Combining Experimental and Computational Methods to Produce Conjugates of Anticholinesterase and Antioxidant Pharmacophores with Linker Chemistries Affecting Biological Activities Related to Treatment of Alzheimer's Disease.

Effective therapeutics for Alzheimer's disease (AD) are in great demand worldwide. In our previous work, we responded to this need by synthesizing novel drug candidates consisting of 4-amino-2,3-polymethylenequinolines conjugated with butylated hydroxytoluene via fixed-length alkylimine or alkylamine linkers (spacers) and studying their bioactivities pertaining to AD treatment. Here, we report significant extensions of these studies, including the use of variable-length spacers and more detailed biological characterizations. Conjugates were potent inhibitors of acetylcholinesterase (AChE, the most active was 17d IC50 15.1 ± 0.2 nM) and butyrylcholinesterase (BChE, the most active was 18d: IC50 5.96 ± 0.58 nM), with weak inhibition of off-target carboxylesterase. Conjugates with alkylamine spacers were more effective cholinesterase inhibitors than alkylimine analogs. Optimal inhibition for AChE was exhibited by cyclohexaquinoline and for BChE by cycloheptaquinoline. Increasing spacer length elevated the potency against both cholinesterases. Structure-activity relationships agreed with docking results. Mixed-type reversible AChE inhibition, dual docking to catalytic and peripheral anionic sites, and propidium iodide displacement suggested the potential of hybrids to block AChE-induced ß-amyloid (Aß) aggregation. Hybrids also exhibited the inhibition of Aß self-aggregation in the thioflavin test; those with a hexaquinoline ring and C8 spacer were the most active. Conjugates demonstrated high antioxidant activity in ABTS and FRAP assays as well as the inhibition of luminol chemiluminescence and lipid peroxidation in mouse brain homogenates. Quantum-chemical calculations explained antioxidant results. Computed ADMET profiles indicated favorable blood-brain barrier permeability, suggesting the CNS activity potential. Thus, the conjugates could be considered promising multifunctional agents for the potential treatment of AD.

Conjugates of amiridine and thiouracil derivatives as effective inhibitors of butyrylcholinesterase with the potential to block ß-amyloid aggregation.

New amiridine-thiouracil conjugates with different substituents in the pyrimidine fragment (R = CH3 , CF2 Н, CF3 , (CF2 )2 H) and different spacer lengths (n = 1-3) were synthesized. The conjugates rather weakly inhibit acetylcholinesterase (AChE) and exhibit high inhibitory activity (IC50 up to 0.752 ± 0.021 µM) and selectivity to butyrylcholinesterase (BChE), which increases with spacer elongation; the lead compounds are 11c, 12c, and 13c. The conjugates are mixed-type reversible inhibitors of both cholinesterases and practically do not inhibit the structurally related off-target enzyme carboxylesterase. The results of molecular docking to AChE and BChE are consistent with the experiment on enzyme inhibition and explain the structure-activity relationships, including the rather low anti-AChE activity and the high anti-BChE activity of long-chain conjugates. The lead compounds displace propidium from the AChE peripheral anion site (PAS) at the level of the reference compound donepezil, which agrees with the mixed-type mechanism of AChE inhibition and the main mode of binding of conjugates in the active site of AChE due to the interaction of the pyrimidine moiety with the PAS. This indicates the ability of the studied conjugates to block AChE-induced aggregation of ß-amyloid, thereby exerting a disease-modifying effect. According to computer calculations, all synthesized conjugates have an ADME profile acceptable for drugs.

Derivatives of 9-phosphorylated acridine as butyrylcholinesterase inhibitors with antioxidant activity and the ability to inhibit ß-amyloid self-aggregation: potential therapeutic agents for Alzheimer's disease.

