Cs2CO3-Promoted Alkylation of 3-Cyano-2(1H)-Pyridones: Anticancer Evaluation and Molecular Docking.

Herein, a Cs2CO3-promoted N-alkylation of 3-cyano-2(1H)-pyridones containing alkyl groups with diverse alkyl halides to synthesize N-alkyl-2-pyridones over O-alkylpyridines is reported. The use of alkyl dihalides resulted in complex mixtures of N- and O-alkylated products. The primary factor influencing regioselectivity in these reactions is the electronic effects of substituents on the 2(1H)-pyridone ring, as evidenced by the preferential formation of O-alkylpyridines upon the introduction of aryl groups. Remarkably, we efficiently employed CuAAC and Ti(Oi-Pr)4-catalyzed amidation reactions to functionalize N-alkyl-2-pyridones containing propargyl and ester groups, leading to the synthesis of 1,2,3-triazoles and amides, respectively. Moreover, O-alkylpyridines 10 b and 10 d displayed remarkable selectivity toward the A-498 renal cancer cell line with growth inhibition percentages (%GI) of 54.75 and 67.64, respectively. The binding modes of compounds 10 b and 10 d to the PIM-1 kinase enzyme were determined through molecular docking studies.

Synthesis and Antioxidant Properties of HeteroBisNitrones Derived from Benzene Dicarbaldehydes.

We report herein the synthesis and antioxidant profile of nine novel heterobisnitrones (hBNs) as new α-phenyl-tert-butylnitrone (PBN) analogues. The synthesized hBNs 1-9 were evaluated for their antioxidant activity using different in vitro techniques, while they were also tested as inhibitors of soybean LOX, as an indication of their anti-inflammatory effect. Nitrone hBN9 is the most potent antioxidant presenting higher anti-lipid peroxidation and hydroxyl radicals scavenging activities as well as higher lipoxygenase inhibition. In silico calculations reveal that hBN9 follows Lipinski's rule of five and that the molecule is able to penetrate theoretically the brain. All these results led us to propose hBN9 as a new potent antioxidant nitrone.

Tacrines as Therapeutic Agents for Alzheimer's Disease. V. Recent Developments.

Herein we have reviewed our recent developments for the identification of new tacrine analogues for Alzheimer's disease (AD) therapy. Tacrine, the first cholinesterase inhibitor approved for AD treatment, did not stop the progression of AD, producing only some cognitive improvements, but exhibited secondary effects mainly due to its hepatotoxicity. Thus, the drug was withdrawn from the clinics administration. Since then, many publications have described non-hepatotoxic tacrines, and in addition, important efforts have been made to design multitarget tacrines by combining their cholinesterase inhibition profile with the modulation of other biological targets involved in AD.

Synthesis of Hantzsch Adducts as Cholinesterases and Calcium Flux inhibitors, Antioxidants and Neuroprotectives.

We report herein the design, synthesis, biological evaluation, and molecular modelling of new inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), able to block Ca+2 channels also showing antioxidant and neuroprotective activities. The new MTDL, dialkyl 2,6-dimethyl-4-(4-((5-aminoalkyl)oxy)phenyl)-1,4-dihydropyridine-3,5-dicarboxylate 3a-p, have been obtained via Hantzsch reaction from appropriate and commercially available precursors. Pertinent biological analysis has prompted us to identify MTDL 3h [dimethyl-4-(4-((5-(4-benzylpiperidin-1-yl)pentyl)oxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate] as an attractive inhibitor of AChE (1.8 µM) and BuChE (2 µM), Ca+2 channel antagonist (47.72% at 10 µM), and antioxidant (2.54 TE) agent, showing significant neuroprotection 28.68% and 38.29% against H2O2, and O/R, respectively, at 0.3 µM, thus being considered a hit-compound for further investigation in our search for anti-Alzheimer's disease agents.

Synthesis, antioxidant properties and neuroprotection of α-phenyl-tert-butylnitrone derived HomoBisNitrones in in vitro and in vivo ischemia models.

