Electric Eel Acetylcholinesterase Research Articles

Structural Insights into the Marine Alkaloid Discorhabdin G as a Scaffold towards New Acetylcholinesterase Inhibitors.

In this study, Antarctic Latrunculia sponge-derived discorhabdin G was considered a hit for developing potential lead compounds acting as cholinesterase inhibitors. The hypothesis on the pharmacophore moiety suggested through molecular docking allowed us to simplify the structure of the metabolite. ADME prediction and drug-likeness consideration provided valuable support in selecting 5-methyl-2H-benzo[h]imidazo[1,5,4-de]quinoxalin-7(3H)-one as a candidate molecule. It was synthesized in a four-step sequence starting from 2,3-dichloronaphthalene-1,4-dione and evaluated as an inhibitor of electric eel acetylcholinesterase (eeAChE), human recombinant AChE (hAChE), and horse serum butyrylcholinesterase (BChE), together with other analogs obtained by the same synthesis. The candidate molecule showed a slightly lower inhibitory potential against eeAChE but better inhibitory activity against hAChE than discorhabdin G, with a higher selectivity for AChEs than for BChE. It acted as a reversible competitive inhibitor, as previously observed for the natural alkaloid. The findings from the in vitro assay were relatively consistent with the data available from the AutoDock Vina and Protein-Ligand ANTSystem (PLANTS) calculations.

Design, synthesis and biological evaluation of new multi-target scutellarein hybrids for treatment of Alzheimer’s disease

Scutellarein hybrids were designed, synthesized and evaluated as multifunctional therapeutic agents for the treatment of Alzheimer's disease (AD). Compounds 11a–i, containing a 2-hydroxymethyl-3,5,6-trimethylpyrazine fragment at the 7-position of scutellarein, were found to have balanced and effective multi-target potencies against AD. Among them, compound 11e exhibited the most potent inhibition of electric eel and human acetylcholinesterase enzymes with IC50 values of 6.72 ± 0.09 and 8.91 ± 0.08 μM, respectively. In addition, compound 11e displayed not only excellent inhibition of self- and Cu2+-induced Aβ1–42 aggregation (91.85% and 85.62%, respectively) but also induced disassembly of self- and Cu2+-induced Aβ fibrils (84.54% and 83.49% disaggregation, respectively). Moreover, 11e significantly reduced tau protein hyperphosphorylation induced by Aβ25–35, and also exhibited good inhibition of platelet aggregation. A neuroprotective assay demonstrated that pre-treatment of PC12 cells with 11e significantly decreased lactate dehydrogenase levels, increased cell viability, enhanced expression of relevant apoptotic proteins (Bcl-2, Bax and caspase-3) and inhibited RSL3-induced PC12 cell ferroptosis. Furthermore, hCMEC/D3 and hPepT1-MDCK cell line permeability assays indicated that 11e would have optimal blood–brain barrier and intestinal absorption characteristics. In addition, in vivo studies revealed that compound 11e significantly attenuated learning and memory impairment in an AD mice model. Toxicity experiments with the compound did not reveal any safety concerns. Notably, 11e significantly reduced β-amyloid precursor protein (APP) and β-site APP cleaving enzyme-1 (BACE-1) protein expression in brain tissue of scopolamine-treated mice. Taken together, these outstanding properties qualified compound 11e as a promising multi-target candidate for AD therapy, worthy of further studies.

A series of 1,2,3-triazole compounds: Synthesis, characterization, and investigation of the cholinesterase inhibitory properties via in vitro and in silico studies

