Author Report for: Makhaeva GF

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: IC(50) values of the lead compound 10c were 0.0826 microM (AChE) and 0.0156 microM (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 beta-amyloid aggregation. All conjugates inhibited Abeta(42) self-aggregation in the thioflavin test, and inhibition increased with spacer elongation. Salicylimine 10c and salicylamide 5c with (CH(2))(8) spacers were the lead compounds for inhibiting Abeta(42) self-aggregation, which was corroborated by molecular docking to Abeta(42). 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 Cu(2+), Fe(2+), and Zn(2+), with molar compound/metal (Cu(2+)) 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.

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 beta-amyloid (Abeta(42)) 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 (IC(50) = 2.90 +/- 0.23 microM and 3.22 +/- 0.25 microM, respectively). Only one acridine, 2d, an analog of dihydroacridine, 1d, was an effective BChE inhibitor (IC(50) = 6.90 +/- 0.55 microM), consistent with docking results. Dihydroacridines inhibited Abeta(42) 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 Abeta(42) 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.

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.

4-Arylhydrazinylidene-5-(polyfluoroalkyl)pyrazol-3-ones (4-AHPs) were found to be obtained by the regiospecific cyclization of 2-arylhydrazinylidene-3-(polyfluoroalkyl)-3-oxoesters with hydrazines, by the azo coupling of 4-nonsubstituted pyrazol-5-oles with aryldiazonium chlorides or by the firstly discovered acid-promoted self-condensation of 2-arylhydrazinylidene-3-oxoesters. All the 4-AHPs had an acceptable ADME profile. Varying the substituents in 4-AHPs promoted the switching or combining of their biological activity. The polyfluoroalkyl residue in 4-AHPs led to the appearance of an anticarboxylesterase action in the micromolar range. An NH-fragment and/or methyl group instead of the polyfluoroalkyl one in the 4-AHPs promoted antioxidant properties in the ABTS, FRAP and ORAC tests, as well as anti-cancer activity against HeLa that was at the Doxorubicin level coupled with lower cytotoxicity against normal human fibroblasts. Some Ph-N-substituted 4-AHPs could inhibit the growth of N. gonorrhoeae bacteria at MIC 0.9 microg/mL. The possibility of using 4-AHPs for cell visualization was shown. Most of the 4-AHPs exhibited a pronounced analgesic effect in a hot plate test in vivo at and above the diclofenac and metamizole levels except for the ones with two chlorine atoms in the aryl group. The methylsulfonyl residue was proved to raise the anti-inflammatory effect also. A mechanism of the antinociceptive action of the 4-AHPs through blocking the TRPV1 receptor was proposed and confirmed using in vitro experiment and molecular docking.

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, IC(50) 0.24-0.34 M) and butyrylcholinesterase (BChE, IC(50) 0.036-0.0745 M), 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 beta-amyloid aggregation, a disease-modifying effect. All of the conjugates demonstrated metal chelating ability for Cu(2+), Fe(2+), and Zn(2+), as well as high antiradical activity in the ABTS test. Non-fluorinated hybrid compounds 6 and 7 also showed Fe(3+) 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.

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, IC(50) to 0.224microM) and butyrylcholinesterase (BChE, IC(50) to 0.0104microM) 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 beta-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 Cu(2+) , Fe(2+) and Zn(2+) . Thus, the new conjugates have displayed the potential to be multifunctional anti-AD agents for further development.

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 IC(50)(AChE) = 2.9-1.4 microM, IC(50)(BChE) = 0.13-0.067 microM, and 14-18% propidium displacement at 20 microM. 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 Abeta(42) aggregation. Conjugates 3 had no effect on Abeta(42) 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.

A new series of conjugates of aminoadamantane and gamma-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 beta-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.

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.

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 IC(50) for AChE = 1.83 +/- 0.03 microM (K(i) = 1.50 +/- 0.12 and alphaK(i) = 2.58 +/- 0.23 microM). 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 beta-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 pK(a) 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.

Cholines acylated with unsaturated fatty acids are a recently discovered family of endogenous lipids. However, the data on the biological activity of acylcholines remain very limited. We hypothesized that acylcholines containing residues of arachidonic (AA-CHOL), oleic (Ol-CHOL), linoleic (Ln-CHOL), and docosahexaenoic (DHA-CHOL) acids act as modulators of the acetylcholine signaling system. In the radioligand binding assay, acylcholines showed inhibition in the micromolar range of both alpha7 neuronal nAChR overexpressed in GH4C1 cells and muscle type nAChR from Torpedo californica, as well as Lymnaea stagnalis acetylcholine binding protein. Functional response was checked in two cell lines endogenously expressing alpha7 nAChR. In SH-SY5Y cells, these compounds did not induce Ca(2+) rise, but inhibited the acetylcholine-evoked Ca(2+) rise with IC50 9 to 12 muM. In the A549 lung cancer cells, where alpha7 nAChR activation stimulates proliferation, Ol-CHOL, Ln-CHOL, and AA-CHOL dose-dependently decreased cell viability by up to 45%. AA-CHOL inhibited human erythrocyte acetylcholinesterase (AChE) and horse serum butyrylcholinesterase (BChE) by a mixed type mechanism with Ki = 16.7 +/- 1.5 muM and alphaKi = 51.4 +/- 4.1 muM for AChE and Ki = 70.5 +/- 6.3 muM and alphaKi = 214 +/- 17 muM for BChE, being a weak substrate of the last enzyme only, agrees with molecular docking results. Thus, long-chain unsaturated acylcholines could be viewed as endogenous modulators of the acetylcholine signaling system.

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 IC(50) (AChE) = 0.131 +/- 0.01 muM (five times more potent than tacrine), IC(50)(BChE) = 0.0680 +/- 0.0014 muM, and 17.5 +/- 1.5% propidium displacement at 20 muM. 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 beta-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.

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 IC(50)(AChE) = 1.90 +/- 0.16 microM, IC(50)(BChE) = 0.084 +/- 0.008 microM, and 13.6 +/- 1.2% propidium displacement at 20 M. 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 beta-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.

Systemic inhibition of neuropathy target esterase (NTE) with certain organophosphorus (OP) compounds produces OP compound-induced delayed neurotoxicity (OPIDN), a distal degeneration of axons in the central nervous system (CNS) and peripheral nervous system (PNS), thereby providing a powerful model for studying a spectrum of neurodegenerative diseases. Axonopathies are important medical entities in their own right, but in addition, illnesses once considered primary neuronopathies are now thought to begin with axonal degeneration. These disorders include Alzheimer's disease, Parkinson's disease, and motor neuron diseases such as amyotrophic lateral sclerosis (ALS). Moreover, conditional knockout of NTE in the mouse CNS produces vacuolation and other degenerative changes in large neurons in the hippocampus, thalamus, and cerebellum, along with degeneration and swelling of axons in ascending and descending spinal cord tracts. In humans, NTE mutations cause a variety of neurodegenerative conditions resulting in a range of deficits including spastic paraplegia and blindness. Mutations in the Drosophila NTE orthologue SwissCheese (SWS) produce neurodegeneration characterized by vacuolization that can be partially rescued by expression of wild-type human NTE, suggesting a potential therapeutic approach for certain human neurological disorders. This chapter defines NTE and OPIDN, presents an overview of OP compounds, provides a rationale for NTE research, and traces the history of discovery of NTE and its relationship to OPIDN. It then briefly describes subsequent studies of NTE, including practical applications of the assay; aspects of its domain structure, subcellular localization, and tissue expression; abnormalities associated with NTE mutations, knockdown, and conventional or conditional knockout; and hypothetical models to help guide future research on elucidating the role of NTE in OPIDN.