We investigated the inhibitory activities of novel 9-phosphoryl-9,10-dihydroacridines and 9-phosphorylacridines against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and carboxylesterase (CES). We also studied the abilities of the new compounds to interfere with the self-aggregation of ß-amyloid (Aß42) in the thioflavin test as well as their antioxidant activities in the ABTS and FRAP assays. We used molecular docking, molecular dynamics simulations, and quantum-chemical calculations to explain experimental results. All new compounds weakly inhibited AChE and off-target CES. Dihydroacridines with aryl substituents in the phosphoryl moiety inhibited BChE; the most active were the dibenzyloxy derivative 1d and its diphenethyl bioisostere 1e (IC50 = 2.90 ± 0.23 µM and 3.22 ± 0.25 µM, respectively). Only one acridine, 2d, an analog of dihydroacridine, 1d, was an effective BChE inhibitor (IC50 = 6.90 ± 0.55 µM), consistent with docking results. Dihydroacridines inhibited Aß42 self-aggregation; 1d and 1e were the most active (58.9% ± 4.7% and 46.9% ± 4.2%, respectively). All dihydroacridines 1 demonstrated high ABTS•+-scavenging and iron-reducing activities comparable to Trolox, but acridines 2 were almost inactive. Observed features were well explained by quantum-chemical calculations. ADMET parameters calculated for all compounds predicted favorable intestinal absorption, good blood-brain barrier permeability, and low cardiac toxicity. Overall, the best results were obtained for two dihydroacridine derivatives 1d and 1e with dibenzyloxy and diphenethyl substituents in the phosphoryl moiety. These compounds displayed high inhibition of BChE activity and Aß42 self-aggregation, high antioxidant activity, and favorable predicted ADMET profiles. Therefore, we consider 1d and 1e as lead compounds for further in-depth studies as potential anti-AD preparations.

Conjugates of Tacrine and Salicylic Acid Derivatives as New Promising Multitarget Agents for Alzheimer's Disease.

A series of previously synthesized conjugates of tacrine and salicylamide was extended by varying the structure of the salicylamide fragment and using salicylic aldehyde to synthesize salicylimine derivatives. The hybrids exhibited broad-spectrum biological activity. All new conjugates were potent inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with selectivity toward BChE. The structure of the salicylamide moiety exerted little effect on anticholinesterase activity, but AChE inhibition increased with spacer elongation. The most active conjugates were salicylimine derivatives: IC50 values of the lead compound 10c were 0.0826 µM (AChE) and 0.0156 µM (BChE), with weak inhibition of the off-target carboxylesterase. The hybrids were mixed-type reversible inhibitors of both cholinesterases and displayed dual binding to the catalytic and peripheral anionic sites of AChE in molecular docking, which, along with experimental results on propidium iodide displacement, suggested their potential to block AChE-induced ß-amyloid aggregation. All conjugates inhibited Aß42 self-aggregation in the thioflavin test, and inhibition increased with spacer elongation. Salicylimine 10c and salicylamide 5c with (CH2)8 spacers were the lead compounds for inhibiting Aß42 self-aggregation, which was corroborated by molecular docking to Aß42. ABTS•+-scavenging activity was highest for salicylamides 5a-c, intermediate for salicylimines 10a-c, low for F-containing salicylamides 7, and non-existent for methoxybenzoylamides 6 and difluoromethoxybenzoylamides 8. In the FRAP antioxidant (AO) assay, the test compounds displayed little or no activity. Quantum chemical analysis and molecular dynamics (MD) simulations with QM/MM potentials explained the AO structure-activity relationships. All conjugates were effective chelators of Cu2+, Fe2+, and Zn2+, with molar compound/metal (Cu2+) ratios of 2:1 (5b) and ~1:1 (10b). Conjugates exerted comparable or lower cytotoxicity than tacrine on mouse hepatocytes and had favorable predicted intestinal absorption and blood-brain barrier permeability. The overall results indicate that the synthesized conjugates are promising new multifunctional agents for the potential treatment of AD.

