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N-Methyl-D-aspartate receptors (NMDARs) are crucial for the normal function of the central nervous system (CNS), and fundamental in memory and learning-related processes. The overactivation of these receptors is associated with numerous neurodegenerative and psychiatric disorders. Therefore, NMDAR is considered a relevant therapeutic target for many CNS disorders. Herein, we report the synthesis and pharmacological evaluation of a new scaffold with antagonistic activity for NMDAR. Specifically, a chemical library of eighteen 1-aminoindan-2-ol tetracyclic lactams was synthesized and screened as NMDAR antagonists. The compounds were obtained by chiral pool synthesis using enantiomerically pure 1-aminoindan-2-ols as chiral inductors, and their stereochemistry was proven by X-ray crystallographic analysis of two target compounds. Most compounds reveal NMDAR antagonism, and eleven compounds display IC values in a Ca entry-sensitive fluo-4 assay in the same order of magnitude of memantine, a clinically approved NMDAR antagonist. Docking studies suggest that the novel compounds can act as NMDAR channel blockers since there is a compatible conformation with MK-801 co-crystallized with NMDAR channel. In addition, we show that the tetracyclic 1-aminoindan-2-ol derivatives are brain permeable and non-toxic, and we identify promising hits for further optimization as modulators of the NMDAR function.

New multi-target indole and naphthalene derivatives containing the oxadiazolone scaffold as a bioisostere of the melatonin acetamido group have been developed. The novel compounds were characterized at melatonin receptors MTR and MTR, quinone reductase 2 (QR2), lipoxygenase-5 (LOX-5), and monoamine oxidases (MAO-A and MAO-B), and also as radical scavengers. We found that selectivity within the oxadiazolone series can be modulated by modifying the side chain functionality and co-planarity with the indole or naphthalene ring. In phenotypic assays, several oxadiazolone-based derivatives induced signalling mediated by the transcription factor NRF2 and promoted the maturation of neural stem-cells into a neuronal phenotype. Activation of NRF2 could be due to the binding of indole derivatives to KEAP1, as deduced from surface plasmon resonance (SPR) experiments. Molecular modelling studies using the crystal structures of QR2 and the KEAP1 Kelch-domain, as well as the recently described X-ray free-electron laser (XFEL) structures of chimeric MTR and MTR, provided a rationale for the experimental data and afforded valuable insights for future drug design endeavours.

Alzheimer´s disease (AD) is a complex multifactorial illness with no effective treatment, characterized by irreversible global cognitive impairment. As only symptomatic treatment sare available with drugs acting at one single target, exploration of molecules active indifferent pathological targets is required. In this chapter, our objective was to develop new families of multitarget directed ligands (MTDLs) focused on some pathological pathways underlying AD, namely, oxidative stress and neuro inflammation. Thus, we design melatonin- and resveratrol-based MTDLs looking for activity in melatonin receptors (MT1-3Rs), monoaminoxidases (MAO-A/B), lipoxygenase-5 (LOX-5), and nuclear factor erythroid 2-related factor 2 (Nrf2). Considering the neuroprotective and neurogenic properties found in melatonin and resveratrol, we reasonably expected these activities in our MTDLs..., La enfermedad de Alzheimer (EA) es un desorden multifactorial sin cura efectiva,caracterizada por un deterioro cognitivo global. Los tratamientos disponibles son sólo sintomáticos con fármacos que actúan sobre una sola diana, siendo necesarias moléculas activas por diferentes vías. En este capítulo, nuestro objetivo es desarrollar nuevas familias de ligandos multidiana (MTDL) enfocados hacia vías patológicas de la EA, concretamente, el estrés oxidativo y neuroinflamación. Así, desarrollamos MTDLs basados en melatoninay resveratrol buscando actividad en receptores de melatonina (MT1-3Rs), monoaminooxidasas (MAO-A/B), lipoxigenasa-5 (LOX-5) y el factor nuclear (erythroid-derived 2)-like 2 (Nrf2). Considerando las propiedades neuroprotectoras y neurogénicas de la melatonina y el resveratrol, esperamos razonablemente estas actividades en nuestros MTDLs...