We studied the inhibitory activity of methylene blue (MB) gamma-carbolines (gC) conjugates (MB-gCs) against human erythrocyte acetylcholinesterase (AChE), equine serum butyrylcholinesterase (BChE), and a structurally related enzyme, porcine liver carboxylesterase (CaE). In addition, we determined the ability of MB-gCs to bind to the peripheral anionic site (PAS) of Electrophorus electricus AChE (EeAChE) and competitively displace propidium iodide from this site. Moreover, we examined the ability of MB-gCs to scavenge free radicals as well as their influence on mitochondrial potential and iron-induced lipid peroxidation. We found that MB-gCs effectively inhibited AChE and BChE with IC50 values in the range 1.73-10.5 muM and exhibited low potencies against CaE (9.8-26% inhibition at 20 muM). Kinetic studies showed that MB-gCs were mixed-type reversible inhibitors of both cholinesterases. Molecular docking results showed that the MB-gCs could bind both to the catalytic active site and to the PAS of human AChE and BChE. Accordingly, MB-gCs effectively displaced propidium from the peripheral anionic site of EeAChE. In addition, MB-gCs were extremely active in both radical scavenging tests. Quantum mechanical DFT calculations suggested that free radical scavenging was likely mediated by the sulfur atom in the MB fragment. Furthermore, the MB-gCs, in like manner to MB, can restore mitochondrial membrane potential after depolarization with rotenone. Moreover, MB-gCs possess strong antioxidant properties, preventing iron-induced lipid peroxidation in mitochondria. Overall, the results indicate that MB-gCs are promising candidates for further optimization as multitarget therapeutic agents for neurodegenerative diseases.

A series of 2-arylhydrazinylidene-3-oxo-4,4,4-trifluorobutanoic acids was synthesized via dealkylation of ethyl 2-arylhydrazinylidene-3-oxo-4,4,4-trifluorobutanoates under the action of a Lewis acid. Under the same conditions, ethyl 2-arylhydrazinylidene-3-oxobutanoates were also found to undergo dealkylation rather than the previously described cyclization into cinnolones. Study of the esterase profile of these compounds showed that trifluoromethyl-containing acids, in contrast to non-fluorinated analogs, were effective and selective inhibitors of carboxylesterase (CES), without substantially inhibiting structurally related cholinesterases (acetylcholinesterase and butyrylcholinesterase). Moreover, both 3-oxo-4,4,4-trifluorobutanoic and 3-oxobutanoic acids having methyl or methoxy substituent in the arylhydrazinylidene fragment showed high antioxidant activity in the ABTS test. Thus, 2-arylhydrazinylidene-3-oxo-4,4,4-trifluorobutanoic acids were found to constitute a new class of effective and selective CES inhibitors that also possess high radical-scavenging activity.

The synthesized new binary conjugates of tetrahydro-gamma-carbolines, which contained ditriazole spacers of different length, exhibited considerable anticholinesterase and antioxidant activity as well as the potential ability to block the acetylcholinesterase-induced aggregation of beta-amyloid in contrast to the original prototype Dimebon. This makes the compounds promising candidates for further investigation as drugs for the treatment of Alzheimer's disease. Special attention should be given to the conjugate containing the hexamethylene intertriazole spacer, which can be considered as a leader in this series of compounds.

To search for effective and selective inhibitors of carboxylesterase (CES), a series of 3-oxo-2-tolylhydrazinylidene-4,4,4-trifluorobutanoates bearing higher or natural alcohol moieties was synthesized via pre-transesterification of ethyl trifluoroacetylacetate with alcohols to isolate transesterificated oxoesters as lithium salts, which were then subjected to azo coupling with tolyldiazonium chloride. Inhibitory activity against porcine liver CES, along with two structurally related serine hydrolases, acetylcholinesterase and butyrylcholinesterase, were investigated using enzyme kinetics and molecular docking. Kinetics studies demonstrated that the tested keto-esters are reversible and selective mixed-type CES inhibitors. Analysis of X-ray crystallographic data together with our IR and NMR spectra and QM calculations indicated that the Z-isomers were the most stable. The kinetic data were well explained by the molecular docking results of the Z-isomers, which showed specific binding of the compounds in the CES catalytic active site with carbonyl oxygen atoms in the oxyanion hole and non-specific binding outside it. Some compounds were studied as inhibitors of the main human isozymes involved in biotransformation of ester-containing drugs, hCES1 and hCES2. Esters of geraniol (3d) and adamantol (3e) proved to be highly active and selective inhibitors of hCES2, inhibiting the enzyme in the nanomolar range, whereas esters of borneol (3f) and isoborneol (3g) were more active and selective against hCES1. Computational ADMET studies revealed that all test compounds had excellent intestinal absorption, medium blood-brain barrier permeability, and low hERG liability risks. Moreover, all test compounds possessed radical-scavenging properties and low acute toxicity. Overall, the results indicate that members of this novel series of esters have the potential to be good candidates as hCES1 or hCES2 inhibitors for biomedicinal applications.

Alzheimer's disease (AD) is a multifactorial neurodegenerative process whose effective treatment will require drugs that can act simultaneously on multiple pathogenic targets. Here, we present an overview of our previous multitarget studies of five groups of novel hybrid structures that combine, through spacers, five pharmacophores that have been found promising for AD treatment: gamma-carbolines, carbazoles, tetrahydrocarbazoles, phenothiazines, and aminoadamantanes. Biological activity of the compounds was assessed by a battery of assays. These included inhibitory potency against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) as indicators of potential for cognition enhancement and against carboxylesterase (CaE) to exclude unwanted inhibition of this biotransformation pathway. Displacement of propidium from the peripheral anionic site of AChE was determined as a predictor of anti-aggregation activity. Binding to the two sites of the NMDA subtype of the glutamate receptor was conducted as an additional indicator of potential cognition enhancement and neuroprotection. Propensity to protect against mitochondrial triggers of cell death was evaluated by tests of mitochondrial potential and calcium-induced swelling as indicators of mitochondrial permeability transition. Antioxidant potential was measured to evaluate the tendency to prevent oxidative stress. Potential for disease modification was gauged by the ability to stimulate microtubule assembly. Finally, binding modes of conjugates to AChE and BChE were studied using quantum mechanical-assisted molecular docking. We found selective BChE inhibitors (conjugates of gamma-carbolines and phenothiazine I, gamma-carbolines and carbazoles II, and aminoadamantanes and carbazoles III) as well as inhibitors of both cholinesterases (conjugates of gamma-carbolines and methylene blue IV and bis-gamma-carbolines with ditriazole-containing spacers V). These compounds combined potentials for cognition enhancement, neuroprotection, and disease modification. None of the conjugates exhibited high potency against CaE, thereby precluding potential drug-drug interactions from CaE inhibition. Thus, the studied compounds exhibited positive characteristics of multitarget drugs, indicating their potential for the next generation of AD therapeutics.