We herein report the synthesis, antioxidant power and neuroprotective properties of nine homo-bis-nitrones HBNs 1-9 as alpha-phenyl-N-tert-butylnitrone (PBN) analogues for stroke therapy. In vitro neuroprotection studies of HBNs 1-9 against Oligomycin A/Rotenone and in an oxygen-glucose-deprivation model of ischemia in human neuroblastoma cell cultures, indicate that (1Z,1'Z)-1,1'-(1,3-phenylene)bis(N-benzylmethanimine oxide) (HBN6) is a potent neuroprotective agent that prevents the decrease in neuronal metabolic activity (EC50 = 1.24 ± 0.39 µM) as well as necrotic and apoptotic cell death. HBN6 shows strong hydroxyl radical scavenger power (81%), and capacity to decrease superoxide production in human neuroblastoma cell cultures (maximal activity = 95.8 ± 3.6%), values significantly superior to the neuroprotective and antioxidant properties of the parent PBN. The higher neuroprotective ability of HBN6 has been rationalized by means of Density Functional Theory calculations. Calculated physicochemical and ADME properties confirmed HBN6 as a hit-agent showing suitable drug-like properties. Finally, the contribution of HBN6 to brain damage prevention was confirmed in a permanent MCAO setting by assessing infarct volume outcome 48 h after stroke in drug administered experimental animals, which provides evidence of a significant reduction of the brain lesion size and strongly suggests that HBN6 is a potential neuroprotective agent against stroke.

New Dual Small Molecules for Alzheimer's Disease Therapy Combining Histamine H3 Receptor (H3R) Antagonism and Calcium Channels Blockade with Additional Cholinesterase Inhibition.

New tritarget small molecules combining Ca2+ channels blockade, cholinesterase, and H3 receptor inhibition were obtained by multicomponent synthesis. Compound 3p has been identified as a very promising lead, showing good Ca2+ channels blockade activity (IC50 = 21 ± 1 µM), potent affinity against hH3R (Ki = 565 ± 62 nM), a moderate but selective hBuChE inhibition (IC50 = 7.83 ± 0.10 µM), strong antioxidant power (3.6 TE), and ability to restore cognitive impairment induced by lipopolysaccharide.

Multi-target 1,4-dihydropyridines showing calcium channel blockade and antioxidant capacity for Alzheimer's disease therapy.

In this work we describe the synthesis, Ca+2 channel blockade capacity and antioxidant power of N3,N5-bis(2-(5-methoxy-1H-indol-3-yl)ethyl)-2,6-dimethyl-4-aryl-1,4-dihydropyridine-3,5-dicarboxamides 1-9, a number of multi-target small 1,4-dihydropyridines (DHP), designed by juxtaposition of melatonin and nimodipine. As a result, we have identified antioxidant DHP 7 (Ca2+ channel blockade: 55%, and 8.78 Trolox/Equivalents), the most balanced DHP analyzed here, for potential Alzheimer's disease therapy.

Discovery of novel benzofuran-based compounds with neuroprotective and immunomodulatory properties for Alzheimer's disease treatment.

To address the multifactorial nature of Alzheimer's Disease (AD), a multi-target-directed ligand approach was herein developed. As a follow-up of our previous studies, a small library of newly designed 2-arylbenzofuran derivatives was evaluated towards cholinesterases and cannabinoid receptors. The two most promising compounds, 8 and 10, were then assessed for their neuroprotective activity and for their ability to modulate the microglial phenotype. Compound 8 emerged as able to fight AD from several directions: it restored the cholinergic system by inhibiting butyrylcholinesterase, showed neuroprotective activity against Aß1-42 oligomers, was a potent and selective CB2 ligand and had immunomodulatory effects, switching microglia from the pro-inflammatory M1 to the neuroprotective M2 phenotype. Derivative 10 was a potent CB2 inverse agonist with promising immunomodulatory properties and could be considered as a tool for investigating the role of CB2 receptors and for developing potential immunomodulating drugs addressing the endocannabinoid system.

QuinoxalineTacrine QT78, a Cholinesterase Inhibitor as a Potential Ligand for Alzheimer's Disease Therapy.

