Design, synthesis and pharmacological evaluation of hybrid molecules out of quinazolinimines and lipoic acid lead to highly potent and selective butyrylcholinesterase inhibitors with antioxidant properties

A set of hybrid molecules were synthesized out of lipoic acid, alpha,omega-diamines of different lengths serving as spacers, and cholinesterase (ChE) inhibiting [2,1-b]quinazolinimines. Depending on the length of the alkylene spacer the amide hybrids are inhibitors of acetylcholinesterase (AChE) with inhibitory activities of 0.5-4.6microM and inhibitors of butyrylcholinesterase (BChE) with activities down to 5.7nM, therefore greatly exceeding the inhibitory activities of the parent quinazolinimines by factors of up to 1000. Due to increasing activity at BChE with increasing length of the alkylene spacer approximately 100-fold selectivity toward BChE is reached with a hepta- and an octamethylene spacer. Kinetic measurements reveal competitive and reversible inhibition of both ChEs by the hybrids. Furthermore, cell viability and antioxidant activity (using the ORAC-fluorescein assay) of several hybrids were evaluated, showing cytotoxicity at concentrations from 3.7 to 10.2microM and antioxidant properties are in the range of 0.4-0.8 Trolox equivalents (lipoic acid=0.6).     

Synthesis,cholinesterase inhibition and molecular modelling studies of coumarin linked thiourea derivatives

Alzheimer’s disease is among the most widespread neurodegenerative disorder. Cholinesterases (ChEs) play an indispensable role in the control of cholinergic transmission and thus the acetylcholine level in the brain is enhanced by inhibition of ChEs. Coumarin linked thiourea derivatives were designed, synthesized and evaluated biologically in order to determine their inhibitory activity against acetylcholinesterases (AChE) and butyrylcholinesterases (BChE). The synthesized derivatives of coumarin linked thiourea compounds showed potential inhibitory activity against AChE and BChE. Among all the synthesized compounds, 1-(2-Oxo-2H-chromene-3-carbonyl)-3-(3-chlorophenyl)thiourea (2e) was the most potent inhibitor against AChE with an IC₅₀ value of 0.04 ± 0.01 μM, while 1-(2-Oxo-2H-chromene-3-carbonyl)-3-(2-methoxyphenyl)thiourea (2b) showed the most potent inhibitory activity with an IC₅₀ value of 0.06 ± 0.02 μM against BChE. Molecular docking simulations were performed using the homology models of both cholinesterases in order to explore the probable binding modes of inhibitors. Results showed that the novel synthesized coumarin linked thiourea derivatives are potential candidates to develop for potent and efficacious acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors.     

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.     

Prenylated xanthones from mangosteen as promising cholinesterase inhibitors and their molecular docking studies

Garcinia mangostana is a well-known tropical plant found mostly in South East Asia. The present study investigated acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities of G. mangostana extract and its chemical constituents using Ellman's colorimetric method. Cholinesterase inhibitory-guided approach led to identification of six bioactive prenylated xanthones showing moderate to potent cholinesterases inhibition with IC₅₀ values of lower than 20.5 μM. The most potent inhibitor of AChE was garcinone C while γ-mangostin was the most potent inhibitor of BChE with IC₅₀ values of 1.24 and 1.78 μM, respectively. Among the xanthones, mangostanol, 3-isomangostin, garcinone C and α-mangostin are AChE selective inhibitors, 8-deoxygartanin is a BChE selective inhibitor while γ-mangostin is a dual inhibitor. Preliminary structure-activity relationship suggests the importance of the C-8 prenyl and C-7 hydroxy groups for good AChE and BChE inhibitory activities. The enzyme kinetic studies indicate that both α-mangostin and garcinone C are mixed-mode inhibitors, while γ-mangostin is a non-competitive inhibitor of AChE. In contrast, both γ-mangostin and garcinone C are uncompetitive inhibitors, while α-mangostin is a mixed-mode inhibitor of BChE. Molecular docking studies revealed that α-mangostin, γ-mangostin and garcinone C interacts differently with the five important regions of AChE and BChE. The nature of protein–ligand interactions is mainly hydrophobic and hydrogen bonding. These bioactive prenylated xanthones are worthy for further investigations.     

