Synthesis of monooxime-monocarbamoyl bispyridinium compounds bearing (E)-but-2-ene linker and evaluation of their reactivation activity against tabun- and paraoxon-inhibited acetylcholinesterase

Six AChE monooxime-monocarbamoyl reactivators with an (E)-but-2-ene linker were synthesized using modification of currently known synthetic pathways. Their potency to reactivate AChE inhibited by the nerve agent tabun and insecticide paraoxon was tested in vitro. The reactivation efficacies of pralidoxime, HI-6, obidoxime, K048, K075 and the newly prepared reactivators were compared. According to the results obtained, one reactivator seems to be promising against tabun-inhibited AChE and two reactivators against paraoxon-inhibited AChE. The best results were obtained for bisquaternary substances with at least one oxime group in position four.     

New group of xylene linker-containing acetylcholinesterase reactivators as antidotes against the nerve agent cyclosarin

Nerve agents such as sarin, cyclosarin and tabun are organophosphorus substances able to inhibit the enzyme acetylcholinesterase (AChE; EC 3.1.1.7). AChE reactivators and anticholinergics are generally used as antidotes in the case of intoxication with these agents. None of the known AChE reactivators is able to reactivate AChE inhibited by all nerve agents used. In this work, reactivation potency of nine newly developed AChE reactivators with an incorporated xylene ring in their structure was measured in vitro. Cyclosarin was chosen as an appropriate member of the nerve agent family. Reactivation potency of the tested AChE reactivators was compared with the gold standard of AChE reactivators – pralidoxime. Two oximes (K107 and K108) surpassed the reactivation potency of pralidoxime. Moreover, from the obtained results it could be deduced that AChE reactivators with a functional oxime group in position-2 are the most potent AChE reactivators in the case of cyclosarin intoxications.     

Synthesis of a novel series of non-symmetrical bispyridinium compounds bearing a xylene linker and evaluation of their reactivation activity against tabun and paraoxon-inhibited acetylcholinesterase

Nine potential non-symmetrical xylene-bridged AChE reactivators were synthesized using modifications of currently known synthetic pathways. Their potency to reactivate AChE inhibited by the nerve agent tabun and the insecticide paraoxon together with nine symmetrical xylene-bridged compounds, was tested in vitro. Seven compounds were promising against paraoxon-inhibited AChE. Two compounds were found to be more potent against tabun-inhibited AChE than obidoxime at a concentration applicable in vivo.     

Synthesis of asymmetrical bispyridinium compounds bearing cyano-moiety and evaluation of their reactivation activity against tabun and paraoxon-inhibited acetylcholinesterase

Three asymmetrical AChE reactivators with cyano-moiety and propane linker were synthesized using modification of currently known synthetic pathways. Their potency to reactivate AChE inhibited by nerve agent tabun and insecticide paraoxon was tested in vitro and compared to pralidoxime, HI-6, obidoxime, K027, and K048. According to the results, three compounds seem to be promising against paraoxon-inhibited AChE. Better results were obtained for bisquaternary substances at least with one oxime group in position four. None of tested substances was able to satisfactorily reactivate tabun-inhibited AChE at concentration applicable for in vivo experiments.     

New group of xylene linker-containing acetylcholinesterase reactivators as antidotes against the nerve agent cyclosarin

Nerve agents such as sarin, cyclosarin and tabun are organophosphorus substances able to inhibit the enzyme acetylcholinesterase (AChE; EC 3.1.1.7). AChE reactivators and anticholinergics are generally used as antidotes in the case of intoxication with these agents. None of the known AChE reactivators is able to reactivate AChE inhibited by all nerve agents used. In this work, reactivation potency of nine newly developed AChE reactivators with an incorporated xylene ring in their structure was measured in vitro. Cyclosarin was chosen as an appropriate member of the nerve agent family. Reactivation potency of the tested AChE reactivators was compared with the gold standard of AChE reactivators--pralidoxime. Two oximes (K107 and K108) surpassed the reactivation potency of pralidoxime. Moreover, from the obtained results it could be deduced that AChE reactivators with a functional oxime group in position-2 are the most potent AChE reactivators in the case of cyclosarin intoxications.     

