Bartolini ManuelaUniversity of Bologna; Department of Pharmaceutical Sciences; via Belmeloro 6; 40126 Bologna ItalyPhone : +390512099729 Fax : Send E-Mail to Bartolini Manuela
Multi-target drug discovery is one of the most active fields in the search for new drugs against Alzheimer's disease (AD). This is because the complexity of AD pathological network might be adequately tackled by multi-target-directed ligands (MTDLs) aimed at modulating simultaneously multiple targets of such a network. In a continuation of our efforts to develop MTDLs for AD, we have been focusing on the molecular hybridization of the acetylcholinesterase inhibitor tacrine with the aim of expanding its anti-AD profile. Herein, we manipulated the structure of a previously developed tacrine-quinone hybrid (1). We designed and synthesized a novel set of MTDLs (2-6) by replacing the naphthoquinone scaffold of 1 with that of 2,5,8-quinolinetrione. The most interesting hybrid 3 inhibited cholinesterase enzymes at nanomolar concentrations. In addition, 3 exerted antioxidant effects in menadione-induced oxidative stress of SH-SY5Y cells. Importantly, 3 also showed low hepatotoxicity and good anti-amyloid aggregation properties. Remarkably, we uncovered the potential of the quinolinetrione scaffold, as a novel anti-amyloid aggregation and antioxidant motif to be used in further anti-AD MTDL drug discovery endeavors.
With innumerable clinical failures of target-specific drug candidates for multifactorial diseases, such as Alzheimer's disease (AD), which remains inefficiently treated, the advent of multitarget drug discovery has brought a new breath of hope. Here, we disclose a class of 6-chlorotacrine (huprine)-TPPU hybrids as dual inhibitors of the enzymes soluble epoxide hydrolase (sEH) and acetylcholinesterase (AChE), a multitarget profile to provide cumulative effects against neuroinflammation and memory impairment. Computational studies confirmed the gorge-wide occupancy of both enzymes, from the main site to a secondary site, including a so far non-described AChE cryptic pocket. The lead compound displayed in vitro dual nanomolar potencies, adequate brain permeability, aqueous solubility, human microsomal stability, lack of neurotoxicity, and it rescued memory, synaptic plasticity, and neuroinflammation in an AD mouse model, after low dose chronic oral administration.
Since 2002, no clinical candidate against Alzheimer's disease has reached the market; hence, an effective therapy is urgently needed. We followed the so-called "multitarget directed ligand" approach and designed 36 novel tacrine-phenothiazine heterodimers which were in vitro evaluated for their anticholinesterase properties. The assessment of the structure-activity relationships of such derivatives highlighted compound 1dC as a potent and selective acetylcholinesterase inhibitor with IC(50) = 8 nM and 1aA as a potent butyrylcholinesterase inhibitor with IC(50) = 15 nM. Selected hybrids, namely, 1aC, 1bC, 1cC, 1dC, and 2dC, showed a significant inhibitory activity toward tau((306-336)) peptide aggregation with percent inhibition ranging from 50.5 to 62.1%. Likewise, 1dC and 2dC exerted a remarkable ability to inhibit self-induced Abeta(1-42) aggregation. Notwithstanding, in vitro studies displayed cytotoxicity toward HepG2 cells and cerebellar granule neurons; no pathophysiological abnormality was observed when 1dC was administered to mice at 14 mg/kg (i.p.). 1dC was also able to permeate to the CNS as shown by in vitro and in vivo models. The maximum brain concentration was close to the IC(50) value for acetylcholinesterase inhibition with a relatively slow elimination half-time. 1dC showed an acceptable safety and good pharmacokinetic properties and a multifunctional biological profile.
Multitarget-directed ligands (MTDLs) are considered a promising therapeutic strategy to address the multifactorial nature of Alzheimer's disease (AD). Novel MTDLs have been designed as inhibitors of human acetylcholinesterases/butyrylcholinesterases, monoamine oxidase A/B, and glycogen synthase kinase 3beta and as calcium channel antagonists via the Biginelli multicomponent reaction. Among these MTDLs, (+/-)-BIGI-3h was identified as a promising new hit compound showing in vitro balanced activities toward the aforementioned recognized AD targets. Additional in vitro studies demonstrated antioxidant effects and brain penetration, along with the ability to inhibit the aggregation of both tau protein and beta-amyloid peptide. The in vivo studies have shown that (+/-)-BIGI-3h (10 mg/kg intraperitoneally) significantly reduces scopolamine-induced cognitive deficits.
In the last years, the connection between the endocannabinoid system (eCS) and neuroprotection has been discovered, and evidence indicates that eCS signaling is involved in the regulation of cognitive processes and in the pathophysiology of Alzheimer's disease (AD). Accordingly, pharmacotherapy targeting eCS could represent a valuable contribution in fighting a multifaceted disease such as AD, opening a new perspective for the development of active agents with multitarget potential. In this paper, a series of coumarin-based carbamic and amide derivatives were designed and synthesized as multipotent compounds acting on cholinergic system and eCS-related targets. Indeed, they were tested with appropriate enzymatic assays on acetyl and butyryl-cholinesterases and on fatty acid amide hydrolase (FAAH), and also evaluated as cannabinoid receptor (CB1 and CB2) ligands. Moreover, their ability to reduce the self-aggregation of beta amyloid protein (Abeta(42)) was assessed. Compounds 2 and 3, bearing a carbamate function, emerged as promising inhibitors of hAChE, hBuChE, FAAH and Abeta(42) self-aggregation, albeit with moderate potencies, while the amide 6 also appears a promising CB1/CB2 receptors ligand. These data prove for the new compounds an encouraging multitarget profile, deserving further evaluation.
A combination of flash chromatography, solid phase extraction, high-performance liquid chromatography, and in vitro bioassays was used to isolate phytocomponents endowed with anticholinesterase activity in extract from Phyllanthus muellarianus. Phytocomponents responsible for the anti-cholinesterase activity of subfractions PMF1 and PMF4 were identified and re-assayed to confirm their activity. Magnoflorine was identified as an active phytocomponent from PMF1 while nitidine was isolated from PMF4. Magnoflorine was shown to be a selective inhibitor of human butyrylcholinesterase-hBChE (IC(50) = 131 +/- 9 microM and IC(50) = 1120 +/- 83 microM, for hBuChE and human acetylcholinesterase-hAChE, respectively), while nitidine showed comparable inhibitory potencies against both enzymes (IC(50) = 6.68 +/- 0.13 microM and IC(50) = 5.31 +/- 0.50 microM, for hBChE and hAChE, respectively). When compared with the commercial anti-Alzheimer drug galanthamine, nitidine was as potent as galanthamine against hAChE and one order of magnitude more potent against hBuChE. Furthermore, nitidine also showed significant, although weak, antiaggregating activity towards amyloid-beta self-aggregation.
Thanks to the widespread use and safety profile of donepezil (1) in the treatment of Alzheimer's disease (AD), one of the most widely adopted multi-target-directed ligand (MTDL) design strategies is to modify its molecular structure by linking a second fragment carrying an additional AD-relevant biological property. Herein, supported by a proposed combination therapy of 1 and the quinone drug idebenone, we rationally designed novel 1-based MTDLs targeting Abeta and oxidative pathways. By exploiting a bioisosteric replacement of the indanone core of 1 with a 1,4-naphthoquinone, we ended up with a series of highly merged derivatives, in principle devoid of the "physicochemical challenge" typical of large hybrid-based MTDLs. A preliminary investigation of their multi-target profile identified 9, which showed a potent and selective butyrylcholinesterase inhibitory activity, together with antioxidant and antiaggregating properties. In addition, it displayed a promising drug-like profile.
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 Abeta42 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.
The multifactorial nature of Alzheimer's disease (AD) is a reason for the lack of effective drugs as well as a basis for the development of "multi-target-directed ligands" (MTDLs). As cases increase in developing countries, there is a need of new drugs that are not only effective but also accessible. With this motivation, we report the first sustainable MTDLs, derived from cashew nutshell liquid (CNSL), an inexpensive food waste with anti-inflammatory properties. We applied a framework combination of functionalized CNSL components and well-established acetylcholinesterase (AChE)/butyrylcholinesterase (BChE) tacrine templates. MTDLs were selected based on hepatic, neuronal, and microglial cell toxicity. Enzymatic studies disclosed potent and selective AChE/BChE inhibitors (5, 6, and 12), with subnanomolar activities. The X-ray crystal structure of 5 complexed with BChE allowed rationalizing the observed activity (0.0352 nM). Investigation in BV-2 microglial cells revealed antineuroinflammatory and neuroprotective activities for 5 and 6 (already at 0.01 microM), confirming the design rationale.
The combination of the scaffolds of the cholinesterase inhibitor huprine Y and the antioxidant capsaicin results in compounds with nanomolar potencies toward human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) that retain or improve the antioxidant properties of capsaicin. Crystal structures of their complexes with AChE and BChE revealed the molecular basis for their high potency. Brain penetration was confirmed by biodistribution studies in C57BL6 mice, with one compound (5i) displaying better brain/plasma ratio than donepezil. Chronic treatment of 10 month-old APP/PS1 mice with 5i (2 mg/kg, i.p., 3 times per week, 4 weeks) rescued learning and memory impairments, as measured by three different behavioral tests, delayed the Alzheimer-like pathology progression, as suggested by a significantly reduced Abeta42/Abeta40 ratio in the hippocampus, improved basal synaptic efficacy, and significantly reduced hippocampal oxidative stress and neuroinflammation. Compound 5i emerges as an interesting anti-Alzheimer lead with beneficial effects on cognitive symptoms and on some underlying disease mechanisms.
As part of our efforts to develop sustainable drugs for Alzheimer's disease (AD), we have been focusing on the inexpensive and largely available cashew nut shell liquid (CNSL) as a starting material for the identification of new acetylcholinesterase (AChE) inhibitors. Herein, we decided to investigate whether cardanol, a phenolic CNSL component, could serve as a scaffold for improved compounds with concomitant anti-amyloid and antioxidant activities. Ten new derivatives, carrying the intact phenolic function and an aminomethyl functionality, were synthesized and first tested for their inhibitory potencies towards AChE and butyrylcholinesterase (BChE). 5 and 11 were found to inhibit human BChE at a single-digit micromolar concentration. Transmission electron microscopy revealed the potential of five derivatives to modulate Abeta aggregation, including 5 and 11. In HORAC assays, 5 and 11 performed similarly to standard antioxidant ferulic acid as hydroxyl scavenging agents. Furthermore, in in vitro studies in neuronal cell cultures, 5 and 11 were found to effectively inhibit reactive oxygen species production at a 10 microM concentration. They also showed a favorable initial ADME/Tox profile. Overall, these results suggest that CNSL is a promising raw material for the development of potential disease-modifying treatments for AD.
Alzheimer's disease (AD) is a neurodegenerative disorder associated with cholinergic dysfunction, provoking memory loss and cognitive dysfunction in elderly patients. The cholinergic hypothesis provided over the years with molecular targets for developing palliative treatments for AD, acting on the cholinergic system, namely, acetylcholinesterase and alpha7 nicotinic acetylcholine receptor (alpha7 nAChR). In our synthetic work, we used "click-chemistry" to synthesize two Multi Target Directed Ligands (MTDLs) MB105 and MB118 carrying tacrine and quinuclidine scaffolds which are known for their anticholinesterase and alpha7 nicotinic acetylcholine receptor agonist activities, respectively. Both, MB105 and MB118, inhibit human acetylcholinesterase and human butyrylcholinesterase in the nanomolar range. Electrophysiological recordings on Xenopus laevis oocytes expressing human alpha7 nAChR showed that MB105 and MB118 acted as partial agonists of the referred nicotinic receptor, albeit, with different potencies despite their similar structure. The different substitution at C-3 on the 2,3-disubtituted quinuclidine scaffold may account for the significantly lower potency of MB118 compared to MB105. Electrophysiological recordings showed that the tacrine precursor MB320 behaved as a competitive antagonist of human alpha7 nAChR, in the micromolar range, while the quinuclidine synthetic precursor MB099 acted as a partial agonist. Taken all together, MB105 behaved as a partial agonist of alpha7 nAChR at concentrations where it completely inhibited human acetylcholinesterase activity paving the way for the design of novel MTDLs for palliative treatment of AD.