The design of multi-target directed ligands (MTDLs) is a valid approach for obtaining effective drugs for complex pathologies. MTDLs that combine neuro-repair properties and block the first steps of neurotoxic cascades could be the so long wanted remedies to treat neurodegenerative diseases (NDs). By linking two privileged scaffolds with well-known activities in ND-targets, the flavonoid and the N,N-dibenzyl(N-methyl)- amine (DBMA) fragments, new CNS-permeable flavonoid – DBMA hybrids (1–13) were obtained. They were subjected to biological evaluation in a battery of targets involved in Alzheimer’s disease (AD) and other NDs, namely human cholinesterases (hAChE/hBuChE), b-secretase (hBACE-1), monoamine oxidases (hMAO-A/B), lipoxygenase-5 (hLOX-5) and sigma receptors (r1R/r2R). After a funnel-type screening, 6,7- dimethoxychromone – DBMA (6) was highlighted due to its neurogenic properties and an interesting MTD-profile in hAChE, hLOX-5, hBACE-1 and r1R. Molecular dynamic simulations showed the most relevant drug-protein interactions of hybrid 6, which could synergistically contribute to neuronal regeneration and block neurodegeneration.

This study has been developed within a multidisciplinary project designed to search for multitarget compounds with NRF2 inducing capacity, by inhibiting the Keap1-NRF2 interaction, for the treatment of Alzheimer¿s disease. We have focused on melatonin and resveratrol derivatives, which additionally induce NRF2 transcription factor, master regulator of oxidative stress. For this study, two melatonin (CH213 and CH507) and one resveratrol (PL119) derivatives, which were the ones that duplicated NRF2 expression at concentrations below 10 ¿M, were selected. We found that these compounds were not neurotoxic in the human neuroblastoma cell line SH-SY5Y at the concentration of 100 ¿M. Thereafter, neuroprotection in a tau hyperphosphorylation in vitro model induced by okadaic acid (OA) was studied; compounds CH507 and PLC119 presented significant neuroprotection, while CH213 did not. Nevertheless, neuroprotection of compound CH213 against OA was studied in primary neuronal cultures achieving significant results. Additionally, their potential anti neuroinflammatory effects were studied in primary rat glial cultures exposed to LPS; in this model, only compound CH213 reduced nitric oxide production and IL-1b. CH123 was further studied in an acute model of hippocampal slices, where toxicity was induced by incubation with OA for 6 h and in organotypic hippocampal cultures of 14 months old APPTau mice; in these models, CH213 was able to provide neuroprotection and reduce oxidative stress. Finally, since QR2, also known as MT3, is overexpressed in AD patients, we sought of interest to perform a molecular docking study to evaluate how the compounds could interact with this receptor. In conclusion, CH213 is a melatonin derivative that induces NRF2 with antineuroinflammatory, antioxidant and neuroprotective properties; however, future studies in in vivo AD models need to be conducted to validate the in vitro results here obtained.

Muscular nicotinic acetylcholine receptors (nAChR) are ligand-gated ion channels located in the plasma membrane of the post-junctional motor endplate in skeletal muscles. These receptors are responsible for triggering the electrical signal that leads to muscle contraction. Here, we have developed a family of photoisomerizable nAChR inhibitors, using the azobenzene scaffold. Biochemical assays showed that all these novel compounds have higher affinity for muscular nAChR (up to 60-fold) than for either ¿7 or ¿4ß2 nAChRs. Thus, we proceeded to characterize the action of these compounds on the activity of the embryonic muscular nAChR expressed in Xenopus oocytes. To do so, we recorded currents evoked by acetylcholine, using the Two-Electrode Voltage-Clamp technique. In doing so, we observed that all but one of the compounds were effective inhibitors of the receptor¿s activity. As reported elsewhere, azobenzene-based molecules can be as either cis- or trans-isomers, displaying remarkable difference in their pharmacology. To test whether the isomer had different inhibitory activity, we proceeded to irradiate the drugs with either near UV (335-365 nm) or blue light (400-450 nm) while recording currents. In all cases, we observed that the trans-isomers were the most potent form of these molecules. The main difference among the compounds of this family is the ¿molecular volume¿ of the functional group. The molecules were effective inhibitors of the receptor with nano- to micromolar activity, decreasing their potency as their size decrease. In fact, the trans-isomer of the smallest derivative reverting its behavior, acting as an agonist drug, with the cis-isomer being unable to activate the channels. In terms structure-activity relationship (SAR) parameters, these observations led us to infer the that decreasing the bulkiness of the molecule is a critical determinant of the character and potency of these novel azobenzene-based inhibitors.