New quaternary pyridine aldoximes as casual antidotes against nerve agents intoxications

In this work, the ability of four newly synthesized oximes--K005 (1,3-bis(2-hydroxyiminomethylpyridinium) propane dibromide), K027 (1-(4-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium) propane dibromide), K033 (1,4-bis(2-hydroxyiminomethylpyridinium) butane dibromide) and K048 (1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide) to reactivate acetylcholinesterase (AChE, EC 3.1.1.7) inhibited by nerve agents is summarized. Reactivation potency of these compounds was tested using standard in vitro reactivation test. Tabun, sarin, cyclosarin and VX agent were used as appropriate testing nerve agents. Rat brain AChE was used as a source of the enzyme. Efficacies of new reactivators to reactivate tabun-, sarin-, cyclosarin- and VX-inhibited AChE were compared with the currently used AChE reactivators (pralidoxime, obidoxime and HI-6). Oxime K048 seems to be promising reactivator of tabun-inhibited AChE. Its reactivation potency is significantly higher than that of HI-6 and pralidoxime and comparable with the potency of obidoxime. The best reactivator of sarin-inhibited AChE seems to be oxime HI-6. None of the new AChE reactivators reached comparable reactivation potency. The same results were obtained for cyclosarin-inhibited AChE. However, oxime K033 is also potent reactivator of AChE inhibited by this nerve agent. In the case of VX inhibition, obidoxime and new oximes K027 and K048 seem to be the best AChE reactivators. None from the currently tested AChE reactivators is able to reactivate AChE inhibited by all nerve agents used and, therefore, the search for new potential broad spectrum AChE reactivators is needed.     

Novel tacrine-pyridinium hybrid reactivators of organophosphorus-inhibited acetylcholinesterase: Synthesis,molecular docking,and in vitro reactivation study

First-line medical treatment against nerve agents consists of co-administration of anticholinergic agents and oxime reactivators, which reactivate inhibited AChE. Pralidoxime, a commonly used oxime reactivator, is effective against some nerve agents but not against others; thus, new oxime reactivators are needed. Novel tacrine-pyridinium hybrid reactivators in which 4-pyridinealdoxime derivatives are connected to tacrine moieties by linear carbon chains of different lengths (C2–C7) were prepared (Scheme 1, 5a–f). Their binding affinities to electric eel AChE were tested because oximes can inhibit free AChE, and the highest AChE activity (95%, 92%, and 90%) was observed at 1?μM concentrations of the oximes (5a, 5b, and 5c, respectively). Based on their inhibitory affinities towards free AChE, 1?μM concentrations of the oxime derivatives (5) were used to examine reactivation of paraoxon-inhibited AChE. Reactivation ability increased as the carbon linker chains lengthened (n?=?2–5), and 5c and 5d showed remarkable reactivation ability (41%) compared to that of 2-PAM (16%) and HI-6 (4%) against paraoxon-inhibited electric eel AChE at 1?μM concentrations. Molecular docking simulation showed that the most stable binding free energy was observed in 5c at 73.79?kcal?mol^?1, and the binding mode of 5c is acceptable for the oxygen atom of oximate to attack the phosphorus atom of paraoxon and reactivate paraoxon-inhibited eel AChE model structure.     

Synthesis and in vitro reactivation study of isonicotinamide derivatives of 2-(hydroxyimino)-N-(pyridin-3-yl)acetamide as reactivators of Sarin and VX inhibited human acetylcholinesterase (hAChE)

Previously (Karade et al., 2014), we have reported the synthesis and in vitro evaluation of bis-pyridinium derivatives of pyridine-3-yl-(2-hydroxyimino acetamide), as reactivators of sarin and VX inhibited hAChE. Few of the molecules showed superior in vivo protection efficacy (mice model) (Kumar et al., 2014; Swami et al., 2016) in comparison to 2-PAM against DFP and sarin poisoning. Encouraged by these results, herein we report the synthesis and in vitro evaluation of isonicotinamide derivatives of pyridine-3-yl-(2-hydroxyimino acetamide) (4a–4d) against sarin and VX inhibited erythrocyte ghost hAChE. Reactivation kinetics of these compounds was studied and the determined kinetic parameters were compared with that of commercial reactivators viz. 2-PAM and obidoxime. In comparison to 2-PAM and obidoxime, oxime 4a and 4b exhibited enhanced reactivation efficacy toward sarin inhibited hAChE while oxime 4c showed far greater reactivation efficacy toward VX inhibited hAChE. The acid dissociation constant and IC₅₀ values of these oximes were determined and correlated with the observed reactivation potential.     

