Synthesis,Biological Evaluation,and In Silico Studies of New Acetylcholinesterase Inhibitors Based on Quinoxaline Scaffold

A quinoxaline scaffold exhibits various bioactivities in pharmacotherapeutic interests. In this research, twelve quinoxaline derivatives were synthesized and evaluated as new acetylcholinesterase inhibitors. We found all compounds showed potent inhibitory activity against acetylcholinesterase (AChE) with IC₅₀ values of 0.077 to 50.080 µM, along with promising predicted drug-likeness and blood–brain barrier (BBB) permeation. In addition, potent butyrylcholinesterase (BChE) inhibitory activity with IC₅₀ values of 14.91 to 60.95 µM was observed in some compounds. Enzyme kinetic study revealed the most potent compound (6c) as a mixed-type AChE inhibitor. No cytotoxicity from the quinoxaline derivatives was noticed in the human neuroblastoma cell line (SHSY5Y). In silico study suggested the compounds preferred the peripheral anionic site (PAS) to the catalytic anionic site (CAS), which was different from AChE inhibitors (tacrine and galanthamine). We had proposed the molecular design guided for quinoxaline derivatives targeting the PAS site. Therefore, the quinoxaline derivatives could offer the lead for the newly developed candidate as potential acetylcholinesterase inhibitors.     

Monitoring enzyme reaction and screening enzyme inhibitor based on MALDI-TOF-MS platform with a matrix of oxidized carbon nanotubes

A matrix assisted laser desorption/ionization time-of-flight mass spectrometry platform for quantitatively monitoring enzyme activity and screening enzyme inhibitors has been demonstrated. The described method employs a new matrix of oxidized carbon nanotubes. Compared with the traditional fluorescence approach, this label-free method has the advantage of directly identifying the substrates and products in enzymatic reactions. Moreover, the method could be conveniently carried out with any commercial mass spectrometer without modification. We quantitatively monitored the acetylcholinesterase activity and screened acetylcholinesterase inhibitors with a detection rate of about 3.3 s per sample.     

Novel sulfenamides as promising acetylcholinesterase inhibitors

Several sulfenamide derivatives were designed as possible acetylcholinesterase (AChE) inhibitors. New sulfenamides were synthesized and proved to be stable under the physiological conditions used in the enzymatic assays. N‐benzyl‐2‐benzoxazolylsulfenamide (8) and N‐benzyl‐2‐benzimidazolylsulfenamide (9) revealed anti‐AChE activity with IC₅₀ values of 0.6 and 0.8 μM, respectively, values of the same magnitude as those reported for galantamine and tacrine. The affinity for the biological site was evaluated in terms of interaction/partition toward sodium dodecyl sulfate (SDS) micelles. The inhibitory activity profiles were reasoned in terms of both partition toward a hydrophobic anionic environment and molecular geometry. The X? CSN dihedral angle deviations from collinearity stood out as a major parameter linked to enzyme specificity. J. Heterocyclic Chem., (2011).     

Synthesis of new indole derivatives structurally related to donepezil and their biological evaluation as acetylcholinesterase inhibitors

New series of indole derivatives analogous to donepezil, a well known anti-Alzheimer and acetylcholinesterase inhibitor drug, was synthesized. A full chemical characterization of the new compounds is provided. Biological evaluation of the new compounds as acetylcholinesterase inhibitors was performed. Most of the compounds were found to have potent acetylcholinesterase inhibitor activity compared to donepezil as standard. The compound 1-(2-(4-(2-fluorobenzyl) piperazin-1-yl)acetyl)indoline-2,3-dione (IIId) was found to be the most potent.     

6-羟基石松碱与乙酰胆碱酯酶的相互作用

目前治疗阿尔茨海默症(Alzheimer’s disease,AD)的主要药物为乙酰胆碱酯酶抑制剂(AChEI).为探讨石松碱类化合物对AChE的抑制作用,以6-羟基石松碱(6-hydroxylycopodine,HLD)为对象,应用核磁共振、分子对接、酶活性测定、分子动力学模拟及自由能分析,研究了HLD与AChE的相互作用.研究发现6-羟基石松碱对乙酰胆碱酯酶具有混合型抑制作用,其结合作用主要来自氢键和范德华作用.     

