Synthesis, in vitro assay, and molecular modeling of new piperidine derivatives having dual inhibitory potency against acetylcholinesterase and Abeta1-42 aggregation for Alzheimer's disease therapeutics

With the goal of developing Alzheimer's disease therapeutics, we have designed and synthesized new piperidine derivatives having dual action of acetylcholinesterase (AChE) and beta-amyloid peptide (Abeta) aggregation inhibition. For binding with the catalytic site of AChE, an ester with aromatic group was designed, and for the peripheral site, another aromatic group was considered. And for intercalating amyloid-beta oligomerization, long and linear conformation with a lipophilic group was considered. The synthetic methods employed for the structure with dual action depended on alcohols with an aromatic ring and the substituted benzoic acids, which are esterificated in the last step of the synthetic pathway. We screened these new derivatives through inhibition tests of acetylcholinesterase, butyrylcholinesterase (BChE), and Abeta(1-42) peptide aggregation, AChE-induced Abeta(1-42) aggregation. Our results displayed that compound 12 showed the best inhibitory potency and selectivity of AChE, and 29 showed the highest selectivity of BChE inhibition. Compounds 15 and 12 had inhibitory activities against Abeta(1-42) aggregation and AChE-induced Abeta aggregation. In the docking model, we confirmed that 4-chlorobenzene of 12 plays the parallel pi-pi stacking against the indole ring of Trp84 in the bottom gorge of AChE. Because the benzyhydryl moiety of 12 covered the peripheral site of AChE in a funnel-like shape, 12 showed good inhibitory potency against AChE and could inhibit AChE-induced Abeta(1-42) peptide aggregation.     

Synthesis and biological evaluation of novel quinazoline-triazole hybrid compounds with potential use in Alzheimer’s disease

A library of twelve quinazoline-triazole hybrid compounds were designed, synthesized and evaluated as a novel class of acetylcholinesterase inhibitors to treat Alzheimer’s disease (AD). The biological assay results demonstrated the ability of several hybrid compounds to inhibit AChE enzyme (IC₅₀ range = 0.2–83.9 µM). To understand the high potential activity of these compounds, molecular docking simulations were performed to get better insights into the mechanism of binding of quinazoline-triazole hybrid compounds. As expected, compounds 8a and 9a-b bind to both catalytic anionic site (CAS) and peripheral anionic site (PAS) in the active site of AChE enzyme, which implicates that these compounds could act as dual binding site inhibitors. These compounds were not cytotoxic and they also displayed appropriated physicochemical as well as pharmacokinetic profile to be developed as novel anti-AD drug candidates.     

Mono- or di-substituted imidazole derivatives for inhibition of acetylcholine and butyrylcholine esterases

Mono- or di-substituted imidazole derivatives were synthesized using a one-pot, two-step strategy. All imidazole derivatives were tested for AChE and BChE inhibition and showed nanomolar activity similar to that of the test compound donepezil and higher than that of tacrine. Structure activity relationship studies, docking studies to on X-ray crystal structure of AChE with PDB code 1B41, and adsorption, distribution, metabolism, and excretion (ADME) predictions were performed. The synthesized core skeleton was bound to important regions of the active site of AChE such as the peripheral anionic site (PAS), oxyanion hole (OH), and anionic subsite (AS). Selectivity of the reported test compounds was calculated and enzyme kinetic studies revealed that they behave as competitive inhibitors, while two of the test compounds showed noncompetitive inhibitory behavior. ADME predictions revealed that the synthesized molecules might pass through the blood brain barrier and intestinal epithelial barrier and circulate freely in the blood stream without binding to human serum albumin. While the toxicity of one compound on the WS1 (skin fibroblast) cell line was 1790 µM, its toxicity on the SH-SY5Y (neuroblastoma) cell line was 950 µM.     