A series of 1,2,3-triazole compounds derived from a salicylaldehyde moiety were synthesized in high yields using copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) reactions. The structures of the newly synthesized 1,2,3-triazole compounds were characterized by FT-IR, NMR, and HRMS analyses. The inhibitory activities of all the target compounds were investigated against electric eel acetylcholinesterase (eeAChE) and equine serum butyrylcholinesterase (eqBChE) enzymes. The activity results were compared with galantamine used as a reference compound. The in vitro biological assays showed that most of the target compounds had quite good inhibition activities for eeAChE, while weaker towards eqBChE. Among them, compound 23f exhibited better activity than both other target compounds and galantamine against both eeAChE (IC50: 0.458 µM) and eqBChE (IC50:1.721 µM). Molecular docking studies of all target compounds were performed on the 3D crystallographic structures of eeAChE, Homo sapiens AChE, and BChE enzymes. The binding energies appear to be consistent with the activity results. Moreover, it was determined that 23f interacted with crucial residues of the catalytic active site (CAS) and peripheral anionic site (PAS) of both AChE and BChE due to its dual binding feature. Finally, in silico ADME and toxicity properties of the compounds were investigated using the Swiss ADME and ProTox-II websites. The compounds have appropriate drug-likeness scores, Additionally, it has been estimated that 23f, the most active compound, and 23c are within the best limits in terms of oral bioavailability and exhibit low levels of toxicity. Consequently, the target compounds, especially 23f, can be considered promising inhibitors for the cholinesterase enzymes.

Synthesis and evaluation of small organic molecule as reactivator of organophosphorus inhibited acetylcholinesterase

A series of uncharged salicylaldehyde oximes were synthesized and evaluated for the reactivation of organophosphorus (OP) nerve agents simulants Diethylchlorophosphonate (DCP) & Diethylcyanophosphonate (DCNP) and pesticides (paraoxon & malaoxon) inhibited electric eel Acetylcholinesterase (AChE). The computational software Swiss ADME and molinspiration were used to unfold the probability of drug-likeness properties of the oximes derivatives. Substituted aromatic oximes with diethylamino or bromo group with free hydroxyl group ortho to oxime moiety were found efficient to regenerate the enzymatic activity in in-vitro AChE assay. The alkylation of the ortho hydroxyl group of derivatives led to the loss of reactivation potential. The derivatives with a hydroxyl group and without oxime group and vice versa did not show significant reactivation potency against tested OP toxicants. Further, we also evaluated the reactivation potential of these selected molecules on the rat brain homogenate against different OPs inhibited ChE and found maximum reactivation potency of oxime 2e. The in-vitro results were further validated by molecular docking and dynamic studies which showed that the hydroxyl group interacted with serine amino acids in the catalytic anionic site of AChE enzyme and was stable up to 200 ns consequently providing proper orientation to oxime moiety for reactivating the OP inhibited enzyme. It has thus been proved by the structure-activity relationship of oximes derivatives that hydroxyl group ortho to oxime is essential for reactivating OP inhibited electric eel AChE. Amongst the twenty-one oximes derivatives, 2e was found to be most active in regenerating the paraoxon, malaoxon, DCP and DCNP inhibited AChE enzyme.

Multitargeted C9-substituted ester and ether derivatives of berberrubine for Alzheimer's disease: Design, synthesis, biological evaluation, metabolic stability, and pharmacokinetics.

Berberrubine is a naturally occurring isoquinoline alkaloid and a bioactive metabolite of berberine. Berberine exhibits a wide range of pharmacological activities, including cholinesterase inhibition. The cholinesterase inhibitors provide symptomatic treatment for Alzheimer's disease; however, multitarget-directed ligands have the potential as disease-modifying therapeutics. Herein, we prepared a series of C9-substituted berberrubine derivatives intending to discover dual cholinesterase and beta-site amyloid-precursor protein cleaving enzyme 1 (BACE-1) inhibitors. Most synthesized derivatives possessed balanced dual inhibition (AChE and BChE) activity in the submicromolar range and a moderate inhibition against BACE-1. Two most active ester derivatives, 12a and 11d, display inhibition of AChE, BChE, and BACE-1. The 3-methoxybenzoyl ester derivative, 12a, inhibits electric eel acetylcholinesterase (EeAChE), equine serum butyrylcholinesterase (eqBChE), and human hBACE-1 with IC50 values of 0.5, 4.3, and 11.9 μM, respectively and excellent BBB permeability (Pe = 8 × 10-6 cm/s). The ester derivative 12a is metabolically unstable; however, its ether analog 13 is stable in HLM and exhibits inhibition of AChE, BChE, and BACE-1 with IC50 values of 0.44, 3.8, and 17.9 μM, respectively. The ether analog also inhibits self-aggregation of Aβ and crosses BBB (Pe = 7.3 × 10-6 cm/s). Administration of 13 at 5 mg/kg (iv) in Wistar rats showed excellent plasma exposure with AUC0-∞ of 28,834 ng min/ml. In conclusion, the multitargeted berberrubine ether derivative 13 is CNS permeable and has good ADME properties.