We report the synthesis and relevant pharmacological properties of the quinoxalinetacrine (QT) hybrid QT78 in a project targeted to identify new non-hepatotoxic tacrine derivatives for Alzheimer's disease therapy. We have found that QT78 is less toxic than tacrine at high concentrations (from 100 µM to 1 mM), less potent than tacrine as a ChE inhibitor, but shows selective BuChE inhibition (IC50 (hAChE) = 22.0 ± 1.3 µM; IC50 (hBuChE) = 6.79 ± 0.33 µM). Moreover, QT78 showed effective and strong neuroprotection against diverse toxic stimuli, such as rotenone plus oligomycin-A or okadaic acid, of biological significance for Alzheimer's disease.

Tackling neuroinflammation and cholinergic deficit in Alzheimer's disease: Multi-target inhibitors of cholinesterases, cyclooxygenase-2 and 15-lipoxygenase.

Neuroinflammation and cholinergic deficit are key detrimental processes involved in Alzheimer's disease. Hence, in the search for novel and effective treatment strategies, the multi-target-directed ligand paradigm was applied to the rational design of two series of new hybrids endowed with anti-inflammatory and anticholinesterase activity via triple targeting properties, namely able to simultaneously hit cholinesterases, cyclooxygenase-2 (COX-2) and 15-lipoxygenase (15-LOX) enzymes. Among the synthesized compounds, triazoles 5b and 5d, and thiosemicarbazide hybrid 6e emerged as promising new hits, being able to effectively inhibit human butyrylcholinesterase (hBChE), COX-2 and 15-LOX enzymes with a higher inhibitory potency than the reference inhibitors tacrine (for hBChE inhibition), celecoxib (for COX-2 inhibition) and both NDGA and Zileuton (for 15-LOX inhibition). In addition, compound 6e proved to be a submicromolar mixed-type inhibitor of human acetylcholinesterase (hAChE). The anti-neuroinflammatory activity of the three most promising hybrids was confirmed in a cell-based assay using PC12 neuron cells, showing decreased expression levels of inflammatory cytokines IL-1ß and TNF-α. Importantly, despite the structural resemblance to tacrine, they showed ideal safety profiles on hepatic and murine brain cell lines and were safe up to 100 µM when assayed in PC12 cells. All three hybrids were also predicted to have superior BBB permeability than tacrine in the PAMPA assay, and good physicochemical properties, drug-likeness and ligand efficiency indices. Finally, molecular docking studies highlighted key structural elements impacting selectivity and activity toward the selected target enzymes. To the best of our knowledge, compounds 5b, 5d and 6e are the first balanced, safe and multi-target compounds hitting the disease at the three mentioned hubs.

Synthesis, neuroprotective and antioxidant capacity of PBN-related indanonitrones.

In this work six PBN-related indanonitrones 1-6 have been designed, synthesized, and their neuroprotection capacity tested in vitro, under OGD conditions, in SH-SY5Y human neuroblastoma cell cultures. As a result, we have identified indanonitrones 1, 3 and 4 (EC50 = 6.64 ±â€¯0.28 µM) as the most neuroprotective agents, and in particular, among them, indanonitrone 4 was also the most potent and balanced nitrone, showing antioxidant activity in three experiments [LOX (100 µM), APPH (51%), DPPH (36.5%)], being clearly more potent antioxidant agent than nitrone PBN. Consequently, we have identified (Z)-5-hydroxy-N-methyl-2,3-dihydro-1H-inden-1-imine oxide (4) as a hit-molecule for further investigation.

Exploiting the Chalcone Scaffold to Develop Multifunctional Agents for Alzheimer's Disease.

Alzheimer's disease still represents an untreated multifaceted pathology, and drugs able to stop or reverse its progression are urgently needed. In this paper, a series of naturally inspired chalcone-based derivatives were designed as structural simplification of our previously reported benzofuran lead compound, aiming at targeting both acetyl (AChE)- and butyryl (BuChE) cholinesterases that, despite having been studied for years, still deserve considerable attention. In addition, the new compounds could also modulate different pathways involved in disease progression, due to the peculiar trans-α,ß-unsaturated ketone in the chalcone framework. All molecules presented in this study were evaluated for cholinesterase inhibition on the human enzymes and for antioxidant and neuroprotective activities on a SH-SY5Y cell line. The results proved that almost all the new compounds were low micromolar inhibitors, showing different selectivity depending on the appended substituent; some of them were also effective antioxidant and neuroprotective agents. In particular, compound 4, endowed with dual AChE/BuChE inhibitory activity, was able to decrease ROS formation and increase GSH levels, resulting in enhanced antioxidant endogenous defense. Moreover, this compound also proved to counteract the neurotoxicity elicited by Aß1⁻42 oligomers, showing a promising neuroprotective potential.

