Reactivators of acetylcholinesterase inhibited by organophosphorus nerve agents

Since the September 11, 2001, terrorist attacks in the United States, the specter of a chemical threat against civilian populations has renewed research interest in chemical warfare agents, their mechanisms of action, and treatments that reverse their effects. In this Account, we focus specifically on organophosphorus nerve agents (OPNAs). Although some OPNAs are used as pest control, the most toxic chemicals in this class are used as chemical warfare agents in armed conflicts. The acute toxicity of OPNAs results from the irreversible inhibition of acetylcholinesterase (AChE, EC 3.1.1.7) via the formation of a covalent P-O bond at the serine hydroxyl group in the enzyme active site. AChE breaks down the neurotransmitter acetylcholine at neuronal synapses and neuromuscular junctions. The irreversible inhibition of AChE causes the neurotransmitter to accumulate in the synaptic cleft, leading to overstimulation of cholinergic receptors, seizures, respiratory arrest, and death. The current treatment for OPNA poisoning combines an antimuscarinic drug (e.g., atropine), an anticonvulsant drug (e.g., diazepam), and an AChE reactivator of the pyridinium aldoxime family (pralidoxime, trimedoxime, obidoxime, HI-6, HL?-7). Because of their high nucleophilicity, oximes can displace the phosphyl group from the catalytic serine, thus restoring the enzyme's catalytic activity. During 50 years of research in the reactivator field, researchers have synthesized and tested numerous structural modifications of monopyridinium oximes and bispyridinium oximes. In the past decade, medicinal chemists have focused their research on the more efficient bispyridinium reactivators, but all known reactivators have several drawbacks. First, due to their permanent positive charge, they do not cross the blood-brain barrier (BBB) efficiently and do not readily reactivate AChE in the central nervous system. Second, no single oxime is efficient against a wide variety of OPNAs. Third, oximes cannot reactivate "aged" AChE. This Account summarizes recent strategies for the development of AChE reactivators capable of crossing the BBB. The use of nanoparticulate transport and inhibition of P-glycoprotein efflux pumps improves BBB transport of these AChE reactivators. Chemical modifications that increased the lipophilicity of the pyridinium aldoximes, the addition of a fluorine atom and the replacement of a pyridyl ring with a dihydropyridyl moiety, enhances BBB permeability. The glycosylation of pyridine aldoximes facilitates increased BBB penetration via the GLUT-1 transport system. The development of novel uncharged reactivators that can move efficiently across the BBB represents one of the most promising of these new strategies.     

Reactivation of human acetylcholinesterase and butyrylcholinesterase inhibited by leptophos-oxon with different oxime reactivators in vitro

We have evaluated in vitro the potency of 23 oximes to reactivate human erythrocyte acetylcholinesterase (AChE) and plasma butyrylcholinesterase (BChE) inhibited by racemic leptophos-oxon (O-[4-bromo-2,5-dichlorophenyl]-O-methyl phenyl-phosphonate), a toxic metabolite of the pesticide leptophos. Compounds were assayed in concentrations of 10 and 100 μM. In case of leptophos-oxon inhibited AChE, the best reactivation potency was achieved with methoxime, trimedoxime, obidoxime and oxime K027. The most potent reactivators of inhibited BChE were K033, obidoxime, K117, bis-3-PA, K075, K074 and K127. The reactivation efficacy of tested oximes was lower in case of leptophos-oxon inhibited BChE.     