Our goal was the evaluation of a series of N-1,2,3-triazole-isatin derivatives for multi-target activity which included cholinesterase (ChE) inhibition and beta-amyloid (Abeta) peptide anti-aggregation. The compounds have shown considerable promise as butyrylcholinesterase (BuChE) inhibitors. Although the inhibition of eel acetylcholinesterase (eeAChE) was weak, the inhibitions against equine BuChE (eqBuChE) and human BuChE (hBuChE) were more significant with a best inhibition against eqBuChE of 0.46 muM. In some cases, these molecules gave better inhibitions for hBuChE than eqBuChE. For greater insights into their mode of action, molecular docking studies were carried out, followed by STD-NMR validation. In addition, some of these compounds showed weak Abeta anti-aggregation activity. Hepatotoxicity studies showed that they were non-hepatoxic and neurotoxicity studies using neurite outgrowth experiments led to the conclusion that these compounds are only weakly neurotoxic.
Oxidative stress is a major pathogenic factor in Alzheimer's disease, but it should not be tackled alone rather together with other key targets to derive effective treatments. The combination of the scaffold of the polar antioxidant lead 7-methoxy-2,2-dimethylchroman-6-ol (CR-6) with that of the lipophilic cholinesterase inhibitor 6-chlorotacrine results in compounds with favorable brain permeability and multiple activities in vitro (acetylcholinesterase, butyrylcholinesterase, beta-site amyloid precursor protein (APP) cleaving enzyme-1 (BACE-1), and Abeta42 and tau aggregation inhibition). In in vivo studies on wild-type and APP/presenilin 1 (PS1) mice, two selected compounds were well tolerated and led to positive trends, albeit statistically nonsignificant in some cases, on memory performance, amyloid pathology (reduced amyloid burden and potentiated non-amyloidogenic APP processing), and oxidative stress (reduced cortical oxidized proteins and increased antioxidant enzymes superoxide dismutase 2 (SOD2), catalase, glutathione peroxidase 1 (GPX1), and heme oxygenase 1 (Hmox1) and transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2)). These compounds emerge as interesting brain-permeable multitarget compounds, with a potential as anti-Alzheimer agents beyond that of the original lead CR-6.
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-1beta and TNF-alpha. Importantly, despite the structural resemblance to tacrine, they showed ideal safety profiles on hepatic and murine brain cell lines and were safe up to 100muM when assayed in PC12cells. 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.
Title: Tacrines as Therapeutic Agents for Alzheimer's Disease. IV. The Tacripyrines and Related Annulated Tacrines Bartolini M, Marco-Contelles J Ref: Chem Rec, 19:927, 2019 : PubMed
Notwithstanding the clinical use of tacrine was hampered by severe hepatotoxicity, tacrine still remains a reference scaffold in the search for new efficient drugs for Alzheimer's disease therapy. In this account we summarize the efforts toward the development and characterization of non-hepatotoxic tacripyrines and related tacrine analogues resulting from the substitution of the benzene ring by a 1,4-dihydropyridine, a 1,2,3,4-tetrahydropyrimidine or a pyridone nucleus. These efforts have successfully led to the identification of a number of promising hits endowed with interesting multifunctional profiles. These include the 4'-metoxytacripyrine (S)-ITH122, able to target cholinesterases (ChEs), beta-amyloid (Abeta) and Ca(2+) channels, the racemic 3'-methoxytacripyrimidine EB65F2, the first fully balanced micromolar inhibitor of ChEs and Ca(2+) channels, and tacripyrine (-)-SCR1693 a GSK-3beta (enzyme involved in tau phosphorylation) inhibitor able to also lower Abeta production in N2a cells.
A combination of tacrine and tryptophan led to the development of a new family of heterodimers as multi-target agents with potential to treat Alzheimer's disease. Based on the in vitro biological profile, compound S-K1035 was found to be the most potent inhibitor of human acetylcholinesterase (hAChE) and human butyrylcholinesterase (hBChE), demonstrating balanced IC50 values of 6.3 and 9.1nM, respectively. For all the tacrine-tryptophan heterodimers, favorable inhibitory effect on hAChE as well as on hBChE was coined to the optimal spacer length ranging from five to eight carbon atoms between these two pharmacophores. S-K1035 also showed good ability to inhibit Abeta42 self-aggregation (58.6+/-5.1% at 50muM) as well as hAChE-induced Abeta40 aggregation (48.3+/-6.3% at 100muM). The X-ray crystallographic analysis of TcAChE in complex with S-K1035 pinpointed the utility of the hybridization strategy applied and the structures determined with the two K1035 enantiomers in complex with hBChE could explain the higher inhibition potency of S-K1035. Other in vitro evaluations predicted the ability of S-K1035 to cross blood-brain barrier and to exert a moderate inhibition potency against neuronal nitric oxide synthase. Based on the initial promising biochemical data and a safer in vivo toxicity compared to tacrine, S-K1035 was administered to scopolamine-treated rats being able to dose-dependently revert amnesia.
Despite the constant progress in the understanding of the etiopathogenesis of Alzheimer's disease (AD) over the last 50 years, just four long-standing drugs are currently used for AD therapy. This article reviews the analytical methodologies developed and applied in the last five years to address the early-stage tasks of the AD drug discovery process: the fast selection of active compounds (hits) and the comprehension of the ligand binding mechanism of the compound chosen to be the lead in the forthcoming development. The reviewed analytical methodologies face the most investigated pharmacological protein targets (amyloids, secretases, kinases, cholinesterases) and specific receptor- and enzyme-mediated effects in neurotransmission, neuroprotection and neurodegeneration. Some of these methodologies are noteworthy for their use in middle/high-throughput screening campaigns during hit selection (e.g. surface plasmon resonance biosensing, fluorescence resonance energy transfer assays), whereas some others (circular dichroism and nuclear magnetic resonance spectroscopies, ion mobility-mass spectrometry) can provide in-depth information about the structure, conformation and ligand binding properties of target proteins.
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 Abeta1-42 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.
Both cholinesterases (AChE and BChE) and kinases, such as GSK-3alpha/beta, are associated with the pathology of Alzheimer's disease. Two scaffolds, targeting AChE (tacrine) and GSK-3alpha/beta (valmerin) simultaneously, were assembled, using copper(I)-catalysed azide alkyne cycloaddition (CuAAC), to generate a new series of multifunctional ligands. A series of eight multi-target directed ligands (MTDLs) was synthesized and evaluated in vitro and in cell cultures. Molecular docking studies, together with the crystal structures of three MTDL/TcAChE complexes, with three tacrine-valmerin hybrids allowed designing an appropriate linker containing a 1,2,3-triazole moiety whose incorporation preserved, and even increased, the original inhibitory potencies of the two selected pharmacophores toward the two targets. Most of the new derivatives exhibited nanomolar affinity for both targets, and the most potent compound of the series displayed inhibitory potencies of 9.5nM for human acetylcholinesterase (hAChE) and 7nM for GSK-3alpha/beta. These novel dual MTDLs may serve as suitable leads for further development, since, in the micromolar range, they exhibited low cytotoxicity on a panel of representative human cell lines including the human neuroblastoma cell line SH-SY5Y. Moreover, these tacrine-valmerin hybrids displayed a good ability to penetrate the blood-brain barrier (BBB) without interacting with efflux pumps such as P-gp.
The development of multitarget compounds against multifactorial diseases, such as Alzheimer's disease, is an area of very intensive research, due to the expected superior therapeutic efficacy that should arise from the simultaneous modulation of several key targets of the complex pathological network. Here we describe the synthesis and multitarget biological profiling of a new class of compounds designed by molecular hybridization of an NMDA receptor antagonist fluorobenzohomoadamantanamine with the potent acetylcholinesterase (AChE) inhibitor 6-chlorotacrine, using two different linker lengths and linkage positions, to preserve or not the memantine-like polycyclic unsubstituted primary amine. The best hybrids exhibit greater potencies than parent compounds against AChE (IC50 0.33nM in the best case, 44-fold increased potency over 6-chlorotacrine), butyrylcholinesterase (IC50 21nM in the best case, 24-fold increased potency over 6-chlorotacrine), and NMDA receptors (IC50 0.89muM in the best case, 2-fold increased potency over the parent benzohomoadamantanamine and memantine), which suggests an additive effect of both pharmacophoric moieties in the interaction with the primary targets. Moreover, most of these compounds have been predicted to be brain permeable. This set of biological properties makes them promising leads for further anti-Alzheimer drug development.
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 muM to 1 mM), less potent than tacrine as a ChE inhibitor, but shows selective BuChE inhibition (IC50 (hAChE) = 22.0 +/- 1.3 muM; IC50 (hBuChE) = 6.79 +/- 0.33 muM). 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.
We have designed and synthesized a series of 14 hybrid molecules out of the cholinesterase (ChE) inhibitor tacrine and a benzimidazole-based human cannabinoid receptor subtype 2 (hCB2R) agonist and investigated them in vitro and in vivo. The compounds are potent ChE inhibitors, and for the most promising hybrids, the mechanism of human acetylcholinesterase (hAChE) inhibition as well as their ability to interfere with AChE-induced aggregation of beta-amyloid (Abeta), and Abeta self-aggregation was assessed. All hybrids were evaluated for affinity and selectivity for hCB1R and hCB2R. To ensure that the hybrids retained their agonist character, the expression of cAMP-regulated genes was quantified, and potency and efficacy were determined. Additionally, the effects of the hybrids on microglia activation and neuroprotection on HT-22 cells were investigated. The most promising in vitro hybrids showed pronounced neuroprotection in an Alzheimer's mouse model at low dosage (0.1 mg/kg, i.p.), lacking hepatotoxicity even at high dose (3 mg/kg, i.p.).
INTRODUCTION: Butyrylcholinesterase (BuChE) has obtained a renewed interest as therapeutic target in Alzheimer's disease (AD), when changes in BuChE activity and expression along disease progression were highlighted as well as correlation between BuChE levels and cognitive function. Areas covered: During the last eight years, fourteen patents on BuChE inhibitors (BuChEI) have been submitted. Only three of them relate to BuChE selective inhibitors, while four of them focus on multitarget inhibitors which address different key pathological factors other than BuChE. Two patents report on non-selective acetylcholinesterase (AChE)/BuChE inhibitors, while four patents deal with natural compounds and their derivatives. One patent relates to antitoxic agents to treat exposure to ChEI pesticides and nerve agents. Expert opinion: Increasing evidence supports BuChE as a more beneficial target in moderate-to-severe forms of AD in comparison to the well-known AChE. However, hitting a single pathological target is likely not sufficient to halt the disease progression. Therefore, patented BuChE inhibitors with a multifunctional profile may open new therapeutic avenues, since the additional activities could reinforce the therapeutic effects. Unfortunately, in vivo studies are limited and key parameters, such as ADMET data, are missing. This lack of information makes difficult to forecast the development of patented BuChEIs into effective drug candidates.
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 (3.05muM and 3.19muM on hAChE and hBuChE, respectively) and moderate CCB (30.4% at 1muM) with no significant hepatotoxicity toward HepG2 cells and good predicted oral absorption and blood brain barrier permeability.
Title: Combination of human acetylcholinesterase and serum albumin sensing surfaces as highly informative analytical tool for inhibitor screening Fabini E, Tramarin A, Bartolini M Ref: J Pharm Biomed Anal, 155:177, 2018 : PubMed
In the continuous research for potential drug lead candidates, the availability of highly informative screening methodologies may constitute a decisive element in the selection of best-in-class compounds. In the present study, a surface plasmon resonance (SPR)-based assay was developed and employed to investigate interactions between human recombinant AChE (hAChE) and four known ligands: galantamine, tacrine, donepezil and edrophonium. To this aim, a sensor chip was functionalized with hAChE using mild immobilization conditions to best preserve enzyme integrity. Binding affinities and, for the first time, kinetic rate constants for all drug-hAChE complexes formation/disruption were determined. Inhibitors were classified in two groups: slow-reversible and fast-reversible binders according to respective target residence time. Combining data obtained on drug-target residence time with data obtained on serum albumin binding levels, a good correlation with potency, plasma protein binding in vivo, and administration regimen was found. The outcomes of this work demonstrated that the developed SPR-based assay is suitable for the screening, the binding affinity ranking and the kinetic evaluation of hAChE inhibitors. The method proposed ensures a simpler and cost-effective assay to quantify kinetic rate constants for inhibitor-hAChE interaction as compared with other proposed and published methods. Eventually, the determination of residence time in combination with preliminary ADME studies might constitute a better tool to predict in vivo behaviour, a key information for the research of new potential drug candidates.
