Discovery of potent carbonic anhydrase,acetylcholinesterase, and butyrylcholinesterase enzymes inhibitors: The new amides and thiazolidine‐4‐ones synthesized on an acetophenone base

Compounds containing nitrogen and sulfur atoms can be widely used in various fields, including industry, medicine, biotechnology, and chemical technology. Among them, amides of acids and heterocyclic compounds have an important place. These amides and thiazolidine‐4‐ones showed good inhibitory action against butyrylcholinesterase (BChE), acetylcholinesterase (AChE), and human carbonic anhydrase isoforms. AChE exists at high concentrations in the brain and red blood cells. BChE is an important enzyme that is plentiful in the liver, and it is released into the blood in a soluble form. They were demonstrated to have effective inhibition profiles with K_i values of 23.76–102.75 nM against hCA I, 58.92–136.64 nM against hCA II, 1.40–12.86 nM against AChE, and 9.82–52.77 nM against BChE. On the other hand, acetazolamide showed K_i value of 482.63 ± 56.20 nM against hCA I, and 1019.60 ± 163.70 nM against hCA II. Additionally, Tacrine inhibited AChE and BChE, showing K_i values of 397.03 ± 31.66 and 210.21 ± 15.98 nM, respectively.     

The impact of some natural phenolic compounds on carbonic anhydrase,acetylcholinesterase, butyrylcholinesterase,and α‐glycosidase enzymes: An antidiabetic,anticholinergic, and antiepileptic study

Natural products from food and plant sources have been used for medicinal usage for ages. Also, natural products with therapeutic significance are compounds derived from animals, plants, or any microorganism. In this study, chrysin, carvacrol, hesperidin, zingerone, and naringin as natural phenols showed excellent inhibitory effects against human (h) carbonic anhydrase (CA) isoforms I and II (hCA I and II), α‐glucosidase (α‐Gly), acetylcholinesterase (AChE), and butyrylcholinesterase (BChE). These phenolic compounds were tested for the inhibition of α‐glycosidase, hCA I, hCA II, AChE, and BChE enzymes and demonstrated efficient inhibition profiles with K_i values in the range of 3.70 ± 0.92–79.66 ± 20.81 nM against hCA I, 2.98 ± 0.33–84.88 ± 40.32 nM against hCA II, 4.93 ± 2.01–593.60 ± 134.74 nM against α‐Gly, 0.52 ± 0.18–46.80 ± 17.15 nM against AChE, and 1.25 ± 0.22–32.08 ± 2.68 against BChE.     

Stereoselective Inhibition of Cholesterol Esterase by Enantiomers of <Emphasis Type="Italic">exo</Emphasis>- and <Emphasis Type="Italic">endo</Emphasis>-2-Norbornyl-<Emphasis Type="Italic">N</Emphasis>–<Emphasis Type="Italic">n</Emphasis>-butylcarbamates

Four stereoisomers of 2-norbornyl-N–n-butylcarbamates are characterized as the pseudo substrate inhibitors of cholesterol esterase. Cholesterol esterase shows enantioselective inhibition for enantiomers of exo- and endo-2-norbornyl-N–n-butylcarbamates. For the inhibitions by (R)-(+)- and (S)-(−)-exo-2-norbornyl-N–n-butylcarbamates, the R-enantiomer is 6.8 times more potent than the S-enantiomer. For the inhibitions by (R)-(+)- and (S)-(−)-endo-2-norbornyl-N–n-butyl-carbamates, the S-enantiomer is 4.6 times more potent than the R-enantiomer. The enzyme-inhibitor complex models have been proposed to explain these different enantioselectivities.     