Literature data and authors' own results on the role of serine hydrolases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), as drug targets for treatment of neurodegenerative diseases and carboxylesterase (CaE) inhibitors as modulators of CaE-hydrolysis of ester-containing drugs are analyzed. Today, a promising approach is the development of cholinesterase inhibitors with additional neuroprotective and disease-modifying properties. The developed esterase profile approach, that is, comparative assessment of the inhibitory activity against AChE, BChE, and CaE, can be used to evaluate both the main potential pharmacological effect and possible side effects of a new compound. Analysis of the esterase profile, in combination with computer modeling and assessment of radical-scavenging ability of the synthesized compounds and their potential ability to block AChE-induced beta-amyloid aggregation revealed highly active multifunctional compounds for the treatment of Alzheimer's disease: selective inhibitors of BChE and inhibitors of both cholinesterases without potential side effects associated with CaE inhibition. A number of effective and selective inhibitors of CaE, free from cholinergic side effects, were also found for modulation of the rate of hydrolytic metabolism and for rational use of ester-containing drugs.

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 beta-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.

Using the acylation reaction with tosyl chloride of N-aminopropyl analogues of tacrine and its cyclic homologues with different size of the aliphatic cycle (5-8), we synthesized a number of new derivatives of p-toluenesulfonamide. It is shown that the synthesized hybrid compounds of tacrine and p-toluenesulfonamide are effective inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with the preferential inhibition of BChE. They also displace propidium from the peripheral anionic site of the electric eel AChE (Electrophorus electricus). The characteristics of the efficiency and selectivity of cholinesterase inhibition by the test compounds were confirmed by the results of molecular docking.

We investigated the biological activity of a series of substituted chromeno[3,2-c]pyridines, including compounds previously synthesized by our group and novel compounds whose syntheses are reported here. Tandem transformation of their tetrahydropyridine ring under the action of activated alkynes yielding 2-vinylsubstituted chromones was used to prepare nitrogen-containing derivatives of a biologically active chromone system. The inhibitory activity of these chromone derivatives against acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and carboxylesterase (CaE) was investigated using the methods of enzyme kinetics and molecular docking. Antioxidant (antiradical) activity of the compounds was assessed in the ABTS assay. The results demonstrated that a subset of the studied chromone derivatives selectively inhibit BChE but do not exhibit antiradical activity. In addition, the results of molecular docking effectively explained the observed features in the efficacy, selectivity, and mechanism of BChE inhibition by the chromone derivatives.

A new group of compounds, promising for the design of original multitarget therapeutic agents for treating neurodegenerative diseases, based on conjugates of aminoadamantane and carbazole derivatives was synthesized and investigated. Compounds of these series were found to interact with a group of targets that play an important role in the development of this type of diseases. First of all, these compounds selectively inhibit butyrylcholinesterase, block NMDA receptors containing NR2B subunits while maintaining the properties of MK-801 binding site blockers, exert microtubules stabilizing properties, and possess the ability to protect nerve cells from death at the calcium overload conditions. The leading compound C-2h has been shown the most promising effects on all analyzed parameters. Thus, these compounds can be regarded as promising candidates for the design of multi-target disease-modifying drugs for treatment of AD and/or similar neuropathologies.

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.

Conjugates of tacrine with 1,2,4-thiadiazole derivatives were synthesized for the first time. Their esterase profile and effects on the key NMDA receptor-binding sites as well as antioxidant activity were investigated. The obtained compounds effectively inhibited cholinesterases (with a predominant effect on butyrylcholinesterase), simultaneously blocked two NMDA receptor-binding sites (allosteric and intrachannel sites, and exhibited a high radical-scavenging activity. Our study shows that the obtained compounds are promising to design drugs for the treatment of Alzheimer's disease and other multifactorial 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.

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 0.22-2.3muM. 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 (<30muM), none of the tested compounds significantly affected viability of human fetal mesenchymal stem cells. The results indicate that a new series of N,N-disubstituted 2-aminothiazolines could serve as BChE and CaE inhibitors for potential medicinal applications.

We studied 4 serine esterases (EOHs) that are associated with the following consequences from their inhibition by organophosphorus compounds (OPCs): acetylcholinesterase (AChE: acute neurotoxicity; cognition enhancement), butyrylcholinesterase (BChE: inhibition of drug metabolism and/or stoichiometric scavenging of EOH inhibitors; cognition enhancement), carboxylesterase (CaE; inhibition of drug metabolism and/or stoichiometric scavenging of EOH inhibitors), and neuropathy target esterase (NTE: delayed neurotoxicity, OPIDN). The relative degree of inhibition of these EOHs constitutes the "esterase profile" of an OPC, which we hypothesize can serve as a predictor of its overall physiological effects. To test this hypothesis, we selected 3 OPCs known from previous work on reference enzymes to span a wide range of esterase profiles, neuropathic potential, and acute cholinergic toxicity. For each compound, we determined in vitro IC50 and in vivo ED50 values for inhibition of AChE, BChE, CaE, and NTE in mouse brain and blood. The results showed good correlations between in vitro and in vivo measures of potency and selectivity except for brain CaE, a tissue-specific isoform of the enzyme that was less sensitive to the test compounds than expected. Thus, this synthesis of new and previously published results indicates that the concept of the esterase profile of OPCs is useful for the prediction of therapeutic and toxic effects in vivo.

The adult hen is the standard animal model for testing organophosphorus (OP) compounds for organophosphorus compound-induced delayed neurotoxicity (OPIDN). Recently, we developed a mouse model for biochemical assessment of the neuropathic potential of OP compounds based on brain neuropathy target esterase (NTE) and acetylcholinesterase (AChE) inhibition. We carried out the present work to further develop the mouse model by testing the hypothesis that whole blood NTE inhibition could be used as a biochemical marker for exposure to neuropathic OP compounds. Because brain NTE and AChE inhibition are biomarkers of OPIDN and acute cholinergic toxicity, respectively, we compared NTE and AChE 20-min IC50 values as well as ED50 values 1 h after single intraperitoneal (i.p.) injections of increasing doses of two neuropathic OP compounds that differed in acute toxicity potency. We found good agreement between the brain and blood for in vitro sensitivity of each enzyme as well for the ratios IC50 (AChE)/IC50 (NTE). Both OP compounds inhibited AChE and NTE in the mouse brain and blood dose-dependently, and brain and blood inhibitions in vivo were well correlated for each enzyme. For both OP compounds, the ratio ED50 (AChE)/ED50 (NTE) in blood corresponded to that in the brain despite the somewhat higher sensitivity of blood enzymes. Thus, our results indicate that mouse blood NTE could serve as a biomarker of exposure to neuropathic OP compounds. Moreover, the data suggest that relative inhibition of blood NTE and AChE provide a way to assess the likelihood that OP compound exposure in a susceptible species would produce cholinergic and/or delayed neuropathic effects. Copyright (c) 2016 John Wiley & Sons, Ltd.