Symptomatic treatment of myasthenia gravis is based on the use of peripherally-acting acetylcholinesterase (AChE) inhibitors that, in some cases, must be discontinued due to the occurrence of a number of side-effects. Thus, new AChE inhibitors are being developed and investigated for their potential use against this disease. Here, we have explored two alternative approaches to get access to peripherally-acting AChE inhibitors as new agents against myasthenia gravis, by structural modification of the brain permeable anti-Alzheimer AChE inhibitors tacrine, 6-chlorotacrine, and huprine Y. Both quaternization upon methylation of the quinoline nitrogen atom, and tethering of a triazole ring, with, in some cases, the additional incorporation of a polyphenol-like moiety, result in more polar compounds with higher inhibitory activity against human AChE (up to 190-fold) and butyrylcholinesterase (up to 40-fold) than pyridostigmine, the standard drug for symptomatic treatment of myasthenia gravis. The novel compounds are furthermore devoid of brain permeability, thereby emerging as interesting leads against myasthenia gravis.
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-alpha,beta-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 Abeta1(-)42 oligomers, showing a promising neuroprotective potential.
A new series of pyridine derivatives with carbamic or amidic function has been designed and synthesized to act as cholinesterase inhibitors. The synthesized compounds were tested toward EeAChE and hAChE and toward eqBChE and hBChE. The carbamate 8 was the most potent hAChE inhibitor (IC50 = 0.153 +/- 0.016 muM) while the carbamate 11 was the most potent inhibitor of hBChE (IC50 = 0.828 +/- 0.067 muM). A molecular docking study indicated that the carbamate 8 was able to bind AChE by interacting with both CAS and PAS, in agreement with the mixed inhibition mechanism. Furthermore, the carbamates 8, 9 and 11 were able to inhibit Abeta42 self-aggregation and possessed quite low toxicity against human astrocytoma T67 and HeLa cell lines, being the carbamate 8 the less toxic compound on both cell lines.
AIM: Simultaneous modulation of several key targets of the pathological network of Alzheimer's disease (AD) is being increasingly pursued as a promising option to fill the critical gap of efficacious drugs against this condition. MATERIALS & METHODS: A short series of compounds purported to hit multiple targets of relevance in AD has been designed, on the basis of their distinct basicities estimated from high-level quantum mechanical computations, synthesized, and subjected to assays of inhibition of cholinesterases, BACE-1, and Abeta42 and tau aggregation, of antioxidant activity, and of brain permeation. RESULTS: Using, as a template, a lead rhein-huprine hybrid with an interesting multitarget profile, we have developed second-generation compounds, designed by the modification of the huprine aromatic ring. Replacement by [1,8]-naphthyridine or thieno[3,2-e]pyridine systems resulted in decreased, although still potent, acetylcholinesterase or BACE-1 inhibitory activities, which are more balanced relative to their Abeta42 and tau antiaggregating and antioxidant activities. CONCLUSION: Second-generation naphthyridine- and thienopyridine-based rhein-huprine hybrids emerge as interesting brain permeable compounds that hit several crucial pathogenic factors of AD.
AIM: Alzheimer's disease is a still untreatable multifaceted pathology, and drugs able to stop or reverse its progression are urgently needed. In this picture, the recent reformulation of the cholinergic hypothesis renewed the interest for acetylcholinesterase inhibitors. In this paper, a series of naturally inspired chalcone-based carbamates was designed to target cholinesterase enzymes and possibly generate fragments endowed with neuroprotective activity in situ. Results & methodology: All compounds presented in this study showed nanomolar potency for cholinesterase inhibition. Notably, fragment 11d also displayed an interesting neuroprotective profile. CONCLUSION: These new derivatives are able to simultaneously modulate different key targets involved in Alzheimer's disease, and could be regarded as promising starting points for the development of disease-modifying drug candidates. [Formula: see text].
BACKGROUND: Alzheimer s disease, a progressive and degenerative disorder of the brain, is the most common cause of dementia among the elderly. To face its multifactorial nature, the use of single compounds that can simultaneously modulate different targets involved in the neurodegenerative cascade has emerged as an interesting therapeutic approach. OBJECTIVE: This work investigated the ability of uleine, the major indole alkaloid purified from stem barks of the Brazilian medicinal plant Himatanthus lancifolius, to interact with crucial Alzheimer s disease disruptive targets associated with two of its major neurodegenerative pathways: acetylcholinesterase and butyrylcholinesterase (cholinergic pathway) and beta-secretase and beta-amyloid peptide (amyloidogenic pathway). METHODS: Uleine's capacity to inhibit human acetylcholinesterase and butyrylcholinesterase enzymes was determined measuring the difference between reaction rates with and without uleine monitored at 412 nm using 5,5'- dithiobis-(2- nitrobenzoic acid) as colorimetric agent. FRET based assay was used to evaluate beta-secretase inhibition using DABCYL- Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-EDANS as substrate and beta-amyloid peptide spontaneous aggregation assay was performed using the thioflavin T spectroscopy assay. Cell viability and toxicity experiments with PC12 and SH-SY5Y cell lines were performed using the MTT colorimetric assay. RESULTS: Uleine demonstrated strong inhibitory activities for both cholinesterases (IC50 279.0+/-4.5 and 24.0+/-1.5 muM, respectively) and beta-secretase (IC50 180+/-22 nM). Above all, uleine significantly inhibited the self-aggregation of amyloid- beta peptide and was not toxic for PC12 or SH-SY5Y neuronal cells. CONCLUSION: These data show for the first time that the natural compound uleine has a novel, multieffective ability to decelerate or even inhibit the development of Alzheimer s disease.
AIM: Alzheimer pathogenesis has been associated with a network of processes working simultaneously and synergistically. Over time, much interest has been focused on cholinergic transmission and its mutual interconnections with other active players of the disease. Besides the cholinesterase mainstay, the multifaceted interplay between nicotinic receptors and amyloid is actually considered to have a central role in neuroprotection. Thus, the multitarget drug-design strategy has emerged as a chance to face the disease network. METHODS: By exploiting the multitarget approach, hybrid compounds have been synthesized and studied in vitro and in silico toward selected targets of the cholinergic and amyloidogenic pathways. RESULTS: The new molecules were able to target the cholinergic system, by joining direct nicotinic receptor stimulation to acetylcholinesterase inhibition, and to inhibit amyloid-beta aggregation. CONCLUSION: The compounds emerged as a suitable starting point for a further optimization process.
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 muM), and a very potent antioxidant agent on the basis of the ORAC test (2.31 +/- 0.29 mumol.Trolox/mumol compound).
Multi-target drug discovery is one of the most followed approaches in the active central nervous system (CNS) therapeutic area, especially in the search for new drugs against Alzheimer's disease (AD). This is because innovative multi-target-directed ligands (MTDLs) could more adequately address the complexity of this pathological condition. In a continuation of our efforts aimed at a new series of anti-AD MTDLs, we combined the structural features of the cholinesterase inhibitor drug tacrine with that of resveratrol, which is known for its purported antioxidant and anti-neuroinflammatory activities. The most interesting hybrid compounds (5, 8, 9 and 12) inhibited human acetylcholinesterase at micromolar concentrations and effectively modulated Abeta self-aggregation in vitro. In addition, 12 showed intriguing anti-inflammatory and immuno-modulatory properties in neuronal and glial AD cell models. Importantly, the MTDL profile is accompanied by high-predicted blood-brain barrier permeability, and low cytotoxicity on primary neurons.
Alzheimer's disease (AD) is a major public health challenge that faces an aging global population. Current drug treatment has demonstrated only symptomatic efficacy, leaving an unmet medical need for a new generation of disease-modifying therapies. Following the multitarget-directed ligand approach, a small library of coumarin-based derivatives was designed and synthesized as a follow-up to our studies on AP2238, aimed at expanding its biological profile. The coumarin substitution pattern at the 6- or 7-position was modified by introducing alkyl chains of variable lengths and with different terminal amino functional groups. 3-(4-{[Benzyl(ethyl)amino]methyl}phenyl)-6-({5-[(7-methoxy-6H-indeno[2,1-b]quinol in-11-yl)amino]pentyl}oxy)-2H-chromen-2-one, bearing the bulkiest amine, emerged as a non-neurotoxic dual acetylcholinesterase (AChE)/butyrylcholinesterase (BuChE) inhibitor, potentially suitable for the treatment of the middle stage of AD. Furthermore, the introduction of a diethylamino spacer, as in 3-(4-{[benzyl(ethyl)amino]methyl}phenyl)-6-{[5-(diethylamino)pentyl]oxy}-2H-chrom en-2-one and 3-(4-{[benzyl(ethyl)amino]methyl}phenyl)-7-[4-(diethylamino)butoxy]-2H-chromen-2- one, led to nanomolar human AChE inhibitors endowed with significant inhibitory activity toward Abeta42 self-aggregation, whereas the reference compound was completely ineffective. Furthermore, 3-(4-{[benzyl(ethyl)amino]methyl}phenyl)-7-[4-(diethylamino)butoxy]-2H-chromen-2- one also showed promising neuroprotective behavior, which makes it a potential candidate for development into a disease-modifying agent.
The modulation of the endocannabinoid system is emerging as a viable avenue for the treatment of neurodegeneration, being involved in neuroprotective and anti-inflammatory processes. In particular, indirectly enhancing endocannabinoid signaling to therapeutic levels through FAAH inhibition might be beneficial for neurodegenerative disorders such as Alzheimer's disease, effectively preventing or slowing the progression of the disease. Hence, in the search for a more effective treatment for Alzheimer's disease, in this paper, the multitarget-directed ligand paradigm was applied to the design of carbamates able to simultaneously target the recently proposed endocannabinoid system and the classic cholinesterase system, and achieve effective dual FAAH/cholinesterase inhibitors. Among the two series of synthesized compounds, while some derivatives proved to be extremely potent on a single target, compounds 9 and 19 were identified as effective dual FAAH/ChE inhibitors, with well-balanced nanomolar activities. Thus, 9 and 19 might be considered as new promising candidates for Alzheimer's disease treatment.
We discovered a small series of hit compounds that show multitargeting activities against key targets in Alzheimer's disease (AD). The compounds were designed by combining the structural features of the anti-AD drug donepezil with clioquinol, which is able to chelate redox-active metals, thus decreasing metal-driven oxidative phenomena and beta-amyloid (Abeta)-mediated neurotoxicity. The majority of the new hybrid compounds selectively target human butyrylcholinesterase at micromolar concentrations and effectively inhibit Abeta self-aggregation. In addition, compounds 5-chloro-7-((4-(2-methoxybenzyl)piperazin-1-yl)methyl)-8-hydroxyquinoline (1 b), 7-((4-(2-methoxybenzyl)piperazin-1-yl)methyl)-8-hydroxyquinoline (2 b), and 7-(((1-benzylpiperidin-4-yl)amino)methyl)-5-chloro-8-hydroxyquinoline (3 a) are able to chelate copper(II) and zinc(II) and exert antioxidant activity in vitro. Importantly, in the case of 2 b, the multitarget profile is accompanied by high predicted blood-brain barrier permeability, low cytotoxicity in T67 cells, and acceptable toxicity in HUVEC primary cells.
Twenty-six new tacrine-benzofuran hybrids were designed, synthesized, and evaluated in vitro on key molecular targets for Alzheimer's disease. Most hybrids exhibited good inhibitory activities on cholinesterases and beta-amyloid self-aggregation. Selected compounds displayed significant inhibition of human beta-secretase-1 (hBACE-1). Among the 26 hybrids, 2e showed the most interesting profile as a subnanomolar selective inhibitor of human acetylcholinesterase (hAChE) (IC50 = 0.86 nM) and a good inhibitor of both beta-amyloid aggregation (hAChE- and self-induced, 61.3% and 58.4%, respectively) and hBACE-1 activity (IC50 = 1.35 muM). Kinetic studies showed that 2e acted as a slow, tight-binding, mixed-type inhibitor, while X-ray crystallographic studies highlighted the ability of 2e to induce large-scale structural changes in the active-site gorge of Torpedo californica AChE (TcAChE), with significant implications for structure-based drug design. In vivo studies confirmed that 2e significantly ameliorates performances of scopolamine-treated ICR mice. Finally, 2e administration did not exhibit significant hepatotoxicity.