In vitro ability of currently available oximes to reactivate organophosphate pesticide-inhibited human acetylcholinesterase and butyrylcholinesterase

We have in vitro tested the ability of common, commercially available, cholinesterase reactivators (pralidoxime, obidoxime, methoxime, trimedoxime and HI-6) to reactivate human acetylcholinesterase (AChE), inhibited by five structurally different organophosphate pesticides and inhibitors (paraoxon, dichlorvos, DFP, leptophos-oxon and methamidophos). We also tested reactivation of human butyrylcholinesterase (BChE) with the aim of finding a potent oxime, suitable to serve as a "pseudocatalytic" bioscavenger in combination with this enzyme. Such a combination could allow an increase of prophylactic and therapeutic efficacy of the administered enzyme. According to our results, the best broad-spectrum AChE reactivators were trimedoxime and obidoxime in the case of paraoxon, leptophos-oxon, and methamidophos-inhibited AChE. Methamidophos and leptophos-oxon were quite easily reactivatable by all tested reactivators. In the case of methamidophos-inhibited AChE, the lower oxime concentration (10(-5) M) had higher reactivation ability than the 10(-4) M concentration. Therefore, we evaluated the reactivation ability of obidoxime in a concentration range of 10(-3)-10(-7) M. The reactivation of methamidophos-inhibited AChE with different obidoxime concentrations resulted in a bell shaped curve with maximum reactivation at 10(-5) M. In the case of BChE, no reactivator exceeded 15% reactivation ability and therefore none of the oximes can be recommended as a candidate for "pseudocatalytic" bioscavengers with BChE.     

The Extraction of Nickel with Aliphatic Oximes

Abstract

U.S. Bureau of Mines researchers investigated the use of aliphatic oximes to extract nickel from acidic, nickelous-chromic-chloride solutions. Batch solvent extraction tests were conducted to characterize the nickel extraction and stripping properties of several oximes. Results indicated that the oximes selectively extracted nickel over chromium by an ion pair mechanism. Nickel was most effectively stripped from the loaded oxime extractants with dilute hydrochloric acid; however, these acidic strip solutions partially degraded the oximes to their parent aldehydes by hydrolysis. The oximes could, however, be regenerated by contacting the solvent phase with a neutralized hydroxylamine-hydrochloride solution. Decyl oxime was successfully used in a continuous counter-current solvent extraction circuit that incorporated loading, stripping, and regeneration stages. The decyl oxime degraded 12 pct per loading and stripping cycle, but was effectively regenerated prior to recycling to the head of the circuit.     

Two reduction waves of oximes and imine formation in acidic media

The separation of the two two-electron waves in acidic media in solutions of some aromatic aldoximes and ketoximes is attributed to differences between the pK_a-values of oximes (which are smaller than about 1.0) and those of protonated forms of corresponding imines. Dependences of half-wave potentials on pH show that the lower limit pK_a of imine varies from −0.25 for p-formylbenzaldehyde oxime to 2.2 for p-fluorobenzaldehyde oxime. For the benzaldehyde and acetophenone oximes bearing substituents, which have pK_a higher than −1.50, a good agreement was observed for pK_a-values obtained spectrophotometrically and pK_ox values obtained from the change in the slope of plots. Past misrepresentations of the electroreduction mechanism of protonated forms of imines is attributed to insufficiently controlled compositions of supporting electrolytes. The reduction potential of protonated forms of oximes in the more positive wave i₁ is affected by the concentration of halides in the supporting electrolyte. This is attributed to an ion-pair formation, between these anions and protonated forms of oximes. The reduction of the protonated form of imines at acidity, where in the bulk predominates the same protonated form that is reduced, is independent of the nature and concentration of anions of the supporting electrolyte.     

Preparation and surface active properties of alcohol ethoxylates with an amide oxime terminal group

Amide oxime compounds have become of major interest because they can complex with uranium, gallium and various transition metal ions. In this work surface active amide oximes were prepared to make some functions of the amide oxime group exhibit as molecular aggregates in aqueous media. Amide oximes were obtained from nitriles which were prepared by the cyanoethylation of alcohol ethoxylates with a monodispersed oligo(oxyethylene) group. Surface active properties of these compounds were measured under various conditions and were compared with nonionics of the alcohol ethoxylate type. The pKa₁ values of these amide oximes were about five, and they acted as cationic surfactants under acidic conditions and nonionic ones under neutral and basic conditions. When they served as nonionics, the cloud point, CMC, γ_CMC and foaming properties of surface active amide oximes were similar to other alcohol ethoxylate nonionics.     