New Multifunctional Agents for Potential Alzheimer's Disease Treatment Based on Tacrine Conjugates with 2-Arylhydrazinylidene-1,3-Diketones.

Alzheimer's disease (AD) is considered a modern epidemic because of its increasing prevalence worldwide and serious medico-social consequences, including the economic burden of treatment and patient care. The development of new effective therapeutic agents for AD is one of the most urgent and challenging tasks. To address this need, we used an aminoalkylene linker to combine the well-known anticholinesterase drug tacrine with antioxidant 2-tolylhydrazinylidene-1,3-diketones to create 3 groups of hybrid compounds as new multifunctional agents with the potential for AD treatment. Lead compounds of the new conjugates effectively inhibited acetylcholinesterase (AChE, IC50 0.24-0.34 µM) and butyrylcholinesterase (BChE, IC50 0.036-0.0745 µM), with weak inhibition of off-target carboxylesterase. Anti-AChE activity increased with elongation of the alkylene spacer, in agreement with molecular docking, which showed compounds binding to both the catalytic active site and peripheral anionic site (PAS) of AChE, consistent with mixed type reversible inhibition. PAS binding along with effective propidium displacement suggest the potential of the hybrids to block AChE-induced ß-amyloid aggregation, a disease-modifying effect. All of the conjugates demonstrated metal chelating ability for Cu2+, Fe2+, and Zn2+, as well as high antiradical activity in the ABTS test. Non-fluorinated hybrid compounds 6 and 7 also showed Fe3+ reducing activity in the FRAP test. Predicted ADMET and physicochemical properties of conjugates indicated good CNS bioavailability and safety parameters acceptable for potential lead compounds at the early stages of anti-AD drug development.

Novel Uracil-Based Inhibitors of Acetylcholinesterase with Potency for Treating Memory Impairment in an Animal Model of Alzheimer's Disease.

Novel derivatives based on 6-methyluracil and condensed uracil, 2,4-quinazoline-2,4-dione, were synthesized with terminal meta- and para-benzoate moieties in polymethylene chains at the N atoms of the pyrimidine ring. In the synthesized compounds, the polymethylene chains were varied from having tris- to hexamethylene chains and quaternary ammonium groups; varying substituents (ester, salt, acid) at benzene ring were introduced into the chains and benzoate moieties. In vivo biological experiments demonstrated the potency of these compounds in decreasing the number of ß-amyloid plaques and their suitability for the treatment of memory impairment in a transgenic model of Alzheimer's disease.

Conjugates of Methylene Blue with Cycloalkaneindoles as New Multifunctional Agents for Potential Treatment of Neurodegenerative Disease.

The development of multi-target-directed ligands (MTDLs) would provide effective therapy of neurodegenerative diseases (ND) with complex and nonclear pathogenesis. A promising method to create such potential drugs is combining neuroactive pharmacophoric groups acting on different biotargets involved in the pathogenesis of ND. We developed a synthetic algorithm for the conjugation of indole derivatives and methylene blue (MB), which are pharmacophoric ligands that act on the key stages of pathogenesis. We synthesized hybrid structures and performed a comprehensive screening for a specific set of biotargets participating in the pathogenesis of ND (i.e., cholinesterases, NMDA receptor, mitochondria, and microtubules assembly). The results of the screening study enabled us to find two lead compounds (4h and 4i) which effectively inhibited cholinesterases and bound to the AChE PAS, possessed antioxidant activity, and stimulated the assembly of microtubules. One of them (4i) exhibited activity as a ligand for the ifenprodil-specific site of the NMDA receptor. In addition, this lead compound was able to bypass the inhibition of complex I and prevent calcium-induced mitochondrial depolarization, suggesting a neuroprotective property that was confirmed using a cellular calcium overload model of neurodegeneration. Thus, these new MB-cycloalkaneindole conjugates constitute a promising class of compounds for the development of multitarget neuroprotective drugs which simultaneously act on several targets, thereby providing cognitive stimulating, neuroprotective, and disease-modifying effects.