Synthesis and Anticholinesterase Evaluation of Cassine, Spectaline and Analogues

In this work, twelve analogues of piperidine alkaloids (-)-cassine and (-)-spectaline were synthesized, as well as the racemic forms of these natural products. The compounds were evaluated for their inhibition of electric eel acetylcholinesterase (AChEee) and human butyrylcholinesterase (BChEhu) by on-flow mass-spectrometry-based dual-enzyme assay, and the inhibition mechanisms for the most potent analogues were also determined. Our results showed a preference for BChEhu inhibition with compounds 10c (Ki = 5.24 μM), 12b (Ki = 17.4 μM), 13a (Ki = 13.2 μM) and 3 (Ki = 11.3 μM) displaying the best inhibitory activities.

Enantioselective Total Synthesis of (+)-Sieboldine A and Analogues Thereof.

An 11-step enantioselective total synthesis of (+)-sieboldine A (1) has been accomplished from (5R)-methylcyclohex-2-en-1-one (16), in which an intramolecular ketone/ester reductive coupling followed by one-pot acidic treatment to quickly construct the unique oxa-spiroacetal and a TsOH-catalyzed displacement to directly form the characteristic N-hydroxyazacyclononane ring successfully served as the key methodologies. Moreover, several full-skeleton analogues of 1 were also synthesized on the basis of the advanced intermediates, and their inhibitory effects on electric eel acetylcholinesterase were examined.

In vitro and in silico evaluation of inhibitory effects of bisphenol derivatives on acetylcholinesterase of electric eel (Electrophorus electricus L.)

The inhibitory effects of bisphenol A (BPA) and bisphenol S (BPS), which are common pollutants, especially in marine and freshwater, on the electric eel acetylcholinesterase (AChE) activity were studied in vitro and in silico. Both produced full non-competitive inhibition, but the Ki value of BPA was half that of BPS. Molecular docking analyses revealed that both interact with residues W286, F297, Y337, F338 in the PAS and ABS regions in the middle and entrance of the active site gorge, and that BPS also has hydrogen bond with S203 of the catalytic triad. The surge at IC50 values of both compounds with an inflection point at pH: 8.2 suggested that Y124 and/or Y337 in the narrow gorge are primary structural factors in binding. Less effective inhibition of BPS, especially at 25–30 °C, the temperature at which enzyme activity peaks, was attributed to the conformation of the narrow gorge. Homology analyses for AChE initially revealed a significant degree of identity, particularly in the alpha/beta hydrolase domain, which also comprises the active site, with sequences from seven distinct teleost species of various environments. Finally, it was discovered for the first time that BPS, like BPA, is a significant inhibitor of AChE, and this was confirmed by in vitro and in silico analyses done at various pH and temperature levels. It was concluded that this effect might also apply to AChE of most other bony fish.

Synthesis, kinetic evaluation and molecular docking studies of donepezil-based acetylcholinesterase inhibitors

In an effort to develop new therapeutic agents to treat Alzheimer's disease, a series of donepezil-based analogs were designed, synthesized using an environmentally friendly route, and biologically evaluated for their inhibitory activity against electric eel acetylcholinesterase (AChE) enzyme. In vitro studies revealed that the phenyl moiety of donepezil can be successfully replaced with a pyridine ring leading to equally potent inhibitors of electric eel AChE. Further kinetic evaluations of the most potent inhibitor showed a dual-binding (mixed inhibition) mode, similar to donepezil. Molecular modeling studies suggest that several additional residues could be involved in the binding of this inhibitor in the human AChE enzyme active site compared to donepezil.