A series of alkyl 2-Arylhydrazinylidene-3-oxo-3-polyfluoroalkylpropionates was synthesized and their inhibitory activity with respect to porcine liver carboxylesterase (CaE, EC 3.1.1.1), human erythrocyte acetylcholinesterase (AChE, EC 3.1.1.7), and horse serum butyrylcholinesterase (BChE, EC 3.1.1.8) was studied. The molecular docking method was used to study the binding mode of the compounds in the active site of CaE. It was found that compounds containing the trifluoromethyl group in the third position of carbonyl chain are highly effective and selective inhibitors of CaE with nanomolar IC50 values, which agrees well with the results of molecular docking.

Alzheimer disease is a multifactorial pathology and the development of new multitarget neuroprotective drugs is promising and attractive. We synthesized a group of original compounds, which combine in one molecule gamma-carboline fragment of dimebon and phenothiazine core of methylene blue (MB) linked by 1-oxo- and 2-hydroxypropylene spacers. Inhibitory activity of the conjugates toward acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and structurally close to them carboxylesterase (CaE), as well their binding to NMDA-receptors were evaluated in vitro and in silico. These newly synthesized compounds showed significantly higher inhibitory activity toward BChE with IC50 values in submicromolar and micromolar range and exhibited selective inhibitory action against BChE over AChE and CaE. Kinetic studies for the 9 most active compounds indicated that majority of them were mixed-type BChE inhibitors. The main specific protein-ligand interaction is pi-pi stacking of phenothiazine ring with indole group of Trp82. These compounds emerge as promising safe multitarget ligands for the further development of a therapeutic approach against aging-related neurodegenerative disorders such as Alzheimer and/or other pathological conditions.

Inhibition and aging of neuropathy target esterase (NTE) by neuropathic organophosphorus (OP) compounds triggers OP compound-induced delayed neuropathy (OPIDN), whereas inhibition of acetylcholinesterase (AChE) produces cholinergic toxicity. The neuropathic potential of an OP compound is defined by its relative inhibitory potency toward NTE vs. AChE assessed by enzyme assays following dosing in vivo or after incubations of direct-acting compounds or active metabolites with enzymes in vitro. The standard animal model of OPIDN is the adult hen, but its large size and high husbandry costs make this species a burdensome model for assessing neuropathic potential. Although the mouse does not readily exhibit clinical signs of OPIDN, it displays axonal lesions and expresses brain AChE and NTE. Therefore, the present research was performed as a further test of the hypothesis that inhibition of mouse brain AChE and NTE could be used to assess neuropathic potential using mouse brain preparations in vitro or employing mouse brain assays following dosing of OP compounds in vivo. Excellent correlations were obtained for inhibition kinetics in vitro of mouse brain enzymes vs. hen brain and human recombinant enzymes. Furthermore, inhibition of mouse brain AChE and NTE after dosing with OP compounds afforded ED50 ratios that agreed with relative inhibitory potencies assessed in vitro. Taken together, results with mouse brain enzymes demonstrated consistent correspondence between in vitro and in vivo predictors of neuropathic potential, thus adding to previous studies supporting the validity of a mouse model for biochemical assessment of the ability of OP compounds to produce OPIDN. Copyright (c) 2014 John Wiley & Sons, Ltd.

Certain organophosphorus compounds (OPCs) inhibit various serine esterases (EOHs) via phosphorylation of their active site serines. We focused on 4 EOHs of particular toxicological interest: acetylcholinesterase (AChE: acute neurotoxicity; cognition enhancement), butyrylcholinesterase (BChE: inhibition of drug metabolism and/or stoichiometric scavenging of EOH inhibitors; cognition enhancement), carboxylesterase (CaE: inhibition of drug metabolism and/or stoichiometric scavenging of EOH inhibitors), and neuropathy target esterase (NTE: delayed neurotoxicity, OPIDN). The relative degree of inhibition of these EOHs constitutes the "esterase profile" of an OPC and serves as a major determinant of its net physiological effects. Thus, understanding and controlling the esterase profile of OPC activity and selectivity toward these 4 target enzymes is a significant undertaking. In the present study, we analyzed the inhibitor properties of 52 OPCs against the 4 EOHs, along with pairwise and multitarget selectivities between them, using 2 QSAR approaches: Hansch modeling and Molecular Field Topology Analysis (MFTA). The general formula of the OPCs was (RO)2P(O)X, where R=alkyl, X=- SCH(Hal)COOEt (Hal=Cl, Br), -SCHCl2, -SCH2Br, -OCH(CF3)R(1) (R(1)=C6H5, CF3, COOEt, COOMe). The Hansch model showed that increasing neuropathic potential correlated with rising R hydrophobicity; moreover, OPC binding to scavenger EOHs (BChE and CaE) had different effects on potential acute and delayed neurotoxicity. Predicted protective roles of BChE and CaE against acute toxicity were enhanced with increasing hydrophobicity, but projected protection against OPIDN was decreased. Next, Molecular Field Topology Analysis (MFTA) models were built, considering atomic descriptors, e.g., effective charge, van der Waals radius of environment, and group lipophilicity. Activity/selectivity maps confirmed predictions from Hansch models and revealed other structural factors affecting activity and selectivity. Virtual screening based on multitarget selectivity MFTA models was used to design libraries of OPCs with favorable esterase profiles for potential application as selective inhibitors of CaE without untoward side effects.

Neuropathy target esterase (NTE) was discovered by M.K. Johnson in his quest for the entity responsible for the striking and mysterious paralysis brought about by certain organophosphorus (OP) esters. His pioneering work on OP neuropathy led to the view that the biochemical lesion consisted of NTE that had undergone OP inhibition and aging. Indeed, nonaging NTE inhibitors failed to produce disease but protected against neuropathy from subsequently administered aging inhibitors. Thus, inhibition of NTE activity was not the culprit; rather, formation of an abnormal protein was the agent of the disorder. More recently, however, Paul Glynn and colleagues showed that whereas conventional knockout of the NTE gene was embryonic lethal, conditional knockout of central nervous system NTE produced neurodegeneration, suggesting to these authors that the absence of NTE rather than its presence in some altered form caused disease. We now know that NTE is the 6th member of a 9-protein family called patatin-like phospholipase domain-containing proteins, PNPLA1-9. Mutations in the catalytic domain of NTE (PNPLA6) are associated with a slowly developing disease akin to OP neuropathy and hereditary spastic paraplegia called NTE-related motor neuron disorder (NTE-MND). Furthermore, the NTE protein from affected individuals has altered enzymological characteristics. Moreover, closely related PNPLA7 is regulated by insulin and glucose. These seemingly disparate findings are not necessarily mutually exclusive, but we need to reconcile recent genetic findings with the historical body of toxicological data indicating that inhibition and aging of NTE are both necessary in order to produce neuropathy from exposure to certain OP compounds. Solving this mystery will be satisfying in itself, but it is also an enterprise likely to pay dividends by enhancing our understanding of the physiological and pathogenic roles of the PNPLA family of proteins in neurological health and disease, including a potential role for NTE in diabetic neuropathy.

