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A series of novel conjugates of 4-amino-2,3-polymethylene quinolines and phenolic antioxidant vanillin was synthesized by the condensation of aminoquinolines with vanillin followed by reduction of imines with sodium borohydride. The conjugates effectively inhibit AChE and BChE with preferable BChE inhibition and displace propidium from the PAS AChE. Compounds with aminoalkyl spacer have preferable esterase profile being more potent cholinesterases inhibitors with lower anti-CES activity and are the most potent antioxidants in ABTS and FRAP tests.

Refluxing of polyfluoroalkyl-containing 2-(arylhydrazinylidene)-1,3-diketones in dichloroalkanes in the presence of TiCl4 gives 3-acyl-4-polyfluoroalkylcinnolines as a result of Friedel-Crafts intramolecular cyclization with the participation of polyfluoroacyl and arylhydrazone moieties. 2-(Arylhydrazinylidene)-3-phenyl-1-polyfluoroalkylpropan-1,3-diones in the presence of CF3SO3H in dichloromethane at −20…−30 °C undergo the competitive cyclization with the participation of polyfluoroacyl and benzoyl groups to form 3-hydroxy-2-(arylhydrazinylidene)-3-polyfluoroalkylindan-1-ones. The heating of 2-(arylhydrazinylidene)-1,3-diketones in polyphosphoric acid at 115…120 °C resulted in 2-polyfluoroalkyl-1-(2-arylhydrazinylidene)ethan-2-ones. The tuberculostatic activity of the obtained cinnolines and indanone was found to be at the level of pyrazinamide or higher. It was established that these compounds do not inhibit cholinesterases and have rather weak inhibitory activity against carboxylesterase. A high antiradical activity was shown for indanone.

The butyrylcholinesterase activity in human blood is an important marker of healthiness. Either level of this enzyme – increased or otherwise – can indicate disease or intoxication. The most popular technique to assess the cholinesterase activity is the detection of thiocholine, a product of enzymatic hydrolysis of butyrylthiocholine. In this work, Ag-paste was used as SERS-substrate and a new technique was created for thiocholine detection. As explained below, the designed technique facilitates also determination of butyrylcholinesterase (BChE) activity both in the buffer and in spike solution with human plasma. Ellman’s assay was used as a reference method, a good correlation between spectrophotometric detection and Raman detection was shown.

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 β-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.

We investigated the inhibitory activity of 4 groups of novel acridine derivatives against acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and carboxylesterase (CaE) using the methods of enzyme kinetics and molecular docking. Antioxidant activity of the compounds was determined using the 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+) radical decolorization assay as their ability to scavenge free radicals. Analysis of the esterase profiles and antiradical activities of the acridine derivatives showed that 9-aryl(heteroaryl)-N-methyl-9,10-dihydroacridines have a high radical-scavenging activity but low potency as AChE and BChE inhibitors, whereas 9-aryl(heteroaryl)-N-methyl-acridinium tetrafluoroborates effectively inhibit cholinesterases but do not exhibit antiradical activity. In contrast, a group of derivatives of 9-heterocyclic amino-N-methyl-9,10-dihydroacridine has been found that combine effective inhibition of AChE and BChE with rather high radical-scavenging activity. The results of molecular docking well explain the observed features in the efficacy, selectivity, and mechanism of cholinesterase inhibition by the acridine derivatives. Thus, in a series of acridine derivatives we have found compounds possessing dual properties of effective and selective cholinesterase inhibition together with free radical scavenging, which makes promising the use of the acridine scaffold to create multifunctional drugs for the therapy of neurodegenerative diseases.

To search for effective and selective inhibitors of carboxylesterase (CaE), a series of 7-hydroxy-7-polyfluoroalkyl-4,7-dihydroazolo[5,1-c][1,2,4]triazines has been synthesized. Their inhibitory activity against acetylcholinesterase, butyrylcholinesterase, and CaE were investigated using the methods of enzyme kinetics and molecular docking. It was shown that the tested compounds are reversible selective CaE inhibitors of mixed type. Elongation of the polyfluoroalkyl substituent and the presence of an ester, preferably the ethoxycarbonyl group, enhance inhibitory activity toward CaE. Furthermore, the compounds with a tetrazole ring are more active against CaE than their triazole analogues. The obtained kinetic data are well explained by the results of molecular docking, according to which there is a similar orientation of triazolo- and tetrazolotriazines in the active site of CaE and the opposite one for pyrazolotriazines. In the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assay, all of the studied tetrazolotriazines and some pyrazolotriazines demonstrated good antiradical activity comparable with a standard antioxidant, Trolox. The leading compounds were nonafluorobutyl substituted tetrazolo- and 7-phenylpyrazolotriazines, which possess effective and selective CaE inhibitory activity as well as additional useful radical-scavenging properties.