Novel aromatic–polyamine conjugates as cholinesterase inhibitors with notable selectivity toward butyrylcholinesterase

Three types of aromatic–polyamine conjugates (6a–6s) were designed, synthesized and evaluated as potential inhibitors for cholinesterases (ChEs). The results showed that anthraquinone–polyamine conjugates (AQPCs) exhibited the most potent acetylcholinesterase (AChE) inhibitory activity with IC₅₀ values from 1.50 to 11.13 μM. Anthracene–polyamine conjugates (APCs) showed a surprising selectivity (from 76- to 3125-fold) and were most potent at inhibiting butyrylcholinesterase (BChE), with IC₅₀ values from 0.016 to 0.657 μM. A Lineweaver–Burk plot and molecular modeling studies indicated that the representative compounds, 6l and 6k, targeted both the catalytic active site (CAS) and the peripheral anionic site (PAS) of ChEs. Furthermore, APCs did not affect HepG2 cell viability at the concentration of 100 μM. Consequently, these polyamine conjugates could be thoroughly and systematically studied for the treatment of AD.     

Design,synthesis and cholinesterase inhibitory activity of novel spiropyrrolidine tethered imidazole heterocyclic hybrids

A small library of structurally fascinating spiropyrrolidine tethered imidazole heterocylic hybrids has been synthesized regioselectively in good yields employing [bmim]Br mediated 1,3-diplar cycloaddition strategy. The new class of azomethine ylide generated in situ from l-histidine and 11H-indeno[1,2-b]quinoxalin-11-one reacts with various substituted β-nitrostyrenes affording the spiropyrrolidine tethered imidazole heterocylic hybrids. Compounds thus synthesized were assessed for their in vitro cholinesterase (ChEs) inhibitory activities, among them compounds possessing 4-methyl and 4-methoxy substituents on the aryl ring showed potent activities with IC₅₀ values of 2.02 ± 0.05 and 2.05 ± 0.06 μM against AChE and 12.40 ± 0.14 and 11.45 ± 0.28 μM against BChE enzyme, respectively. In addition, the most active compounds were performed for their molecular docking simulation and the results revealed interesting binding templates to the active site channel of cholinesterase enzymes.     

Novel tricyclic quinazolinimines and related tetracyclic nitrogen bridgehead compounds as cholinesterase inhibitors with selectivity towards butyrylcholinesterase

Tetracyclic nitrogen bridgehead compounds, dibenzodiazecines and tricyclic quinazolinimines, in which the size of the alicyclic ring system and the length of the alkyl chain between the quinazolinimine moiety and a phenyl ring connected to the imine nitrogen atom were changed systematically, were synthesized and their ability to inhibit acetyl- and butyrylcholinesterase (AChE/BChE), respectively, was evaluated. Moderate and strong inhibitors of BChE--compared to galanthamine and rivastigmine--were identified, which show mixed affinities or are moderately or highly selective towards BChE, respectively.     

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

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

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

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

Alkylammonium chlorobenzoates are a new group of ester-containing reversible inhibitors of cholinesterases of different animals

Interaction with cholinesterases (ChEs) of nine specially synthesized derivatives of dimethylaminoalkyl esters of 2-chloro-and 2,4-dichlorobenzoic acids and their iodoalkylates is studied. Used as enzyme sources were partially purified preparations of acetylcholinesterase (AChE) from human erythrocytes and butyrylcholinesterase (BChE) from horse blood serum, as well as water homogenates of the frog Rana temporaria brain and of the Pacific squid Todarodes pacificus optical ganglia. The studied benzoates failed to be hydrolyzed by the studied ChEs at the enzyme concentrations exceeding 10 times those used for determination of the acetylthiocholine hydrolysis rate. These compounds have turned out to be reversible inhibitors of ChEs of the mixed-noncompetitive type of action. Effects on the anticholinesterase activity of such structural elements of the inhibitors as the acidic part of the benzoate molecule, length of polymethylene chain in the molecule alcoholic part, and the structure of ammonium group are studied. This study has allowed revealing some peculiarities of the reaction capability of vertebrate and invertebrate ChEs.     

Synthesis of physostigmine analogues and evaluation of their anticholinesterase activities

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

Cholinesterase inhibitory activity versus aromatic core multiplicity: A facile green synthesis and molecular docking study of novel piperidone embedded thiazolopyrimidines

Novel thiazolopyrimidine derivatives have been synthesized via microwave assisted, domino cascade methodology in ionic liquid and evaluated in vitro for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities. Among the newly synthesized compounds 6d, 6a, 6e and 6b displayed higher AChE inhibitory activity than standard drug, galanthamine, with IC₅₀ values of 0.53, 1.47, 1.62 and 2.05 μM, respectively. Interestingly, all the compounds except for 6m–r and 6x displayed higher BChE inhibitory potentials than galanthamine with IC₅₀ values ranging from 1.09 to 18.56 μM. Molecular docking simulations for 6d possessing the most potent AChE and BChE inhibitory activities, disclosed its binding interactions at the active site gorge of AChE and BChE enzymes.     