Conjugates of Tacrine with Salicylamide as Promising Multitarget Agents for Alzheimer's Disease.

New conjugates of tacrine and salicylamide with alkylene spacers were synthesized and evaluated as potential multifunctional agents for Alzheimer's disease (AD). The compounds exhibited high acetylcholinesterase (AChE, IC50 to 0.224 µM) and butyrylcholinesterase (BChE, IC50 to 0.0104 µM) inhibitory activities. They were also rather poor inhibitors of carboxylesterase, suggesting a low tendency to exert potential unwanted drug-drug interactions in clinical use. The conjugates were mixed-type reversible inhibitors of both cholinesterases and demonstrated dual binding to the catalytic and peripheral anionic sites of AChE in molecular docking that, along with experimental results on propidium iodide displacement, suggest their potential to block AChE-induced ß-amyloid aggregation. The new conjugates exhibited high ABTS.+ -scavenging activity. N-(6-(1,2,3,4-Tetrahydroacridin-9-ylamino)hexyl)salicylamide is a lead compound that also demonstrates metal chelating ability toward Cu2+ , Fe2+ and Zn2+ . Thus, the new conjugates have displayed the potential to be multifunctional anti-AD agents for further development.

Bis-Amiridines as Acetylcholinesterase and Butyrylcholinesterase Inhibitors: N-Functionalization Determines the Multitarget Anti-Alzheimer's Activity Profile.

Using two ways of functionalizing amiridine-acylation with chloroacetic acid chloride and reaction with thiophosgene-we have synthesized new homobivalent bis-amiridines joined by two different spacers-bis-N-acyl-alkylene (3) and bis-N-thiourea-alkylene (5) -as potential multifunctional agents for the treatment of Alzheimer's disease (AD). All compounds exhibited high inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with selectivity for BChE. These new agents displayed negligible carboxylesterase inhibition, suggesting a probable lack of untoward drug-drug interactions arising from hydrolytic biotransformation. Compounds 3 with bis-N-acyl-alkylene spacers were more potent inhibitors of both cholinesterases compared to compounds 5 and the parent amiridine. The lead compounds 3a-c exhibited an IC50(AChE) = 2.9-1.4 µM, IC50(BChE) = 0.13-0.067 µM, and 14-18% propidium displacement at 20 µM. Kinetic studies of compounds 3a and 5d indicated mixed-type reversible inhibition. Molecular docking revealed favorable poses in both catalytic and peripheral AChE sites. Propidium displacement from the peripheral site by the hybrids suggests their potential to hinder AChE-assisted Aß42 aggregation. Conjugates 3 had no effect on Aß42 self-aggregation, whereas compounds 5c-e (m = 4, 5, 6) showed mild (13-17%) inhibition. The greatest difference between conjugates 3 and 5 was their antioxidant activity. Bis-amiridines 3 with N-acylalkylene spacers were nearly inactive in ABTS and FRAP tests, whereas compounds 5 with thiourea in the spacers demonstrated high antioxidant activity, especially in the ABTS test (TEAC = 1.2-2.1), in agreement with their significantly lower HOMO-LUMO gap values. Calculated ADMET parameters for all conjugates predicted favorable blood-brain barrier permeability and intestinal absorption, as well as a low propensity for cardiac toxicity. Thus, it was possible to obtain amiridine derivatives whose potencies against AChE and BChE equaled (5) or exceeded (3) that of the parent compound, amiridine. Overall, based on their expanded and balanced pharmacological profiles, conjugates 5c-e appear promising for future optimization and development as multitarget anti-AD agents.

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 (≪Km) 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.

Conjugation of Aminoadamantane and γ-Carboline Pharmacophores Gives Rise to Unexpected Properties of Multifunctional Ligands.

