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Aims: Alzheimer's disease (AD) is a devastating dementia characterized by extracellular amyloid-β (Aβ) protein aggregates and intracellular tau protein deposition. Clinically available drugs mainly target acetylcholinesterase (AChE) and indirectly sustain cholinergic neuronal tonus. Butyrylcholinesterase (BChE) also controls acetylcholine (ACh) turnover and is involved in the formation of Aß aggregates and senile plaques. UW-MD-95 is a novel carbamate-based compound acting as a potent pseudo-irreversible BChE inhibitor, with high selectivity versus AChE, and showing promising protective potentials in AD., Methods: We characterized the neuroprotective activity of UW-MD-95 in mice treated intracerebroventricularly with oligomerized Aβ 25-35 peptide using behavioral, biochemical, and immunohistochemical approaches., Results: When injected acutely 30 min before the behavioral tests (spontaneous alternation in the Y-maze, object recognition, or passive avoidance), UW-MD-95 (0.3-3 mg/kg) showed anti-amnesic effects in Aβ 25-35 -treated mice. When injected once a day over 7 days, it prevented Aβ 25-35 -induced memory deficits. This effect was lost in BChE knockout mice. Moreover, the compound prevented Aβ 25-35 -induced oxidative stress (assessed by lipid peroxidation or cytochrome c release), neuroinflammation (IL-6 and TNFα levels or GFAP and IBA1 immunoreactivity) in the hippocampus and cortex, and apoptosis (Bax level). Moreover, UW-MD-95 significantly reduced the increase in soluble Aβ 1-42 level in the hippocampus induced by Aβ 25-35 ., Conclusion: UW-MD-95 appeared as a potent neuroprotective compound in the Aβ 25-35 model of AD, with potentially an impact on Aβ 1-42 accumulation that could suggest a novel mechanism of neuroprotection., (© 2024 The Author(s). CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

We present the synthesis and characterization of merged human butyrylcholinesterase ( h BChE) inhibitor/cannabinoid receptor 2 ( h CB 2 R) ligands for the treatment of neurodegeneration. In total, 15 benzimidazole carbamates were synthesized and tested for their inhibition of human cholinesterases, also with regard to their pseudoirreversible binding mode and affinity toward both cannabinoid receptors in radioligand binding studies. After evaluation in a calcium mobilization assay as well as a β-arrestin 2 (βarr2) recruitment assay, two compounds with balanced activities on both targets were tested for their immunomodulatory effect on microglia activation and regarding their pharmacokinetic properties and blood-brain barrier penetration. Compound 15d , containing a dimethyl carbamate motif, was further evaluated in vivo, showing prevention of Aβ 25-35 -induced learning impairments in a pharmacological mouse model of Alzheimer's disease for both short- and long-term memory responses. Additional combination studies proved a synergic effect of BChE inhibition and CB 2 R activation in vivo.

Flavonoids are polyphenolic natural products and have shown significant potential as disease-modifying agents against neurodegenerative disorders like Alzheimer's disease (AD), with activities even in vivo. Hybridization of the natural products taxifolin and silibinin with cinnamic acid led to an overadditive effect of these compounds in several phenotypic screening assays related to neurodegeneration and AD. Therefore, we have exchanged the flavonoid part of the hybrids with different flavonoids, which show higher efficacy than taxifolin or silibinin, to improve the activity of the respective hybrids. Chemical connection between the flavonoid and cinnamic acid was realized by an amide instead of a labile ester bond to improve stability towards hydrolysis. To investigate the influence of a double bond at the C-ring of the flavonoid, the dehydro analogues of the respective hybrids were also synthesized. All compounds obtained show neuroprotection against oxytosis, ferroptosis and ATP-depletion, respectively, in the murine hippocampal cell line HT22. Interestingly, the taxifolin and the quercetin derivatives are the most active compounds, whereby the quercetin derivate shows even more pronounced activity than the taxifolin one in all assays applied. As aimed for, no hydrolysis product was found in cellular uptake experiments after 4 h whereas different metabolites were detected. Furthermore, the quercetin-cinnamic acid amide showed pronounced activity in an in vivo AD mouse model at a remarkably low dose of 0.3 mg/kg., (© 2022 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

To develop tools to investigate the biological functions of butyrylcholinesterase (BChE) and the mechanisms by which BChE affects Alzheimer's disease (AD), we synthesized several selective, nanomolar active, pseudoirreversible photoswitchable BChE inhibitors. The compounds were able to specifically influence different kinetic parameters of the inhibition process by light. For one compound, a 10-fold difference in the IC 50 -values (44.6 nM cis , 424 nM trans ) in vitro was translated to an "all or nothing" response with complete recovery in a murine cognition-deficit AD model at dosages as low as 0.3 mg/kg.