Comparison of methods used for the determination of cholinesterase activity in whole blood

Cholinesterases (ChEs) are classified as either acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) based on their substrate and inhibitor specificity. Organophosphate and carbamate compounds commonly represented by herbicides, pesticides, and nerve gases irreversibly inhibit ChEs. Therefore, exposure to organophosphates and carbamates is normally assessed by measuring ChE activity in blood. There are two approaches for measuring AChE and BChE activity present in whole blood: (1) separating blood into erythrocytes, which contain only AChE, and plasma which contains only BChE, to measure their activity individually, or (2) use a BChE-specific inhibitor to measure the activity of AChE in whole blood. A number of studies have reported the use of different inhibitors for the simultaneous measurement of AChE and BChE activities. However, the inhibitors used for completely inhibiting BChE activity also inhibited AChE activity leading to errors in reported values. The goal of this study was to find the most accurate and simple method for the simultaneous determination of AChE and BChE activity in animal whole blood. Solutions containing human AChE and BChE in various proportions were prepared and AChE and BChE activities were measured using three reported methods. Results demonstrate that ethopropazine and (-) huperzine A appear to be the most specific ChE inhibitors. Preliminary results with human and animal whole blood suggest that 20muM ethopropazine and 500nM (-) huperzine A can be used for measuring AChE and BChE activities across species.     

Synthesis and discovery of potent carbonic anhydrase,acetylcholinesterase, butyrylcholinesterase,and α‐glycosidase enzymes inhibitors: The novel N,N′‐bis‐cyanomethylamine and alkoxymethylamine derivatives

During this investigation, N,N′‐bis‐azidomethylamines, N,N′‐bis‐cyanomethylamine, new alkoxymethylamine and chiral derivatives, which are considered to be a new generation of multifunctional compounds, were synthesized, functional properties were investigated, and anticholinergic and antidiabetic properties of those compounds were studied through the laboratory tests, and it was approved that they contain physiologically active compounds rather than analogues. Novel N‐bis‐cyanomethylamine and alkoxymethylamine derivatives were effective inhibitors of the α‐glycosidase, cytosolic carbonic anhydrase I and II isoforms, butyrylcholinesterase (BChE), and acetylcholinesterase (AChE) with K_i values in the range of 0.15–13.31 nM for α‐glycosidase, 2.77–15.30 nM for human carbonic anhydrase isoenzymes I (hCA I), 3.12–21.90 nM for human carbonic anhydrase isoenzymes II (hCA II), 23.33–73.23 nM for AChE, and 3.84–48.41 nM for BChE, respectively. Indeed, the inhibition of these metabolic enzymes has been considered as a promising factor for pharmacologic intervention in a diversity of disturbances.     

Steady-state kinetic analysis of human cholinesterases over wide concentration ranges of competing substrates

Substrate competition for human acetylcholinesterase (AChE) and human butyrylcholinesterase (BChE) was studies under steady-state conditions using wide range of substrate concentrations. Competing couples of substates were acetyl-(thio)esters. Phenyl acetate (PhA) was the reporter substrate and competitor were either acetylcholine (ACh) or acetylthiocholine (ATC). The common point between investigated substrates is that the acyl moiety is acetate, i.e. same deacylation rate constant for reporter and competitor substrate.Steady-state kinetics of cholinesterase-catalyzed hydrolysis of PhA in the presence of ACh or ATC revealed 3 phases of inhibition as concentration of competitor increased: a) competitive inhibition, b) partially mixed inhibition, c) partially uncompetitive inhibition for AChE and partially uncompetitive activation for BChE. This sequence reflects binding of competitor in the active centrer at low concentration and on the peripheral anionic site (PAS) at high concentration. In particular, it showed that binding of a competing ligand on PAS may affect the catalytic behavior of AChE and BChE in an opposite way, i.e. inhibition of AChE and activation of BChE, regardless the nature of the reporter substrate.For both enzymes, progress curves for hydrolysis of PhA at very low concentration (?K_m) in the presence of increasing concentration of ATC showed that: a) the competing substrate and the reporter substrate are hydrolyzed at the same time, b) complete hydrolysis of PhA cannot be reached above 1 mM competing substrate. This likely results from accumulation of hydrolysis products (P) of competing substrate and/or accumulation of acetylated enzyme·P complex that inhibit hydrolysis of the reporter substrate.     

Kinetic model of ethopropazine interaction with horse serum butyrylcholinesterase and its docking into the active site.