Tacripyrimidines, the first tacrine-dihydropyrimidine hybrids, as multi-target-directed ligands for Alzheimer's disease.

Notwithstanding the combination of cholinesterase (ChE) inhibition and calcium channel blockade within a multitarget therapeutic approach is envisaged as potentially beneficial to confront Alzheimer's disease (AD), this strategy has been scarcely investigated. To explore this promising line, a series of 5-amino-4-aryl-3,4,6,7,8,9-hexahydropyrimido [4,5-b]quinoline-2(1H)-thiones (tacripyrimidines) (4a-l) were designed by juxtaposition of tacrine, a ChE inhibitor (ChEI), and 3,4-dihydropyrimidin-2(1H)-thiones, as efficient calcium channel blockers (CCBs). In agreement with their design, all tacripyrimidines, except the unsubstituted parent compound and its p-methoxy derivative, acted as moderate to potent CCBs with activities generally similar or higher than the reference CCB drug nimodipine and were modest-to-good ChEIs. Most interestingly, the 3'-methoxy derivative (4e) emerged as the first well balanced ChEI/CCB agent, acting as low micromolar hChEI (3.05 µM and 3.19 µM on hAChE and hBuChE, respectively) and moderate CCB (30.4% at 1 µM) with no significant hepatotoxicity toward HepG2 cells and good predicted oral absorption and blood brain barrier permeability.

Synthesis and biological assessment of racemic benzochromenopyrimidinetriones as promising agents for Alzheimer's disease therapy.

AIM: Due to the complex nature of Alzheimer's disease, there is a renewed search for multitarget directed drugs. RESULTS: This paper describes the synthesis and in vitro biological evaluation of six racemic 13-aryl-2,3,4,13-tetrahydro-1H,12H-benzo[6,7]chromeno[2,3-d]pyrido[1,2-a]pyrimidine-7,12,14-triones (1a-6a), and six racemic 15-aryl-8,9,10,11,12,15-hexahydro-14H-benzo[6',7']chromeno[2',3:4,5] pyr-imido [1,2-a]azepine-5,14,16-triones (1b-6b), showing antioxidant and cholinesterase inhibitory capacity. Among these compounds, 13-phenyl-2,3,4,13-tetrahydro-1H,12H-benzo[6,7]chromeno[2,3-d]pyrido[1,2-a]pyrimidine-7,12,14-trione (1a) is a nonhepatotoxic at 300 µmol/l dose concentration, and a selective EeAChE inhibitor showing good antioxidant power. CONCLUSION: A new family of racemic benzochromenopyrimidinetriones has been investigated for their potential use in the treatment of Alzheimer's disease.

Poly(amidoamine) dendrimers grafted on electrospun poly(acrylic acid)/poly(vinyl alcohol) membranes for host-guest encapsulation of antioxidant thymol.

Amino-terminated fifth generation poly(amidoamine) (PAMAM G5-NH2) dendrimers were grafted onto the surface of poly(acrylic acid)/poly(vinyl alcohol) (PAA/PVA) electrospun fibres with the purpose of creating a host-guest architecture for the controlled delivery of a natural antioxidant, thymol. The nanofibers were stabilized by esterification crosslinking to produce a water insoluble non-woven membrane. The functionalization with PAMAM G5-NH2 led to dendrimer loadings in the 7.4 × 10-7-2.25 × 10-6 mol dendrimer per g membrane range. The resulting materials were characterized using SEM, ATR-FTIR and surface ζ-potential measurements. The loading capacity for thymol reached 2.5 × 10-4 mol thymol per g membrane. The membranes were tested for thymol release in different aqueous and non-aqueous food simulants. Computational modelling was used to get a further insight into the host-guest association of thymol and PAMAM G5-NH2 molecules through docking studies. For this purpose, we examined the molecular level details of the dendrimer-guest complex, calculated the number of included or attached molecules, the exact location of thymol in host-guest complexes and the local environment around the thymol molecules. Docking studies showed that PAMAM-G5-NH2 dendrimers can encapsulate thymol molecules through hydrophobic interactions and hydrogen bonding. The maximum amount of thymol molecules theoretically encapsulated was 16, while another 25 could be hosted at the dendrimer surface through interaction with the outer part or the dendritic branches. The experimental value was 37 ± 5, in agreement with theoretical predictions.

