF9404C and seems to be mediated by a mechanism different to that of nitrates. This makes PF9404C a nice pharmacological tool to further develop novel NO-donor compounds with a lesser degree of vascular tolerance than those now available.

Field-incurred fenitrothion residues in kakis: comparison of individual fruits, composite samples, and peeled and cooked fruits.

Field trials have been carried out to determine the variability of residue levels of fenitrothion and its main metabolites fenitrothion-oxon and 3-methyl-4-nitrophenol in individual kaki fruits versus composite samples, in peel versus flesh, and in whole uncooked versus whole cooked fruits. Residue levels have been determined by gas chromatography with thermionic specific detection after extraction with ethyl acetate and without further cleanup. At harvest, residue levels of fenitrothion were below maximum residue levels (MRLs) and the two metabolites 3-methyl-4-nitrophenol and fenitrothion-oxon could be quantified with average amounts of 0.080 and 0.012 mg/kg, respectively. Levels of fenitrothion decreased 88% after peeling, whereas temperature did not result in a high variation. The ratios of the highest residue level in the individual fruits to the corresponding mean of residue levels in the composite samples for fenitrothion were <3. This value is lower than that recommended by the World Health Organization as default value for consumer risk assessment.

On the antiatherogenity of calcium channel blockers: studies in proliferating vascular smooth muscle cells on age sensitivity, dose dependent inhibitory effect, and time of action.

We study the effect of diltiazem on cultured vascular smooth muscle cells from humans and rats, paying special attention to its activity in relation to the concentrations applied, incubation times after addition and the capacity to act against the mitogenic activity of insulin and insulin-like growth factor 1 (IGF-1). The mitotic activity was measured by means of bromodeoxyuridine DNA incorporation. Smooth muscle cells from old individuals showed a dose-dependent regression of the inhibitory level but not those from the young subjects, which showed a remarkable inhibition of mitosis at all concentrations tested. Around 8 h after addition, diltiazem inhibited cell proliferation at all concentrations tested. The inhibition exerted by 10(-7) M rapidly disappeared, reaching values higher than those initially registered and returning to basal rates after 72 h. The inhibition by 10(-6) and 10(-5) M remained after 30 and 72 h, respectively. Insulin (100 nM) or IGF-1 (1 nM) did not counteract the inhibitory effect of diltiazem (10(-5) M). Despite differences related to doses and age of cells, we conclude that diltiazem--as an L-type calcium channels blocker--is a potent inhibitor of vascular smooth muscle cell proliferation.

Dibenzo[1,4,5]thiadiazepine: a hardly-known heterocyclic system with neuroprotective properties of potential usefulness in the treatment of neurodegenerative diseases.

In this work we describe a new family of dibenzo[1,4,5]thiadiazepines (2-12) that showed an interesting in vitro biological profile, namely neuroprotective and antioxidant properties, as well as blockade of cytosolic calcium entry. They showed no cytotoxic effects and the majority were predicted as CNS-permeable compounds. In human neuroblastoma cells they displayed good neuroprotective properties against mitochondrial oxidative stress which, in many cases, almost reached the full protection (>90%) when compounds were incubated with cells 24 h before the addition of toxic stressors. In co-incubation conditions these figures were smaller, although some compounds maintained an interesting level of neuroprotection, higher than 50%. Four selected compounds (2, 5, 8, and 11) were found to be effective antioxidant agents by sequestering mitochondrial radical oxygen species (ROS). Moreover, compound 2 showed a remarkable calcium-channel modulating activity. The interest of these compounds is increased by the fact that dibenzo[1,4,5]thiadiazepine is a barely known structure that is not difficult to synthesize and presents very few described derivatives, opening a new and broad line of research in Medicinal Chemistry.

ITH12410/SC058: a new neuroprotective compound with potential in the treatment of Alzheimer's disease.

The neuroprotective profile of the dibenzothiadiazepine ITH12410/SC058 (2-chloro-5,6-dihydro-5,6-diacetyldibenzo[b,f][1,4,5]thiadiazepine) against several neurotoxicity models related to neurodegenerative diseases is herein described. ITH12410/SC058 protected SH-SY5Y cells against the loss of cell viability elicited by amyloid beta peptide and okadaic acid, a selective inhibitor of phosphoprotein phosphatase 2A that induces neurofibrillary tangle formation. Furthermore, ITH12410/SC058 is neuroprotective against several in vitro models of oxidative stress, that is, H2O2 exposure or incubation with rotenone plus oligomycin A in SH-SY5Y cells, and oxygen and glucose deprivation followed by reoxygenation in rat hippocampal slices. By contrast, ITH12410/SC058 was unable to significantly protect SH-SY5Y neuroblastoma cells against the toxicity elicited by Ca(2+) overload. Our results confirm the hypothesis that the dibenzothiadiazepine ITH12410/SC058 features its neuroprotective actions in a multitarget fashion, and is a promising drug for the treatment of neurodegenerative diseases.

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.

Here, we describe a new family of melatonin-N,N-dibenzyl(N-methyl)amine hybrids that show a balanced multifunctional profile covering neurogenic, antioxidant, cholinergic, and neuroprotective properties at low-micromolar 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 ß-amyloid 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.