Enzymatic determination of phenols using peanut peroxidase

The influence of phenol and its derivatives on the kinetics of oxidation of aryldiamines (indicator-substrates) catalyzed by novel plant peroxidase—cationic peanut peroxidase—was studied. The character of influence of phenols on the kinetics of enzymatic oxidation of benzidine, o-dianisidine, and 3,3′,5,5′-tetramethylbenzidine (TMB) with hydrogen peroxide was found to depend on a correlation between redox properties of phenols and the indicator-substrate of peroxidase. Thus, the catalytic activity of peanut peroxidase is inhibited by phenols with redox potentials higher than that of aryldiamines mentioned above, whereas phenols with potentials below those of aryldiamines, play the role of second substrates of the enzyme. The enzymatic procedures for the determination of numerous phenols on the level of their concentrations 0.05–80 μM were developed using the reactions of benzidine, o-dianisidine, and TMB oxidation. Different analytical signals—the indicator reaction rate and the induction period duration—were used for the determination of phenols, belonging to various groups—the inhibitors and second substrates of the enzyme, respectively.     

A Biotest Based on an Acetylcholinesterase Tissue Preparation Immobilized on Paper

A sensitive biotest for determining acetylcholinesterase inhibitors has been developed on the basis of a paper matrix and an electric eel (Electrophorus electricus) tissue preparation with high enzymatic activity. Photometric detection at λ = 480 nm based on the ferricyanide reaction (resulting in the formation of copper ferricyanide) was used. The biotest allows the determination of carbamate inhibitors (eserine and neostigmine) and sanguinarine (an isoquinoline alkaloid) at levels of 10^?6 to 10^?5 M.     

Development of nano Bio LC columns for the search of acetylcholinesterase molecular targets

A novel acetylcholinesterase Nano liquid chromatography capillary column (75 μm i.d. × 50 mm length) was developed for the fast screening of acetylcholinesterase inhibitors and the evaluation of their molecular recognition mechanism. Biotinylated acetylcholinesterase was immobilized on a streptavidin Nano liquid chromatography capillary column. Because of the very strong streptavidin-biotin interaction, the acetylcholinesterase immobilization step performed by frontal analysis is very fast (required less than 10 min), and the amount of immobilized acetylcholinesterase was in the microgram range (1 μg). The yellow anion obtained from the enzymatic reaction detected at 412 nm was achieved within 60 s, and the immobilized acetylcholinesterase retained 96% of the initial activity beyond 90 days. This column was successfully applied for the discrimination of weak affinity ligands to acetylcholinesterase from nonbinders, which is the heart of fragment-based drug design. This column was used for the determination of the IC₅₀ values of a series of inhibitor molecules. In addition, it was demonstrated that competitive experiments could be performed with our miniaturized system to confirm the existence and binding pocket of a ligand to acetylcholinesterase contained in a methanol plant extract. The results revealed that our acetylcholinesterase Nano liquid chromatography capillary column developed in this work represents a useful tool for the rapid screening of inhibitor candidates and evaluation of the action mechanism.     

Synthesis, Biological Activity Evaluation and Molecular Modeling Study on the New Isoconessimine Derivatives as Acetylcholinesterase Inhibitors

New isoconessimine derivatives were synthesized from conessine （1） and evaluated as acetylcholinesterase （ACHE） inhibitors. The derivatives were prepared via two reaction steps, N-demethylation and nuc]eophilic substi- tution. All of the synthesized derivatives exhibited more potential anti-acetylcholinesterase activities than conessine （1） （IC50=16μmol·L^-1） and isoconessimine （2） （IC50〉300 μmol·L ^-1）. Compound 7b （3fl-[methyl-[2-（4-nitro- phenoxy）ethyl]amino]con-5-enine） showed the most potent inhibitory activity with an IC50 of 110 μmol/L which is close to that of reference compound huperzine A （IC50= 70 μmol/L）. The mode of AChE inhibition by 7h was re- versible and non-competitive. In addition, molecular modeling was performed to explore the binding mode of in- hibitor 7b at the active site of AChE and the results showed that 7b could be docked into the acetylcholinesterase active site and compound 7h had hydrophobic interactions with Trp279 and Leu282.     