Nantenine as an acetylcholinesterase inhibitor: SAR,enzyme kinetics and molecular modeling investigations

Nantenine, as well as a number of flexible analogs, were evaluated for acetylcholinesterase (AChE) inhibitory activity in microplate spectrophotometric assays based on Ellman’s method. It was found that the rigid aporphine core of nantenine is an important structural requirement for its anticholinesterase activity. Nantenine showed mixed inhibition kinetics in enzyme assays. Molecular docking experiments suggest that nantenine binds preferentially to the catalytic site of AChE but is also capable of interacting with the peripheral anionic site (PAS) of the enzyme, thus accounting for its mixed inhibition profile. The aporphine core of nantenine may thus be a useful template for the design of novel PAS or dual-site AChE inhibitors. Inhibiting the PAS is desirable for prevention of aggregation of the amyloid peptide Aβ, a major causative factor in the progression of Alzheimer’s disease (AD).     

Design,synthesis and neuroprotective evaluation of novel tacrine–benzothiazole hybrids as multi-targeted compounds against Alzheimer’s disease

Alzheimer’s disease (AD) is a multifactorial disorder with several target proteins contributing to its etiology. In search for multifunctional anti-AD drug candidates, taking into account that the acetylcholinesterase (AChE) and beta-amyloid (Aβ) aggregation are particularly important targets for inhibition, the tacrine and benzothiazole (BTA) moieties were conjugated with suitable linkers in a novel series of hybrids. The designed compounds (7a–7e) were synthesized and in vitro as well as in ex vivo evaluated for their capacity for the inhibition of acetylcholinesterase (AChE) and Aβ self-induced aggregation, and also for the protection of neuronal cells death (SHSY-5Y cells, AD and MCI cybrids). All the tacrine–BTA hybrids displayed high in vitro activities, namely with IC₅₀ values in the low micromolar to sub-micromolar concentration range towards the inhibition of AChE, and high percentages of inhibition of the self-induced Aβ aggregation. Among them, compound 7a, with the shortest linker, presented the best inhibitory activity of AChE (IC₅₀ = 0.34 μM), while the highest activity as anti-Aβ₄₂ self-aggregation, was evidenced for compound 7b (61.3%, at 50 μM. The docking studies demonstrated that all compounds are able to interact with both catalytic active site (CAS) and peripheral anionic site (PAS) of AChE. Our results show that compounds 7d and 7e improved cell viability in cells treated with Aβ₄₂ peptide. Overall, these multi-targeted hybrid compounds appear as promising lead compounds for the treatment of Alzheimer’s disease.     

Synthesis and inhibitory activity of ureidophosphonates,against acetylcholinesterase: Pharmacological assay and molecular modeling

A novel method has been developed for the synthesis of 1-ureidophosphonates through a three components condensation of aldehyde with amine and diethylphosphite in the presence of sulfanilic acid as catalyst followed by subsequent reaction of the product with isocyanate. This method is easy, rapid, and good yielding. The anticholinesterase (AChE) activities (inhibition potency through IC₅₀) of newly synthesized 1-ureidophosphonates were also investigated. The activities of the synthesized compounds toward the enzyme AChE were determined and compared in terms of their molecular structures and it was found, through molecular docking simulations, that the most potent derivative (compound 3i) inhibited the enzyme through binding to the peripheral anionic site (PAS) and not to its acylation site (A site).     

Exploration of natural compounds as sources of new bifunctional scaffolds targeting cholinesterases and beta amyloid aggregation: The case of chelerythrine