Starting from six potential hits identified in a virtual screening campaign directed to a cryptic pocket of BACE-1, at the edge of the catalytic cleft, we have synthesized and evaluated six hybrid compounds, designed to simultaneously reach BACE-1 secondary and catalytic sites and to exert additional activities of interest for Alzheimer's disease (AD). We have identified a lead compound with potent in vitro activity towards human BACE-1 and cholinesterases, moderate Aβ42 and tau antiaggregating activity, and brain permeability, which is nontoxic in neuronal cells and zebrafish embryos at concentrations above those required for the in vitro activities. This compound completely restored short- and long-term memory in a mouse model of AD (SAMP8) relative to healthy control strain SAMR1, shifted APP processing towards the non-amyloidogenic pathway, reduced tau phosphorylation, and increased the levels of synaptic proteins PSD95 and synaptophysin, thereby emerging as a promising disease-modifying, cognition-enhancing anti-AD lead., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

A series of multitarget-directed ligands (MTDLs) was designed by functionalizing a pseudo-irreversible butyrylcholinesterase (BChE) inhibitor. The obtained hybrids were investigated in vitro regarding their h BChE and h AChE inhibition, their enzyme kinetics, and their antioxidant physicochemical properties (DPPH, ORAC, metal chelating). In addition, in vitro assays were applied to investigate antioxidant effects using murine hippocampal HT22 cells and immunomodulatory effects on the murine microglial N9 cell line. The MTDLs retained their antioxidative properties compared to the parent antioxidant-moieties in vitro and the inhibition of h BChE was maintained in the submicromolar range. Representative compounds were tested in a pharmacological Alzheimer's disease (AD) mouse model and demonstrated very high efficacy at doses as low as 0.1 mg/kg. The most promising compound was also tested in BChE -/- mice and showed reduced efficacy. In vivo neuroprotection by BChE inhibition can be effectively enhanced by incorporation of structurally diverse antioxidant moieties.

The structures of melatonin and ferulic acid were merged into tertiary amide-based histone deacetylase 6 (HDAC6) inhibitors to develop multi-target-directed inhibitors for neurodegenerative diseases to incorporate antioxidant effects without losing affinity and selectivity at HDAC6. Structure-activity relationships led to compound 10b as a hybrid molecule showing pronounced and selective inhibition of HDAC6 (IC50 = 30.7 nM, > 25-fold selectivity over other subtypes). This compound shows comparable DPPH radical scavenging ability to ferulic acid, comparable ORAC value to melatonin and comparable Cu 2+ chelating ability to EDTA. It also lacks neurotoxicity on HT-22 cells, exhibits a pronounced immunomodulatory effect, and is active in vivo showing significantly higher efficacy in an AD mouse model to prevent both Aβ25-35-induced spatial working and long-term memory dysfunction at lower dose (0.3 mg/kg) compared to positive control HDAC6 inhibitor ACY1215 and an equimolar mixture of the three entities ACY1215, melatonin and ferulic acid, suggesting potentially disease-modifying properties.

Alzheimer's disease (AD) is the most common form of dementia, and up to now, there are no disease-modifying drugs available. Natural product hybrids based on the flavonoid taxifolin and phenolic acids have shown a promising pleiotropic neuroprotective profile in cell culture assays and even disease-modifying effects in vivo . However, the detailed mechanisms of action remain unclear. To elucidate the distinct intracellular targets of 7- O -esters of taxifolin, we present in this work the development and application of a chemical probe, 7- O -cinnamoyltaxifolin-alkyne, for target identification using activity-based protein profiling. 7- O -Cinnamoyltaxifolin-alkyne remained neuroprotective in all cell culture assays. Western blot analysis showed a comparable influence on the same intracellular pathways as that of the lead compound 7- O -cinnamoyltaxifolin, thereby confirming its suitability as a probe for target identification experiments. Affinity pulldown and MS analysis revealed adenine nucleotide translocase 1 (ANT-1) and sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) as intracellular interaction partners of 7- O -cinnamoyltaxifolin-alkyne and thus of 7- O -esters of taxifolin.