Effect of fluorinated tetrahydrocarbazole derivatives on the enzymes of oxidative deamination of biogenic amines and on the process of lipid peroxidation

The effect of two fluorinated derivatives of tetrahydrocarbazoles on mitochondrial en-zymes catalyzing the oxidative deamination of biogenic amines such as monoamine oxidase A and B (MAO-A and MAO-B) has been investigated. A dose-dependent effect of the studied compounds on the enzymatic activity MAO-A was shown. It was revealed that both com-pounds are MAO-B inhibitors. Inhibition mechanisms were established and MAO-B inhibi-tion constants were calculated. The above derivatives exhibit strong antioxidant and antirad-ical activity.     

Synthesis and structure activity relationship studies of benzothieno[3,2-b]furan derivatives as a novel class of IKKbeta inhibitors

As a novel class of IKKbeta inhibitors, a series of tricyclic furan derivatives was designed and synthesized based on the structure of known thiophene IKKbeta inhibitors. Among the various fused furan derivatives synthesized, a benzothieno[3,2-b]furan derivative 13a displayed potent inhibitory activity towards IKKbeta in enzymatic and cellular assays. The potent inhibitory activity originates from an intramolecular non-bonded S...O interaction which was confirmed by the X-ray structure of JNK3 with 16k. The introduction of further substituents on the core structure led to the discovery of the 6-alkoxy derivatives, which possessed a comparable IKKbeta inhibitory activity to 13a and an improved metabolic stability. Among these, appropriately lipophilic compounds 16a, h, i, and 13g (log D>2) were found to possess good oral bioavailability.     

Synthesis and biological evaluation of thiophene derivatives as acetylcholinesterase inhibitors

A series of new thiophene derivatives has been synthesized using the Gewald protocol. The acetylcholinesterase inhibition activity was assayed according to Ellman's method using donepezil as reference. Some of the compounds were found to be more potent inhibitors than the reference. 2-(2-(4-(4-Methoxyphenyl)piperazin-1-yl)acetamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (IIId) showed 60% inhibition, compared to only 40% inhibition by donepezil.     

Effect of a hydrophobic interaction in the reaction of S-(β-alkylthioethyl) O-propyl methylphosphonothionates and their methyl methosulfates with cholinesterases of warm-blooded animals

The action of S-(β-alkylthioethyl) O-propyl methylphosphonates and their methyl methosulfates with different lengths of the S-alkyl radical on the enzymatic activity of acetylcholinesterase from human blood erythrocytes and butyrylcholinesterase from horse blood serum has been investigated. The existence of a hydrophobic interaction on the sorption of inhibitors in the active site of the enzymes has been established. For the enzymes investigated a definite dependence has been observed of the antienzymatic activity of the compounds on the nature in the region of the anionic point and indicates differences in their length and structure.     

Synthesis and biological evaluation of lycorine derivatives as dual inhibitors of human acetylcholinesterase and butyrylcholinesterase

ABSTRACT: BACKGROUND: Alzheimer's disease (AD) is a neurologically degenerative disorder that affects more than 20 million people worldwide. The selective butyrylcholinesterase (BChE) inhibitors and bivalent cholinesterase (ChE) inhibitors represent new treatments for AD. FINDINGS: A series of lycorine derivatives (1--10) were synthesized and evaluated for anti-cholinesterase activity. Result showed that the novel compound 2-O-tert-butyldimethylsilyl-1-O-(methylthio)methyllycorine (7) was a dual inhibitor of human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBChE) with IC50 values of 11.40 [PLUS-MINUS SIGN] 0.66 muM and 4.17 [PLUS-MINUS SIGN] 0.29 muM, respectively. The structure-activity relationships indicated that (i) the 1-O-(methylthio)methyl substituent in lycorine was better than the 1-O-acetyl group for the inhibition of cholinesterase; (ii) the acylated or etherified derivatives of lycorine and lycorin-2-one were more potent against hBChE than hAChE; and (iii) the oxidation of lycorine at C-2 decreases the activity. CONCLUSION: Acylated or etherified derivatives of lycorine are potential dual inhibitors of hBChE and hAChE. Hence, further study on the modification of lycorine for ChE inhibition is necessary.     

