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.     

Reactivation of DFP- and paraoxon-inhibited acetylcholinesterases by pyridinium oximes

Exposure to the organophosphorus nerve agents such as sarin, soman, cyclosarin, and VX causes acute intoxication by inhibiting acetylcholinesterase (AChE), where the serine residue of the active site can attack the phosphorous atom of the organophosphorus agents to form a strong P–O bond. The purpose of the present study was to evaluate new oxime antidotes to reactivate the inhibited AChE. We have designed and synthesized several new oximes, and have evaluated the substances that differ from the currently used oximes in linker between the two pyridinium rings. The potency of newly synthesized oximes was compared with two currently used AChE reactivators (2-PAM, HI-6). The reactivation potencies of the bis-pyridinium oximes connected with a (CH₂)_n linker between the two quaternary nitrogen atoms were evaluated with housefly (HF) AChE inhibited by diisopropyl fluorophosphates (DFP) and by paraoxon. The bis-pyridinium oximes showed stronger activity compared with mono-pyridinium oxime, and the magnitude of reactivation potency depended on the length of the methylene linker. The potency order was (CH₂) < (CH₂)₂ < (CH₂)₃ > (CH₂)₄ > (CH₂)₇. A (CH₂)₃ linker was optimal in HF AChE inhibited by either DFP or paraoxon. Thus, bis-pyridinium oxime 5 which has (CH₂)₃ linker showed the highest activity in this series of compounds. Interestingly, 5 was not as active as 2-PAM, showing that the position of the oxime group on the pyridinium ring is also very important for the reactivation potency.     

Synthesis,in vitro screening and molecular docking of isoquinolinium-5-carbaldoximes as acetylcholinesterase and butyrylcholinesterase reactivators

Abstract

The series of symmetrical and unsymmetrical isoquinolinium-5-carbaldoximes was designed and prepared for cholinesterase reactivation purposes. The novel compounds were evaluated for intrinsic acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) inhibition, when the majority of novel compounds resulted with high inhibition of both enzymes and only weak inhibitors were selected for reactivation experiments on human AChE or BChE inhibited by sarin, VX, or paraoxon. The AChE reactivation for all used organophosphates was found negligible if compared to the reactivation ability of obidoxime. Importantly, two compounds were found to reactivate BChE inhibited by sarin or VX better to obidoxime at human attainable concentration. One compound resulted as better reactivator of NEMP (VX surrogate)-inhibited BChE than obidoxime. The in vitro results were further rationalized by molecular docking studies showing future directions on designing potent BChE reactivators.     

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.     

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.     

Design,synthesis and evaluation of new classes of nonquaternary reactivators for acetylcholinesterase inhibited by organophosphates

A new series of nonquaternary conjugates for reactivation of both nerve agents and pesticides inhibited hAChE were described in this paper. It was found that substituted salicylaldehydes conjugated to aminobenzamide through piperidine would produce efficient reactivators for sarin, VX and tabun inhibited hAChE, such as L6M1R3, L6M1R5 to L6M1R7, L4M1R3 and L4M1R5 to L4M1R7. The in vitro reactivation experiment for pesticides inhibited hAChE of these new synthesized oximes were conducted for the first time. Despite they were less efficient than obidoxime, some of them were highlighted as equal or more efficient reactivators in comparison to 2-PAM. It was found that introduction of peripheral site ligands could increase oximes’ binding affinity for inhibited hAChE in most cases, which resulted in greater reactivation ability.     

A comparison of the ability of a new bispyridinium oxime--1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium)butane dibromide and currently used oximes to reactivate nerve agent-inhibited rat brain acetylcholinesterase by in vitro methods

The efficacy of a new bispyridinium oxime 1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium)butane dibromide, called K048, and currently used oximes (pralidoxime, obidoxime, the oxime HI-6) to reactivate acetylcholinesterase inhibited by various nerve agents (sarin, tabun, cyclosarin, VX) was tested by in vitro methods. The new oxime K048 was found to be a more efficacious reactivator of nerve agent-inhibited acetylcholinesterase than pralidoxime (in the case of VX, tabun and cyclosarin), obidoxime (cyclosarin and tabun) and HI-6 (tabun) but it did not reach the efficacy of currently used oximes for the reactivation of acetylcholinesterase inhibited by sarin. Thus, the oxime K048 seems to be a relatively efficacious broad spectrum acetylcholinesterase reactivator and, therefore, it could be useful for the treatment of a nerve agent-exposed population if information about detection of the type of nerve agent is not available.     

Reactivation of organophosphate-inhibited human acetylcholinesterase by isonitrosoacetone (MINA): a kinetic analysis

Treatment of poisoning by highly toxic organophosphorus compounds (OP) with atropine and an acetylcholinesterase (AChE) reactivator (oxime) is of limited effectiveness in case of different nerve agents and pesticides. One challenge is the reactivation of OP-inhibited brain AChE which shows inadequate success with charged pyridinium oximes. Recent studies with high doses of the tertiary oxime isonitrosoacetone (MINA) indicated a beneficial effect on central and peripheral AChE and on survival in nerve agent poisoned guinea pigs. Now, an in vitro study was performed to determine the reactivation kinetics of MINA with tabun-, sarin-, cyclosarin-, VX- and paraoxon-inhibited human AChE. MINA showed an exceptionally low affinity to inhibited AChE but, with the exception of tabun-inhibited AChE, a moderate to high reactivity. In comparison to the pyridinium oximes obidoxime, 2-PAM and HI-6 the affinity and reactivity of MINA was in most cases lower and in relation to the most effective reactivators, the second order reactivation constant of MINA was 500 to 3400-fold lower. Hence, high in vivo MINA concentrations would be necessary to achieve at least partial reactivation. This assumption corresponds to in vivo data showing a dose-dependent effect on reactivation and survival in animals. In view, of the toxic potential of MINA in animals human studies would be necessary to determine the tolerability and pharmacokinetics of MINA in order to enable a proper assessment of the value of this oxime as an antidote in OP poisoning.     