The preparation,in vitro screening and molecular docking of symmetrical bisquaternary cholinesterase inhibitors containing a but-(2E)-en-1,4-diyl connecting linkage

Carbamate inhibitors (e.g. pyridostigmine bromide) are used as a pre-treatment for the prevention of organophosphorus poisoning. They work by blocking the native function of acetylcholinesterases (AChE) and thus protect AChE against irreversible inhibition by organophosphorus compounds. However, carbamate inhibitors are known for their many undesirable side effects related to the carbamylation of AChE. In this paper, we describe 17 novel bisquaternary compounds and have analysed their effect on AChE inhibition. The newly prepared compounds were evaluated in vitro using both human erythrocyte AChE and human plasmatic butyrylcholinesterase. Their inhibitory ability was expressed as the half maximal inhibitory concentration (IC₅₀) and then compared to the standard carbamate drugs and two AChE reactivators. One of these novel compounds showed promising AChE inhibition in vitro (nM range) and was better than the currently used standards. Additionally, a kinetic assay confirmed the non-competitive inhibition of hAChE by this novel compound. Consequently, the docking results confirmed the apparent π-π or π-cationic interactions with the key amino acid residues of hAChE and the binding of the chosen compound at the enzyme catalytic site.     

Photometric microplate assay for estimation of the efficacy of paraoxon-inhibited acetylcholinesterase reactivation

Photometric microplate assay was performed for testing of paraoxon-inhibited acetylcholinesterase (AChE) using three reactivators for reactivation purposes: obidoxime, pralidoxime, and HI-6. 3-D graphs (percent of reactivation vs. concentration of reactivator and vs. time of reactivator effecting) were constructed for each reactivator to compare their efficacy. The best results were obtained using obidoxime where reactivation was near to 80%. Suitability of photometric microplates for following of reactivation procedures is discussed.     

Potency of new structurally different oximes to reactivate cyclosarin-inhibited human brain acetylcholinesterases

Antidotes currently used for organophosphorus pesticide and nerve agent intoxications consist of anticholinergics (atropine mainly) and acetylcholinesterase (AChE, EC 3.1.1.7) reactivators called oximes. Owing to the wide-spread of these toxic compounds worldwide, development of antidotes in the case of first aid is needed. To select the most promising AChE reactivators is a very time consuming process, which is necessary before approval of these compounds to be used as human antidotes. Because of ethical reasons, many developing experiments have been conducted on laboratory animals. However, these results often could not be transferred directly to human. Here, we have tested five newly developed AChE reactivators--K027, K033, K048, K074 and K075, which showed promising reactivation activity on rodents, as reactivators of inhibited human brain cholinesterases. For this purpose, cyclosarin was used as member of the nerve agent family. Oxime HI-6 and pralidoxime were used as AChE reactivator standards. Two AChE reactivators, K027 and K033, achieved comparable reactivation potency as HI-6. Moreover, oxime K033 reached its maximal reactivation potency at the lowest concentration which could be attained in humans.     

Potency of new structurally different oximes to reactivate cyclosarin-inhibited human brain acetylcholinesterases

Antidotes currently used for organophosphorus pesticide and nerve agent intoxications consist of anticholinergics (atropine mainly) and acetylcholinesterase (AChE, EC 3.1.1.7) reactivators called oximes. Owing to the wide-spread of these toxic compounds worldwide, development of antidotes in the case of first aid is needed. To select the most promising AChE reactivators is a very time consuming process, which is necessary before approval of these compounds to be used as human antidotes. Because of ethical reasons, many developing experiments have been conducted on laboratory animals. However, these results often could not be transferred directly to human. Here, we have tested five newly developed AChE reactivators – K027, K033, K048, K074 and K075, which showed promising reactivation activity on rodents, as reactivators of inhibited human brain cholinesterases. For this purpose, cyclosarin was used as member of the nerve agent family. Oxime HI-6 and pralidoxime were used as AChE reactivator standards. Two AChE reactivators, K027 and K033, achieved comparable reactivation potency as HI-6. Moreover, oxime K033 reached its maximal reactivation potency at the lowest concentration which could be attained in humans.     