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

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

LC-HRMS Profiling and Phenolic Content,Cholinesterase, and Antioxidant Activities of Terminalia citrina

Terminalia citrina (T. citrina) belongs to the Combretaceae family and is included in the class of medicinal plants in tropical countries such as Bangladesh, Myanmar, and India. The antioxidant activities of lyophilized water (WTE) and alcohol extracts (ETE) of T. citrina fruits, their phenolic content by LC-HRMS, and their effects on cholinesterases (ChEs; AChE, acetylcholinesterase, and BChE, butyrylcholinesterase) were investigated. Especially ten different analytical methods were applied to determine the antioxidant capacity. Compared with similar studies for natural products in the literature, it was determined that both WTE and ETE exhibited strong antioxidant capacity. Syringe and ellagic acids were higher than other acids in ETE and WTE. IC₅₀ values for ETE and WTE in DPPH radical and ABTS⋅⁺ scavenging activities were calculated as 1.69–1.68 μg mL⁻¹ and 6.79–5.78 μg mL⁻¹, respectively. The results of the biological investigations showed that ETE and WTE had an inhibition effect against ChEs, with IC₅₀ values of 94.87 and 130.90 mg mL⁻¹ for AChE and 262.55 and 279.70 mg mL⁻¹ for BChE, respectively. These findings indicate that with the prominence of herbal treatments, T. citrina plant may guide the literature in treating Alzheimer's Disease, preventing oxidative damage, and mitochondrial dysfunction.     

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

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

Highly selective carbamate-based butyrylcholinesterase inhibitors derived from a naturally occurring pyranoisoflavone

This current study described the design and synthesis of a series of derivatives based on a natural pyranoisaflavone, which was obtained from the seeds of Millettia pachycarpa and displayed attractive BChE inhibition and high selectivity in our previous study. The inhibitory potential of all derivatives against two cholinesterases was evaluated. Only a few compounds demonstrated AChE inhibitory activity at the tested concentrations, while 26 compounds showed significant inhibition on BChE (the IC₅₀ values varied from 9.34 μM to 0.093 μM), most of them presented promising selectivity to ward BChE. Prediction of ADME properties for 7 most active compounds was performed. Among them, 9g (IC₅₀ = 222 nM) and 9h (IC₅₀ = 93 nM) were found to be the most potent BChE inhibitors with excellent selectivity over AChE (SI ratio = 1339 and 836, respectively). The kinetic analysis demonstrated both of them acted as mixed-type BChE inhibitors, while the molecular docking results indicated that they interacted with both residues in the catalytic active site. A cytotoxicity test on PC12 cells showed that both 9g and 9h had a therapeutic safety range similar to tacrine. Overall, the results indicate that 9h could be a good candidate of BChE inhibitors.     

Aromatic amino-acid residues at the active and peripheral anionic sites control the binding of E2020 (Aricept) to cholinesterases.

E2020 (R,S)-1-benzyl-4-[(5,6-dimethoxy-1-indanon)-2-yl]methyl)piperidine hydrochloride is a piperidine-based acetylcholinesterase (AChE) inhibitor that was approved for the treatment of Alzheimer's disease in the United States. Structure-activity studies of this class of inhibitors have indicated that both the benzoyl containing functionality and the N-benzylpiperidine moiety are the key features for binding and inhibition of AChE. In the present study, the interaction of E2020 with cholinesterases (ChEs) with known sequence differences, was examined in more detail by measuring the inhibition constants with Torpedo AChE, fetal bovine serum AChE, human butyrylcholinesterase (BChE), and equine BChE. The basis for particular residues conferring selectivity was then confirmed by using site-specific mutants of the implicated residue in two template enzymes. Differences in the reactivity of E2020 toward AChE and BChE (200- to 400-fold) show that residues at the peripheral anionic site such as Asp74(72), Tyr72(70), Tyr124(121), and Trp286(279) in mammalian AChE may be important in the binding of E2020 to AChE. Site-directed mutagenesis studies using mouse AChE showed that these residues contribute to the stabilization energy for the AChE-E2020 complex. However, replacement of Ala277(Trp279) with Trp in human BChE does not affect the binding of E2020 to BChE. Molecular modeling studies suggest that E2020 interacts with the active-site and the peripheral anionic site in AChE, but in the case of BChE, as the gorge is larger, E2020 cannot simultaneously interact at both sites. The observation that the KI value for mutant AChE in which Ala replaced Trp286 is similar to that for wild-type BChE, further confirms our hypothesis.     

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

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