EFFECT OF HYDROXYOXIME HYDROPHOBICITY UPON RATE OF COPPER EXTRACTION WITH 2-HYDR0XY-5-ALKYLBENZ0PHEN0NE OXIMES IN THE PRESENCE OF α-ACYLOIN OXIMES

ABSTRACT

Effects of α-acyloin oximes of various hydrophobicity upon copper(II) extraction rates with 2-hydroxy-5-alkylbenzophenone oximes was studied. Extraction rate depends mainly upon the hydrophobicity of 2-hydroxy-5-alkylbenzophenone oxime and sharply decreases when its hydrophobicity rises. The effect of a-acyloln oximes is small and more pronounced for more and less hydrophobic 2-hydroxy-5-alkylbenzophenone oximes and a-acyloin oximes, respectively, rising with the decrease of the aqueous phase acidity.     

Extraction of Copper(II) Ions from Chloride and Sulphate Solutions Using Hydrophobic Pyridyl Ketoximes

Hydrophobic pyridyl ketoximes: 1-(2-pyridyl)tridecan-1-one oxime, 1-(3-pyridyl)tridecan-1-one oxime and 1-(4-pyridyl)tridecan-1-one oxime have been synthesized and investigated as extractants of copper(II) ions. Removal of metal ions was conducted from chloride, sulphate, and sulphate/chloride solutions. The influence of pH of aqueous solutions, copper(II), chloride, and sulphate ions and ligand concentration for extraction process were studied. Copper(II) extraction by hydrophobic 2-, 3-, and 4-pyridyl ketoximes from sulphate solutions is not possible. However, addition of chloride ions to initial sulphate media enables metal removal. The oxime of 1-(2-pyridyl)tridecane-1-one was determined as the strongest extractant of the tested oximes, but metal stripping was impossible. For the rest of the studied extractants the stripping process could be done using water or diluted mineral acid.     

Synthesis of New Biscoumarin Derivatives,In Vitro Cholinesterase Inhibition,Molecular Modelling and Antiproliferative Effect in A549 Human Lung Carcinoma Cells

A series of novel C4-C7-tethered biscoumarin derivatives (12a–e) linked through piperazine moiety was designed, synthesized, and evaluated biological/therapeutic potential. Biscoumarin 12d was found to be the most effective inhibitor of both acetylcholinesterase (AChE, IC₅₀ = 6.30 µM) and butyrylcholinesterase (BChE, IC₅₀ = 49 µM). Detailed molecular modelling studies compared the accommodation of ensaculin (well-established coumarin derivative tested in phase I of clinical trials) and 12d in the human recombinant AChE (hAChE) active site. The ability of novel compounds to cross the blood–brain barrier (BBB) was predicted with a positive outcome for compound 12e. The antiproliferative effects of newly synthesized biscoumarin derivatives were tested in vitro on human lung carcinoma cell line (A549) and normal colon fibroblast cell line (CCD-18Co). The effect of derivatives on cell proliferation was evaluated by MTT assay, quantification of cell numbers and viability, colony-forming assay, analysis of cell cycle distribution and mitotic activity. Intracellular localization of used derivatives in A549 cells was confirmed by confocal microscopy. Derivatives 12d and 12e showed significant antiproliferative activity in A549 cancer cells without a significant effect on normal CCD-18Co cells. The inhibition of hAChE/human recombinant BChE (hBChE), the antiproliferative activity on cancer cells, and the ability to cross the BBB suggest the high potential of biscoumarin derivatives. Beside the treatment of cancer, 12e might be applicable against disorders such as schizophrenia, and 12d could serve future development as therapeutic agents in the prevention and/or treatment of Alzheimer’s disease.     