In this study, the carbamate structure of pseudo-irreversible butyrylcholinesterase (BChE) inhibitors was optimized with regard to a longer binding to the enzyme. A set of compounds bearing different heterocycles (e.g., morpholine, tetrahydroisoquinoline, benzimidazole, piperidine) and alkylene spacers (2 to 10 methylene groups between carbamate and heterocycle) in the carbamate residue was synthesized and characterized in vitro for their binding affinity, binding kinetics, and carbamate hydrolysis. These novel BChE inhibitors are highly selective for h BChE over human acetycholinesterase ( h AChE), yielding short-, medium-, and long-acting nanomolar h BChE inhibitors (with a half-life of the carbamoylated enzyme ranging from 1 to 28 h). The inhibitors show neuroprotective properties in a murine hippocampal cell line and a pharmacological mouse model of Alzheimer's disease (AD), suggesting a significant benefit of BChE inhibition for a disease-modifying treatment of AD.

The enzyme butyrylcholinesterase (BChE) and the human cannabinoid receptor 2 (hCB 2 R) represent promising targets for pharmacotherapy in the later stages of Alzheimer's disease. We merged pharmacophores for both targets into small benzimidazole-based molecules, investigated SARs, and identified several dual-acting ligands with a balanced affinity/inhibitory activity and an excellent selectivity over both hCB 1 R and hAChE. A homology model for the hCB 2 R was developed based on the hCB 1 R crystal structure and used for molecular dynamics studies to investigate binding modes. In vitro studies proved hCB 2 R agonism. Unwanted μ-opioid receptor affinity could be designed out. One well-balanced dual-acting and selective hBChE inhibitor/hCB 2 R agonist showed superior in vivo activity over the lead CB 2 agonist with regards to cognition improvement. The data shows the possibility to combine a small molecule with selective and balanced GPCR-activity/enzyme inhibition and in vivo activity for the therapy of AD and may help to rationalize the development of other dual-acting ligands.

Given the complex nature of Alzheimer's disease (AD), compounds that are able to simultaneously address two or more AD-associated targets show greater promise for development into drugs for AD therapy. Herein we report an efficient two-step synthesis and biological evaluation of new racemic benzochromene derivatives as antioxidants, inhibitors of cholinesterase and β-amyloid (Aβ1-42 ) aggregation. Based on the results of the primary screening, we identified 15-(3-methoxyphenyl)-9,11,12,15-tetrahydro-10H,14H-benzo[5,6]chromeno[2,3-d]pyrido[1,2-a]pyrimidin-14-imine (3 e) and 16-(3-methoxyphenyl)-9,10,11,12,13,16-hexahydro-15H-benzo[5',6']chromeno[2',3':4,5]pyrimido[1,2-a]azepin-15-imine (3 f) as new potential multitarget-directed ligands for AD therapy. Further in-depth biological analysis showed that compound 3 f is a good human acetylcholinesterase inhibitor [IC50 =(0.36±0.02) μm], has strong antioxidant activity (3.61 μmol Trolox equivalents), and moderate Aβ1-42 antiaggregating power (40.3 %)., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Herein we describe the design, multicomponent synthesis, and biological, molecular modeling and ADMET studies, as well as in vitro PAMPA-blood-brain barrier (BBB) analysis of new tacrine-ferulic acid hybrids (TFAHs). We identified (E)-3-(hydroxy-3-methoxyphenyl)-N-{8[(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl)amino]octyl}-N-[2-(naphthalen-2-ylamino)2-oxoethyl]acrylamide (TFAH 10 n) as a particularly interesting multipotent compound that shows moderate and completely selective inhibition of human butyrylcholinesterase (IC50 =68.2 nM), strong antioxidant activity (4.29 equiv trolox in an oxygen radical absorbance capacity (ORAC) assay), and good β-amyloid (Aβ) anti-aggregation properties (65.6 % at 1:1 ratio); moreover, it is able to permeate central nervous system (CNS) tissues, as determined by PAMPA-BBB assay. Notably, even when tested at very high concentrations, TFAH 10 n easily surpasses the other TFAHs in hepatotoxicity profiling (59.4 % cell viability at 1000 μM), affording good neuroprotection against toxic insults such as Aβ1-40 , Aβ1-42 , H2 O2 , and oligomycin A/rotenone on SH-SY5Y cells, at 1 μM. The results reported herein support the development of new multipotent TFAH derivatives as potential drugs for the treatment of Alzheimer's disease., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Alzheimer disease (AD) is a progressive neurodegenerative disorder with associated memory loss, spatial disorientation, and other psychiatric problems. Cholinergic system dysfunction is an early and salient feature of AD, and enhancing cholinergic signaling with acetylcholinesterase inhibitors is currently the primary strategy for improving cognition. The beneficial effects of acetylcholinesterase inhibitors, however, are typically short-lived and accompanied by adverse effects. Recent evidence suggests that activating α7 nicotinic acetylcholine receptors (α7 nAChR) may facilitate the specific modulation of brain cholinergic signaling, leading to cognitive enhancement and possibly to amelioration of AD pathologic findings. In the present study, we determined the effect of long-term treatment with the selective α7 nAChR agonist A-582941 in aged 3xTg-AD mice with robust AD-like pathology, which is particularly significant not only because this is the only mouse model that co-develops amyloid plaques and neurofibrillary tangles but also because it enabled us to explore whether A-582941 is able to restore brain function after the severe damage associated with AD. Analysis of β-amyloid deposits, tau phosphorylation, and inflammatory cells revealed that, overall, pathologic findings were unchanged. Rather, α7 nAChR activation induced expression of c-Fos and brain-derived neurotrophic factor and phosphorylation of cyclic adenosine monophosphate response element binding and neurotrophic tyrosine receptor kinase type 2. More important, A-582941 completely restored cognition in aged 3xTg-AD mice to the level of that in age-matched nontransgenic mice. These novel findings indicate that activating α7 nAChR is a promising treatment for cognitive impairment in AD., (Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