Potent anticholinesterasic and neuroprotective pyranotacrines as inhibitors of beta-amyloid aggregation, oxidative stress and tau-phosphorylation for Alzheimer's disease.

Herein we describe the synthesis and in vitro biological evaluation of thirteen new, racemic, diversely functionalized 2-chloroquinolin-3-yl substituted PyranoTacrines (PTs) as multipotent tacrine analogues for Alzheimer's disease (AD) therapy. Among these compounds, 1-(5-amino-4-(2-chloro-7-methoxyquinolin-3-yl)-2-methyl-6,7,8,9-tetrahydro-4H-pyrano [2,3-b]quinolin-3-yl)éthanone (9) and ethyl 5-amino-4-(2-chloroquinolin-3-yl)-2-methyl-6,7,8,9-tetrahydro-4H-pyrano[2,3-b]quinoline-3-carboxylate (4) were found to be non-neurotoxic agents in human neuroblastoma SHSY5Y cells. Compounds 9 (IC50 = 0.47 ± 0.13 µM) and 4 (IC50 = 0.48 ± 0.05 µM) are potent, mixed-type (9: Ki = 0.0142 ± 0.003 µM), and selective EeAChE inhibitors, binding at the both catalytic and peripheral anionic site of the enzyme. Compounds 9 and 4 are neuroprotective agents at low µM concentrations upon decreased viability of SHSY5Y cells induced by oxidative stress, and stimulators of GSK3ß-dependent tau phosphorylation. In addition, molecules 9 and 4 effectively counteract Aß-aggregation on exposure to Aß1-40, as well as Aß1-40 aggregation-dependent tau-oligomerization and phosphorylation in (396)Ser, which could be ascribed to the anti-aggregating properties shown in vitro. Thus, a new family of tacrine analogues, whose potent AChEI activity is linked to both their Aß-aggregating and tau-phosphorylation inhibitory capacities, has been discovered for the potential treatment of AD.

Imidazopyranotacrines as Non-Hepatotoxic, Selective Acetylcholinesterase Inhibitors, and Antioxidant Agents for Alzheimer's Disease Therapy.

Herein we describe the synthesis and in vitro biological evaluation of thirteen new, racemic, diversely functionalized imidazo pyranotacrines as non-hepatotoxic, multipotent tacrine analogues. Among these compounds, 1-(5-amino-2-methyl-4-(1-methyl-1H-imidazol-2-yl)-6,7,8,9-tetrahydro-4H-pyrano[2,3-b]quinolin-3-yl)ethan-1-one (4) is non-hepatotoxic (cell viability assay on HepG2 cells), a selective but moderately potent EeAChE inhibitor (IC50 = 38.7 ± 1.7 µM), and a very potent antioxidant agent on the basis of the ORAC test (2.31 ± 0.29 µmol·Trolox/µmol compound).

Pyranopyrazolotacrines as nonneurotoxic, Aß-anti-aggregating and neuroprotective agents for Alzheimer's disease.

AIM: Due to the complex nature of Alzheimer's disease, there is a renewed search for multipotent, nonhepatotoxic tacrines. RESULTS: This paper describes the synthesis and in vitro biological evaluation of eight new racemic 3-methyl-4-aryl-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-amines (pyranopyrazolotacrines, PPT) as nonhepatotoxic multipotent tacrine analogs. Among these compounds, PPT4 is the less hepatotoxic in the cell viability assay on HepG2 cells, showing a good neuroprotective effect in the decreased cortical neuron viability induced by oligomycin A/rotenone analysis. PPT4 is a selective and good, noncompetitive EeAChE inhibitor, able to completely inhibit the Aß1-40 aggregation induced by acetylcholinesterase. CONCLUSION: A new family of nonhepatotoxic showing selective acetylcholinesterase inhibition, permeable tacrine analogs have been discovered for the potential treatment of Alzheimer's disease.