Fine-Tuning the Biological Profile of Multitarget Mitochondriotropic Antioxidants for Neurodegenerative Diseases.

Neurotransmitter depletion and mitochondrial dysfunction are among the multiple pathological events that lead to neurodegeneration. Following our previous studies related with the development of multitarget mitochondriotropic antioxidants, this study aims to evaluate whether the π-system extension on the chemical scaffolds of AntiOXCIN2 and AntiOXCIN3 affects their bioactivity and safety profiles. After the synthesis of four triphenylphosphonium (TPP+) conjugates (compounds 2–5), we evaluated their antioxidant properties and their effect on neurotransmitter-metabolizing enzymes. All compounds were potent equine butyrylcholinesterase (eqBChE) and moderate electric eel acetylcholinesterase (eeAChE) inhibitors, with catechols 4 and 5 presenting lower IC50 values than AntiOXCIN2 and AntiOXCIN3, respectively. However, differences in the inhibition potency and selectivity of compounds 2–5 towards non-human and human cholinesterases (ChEs) were observed. Co-crystallization studies with compounds 2–5 in complex with human ChEs (hChEs) showed that these compounds exhibit different binging modes to hAChE and hBChE. Unlike AntiOXCINs, compounds 2–5 displayed moderate human monoamine oxidase (hMAO) inhibitory activity. Moreover, compounds 4 and 5 presented higher ORAC-FL indexes and lower oxidation potential values than the corresponding AntiOXCINs. Catechols 4 and 5 exhibited broader safety windows in differentiated neuroblastoma cells than benzodioxole derivatives 2 and 3. Compound 4 is highlighted as a safe mitochondria-targeted antioxidant with dual ChE/MAO inhibitory activity. Overall, this work is a contribution for the development of dual therapeutic agents addressing both mitochondrial oxidative stress and neurotransmitter depletion.

Identification of aplysinopsin as a blood-brain barrier permeable scaffold for anti-cholinesterase and anti-BACE-1 activity

Aplysinopsins are a group of marine-derived indole alkaloids that display diverse array of pharmacological effects. However, their effect on anti-Alzheimer targets has not been reported. Herein, we report the synthesis of aplysinopsin (1) and its effect on cholinesterases and beta-site amyloid-precursor protein cleaving enzyme 1 (BACE-1). It inhibits electric eel acetylcholinesterase (AChE), equine serum butyrylcholinesterase (BChE), and human BACE-1 with IC50 values of 33.9, 30.3, and 33.7 µM, respectively, and excellent BBB permeability (Pe 8.92 × 10−6 cm/s). To optimize its sub-micromolar activity, the first-generation analogs were prepared and screened. Two most active analogs 5b and (Z)-8g were found to effectively permeate the BBB (Pe > 5 × 10−6 cm/s). The N-sulphonamide derivative 5b display better cholinesterase inhibition, whereas the other analog (Z)-8g strongly inhibits BACE-1 (IC50 0.78 µM) activity. The analog 5b interacts primarily with PAS of AChE, and thus exhibit a mixed-type of inhibition. In addition, aplysinopsin along with new analogs inhibited the self-induced Aβ1-42 aggregation. The data presented herein indicate that the aplysinopsin-scaffold holds a potential for further investigation as a multi-targeted anti-Alzheimer agent.