In this study, the binding points of MethB and two structurally-related cationic phenoxazine dyes [meldola blue (MB) and nile blue (NB)] to human butyrylcholinesterase (BChE) were investigated by molecular docking and site directed mutagenesis. The comparative inhibitory effects of MethB, MB and NB on recombinant wild type BChE and six human BChE mutants were spectrophotometrically studied. Kinetic analyses yielded the following information: MethB and MB were found to cause nonlinear inhibition of all recombinant BChEs except Y332A, compatible with a multi-site binding model. On the other hand, MethB and MB caused linear mixed inhibition of Y332A mutant, compatible with a single binding mode. Comparing the inhibitory effects in aspect of Ki values with recombinant wild type BChE (Ki=0.042 muM), MethB was found to be approximately 30, 80 and 270-fold less effective as an inhibitor of Y332A, F329A and T120F, respectively. NB caused nonlinear inhibition of all recombinant BChEs. The inhibitory effect of NB on Y332A mutant was approximately 370-fold lower, compared to recombinant wild type BChE (Ki=0.006 muM). Considering both kinetic and molecular docking results together, it was concluded that threonine 120, phenylalanine 329 and tyrosine 332 are critical amino acids in binding of cationic phenoxazine/phenothiazine structured ligands to human BChE.

Oxime reactivation of serine esterases (EOHs) inhibited by organophosphorus (OP) compounds can produce O-phosphorylated oximes (POXs). Such oxime derivatives are of interest, because some of them can have greater anti-EOH potencies than the OP inhibitors from which they were derived. Accordingly, inhibitor properties of 58 POXs against four EOHs, along with pair-wise selectivities between them, have been analysed using different QSAR approaches. EOHs (with their abbreviations and consequences of inhibition in parentheses) comprised acetylcholinesterase (AChE: acute neurotoxicity; cognition enhancement), butyrylcholinesterase (BChE: inhibition of drug metabolism or stoichiometric scavenging of EOH inhibitors; cognition enhancement), carboxylesterase (CaE: inhibition of drug metabolism or stoichiometric scavenging of EOH inhibitors), and neuropathy target esterase (NTE: delayed neurotoxicity). QSAR techniques encompassed linear regression and backpropagation neural networks in conjunction with fragmental descriptors containing labelled atoms, Molecular Field Topology Analysis (MFTA), Comparative Molecular Similarity Index Analysis (CoMSIA), and molecular modelling. All methods provided mostly consistent and complementary information, and they revealed structural features controlling the 'esterase profiles', i.e. patterns of anti-EOH activities and selectivities of the compounds of interest. In addition, MFTA models were used to design a library of compounds having a cognition-enhancement esterase profile suitable for potential application to the treatment of Alzheimer's disease.

A series of O-carbamoylated 1,1,1,3,3,3-hexafluoroisopropanols of general formula RNHC(O)OCH(CF3)2, where R = CH3, n-C3H7, tert-C4H9, cyclo-C6H11, C6H5-CH2, C6H5, 4-Cl-C6H4, 3-Cl-C6H4, 3,4-Cl2-C6H3, and naphthylen-2-yl were synthesized. The reaction kinetics of the synthesized carbamates with human erythrocyte acetylcholinesterase (EC 3.1.1.7), horse serum butyrylcholinesterase (EC 3.1.1.8), and porcine liver carboxylesterase (EC 3.1.1.1) were studied. It was shown that the synthesized carbamates did not inhibit acetylcholinesterase, inhibited weakly butyrylcholinesterase, and inhibited selectively the activity of carboxylesterase. A new selective irreversible inhibitor of carboxylesterase, 2,2,2-trifluoro-1-trifluoromethylethyl cyclohexylcarbamate, which had low acute toxicity, was obtained.

Acetylcholinesterase, butyrylcholinesterase, carboxylesterase, and paraoxonase activities in human, mouse, and rat blood were measured. The proportions of these enzymes activities differed significantly. In humans, the most significant were cholinesterase activities, while in rats and mice the contribution of carboxylesterase activity was the greatest. High arylesterase activity of paraoxonase was observed in all cases. Species-specific differences should be taken into consideration when carrying out preclinical trials on rodents for optimization of the pharmacokinetic characteristics of drugs containing complex ester groups.

A modification of novel fluorinated organophosphorous compounds containing terminal alkyne group by different azidopeptides via Cu(I)-catalyzed click chemistry has been described. The inhibitor activity of trifluoromethyl-containing methylphosphonates and their peptide-conjugates towards acetylcholinesterase, butyrylcholinesterase, and carboxylesterase has been investigated. It was shown that the incorporation of peptide fragments significantly modulates the esterase profile of starting methylphosphonates.

This paper reviews previously published data and presents new results to address the hypothesis that fluorinated aminophosphonates (FAPs), (RO)(2)P(O)C(CF(3))(2)NHS(O)(2)C(6)H(5), R=alkyl, inhibit serine esterases by scission of the P-C bond. Kinetics studies demonstrated that FAPs are progressive irreversible inhibitors of acetylcholinesterase (AChE, EC 3.1.1.7.), butyrylcholinesterase (BChE, EC 3.1.1.8.), carboxylesterase (CaE, EC 3.1.1.1.), and neuropathy target esterase (NTE, EC 3.1.1.5.), consistent with P-C bond breakage. Chemical reactivity experiments showed that diMe-FAP and diEt-FAP react with water to yield the corresponding dialkylphosphates and (CF(3))(2)CHNHS(O)(2)C(6)H(5), indicating lability of the P-C bond. X-ray crystallography of diEt-FAP revealed an elongated (and therefore weaker) P-C bond (1.8797 (13)A) compared to P-C bonds in dialkylphosphonates lacking alpha-CF(3) groups (1.805-1.822A). Semi-empirical and non-empirical molecular modeling of diEt-FAP and (EtO)(2)P(O)C(CH(3))(2)NHS(O)(2)C(6)H(5) (diEt-AP), which lacks CF(3) groups, indicated lengthening and destabilization of the P-C bond in diEt-FAP compared to diEt-AP. Active site peptide adducts formed by reacting diEt-FAP with BChE and diBu-FAP with NTE catalytic domain (NEST) were identified using peptide mass mapping with mass spectrometry (MS). Mass shifts (mean+/-SE, average mass) for peaks corresponding to active site peptides with diethylphosphoryl and monoethylphosphoryl adducts on BChE were 136.1+/-0.1 and 108.0+/-0.1Da, respectively. Corresponding mass shifts for dibutylphosphoryl and monobutylphosphoryl adducts on NEST were 191.8+/-0.2 and 135.5+/-0.1Da, respectively. Each of these values was statistically identical to the theoretical mass shift for each dialkylphosphoryl and monoalkylphosphoryl species. The MS results demonstrate that inhibition of BChE and NEST by FAPs yields dialkylphosphoryl and monoalkylphosphoryl adducts, consistent with phosphorylation via P-C bond cleavage and aging by net dealkylation. Taken together, predictions from enzyme kinetics, chemical reactivity, X-ray crystallography, and molecular modeling were confirmed by MS and support the hypothesis that FAPs inhibit serine esterases via scission of the P-C bond.

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.

Acetylcholinesterase and butyrylcholinesterase inhibitors are potential cognition enhancers in Alzheimer disease. O,O-Dialkylphosphate inhibitors with 1-substituted 2,2,2-trifluoroethoxy leaving groups were synthesized by phosphonate-phosphate rearrangement. Substituents in the 1-position of the leaving group along with the O-alkyl groups modulated potency and selectivity against acetylcholinesterase, butyrylcholinesterase, and carboxylesterase.