Novel series of bispyridinium compounds bearing a (Z)-but-2-ene linker--synthesis and evaluation of their reactivation activity against tabun and paraoxon-inhibited acetylcholinesterase

Six novel AChE reactivators with a (Z)-but-2-ene linker were synthesized using the known synthetic pathways. Their ability to reactivate AChE, which had been previously inhibited by nerve agent tabun or pesticide paraoxon, was tested in vitro and compared to pralidoxime, HI-6, obidoxime, and K075. The novel synthesized compounds were found to be ineffective against GA-inhibited AChE but the ability of (Z)-1,4-bis(4-hydroxyiminomethylpyridinium)-but-2-ene dibromide to reactivate paraoxon-inhibited AChE was comparable with that of oxime K075. Notably, the oxime group in position four substantially increased the ability of the novel compounds to reactivate paraoxon-inhibited AChE.     

New efficient imidazolium aldoxime reactivators for nerve agent-inhibited acetylcholinesterase

Herein, we described a new class of uncharged non-pyridinium reactivators for nerve agent-inhibited acetylcholinesterase (AChE). Based on a dual site binding strategy, we conjugated the imidazolium aldoxime to different peripheral site ligands (PSLs) of AChE through alkyl chains. Compared with the known quaternary pyridinium reactivators, two of the resulting conjugates (7g and 7h) were highlighted to be the first efficient non-pyridinium oxime conjugates exhibiting similar or superior ability to reactivate sarin-, VX- and tabun-inhibited AChE. Moreover, they were more broad-spectrum reactivators.     

Pyridinium-2-carbaldoximes with quinolinium carboxamide moiety are simultaneous reactivators of acetylcholinesterase and butyrylcholinesterase inhibited by nerve agent surrogates

The pyridinium-2-carbaldoximes with quinolinium carboxamide moiety were designed and synthesised as cholinesterase reactivators. The prepared compounds showed intermediate-to-high inhibition of both cholinesterases when compared to standard oximes. Their reactivation ability was evaluated in vitro on human recombinant acetylcholinesterase (hrAChE) and human recombinant butyrylcholinesterase (hrBChE) inhibited by nerve agent surrogates (NIMP, NEMP, and NEDPA) or paraoxon. In the reactivation screening, one compound was able to reactivate hrAChE inhibited by all used organophosphates and two novel compounds were able to reactivate NIMP/NEMP-hrBChE. The reactivation kinetics revealed compound 11 that proved to be excellent reactivator of paraoxon-hrAChE better to obidoxime and showed increased reactivation of NIMP/NEMP-hrBChE, although worse to obidoxime. The molecular interactions of studied reactivators were further identified by in silico calculations. Molecular modelling results revealed the importance of creation of the pre-reactivation complex that could lead to better reactivation of both cholinesterases together with reducing particular interactions for lower intrinsic inhibition by the oxime.     

Assessment of antidotal efficacy of cholinesterase reactivators against paraoxon: In vitro reactivation kinetics and physicochemical properties

The search of proficient oximes as reactivators of irreversibly inhibited-AChE by organophosphate poisoning necessitates an appropriate assessment of their physicochemical properties and reactivation kinetics. Therefore, herein acid dissociation constant; pK_a, lipophilicity; log P, polar surface area, hydrogen bond donor and acceptor counts of structurally different oximes (two tertiary oximes and thirteen pyridinium aldoxime derivatives) have been evaluated. The experimentally obtained data for pK_a has been comparatively analyzed by using non-linear regression. Further the tested oximes were screened through in vitro reactivation kinetics against paraoxon-inhibited AChE. The pK_a values of all the examined oximes were within the range of 7.50–9.53. pK_a values of uncharged and mono-pyridinium oximes were in good correlation with their reactivation potency. The high negative log P values of pyridinium oxime reactivators indicate their high hydrophilic character; hence oximes with improved lipophilicity should be designed for the development of novel and more potent antidotes. Propane and butane linked oximes were superior reactivators than xylene linked bis-oxime reactivators. It is concluded from the present study that pK_a value is not only ruled by the position of oximino functionality in the pyridinium ring, but also by the position of linker. Although, pyridinium oximes are proved to be better reactivators but their lipophilicity has to be improved.     