A new series of conjugates of aminoadamantane and γ-carboline, which are basic scaffolds of the known neuroactive agents, memantine and dimebon (Latrepirdine) was synthesized and characterized. Conjugates act simultaneously on several biological structures and processes involved in the pathogenesis of Alzheimer's disease and some other neurodegenerative disorders. In particular, these compounds inhibit enzymes of the cholinesterase family, exhibiting higher inhibitory activity against butyrylcholinesterase (BChE), but having almost no effect on the activity of carboxylesterase (anti-target). The compounds serve as NMDA-subtype glutamate receptor ligands, show mitoprotective properties by preventing opening of the mitochondrial permeability transition (MPT) pore, and act as microtubule stabilizers, stimulating the polymerization of tubulin and microtubule-associated proteins. Structure-activity relationships were studied, with particular attention to the effect of the spacer on biological activity. The synthesized conjugates showed new properties compared to their prototypes (memantine and dimebon), including the ability to bind to the ifenprodil-binding site of the NMDA receptor and to occupy the peripheral anionic site of acetylcholinesterase (AChE), which indicates that these compounds can act as blockers of AChE-induced ß-amyloid aggregation. These new attributes of the conjugates represent improvements to the pharmacological profiles of the separate components by conferring the potential to act as neuroprotectants and cognition enhancers with a multifunctional mode of action.

Amiridine-piperazine hybrids as cholinesterase inhibitors and potential multitarget agents for Alzheimer's disease treatment.

We synthesized eleven new amiridine-piperazine hybrids 5a-j and 7 as potential multifunctional agents for Alzheimer's disease (AD) treatment by reacting N-chloroacetylamiridine with piperazines. The compounds displayed mixed-type reversible inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Conjugates were moderate inhibitors of equine and human BChE with negligible fluctuation in anti-BChE activity, whereas anti-AChE activity was substantially dependent on N4-substitution of the piperazine ring. Compounds with para-substituted aromatic moieties (5g, 5h, and bis-amiridine 7) had the highest anti-AChE activity in the low micromolar range. Top-ranked compound 5h, N-(2,3,5,6,7,8-hexahydro-1H-cyclopenta[b]quinolin-9-yl)-2-[4-(4-nitro-phenyl)-piperazin-1-yl]-acetamide, had an IC50 for AChE = 1.83 ± 0.03 µM (Ki = 1.50 ± 0.12 and αKi = 2.58 ± 0.23 µM). The conjugates possessed low activity against carboxylesterase, indicating a likely absence of unwanted drug-drug interactions in clinical use. In agreement with analysis of inhibition kinetics and molecular modeling studies, the lead compounds were found to bind effectively to the peripheral anionic site of AChE and displace propidium, indicating their potential to block AChE-induced ß-amyloid aggregation. Similar propidium displacement activity was first shown for amiridine. Two compounds, 5c (R = cyclohexyl) and 5e (R = 2-MeO-Ph), exhibited appreciable antioxidant capability with Trolox equivalent antioxidant capacity values of 0.47 ± 0.03 and 0.39 ± 0.02, respectively. Molecular docking and molecular dynamics simulations provided insights into the structure-activity relationships for AChE and BChE inhibition, including the observation that inhibitory potencies and computed pKa values of hybrids were generally lower than those of the parent molecules. Predicted ADMET and physicochemical properties of conjugates indicated good CNS bioavailability and safety parameters comparable to those of amiridine and therefore acceptable for potential lead compounds at the early stages of anti-AD drug development.

Novel potent bifunctional carboxylesterase inhibitors based on a polyfluoroalkyl-2-imino-1,3-dione scaffold.