The action of a potent tricyclic cholinesterase inhibitor ethopropazine on the hydrolysis of acetylthiocholine and butyrylthiocholine by purified horse serum butyrylcholinesterase (EC 3.1.1.8) was investigated at 25 and 37 degrees C. The enzyme activities were measured on a stopped-flow apparatus and the analysis of experimental data was done by applying a six-parameter model for substrate hydrolysis. The model, which was introduced to explain the kinetics of Drosophila melanogaster acetylcholinesterase [Stojan et al. (1998) FEBS Lett. 440, 85-88], is defined with two dissociation constants and four rate constants and can describe both cooperative phenomena, apparent activation at low substrate concentrations and substrate inhibition by excess of substrate. For the analysis of the data in the presence of ethopropazine at two temperatures, we have enlarged the reaction scheme to allow primarily its competition with the substrate at the peripheral site, but the competition at the acylation site was not excluded. The proposed reaction scheme revealed, upon analysis, competitive effects of ethopropazine at both sites; at 25 degrees C, three enzyme-inhibitor dissociation constants could be evaluated; at 37 degrees C, only two constants could be evaluated. Although the model considers both cooperative phenomena, it appears that decreased enzyme sensitivity at higher temperature, predominantly for the ligands at the peripheral binding site, makes the determination of some expected enzyme substrate and/or inhibitor complexes technically impossible. The same reason might also account for one of the paradoxes in cholinesterases: activities at 25 degrees C at low substrate concentrations are higher than at 37 degrees C. Positioning of ethopropazine in the active-site gorge by molecular dynamics simulations shows that A328, W82, D70, and Y332 amino acid residues stabilize binding of the inhibitor.     

Evolution of acetylcholinesterase and butyrylcholinesterase in the vertebrates: an atypical butyrylcholinesterase from the Medaka Oryzias latipes

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are thought to be the result of a gene duplication event early in vertebrate evolution. To learn more about the evolution of these enzymes, we expressed in vitro, characterized, and modeled a recombinant cholinesterase (ChE) from a teleost, the medaka Oryzias latipes. In addition to AChE, O. latipes has a ChE that is different from either vertebrate AChE or BChE, which we are classifying as an atypical BChE, and which may resemble a transitional form between the two. Of the fourteen aromatic amino acids in the catalytic gorge of vertebrate AChE, ten are conserved in the atypical BChE of O. latipes; by contrast, only eight are conserved in vertebrate BChE. Notably, the atypical BChE has one phenylalanine in its acyl pocket, while AChE has two and BChE none. These substitutions could account for the intermediate nature of this atypical BChE. Molecular modeling supports this proposal. The atypical BChE hydrolyzes acetylthiocholine (ATCh) and propionylthiocholine (PTCh) preferentially but butyrylthiocholine (BTCh) to a considerable extent, which is different from the substrate specificity of AChE or BChE. The enzyme shows substrate inhibition with the two smaller substrates but not with the larger substrate BTCh. In comparison, AChE exhibits substrate inhibition, while BChE does not, but may instead show substrate activation. The atypical BChE from O. latipes also shows a mixed pattern of inhibition. It is effectively inhibited by physostigmine, typical of all ChEs. However, although the atypical BChE is efficiently inhibited by the BChE-specific inhibitor ethopropazine, it is not by another BChE inhibitor, iso-OMPA, nor by the AChE-specific inhibitor BW284c51. The atypical BChE is found as a glycophosphatidylinositol-anchored (GPI-anchored) amphiphilic dimer (G(2) (a)), which is unusual for any BChE. We classify the enzyme as an atypical BChE and discuss its implications for the evolution of AChE and BChE and for ecotoxicology.     