The presented project started by screening a library consisting of natural and natural based compounds for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity. Active compounds were chemically clustered into groups and further tested on the human cholinesterases isoforms. The aim of the presented study was to identify compounds that could be used as leads to target two key mechanisms associated with the AD’s pathogenesis simultaneously: cholinergic depletion and beta amyloid (Aβ) aggregation. Berberin, palmatine and chelerythrine, chemically clustered in the so-called isoquinoline group, showed promising cholinesterase inhibitory activity and were therefore further investigated. Moreover, the compounds demonstrated moderate to good inhibition of Aβ aggregation as well as the ability to disaggregate already preformed Aβ aggregates in an experimental set-up using HFIP as promotor of Aβ aggregates. Analysis of the kinetic mechanism of the AChE inhibition revealed chelerythrine as a mixed inhibitor. Using molecular docking studies, it was further proven that chelerythrine binds on both the catalytic site and the peripheral anionic site (PAS) of the AChE. In view of this, we went on to investigate its effect on inhibiting Aβ aggregation stimulated by AChE. Chelerythrine showed inhibition of fibril formation in the same range as propidium iodide. This approach enabled for the first time to identify a cholinesterase inhibitor of natural origin—chelerythrine—acting on AChE and BChE with a dual ability to inhibit Aβ aggregation as well as to disaggregate preformed Aβ aggregates. This compound could be an excellent starting point paving the way to develop more successful anti-AD drugs.     

Design,synthesis and evaluation of flavonoid derivatives as potential multifunctional acetylcholinesterase inhibitors against Alzheimer’s disease

A new series of flavonoid derivatives were designed, synthesized and evaluated as potential multifunctional AChE inhibitors against Alzheimer’s disease. Most of them exhibited potent AChE inhibitory activity, high selectivity for AChE over BuChE, and moderate to good inhibitory potency toward Aβ aggregation. Specifically, compound 12c was the strongest AChE inhibitor, being 20-fold more potent than galanthamine and twofold more potent than tacrine, and it also had ability to inhibit Aβ aggregation (close to the reference compound) and to function as a metal chelator. Molecular modeling and enzyme kinetic study revealed that it targeted both the catalytic active site and the peripheral anionic site of AChE. Consequently, this class of compounds deserved to be thoroughly and systematically studied for the treatment of Alzheimer’s disease.     

New benzyl pyridinium derivatives bearing 2,4-dioxochroman moiety as potent agents for treatment of Alzheimer’s disease: Design,synthesis, biological evaluation,and docking study

A new series of benzyl pyridinium-2,4-dioxochroman derivatives 7a-o was synthesized and evaluated as new anti-Alzheimer agents. Among the synthesized compounds, the compounds 7f and 7i exhibited the most potent anti-AChE and anti-BuChE activities, respectively. The kinetic study of the compound 7f revealed that this compound inhibited AChE in a mixed-type inhibition mode. Furthermore, the docking study of the compounds 7f and 7i showed that these compounds bound to both the catalytic site (CS) and peripheral anionic site (PAS) of AChE and BuChE, respectively. The compound 7f also exhibited a greater self-induced Aβ peptide aggregation inhibitory activity in compare to donepezil. Furthermore, the neuroprotective activity of this compound at 20 μM was comparable to that of the standard neuroprotective agent (quercetin).     

Targeting acetylcholinesterase: identification of chemical leads by high throughput screening, structure determination and molecular modeling

Acetylcholinesterase (AChE) is an essential enzyme that terminates cholinergic transmission by rapid hydrolysis of the neurotransmitter acetylcholine. Compounds inhibiting this enzyme can be used (inter alia) to treat cholinergic deficiencies (e.g. in Alzheimer''s disease), but may also act as dangerous toxins (e.g. nerve agents such as sarin). Treatment of nerve agent poisoning involves use of antidotes, small molecules capable of reactivating AChE. We have screened a collection of organic molecules to assess their ability to inhibit the enzymatic activity of AChE, aiming to find lead compounds for further optimization leading to drugs with increased efficacy and/or decreased side effects. 124 inhibitors were discovered, with considerable chemical diversity regarding size, polarity, flexibility and charge distribution. An extensive structure determination campaign resulted in a set of crystal structures of protein-ligand complexes. Overall, the ligands have substantial interactions with the peripheral anionic site of AChE, and the majority form additional interactions with the catalytic site (CAS). Reproduction of the bioactive conformation of six of the ligands using molecular docking simulations required modification of the default parameter settings of the docking software. The results show that docking-assisted structure-based design of AChE inhibitors is challenging and requires crystallographic support to obtain reliable results, at least with currently available software. The complex formed between C5685 and Mus musculus AChE (C5685•mAChE) is a representative structure for the general binding mode of the determined structures. The CAS binding part of C5685 could not be structurally determined due to a disordered electron density map and the developed docking protocol was used to predict the binding modes of this part of the molecule. We believe that chemical modifications of our discovered inhibitors, biochemical and biophysical characterization, crystallography and computational chemistry provide a route to novel AChE inhibitors and reactivators.     