An expanded series of alkyl 2-arylhydrazinylidene-3-oxo-3-polyfluoroalkylpropionates (HOPs) 3 was obtained via Cu(OAc)2-catalyzed azo coupling. All were nanomolar inhibitors of carboxylesterase (CES), while moderate or weak inhibitors of acetylcholinesterase and butyrylcholinesterase. Steady-state kinetics studies showed that HOPs 3 are mixed type inhibitors of the three esterases. Molecular docking studies demonstrated that two functional groups in the structure of HOPs, trifluoromethyl ketone (TFK) and ester groups, bind to the CES active site suggesting subsequent reactions: formation of a tetrahedral adduct, and a slow hydrolysis reaction. The results of molecular modeling allowed us to explain some structure-activity relationships of CES inhibition by HOPs 3: their selectivity toward CES in comparison with cholinesterases and the high selectivity of pentafluoroethyl-substituted HOP 3p to hCES1 compared to hCES2. All compounds were predicted to have good intestinal absorption and blood-brain barrier permeability, low cardiac toxicity, good lipophilicity and aqueous solubility, and reasonable overall drug-likeness. HOPs with a TFK group and electron-donor substituents in the arylhydrazone moiety were potent antioxidants. All compounds possessed low cytotoxicity and low acute toxicity. Overall, a new promising type of bifunctional CES inhibitors has been found that are able to interact with the active site of the enzyme with the participation of two functional groups. The results indicate that HOPs have the potential to be good candidates as human CES inhibitors for biomedicinal applications.

Impact of Sucrose as Osmolyte on Molecular Dynamics of Mouse Acetylcholinesterase.

The enzyme model, mouse acetylcholinesterase, which exhibits its active site at the bottom of a narrow gorge, was investigated in the presence of different concentrations of sucrose to shed light on the protein and water dynamics in cholinesterases. The study was conducted by incoherent neutron scattering, giving access to molecular dynamics within the time scale of sub-nano to nanoseconds, in comparison with molecular dynamics simulations. With increasing sucrose concentration, we found non-linear effects, e.g., first a decrease in the dynamics at 5 wt% followed by a gain at 10 wt% sucrose. Direct comparisons with simulations permitted us to understand the following findings: at 5 wt%, sugar molecules interact with the protein surface through water molecules and damp the motions to reduce the overall protein mobility, although the motions inside the gorge are enhanced due to water depletion. When going to 10 wt% of sucrose, some water molecules at the protein surface are replaced by sugar molecules. By penetrating the protein surface, they disrupt some of the intra-protein contacts, and induce new ones, creating new pathways for correlated motions, and therefore, increasing the dynamics. This exhaustive study allowed for an explanation of the detail interactions leading to the observed non-linear behavior.

New Multifunctional Agents Based on Conjugates of 4-Amino-2,3-polymethylenequinoline and Butylated Hydroxytoluene for Alzheimer's Disease Treatment.

New hybrids of 4-amino-2,3-polymethylenequinoline with different sizes of the aliphatic ring linked to butylated hydroxytoluene (BHT) by enaminoalkyl (7) or aminoalkyl (8) spacers were synthesized as potential multifunctional agents for Alzheimer's disease (AD) treatment. All compounds were potent inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with selectivity toward BChE. Lead compound 8c, 2,6-di-tert-butyl-4-{[2-(7,8,9,10- tetrahydro-6H-cyclohepta[b]quinolin-11-ylamino)-ethylimino]-methyl}-phenol exhibited an IC50(AChE) = 1.90 ± 0.16 µM, IC50(BChE) = 0.084 ± 0.008 µM, and 13.6 ± 1.2% propidium displacement at 20 µM. Compounds possessed low activity against carboxylesterase, indicating likely absence of clinically unwanted drug-drug interactions. Kinetics were consistent with mixed-type reversible inhibition of both cholinesterases. Docking indicated binding to catalytic and peripheral AChE sites; peripheral site binding along with propidium displacement suggest the potential of the hybrids to block AChE-induced ß-amyloid aggregation, a disease-modifying effect. Compounds demonstrated high antioxidant activity in ABTS and FRAP assays as well as inhibition of luminol chemiluminescence and lipid peroxidation in mouse brain homogenates. Conjugates 8 with amine-containing spacers were better antioxidants than those with enamine spacers 7. Computational ADMET profiles for all compounds predicted good blood-brain barrier distribution (permeability), good intestinal absorption, and medium cardiac toxicity risk. Overall, based on their favorable pharmacological and ADMET profiles, conjugates 8 appear promising as candidates for AD therapeutics.