Preparation of dodecylamine and 6-aminohexanoic acid from petroselinic acid

A procedure is given for the ozonization of petroselinic acid, chemical reduction of the ozonides to aldehydes, formation of oximes, and catalytic hydrogenation of the oximes using Raney nickel in the presence of ammonia, to yield 48% dodecylamine and 44% 6-aminohexanoic acid calculated on the amounts theoretically available from the wt of petroselinic acid. The ultimate analyses, melting points, and distribution coefficients between ethyl ether and water of lauraldehyde and adipaldehydic acid oximes were determined. The infrared spectra of lauraldehyde oxime, adipaldehydic acid oxime, and 6-aminohexanoic acid are given and interpreted. Presented at the AOCS meeting in New Orleans, La., 1962. A laboratory of the Southern Utilization Research and Development Division, Agricultural Research Service, U.S.D.A., New Orleans, La.     

Hydrolysis of active esters by polymer-supported oximes: Influence of macromolecular characters

Hydrolysis of p-nitrophenyl acetate by oximino groups anchored onto two types of supports (i.e., polystyrene and polyacrolein) has been studied. Polystyrene and polyacrolein oximes were prepared using divinylbenzene and tetraethyleneglycol diacrylate as crosslinking agents with a varying extent of crosslinking. The rate of the esterolytic reactions was found to depend on the structural characteristics of the polymer support, which include the hydrophobic or hydrophilic nature of the polymer backbone, solvation of the polymer and the attached functional groups, the nature and extent of crosslinking of the polymer matrix, and also the chemical environments of the oximino groups. The effect of the presence of pyridine residues neighboring the acrolein oxime functionality on the hydrolysis reaction was also studied. © 1998 John Wiley & Sons, Inc. J. Appl. Polym. Sci. 70: 493–500, 1998     

Synthesis,spectral data and extraction of copper by 1-(2′-Hydroxy-5′-alkylphenyl)-1-alkanone Oximes

1-(2′-Hydroxy-5′-alkylphenyl)-1-ethanone oximes with a normal alkyl group containing 2 to 12 carbon atoms and 1-(2′-hydroxy-5′-methylphenyl)-1-alkanone oximes containing 1 to 11 carbon atoms in the hydrocarbon chain were synthesized. Spectral data (u.v., i.r., n.m.r. and ¹³C) of oximes are reported. Four of these oximes were used for copper extraction from acidic solution. The results obtained indicate, that these oximes are better extractants than alkyl derivatives of 2-hydroxybenzophenone oximes.     

EQCN based cholinesterase biosensors

The binding of acetylcholinesterase (AChE) to a propidium-modified piezoelectric quartz crystal and its surface enzymatic activity have been investigated.Propidium binds to a site remote to the active center of AChE - the peripheral anionic site (PAS) - which is located on the rim of the gorge to the active site.The gold electrodes of the quartz crystal were first modified with 11-mercaptoundecanoic acid to which propidium was coupled. AChE binding was monitored by a quartz crystal nanobalance (QCN), followed by amperometric activity evaluation of the AChE loaded on the sensor. Interestingly, the binding is strong but does not inhibit AChE. However, an excess of propidium in solution inhibits the immobilized enzyme. The surface enzymatic activities observed depend on the amount of enzyme and differ according to the type and species, i.e. number of enzyme subunits (Electrophorus electricus tetrameric, Drosophila melanogaster mono- and dimeric form - DmAChE).The operational stability and regeneration, effect of propidium in solution and detection limit for substrate for various AChEs were investigated amperometrically.     

