Novel oxoisoaporphine-based inhibitors of acetyl- and butyrylcholinesterase and acetylcholinesterase-induced beta-amyloid aggregation

A series of novel oxoisoaporphine-based inhibitors (10-aminoalkylamino-1-azabenzanthrone Ar-NH(CH(2))(n)NR(1)R(2)) of acetylcholinesterase (AChE) has been designed, synthesized, and tested for their ability to inhibit AChE, butyrylcholinesterase (BChE) and AChE-induced β-amyloid (Aβ) aggregation. The synthetic compounds exhibited high AChE inhibitory activity with IC(50) values in the submicromolar range in most cases. Non-competitive binding mode was found for these derivatives by the graphical analysis of steady-state inhibition data. Moreover, all compounds exhibit significant inhibitory activity on AChE-induced Aβ aggregation with inhibitory potency from 54.5% to 93.5%. Finally, six out of twelve synthetic compounds were predicted to be able to cross the blood-brain barrier (BBB) to reach their targets in the central nervous system (CNS) according to a parallel artificial membrane permeation assay for BBB. The result encourages us to study this class of compounds thoroughly and systematically.     

Synthesis and in vitro evaluation of novel rhodanine derivatives as potential cholinesterase inhibitors

Based on a broad spectrum of biological activities of rhodanines, we synthesized aromatic amides and esters of 2-(4-oxo-2-thioxothiazolidin-3-yl)acetic acid (rhodanine-3-acetic acid) via carbodiimide- or PCl₃-mediated coupling. Both esters and amides were investigated for their in vitro inhibitory potency and selectivity against acetylcholinesterase (AChE) from electric eel and butyrylcholinesterase (BChE) from equine serum using Ellman’s spectrophotometric method. The derivatives exhibited mostly a moderate activity against both cholinesterases. IC₅₀ values for AChE were in a closer concentration range of 24.05–86.85 μM when compared to BChE inhibition (7.92–227.19 μM). The esters caused the more efficient inhibition of AChE than amides and parent acid. The esterification and amidation of the rhodanine-3-acetic acid increased inhibition of BChE, even up to 26 times. Derivatives of 4-nitroaniline/phenol showed the activity superior to other substituents (H, Cl, CH₃, OCH₃, CF₃). Rhodanines produced a balanced inhibition of both cholinesterases. Seven derivatives produced the more potent inhibition of AChE than rivastigmine, a clinically used drug; additional three compounds were comparable. Two amides exceeded inhibitory potency of rivastigmine towards BChE. Importantly, this is the first evidence that rhodanine-based compounds are able to inhibit BChE.     

Evaluation of short-tether bis-THA AChE inhibitors. A further test of the dual binding site hypothesis

To provide a further test of the dual binding site hypothesis proposed for acetylcholinesterase (AChE) inhibitor heptylene-linked bis-(9-amino-1,2,3,4-tetrahydroacridine) A7A, short-tether (ethylene hexylene) homologs A2A-A6A were prepared. En route to these compounds, convenient and scaleable syntheses of useful pharmaceutical intermediate 9-chloro-1.2,3,4-tetrahydroacridine 3 and A7A were developed. AChE and butyrylcholinesterase (BChE) inhibition assays of A2A-A10A confirm that a seven methylene tether (A7A) optimizes AChE inhibition potency and AChE/BChE selectivity. Finally, these studies indicate that simultaneous binding of alkylene-linked 9-amino-1,2,3,4-tetrahydroacridine dimers to the catalytic and peripheral sites of AChE is possible with a tether length as short as 5 methylenes.     

Organophosphorus Compound O,O-Dimethyl-O-(2,2-dichlorovynyl)-Phosphate as Selective Inhibitor for Separate Determination of Acetylcholinesterase and Butyrylcholinesterase Activities in the Roach Rutilus rutilus

A comparative study is carried out on dependence of degree of activity inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) of a freshwater bony fish, the roach Rutilus rutilus L., on concentration of organophosphates: O,O-dimethyl-O-(2,2-dichlorovynyl)phosphate (DDVP) and tetraisopropylamidopyrophosphate (iso-OMPA). It has been shown that both in roach and in horse the both inhibitors are selective for BChE in comparison with AChE. Their selectivity degree was 2000-fold and 80-fold, respectively. The ranges of effective DDVP concentrations are overlapped for horse AChE and BChE, while they do not for the roach enzymes. A similar regularity is revealed at action of iso-OMPA. It is established that DDVP has a higher inhibitory potency and selectivity in relation to roach BChE, than iso-OMPA. It is suggested to use DDVP as a new selective inhibitor for separate evaluation of AChE and BChE activities in fish tissues.     