1-(3-Tert-Butylphenyl)-2,2,2-Trifluoroethanone as a Potent Transition-State Analogue Slow-Binding Inhibitor of Human Acetylcholinesterase: Kinetic, MD and QM/MM Studies.

Kinetic studies and molecular modeling of human acetylcholinesterase (AChE) inhibition by a fluorinated acetophenone derivative, 1-(3-tert-butylphenyl)-2,2,2-trifluoroethanone (TFK), were performed. Fast reversible inhibition of AChE by TFK is of competitive type with Ki = 5.15 nM. However, steady state of inhibition is reached slowly. Kinetic analysis showed that TFK is a slow-binding inhibitor (SBI) of type B with Ki* = 0.53 nM. Reversible binding of TFK provides a long residence time, τ = 20 min, on AChE. After binding, TFK acylates the active serine, forming an hemiketal. Then, disruption of hemiketal (deacylation) is slow. AChE recovers full activity in approximately 40 min. Molecular docking and MD simulations depicted the different steps. It was shown that TFK binds first to the peripheral anionic site. Then, subsequent slow induced-fit step enlarged the gorge, allowing tight adjustment into the catalytic active site. Modeling of interactions between TFK and AChE active site by QM/MM showed that the "isomerization" step of enzyme-inhibitor complex leads to a complex similar to substrate tetrahedral intermediate, a so-called "transition state analog", followed by a labile covalent intermediate. SBIs of AChE show prolonged pharmacological efficacy. Thus, this fluoroalkylketone intended for neuroimaging, could be of interest in palliative therapy of Alzheimer's disease and protection of central AChE against organophosphorus compounds.

New Hybrids of 4-Amino-2,3-polymethylene-quinoline and p-Tolylsulfonamide as Dual Inhibitors of Acetyl- and Butyrylcholinesterase and Potential Multifunctional Agents for Alzheimer's Disease Treatment.

New hybrid compounds of 4-amino-2,3-polymethylene-quinoline containing different sizes of the aliphatic ring and linked to p-tolylsulfonamide with alkylene spacers of increasing length were synthesized as potential drugs for treatment of Alzheimer's disease (AD). All compounds were potent inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with selectivity toward BChE. The lead compound 4-methyl-N-(5-(1,2,3,4-tetrahydro-acridin-9-ylamino)-pentyl)-benzenesulfonamide (7h) exhibited an IC50 (AChE) = 0.131 ± 0.01 µM (five times more potent than tacrine), IC50(BChE) = 0.0680 ± 0.0014 µM, and 17.5 ± 1.5% propidium displacement at 20 µM. The compounds possessed low activity against carboxylesterase, indicating a likely absence of unwanted drug-drug interactions in clinical use. Kinetics studies were consistent with mixed-type reversible inhibition of both cholinesterases. Molecular docking demonstrated dual binding sites of the conjugates in AChE and clarified the differences in the structure-activity relationships for AChE and BChE inhibition. The conjugates could bind to the AChE peripheral anionic site and displace propidium, indicating their potential to block AChE-induced ß-amyloid aggregation, thereby exerting a disease-modifying effect. All compounds demonstrated low antioxidant activity. Computational ADMET profiles predicted that all compounds would have good intestinal absorption, medium blood-brain barrier permeability, and medium cardiac toxicity risk. Overall, the results indicate that the novel conjugates show promise for further development and optimization as multitarget anti-AD agents.

Novel Acetylcholinesterase Inhibitors Based on Uracil Moiety for Possible Treatment of Alzheimer Disease.