Acetylcholinesterase inhibition in electric eel and human donor blood: an in vitro approach to investigate interspecies differences and human variability in toxicodynamics

In chemical risk assessment, default uncertainty factors are used to account for interspecies and interindividual differences, and differences in toxicokinetics and toxicodynamics herein. However, these default factors come with little scientific support. Therefore, our aim was to develop an in vitro method, using acetylcholinesterase (AChE) inhibition as a proof of principle, to assess both interspecies and interindividual differences in toxicodynamics. Electric eel enzyme and human blood of 20 different donors (12 men/8 women) were exposed to eight different compounds (chlorpyrifos, chlorpyrifos-oxon, phosmet, phosmet-oxon, diazinon, diazinon-oxon, pirimicarb, rivastigmine) and inhibition of AChE was measured using the Ellman method. The organophosphate parent compounds, chlorpyrifos, phosmet and diazinon, did not show inhibition of AChE. All other compounds showed concentration-dependent inhibition of AChE, with IC50s in human blood ranging from 0.2–29 µM and IC20s ranging from 0.1–18 µM, indicating that AChE is inhibited at concentrations relevant to the in vivo human situation. The oxon analogues were more potent inhibitors of electric eel AChE compared to human AChE. The opposite was true for carbamates, pointing towards interspecies differences for AChE inhibition. Human interindividual variability was low and ranged from 5–25%, depending on the concentration. This study provides a reliable in vitro method for assessing human variability in AChE toxicodynamics. The data suggest that the default uncertainty factor of ~ 3.16 may overestimate human variability for this toxicity endpoint, implying that specific toxicodynamic-related adjustment factors can support quantitative in vitro to in vivo extrapolations that link kinetic and dynamic data to improve chemical risk assessment.

Inhibition of Acetylcholinesterases by Stereoisomeric Organophosphorus Compounds Containing Both Thioester and p-Nitrophenyl Leaving Groups.

Studies with acetylcholinesterase (AChE) inhibited by organophosphorus (OP) compounds with two chiral centers can serve as models or surrogates for understanding the rate, orientation, and postinhibitory mechanisms by the nerve agent soman that possesses dual phosphorus and carbon chiral centers. In the current approach, stereoisomers of O-methyl, [S-(succinic acid, diethyl ester), O-(4-nitrophenyl) phosphorothiolate (MSNPs) were synthesized, and the inhibition, reactivation, and aging mechanisms were studied with electric eel AChE (eeAChE) and recombinant mouse brain AChE (rmAChE). The MSNP RPRC isomer was the strongest inhibitor of both eeAChE and rmAChE at 8- and 24-fold greater potency, respectively, than the weakest SPSC isomer. eeAChE inhibited by the RPRC- or RPSC-MSNP isomer underwent spontaneous reactivation ∼10- to 20-fold faster than the enzyme inhibited by SPRC- and SPSC-MSNP, and only 4% spontaneous reactivation was observed from the SPRC-eeAChE adduct. Using 2-pyridine aldoxime methiodide (2-PAM) or trimedoxime (TMB-4), eeAChE inhibited by RPRC- or SPRC-MSNP reactivated up to 90% and 3- to 4-fold faster than eeAChE inhibited by the RPSC- or SPSC-MSNP isomer. Spontaneous reactivation rates for rmAChE were 1.5- to 10-fold higher following inhibition by RPSC- and SPSC-MSNPs than inhibition by either RC isomer, a trend opposite to that found for eeAChE. Oxime reactivation of rmAChE following inhibition by RPRC- and SPRC-MSNPs was 2.5- to 5-fold faster than inhibition by RPSC- or SPSC-MSNPs. Due to structural similarities, MSNPs that phosphylate AChE with the loss of the p-nitrophenoxy (PNP) group form identical, nonreactivatable adducts to those formed from SP-isomalathion; however, all the MSNP isomers inhibited AChE to form adducts that reactivated. Thus, MSNPs inactivate AChE via the ejection of either PNP or thiosuccinyl groups to form a combination of reactivatable and nonreactivatable adducts, and this differs from the mechanism of AChE inhibition by isomalathion.

Structural Isomerism and Enhanced Lipophilicity of Pyrithione Ligands of Organoruthenium(II) Complexes Increase Inhibition on AChE and BuChE.