The title compound, C(13)H(16)F(6)NO(5)PS, is of inter-est with respect to inhibition of serine hydro-lases. Its structure contains a 1.8797 (13) A P-C bond and two inter-molecular N-Hcdots, three dots, centeredO=P hydrogen bonds, resulting in centrosymmetric dimers. An intra-molecular N-Hcdots, three dots, centeredO=P hydrogen bond is also present.

Organophosphates (OPs) that inhibit neuropathy target esterase (NTE) with subsequent ageing can produce OP-induced delayed neuropathy (OPIDN). NTE inhibition in lymphocytes can be used as a biomarker of exposure to neuropathic OPs. An electrochemical method was developed to assay NTE in whole blood. The high sensitivity of the tyrosinase carbon-paste biosensors for the phenol produced by hydrolysis of the substrate, phenyl valerate, allowed NTE activity to be measured in diluted samples of whole blood, which cannot be done using the standard colorimetric assay. The biosensor was used to establish correlations of NTE inhibitions in blood with that in lymphocytes and brain after dosing hens with a neuropathic OP. The results of further studies demonstrated that whole blood NTE is a reliable biomarker of neuropathic OPs for up to 96 hours after exposure. These validation results suggest that the biosensor NTE assay for whole blood could be developed to measure human exposure to neuropathic OPs as a predictor of OPIDN. The small blood volume required (100 microL), simplicity of sample preparation and rapid analysis times indicate that the biosensor should be useful in biomonitoring and epidemiological studies. The present paper is an overview of our previous and ongoing work in this area.

A graphite-paste tyrosinase biosensor was improved by adding 1-methoxyphenazine methosulfate as a mediator. Mediator modification enhanced sensitivity to phenol 4-fold and long-term stability 3-fold. Phenol could be detected at 25 nM (S/N = 2) using an Ag/AgCl reference electrode. The biosensor was used to measure the activity of a toxicologically significant enzyme, neuropathy target esterase (NTE), which yields phenol by hydrolysis of the substrate, phenyl valerate. Using the new biosensor, blood and brain NTE inhibition by organophosphorus (OP) compounds with different neuropathic potencies were well correlated (r = 0.990, n = 7), supporting the use of blood NTE as a biochemical marker of exposure to neuropathic OP compounds.

Neuropathy target esterase (NTE) is the target protein for neuropathic organophosphorus (OP) compounds that produce OP compound-induced delayed neurotoxicity (OPIDN). Inhibition/aging of brain NTE within hours of exposure predicts the potential for development of OPIDN in susceptible animal models. Lymphocyte NTE has also found limited use as a biomarker of human exposure to neuropathic OP compounds. Recently, a highly sensitive biosensor was developed for NTE activity using a tyrosinase carbon-paste electrode for amperometric detection of phenol produced by hydrolysis of the substrate, phenyl valerate. The I50 (20 min at 37 degrees C) for N,N'-di-2-propylphosphorodiamidofluoridate (mipafox) against hen lymphocyte NTE was 6.94 +/- 0.28 microM amperometrically and 6.02 +/- 0.71 microM colorimetrically. For O,O-di1-propyl O-2,2-dichlorvinyl phosphate (PrDChVP), the I50 against hen brain NTE was 39 +/- 8 nM amperometrically and 42 +/- 2 nM colorimetrically. The biosensor enables NTE to be assayed in whole blood, whereas this cannot be done with the usual colorimetric method. Amperometrically, I50 values for PrDChVP against hen and human blood NTE were 66 +/- 3 and 70 +/- 14 nM, respectively. To study the possibility of using blood NTE inhibition as a biochemical marker of neuropathic OP compound exposure, NTE activities in brain and lymphocytes as well in brain and blood were measured 24 h after dosing hens with PrDChVP. Brain, lymphocyte, and blood NTE were inhibited in a dose-responsive manner, and NTE inhibition was highly correlated between brain and lymphocyte (r = .994) and between brain and blood (r = .997). The results suggest that the biosensor NTE assay for whole blood could serve as a biomarker of exposure to neuropathic OP compounds as well as a predictor of OPIDN and an adjunct to its early diagnosis.

Inhibition of acetylcholinesterase (AChE) versus inhibition and aging of neuropathy target esterase (NTE) by organophosphorus (OP) compounds in vivo can give rise to distinct neurological consequences: acute cholinergic toxicity versus OP compound-induced delayed neurotoxicity (OPIDN). Previous work has shown that the relative potency of an OP compound to react with NTE versus AChE in vitro may predict its capability to produce OPIDN. The present study was conducted to evaluate further the validity of such predictions and to enhance them with quantitative structure-activity relationships (QSAR) using a homologous series of alkyl phenylphosphonates (RO)C6H5P(O)ON = CCICH3 (PhP; R = alkyl). Neuropathic potential of PhP was assessed by measuring ki(NTE)ki(AChE) ratios in vitro and comparing these with ED50 ratios in vivo. Selectivity for NTE increased with rising R-group hydrophobicity. The ki(NTE)/ki(AChE) ratios were 0.42 (methyl), 3.6 (ethyl), 15 (isopropyl), 36 (propyl), 69 (isobutyl), 105 (butyl), and 124 (pentyl). Ratios > 1 suggest the potential to produce OPIDN at doses lower than the LD50. Inhibition of NTE and AChE in hen brain in vivo was studied 24 h after i.m. injection of hens with increasing doses of methyl and butyl derivatives. Analysis of dose-response curves yielded ED50(AChE)/ED50(NTE) ratio of 0.86 for methyl PhP and 22.1 for butyl PhP. These results predict that the butyl derivative should be more neuropathic than the methyl analogue. Excellent correspondence between in vivo and in vitro predictions of neuropathic potential indicate that valid predictive QSAR models may be based on the in vitro approach. Adoption of this system would result in reducing experimental animal use, lowering costs, accelerating data production, and enabling standardization of a biochemically based risk assessment of the neuropathic potential of OP compounds.

Bioelectrochemical analysis of neuropathy target esterase (NTE) and its inhibitors is based on the combination of the NTE-catalyzed hydrolysis of phenyl valerate and phenol detection by a tyrosinase carbon-paste electrode. The use of the tyrosinase electrode improves 10-fold the sensitivity of NTE detection in comparison with a spectrophotometric method. The tyrosinase electrode was found to be suitable for measurements in whole human blood where spectrophotometric detection is considerably restricted. The specificity of NTE in blood for mipafox and di-2-propyl phosphorofluoridate was close to that for neuronal NTE. The NTE-like activity in blood was determined to be 0.19 +/- 0.02 nmol/min/mg of protein.