In this study, novel derivatives based on 6-methyluracil and condensed uracil were synthesized, namely, 2,4-quinazoline-2,4-dione with ω-(ortho-nitrilebenzylethylamino) alkyl chains at the N atoms of the pyrimidine ring. In this series of synthesized compounds, the polymethylene chains were varied from having tetra- to hexamethylene chains, and secondary NH, tertiary ethylamino, and quaternary ammonium groups were introduced into the chains. The molecular modeling of the compounds indicated that they could function as dual binding site acetylcholinesterase inhibitors, binding to both the peripheral anionic site and active site. The data from in vitro experiments show that the most active compounds exhibit affinity toward acetylcholinesterase within a nanomolar range, with selectivity for acetylcholinesterase over butyrylcholinesterase reaching four orders of magnitude. In vivo biological assays demonstrated the potency of these compounds in the treatment of memory impairment using an animal model of Alzheimer disease.

Slow-binding inhibitors of acetylcholinesterase of medical interest.

Certain ligands slowly bind to acetylcholinesterase. As a result, there is a slow establishment of enzyme-inhibitor equilibrium characterized by a slow onset of inhibition prior reaching steady state. Three mechanisms account for slow-binding inhibition: a) slow binding rate constant kon, b) slow ligand induced-fit following a fast binding step, c) slow conformational selection of an enzyme form. The slow equilibrium may be followed by a chemical step. This later that can be irreversible has been observed with certain alkylating agents and substrate transition state analogs. Slow-binding inhibitors present long residence times on target. This results in prolonged pharmacological or toxicological action. Through several well-known molecules (e.g. huperzine) and new examples (tocopherol, trifluoroacetophenone and a 6-methyluracil alkylammonium derivative), we show that slow-binding inhibitors of acetylcholinesterase are promising drugs for treatment of neurological diseases such as Alzheimer disease and myasthenia gravis. Moreover, they may be of interest for neuroprotection (prophylaxis) against organophosphorus poisoning. This article is part of the special issue entitled 'Acetylcholinesterase Inhibitors: From Bench to Bedside to Battlefield'.

Bi-functional sterically hindered phenol lipid-based delivery systems as potential multi-target agents against Alzheimer's disease via an intranasal route.

New lipid-based nanomaterials and multi-target directed ligands (MTDLs) based on sterically hindered phenol, containing a quaternary ammonium moiety (SHP-s-R, with s = 2,3) of varying hydrophobicity (R = CH2Ph and CnH2n+1, with n = 8, 10, 12, 16), have been prepared as potential drugs against Alzheimer's disease (AD). SHP-s-R are inhibitors of human cholinesterases with antioxidant properties. The inhibitory potency of SHP-s-R and selectivity ratio of cholinesterase inhibition were found to significantly depend on the length of the methylene spacer (s) and alkyl chain length. The compound SHP-2-16 showed the best IC50 for human AChE and the highest selectivity, being 30-fold more potent than for human BChE. Molecular modeling of SHP-2-16 binding to human AChE suggests that this compound is a dual binding site inhibitor that interacts with both the peripheral anionic site and catalytic active site. The relationship between self-assembly parameters (CMC, solubilization capacity, aggregation number), antioxidant activity and a toxicological parameter (hemolytic action on human red blood cells) was investigated. Two sterically hindered phenols (SHP-2-Bn and SHP-2-R) were loaded into L-α-phosphatidylcholine (PC) nanoparticles by varying the SHP alkyl chain length. For the brain AChE inhibition assay, PC/SHP-2-Bn/SHP-2-16 nanoparticles were administered to rats intranasally at a dose of 8 mg kg-1. The Morris water maze experiment showed that scopolamine-induced AD-like dementia in rats treated with PC/SHP-2-Bn/SHP-2-16 nanoparticles was significantly reduced. This is the first example of cationic SHP-phospholipid nanoparticles for inhibition of brain cholinesterases realized by the use of intranasal administration. This route has promising potential for the treatment of AD.