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)am ino]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 beta-amyloid (Abeta) 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 muM), affording good neuroprotection against toxic insults such as Abeta1-40 , Abeta1-42 , H2 O2 , and oligomycin A/rotenone on SH-SY5Y cells, at 1 muM. The results reported herein support the development of new multipotent TFAH derivatives as potential drugs for the treatment of Alzheimer's disease.
Cardanol is a phenolic lipid component of cashew nut shell liquid (CNSL), obtained as the byproduct of cashew nut food processing. Being a waste product, it has attracted much attention as a precursor for the production of high-value chemicals, including drugs. On the basis of these findings and in connection with our previous studies on cardanol derivatives as acetylcholinesterase (AChE) inhibitors, we designed a novel series of analogues by including a protonable amino moiety belonging to different systems. Properly addressed docking studies suggested that the proposed structural modifications would allow the new molecules to interact with both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE, thus being able to act as dual binding inhibitors. To disclose whether the new molecules showed the desired profile, they were first tested for their cholinesterase inhibitory activity towards EeAChE and eqBuChE. Compound 26, bearing an N-ethyl-N-(2-methoxybenzyl)amine moiety, showed the highest inhibitory activity against EeAChE, with a promising IC50 of 6.6 muM, and a similar inhibition profile of the human isoform (IC50 = 5.7 muM). As another positive feature, most of the derivatives did not show appreciable toxicity against HT-29 cells, up to a concentration of 100 muM, which indicates drug-conform behavior. Also, compound 26 is capable of crossing the blood-brain barrier (BBB), as predicted by a PAMPA-BBB assay. Collectively, the data suggest that the approach to obtain potential anti-Alzheimer drugs from CNSL is worth of further pursuit and development.
In an effort to develop multipotent agents against beta-secretase (BACE-1) and acetylcholinesterase (AChE), able to counteract intracellular ROS formation as well, the structure of the fluorinated benzophenone 3 served as starting point for the synthesis of a small library of 3-fluoro-4-hydroxy- analogues. Among the series, derivatives 5 and 12, carrying chemically different amino functions, showed a balanced micromolar potency against the selected targets. In particular, compound 12, completely devoid of toxic effects, seems to be a promising lead for obtaining effective anti-AD drug candidates.
AIMS: We recently described multifunctional tools (2a-c) as potent inhibitors of human Cholinesterases (ChEs) also able to modulate events correlated with Abeta aggregation. We herein propose a thorough biological and computational analysis aiming at understanding their mechanism of action at the molecular level. METHODS: We determined the inhibitory potency of 2a-c on Abeta1-42 self-aggregation, the interference of 2a with the toxic Abeta oligomeric species and with the postaggregation states by capillary electrophoresis analysis and transmission electron microscopy. The modulation of Abeta toxicity was assessed for 2a and 2b on human neuroblastoma cells. The key interactions of 2a with Abeta and with the Abeta-preformed fibrils were computationally analyzed. 2a-c toxicity profile was also assessed (human hepatocytes and mouse fibroblasts). RESULTS: Our prototypical pluripotent analogue 2a interferes with Abeta oligomerization process thus reducing Abeta oligomers-mediated toxicity in human neuroblastoma cells. 2a also disrupts preformed fibrils. Computational studies highlighted the bases governing the diversified activities of 2a. CONCLUSION: Converging analytical, biological, and in silico data explained the mechanism of action of 2a on Abeta1-42 oligomers formation and against Abeta-preformed fibrils. This evidence, combined with toxicity data, will orient the future design of safer analogues.
We report the identification of multitarget anti-Alzheimer compounds designed by combining a naphthoquinone function and a tacrine fragment. In vitro, 15 compounds displayed excellent acetylcholinesterase (AChE) inhibitory potencies and interesting capabilities to block amyloid-beta (Abeta) aggregation. The X-ray analysis of one of those compounds in complex with AChE allowed rationalizing the outstanding activity data (IC50 = 0.72 nM). Two of the compounds showed negligible toxicity in immortalized mouse cortical neurons Neuro2A and primary rat cerebellar granule neurons. However, only one of them was less hepatotoxic than tacrine in HepG2 cells. In T67 cells, both compounds showed antioxidant activity, following NQO1 induction. Furthermore, in Neuro2A, they were able to completely revert the decrease in viability induced by Abeta. Importantly, they crossed the blood-brain barrier, as demonstrated in ex vivo experiments with rats. When ex vivo results were combined with in vitro studies, these two compounds emerged to be promising multitarget lead candidates worthy of further pursuit.
The development of drugs with different pharmacological properties appears to be an innovative therapeutic approach for Alzheimer's disease. In this article, we describe a simple structural modification of AP2238, a first dual function lead, in particular the introduction of the catechol moiety performed in order to search for multi-target ligands. The new compound AP2469 retains anti-acetylcholinesterase (AChE) and beta-site amyloid precursor protein cleaving enzyme (BACE)1 activities compared to the reference, and is also able to inhibit Abeta 42 self-aggregation, Abeta 42 oligomer-binding to cell membrane and subsequently reactive oxygen species formation in both neuronal and microglial cells. The ability of AP2469 to interfere with Abeta 42 oligomer-binding to neuron and microglial cell membrane gives this molecule both neuroprotective and anti-inflammatory properties. These findings, together with its strong chain-breaking antioxidant performance, make AP2469 a potential drug able to modify the course of the disease.
We have synthesized a family of rhein-huprine hybrids to hit several key targets for Alzheimer's disease. Biological screening performed in vitro and in Escherichia coli cells has shown that these hybrids exhibit potent inhibitory activities against human acetylcholinesterase, butyrylcholinesterase, and BACE-1, dual Abeta42 and tau antiaggregating activity, and brain permeability. Ex vivo studies with the leads (+)- and (-)-7e in brain slices of C57bl6 mice have revealed that they efficiently protect against the Abeta-induced synaptic dysfunction, preventing the loss of synaptic proteins and/or have a positive effect on the induction of long-term potentiation. In vivo studies in APP-PS1 transgenic mice treated ip for 4 weeks with (+)- and (-)-7e have shown a central soluble Abeta lowering effect, accompanied by an increase in the levels of mature amyloid precursor protein (APP). Thus, (+)- and (-)-7e emerge as very promising disease-modifying anti-Alzheimer drug candidates.
Human acetylcholinesterase (AChE) is a widely studied target enzyme in drug discovery for Alzheimer's disease (AD). In this paper we report evaluation of the optimum structure and chemistry of the supporting material for a new AChE-based fluorescence sensing surface. To achieve this objective, multilayered silicon wafers with spatially controlled geometry and chemical diversity were fabricated. Specifically, silicon wafers with silicon oxide patterns (SiO(2)/Si wafers), platinum-coated silicon wafers with SiO(2) patterns (SiO(2)/Pt/Ti/Si wafers), and Pt-coated wafers coated with different thicknesses of TiO(2) and SiO(2) (SiO(2)/TiO(2)/Pt/Ti/Si wafers) were labelled with the fluorescent conjugation agent HiLyte Fluor 555. Selection of a suitable material and the optimum pattern thickness required to maximize the fluorescence signal and maintain chemical stability was performed by confocal laser-scanning microscopy (CLSM). Results showed that the highest signal-to-background ratio was always obtained on wafers with 100 nm thick SiO(2) features. Hence, these wafers were selected for covalent binding of human AChE. Batch-wise kinetic studies revealed that enzyme activity was retained after immobilization. Combined use of atomic-force microscopy and CLSM revealed that AChE was homogeneously and selectively distributed on the SiO(2) microstructures at a suitable distance from the reflective surface. In the optimum design, efficient fluorescence emission was obtained from the AChE-based biosensing surface after labelling with propidium, a selective fluorescent probe of the peripheral binding site of AChE.
In order to identify novel Alzheimer's modifying pharmacological tools, we developed bis-tacrines bearing a peptide moiety for specific interference with surface sites of human acetylcholinesterase (hAChE) binding amyloid-beta (Abeta). Accordingly, compounds 2a-c proved to be inhibitors of hAChE catalytic and noncatalytic functions, binding the catalytic and peripheral sites, interfering with Abeta aggregation and with the Abeta self-oligomerization process (2a). Compounds 2a-c in complex with TcAChE span the gorge with the bis-tacrine system, and the peptide moieties bulge outside the gorge in proximity of the peripheral site. These moieties are likely responsible for the observed reduction of hAChE-induced Abeta aggregation since they physically hamper Abeta binding to the enzyme surface. Moreover, 2a was able to significantly interfere with Abeta self-oligomerization, while 2b,c showed improved inhibition of hAChE-induced Abeta aggregation.
A series of 1,2,3,4-tetrahydrobenzo[h][1,6]naphthyridines differently substituted at positions 1, 5, and 9 have been designed from the pyrano[3,2-c]quinoline derivative 1, a weak inhibitor of acetylcholinesterase (AChE) with predicted ability to bind to the AChE peripheral anionic site (PAS), at the entrance of the catalytic gorge. Fourteen novel benzonaphthyridines have been synthesized through synthetic sequences involving as the key step a multicomponent Povarov reaction between an aldehyde, an aniline and an enamine or an enamide as the activated alkene. The novel compounds have been tested against Electrophorus electricus AChE (EeAChE), human recombinant AChE (hAChE), and human serum butyrylcholinesterase (hBChE), and their brain penetration has been assessed using the PAMPA-BBB assay. Also, the mechanism of AChE inhibition of the most potent compounds has been thoroughly studied by kinetic studies, a propidium displacement assay, and molecular modelling. We have found that a seemingly small structural change such as a double O --> NH bioisosteric replacement from the hit 1 to 16a results in a dramatic increase of EeAChE and hAChE inhibitory activities (>217- and >154-fold, respectively), and in a notable increase in hBChE inhibitory activity (>11-fold), as well. An optimized binding at the PAS besides additional interactions with AChE midgorge residues seem to account for the high hAChE inhibitory potency of 16a (IC50 = 65 nM), which emerges as an interesting anti-Alzheimer lead compound with potent dual AChE and BChE inhibitory activities.
The anti-amyloid properties shared by several quinones inspired the design of a new series of hybrids derived from the multi-target drug candidate memoquin (1). The hybrids consist of a central benzoquinone core and a fragment taken from non-steroidal anti-inflammatory drugs, connected through polyamine linkers. The new hybrids retain the potent anti-aggregating activity of the parent 1, while exhibiting micromolar AChE inhibitory activities. Remarkably, 2, 4, (R)-6 and (S)-6 were Abeta aggregation inhibitors even more potent than 1. The balanced amyloid/cholinesterase inhibitory profile is an added value that makes the present series of compounds promising leads against Alzheimer's disease.
Inspired by the concept of bivalent ligands, we prepared a small set of analogues of the drug candidate dimebon. They were shown to inhibit AChE, Abeta42 aggregation, and NMDA receptor activation to a greater extent than dimebon. Some of these compounds also enhanced the survival of chicken neurons under apoptosis-inducing conditions.
A family of huprine-tacrine heterodimers has been developed to simultaneously block the active and peripheral sites of acetylcholinesterase (AChE). Their dual site binding for AChE, supported by kinetic and molecular modeling studies, results in a highly potent inhibition of the catalytic activity of human AChE and, more importantly, in the in vitro neutralization of the pathological chaperoning effect of AChE toward the aggregation of both the beta-amyloid peptide (Abeta) and a prion peptide with a key role in the aggregation of the prion protein. Huprine-tacrine heterodimers take on added value in that they display a potent in vitro inhibitory activity toward human butyrylcholinesterase, self-induced Abeta aggregation, and beta-secretase. Finally, they are able to cross the blood-brain barrier, as predicted in an artificial membrane model assay and demonstrated in ex vivo experiments with OF1 mice, reaching their multiple biological targets in the central nervous system. Overall, these compounds are promising lead compounds for the treatment of Alzheimer's and prion diseases.