Alzheimer¿s Disease (AD) is a complex multifactorial illness with no effective cure, characterized by the irreversible memory loss and global cognitive impairment. Given that the marketed drugs act only at one single target, being a symptomatic treatment, exploration of new molecules acting in different pathways is required. In this sense, we have developed a large family of multitarget directed ligands (MTDL), which interacts in different targets related to neurodegeneration, such as the nuclear factor (erythroid-derived 2)-like 2 (Nrf2), lipoxygenase-5 (LOX-5), melatonin receptors (MT1, MT2, and MT3), and monoamine oxidases (MAO-A and MAO-B). The affinity or inhibition constants of these compounds in such targets are in the nanomolar and micromolar ranges. In general, these molecules have potent antioxidant properties (ORAC assay) and are able to penetrate into the central nervous system (CNS) by passive diffusion (PAMPA-BBB assay). Moreover, some of them are able to promote neurogenesis in vitro by inducing the maturation of neural stem cells into a neuronal phenotype. A structure-activity relationship has been stablished, reaching an optimized structure as possible candidate to treat AD, able to cross the blood brain barrier, reaching the CNS where it would act in three targets involved in the regulation of oxidative stress, as Nrf2 inducer, MT3 ligand and selective MAO B inhibitor. Furthermore, this MTDL is an effective neuroprotective and neurogenic agent.

In recent years the nuclear transcription factor (erythroid-derived 2)¿like 2 (Nrf2) has been identified as the key factor in the cell auto-protection mechanisms. Under no pathological conditions, Nrf2 is mainly located in the cytosol, bound to the Kelch like ECH associated protein 1 (Keap1). In contrast, under oxidative conditions, Keap1 changes its conformation and breaks the binding with Nrf2 allowing its accumulation and translocation to the nucleus where it induces the transcription of key antioxidant enzymes. Nrf2 dysregulation is involved in different pathologies such as cancer, cardiovascular diseases or neurodegenerative diseases, among others [1]. In recent years, a great amount of new molecules able to disrupt the interaction between Nrf2 and Keap1, have been studied, however most of them are electrophilic molecules and are prone to interact with several non-desired targets. In our group we are applying the surface plasmon resonance (SPR) technique for the identification of new non-electrophilic inhibitors of the Nrf2 - Keap1 protein-protein interaction (PPI). This kind of molecules could exhibit a more specific mechanism for the induction of Nrf2 activity. Thus, the ability of several molecules from our library to interact with the purified Kelch domain of Keap1 attached to a CM5 sensor chip has been evaluated in a SPR assay [2]. With this technique, a new hit with affinity for Keap1 was identified. The affinity value was comparable to that of the positive control ML-334, a well-known inhibitor of the Nrf2 - Keap1 PPI. Starting from this new hit, a larger family with improved potency have been obtained [3]. This new family of bis-heterocyclic compounds do not present any electrophilic character, are able to reach the central nervous system and exhibit other complementary activities such as radical scavenging and neurogenic properties, which make them potential candidates for the treatment of neurodegenerative diseases such as Alzheimer¿s disease.

In this work we describe neurogenic and neuroprotective donepezil-flavonoid hybrids (DFHs), exhibiting nanomolar affinities for the sigma-1 receptor and inhibition of key enzymes in Alzheimer's disease (AD), such as acetylcholinesterase (AChE), 5-lipoxygenase (5-LOX), and monoamine oxidases (MAOs). In general, new compounds scavenge free radical species, are predicted to be brain-permeable, and protect neuronal cells against mitochondrial oxidative stress. N-(2-(1-Benzylpiperidin-4-yl)ethyl)-6,7-dimethoxy-4-oxo-4H-chromene-2-carboxamide (18) is highlighted due to its interesting biological profile in sigma1R, AChE, 5-LOX, MAO-A and MAO-B. In phenotypic assays, it protects a neuronal cell line against mitochondrial oxidative stress and promotes maturation of neural stem cells into a neuronal phenotype, which could contribute to the reparation of neuronal tissues. Molecular modelling studies of 18 in AChE, 5-LOX and sigma-1R revealed the main interactions with these proteins, which will be further exploited in the optimization of new, more efficient DFHs.