Automated docking of 82 N-benzylpiperidine derivatives to mouse acetylcholinesterase and comparative molecular field analysis with 'natural' alignment

Automated docking and three-dimensional Quantitative Structure-Activity Relationship studies (3D QSAR) were performed for a series of 82 reversible, competitive and selective acetylcholinesterase (AChE) inhibitors. The suggested automated docking technique, making use of constraints taken from experimental crystallographic data, allowed to dock all the 82 substituted N-benzylpiperidines to the crystal structure of mouse AChE, because of short computational times. A 3D QSAR model was then established using the CoMFA method. In contrast to conventional CoMFA studies, the compounds were not fitted to a reference molecule but taken in their 'natural' alignment obtained by the docking study. The established and validated CoMFA model was then applied to another series of 29 N-benzylpiperidine derivatives whose AChE inhibitory activity data were measured under different experimental conditions. A good correlation between predicted and experimental activity data shows that the model can be extended to AChE inhibitory activity data measured on another acetylcholinesterase and/or at different incubation times and pH level.     

Effect of dendrimers on pure acetylcholinesterase activity and structure

The effect of polyamidoamine (PAMAM) dendrimers on activity and fluorescence of pure acetylcholinesterase (EC 3.1.1.7.) was studied. It has been shown that all dendrimers studied decreased the enzymatic activity of acetylcholinesterase. This effect depended on the type of dendrimers. The data on the intrinsic fluorescence have shown that the dendrimers changed acetylcholinesterase conformation and the strongest effect was induced by PAMAM G3.5 dendrimer.     

Screening for acetylcholinesterase inhibitors from Amaryllidaceae using silica gel thin-layer chromatography in combination with bioactivity staining

Thin-layer chromatography (TLC) was used to screen for acetylcholinesterase inhibitors from Amaryllidaceae extracts. The TLC plate was developed and then stained using Ellman's reagent, 5,5'-dithiobis-(2-nitrobenzoic acid), to detect acetylcholinesterase activity. The advantages of this TLC assay method were that we could dereplicate the known inhibitor galanthamine, widely occurring in Amaryllidaceae, at an early stage of the isolation procedure. Moreover, there is no disturbance from sample dissolving solvents as in the microplate assay, and it is a very simple method. The detection limits were 10-200 ng for several known acetylcholinesterase inhibitors tested, and it is thus more sensitive than UV or Dragendorff's reagent detection. Also the minimal detectable amount for an acetylcholinesterase inhibitor tested was much less than that needed for the microplate assay. We screened 15 Amaryllidaceae extracts using this TLC method, and chose candidates for acetylcholinesterase inhibitor isolation.     

Interaction of the cholinesterases of warm-blooded animals with esters of N-(β-hydroxypropyl)piperidines and the methiodides

The influence of esters of N-(β-hydroxypropyl)pyridines (alkyl radicals from CH₃ to C₄H₉) on the enzymatic activity of acetylcholinesterase (ACE) from human blood erythrocytes and on that of butyrylcholinesterase (BCE) from horse blood serum has been studied. At pH 7.5 and 25°C the majority of the piperidine derivatives inhibit reversibly (by the competitive type of inhibition) both ACE and BCE, their inhibiting properties depending only slightly on the presence of the corresponding alkyl radicals in the acid part of the molecule.     

Acetylcholinesterase Based Amperometric Sensor. Inhibitor Detection.

Abstract

On the basis of the process of the acetylthiocholine iodide hydrolysis, catalysed by the enzyme acetylcholinesterase and the obtaining of an electroactive product, an amperometric sensor is created. An electrochemically modified graphite electrode is used. with covalently linked directiy on its surface dcetylcholinesterase. It is demonstrated that it can be applied to the quantitative determination of some acetylcholinesterase inhibitors – insecticides and pharmaceuticals and for establishing their influence on the acetylcholinesterase activity.