Alzheimer's disease (AD) is the most common cause of dementia, clinically characterized by loss of memory and progressive deficits in different cognitive domains. An emerging disease-modifying approach to face the multifactorial nature of AD may be represented by the development of Multi-Target Directed Ligands (MTDLs), i.e., single compounds which may simultaneously modulate different targets involved in the neurodegenerative AD cascade. The structure of tacrine, an acetylcholinesterase (AChE) inhibitor (AChEI), has been widely used as scaffold to provide new MTDLs. In particular, its homodimer bis(7)tacrine represents an interesting lead compound to design novel MTDLs. Thus, in the search of new rationally designed MTDLs against AD, we replaced the heptamethylene linker of bis(7)tacrine with the structure of cystamine, leading to cystamine-tacrine dimer. In this study we demonstrated that the cystamine-tacrine dimer is endowed with a lower toxicity in comparison to bis(7)tacrine, it is able to inhibit AChE, butyrylcholinesterase (BChE), self- and AChE-induced beta-amyloid aggregation in the same range of the reference compound and exerts a neuroprotective action on SH-SY5Y cell line against H(2)O(2)-induced oxidative injury. The investigation of the mechanism of neuroprotection showed that the cystamine-tacrine dimer acts by activating kinase 1 and 2 (ERK1/2) and Akt/protein kinase B (PKB) pathways. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.
The treatment of Alzheimer's disease (AD) still remains an area of significant unmet need, with drugs that only target the symptoms of the disease. Therefore, there is considerable need for disease-modifying therapies. The complex etiology of AD prompts scientists to develop multitarget strategies to combat causes and symptoms. To this aim, we designed, synthesized, and tested four new carbamates as dual cholinesterase-FAAH inhibitors. The dual activity of these compounds could lead to a potentially more effective treatment for the counteraction of AD progression, because they would allow regulation of both ACh and eCB signaling and improve neuronal transmission and/or counteract neuroinflammation.
The synthesis, pharmacological evaluation and molecular modeling of heterocyclic substituted alkyl and cycloalkyl propargyl amines 1-7 of type I, and 9-12 of type II, designed as multipotent inhibitors able to simultaneously inhibit monoamine oxidases (MAO-A/B) as well as cholinesterase (AChE/BuChE) enzymes, as potential drugs for the treatment of Alzheimer's disease, are described. Indole derivatives 1-7 of type I are well known MAO inhibitors whose capacity to inhibit AChE and BuChE was here investigated for the first time. As a result, compound 7 was identified as a MAO-B inhibitor (IC(50) = 31 +/- 2 nM) and a moderately selective eqBuChE inhibitor (IC(50) = 4.7 +/- 0.2 muM). Conversely, the new and readily available 5-amino-7-(prop-2-yn-1-yl)-6,7,8,9-tetrahydropyrido[2,3-b][1,6]naphthyridine derivatives 9-13 of type II are poor MAO inhibitors, but showed AChE selective inhibition, compound 12 being the most attractive as it acts as a non-competitive inhibitor on EeAChE (IC(50) = 25 +/- 3 nM, K(i) = 65 nM). The ability of this compound to interact with the AChE peripheral binding site was confirmed by kinetic studies and by molecular modeling investigation. Studies on human ChEs confirmed that 12 is a selective AChE inhibitor with inhibitory potency in the submicromolar range. Moreover, in agreement with its mode of action, 12 was shown to be able to inhibit Abeta aggregation induced by hAChE by 30.6%.
Title: Benzophenone-based derivatives: a novel series of potent and selective dual inhibitors of acetylcholinesterase and acetylcholinesterase-induced beta-amyloid aggregation Belluti F, Bartolini M, Bottegoni G, Bisi A, Cavalli A, Andrisano V, Rampa A Ref: Eur Journal of Medicinal Chemistry, 46:1682, 2011 : PubMed
The leading mechanistic theory of Alzheimer's disease (AD) is the "amyloid hypothesis" which states that the accumulation of the amyloid beta protein (Abeta), and its subsequent aggregation into plaques, is responsible for the initiation of a cascade of events resulting in neurodegeneration and dementia. The anti-amyloid disease-modifying approach, based on the decrease in the production of Abeta, gained thus a paramount importance. The aim of this study was the design and synthesis of a new series of acetylcholinesterase inhibitors (AChEIs) endowed with anti-Abeta aggregating capability. These dual binding inhibitors, being able to interact both with the peripheral anionic site (PAS) of AChE and the catalytic subsite, proved to be able to inhibit the AChE-induced Abeta aggregation. Thus, starting from the lead compound 1, an AChEI composed by a benzophenone scaffold and a N,N'-methylbenzylamino group, a substantial modification aimed at targeting the PAS was performed. To this aim, different amino-terminal side chains were incorporated into this main framework, in order to mimic the diethylmethylammonium alkyl moiety of the pure PAS ligand propidium. The synthesized compounds proved to effectively and selectively inhibit AChE. Moreover, compounds 16a-c and 18a,b, with a propoxy and a hexyloxy tether respectively, showed a good activity against the AChE-induced Abeta aggregation. In particular, molecular modeling studies confirmed that compounds carrying the diethylaminopropoxy and the diethylaminohexyloxy side chains (compounds 16a and 19a, respectively) could suitably contact the PAS pocket of the enzyme.
The synthesis and pharmacological analyses of a number of furo[2,3-b]quinolin-4-amine, and pyrrolo[2,3-b]quinolin-4-amine derivatives are reported. Thus, we synthesized diversely substituted tacrine analogues 1-11 and 12-16 by Friedlander-type reaction of readily available o-amino(furano/pyrrolo)nitriles with suitable and selected cycloalkanones. The biological evaluation of furanotacrines1-11 and pyrrolotacrine13 showed that these are good, in the micromolar range, and highly selective inhibitors of BuChE. In the furanotacrine group, the most interesting inhibitor was 2-(p-tolyl)-5,6,7,8-tetrahydrofuro[2,3-b]quinolin-4-amine (3) [IC(50) (eqBuChE)=2.9 +/- 0.4 muM; IC(50) (hBuChE)=119 +/- 15 muM]. Conversely, pyrrolotacrines 12 and 14 proved moderately equipotent for both cholinesterases, being 1,2-diphenyl-5,6,7,8-tetrahydro-1H-pyrrolo[2,3-b]quinolin-4-amine (12) the most potent for the inhibition of both enzymes [IC(50) (EeAChE)=0.61 +/- 0.04 muM; IC(50) (eqBuChE)=0.074 +/- 0.009 muM]. Moreover, pyrrolotacrine 12, at concentrations as low as 300 nM can afford significant neuroprotective effects against Abeta-induced toxicity. Docking studies show that compounds 3 and 12 bind in the middle of the AChE active site gorge, but are buried deeper inside BuChE active site gorge, as a consequence of larger BuChE gorge void. All these data suggest that these new tacrine analogues could be used for the potential treatment of Alzheimer's disease.
The multifactorial nature of Alzheimer's disease (AD) offers us a textbook example where parental compounds, mostly marketed, are modified with the aim of improving and/or conferring two or even more biological activities to contrast or less frequently revert the disease's symptoms. This is the case of tacrine and its dimeric derivative bis(7)-tacrine which, for instance, paved the way for the development of a broad collection of very interesting homo- and heterodimeric structures, conceived in light of the emerging multi-target approach for AD-related drug discovery. As a contribution to the topic, we report here the design, synthesis and biological evaluation of 12 compounds referable to bis(7)-tacrine. In addition to the cholinesterase activity, some of the selected compounds (7-9 and 12) were capable of inhibiting the non-enzymatic function of AChE and/or showed a remarkable activity against BACE1. Thus, the present study outlines a series of newly synthesized molecules, structurally related to bis(7)-tacrine, endowed with extended biological profile in agreement with the emerging multi-target paradigm.
Lipoic acid (LA) is a natural antioxidant. Its structure was previously combined with that of the acetylcholinesterase inhibitor tacrine to give lipocrine (1), a lead compound multitargeted against Alzheimer's disease (AD). Herein, we further explore LA as a privileged structure for developing multimodal compounds to investigate AD. First, we studied the effect of LA chirality by evaluating the cholinesterase profile of 1's enantiomers. Then, a new series of LA hybrids was designed and synthesized by combining racemic LA with motifs of other known anticholinesterase agents (rivastigmine and memoquin). This afforded 4, which represents a step forward in the search for balanced anticholinesterase and antioxidant capacities.
Two novel families of dual binding site acetylcholinesterase (AChE) inhibitors have been developed, consisting of a tacrine or 6-chlorotacrine unit as the active site interacting moiety, either the 5,6-dimethoxy-2-[(4-piperidinyl)methyl]-1-indanone fragment of donepezil (or the indane derivative thereof) or a 5-phenylpyrano[3,2-c]quinoline system, reminiscent to the tryciclic core of propidium, as the peripheral site interacting unit, and a linker of suitable length as to allow the simultaneous binding at both sites. These hybrid compounds are all potent and selective inhibitors of human AChE, and more interestingly, are able to interfere in vitro both formation and aggregation of the beta-amyloid peptide, the latter effects endowing these compounds with the potential to modify Alzheimer's disease progression.
A new family of dual binding site acetylcholinesterase (AChE) inhibitors has been designed, synthesized, and tested for their ability to inhibit AChE, butyrylcholinesterase (BChE), AChE-induced and self-induced beta-amyloid (Abeta) aggregation and beta-secretase (BACE-1), and to cross the blood-brain barrier. The new heterodimers consist of a unit of racemic or enantiopure huprine Y or X and a donepezil-related 5,6-dimethoxy-2-[(4-piperidinyl)methyl]indane moiety as the active site and peripheral site to mid-gorge-interacting moieties, respectively, connected through a short oligomethylene linker. Molecular dynamics simulations and kinetics studies support the dual site binding to AChE. The new heterodimers are potent inhibitors of human AChE and moderately potent inhibitors of human BChE, AChE-induced and self-induced Abeta aggregation, and BACE-1, and are predicted to be able to enter the central nervous system (CNS), thus constituting promising multitarget anti-Alzheimer drug candidates with the potential to modify the natural course of this disease.
Title: Immobilized butyrylcholinesterase in the characterization of new inhibitors that could ease Alzheimer's disease Bartolini M, Greig NH, Yu QS, Andrisano V Ref: Journal of Chromatography A, 1216:2730, 2009 : PubMed
Focus of this work was the development and characterization of a new immobilized enzyme reactor (IMER) containing human recombinant butyrylcholinesterase (rBChE) for the on-line kinetic characterization of specific, pseudo-irreversible and brain-targeted BChE inhibitors as potential drug candidates for Alzheimer's disease (AD). Specifically, a rBChE-IMER containing 0.99 U of covalently bound target enzyme was purposely developed and inserted into a HPLC system connected to a UV-vis detector. Selected reversible cholinesterase inhibitors, (-)-phenserine and (-)-cymserine analogues, were then kinetically characterized by rBChE-IMER, and by classical in solution assays and their carbamoylation and decarbamoylation constants were determined. The results support the elucidation of the potency, inhibition duration, mode of action and specific structure/activity relations of these agents and allow cross-validation of the two assay techniques.
Starting from a structure-based drug design, new acetylcholinesterase inhibitors were designed and synthesized as analogues of donepezil. The compounds were composed by an aromatic function and a tertiary amino moiety connected by a suitable spacer. In particular, the benzophenone nucleus and the N,N-benzylmethylamine function were selected. The easily accessible three-step synthesis of these compounds resulted to be significantly less difficult and expensive than that of donepezil. Several compounds possess anti-cholinesterase activity in the order of micro and sub-micromolar. Particularly, compounds 1 and 10 were the most potent inhibitors of the series.