We previously reported that alpha7 nicotinic acetylcholine receptor (nAChR) agonism produces efficacy in preclinical cognition models correlating with activation of cognitive and neuroprotective signaling pathways associated with Alzheimer's disease (AD) pathology. In the present studies, the selective and potent alpha7 nAChR agonist 5-(6-[(3R)-1-azabicyclo[2.2.2]oct-3-yloxy] pyridazin-3-yl)-1H-indole (ABT-107) was evaluated in behavioral assays representing distinct cognitive domains. Studies were also conducted to address potential issues that may be associated with the clinical development of an alpha7 nAChR agonist. Specifically, ABT-107 improved cognition in monkey delayed matching to sample, rat social recognition, and mouse two-trial inhibitory avoidance, and continued to improve cognitive performance at injection times when exposure levels continued to decline. Rats concurrently infused with ABT-107 and donepezil at steady-state levels consistent with clinical exposure showed improved short-term recognition memory. Compared with nicotine, ABT-107 did not produce behavioral sensitization in rats or exhibit psychomotor stimulant activity in mice. Repeated (3 days) daily dosing of ABT-107 increased extracellular cortical acetylcholine in rats, whereas acute administration increased cortical extracellular signal-regulated kinase and cAMP response element-binding protein phosphorylation in mice, neurochemical and biochemical events germane to cognitive function. ABT-107 increased cortical phosphorylation of the inhibitory residue (Ser9) of glycogen synthase kinase-3, a primary tau kinase associated with AD pathology. In addition, continuous infusion of ABT-107 in tau/amyloid precursor protein transgenic AD mice reduced spinal tau hyperphosphorylation. These findings show that targeting alpha7 nAChRs may have potential utility for symptomatic alleviation and slowing of disease progression in the treatment AD, and expand the understanding of the potential therapeutic viability associated with the alpha7 nAChR approach in the treatment of AD.

Mutant Tg2576 mice which possess the human "Swedish" APP mutation have been shown to demonstrate both Abeta plaque pathology and memory deficits in behavioral tasks. These mice are routinely maintained on a mixed C57BL/6xSJL genetic background which exhibits a high frequency of retinal degeneration allele and high variability in many behavioral assays. The same APP mutation is also available maintained on a 129 genetic background, providing more genetic homogeneity, but little data are published regarding the effects of the mutation on this background. We investigated whether transgenic mice expressing the Swedish mutation on the 129 background show similar behavioral deficits and Abeta pathology as those on the mixed background. Mice on the 129 background were tested at 6-7, 11-12, or 18-19 months of age in locomotor activity, Y-maze spontaneous alternation, and contextual fear conditioning. Differences were detected between WT and Tg mice in locomotor activity at 6-7 and 18-19 months, Y-maze at 6-7 and 11-12 months, and fear conditioning at 6-7, 11-12, and 18-19 months. In contrast, Tg mice on the mixed B6/SJL background tested at 6-7 months only demonstrated significant impairment in the contextual fear conditioning assay and in the Y-maze in one of 2 cohorts tested. Despite the behavioral differences observed, similar Abeta pathology was observed between Tg mice on the two genetic backgrounds. These results indicate that mice on the 129 genetic background may generate more consistent and robust behavioral differences, providing a useful model for testing therapeutic agents for Alzheimer's disease., (Copyright 2009 Elsevier B.V. All rights reserved.)