New classes of carbazoles as potential multi-functional anti-Alzheimer’s agents

Multi-Target approach is particularly promising way to drug discovery against Alzheimer's disease. In the present study, we synthesized a series of compounds comprising the carbazole backbone linked to the benzyl piperazine, benzyl piperidine, pyridine, quinoline, or isoquinoline moiety through an aliphatic linker and evaluated as cholinesterase inhibitors. The synthesized compounds showed IC₅₀ values of 0.11–36.5 µM and 0.02–98.6 µM against acetyl- and butyrylcholinesterase (AChE and BuChE), respectively. The ligand-protein docking simulations and kinetic studies revealed that compound 3s could bind effectively to the peripheral anionic binding site (PAS) and anionic site of the enzyme with mixed-type inhibition. Compound 3s was the most potent compound against AChE and BuChE and showed acceptable inhibition potency for self- and AChE-induced Aβ_1-42 aggregation. Moreover, compound 3s could significantly protect PC12 cells against H₂O₂-induced toxicity. The results suggested that the compounds 3s could be considered as a promising multi-functional agent for further drug discovery development against Alzheimer's disease.     

Dyhidro-β-agarofurans natural and synthetic as acetylcholinesterase and COX inhibitors: interaction with the peripheral anionic site (AChE-PAS), and anti-inflammatory potentials

In order to find molecules of natural origin with potential biological activities, we isolate and synthesise compounds with agarofuran skeletons (epoxyeudesmanes). From the seeds of Maytenus disticha and Maytenus magellanica we obtained six dihydro-β-agarofurans, and by means of the Robinson annulation reaction we synthesised five compounds with the same skeleton. The structures were established on the basis of NMR, IR, and MS. The evaluated compounds showed inhibitory activity on the acetylcholinesterase enzyme and on the COX enzymes. Compound 4 emerged as the most potent in the acetylcholinesterase inhibition assay with IC₅₀ 17.0 ± 0.016 µM on acetylcholinesterase (AChE). The compounds evaluated were shown to be selective for AChE. The molecular docking, and the propidium displacement assay suggested that the compounds do not bind to the active site of the enzyme AChE, but rather bind to the peripheral anionic site (PAS) of the enzyme, on the other hand, the natural compound 8, showed the best inhibitory activity on the COX-2 enzyme with an IC₅₀ value of 0.04 ± 0.007 µM. The pharmacokinetic profile calculated in silico using the SWISSADME platform shows that these molecules could be considered as potential drugs for the treatment of neurodegenerative diseases such as AD.     

Design, synthesis and evaluation of novel tacrine-multialkoxybenzene hybrids as dual inhibitors for cholinesterases and amyloid beta aggregation

A new series of tacrine-multialkoxybenzene hybrids (9a-9n) were designed, synthesized and evaluated as dual inhibitors of cholinesterases (ChEs) and self-induced β-amyloid (Aβ) aggregation. All the synthesized compounds had high acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activity with IC?? values at the nanomolar range, which were much better than tacrine alone. A Lineweaver-Burk plot and molecular modeling study showed that these hybrids targeted both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. Besides, compounds 9a-9f with methylenedioxybenzene moiety showed higher self-induced Aβ aggregation inhibitory activity than a reference compound, curcumin. These compounds could be selected as multi-potent agents for further investigation to treat AD.     