The interaction of potential pesticides, O,O-dialkyl S-ethoxycarbonylbromomethylthiophosphates (RO)2P(O)SCH(Br)COOC2H5 (R = Et, i-Pr, n-Pr, n-Bu, n-Am, or n-Hx) with the esterases of warm-blooded animals [acetylcholinesterase (ACE), butyryl cholinesterase (BCE), and carboxyl esterase (CE)] was studied. The acute toxicities of these compounds for mice were determined. All the compounds were non-hydrolyzable by CE and capable of irreversible inhibition of all these esterases with ki (M-1 min-1) of 1.2 x 10(5)-6 x 10(6), 2.0 x 10(6)-1.5 x 10(8), and 2.0 x 10(8), respectively. By using multiple regression analysis, we found that the steric factor plays a significant role in the inhibition of ACE, with the steric hindrances manifesting themselves even at the sorption stage. On the other hand, hydrophobic interactions predominate in the case of BCE, while steric properties of its substituents exert a markedly weaker effect and manifest themselves at the phosphorylation stage. We suggested the presence of an electrophilic region in the active site of ACE, which can interact with the ethoxycarbonyl group of the thiophosphates under study. The decrease in toxicities and the affinities to BCE and CE were found to correlate with an increase in the length of n-alkyl substituents of the compounds studied. This suggests that the unspecific esterases play a significant role as a buffer system in the exhibition of toxic effects by the thiophosphates under consideration.

The interaction kinetics of potential pesticides, O,O-dialkyl S-bromomethylthiophosphates (RO)2P(O) SCH2Br (R = Et, i-Pr, n-Pr, n-Bu, or n-Am) with acetylcholinesterase, butyryl cholinesterase, and carboxyl esterase from warm-blooded animals was studied. All the compounds irreversibly inhibit these esterases, with k1 (M-1 min-1) being 1.8 x 10(4) - 1.9 x 10(6) for acetylcholinesterase, 2.0 x 10(6) - 4.1 x 10(7) for the more sensitive butyryl cholinesterase, and 2.3 x 10(7) - 2.3 x 10(8) and higher for the most sensitive carboxyl esterase. By using the Hansch and Kubinyi technique of multiple regression analysis, we quantitatively analyzed the relationship between the structure and inhibiting activity of these substances toward acetylcholinesterase and butyryl cholinesterase. Hydrophobic interactions were found to be important for the inhibition of both enzymes but are more pronounced in the case of butyryl cholinesterase. On the other hand, steric factors were much more significant in the inhibition of acetylcholinesterase. For both enzymes, the steric hindrances affect the phosphorylation stage of the enzyme.

Neuropathy target esterase (NTE) is a molecular target for organophosphate-induced delayed neurotoxicity (OPIDN). This enzyme has proved to be an excellent tool for the assessment of neuropathic potential of organophosphates (OP), in particular by comparison of an OP inhibitory activity in vitro against NTE and acetylcholinesterase. A large-scale OP screening for delayed neurotoxicity was largely prevented by the lack of an available stable preparation of NTE. To obtain a stable NTE preparation the influence of intensive freezing and subsequent lyophilization of paraoxon-preinhibited (P2 + P3) hen brain membrane fraction on NTE properties has been studied using two neuropathic OP: mipafox and O,O-dipropyldichlorovinyl phosphate (PrDChVP). It was shown that lyophilization preserved a high NTE specific activity and did not alter the inhibitor characteristics of the enzyme. A long-term storage study showed that lyophilized NTE preparation exhibited inhibitory features actually identical to those of the native enzyme during 1 year and retained rather high specific activity; in this case some loss of NTE specific activity has been observed. Comparative studies of inhibition of the native and lyophilized NTE preparations by a model series of phenyl phosphonates RO(C6H5)P(O)ON=CClCH3 (R = alkyl), demonstrated a good correlation between the values pI50 obtained with both enzyme preparations as well as identical structure-activity relationships for the lyophilized and native enzymes. The results allow the conclusion that the obtained NTE preparation can be used as a standard, stable and readily available source of NTE for assessing the anti-NTE activity of OP.

The interaction of 2-aryloxy-2-thio-1,3,2-oxazaphosphorinanes exhibiting nematocide, insecticide/acaricide, and synergetic activities with monoamine oxidases and the interaction of the corresponding oxones, 2-aryloxy-2-oxo-1,3,2-oxazaphosphorinanes, with various cholinesterases, carboxyl esterases, and monoamine oxidases were studied. We showed that the thioderivatives inhibited monoamine oxidases, whereas oxones, which are, as a rule, weak cholinesterase inhibitors, strongly inhibited carboxyl esterases of the American cockroach and were transformed with monoamine oxidases into the strong cholinesterase inhibitors, acyclic phosphamidates. This allowed us to explain the low toxicity of the thioderivatives, the high toxicity of the oxoderivatives, and the great difference in toxicities of thio- and oxocompounds in the 1,3,2-oxazaphosphorinane series. The capacity of thioderivatives to inhibit monoamine oxidases and of oxoderivatives and their further activation products to inhibit carboxyl esterases, i.e., both enzymes responsible for pyrethroid detoxication in insects, explains the synergetic activity of the 1,3,2-oxazaphosphorinane series.

Neuropathy target esterase (NTE) was shown to be an excellent biochemical marker for screening of organophosphates (OPs) with respect to their ability to result in organophosphate induced delayed neurotoxicity (OPIDN). This paper describes a new biosensor approach to the analysis of NTE and its inhibitors. The method is based on the combination of NTE enzymatic hydrolysis of phenyl valerate (PV) with phenol detection by the Clark-type oxygen electrode modified by immobilized tyrosinase. The validity of this biosensor method is confirmed by the facts that the calibration curves for NTE obtained by colorimetric and flow-through electrochemical methods were nearly identical and the titration of NTE by test inhibitor mipafox was shown to yield the same pI50 values. The developed electrochemical methods can be considered as a promising approach both for serial express NTE analysis and for kinetic characteristics of NTE.

Acetylcholinesterase (AChE) and neuropathy target esterase (neurotoxic esterase, NTE) are two major target enzymes for organophosphorus (OP) esters. The relative potency of an OP ester to react with AChE or with NTE in vitro correlates with its relative potency in vivo to cause acute toxicity (death) or organopohosphate-induced delayed neurotoxicity (OPIDN). On this basis extrapolation from in vitro to in vivo data now seems justifiable to predict risk of OPIDN. The kinetics of NTE and AChE inhibition by experimental pesticides of the general formula (RO)2P(O)ON=CClCH2Cl, where R = methyl, ethyl, isopropyl, propyl, isobutyl, butyl, pentyl, has been studied. Compounds with short R (methyl, ethyl) were shown to be far more potent inhibitors of AChE than NTE. Both anti-NTE activity, selectivity for NTE and, correspondingly, the propensity of compounds to cause OPIDN rise with increasing their hydrophobicity. A high value of ki(NTE)/ki(AChE) for R = pentyl suggests that this compound would have the potential to cause OPIDN at doses lower than the LD50. A quantitative structure-activity relationships (QSAR) analysis indicated that NTE and AChE have different structural and electronic requirements for their respective OP inhibitors.

In order to validate a rodent biochemical model of delayed neurotoxicity of organophosphates (OP) inhibition of rat and hen brain neurotoxic esterase (NTE) by some dichlorovinyl phosphates and phosphonates was studied in vitro and in vivo. It was shown that compounds investigated exhibited the similar inhibitory potency to NTE from both species in vitro, in addition rat and hen NTE showed the same sensitivity to variation of the structure of OP inhibitors. A good correlation was found between pI50 estimated with enzymes from rat and hen trains: r2 = 0.951, n = 18, p < or = 0.05. NTE activities were also measured in rat and hen brains after acute administration of various dosages of potent axonopathic compound dipropyldichlorovinyl phosphate. The results obtained indicate that difference in species susceptibility to neurotoxic action of OP, in particular the absence of ataxia in rats, is not caused by difference in target enzyme sensitivity to axonopathic organophosphates.