The increasing number of Alzheimer’s disease (AD) cases requires the development of new improved drug candidates, possessing the ability of more efficient treatment as well as less unwanted side effects. Cholinesterase enzymes are highly associated with the development of AD and thus represent important druggable targets. Therefore, we have synthesized eight organoruthenium(II) chlorido complexes 1a–h with pyrithione-type ligands (pyrithione = 1-hydroxypyridine-2(1H)-thione, a), bearing either pyrithione a, its methyl (b-e) or bicyclic aromatic analogues (f–h) and tested them for their inhibition towards electric eel acetylcholinesterase (eeAChE) and horse serum butyrylcholinesterase (hsBuChE). The experimental results have shown that the novel complex 1g with the ligand 1-hydroxyquinoline-2-(1H)-thione (g) improves the inhibition towards eeAChE (IC50 = 4.9 μM) and even more potently towards hsBuChE (IC50 = 0.2 μM) in comparison with the referenced 1a. Moreover, computational studies on Torpedo californica AChE have supported the experimental outcomes for 1g, possessing the lowest energy value among all tested complexes and have also predicted several interactions of 1g with the target protein. Consequently, we have shown that the aromatic ring extension of the ligand a, though only at the appropriate position, is a viable strategy to enhance the activity against cholinesterases.

Synthesis of Novel 2,3-Dihydro-5,6-dimethoxy-2-((piperidin-4-yl)methyl) inden-1-one Derivatives as Acetyl Cholinesterase Inhibitors

Background: Novel carboxamides and thioureas of 2,3-dihydro-5,6-dimethoxy-2-((piperidin- 4-yl)methyl) inden-1-one were synthesized and their potential anticholinesterase activities were evaluated. The inhibition potency of the compounds 17a-j and 19a-j against AChE was measured and evaluated using Ellman’s spectrophotometric method. Among carboxamides series, compound 17f, 17i, 17j and among thiourea series, compound 19a, 19b were found to be the most active. Methods: The scaffold 2,3-dihydro-5,6-dimethoxy-2-((piperidin-4-yl)methyl) inden-1-one 16, key intermediate of drug donepezil has been synthesised in three steps and derivatised as carboxamides and thioureas for SAR studies. Compounds 17a-j and 19a-j were characterised by 1H NMR and LCMS. The inhibitory activity and antiamnesic effect were studied using different sources such as electric eel AChE, human serum AChE and rat brain homogenate AChE. Results: The results of bioassays indicated that among all the synthesized compounds tested, five compounds 17f, 17i, 17j, 19a and 19b shows IC50 at a dose of 67, 42, 64, 52 and 63 nM respectively against electric eel, human serum and rat brain homogenate, which lead to the suggestion that compound 17i might be considered to be a potent AChE inhibitor. Conclusion: Derivatives of 2,3-dihydro-5,6-dimethoxy-2-((piperidin-4-yl)methyl)inden-1-one with different substitutions were synthesised and tested for their AChE activity. The order of potency is 17i>17j>17f and 19a>19b. The other compounds screened failed to elicit any inhibition of acetyl cholinesterase from rat brain homogenate. It may be concluded from this study that, for effective binding and blocking the AChE activity, molecule needs to bind with peripheral site and active site of the enzyme. Therefore, it can be summarized that by changing the functional group and substitution in the scaffold 2,3-dihydro-5,6-dimethoxy-2-((piperidin-4-yl)methyl)inden-1-one needs to be studied for better AChE inhibitory activity in future research.

Dual Action of Dipyridothiazine and Quinobenzothiazine Derivatives-Anticancer and Cholinesterase-Inhibiting Activity.