One photon-two radical-electron transfer photoinitiators. 2-(o-, m-, or p-Methoxypyridine)-p-pyrrolidinestyrilium methyl sulfates as photoinitiators of free radical polymerization

In this paper, the novel hemicyanine dye-borate pair, e.g. 2-((o, m, or p)-methoxypyridine)-p-pyrrolidinestyrilium methyl sulfates (MeOSp)-tetramethylammonium n-butyltriphenyl borate (TBAB) were evaluated and employed as the photoinitiating pairs of multiacrylate monomer polymerization. The kinetic studies clearly demonstrate that the modification of the dye structure by the exchange of the N-alkyl group on N-alkoxy one in pyridinium moiety causes a marked increase in the efficiency of photoinitiation of 2-ethyl-2-(hydroxymethyl)-1,3-propanediol triacrylate (TMPTA) polymerization.     

A simple process to prepare nitrogen-modified few-layer graphene for a supercapacitor electrode

We report a simple approach to prepare the nitrogen-modified few-layer graphene (FLG) directly from graphite flakes. With the aid of melamine, graphite flakes can be directly ultrasonicated into FLG in acetone. The subsequent annealing process further transforms the melamine absorbed on the surface of graphene into melon (C₆N₉H₃)_x, which is one type of condensation product of melamine, and simultaneously dopes the graphene with nitrogen. When tested as a supercapacitor electrode, the nitrogen-modified FLG shows a much higher specific capacitance (e.g., 227 F/g at 1A/g) than that of reduced graphene oxide (rGO) (e.g., 133 F/g at 1A/g).     

Meerwein's Catalytic Beckmann Rearrangement

Meerwein recommended nitrilium salts ( 2 ) as catalysts for the Beckmann rearrangement of oximes ( 1 ) under neutral and water-free conditions. For the first time, primary adducts (Z)–( 3 ) of oximes to nitrilium salts have been isolated. Reported are activation energies for Beckmann rearrangements of these adducts, and an X-ray structural analysis of (Z)– 3a . Rearrangement of 3 produces mixtures of up to four different N-acylamidinium salts 5 – 8 , which arise from fast reactions of primary-formed secondary amides ( 4 ) with nitrilium salts ( 2 ). Because of their ambident electrophilic character the N-acylamidinium salts react with excess of oxime not only to amides ( 4 ) but to mixtures of products. It is shown that nitrilium salts cannot be used as catalysts for the Beckmann rearrangement.     

Experimental and Established Oximes as Pretreatment before Acute Exposure to Azinphos-Methyl

Poisoning with organophosphorus compounds (OPCs) represents an ongoing threat to civilians and rescue personal. We have previously shown that oximes, when administered prophylactically before exposure to the OPC paraoxon, are able to protect from its toxic effects. In the present study, we have assessed to what degree experimental (K-27; K-48; K-53; K-74; K-75) or established oximes (pralidoxime, obidoxime), when given as pretreatment at an equitoxic dosage of 25% of LD₀₁, are able to reduce mortality induced by the OPC azinphos-methyl. Their efficacy was compared with that of pyridostigmine, the only FDA-approved substance for such prophylaxis. Efficacy was quantified in rats by Cox analysis, calculating the relative risk of death (RR), with RR=1 for the reference group given only azinphos-methyl, but no prophylaxis. All tested compounds significantly (p ≤ 0.05) reduced azinphos-methyl-induced mortality. In addition, the efficacy of all tested experimental and established oximes except K-53 was significantly superior to the FDA-approved compound pyridostigmine. Best protection was observed for the oximes K-48 (RR = 0.20), K-27 (RR = 0.23), and obidoxime (RR = 0.21), which were significantly more efficacious than pralidoxime and pyridostigmine. The second-best group of prophylactic compounds consisted of K-74 (RR = 0.26), K-75 (RR = 0.35) and pralidoxime (RR = 0.37), which were significantly more efficacious than pyridostigmine. Pretreatment with K-53 (RR = 0.37) and pyridostigmine (RR = 0.52) was the least efficacious. Our present data, together with previous results on other OPCs, indicate that the experimental oximes K-27 and K-48 are very promising pretreatment compounds. When penetration into the brain is undesirable, obidoxime is the most efficacious prophylactic agent already approved for clinical use.     