Toxicity and insecticide and acaricide activity of compounds (1) is significantly dependent on the nature of amino acid (n = 1 or 2) and substituents in carbamate and amino acid ester groups (R and R1). Investigation of interaction of these compounds with mammalian carboxylesterases, and the appropriate "oxones" with choline esterases of mammal and arthropoda revealed that the lower toxicity and activity of beta-alanine derivatives (n = 2) compared with glycine derivatives (n = 1) are due to the more rapid hydrolysis by carboxylesterases (detoxication). The low toxicity of dithiophosphonate with R = Me, R1 = Bu(i), n = 1 and the high toxicity of its isomer with R = Bu(i), R1 = Me, n = 1 are associated with the more rapid oxidative cleavage of isobutyl group in comparison with the other substituents, because detoxication occurs by the cleavage of R1 and activation--by that of R respectively.

Studies have been made on the interaction of four types of phosphorylated alkylchloroformoximes, i.e. analogues of an insecticide-acaricide valexon, with acetylcholinesterases from human erythrocytes and from the heads of the housefly Musca domestica. Antiacetylcholinesterase activity of the drugs depended both on the structure of the organophosphorus compounds, and the origin of the enzyme, indicating the existence of differences in the active surface of these acetylcholinesterases. Incorporation of one or two chloride atoms into alkylchloroformoxime group of the cleaved part of the organophosphorus compounds increased anticholinesterase activity with respect to both enzymes. Diethyl derivatives of these drugs exhibited higher specificity with respect to housefly enzyme as compared to human acetylcholinesterase.

The interaction of dialkyl (alpha-carbometoxy-beta,beta,beta-trifluoroethyl) phosphates (RO)2P(O) . OCH(CF3)COOMe (R = Me, Et, Pr, Pri, Bu, Bui, Am, Hex) (I-VIII) with human erythrocyte acetylcholinesterase, horse serum butyrylcholinesterase, pig liver carboxylesterase was studied and acute toxicity in mice was estimated. Compounds (I)-(VIII) were not hydrolyzed by carboxylesterase, slowly and irreversibly inhibited acetylcholinesterase (kII = 10(2)-10(4) M-1 X min-1) and more efficiently inhibited butyrylcholinesterase and carboxylesterase (kII = 10(3)-10(7) M-1 X min-1). The structure--antienzymatic activity relationships were investigated. With increasing of hydrophobicity of alkoxy groups, antienzymatic activity to butyrylcholinesterase and carboxylesterase ("sites of loss") rises equally and more significantly, than antiacetylcholinesterase activity (delta lg kII 1.0 and 2.4 for R = CH3 and C5H11 resp.). Branching at the alpha-position of alkoxy groups leads to sharp reducing of acetylcholinesterase and butyrylcholinesterase inhibition constants, the carboxylesterase inhibition mechanism becoming reversible. Multiple regression analysis (the Kubinyi model) showed that influence of steric hindrances is revealed at the phosphorylation stage. It was found that phosphates (I)-(VIII) possess low acute toxicity in mice (900-2000 mg/kg). The toxicity of this homologous series appears to be independent of the hydrophobicity. Role of esterases in toxicological effect of compounds (I)-(VIII) is discussed.

nteraction of insectoacaricide Me (EtO)P(S)SCH2SCH2COOMe (I), its activation metabolites (P = O (II), S = O, and P = O, S = O (III) analogues), and a detoxication product (-COOH analoque (IV) with rat liver carboxylesterase, acetylcholinesterase and butyrylcholinesterase of warm-blooded animals, as well as with cholinesterase and carboxylesterase of American cockroach has been studied. Low toxicity of (I) towards warm-blooded animals and American cockroach is shown to result from its rapid hydrolysis with corresponding carboxylesterases to form (IV). Monothiophosphonates (II) and (III) are not hydrolyzed by carboxylesterases but inhibit them irreversibly. High toxicity of (I) towards aphids can be ascribed to low activity of the carboxylesterase of that insect.

The dependence of antiesteratic activity on the structure of insecticides (RO)2P(O)SCH(COOEt)SP(O)(OR)2 (I) and (RO)2P(O)SCH(COOEt)OP(S)(OR)2 (II) was examined. Nonlinear regression equations (parabolic and bilinear) "hydrophobicity-antiesteratic activity" were derived. Basing on the studies of the relationships between hydrophobicity and individual constants, the detailed mechanisms were proposed for the interaction of type (I) and (II) compounds with the esterase active centers. The mechanisms implicate different kinds of sorbtion for compounds of type I and II. Applicability of bilinear models, similar to that of Kubinyi type, for analyzing the structure-antienzyme activity dependences was demonstrated. Thus, several equations were obtained starting from the literature data on inhibition of esterases with diverse organophosphorus compounds.

The interaction of human erythrocyte acetylcholinesterase, horse serum butyrylcholinesterase and rat liver carboxylesterase with insecticides (RO)2P(O)SCH(COOEt)SP(O)(OR)2 (I) and (RO)2P(O)SCH(COOEt)OP(S)(OR)2 (II) was studied. The type I and II compounds were not hydrolyzed by carboxylesterase and inhibited the esterases irreversibly. A complex pattern of inhibition of acetylcholinesterase and butyrylcholinesterase by these compounds was caused by kinetically-manifested formation of an enzyme-inhibitor complex. The compounds I and II were more selective towards butyrylcholinesterase than towards acetylcholinesterase and carboxylesterase (kII two orders of magnitude higher) because of effective binding in the butyrylcholinesterase active center (K alpha 10(-8)--10(-9) M) due to hydrophobic interaction. An important role of the thion-phosphoryl-containing fragment in the interaction of type II compounds with hydrophobic sites of butyrylcholinesterase and carboxylesterase active centers was established.

The interaction of insecto-acaricides of the general formula (EtO)2P(S)SCH2CONH(CH2)nCH(R1)COOR2 and their activation metabolites (P = O analog) and detoxication products (R2 = H) with rat liver carboxylesterase was studied. The beta-alanine derivative (n = 1, R1 = H, R2 = Et) was rapidly hydrolyzed by carboxylesterase. The valine derivative (n = 0, R1 = H, R2 = Et) was hydrolytically stable, due to steric hindrances imposed by the isopropyl group, and proved to be a reversible competitive inhibitor of carboxylesterase. The corresponding monothiophosphates were not hydrolyzed by carboxylesterase, but inhibited it irreversibly. It was found that monothiophosphate derivatives of R- and S-valine irreversibly inhibit carboxylesterase, R-enantiomer being somewhat more active than S-antipode. On the other hand, under the conditions of reversible inhibition by the corresponding dithiophosphates, S-enantiomer was more active. Using model compounds, (R)- and (S)-N-chloroacetyl valine ethyl esters, it was shown that both on irreversible and reversible inhibition the differences in stereospecificity can be attributed to changes in the inhibitor orientation in the enzyme active site.