The inverse correlation observed between Alzheimer’s disease (AD) and cancer has prompted us to look for cholinesterase-inhibiting activity in phenothiazine derivatives that possess anticancer properties. With the use of in silico and in vitro screening methods, our study found a new biological activity in anticancer polycyclic, tricyclic, and tetracyclic compounds. The virtual screening of a library of 120 ligands, which are the derivatives of azaphenothiazine, led to the identification of 25 compounds that can act as potential inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Biological assays revealed the presence of selective inhibitors of eeAChE (electric eel AChE) or eqBuChE (equine serum BuChE) and nonselective inhibitors of both enzymes among the tested compounds. Their potencies against eeAChE were in a submicromolar-to-micromolar range with IC50 values from 0.78 to 19.32 μM, while their IC50 values against eqBuChE ranged from 0.46 to 10.38 μM. The most potent among the compounds tested was the tetracyclic derivative, 6-(4-diethylaminobut-2-ynyl)-9-methylthioquinobenzothiazine 24, which was capable of inhibiting both enzymes. 9-Fluoro-6-(1-piperidylethyl)quinobenzothiazine 23 was found to act as a selective inhibitor of eqBuChE with an IC50 value of 0.46 μM. Compounds with such a dual antitumor and cholinesterase-inhibitory activity can be considered as a valuable combination for the treatment of both cancer and AD prevention. The results presented in this study might open new directions of research on the group of tricyclic phenothiazine derivatives.

Structural and functional characterization of an organometallic ruthenium complex as a potential myorelaxant drug

In addition to antibacterial and antitumor effects, synthetic ruthenium complexes have been reported to inhibit several medicinally important enzymes, including acetylcholinesterase (AChE). They may also interact with muscle-type nicotinic acetylcholine receptors (nAChRs) and thus affect the neuromuscular transmission and muscle function. In the present study, the effects of the organometallic ruthenium complex of 5-nitro-1,10-phenanthroline (nitrophen) were evaluated on these systems. The organoruthenium-nitrophen complex [(η6-p-cymene)Ru(nitrophen)Cl]Cl; C22H21Cl2N3O2Ru (C1-Cl) was synthesized, structurally characterized and evaluated in vitro for its inhibitory activity against electric eel acetylcholinesterase (eeAChE), human recombinant acetylcholinesterase (hrAChE), horse serum butyrylcholinesterase (hsBChE) and horse liver glutathione-S-transferase. The physiological effects of C1-Cl were then studied on isolated mouse phrenic nerve-hemidiaphragm muscle preparations, by means of single twitch measurements and electrophysiological recordings. The compound C1-Cl acted as a competitive inhibitor of eeAChE, hrAChE and hsBChE with concentrations producing 50 % inhibition (IC50) of enzyme activity ranging from 16 to 26 μM. Moreover, C1-Cl inhibited the nerve-evoked isometric muscle contraction (IC50 = 19.44 μM), without affecting the directly-evoked muscle single twitch up to 40 μM. The blocking effect of C1-Cl was rapid and almost completely reversed by neostigmine, a reversible cholinesterase inhibitor. The endplate potentials were also inhibited by C1-Cl in a concentration-dependent manner (IC50 = 7.6 μM) without any significant change in the resting membrane potential of muscle fibers up to 40 μM. Finally, C1-Cl (5–40 μM) decreased (i) the amplitude of miniature endplate potentials until a complete block by concentrations higher than 25 μM and (ii) their frequency at 10 μM or higher concentrations. The compound C1-Cl reversibly blocked the neuromuscular transmission in vitro by a non-depolarizing mechanism and mainly through an action on postsynaptic nAChRs. The compound C1-Cl may be therefore interesting for further preclinical testing as a new competitive neuromuscular blocking, and thus myorelaxant, drug.

A novel method for the synthesis of 1,2,4-triazole-derived heterocyclic compounds: enzyme inhibition and molecular docking studies