Design,synthesis and evaluation of some new 4-aminopyridine derivatives in learning and memory

Some new anilide and imide derivatives of 4-aminopyridine (4AP) were synthesized and evaluated against antiamnesic, cognition enhancing and anticholinesterase activity through their respective in vitro and in vivo models. These newly synthesized derivatives have illustrated an enhanced cognition effect on elevated plus maze model and also demonstrated a significant reversal in scopolamine-induced amnesia in same model. The IC₅₀ value of synthesized compounds showed maximum activity of 4APMb compared to standard drug donepezil and other derivatives, whereas its enzyme kinetic study revealed a non-competitive inhibition of acetycholinesterase (AChE) and a competetive inhibition of butyrylcholinesterase (BChE). Significant inhibitions in AChE activity by all the synthesized compounds were found in specific brain regions that is prefrontal cortex, hippocampus and hypothalamus. The docking study confirmed their consensual interaction with AChE, showed an affinity and binding with the key peripheral anionic site residues Trp-286, Tyr-124 and Tyr-341 of AChE.     

Tertiary amine derivatives of chlorochalcone as acetylcholinesterase (AChE) and buthylcholinesterase (BuChE) inhibitors: the influence of chlorine,alkyl amine side chain and α,β-unsaturated ketone group

A new series of tertiary amine derivatives of chlorochalcone (4a～4l) were designed, synthesized and evaluated for the effect on acetylcholinesterase (AChE) and buthylcholinesterase (BuChE). The results indicated that all compounds revealed moderate or potent inhibitory activity against AChE, and some possessed high selectivity for AChE over BuChE. The structure–activity investigation showed that the substituted position of chlorine significantly influenced the activity and selectivity. The alteration of tertiary amine group also leads to obvious change in bioactivity. Among them, IC₅₀ of compound 4l against AChE was 0.17?±?0.06?µmol/L, and the selectivity was 667.2 fold for AChE over BuChE. Molecular docking and enzyme kinetic study on compound 4l suggested that it simultaneously binds to the catalytic active site (CAS) and peripheral anionic site (PAS) of AChE. Further study showed that the pyrazoline derivatives synthesized from chlorochalcones had weaker activity and lower selectivity in inhibiting AChE compared to that of chlorochalcone derivatives.     

Structural insight into the inhibition of acetylcholinesterase by 2,3,4, 5-tetrahydro-1, 5-benzothiazepines

Benzothiazepines 1-3 inhibited acetylcholinesterase (AChE; EC 3.1.1.7) enzyme in a concentration-dependent fashion with IC(50) values of 1.0 +/- 0.002, 1.2 +/- 0.005 and 1.3 +/- 0.001 microM, respectively. By using linear-regression equations, Lineweaver-Burk, Dixon plots and their secondary replots were constructed which indicated that compounds 1-3 are non-competitive inhibitors of AChE with K(i) values of 0.8 +/- 0.04, 1.1 +/- 0.002, and 1.5 +/- 0.001 microM, respectively. Molecular docking studies revealed that all the compounds are completely buried inside the aromatic gorge of AChE, extending deep into the gorge of AChE. A comparison of the docking results of compounds 1-3 displayed that these compounds generally adopt the same binding mode in the active site of AChE. The superposition of the docked structures demonstrated that the non-flexible benzothiazepine always penetrate into the aromatic gorge through the six-membered ring A, which allowed the ligands to interact simultaneously with more than one subsites of the active center of AChE. The higher AChE inhibitory potential of compounds 1-3 was found to be the cumulative effect of hydrophobic contacts and pi-pi interactions between the ligands and AChE. The relatively high affinity of benzothiazepine 1 with AChE was found to be due to additional hydrogen bond in benzothiazepine 1-AChE complex. The results indicated that substitution of halogen and methyl groups by hydrogen at aromatic ring of the benzothiazepine decreased the affinity of these molecules towards enzyme that may be due to the polar non-polar repulsions of these moieties with the amino acid residues in the active site of AChE. The observed binding modes of benzothiazepines 1-3 in the active site of AChE explain the affinities of benzothiazepines and provide a rational basis for the structure-based drug design of benzothiazepines with improved pharmacological properties.     