Anatoxin-A(S), a naturally occurring organophosphate,is an irreversible active site-directed inhibitor of acetylcholinesterase (EC 3.1.1.7)

Anatoxin-a(s) is a guanidine methyl phosphate ester (unprotonated molecular ion equals 252 daltons) isolated from the freshwater cyanobac-terium (blue-green alga) Anabaena flos-aquae strain NRC 525–17. Previous work has shown anatoxin-a(s) to be a potent irreversible inhibitor of electric eel ace-tylcholinesterase (EC 3.1.1.7, AChE). In the present study the interaction of anatoxin-a(s) with AChE was investigated by protection studies and since similarities have been noted between anatoxin-a(s) and the synthetic organophosphate anticholinesterases, the ability of reactivators to reactivate the inhibited enzyme was investigated. Treatments directed toward eliminating poisoning symptoms and in vivo protection from anatoxin-a(s) poisonings were investigated using oxime reactivators and atropine or pretreatment with a carbamate and atropine. Anatoxin-a(s) was shown to be an active site-directed inhibitor of acetyl-cholinesterase which is resistant to oxime reactivation due to the structure of its enzyme adduct. In vivo pretreatment with physostigmine and high concentrations of 2-PAM were the only effective antagonists against a lethal dose of anatoxin-a(s).     

Changes of rat plasma total low molecular weight antioxidant level after tabun exposure and consequent treatment by acetylcholinesterase reactivators

These experiments were performed on a rat model. The rats were divided into eight groups and consequently exposed to either a saline solution (control), atropine or a combination of atropine and tabun. The reactivation efficacy of the oximes was estimated on the rats exposed to tabun, atropine and a reactivator of AChE. The oximes HI-6, obidoxime, trimedoxime, K203 and KR-22836 were used as representative compounds of commonly available and new AChE reactivators. Besides the positive effect of the administered reactivators on blood AChE activity, the sizable modulation of low molecular weight antioxidant (LMWA) levels was also determined. The LMWA levels in the the animals treated with the oxime reactivators were decreased in comparison with the animals treated by atropine alone. It was found that the levels of LMWA returned to the level found in the control animals when either trimedoxime, K203 or KR-22836 were administered. The principle of oxime reactivator function and a novel insight into AChE activity regulation and oxidative stress is discussed.     

Design,synthesis, and evaluation of guanylhydrazones as potential inhibitors or reactivators of acetylcholinesterase

Analogs of pralidoxime, which is a commercial antidote for intoxication from neurotoxic organophosphorus compounds, were designed, synthesized, characterized, and tested as potential inhibitors or reactivators of acetylcholinesterase (AChE) using the Ellman’s test, nuclear magnetic resonance, and molecular modeling. These analogs include 1-methylpyridine-2-carboxaldehyde hydrazone, 1-methylpyridine-2-carboxaldehyde guanylhydrazone, and six other guanylhydrazones obtained from different benzaldehydes. The results indicate that all compounds are weak AChE reactivators but relatively good AChE inhibitors. The most effective AChE inhibitor discovered was the guanylhydrazone derived from 2,4-dinitrobenzaldehyde and was compared with tacrine, displaying similar activity to this reference material. These results indicate that guanylhydrazones as well as future similar derivatives may function as drugs for the treatment of Alzheimer's disease.     

A comparison of tabun-inhibited rat brain acetylcholinesterase reactivation by three oximes (HI-6, obidoxime, and K048) in vivo detected by biochemical and histochemical techniques

Tabun belongs to the most toxic nerve agents. Its mechanism of action is based on acetylcholinesterase (AChE) inhibition at the peripheral and central nervous systems. Therapeutic countermeasures comprise administration of atropine with cholinesterase reactivators able to reactivate the inhibited enzyme. Reactivation of AChE is determined mostly biochemically without specification of different brain structures. Histochemical determination allows a fine search for different structures but is performed mostly without quantitative evaluation. In rats intoxicated with tabun and treated with a combination of atropine and HI-6, obidoxime, or new oxime K048, AChE activities in different brain structures were determined using biochemical and quantitative histochemical methods. Inhibition of AChE following untreated tabun intoxication was different in the various brain structures, having the highest degree in the frontal cortex and reticular formation and lowest in the basal ganglia and substantia nigra. Treatment resulted in an increase of AChE activity detected by both methods. The highest increase was observed in the frontal cortex. This reactivation was increased in the order HI-6 < K048 < obidoxime; however, this order was not uniform for all brain parts studied. A correlation between AChE activity detected by histochemical and biochemical methods was demonstrated. The results suggest that for the mechanism of action of the nerve agent tabun, reactivation in various parts of the brain is not of the same physiological importance. AChE activity in the pontomedullar area and frontal cortex seems to be the most important for the therapeutic effect of the reactivators. HI-6 was not a good reactivator for the treatment of tabun intoxication.