Two series of new N-aryl/aralkyl derivatives (9a–q) of 2-(4-ethyl-5-(thiophen-2-ylmethyl)-4H-1,2,4-triazol-3-ylthio)acetamide and N-aryl/aralkyl derivatives (10a–q) of 2-(4-phenyl-5-(thiophen-2-ylmethyl)-4H-1,2,4-triazol-3-ylthio)acetamide were synthesized. The methods included successive conversions of thiophen-2-acetic acid (a) into its respective ester, hydrazide and N-aryl/aralkyl 1,3,4-triazole. The target compounds (9a–q; 10a–q) were obtained by the reaction of N-aryl/aralkyl 1,3,4-triazole (5, 6) with various electrophiles, (8a–q), in N,N-dimethyl formamide (DMF) and sodium hydroxide at room temperature. The characterization of these compounds was done by FTIR, 1H-, 13C-NMR, EI-MS and HR-EI-MS spectral data. All compounds were evaluated for their enzyme inhibitory potentials against electric eel acetylcholinesterase, AChE (10f, 10d; IC50 values 32.26 ± 0.12, 45.72 ± 0.11 µM, respectively), equine butyrylcholinesterase, BChE (9d, 9l, 9b, 10d, 10h; IC50 values 12.52 ± 0.19, 12.52 ± 0.19, 21.72 ± 0.18, 23.62 ± 0.22, 24.52 ± 0.21 µM, respectively), jack bean urease (10i, 10n, 9e; IC50 values 7.27 ± 0.05, 7.35 ± 0.04, 8.79 ± 0.05 µM, respectively) and yeast α-glucosidase enzymes (9o, 10i; IC50 values 62.94 ± 0.19, and 69.46 ± 0.15 µM, respectively). The molecular docking studies supported these findings. This study provides cheaper bioactive triazole amides as promising future lead molecules.

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 (IC50(AChE) = 792 nM, IC50(BChE) = 2.2 nM) and BMC-16 (IC50(AChE) = 266 nM, IC50(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.

A comprehensive study on narcissus tazetta subsp. tazetta L.: Chemo-profiling, isolation, anticholinesterase activity and molecular docking of amaryllidaceae alkaloids

Galanthamine, is the most known Amaryllidaceae alkaloid, which is currently being prescribed for the treatment of mild to moderate Alzheimer's disease (AD) due to its anti-acetylcholinesterase activity. Narcissus L., a genus belonging to the same family, has been subjected to intensive research owing to its broad alkaloidal range.The main objective of the present study was to evaluate Narcissus tazetta L. in terms of its therapeutic potential in AD and to isolate Amaryllidaceae alkaloids with anticholinesterase activity.A qualitative determination of the alkaloidal contents of aerial parts and bulbs of the plant were carried out via Gas chromatography–mass spectroscopy (GC–MS) analysis. Additionally, secondary metabolites were isolated from the ethanolic extract of the plant by means of chromatographic methods. The structural elucidation of isolated compounds was established by spectroscopic techniques. Determination of acetylcholinesterase and butyrylcholinesterase inhibitory activities of the isolated Amaryllidaceae alkaloids were accomplished using a microplate assay modified from Ellman's method. These compounds were also subjected to docking studies to determine the non-covalent interactions formed with the active site residues of cholinesterase enzymes.GC–MS monitoring on the profile of N. tazetta was resulted in the detection of 22 alkaloids. Isolation process on the plant has led to isolate four alkaloids belonging to three different skeleton types. Nuclear Magnetic Resonance (NMR) and MS findings verified that those compounds were identical with (−)-9-O-methylpseudolycorine, (−)-narcissidine, (−)-pancratinine-C and (+)-9-O-demethyl-2-α-hydroxyhomolycorine. Among those; 11-hydroxygalanthine and narcissidine have shown significant acetylcholinesterase (AChE) inhibitory activity based on our bioactivity results. It was observed that the docking scores were found close in general, when the docking scores of the isolated alkaloids calculated inside electric eel acetylcholinesterase (eeAChE) and Equus caballus butyrylcholinesterase (eqBuChE) were examined. Increased anti-acetylcholinesterase activity was observed in lycorine-type.This is the first report on the isolation of (−)-pancratinine-C from the genus Narcissus. Docking scores of all the compounds isolated from the title plant were agreeable with the bioactivity results. The evaluation of the results given in this study, reveals that this particular Turkish Narcissus species represents a promising source of bioactive alkaloids with intriguing chemical structures which may have the potential to be drug leads.