The multifunctional nature of Alzheimer's disease (AD) provides the logical foundation for the development of an innovative drug design strategy centered on multi-target-directed-ligands (MTDLs). In recent years, the MTDL concept has been exploited to design different ligands hitting different biological targets. Our first rationally designed MTDL was the polyamine caproctamine (1), which provided a synergistic cholinergic action against AD by antagonizing muscarinic M(2) autoreceptors and inhibiting acetylcholinesterase (AChE). Lipocrine (7) represented the next step in our research. Due to its ability to inhibit AChE catalytic and non-catalytic functions together with oxidative stress, 7 emerged as an interesting pharmacological tool for investigating the neurodegenerative mechanism underlying AD. Memoquin (9) is a quinone-bearing polyamine endowed with a unique multifunctional profile. With its development, we arrived at the proof of concept of the MTDL drug discovery approach. Experiments in vitro and in vivo confirmed its multimodal mechanisms of action and its interaction with different end-points of the neurotoxic cascade leading to AD. More recently, the MTDL approach led to carbacrine (12). In addition to the multiple activities displayed by 7, 12 displayed an interesting modulation of NMDA receptor activity. The pivotal role played by this target in AD pathogenesis suggests that 12 may be a promising new chemical entity in the MTDL gold rush.
Title: Structure-activity relationships of memoquin: Influence of the chain chirality in the multi-target mechanism of action Bolognesi ML, Bartolini M, Rosini M, Andrisano V, Melchiorre C Ref: Bioorganic & Medicinal Chemistry Lett, 19:4312, 2009 : PubMed
The present article expands on the study of structure-activity relationships of the novel class of quinone-bearing polyamines, as multi-target-directed ligands against Alzheimer's disease. Namely, the effect of inserting a methyl substituent at the alpha position of the terminal benzyl amine moieties of lead candidate 1 (memoquin) was evaluated at the multiple targets involved in the multifunctional mechanism of action. The RR stereoisomer 2 resulted more effective than 1 in reverting two important effects mediated by acetylcholinesterase (AChE), that is, acetylcholine hydrolysis and AChE-induced amyloid-beta aggregation.
Novel multitargeted antioxidants 3-6 were designed by combining the antioxidant features, namely, a benzoquinone fragment and a lipoyl function, of two multifunctional lead candidates. They were then evaluated to determine their profile against Alzheimer's disease. They showed antioxidant activity, improved following enzymatic reduction, in mitochondria and T67 cell line. They also displayed a balanced inhibitory profile against amyloid-beta aggregation and acetylcholinesterase, emerging as promising molecules for neuroprotectant lead discovery.
Two isomeric series of dual binding site acetylcholinesterase (AChE) inhibitors have been designed, synthesized, and tested for their ability to inhibit AChE, butyrylcholinesterase, AChE-induced and self-induced beta-amyloid (Abeta) aggregation, and beta-secretase (BACE-1) and to cross blood-brain barrier. The new hybrids consist of a unit of 6-chlorotacrine and a multicomponent reaction-derived pyrano[3,2-c]quinoline scaffold as the active-site and peripheral-site interacting moieties, respectively, connected through an oligomethylene linker containing an amido group at variable position. Indeed, molecular modeling and kinetic studies have confirmed the dual site binding of these compounds. The new hybrids, and particularly 27, retain the potent and selective human AChE inhibitory activity of the parent 6-chlorotacrine while exhibiting a significant in vitro inhibitory activity toward the AChE-induced and self-induced Abeta aggregation and toward BACE-1, as well as ability to enter the central nervous system, which makes them promising anti-Alzheimer lead compounds.
Structure-activity relationship studies on acetylcholinesterase (AChE) inhibitors were extended to newly synthesized compounds derived from the lead compound xantostigmine (1). The xanthone ring of compound 1 was replaced with several different scaffolds based on the benzopyran skeleton, linked to the tertiary amino nitrogen through an heptyloxy chain. These modifications resulted in 19 new compounds, most of them showing activity in the nanomolar-subnanomolar range. Docking and molecular dynamics simulations were carried out to both define a new computational protocol for the simulation of pseudo-irreversibile AChE covalent inhibitors, and to acquire a better understanding of the structure-activity relationships of the present series of compounds. The results of this computational work prompted us to to evaluate the ability of compounds 5 and 13 to inhibit acetylcholinesterase-induced Abeta aggregation.
A novel series of donepezil-tacrine hybrids designed to simultaneously interact with the active, peripheral and midgorge binding sites of acetylcholinesterase (AChE) have been synthesized and tested for their ability to inhibit AChE, butyrylcholinesterase (BChE), and AChE-induced A beta aggregation. These compounds consist of a unit of tacrine or 6-chlorotacrine, which occupies the same position as tacrine at the AChE active site, and the 5,6-dimethoxy-2-[(4-piperidinyl)methyl]-1-indanone moiety of donepezil (or the indane derivative thereof), whose position along the enzyme gorge and the peripheral site can be modulated by a suitable tether that connects tacrine and donepezil fragments. All of the new compounds are highly potent inhibitors of bovine and human AChE and BChE, exhibiting IC50 values in the subnanomolar or low nanomolar range in most cases. Moreover, six out of the eight hybrids of the series, particularly those bearing an indane moiety, exhibit a significant A beta antiaggregating activity, which makes them promising anti-Alzheimer drug candidates.
BACKGROUND: Paraoxonase is high-density lipoprotein (HDL)-associated esterase/lactonase implicated to play a role in the antioxidant and anti-inflammatory properties exerted by HDL. Increasing evidence support a role of free radicals and oxidative stress in neuronal damage induced by ischemia-reperfusion. The aim of this study was to further investigate the relationship between lipoprotein oxidative damage and stroke. METHODS: We compared the paraoxonase activity and levels of lipid hydroperoxides in plasma isolated from healthy subjects (n=50) and from stroke patients (n=49). Moreover, the correlations between biochemical markers and the National Institute of Health Stroke Scale (NIHSS), which is widely used to study neurological severity, were evaluated. RESULTS: Our results demonstrated, for the first time, that the activity of paraoxonase in plasma of stroke subjects was significantly lower than controls (p<0.001) and the levels of lipid hydroperoxides were significantly higher in plasma from patients (p<0.001). Moreover, using linear regression analysis, significant correlations between the activity of paraoxonase, lipid peroxidation and the severity of neurological deficit at admission were observed. CONCLUSIONS: These results provide further evidence that oxidative stress and impairment of the antioxidant system may play a role in stroke. Antioxidant activity of plasma may be an important factor providing protection from neurological damage caused by stroke-associated oxidative stress.
Alkali cations can affect the catalytic efficiency of enzymes. This is particularly true when dealing with enzymes whose substrate bears a formal positive charge. Computational and biochemical approaches have been combined to shed light on the atomic aspects of the role of Li(+), Na(+), and K(+) on human acetylcholinesterase (hAChE) ligand binding. In this respect, molecular dynamics simulations and our recently developed metadynamics method were applied to study the entrance of the three cations in the gorge of hAChE, and their effect on the dynamical motion of a ligand (tetramethylammonium) from the bulk of the solvent into the deep narrow enzyme gorge. Furthermore, in order to support the theoretical results, K(M) and k(cat) for the acetylcholine hydrolysis in the presence of the three cations were evaluated by using an approach based on the Ellman's method. The combination of computational and biochemical experiments clearly showed that Li(+), Na(+), and K(+) may influence the ligand binding at the hAChE gorge.
The complex etiology of Alzheimer's disease (AD) prompts scientists to develop multifunctional compounds to combat causes and symptoms of such neurodegeneration. To this aim we designed, synthesized, and tested a series of compounds by introducing halophenylalkylamidic functions on the scaffold of AP2238, which is a dual binding site acetylcholinesterase inhibitor. The inhibitory activity was successfully extended to the beta-site amyloid precursor protein cleavage enzyme, leading to the discovery of a potent inhibitor of this enzyme (3) and affording multifunctional compounds (2, 6, 8) for the treatment of AD.
The complex etiology of Alzheimer's disease (AD) prompts scientists to develop multitarget strategies to combat causes and symptoms. We therefore designed, synthesized, and tested new hybrid molecules linking a benzofuran ring to a N-methyl- N-benzylamine through a heptyloxy chain, affording a series of potential multifunctional drugs for AD. The cholinesterase inhibitory activity was extended to the inhibition of Abeta fibril formation for 1, 3, and 5. Compound 3 showed an additional neuroprotective effect.
Alzheimer's disease (AD) is a multifactorial syndrome with several target proteins contributing to its etiology. To confront AD, an innovative strategy is to design single chemical entities able to simultaneously modulate more than one target. Here, we present compounds that inhibit acetylcholinesterase and NMDA receptor activity. Furthermore, these compounds inhibit AChE-induced Abeta aggregation and display antioxidant properties, emerging as lead candidates for treating AD.
Novel multi-target-directed ligands were designed by replacing the inner dipiperidino function of 3 with less flexible or completely rigid moieties to obtain compounds endowed with multiple biological properties that might be relevant to Alzheimer's disease. 15 was the most interesting, inhibiting AChE in the nanomolar range and inhibiting AChE-induced and self-promoted beta-amyloid aggregation in the micromolar range.
Title: Characterization of reversible and pseudo-irreversible acetylcholinesterase inhibitors by means of an immobilized enzyme reactor Bartolini M, Cavrini V, Andrisano V Ref: Journal of Chromatography A, 1144:102, 2007 : PubMed
The aim of the present study was the application of a human AChE-CIM-IMER (enzyme reactor containing acetylcholinesterase immobilized on a monolithic disk) for the rapid evaluation of the thermodynamic and kinetic constants, and the mechanism of action of new selected inhibitors. For this application, human recombinant AChE was covalently immobilized onto an ethylenediamine (EDA) monolithic Convective Interaction Media (CIM) disk and on-line studies were performed by inserting this IMER into a HPLC system. Short analysis time, absence of backpressure, low nonspecific matrix interactions and immediate recovery of enzyme activity were the best characteristics of this AChE-CIM-IMER. Mechanisms of action of selected reversible inhibitors (tacrine, donepezil, edrophonium, ambenonium) were evaluated by means of Lineweaver-Burk plot analysis. Analyses were performed on-line by injecting increasing concentrations of the tested inhibitor and substrate and by monitoring the product peak area. AChE-CIM-IMER kinetic parameters (Km(app) and vmax(app)) were derived as well as inhibitory constants (Ki(app)) of selected compounds. Moreover, noteworthy results were obtained in the application of the AChE-CIM-IMER to the characterization of the carbamoylation and decarbamoylation steps in pseudo-irreversible binding of carbamate derivatives (physostigmine and rivastigmine). AChE-CIM-IMER appeared to be a valid tool to determine simultaneously the kinetic constants in a reliable and fast mode. The obtained values were found in agreement with those obtained with the classical methods with the free enzyme. Furthermore, after inactivation by carbamates, activity could be fully recovered and the AChE-CIM-IMER could be reused for further studies. Results showed that the AChE-CIM-IMER is a valid tool not only for automated fast screening in the first phase of the drug discovery process but also for the finest characterization of the mode of action of new hit compounds with increased accuracy and reproducibility and with saving of time and materials.
A design strategy to convert a dual-binding site AChE inhibitor into triple functional compounds with promising in vitro profile against multifactorial syndromes, such as Alzheimer's disease, is proposed. The lead compound bis(7)-tacrine (2) was properly modified to confer to the new molecules the ability of chelating metals, involved in the neurodegenerative process. The multifunctional compounds show activity against human AChE, are able to inhibit the AChE-induced amyloid-beta aggregation, and chelate metals, such as iron and copper.
In this work, we further investigated a previously introduced class of cholinesterase inhibitors. The removal of the carbamic function from the lead compound xanthostigmine led to a reversible cholinesterase inhibitors 3. Some new 3-[omega-(benzylmethylamino)alkoxy]xanthen-9-one analogs were designed, synthesized, and evaluated for their inhibitory activity against both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The length of the alkoxy chain of compound 3 was increased and different substituents were introduced. From the IC(50) values, it clearly appears that the carbamic residue is crucial to obtain highly potent AChE inhibitors. On the other hand, peculiarity of these compounds is the high selectivity toward BuChE with respect to AChE, being compound 12 the most selective one (6000-fold). The development of selective BuChE inhibitors may be of great interest to clarify the physiological role of this enzyme and to provide novel therapeutics for various diseases.
AP2238 was the first compound published to bind both anionic sites of the human acetylcholinesterase, allowing the simultaneous inhibition of the catalytic and the amyloid-beta pro-aggregating activities of AChE. Here we attempted to derive a comprehensive structure-activity relationship picture for this molecule, affording 28 derivatives for which AChE and BChE inhibitory activities were evaluated. Selected compounds were also tested for their ability to prevent the AChE-induced Abeta-aggregation. Moreover, docking simulations and molecular orbital calculations were performed.