Structural basis of drugs that increase cardiac inward rectifier Kir2.1 currents.

AIMS: We hypothesize that some drugs, besides flecainide, increase the inward rectifier current (IK1) generated by Kir2.1 homotetramers (IKir2.1) and thus, exhibit pro- and/or antiarrhythmic effects particularly at the ventricular level. To test this hypothesis, we analysed the effects of propafenone, atenolol, dronedarone, and timolol on Kir2.x channels. METHODS AND RESULTS: Currents were recorded with the patch-clamp technique using whole-cell, inside-out, and cell-attached configurations. Propafenone (0.1 nM-1 µM) did not modify either IK1 recorded in human right atrial myocytes or the current generated by homo- or heterotetramers of Kir2.2 and 2.3 channels recorded in transiently transfected Chinese hamster ovary cells. On the other hand, propafenone increased IKir2.1 (EC50 = 12.0 ± 3.0 nM) as a consequence of its interaction with Cys311, an effect which decreased inward rectification of the current. Propafenone significantly increased mean open time and opening frequency at all the voltages tested, resulting in a significant increase of the mean open probability of the channel. Timolol, which interacted with Cys311, was also able to increase IKir2.1. On the contrary, neither atenolol nor dronedarone modified IKir2.1. Molecular modelling of the Kir2.1-drugs interaction allowed identification of the pharmacophore of drugs that increase IKir2.1. CONCLUSIONS: Kir2.1 channels exhibit a binding site determined by Cys311 that is responsible for drug-induced IKir2.1 increase. Drug binding decreases channel affinity for polyamines and current rectification, and can be a mechanism of drug-induced pro- and antiarrhythmic effects not considered until now.

Donepezil + propargylamine + 8-hydroxyquinoline hybrids as new multifunctional metal-chelators, ChE and MAO inhibitors for the potential treatment of Alzheimer's disease.

The synthesis, biochemical evaluation, ADMET, toxicity and molecular modeling of novel multi-target-directed Donepezil + Propargylamine + 8-Hydroxyquinoline (DPH) hybrids 1-7 for the potential prevention and treatment of Alzheimer's disease is described. The most interesting derivative was racemic α-aminotrile4-(1-benzylpiperidin-4-yl)-2-(((8-hydroxyquinolin-5-yl)methyl)(prop-2-yn-1-yl)amino) butanenitrile (DPH6) [MAO A (IC50 = 6.2 ± 0.7 µM; MAO B (IC50 = 10.2 ± 0.9 µM); AChE (IC50 = 1.8 ± 0.1 µM); BuChE (IC50 = 1.6 ± 0.25 µM)], an irreversible MAO A/B inhibitor and mixed-type AChE inhibitor with metal-chelating properties. According to docking studies, both DPH6 enantiomers interact simultaneously with the catalytic and peripheral site of EeAChE through a linker of appropriate length, supporting the observed mixed-type AChE inhibition. Both enantiomers exhibited a relatively similar position of both hydroxyquinoline and benzyl moieties with the rest of the molecule easily accommodated in the relatively large cavity of MAO A. For MAO B, the quinoline system was hosted at the cavity entrance whereas for MAO A this system occupied the substrate cavity. In this disposition the quinoline moiety interacted directly with the FAD aromatic ring. Very similar binding affinity values were also observed for both enantiomers with ChE and MAO enzymes. DPH derivatives exhibited moderate to good ADMET properties and brain penetration capacity for CNS activity. DPH6 was less toxic than donepezil at high concentrations; while at low concentrations both displayed a similar cell viability profile. Finally, in a passive avoidance task, the antiamnesic effect of DPH6 was tested on mice with experimentally induced amnesia. DPH6 was capable to significantly decrease scopolamine-induced learning deficits in healthy adult mice.