Structure–activity relationship investigation of benzamide and picolinamide derivatives containing dimethylamine side chain as acetylcholinesterase inhibitors

A series of benzamide and picolinamide derivatives containing dimethylamine side chain (4a–4c and 7a–7i) were synthesised and evaluated for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity in vitro. Structure–activity relationship investigation revealed that the substituted position of dimethylamine side chain markedly influenced the inhibitory activity and selectivity against AChE and BChE. In addition, it seemed that the bioactivity of picolinamide amide derivatives was stronger than that of benzamide derivatives. Among them, compound 7a revealed the most potent AChE inhibitory activity (IC₅₀: 2.49?±?0.19?μM) and the highest selectivity against AChE over BChE (Ratio: 99.40). Enzyme kinetic study indicated that compound 7a show a mixed-type inhibition against AChE. The molecular docking study revealed that this compound can bind with both the catalytic site and the peripheral site of AChE.     

Design,synthesis and structure-activity relationships of dual inhibitors of acetylcholinesterase and serotonin transporter as potential agents for Alzheimer's disease

We have designed and synthesized a dual inhibitor of acetylcholinesterase (AChE) and serotonin transporter (SERT) as a novel class of treatment drugs for Alzheimer's disease on the basis of a hypothetical model of the AChE active site. Dual inhibitions of AChE and SERT would bring about greater therapeutic effects than AChE inhibition alone and avoid adverse peripheral effects caused by excessive AChE inhibition. Compound (S)-6j exhibited potent inhibitory activities against AChE (IC(50)=101 nM) and SERT (IC(50)=42 nM). Furthermore, (S)-6j showed inhibitory activities of both AChE and SERT in mice brain following oral administration.     

Design,Synthesis, and Biological Evaluation of Novel Indanone Derivatives as Cholinesterase Inhibitors for Potential Use in Alzheimer's Disease

Indanone derivatives containing meta/para-substituted aminopropoxy benzyl/benzylidene moieties were designed based on the structures of donepezil and ebselen analogs as the cholinesterase inhibitors. The designed compounds were synthesized and their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities were measured. Inhibitory potencies (IC₅₀ values) for the synthesized compounds ranged from 0.12 to 11.92 μM and 0.04 to 24.36 μM against AChE and BChE, respectively. Compound 5 c showed the highest AChE inhibitory potency with IC₅₀ value of 0.12 μM, whereas the highest BChE inhibition was achieved by structure 7 b (IC₅₀=0.04 μM). Structure-activity relationship (SAR) analysis revealed that there is no significant difference between meta and para-substituted derivatives in AChE and BChE inhibition. However, the most potent AChE inhibitor 5 c belongs to meta-substituted compounds, while the most active BChE inhibitor is para-substituted derivative 7 b . The order of enzyme inhibition potency based on the substituted amine group is dimethyl amine>piperidine>morpholine. Compounds containing C=C linkage are more potent AChE inhibitors than the corresponding saturated structures. Molecular docking studies indicated that 5 c interacts with AChE in a very similar way to that observed experimentally for donepezil. The introduced indanone-aminopropoxy benzylidenes could be used in drug-discovery against Alzheimer's disease.     

Sulphonamides incorporating 1,3,5-triazine structural motifs show antioxidant,acetylcholinesterase, butyrylcholinesterase,and tyrosinase inhibitory profile

Abstract

A series of 16 novel benzenesulfonamides incorporating 1,3,5-triazine moieties substituted with aromatic amines, dimethylamine, morpholine and piperidine were investigated. These compounds were assayed for antioxidant properties by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay, 2,2`-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical decolarisation assay and metal chelating methods. They were also investigated as inhibitors of acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and tyrosinase, which are associated with several diseases such as Alzheimer, Parkinson and pigmentation disorders. These benzenesulfonamides showed moderate DPPH radical scavenging and metal chelating activity, and low ABTS cation radical scavenging activity. Compounds 2?b, 3d and 3?h showed inhibitory potency against AChE with % inhibition values of >90. BChE was also effectively inhibited by most of the synthesised compounds with >90% inhibition potency. Tyrosinase was less inhibited by these compounds.     