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.

A series of 31 N,N-disubstituted 2-amino-5-halomethyl-2-thiazolines was designed, synthesized, and evaluated for inhibitory potential against acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and carboxylesterase (CaE). The compounds did not inhibit AChE; the most active compounds inhibited BChE and CaE with IC50 values of 0.22-2.3μM. Pyridine-containing compounds were more selective toward BChE; compounds with the para-OMe substituent in one of the two dibenzyl fragments were more selective toward CaE. Iodinated derivatives were more effective BChE inhibitors than brominated ones, while there was no influence of halogen type on CaE inhibition. Inhibition kinetics for the 9 most active compounds indicated non-competitive inhibition of CaE and varied mechanisms (competitive, non-competitive, or mixed-type) for inhibition of BChE. Docking simulations predicted key binding interactions of compounds with BChE and CaE and revealed that the best docked positions in BChE were at the bottom of the gorge in close proximity to the catalytic residues in the active site. In contrast, the best binding positions for CaE were clustered rather far from the active site at the top of the gorge. Thus, the docking results provided insight into differences in kinetic mechanisms and inhibitor activities of the tested compounds. A cytotoxicity test using the MTT assay showed that within solubility limits (

We synthesized conjugates of tacrine with 1,2,4-thiadiazole derivatives linked by two different spacers, pentylaminopropene (compounds 4) and pentylaminopropane (compounds 5), as potential drugs for the treatment of Alzheimer's disease (AD). The conjugates effectively inhibited cholinesterases with a predominant effect on butyrylcholinesterase (BChE). They were also effective at displacing propidium from the peripheral anionic site (PAS) of acetylcholinesterase (AChE), suggesting that they could block AChE-induced β-amyloid aggregation. In addition, the compounds exhibited high radical-scavenging capacity. Conjugates 5 had higher anti-BChE activity and greater anti-aggregant potential as well relatively lower potency against carboxylesterase than compounds 4. Quantum-mechanical (QM) characterization agreed with NMR data to identify the most stable forms of conjugates for docking studies, which showed that the compounds bind to both CAS and PAS of AChE consistent with mixed reversible inhibition. Conjugates 4 were more potent radical scavengers, in agreement with HOMO localization in the enamine-thiadiazole system. Computational studies showed that all of the conjugates were expected to have good intestinal absorption, whereas conjugates 4 and 5 were predicted to have medium and high blood-brain barrier permeability, respectively. All conjugates were predicted to have medium cardiac toxicity risks. Overall, the results indicated that the conjugates are promising candidates for further development and optimization as multifunctional therapeutic agents for the treatment of AD.

This paper reviews our previously published data and presents new results on biosensor assay of blood esterases. Tyrosinase and choline oxidase biosensors based on nanostructured polyelectrolyte films were developed for these purposes. Experiments were performed on the quantitative determination of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), carboxylesterase (CaE), and neuropathy target esterase (NTE) in samples of whole blood of rats, mice, and humans. Good agreement was found between biosensor and spectrophotometric assays for AChE, BChE, and CaE. No direct comparison could be made for NTE because its activity cannot be measured spectrophotometrically in whole blood. A new method of simultaneous quantitative determination of AChE and BChE in test mixtures is also described. This method represents a bifunctional biosensor for the simultaneous analysis of choline and phenol based on integration of individual sensors. Algorithms for calculation of separate concentrations of AChE and BChE in the mixture were developed. The mean error of calculated component concentrations was approximately 6% for binary test mixtures. The present work provides a foundation for building multiplexed systems for the simultaneous determination of multiple esterases with applications to biomonitoring for exposures to organophosphorus compounds.