Factors screening to statistical experimental design of racemic atenolol kinetic resolution via transesterification reaction in organic solvent using free Pseudomonas fluorescens lipase

As the (R )‐enantiomer of racemic atenolol has no β‐blocking activity and no lack of side effects, switching from the racemate to the (S )‐atenolol is more favorable. Transesterification of racemic atenolol using free enzymes investigated as a resource to resolve the racemate via this method is limited. Screenings of enzyme, medium, and acetyl donor were conducted first to give Pseudomonas fluorescens lipase, tetrahydrofuran, and vinyl acetate. A statistical design of the experiment was then developed using Central Composite Design on some operational factors, which resulted in the conversions of 11.70–61.91% and substrate enantiomeric excess (ee ) of 7.31–100%. The quadratic models are acceptable with R² of 95.13% (conversion) and 89.63% (ee ). The predicted values match the observed values reasonably well. Temperature, agitation speed, and substrate molar ratio factor have low effects on conversion and ee , but enzyme loading affects the responses highly. The interaction of temperature–agitation speed and temperature–substrate molar ratio show significant effects on conversion, while temperature–agitation speed, temperature–substrate molar ratio, and agitation speed–substrate molar ratio affect ee highly. Optimum conditions for the use of Pseudomonas fluorescens lipase, tetrahydrofuran, and vinyl acetate were found at 45°C, 175 rpm, 2000 U, and 1:3.6 substrate molar ratio.     

Candida parapsilosis ATCC 7330 can also deracemise 1-arylethanols

Abstract

Candida parapsilosis ATCC 7330 grown using different culture conditions (inoculum size 4% (v/v), inoculum age 12 h, and harvest time 14 h) from those previously reported (inoculum size 2% (v/v), inoculum age 24 h, and harvest time 44 h) successfully deracemised racemic 1-arylethanols and 4-phenyl-2-butanol to the (R)-enantiomer (ee up to >99%). The deracemisation of racemic 1-aryl ethanol proceeds via (i) enantioselective oxidation of (S)-enantiomer followed by (ii) reduction of the ketone formed to give the racemic alcohol which gets kinetically resolved thus enriching for the (R)-enantiomer from the racemate. This is the first report on the deracemisation of 1-arylethanols using Candida parapsilosis ATTC 7330 via dynamic kinetic resolution.     

Synthesis of some tetrahydropyrimidine-5-carboxylates,determination of their metal chelating effects and inhibition profiles against acetylcholinesterase,butyrylcholinesterase and carbonic anhydrase

2-(Methacryloyloxy)ethyl 6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate, is a cyclic urea derivative synthesized from urea, 2-(methacryloyloxy) ethyl acetoacetate and substituted benzaldehyde, and tested in terms of the inhibition of two physiologically relevant carbonic anhydrase (CA) isozymes I and II. Acetylcholinesterase (AChE) is found in high concentrations in the red blood cells and brain. Butyrylcholinesterase (BChE) is another enzyme abundantly present in the liver and released into blood in a soluble form. Also, they were tested for the inhibition of AChE and BChE enzymes and demonstrated effective inhibition profiles with Ki values in the range of 429.24–530.80?nM against hCA I, 391.86–530.80?nM against hCA II, 68.48–97.19?nM against AChE and 104.70–214.15?nM against BChE. On the other hand, acetazolamide clinically used as CA inhibitor, showed Ki value of 281.33?nM against hCA I, and 202.70?nM against hCA II. Also, Tacrine inhibited AChE and BChE showed Ki values of 396.03 and 209.21?nM, respectively.     

Stereoselective determination of apomorphine enantiomers in serum with a cellulose-based high-performance liquid chromatographic chiral column using solid-phase extraction and ultraviolet detection

A novel and rapid method for the separation and determination of R-(−)- and S-(+)-enantiomers of apomorphine in serum by high-performance liquid chromatography with UV detection is reported. The method involved a solid-phase extraction of the R-(−)- and S-(+)-enantiomers of apomorphine and the internal standard R-(−)-propylnorapomorphine from serum using a C₈ Bond-Elut column. The HPLC system consisted of a reversed-phase cellulose-based chiral column (Chiralcel OD-R, 250×4.6 mm I.D.) with a mobile phase of 35:65 (v/v) acetonitrile-0.05 M sodium perchlorate (pH 2.0, adjusted with 60–62% perchloric acid) at a flow-rate of 0.5 ml/min with UV detection at 273 nm. The detection and quantitation limits were 10 ng/ml for each enantiomer using 1 ml of serum. Linear calibration curves from 10 to 1000 ng/ml for both R-(−)- and S-(+)-enantiomers show coefficient of determination of more than 0.9995. Precision calculated as %R.S.D. and accuracy calculated as % error were 0.2–4.7 and 3.1–6.9%, respectively, for the R-(−)-enantiomer and 1.3–4.2 and 0.3–6.8%, respectively, for the S-(+)-enantiomer.     