Catalytic hydrolysis of p-nitrophenyl acetate by electrospun polyacrylamidoxime nanofibers

Modification of polyacrylonitrile (PAN) by hydroxylamine resulted in polyacrylamidoxime (PANOx), the oxime groups of which are nucleophilic and capable of hydrolyzing esters. PANOx fiber mats with submicrometer fiber diameters ranging from tens to 300 nm were produced by electrospinning a suspension of PANOx blended with PAN (1:1 by weight) in a mixture of N,N-dimethyl formamide (DMF) and dimethyl sulfoxide (DMSO) (85:15 by weight). Catalytic properties of the PANOx nanofibers were tested by the hydrolysis of p-nitrophenyl acetate (PNPA), which mimics toxic organophosphate nerve agents and insecticides. The presence of PANOx fibers significantly accelerated the hydrolysis of PNPA compared to its spontaneous hydrolysis. The rate constants for the hydrolysis (k₁) and the deacetylation (k₂) reactions for the fibers were obtained using a proposed kinetic model. The effect of the fiber size on reaction rate indicated that intra-fiber diffusional resistances might limit the accessibility of the oxime catalytic sites in the fibers and affect their catalytic activity.     

Multitarget‐Directed Tricyclic Pyridazinones as G Protein‐Coupled Receptor Ligands and Cholinesterase Inhibitors

By following a multitarget ligand design approach, a library of 47 compounds was prepared, and they were tested as binders of selected G protein‐coupled receptors (GPCRs) and inhibitors of acetyl and/or butyryl cholinesterase. The newly designed ligands feature pyridazinone‐based tricyclic scaffolds connected through alkyl chains of variable length to proper amine moieties (e.g., substituted piperazines or piperidines) for GPCR and cholinesterase (ChE) molecular recognition. The compounds were tested at three different GPCRs, namely serotoninergic 5‐HT_1A, adrenergic α_1A, and dopaminergic D₂ receptors. Our main goal was the discovery of compounds that exhibit, in addition to ChE inhibition, antagonist activity at 5‐HT_1A because of its involvement in neuronal deficits typical of Alzheimer’s and other neurodegenerative diseases. Ligands with nanomolar affinity for the tested GPCRs were discovered, but most of them behaved as dual antagonists of α_1A and 5‐HT_1A receptors. Nevertheless, several compounds displaying this GPCR affinity profile also showed moderate to good inhibition of AChE and BChE, thus deserving further investigations to exploit the therapeutic potential of such unusual biological profiles.     

Folgeprodukte halogenierter Aldehyde. XI [1]. Substitutionen am 2,5-Bis-trichlormethyl- und 2-Trichlormethyl-5-dichlormethylen-1,3-dioxolan-4-on-oxim

Derivatives of Halogenated Aldehydes. XI. Substitution Reactions of 2,5-Bis-Trichloromethyl- and 2-Trichloromethyl-5-dichloromethylene-1,3-dioxolan-4-one Oxime 2,5-Bis-trichloromethyl- ( 1 ) and 2-trichloromethyl-5-dichloromethylene-1,3-dioxolan-4-one oxime 2 are acylated with acyl halides, alkyl chlorocarbonates and N,N-dimethylcarbamyl chloride to give the corresponding O-acyl oximes 4–6, 9, 11 and 13 . The addition of the oximes 1 and 2 to isocyanates yields the oxime carbamates 7 and 10 , and the addition to N-acetyl-2,2,2-trichloroacetaldimine leads to the O-(1-acetamino-2,2,2-trichloroethyl) oximes 8 and 12 . Oxime 2 forms a sodium salt 3 which gives the O-alkyl ( 15 ), the O-aryl ( 16 ) and the O-sulphonyl oximes ( 14 ) by reactions with alkyl halides or dialkyl sulfates, with activated aryl halides or with sulphonyl chlorides, respectively.