Title: Analysis of Amaryllidaceae alkaloids from Narcissus by GC-MS and capillary electrophoresis Gotti R, Fiori J, Bartolini M, Cavrini V Ref: J Pharm Biomed Anal, 42:17, 2006 : PubMed
Amaryllidaceae are known as ornamental plants, furthermore some species of this family contain galanthamine, an acetylcholinesterase inhibitor approved for the treatment of Alzheimer's disease, and other alkaloids with interesting pharmacological activity. In the present work, the quali- and quantitative analysis of Amaryllidaceae-type alkaloids in the bulbs of Narcissus species is presented using different analytical approaches. Extracts of Narcissus pseudonarcissus cv. Carlton and Narcissus jonquilla Quail, were first examined by GC-MS using a Rtx-5 MS (programmed temperature) and the major alkaloids were identified. Together with galanthamine, high contents of haemanthamine, were found. Galanthamine was reliably quantified by GC-MS, whereas haemanthamine partly decomposed under the GC conditions, thus alternative analytical methods were investigated. Firstly, reversed-phase HPLC-ESI-MS was applied to identify and isolate at semipreparative levels haemanthamine. The compound was fully characterized by MS/MS and (1)H NMR and then used as a reference substance. The quantitation of both galanthamine and haemanthamine was then accomplished by capillary electrophoresis with spectrophotometric detection. A non-aqueous (NACE) approach was selected in order to use a running buffer fully compatible with samples in organic solvent. In particular, a mixture methanol-acetonitrile (75:25, v/v) containing ammonium acetate (90 mM) was used as a background electrolyte. The same analytical sample was subjected to GC-MS and NACE analysis; the different selectivity displayed by these techniques allowed different separation profiles that can be useful in phytochemical characterization of the extracts. The GC-MS and NACE methods were validated and applied to the quantitation of galanthamine (GC-MS and NACE) and haemanthamine (NACE) in bulbs of N. jonquilla.
Title: Batchwise covalent immobilization of human acetylcholinesterase: Kinetic and inhibition spectrophotometric studies Bartolini M, Cavrini V, Andrisano V Ref: Analytical Biochemistry, 342:163, 2005 : PubMed
Title: Choosing the right chromatographic support in making a new acetylcholinesterase-micro-immobilised enzyme reactor for drug discovery Bartolini M, Cavrini V, Andrisano V Ref: Journal of Chromatography A, 1065:135, 2005 : PubMed
The aim of the present study was to optimize the preparation of an immobilized acetylcholinesterase (AChE)-based micro-immobilized enzyme reactor (IMER) for inhibition studies. For this purpose two polymeric monolithic disks (CIM, 3 mm x 12 mm i.d.) with different reactive groups (epoxy and ethylendiamino) and a packed silica column (3 mm x 5 mm i.d.; Glutaraldehyde-P, 40 microm) were selected as solid chromatographic supports. All these reactors were characterized in terms of rate of immobilization, stability, conditioning time for HPLC analyses, optimum mobile phase and peak shape, aspecific interactions and costs. Advantages and disadvantages were defined for each system. Immobilization through Schiff base linkage gave more stable reactors without any significant change in the enzyme behaviour; monolithic matrices showed very short conditioning time and fast recovery of the enzymatic activity that could represent very important features in high throughput analysis and satisfactory reproducibility of immobilization yield. Unpacked silica material allowed off-line low costs studies for the optimization of the immobilization step.
In continuing research that led us to identify a new class of carbamate derivatives acting as potent (Rampa et al. J. Med. Chem. 1998, 41, 3976) and long-lasting (Rampa et al. J. Med. Chem. 2001, 44, 3810) acetylcholinesterase (AChE) inhibitors, we obtained some analogues able to simultaneously block both the catalytic and the beta-amyloid (Abeta) proaggregatory activities of AChE. The key feature of these derivatives is a 2-arylidenebenzocycloalkanone moiety that provides the ability to bind at the AChE peripheral site responsible for promoting the Abeta aggregation. The new carbamates were tested in vitro for the inhibition of both cholinesterases and also for the ability to prevent the AChE-induced Abeta aggregation. All of the compounds had AChE IC(50) values in the nanomolar range and showed the ability to block the AChE-induced Abeta aggregation, thus supporting the feasibility of this new strategy in the search of compounds for the treatment of Alzheimer's disease.
Title: Propidium-based polyamine ligands as potent inhibitors of acetylcholinesterase and acetylcholinesterase-induced amyloid-beta aggregation Bolognesi ML, Andrisano V, Bartolini M, Banzi R, Melchiorre C Ref: Journal of Medicinal Chemistry, 48:24, 2005 : PubMed
Heterodimers 4 and 5 were effective inhibitors of acetylcholinesterase (AChE) activity and AChE-induced amyloid-beta (A beta) aggregation. The peculiar biological profile of 4 can be relevant in studying the molecular basis underlying the nonclassical action of AChE and in addressing the question whether AChE inhibitors can affect the neurotoxic cascade leading to Alzheimer's disease. Compound 4 emerged as the most potent heterodimer so far available to inhibit AChE-induced A beta aggregation.
Notwithstanding the criticism to the so called " cholinergic hypothesis", the therapeutic strategies for the treatment of Alzheimer's disease (AD) have been mainly centered on the restoration of cholinergic functionality and, until the last year, the only drugs licensed for the management of AD were the acetycholinesterase (AChE) inhibitors. Target enzyme AChE consists of a narrow gorge with two separate ligand binding sites: an acylation site at the bottom of the gorge containing the catalytic triad and a peripheral site located at the gorge rim, which encompasses binding sites for allosteric ligands. The aim of this short review is to update the knowledge on heterocyclic AChE inhibitors able to interact with the two sites of enzymes, structurally related to the well known inhibitors physostigmine, rivastigmine and propidium. The therapeutic potential of the dual site inhibithors in inhibiting amyloid-beta aggregatrion and deposition is also briefly summarised.
New dual binding site acetylcholinesterase (AChE) inhibitors have been designed and synthesized as new potent drugs that may simultaneously alleviate cognitive deficits and behave as disease-modifying agents by inhibiting the beta-amyloid (A beta) peptide aggregation through binding to both catalytic and peripheral sites of the enzyme. Particularly, compounds 5 and 6 emerged as the most potent heterodimers reported so far, displaying IC50 values for AChE inhibition of 20 and 60 pM, respectively. More importantly, these dual AChE inhibitors inhibit the AChE-induced A beta peptide aggregation with IC50 values 1 order of magnitude lower than that of propidium, thus being the most potent derivatives with this activity reported up to date. We therefore conclude that these compounds are very promising disease-modifying agents for the treatment of Alzheimer's disease (AD).
The coupling of two different pharmacophores, each endowed with different biological properties, afforded the hybrid compound lipocrine (7), whose biological profile was markedly improved relative to those of prototypes tacrine and lipoic acid. Lipocrine is the first compound that inhibits the catalytic activity of AChE and AChE-induced amyloid-beta aggregation and protects against reactive oxygen species. Thus, it emerged as a valuable pharmacological tool to investigate Alzheimer's disease and as a promising lead compound for new anti-Alzheimer drugs.
Title: Monolithic micro-immobilized-enzyme reactor with human recombinant acetylcholinesterase for on-line inhibition studies Bartolini M, Cavrini V, Andrisano V Ref: Journal of Chromatography A, 1031:27, 2004 : PubMed
The development and characterization of a human recombinant acetylcholinesterase (hrAChE) micro-immobilized-enzyme reactor (IMER), prepared by using an in situ immobilization procedure is reported. hrAChE was covalently immobilized on an ethylenediamine (EDA) monolithic convective interaction media (CIM) disk (12 mm x 3 mm i.d.), previously derivatized with glutaraldehyde. The optimal conditions for the immobilization were: 12 microg of enzyme dissolved in 800 microl of phosphate buffer (50 mM, pH 6.0). The mixture was gently agitated overnight at 4 degrees C. The resulting Schiff bases were reduced by cyanoborohydride and the remaining aldehydic groups were condensed with monoethanolamine. Under these conditions, 0.22 U of hrAChE were immobilized with retention of 3.0% of the initial enzymatic activity. The activity of the immobilized hrAChE was stable for over 60 days. The activity and kinetic parameters of the hrAChE micro-IMER were investigated by inserting the micro-IMER in a HPLC system and it was demonstrated that the enzyme retained its activity. The micro-IMER was characterized in terms of units of immobilized enzyme and best conditions for immobilization yield. IMERs were compared for their relative enzyme stability, immobilized units, yield and aspecific matrix interactions. The effect of AChE inhibitors was evaluated by the simultaneous injection of each inhibitor with the substrate. The relative IC50 values were found in agreement with those derived by the conventional kinetic spectrophotometric method. In comparison with previously developed AChE-based IMERs, AChE monolithic micro-IMER showed advantages in terms of reduction of analysis time (2 min), lower aspecific matrix interactions and lower backpressure. Included in a HPLC system, it can be used for the rapid screening of new compounds' inhibitory potency. The advantages over the conventional methods are the increased enzyme stability and system automation which allows a large number of compounds to be analyzed in continuous.
Rivastigmine (1), an acetylcholinesterase (AChE) inhibitor approved in 2000 for the treatment of Alzheimer disease, bears a carbamate moiety in its structure, which is able to react covalently with the active site of the enzyme. Kinetic and structural studies on the interaction of 1 with different cholinesterases have been published, giving deeper, but not definitive, insights on the catalysis mechanism. On the basis of these findings and in connection with our previous studies on a series of benzopyrano[4,3-b]pyrrole carbamates as AChE inhibitors, we designed a series of conformationally restricted analogues of 1 by including the dimethylamino-alpha-methylbenzyl moiety in different tricyclic systems. A superimposition between the conformation of 1 and the carbon derivative 4, as obtained from Monte Carlo simulations, supported the idea that the tricyclic derivatives might act as rigid analogues of 1. The biological profile of 4-9, assessed in vitro against human AChE and BChE, validated our rational design. Compound 5, bearing a sulfur-containing system, showed the highest inhibitory activity, being 192-fold more potent than 1. In the present study, the most potent inhibitors were always methyl derivatives 3-5, endowed with a nanomolar range potency, whereas the ethyl ones were 40 times less potent. A reasonable explanation for this finding might be a steric hindrance effect between the ethyl group of 1 and His440 in the active site, as already suggested by the crystal structure of the complex AChE/1. The unfavorable influence of the carbamic N-alkyl chain on AChE inhibition is less striking when considering BChE inhibition, since BChE is characterized by a bigger acyl binding pocket than AChE. In fact, methyl carbamates 3-5 did not show AChE/BChE selectivity, whereas compounds 6-9 were significantly more potent in inhibiting BChE than AChE activity. At 100 microM, 5 was found to inhibit the AChE-induced aggregation only by 19% likely because it is not able to strongly interact with the peripheral anionic site of AChE, which plays an essential role in the Abeta aggregation mediated by the enzyme but is lacking in BChE structure.
Title: Structure-activity relationships of acetylcholinesterase noncovalent inhibitors based on a polyamine backbone. 3. Effect of replacing the inner polymethylene chain with cyclic moieties Tumiatti V, Andrisano V, Banzi R, Bartolini M, Minarini A, Rosini M, Melchiorre C Ref: Journal of Medicinal Chemistry, 47:6490, 2004 : PubMed
In the present paper we expanded SAR studies of 3, the ethyl analogue of the AChE inhibitor caproctamine (2), by investigating the role of its octamethylene spacer separating the two amide functions through the replacement with dipiperidine and dianiline moieties. Compounds 4 and 8 were the most interesting of the two series of compounds. Compound 4 was the most potent AChE inhibitor with a pIC50 value of 8.48 +/- 0.02, while displaying also significant muscarinic M2 antagonistic activity (pKb value of 6.18 +/- 0.20). The availability of a suitable assay allowed us to verify whether 2, 3, 4, and 8 inhibit AChE-induced Abeta aggregation. Although all four derivatives caused a mixed type of AChE inhibition (active site and PAS), only 4 and 8, which bear an inner constrained spacer, were able to inhibit AChE-induced Abeta aggregation to a greater extent than donepezil. Clearly, the ability of an AChE inhibitor, based on a linear polyamine backbone, to bind both AChE sites may not be a sufficient condition to inhibit also AChE-induced Abeta aggregation. Dipiperidine derivative 4 emerged as a valuable pharmacological tool and a promising lead compound for new ligands to investigate and, hopefully, treat Alzheimer's disease.