Enantioselective resolution of 3-phenylthio-2-propanol with Humicola lanuginosa lipase

The acetylation of 3-phenylthio-2-propanol (168 mg) was performed with vinyl acetate (1 ml) using different lipases from 15°C to 51°C. As a result, the (R)-enantiomer was selectively acetylated and the (S)-enantiomer was non-reactive in all the cases. An appropriate choice of conditions can be made to isolate both (R)-alcohol (ee 99%, 36 h, conversion 46%, sub/enz: 1/2) and (S)-alcohol (ee 93%, 38 h, conversion 46%, THF, sub/enz: 1 l^–1) using Humicola lanuginosalipase (Lipolase). Increasing the amount of enzyme increased the ee.     

Pharmacokinetic behaviour of R-(+)- and S-(−)-amlodipine after single enantiomer administration

Amlodipine, 3-ethyl 5-methyl-2-[(2-aminoethoxymethyl]-4-(2-chlorophenyl)-1,4-dihydro-6-methyl-3,5-pyridinedicarboxylate, is a chiral calcium antagonist, currently on the market and in therapeutic use as a racemate. The pharmacokinetic behaviour of R-(+)- and S-(−)-amlodipine after single enantiomer administration to healthy male human volunteers together with comparative administration of the racemic mixture of both enantiomers were studied. Plasma levels were studied as a function of time and assayed using an enantioselective chromatographic method (coupled chiral and achiral HPLC) with on-line solid-phase extraction and UV absorbance detection. The method was validated separately for the R-(+)- and S-(−)-enantiomer, respectively. Results of the study indicate that the pharmacokinetic behaviour of R-(+)- and S-(−)-amlodipine after single enantiomer administration is comparable to that of each enantiomer after administration of the racemate. No racemization occurs in vivo in human plasma after single enantiomer administration.     

Human serum butyrylcholinesterase interactions with cisplatin and cyclophosphamide

Human serum Butyrylcholinesterase (BChE) is an important enzyme in detoxification with its capacity for hydrolyzing esters. The inhibitory effect of cisplatin (CDDP) and cyclophosphamide (CY) on BChE is characterized. Time dependent inhibition of BChE with both chemotherapeutics was rapid, reversible. CY was found as non-competitive inhibitor with Ki of 503.6 ± 50.4 μM. Time dependent CDDP studies displayed progressive inhibition. The constants for apparent dissociation (Ka), first order constant for the break down of the Michaelis complex (k + 2), and bimolecular rate (ka) were calculated as 6.38 × 10⁻⁵ M⁻¹ min⁻¹, 0.063 min⁻¹, and 9.83 × 10⁻⁴ M, respectively. Enzyme protection could be achieved with moderate butyrylthiocholine concentrations (0.3 mM) but higher concentrations increased CDDP inhibition. Apparent Ki value for CDDP was 191.8 ± 71.2 μM. These results suggest that used in combination therapy, CY and CDDP cause considerable BChE inhibition and may aggravate conditions observed during chemotherapy.     

Preparation, in vitro screening and molecular modelling of symmetrical bis-quinolinium cholinesterase inhibitors--implications for early myasthenia gravis treatment

This paper describes the preparation and in vitro evaluation of 18 newly prepared bis-quinolinium inhibitors on human recombinant acetylcholinesterase (AChE) and human plasmatic butyrylcholinesterase (BChE). Their inhibitory (IC₅₀) and was compared to the chosen standards ambenonium dichloride, edrophonium chloride, BW284c51 and ethopropazine hydrochloride. One novel compound was found to be a promising inhibitor of hAChE (in nM range) and was better than edrophonium chloride or BW284c51, but was worse than ambenonium chloride. This compound also showed selectivity towards hAChE and it was confirmed as a non-competitive inhibitor of hAChE by kinetic analysis. A molecular modelling study further confirmed its binding to the peripheral active site of hAChE via apparent π-π or π-cationic interactions.     