The highly complex pathophysiology of Alzheimer's disease (AD) and other neurodegenerative illnesses have led to replace the traditional one-drug - one-target by the multi-target-directed ligands (MTDLs) paradigm, in which a single molecule is designed to be active against several pharmacological targets. Continuing with our interest in neuroprotective and neurogenic compounds, in this work we describe a new family of donepezil flavonoid hybrids exhibiting nanomolar affinities for the sigma-1 receptor and a combined inhibition of key enzymes in AD, such as 5-lipoxygenase, acetylcholinesterase, and monoaminoxidases. In general, they scavenge free radical species and are predicted to be brain-permeable. In phenotypic assays, new hybrids protect neuronal cells against mitochondrial oxidative stress and promote maturation of neural stem cells into a neuronal phenotype. Therefore, new donepezil - flavonoid hybrids could contribute to the protection and even, the reparation of neuronal tissues, of great therapeutic interest in AD and neurodegenerative diseases.

N-Methyl-d-aspartate (NMDA) receptors are fundamental for the normal function of the central nervous system (CNS), and play an important role in memory and learning. Over-activation of these receptors leads to neuronal loss associated with major neurological disorders such as Parkinson's disease, Alzheimer's disease, schizophrenia, and epilepsy. In this study, 22 novel enantiopure bicyclic lactams were designed, synthesized, and evaluated as NMDA receptor antagonists. Most of the new compounds displayed NMDA receptor antagonism, and the most promising compound showed an IC value on the same order of magnitude as that of memantine, an NMDA receptor antagonist in clinical use for the treatment of Alzheimer's disease. Further biological evaluation indicated that this compound is brain permeable (determined by an in vitro assay) and non-hepatotoxic. All these results indicate that (3S,7aS)-7a-(4-chlorophenyl)-3-(4-hydroxybenzyl)tetrahydropyrrolo[2,1-b]oxazol-5(6H)-one (compound 5 b) is a potential candidate for the treatment of pathologies associated with the over-activation of NMDA receptors.

Here we describe new families of multi-target directed ligands obtained by linking antioxidant cinnamic-related structures with N-benzylpiperidine (NBP) or N,N-dibenzyl(N-methyl)amine (DBMA) fragments. Resulting hybrids, in addition to their antioxidant and neuroprotective properties against mitochondrial oxidative stress, are active at relevant molecular targets in Alzheimer’s disease, such as cholinesterases (hAChE and hBuChE) and monoamine oxidases (hMAO-A and hMAO-B). Hybrids derived from umbellic – NBP (8), caffeic – NBP (9), and ferulic – DBMA (12) displayed balanced biological profiles, with IC50s in the low-micromolar and submicromolar range for hChEs and hMAOs, and an antioxidant potency comparable to vitamin E. Moreover, the caffeic – NBP hybrid 9 is able to improve the differentiation of adult SGZ-derived neural stem cells into a neuronal phenotype in vitro.

The design and synthesis of a series of new fluorescent coumarin-containing melatonin analogues is presented. The combination of high-binding affinities for human melatonergic receptors (h-MT1R and h-MT2R) and fluorescent properties, derived from the inclusion of melatonin pharmacophoric elements in the coumarin scaffold, yielded suitable candidates for the development of MT1R and MT2R fluorescent probes for imaging in biological media.

Herein we present a new family of melatonin-based compounds, in which the acetamido group of melatonin has been bioisosterically replaced by a series of reversed amides and azoles, such as oxazole, 1,2,4-oxadiazole, and 1,3,4-oxadiazole, as well as other related five-membered heterocycles, namely, 1,3,4-oxadiazol(thio)ones, 1,3,4-triazol(thio)ones, and an 1,3,4-thiadiazole. New compounds were fully characterized at melatonin receptors (MT1R and MT2R), and results were rationalized by superimposition studies of their structures to the bioactive conformation of melatonin. We also found that several of these melatonin-based compounds promoted differentiation of rat neural stem cells to a neuronal phenotype in vitro, in some cases to a higher extent than melatonin. This unique profile constitutes the starting point for further pharmacological studies to assess the mechanistic pathways and the relevance of neurogenesis induced by melatonin-related structures.