Tacrine (CAS 321-64-2) is a reversible acetylcholine esterase inhibitor that, despite exerting beneficial effects on Alzheimer's disease (AD), displays marked hepatotoxicity. Searching for safer drugs and taking into account the multi-pathogenesis of AD, two tacrine-NO donor hybrid molecules (FL16, FL38) as well as a tacrine-ferulic acid hybrid (FL67) were synthesized. NO donors coupled to the tacrine moiety may exert an additional beneficial effect on AD via an increased blood supply to the brain and by reducing inflammation. Ferulic acid is an antioxidant. To investigate the hepatotoxicity and effects on the cytochrome P450 (CYP) system of the liver, female rats were treated with the highest tolerated dose of tacrine or equimolar doses of the novel compounds 24 or 36 h, respectively, before sacrifice. Tacrine caused pericentral necrosis and fatty degeneration of the hepatocytes, loss of liver glycogen and (indicating oxidative stress) induction of heme oxygenase (HO)-1. No histopathological changes were observed with the hybrids, but a glycogen deficit and an elevation of HO-1 was noticed after FL38 or FL67 treatment. Both tacrine and the hybrids, but mainly FL38 and FL67, caused an induction of CYP1A1, 2B1 and 3A2 expression. CYP activity was noticeably increased after treatment with FL38 and FL67 only. Thus, since it displays much less hepatotoxicity and interaction potential at the CYP system than tacrine and the other hybrids, FL16 is a good candidate for further investigations.

In search of safer anti-Alzheimer drugs, 14 NO-donor-tacrine hybrids (1- 14) were synthesized and evaluated for their ability to inhibit cholinesterases and for vasorelaxation effects. Compounds 1- 13 showed good cholinesterases inhibitory activities in vitro, while 14, particularly, was highly selective, preferring butyrylcholinesterase rather than acetylcholinesterase. Four selected compounds (1, 9, 11, and 14) moderately relaxed the porcine pulmonary arteries in organ bath. In the hepatotoxicity study, significant hepatotoxicity was caused by tacrine but not by 9.

Six new tri- and tetracyclic nitrogen bridgehead compounds known to be moderate to potent inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in vitro were tested in vivo as experimental therapeutics for treatment of Alzheimer's disease. Cognitive impairment in rats was reversibly induced by scopolamine (CAS 51-34-3). The effect of the new substances was evaluated in an eight-arm radial maze and run times (1), errors (2), correct choices (3), correct choices per second (4), speed (5), and running distance (6) were recorded. For optimisation of data analysis a new strategy was used: A score was created on the basis of the 6 parameters described with score 1 for controls and score 4 for scopolamine rats. Scores above 4 indicate an impairment of cognition function compared to scopolamine. After equimolar dosage compared to the reference drug rivastigmine (CAS 123441-03-2), two of the new substances slightly improved cognition in rats, but only to a significantly lower degree compared to the irreversible inhibitor rivastigmine. Surprisingly, the other four compounds did not improve or even worsened the scopolamine effect on working memory.

Novel compounds have been developed that (in many cases) inhibit cholinesterases and concomitantly interact with at least one further pharmacological target, such as 5-HT3 or H3 receptors. But also enzymes like monoamine oxidase and the serotonin transporter have been targeted. Hybrid molecules can also incorporate antioxidant or neuronal Ca2+-channel-blocking structures.

In the course of studies directed toward the discovery of novel acetyl- and butyrylcholinesterase (AChE and BChE) inhibitors for the treatment of Alzheimer's disease, we focused on beta-carbolines (BCs). 6-Oxygenated beta-carboline and beta-carbolinium derivatives based on the serotonin template were synthesized and tested in vitro for their ability to inhibit AChE and BChE, respectively. Particularly the carbolinium salts, which can be formed by intracerebral methylation out of the tertiary-BC prodrugs, show inhibitory activity levels reaching those of galantamine, physostigmine, and rivastigmine.

The cerebral deposition of Abeta-peptide as amyloid fibrils and plaques represents a hallmark characteristic of Alzheimer's disease (AD). AD plaques are defined by their green birefringence after Congo red staining, their spherulite-like superstructure and their association with specific secondary components. Here we show that primary human macrophages promote the formation of amyloid plaques that correspond in all aforementioned characteristics to typical amyloid plaques from diseased tissues: they consist of aggregated Abeta-peptide, they reveal the typical ''Maltese cross" structure and they are associated with the secondary components glycosaminoglycanes, apolipoprotein E (apoE) and the raft lipids cholesterol and sphingomyelin. Plaque formation can be impaired in this cell system by addition of small molecules, such as Congo red, melantonine and lovastatin, suggesting potential applications for the study of cellular amyloid formation and for the identification or validation of drug candidates.