Design,synthesis and biological evaluation of novel carbamates as potential inhibitors of acetylcholinesterase and butyrylcholinesterase

Rivastigmine, a dual inhibitor of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), has been approved by U.S. Food and Drug Administration to treat Alzheimer’s disease (AD) and Parkinson’s disease (PD) dementia. In the current work, a bambuterol derivative lacking one of the carbamoyloxy groups on the benzene ring (BMC-1) and its analogues were synthesized using 1-(3-hydroxyphenyl) ethan-1-one and 1-(4-hydroxyphenyl) ethan-1-one as starting materials. In-vitro cholinesterase assay established that nine compounds were more potent to inhibit both electric eel AChE and equine serum BChE than rivastigmine under the same experimental conditions. Further study confirmed that among the nine carbamates, BMC-3 (IC_50(AChE) = 792 nM, IC_50(BChE) = 2.2 nM) and BMC-16 (IC_50(AChE) = 266 nM, IC_50(BChE) = 10.6 nM) were excellent cholinesterase inhibitors with potential of permeating through the blood-brain barrier. These carbamates could be used as potential dual inhibitors of AChE and BChE and to discover novel drugs for the treatment of AD and PD dementia.     

Cation-π and π-π stacking interactions allow selective inhibition of butyrylcholinesterase by modified quinine and cinchonidine alkaloids

Scaffold varied quaternized quinine and cinchonidine alkaloid derivatives were evaluated for their selective butyrylcholinesterase (BChE) inhibitory potential. K_i values were between 0.4–260.5 μM (non-competitive inhibition) while corresponding K_ivalues to acetylcholinesterase (AChE) ranged from 7.0–400 μM exhibiting a 250-fold selectivity for BChE.Docking arrangements (GOLD, PLANT) revealed that the extended aromatic moieties and the quaternized nitrogen of the inhibitors were responsible for specific π–π stacking and π–cation interactions with the choline binding site and the peripheral anionic site of BChE’s active site.     

Design, synthesis and evaluation of flavonoid derivatives as potent AChE inhibitors

A new series of flavonoid derivatives have been designed, synthesized and evaluated as potent AChE inhibitors. Most of them showed more potent inhibitory activities to AChE than rivastigmine. The most potent inhibitor isoflavone derivative 10d inhibit AChE with a IC₅₀ of 4 nM and showed high BChE/AChE inhibition ratio (4575-fold), superior to donepezil (IC₅₀ = 12 nM, 389-fold). Molecular docking studies were also performed to explore the detailed interaction with AChE.     

Synthesis and bioactivity of several new hetaryl sulfonamides

1-(4-Methylsulfonyl)-2-thione-4-aryl-5-Z-6-methyl and oxyalkyl-imidazoles were synthesized from different tetrahydropyrimidinethiones and aryl sulfonyl chloride. These compunds were tested for metal chelating effects and to determine the phrase in which inhibition occured between two physiologically pertinent compunds and carbonic anhydrase (CA) isozymes I and II (hCA I and II), butyrylcholinesterase (BChE) and acetylcholinesterase (AChE). AChE was detected in high concentrations in the brain and red blood cells. BChE is another enzymes that is abundant available in the liver and released into the blood in a soluble form. Newly synthesized hetaryl sulfonamides exhibited impressive inhibition profiles with Ki values in the range of 1.42–6.58?nM against hCA I, 1.72–7.41?nM against hCA II, 0.20–1.14?nM against AChE and 1.55–5.92?nM against BChE. Moreover, acetazolamide showed Ki values of 43.69?±?6.44?nM against hCA I and 31.67?±?8.39?nM against hCA II. Additionally, tacrine showed Ki values of 25.75?±?3.39?nM and 37.82?±?2.08 against AChE and BChE, respectively.