Discovery of dual binding site acetylcholinesterase inhibitors identified by pharmacophore modeling and sequential virtual screening techniques

Dual binding site acetylcholinesterase (AChE) inhibitors are promising for the treatment of Alzheimer’s disease (AD). They alleviate the cognitive deficits and AD-modifying agents, by inhibiting the β-amyloid (Aβ) peptide aggregation, through binding to both the catalytic and peripheral anionic sites, the so called dual binding site of the AChE enzyme. In this Letter, chemical features based 3D-pharmacophore models were developed based on the eight potent and structurally diverse AChE inhibitors (I-VIII) obtained from high-throughput in vitro screening technique. The best 3D-pharmacophore model, Hypo1, consists of two hydrogen-bond acceptor lipid, one hydrophobe, and two hydrophobic aliphatic features obtained by Catalyst/HIPHOP algorithm adopted in Discovery studio program. Hypo1 was used as a 3D query in sequential virtual screening study to filter three small compound databases. Further, a total of nine compounds were selected and followed on in vitro analysis. Finally, we identified two leads—Specs1 (IC₅₀ = 3.279 μM) and Spec2 (IC₅₀ = 5.986 μM) dual binding site compounds from Specs database, having good AChE enzyme inhibitory activity.     

Inhibition of acetylcholinesterase from Electrophorus electricus (L.) by tricyclic antidepressants

The effects of tricyclic antidepressants drugs (TCA) amitriptyline, imipramine and nortriptyline, on purified Electrophorus electricus (L.) acetylcholinesterase (AChE; acetylcholine hydrolase, EC 3.1.1.7) were studied using kinetic methods and specific fluorescent probe propidium. The antidepressants inhibited AChE activity by a non-competitive mechanism. Inhibition constants range from 200 to 400 microM. Dimethylated amitriptyline and imipramine were more potent inhibitors than the monomethylated nortriptyline. Fluorescence measurements using bis-quaternary ligand propidium were used to monitor ligand-binding properties of these cationic antidepressants to the AChE peripheral anionic site (PAS). This ligand exhibited an eight-fold fluorescence enhancement upon binding to the peripheral anionic site of AChE from E. electricus (L.) with K(D)=7 x 10(-7)M. It was observed that TCA drugs displaced propidium from the enzyme. On the basis of the displacement experiments antidepressant dissociation constants were determined. Similar values for the inhibition constants suggest that these drugs have similar affinity to the peripheral anionic site. The results also indicate that the catalytic active center of AChE does not participate in the interaction of enzyme with tricyclic antidepressants. These studies suggest that the binding site for tricyclic antidepressants is located at the peripheral anionic site of E. electricus (L.) acetylcholinesterase.     

New multipotent tetracyclic tacrines with neuroprotective activity

The synthesis and the biological evaluation (neuroprotection, voltage dependent calcium channel blockade, AChE/BuChE inhibitory activity and propidium binding) of new multipotent tetracyclic tacrine analogues (5–13) are described. Compounds 7, 8 and 11 showed a significant neuroprotective effect on neuroblastoma cells subjected to Ca²⁺ overload or free radical induced toxicity. These compounds are modest AChE inhibitors [the best inhibitor (11) is 50-fold less potent than tacrine], but proved to be very selective, as for most of them no BuChE inhibition was observed. In addition, the propidium displacement experiments showed that these compounds bind AChE to the peripheral anionic site (PAS) of AChE and, consequently, are potential agents that can prevent the aggregation of β-amyloid. Overall, compound 8 is a modest and selective AChE inhibitor, but an efficient neuroprotective agent against 70 mM K⁺ and 60 μM H₂O₂. Based on these results, some of these molecules can be considered as lead candidates for the further development of anti-Alzheimer drugs.