Efficient synthesis of novel dialkyl-3-cyanopropylphosphate derivatives and evaluation of their anticholinesterase activity

Based on the broad spectrum of biological activities associated with organophosphates, a novel type of this class of compounds was synthesized, bearing a nitrile group, from the sodium alkoxide-catalyzed reaction of dialkylphosphites with γ-ketonitriles at 80 °C under solvent-free conditions. A reaction mechanism involving a phospha-Brook type rearrangement is proposed. Eight title compounds were investigated for their in vitro inhibitory potency and selectivity against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) using Ellman’s spectrophotometric method. The synthesized derivatives exhibited mostly a moderate activity against both cholinesterases. The IC₅₀ values for BChE were in a smaller concentration range (5.96–23.35 µM) compared to those for AChE inhibition (9.61–53.74 µM). The diethyl-3-cyano-1-p-tolylpropylphosphate which displayed the higher dual inhibitory potency towards both cholinesterases could be considered as a potential candidate for developing new drugs to treat Alzheimer’s disease.     

Design,synthesis and bioevaluation of tricyclic fused ring system as dual binding site acetylcholinesterase inhibitors

Due to recently discovered non-classical acetylcholinesterase (AChE) function, dual binding-site AChE inhibitors have acquired a paramount attention of drug designing researchers. The unique structural arrangements of AChE peripheral anionic site (PAS) and catalytic site (CAS) joined by a narrow gorge, prompted us to design the inhibitors that can interact with dual binding sites of AChE. Eighteen homo- and heterodimers of desloratadine and carbazole (already available tricyclic building blocks) were synthesized and tested for their inhibition potential against electric eel acetylcholinesterase (eeAChE) and equine serum butyrylcholinesterase (eqBChE). We identified a six-carbon tether heterodimer of desloratadine and indanedione based tricyclic dihydropyrimidine (4c) as potent and selective inhibitor of eeAChE with IC₅₀ value of 0.09 ± 0.003 μM and 1.04 ± 0.08 μM (for eqBChE) with selectivity index of 11.1. Binding pose analysis of potent inhibitors suggest that tricyclic ring is well accommodated into the AChE active site through hydrophobic interactions with Trp84 and Trp279. The indanone ring of most active heterodimer 4b is stabilized into the bottom of the gorge and forms hydrogen bonding interactions with the important catalytic triad residue Ser200.     

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.     

Synthesis of derivatives of cleistopholine and their anti-acetylcholinesterase and anti-β-amyloid aggregation activity

A series of 6- and 9-substituted cleistopholine derivatives has been designed, synthesized and investigated to inhibit the aggregation of acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and β-myloid (A β). Results showed that these synthetic compounds had excellent AChE inhibitory activity and a significant in vitro inhibitory potency toward the self-induced A β aggregation. When SH-SY5Y cells were treated with these substituted cleistopholine derivatives during they overexpressed the Swedish mutant form of human β -amyloid precursor protein (APPsw), A β 42 secretion levels were significantly reduced. According to a parallel artificial membrane permeation assay for BBB, seven out of these sixteen synthetic compounds probably could cross the blood-brain barrier (BBB) to reach their targets in the central nervous system (CNS).     

Stereoselective inhibition of human, mouse, and horse cholinesterases by bambuterol enantiomers

Bambuterol is a chiral carbamate and a selective inhibitor of butyrylcholinesterase (BChE, EC 3.1.1.8). In order to relate bambuterol selectivity and stereoselectivity of BChE and acetylcholinesterase (AChE, EC 3.1.1.7) of different species, we studied the inhibition of human, mouse, and horse BChE, as well as AChE of human and mouse by (R)- and (S)-bambuterol. AChE and BChE of all studied species were progressively inhibited by both bambuterol enantiomers, with a preference for the (R)-bambuterol whose inhibition rate constants were about five times higher than that of (S)-bambuterol. We observed no significant difference between human and mouse in bambuterol enantiomer BChE inhibition. However, (R)-bambuterol inhibited horse BChE about 14 times slower than human and mouse BChE, and the inhibition rate for (S)-bambuterol was about 18 times slower. Although the primary structure of horse BChE differs from the other two species in 15 amino acids, we presumed that differences in inhibition rates could be attributed to threonine at position 69 located close to the peripheral site of BChE. Since BChE inhibition by bambuterol enantiomers was at least 8000 times faster than that of AChE, both bambuterol enantiomers proved to be selective BChE inhibitors, as was previously shown for racemate.     