Title: beta-Amyloid aggregation induced by human acetylcholinesterase: inhibition studies Bartolini M, Bertucci C, Cavrini V, Andrisano V Ref: Biochemical Pharmacology, 65:407, 2003 : PubMed
The aggregation of beta-amyloid (1-40) (Abeta) induced by human recombinant acetylcholinesterase (HuAChE) was studied by means of circular dichroism (CD) and by thioflavin T fluorescence spectroscopy. Abeta was incubated alone and with HuAChE. The kinetic of fibrils formation was followed for 48 hr. The increasing beta-conformation content induced by HuAChE, preliminary to the formation of Abeta fibrils, was determined by circular dichroism. This phenomenon was found to be related to the thioflavin T emission of fluorescence at 490 nm. Incubation experiments were performed in the presence of known AChE inhibitors (physostigmine, edrophonium, decamethonium, propidium) and drugs used for Alzheimer's disease (AD) (tacrine, donepezil), to test their capability of preventing the HuAChE-induced Abeta aggregation. The non-competitive or mixed mode of AChE inhibition was confirmed to be an essential feature. At 100 microM propidium, decamethonium, donepezil and physostigmine were found to inhibit the HuAChE-induced Abeta aggregation by 82, 25, 22 and 30%, respectively.
Title: 3-(4-[[Benzyl(methyl)amino]methyl]phenyl)-6,7-dimethoxy-2H-2-chromenone (AP2238) inhibits both acetylcholinesterase and acetylcholinesterase-induced beta-amyloid aggregation: a dual function lead for Alzheimer's disease therapy Piazzi L, Rampa A, Bisi A, Gobbi S, Belluti F, Cavalli A, Bartolini M, Andrisano V, Valenti P, Recanatini M Ref: Journal of Medicinal Chemistry, 46:2279, 2003 : PubMed
In recent years, the investigation of acetylcholinesterase (AChE) inhibitors has gained further interest, because the involvement of the peripheral site of the enzyme in the beta-amyloid (Abeta) aggregation process has been disclosed. We present here, for the first time, a direct evidence of the Abeta antiaggregating action of an AChE inhibitor (AP2238) purposely designed to bind at both the catalytic and the peripheral sites of the human enzyme.
Continuing our studies on polyamine-based compounds of potential interest in the field of Alzheimer's disease therapeutics, we investigated the structure-activity relationships (SAR) of a lead compound (caproctamine, 3) identified in a previous work. In particular, we varied the substituents on the phenyl ring and on the nitrogen functions (both the amine and the amide), and studied the effects of such modifications on the inhibitory potency against isolated acetyl- and butyryl-cholinesterase (AChE and BChE). Moreover, the ability of selected compounds to reverse the d-tubocurarine-induced neuromuscular blockade and their antagonism toward muscarinic M(2) receptors in guinea pig left atrium were assayed. The most interesting SAR result was the identification of a relationship between the electronic characteristics of 2-substituents (measured by pK(a)) and the AChE inhibitory potency (pIC(50)) of tertiary amine compounds 6-12, which was confirmed by the invariance of the pIC(50) values of the corresponding methiodide derivatives 14-20. With regard to the biological profile, the most interesting compound was the N-ethyl-analogue of caproctamine (9), that showed pIC(50) values of 7.73 (+/-0.02) and 5.65 (+/-0.03) against AChE and BChE, respectively. The ability to increase the acetylcholine level was maintained in the functional assay (pAI(50) for reversing the neuromuscular blockade was 6.45 (+/-0.07)), as well as the ability to antagonize the M(2) receptors (pK(b) = 5.65 (+/-0.06)). Moreover, 9 showed a long duration of action as AChE inhibitor, an useful property in view of a possible development of this compound as a therapeutic agent.
An RP-HPLC study for the pKa determination of a series of basic compounds related to caproctamine, a dibenzylaminediamide reversible inhibitor of acetylcholinesterase, is reported. The 2-substituted analogues, bearing substituents with different electronegativity, were analysed by RP-HPLC by using C18 C4 stationary phases with a mobile phase consisting of mixture of acetonitrile and triethylamine phosphate buffer (pH range comprised between 4 and 10). Typical sigmoidal curves were obtained, showing the dependence of the capacity factors upon pH. In general, the retention of the investigated basic analytes increased with increasing of the pH. The inflection point of the pH sigmoidal dependence was used for the dissociation constant determination at a fixed acetonitrile percentage. When plotting pKa vs. percent of acetonitrile in the mobile phase for two representative compounds, linear regression were obtained: the y intercept gave the aqueous pKa(w). The pKa estimation by HPLC method was found to be useful to underline the difference of benzylamine basicity produced by the ortho aromatic substituents. The variation of pKa values (6.15-7.80) within the series of compounds was correlated with the electronic properties of the ortho-substituents through the Hammett sigma parameter, whereas the ability of substituents to accept H-bond was found to play a role in determining the conformational behavior of the molecules.
The present article expands on the study of another aspect of structure-activity relationships of the polymethylene tetraamines, namely, the effect of inserting the tetraamine backbone into a macrocyclic structure. To this end, compounds 8-12 were designed by linking the two terminal nitrogen atoms of prototype methoctramine 2 to an aryl moiety. Alternatively, 2 was first modified to achieve compounds 6 and 7, which in turn were cyclized by linking the two terminal primary amine functions to a polyphenyl spacer, affording 13-20. All the compounds were tested on muscle-type nAChRs and most of them as well on AChE. Furthermore, selected compounds were tested also on peripheral M(2) and M(3) mAChRs. All these cyclic derivatives, like prototypes, were potent noncompetitive antagonists at both frog and Torpedo nAChRs, suggesting that polyamines do not need to be linear or in extended conformation to optimally interact with the nicotinic channel; rather, they may bind in a U-shaped conformation. Relative to muscarinic activity, macrocyclic compounds 10, 13, 14, and 20, in contrast with the profile displayed by 2, were almost devoid of affinity. It is derived that an aryl spacer is detrimental to the interaction of polyamines with mAChRs. Finally, all the diamine diamides investigated in this study were much less potent in inhibiting AChE activity than prototype 3, suggesting that a macrocyclic structure may not be suitable for AChE inhibition.
Title: Determination of inhibitors' potency (IC50) by a direct high-performance liquid chromatographic method on an immobilised acetylcholinesterase column Andrisano V, Bartolini M, Gotti R, Cavrini V, Felix G Ref: Journal of Chromatography B Biomed Sci Appl, 753:375, 2001 : PubMed
An immobilised acetylcholinesterase (AChE) stationary phase was prepared by using an in situ AChE immobilisation procedure. A stainless steel column packed with epoxide silica was connected to the HPLC system and the enzyme solution at pH 5.8 was recycled through the column at a flow-rate of 0.5 ml/min for 24 h. The activity of the immobilised AChE was determined by injecting the substrate acetylthiocholine, using as mobile phase 0.1 M phosphate buffer (pH 7.4) containing Ellman's reagent [5,5'-dithio-bis(2-nitrobenzoic acid)] and measuring the area of the obtained peak with UV detection at 412 nm. The effect of AChE inhibitors tacrine, edrophonium and donepezil were evaluated by the simultaneous injection of each inhibitor with the substrate. The resulting decrease in the AChE activity, as expressed by the decrease of the peak area detected at 412 nm, was related to the concentration and potency of the solutes. The obtained IC50 values were compared with those derived by the conventional spectrophotometric method. This immobilized enzyme reactor, included in a chromatographic system, can be used for the rapid screening for new inhibitors allowing for the on-line determination of a compound's inhibitory potency. The advantages over the conventional methods are the increased enzyme stability and system automation which allows a large number of compounds to be analysed continuously.
In a search for less flexible analogues of caproctamine (1), a diamine diamide endowed with an interesting AChE affinity profile, we discovered compound 2, in which the terminal 2-methoxybenzyl groups of 1 have been incorporated into a tricyclic system. Since this compound retains good AChE inhibitory activity and its hexahydrochromeno[4,3-b]pyrrole moiety is reminiscent of the hexahydropyrrolo[2,3-b]indole of physostigmine (3), we have designed and synthesized carbamates 4-6, and their biological evaluation has been assessed in vitro against human AChE and BChE. The 6-carbamate 4 was almost as potent as physostigmine and was 60- and 550-fold more potent than the 7-carbamate 5 and the 8-carbamate 6, respectively. The two enantiomers of 4, (-)-4 and (+)-4, did not show a marked enantioselectivity. Finally, a similar time-dependent pattern of inhibition of AChE was observed for 3 and 4.
In this work, we further investigated a class of carbamic cholinesterase inhibitors introduced in a previous paper (Rampa et al. J. Med. Chem. 1998, 41, 3976). Some new omega-[N-methyl-N-(3-alkylcarbamoyloxyphenyl)methyl]aminoalkoxyaryl analogues were designed, synthesized, and evaluated for their inhibitory activity against both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The structure of the lead compound (xanthostigmine) was systematically varied with the aim to optimize the different parts of the molecule. Moreover, such a structure-activity relationships (SAR) study was integrated with a kinetic analysis of the mechanism of AChE inhibition for two representative compounds. The structural modifications lead to a compound (12b) showing an IC(50) value for the AChE inhibition of 0.32 +/- 0.09 nM and to a group of BuChE inhibitors also active at the nanomolar level, the most potent of which (15d) was characterized by an IC(50) value of 3.3 +/- 0.4 nM. The kinetic analysis allowed for clarification of the role played by different molecular moieties with regard to the rate of AChE carbamoylation and the duration of inhibition. On the basis of the results presented here, it was concluded that the cholinesterase inhibitors of this class possess promising characteristics in view of a potential development as drugs for the treatment of Alzheimer's disease.
In this study, we attempted to derive a comprehensive SAR picture for the class of acetylcholinesterase (AChE) inhibitors related to tacrine, a drug currently in use for the treatment of the Alzheimer's disease. To this aim, we synthesized and tested a series of 9-amino-1,2,3,4-tetrahydroacridine derivatives substituted in the positions 6 and 7 of the acridine nucleus and bearing selected groups on the 9-amino function. By means of the Hansch approach, QSAR equations were obtained, quantitatively accounting for both the detrimental steric effect of substituents in position 7 and the favorable electron-attracting effect exerted by substituents in positions 6 and 7 of the 9-amino-1,2,3,4-tetrahydroacridine derivatives. The three-dimensional (3D) properties of the inhibitors were taken into consideration by performing a CoMFA analysis on the series of AChE inhibitors made by 12 9-amino-1,2,3, 4-tetrahydroacridines and 13 11H-indeno[1,2-b]quinolin-10-ylamines previously developed in our laboratory. The alignment of the molecules to be submitted to the CoMFA procedure was carried out by taking advantage of docking models calculated for the interactions of both the unsubstituted 9-amino-1,2,3,4-tetrahydroacridine and 11H-indeno[1,2-b]quinolin-10-ylamine with the target enzyme. A highly significant CoMFA model was obtained using the steric field alone, and the features of such a 3D QSAR model were compared with the classical QSAR equations previously calculated. The two models appeared consistent, the main aspects they had in common being (a) the individuation of the strongly negative contribution of the substituents in position 7 of tacrine and (b) a tentative assignment of the hydrophobic character to the favorable effect exerted by the substituents in position 6. Finally, a new previously unreported tacrine derivative designed on the basis of both the classical and the 3D QSAR equations was synthesized and kinetically evaluated, to test the predictive ability of the QSAR models. The 6-bromo-9-amino-1,2,3,4-tetrahydroacridine was predicted to have a pIC(50) value of 7.31 by the classical QSAR model and 7.40 by the CoMFA model, while its experimental IC(50) value was equal to 0.066 (+/-0.009) microM, corresponding to a pIC(50) of 7.18, showing a reasonable agreement between predicted and observed AChE inhibition data.