Design,synthesis and biological evaluation of coumarin alkylamines as potent and selective dual binding site inhibitors of acetylcholinesterase

Acetylcholinesterase inhibitors (AChEIs) are currently the drugs of choice, although only symptomatic and palliative, for the treatment of Alzheimer’s disease (AD). Donepezil is one of most used AChEIs in AD therapy, acting as a dual binding site, reversible inhibitor of AChE with high selectivity over butyrylcholinesterase (BChE). Through a combined target- and ligand-based approach, a series of coumarin alkylamines matching the structural determinants of donepezil were designed and prepared. 6,7-Dimethoxycoumarin derivatives carrying a protonatable benzylamino group, linked to position 3 by suitable linkers, exhibited fairly good AChE inhibitory activity and a high selectivity over BChE. The inhibitory potency was strongly influenced by the length and shape of the spacer and by the methoxy substituents on the coumarin scaffold. The inhibition mechanism, assessed for the most active compound 13 (IC₅₀ 7.6 nM) resulted in a mixed-type, thus confirming its binding at both the catalytic and peripheral binding sites of AChE.     

Neurotoxicity of acetylcholinesterase amyloid beta-peptide aggregates is dependent on the type of Abeta peptide and the AChE concentration present in the complexes.

Alzheimer's disease (AD) is a neurodegenerative disorder whose hallmark is the presence of senile plaques and neurofibrillary tangles. Senile plaques are mainly composed of amyloid beta-peptide (Abeta) fibrils and several proteins including acetylcholinesterase (AChE). AChE has been previously shown to stimulate the aggregation of Abeta1-40 into amyloid fibrils. In the present work, the neurotoxicity of different amyloid aggregates formed in the absence or presence of AChE was evaluated in rat pheochromocytoma PC12 cells. Stable AChE-Abeta complexes were found to be more toxic than those formed without the enzyme, for Abeta1-40 and Abeta1-42, but not for amyloid fibrils formed with AbetaVal18-Ala, a synthetic variant of the Abeta1-40 peptide. Of all the AChE-Abeta complexes tested the one containing the Abeta1-40 peptide was the most toxic. When increasing concentrations of AChE were used to aggregate the Abeta1-40 peptide, the neurotoxicity of the complexes increased as a function of the amount of enzyme bound to each complex. Our results show that AChE-Abeta1-40 aggregates are more toxic than those of AChE-Abeta1-42 and that the neurotoxicity depends on the amount of AChE bound to the complexes, suggesting that AChE may play a key role in the neurodegeneration observed in Alzheimer brain.     

Novel N-benzylpyridinium moiety linked to arylisoxazole derivatives as selective butyrylcholinesterase inhibitors: Synthesis,biological evaluation,and docking study

A novel series of N-benzylpyridinium moiety linked to arylisoxazole ring were designed, synthesized, and evaluated for their in vitro acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities. Synthesized compounds were classified into two series of 5a-i and 5j-q considering the position of positively charged nitrogen of pyridinium moiety (3- or 4- position, respectively) connected to isoxazole carboxamide group. Among the synthesized compounds, compound 5n from the second series of compounds possessing 2,4-dichloroaryl group connected to isoxazole ring was found to be the most potent AChE inhibitor (IC₅₀ = 5.96 µM) and compound 5j also from the same series of compounds containing phenyl group connected to isoxazole ring demonstrated the most promising inhibitory activity against BChE (IC₅₀ = 0.32 µM). Also, kinetic study demonstrated competitive inhibition mode for both AChE and BChE inhibitory activity. Docking study was also performed for those compounds and desired interactions with those active site amino acid residues were confirmed through hydrogen bonding as well as π-π and π-anion interactions. In addition, the most potent compounds were tested against BACE1 and their neuroprotectivity on Aβ-treated neurotoxicity in PC12 cells which depicted negligible activity. It should be noted that most of the synthesized compounds from both categories 5a-i and 5j-q showed a significant selectivity toward BChE. However, series 5j-q were more active toward AChE than series 5a-i.     

Cholinesterase inhibitory activities of some flavonoid derivatives and chosen xanthone and their molecular docking studies

Flavonoids are one of the largest classes of plant secondary metabolites and are known to possess a number of significant biological activities for human health. In this study, we examined in vitro acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities of four flavonoid derivatives - quercetin, rutin, kaempferol 3-O-β-d-galactoside and macluraxanthone. The in vitro results showed that quercetin and macluraxanthone displayed a concentration-dependant inhibition of AChE and BChE. Macluraxanthone showed to be the most potent and specific inhibitor of both the enzymes having the IC₅₀ values of 8.47 and 29.8 μM, respectively. The enzyme kinetic studies revealed that quercetin inhibited both the enzymes in competitive manner, whereas the mode of inhibition of macluraxanthone was non-competitive against AChE and competitive against BChE. The inhibitory profiles of the compounds have been compared with standard AChE inhibitor galanthamine. To get insight of the intermolecular interactions, the molecular docking studies of these two compounds were performed at the active site 3D space of both the enzymes, using ICM-Dock™ module. Docking studies exhibited that macluraxanthone binds much more tightly with both the enzymes than quercetin. The calculated docking and binding energies also supported the in vitro inhibitory profiles (IC₅₀ values). Both the compounds showed several strong hydrogen bonds to several important amino acid residues of both the enzymes. A number of hydrophobic interactions could also explain the potency of the compounds to inhibit AChE and BChE.     

Amino acid residues involved in the interaction of acetylcholinesterase and butyrylcholinesterase with the carbamates Ro 02-0683 and bambuterol, and with terbutaline.

In order to identify amino acids involved in the interaction of acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BChE; EC 3.1.1.8) with carbamates, the time course of inhibition of the recombinant mouse enzymes BChE wild-type (w.t.), AChE w.t. and of 11 site-directed AChE mutants by Ro 02-0683 and bambuterol was studied. In addition, the reversible inhibition of cholinesterases by terbutaline, the leaving group of bambuterol, was studied. The bimolecular rate constant of AChE w.t. inhibition was 6.8 times smaller by Ro 02-0683 and 16000 times smaller by bambuterol than that of BChE w.t. The two carbamates were equipotent BChE inhibitors. Replacement of tyrosine-337 in AChE with alanine (resembling the choline binding site of BChE) resulted in 630 times faster inhibition by bambuterol. The same replacement decreased the inhibition by Ro 02-0683 ten times. The difference in size of the choline binding site in the two w.t. enzymes appeared critical for the selectivity of bambuterol and terbutaline binding. Removal of the charge with the mutation D74N caused a reduction in the reaction rate constants for Ro 02-0683 and bambuterol. Substitution of tyrosine-124 with glutamine in the AChE peripheral site significantly increased the inhibition rate for both carbamates. Substitution of phenylalanine-297 with alanine in the AChE acyl pocket decreased the inhibition rate by Ro 02-0683. Computational docking of carbamates provided plausible orientations of the inhibitors inside the active site gorge of mouse AChE and human BChE, thus substantiating involvement of amino acid residues in the enzyme active sites critical for the carbamate binding as derived from kinetic studies.     

Amino acid residues involved in stereoselective inhibition of cholinesterases with bambuterol

Bambuterol is a chiral carbamate known as selective inhibitor of butyrylcholinesterase (BChE). In order to relate bambuterol selectivity and stereoselectivity of cholinesterases to the active site residues, we studied the inhibition of recombinant mouse BChE, acetylcholinesterase (AChE) and six AChE mutants, employed to mimic BChE active site residues, by bambuterol enantiomers. Both enantiomers selectively inhibited BChE about 8000 times faster than AChE. The largest inhibition rate increase in comparison to AChE w.t. was observed with the F295L/Y337A mutant, showing that leucine 295 and alanine 337 are crucial residues in BChE for high bambuterol selectivity. All studied enzymes preferred inhibition by the R- over the S-bambuterol. The enlargement of the AChE choline binding site and of the acyl pocket by single or double mutations (Y337A, F295L/Y337A and F297I/Y337A) increased, in comparison to w.t. enzymes, inhibition rate constants of R- bambuterol more than that of S- bambuterol resulting in four times higher stereoselectivity. Peripheral site mutations (Y124Q and Y72N/Y124Q/Y337A) increased inhibition rate by S- more than R-bambuterol and consequently diminished the stereoselectivity.     

Synthesis of physostigmine analogues and evaluation of their anticholinesterase activities

A series of physostigmine analogues were prepared and evaluated for cholinesterase inhibition activities, including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Most of them showed potent inhibition activities against AChE, in which compound 17 especially exhibited significantly higher selectivity over BChE than phenserine, a compound currently on clinical trial. Discussion about the relationships between structure and activity of these derivatives was also presented.