Bis-pyridiumaldoxime reactivators connected with CH2O(CH2)n OCH2 linkers between pyridinium rings and their reactivity against VX

New bis-pyridinium oxime reactivators connected with CH2O(CH2)n OCH2 linkers between two pyridinium rings were designed and synthesized, and their reactivation potency was evaluated for AChE inhibited by organophosphorus VX agent. Among the prepared compounds, 1,2-dimethoxy-ethylene-bis-N,N'-4-pyridiumaldoxime dichloride 5a was the most potent and appeared to be the most promising compound as a potential reactivator for AChE inhibited by organophosphorus VX agent.     

Reactivation of model cholinesterases by oximes and intermediate phosphyloximes: a computational study

Phosphyloximes (POX) are generated upon the reactivation of organophosphorus (OP)-inhibited cholinesterases (ChEs) by pyridinium oximes. These POXs are known to be potent inhibitors of the ChEs following reactivation. However, they can also decompose to give an OP derivative and a cyano derivative of the oxime when a base abstracts the benzylic proton. Using density functional theory, thermodynamic properties were calculated for the reactivation and decomposition pathways of three different oximes (2-PAM, 3-PAM and 4-PAM) with six different OPs (cyclosarin, paraoxon, sarin, tabun, VR and VX). For reactivation purposes, 2-PAM is predicted to be more efficient than 3- and 4-PAM. Based on atomic charges and relative energies, 2-POXs were found to be more inclined towards the decomposition process.     

Bisquaternary pyridinium oximes: Comparison of in vitro reactivation potency of compounds bearing aliphatic linkers and heteroaromatic linkers for paraoxon-inhibited electric eel and recombinant human acetylcholinesterase

Oxime reactivators are the drugs of choice for the post-treatment of OP (organophosphorus) intoxication and used widely for mechanistic and kinetic studies of OP-inhibited cholinesterases. The purpose of the present study was to evaluate new oxime compounds to reactivate acetylcholinesterase (AChE) inhibited by the OP paraoxon. Several new bisquaternary pyridinium oximes with heterocyclic linkers along with some known bisquaternary pyridinium oximes bearing aliphatic linkers were synthesized and evaluated for their in vitro reactivation potency against paraoxon-inhibited electric eel acetylcholinesterase (EeAChE) and recombinant human acetylcholinesterase (rHuAChE). Results herein indicate that most of the compounds are better reactivators of EeAChE than of rHuAChE. The reactivation potency of two different classes of compounds with varying linker chains was compared and observed that the structure of the connecting chain is an important factor for the activity of the reactivators. At a higher concentration (10^?3 M), compounds bearing aliphatic linker showed better reactivation than compounds with heterocyclic linkers. Interestingly, oximes with a heterocyclic linker inhibited AChE at higher concentration (10^?3 M), whereas their ability to reactivate was increased at lower concentrations (10^?4 M and 10^?5 M). Compounds bearing either a thiophene linker 26, 46 or a furan linker 31 showed 59%, 49% and 52% reactivation of EeAChE, respectively, at 10^?5 M. These compounds showed 14%, 6% and 15% reactivation of rHuAChE at 10^?4 M. Amongst newly synthesized analogs with heterocyclic linkers (26–35 and 45–46), compound 31, bearing furan linker chain, was found to be the most effective reactivator with a k_r 0.042 min^?1, which is better than obidoxime (3) for paraoxon-inhibited EeAChE. Compound 31 showed a k_r 0.0041 min^?1 that is near equal to pralidoxime (1) for paraoxon-inhibited rHuAChE.     

A Comparison of the Ability of a New Bispyridinium Oxime—1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium)butane Dibromide and Currently used Oximes to Reactivate Nerve Agent-inhibited Rat Brain Acetylcholinesterase by In Vitro Methods

The efficacy of a new bispyridinium oxime 1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium)butane dibromide, called K048, and currently used oximes (pralidoxime, obidoxime, the oxime HI-6) to reactivate acetylcholinesterase inhibited by various nerve agents (sarin, tabun, cyclosarin, VX) was tested by in vitro methods. The new oxime K048 was found to be a more efficacious reactivator of nerve agent-inhibited acetylcholinesterase than pralidoxime (in the case of VX, tabun and cyclosarin), obidoxime (cyclosarin and tabun) and HI-6 (tabun) but it did not reach the efficacy of currently used oximes for the reactivation of acetylcholinesterase inhibited by sarin. Thus, the oxime K048 seems to be a relatively efficacious broad spectrum acetylcholinesterase reactivator and, therefore, it could be useful for the treatment of a nerve agent-exposed population if information about detection of the type of nerve agent is not available.     

Potency of several oximes to reactivate human acetylcholinesterase and butyrylcholinesterase inhibited by paraoxon in vitro

Organophosphorus pesticides (e.g. chlorpyrifos, malathion, and parathion) and nerve agents (sarin, tabun, and VX) are highly toxic organophosphorus compounds with strong inhibition potency against two key enzymes in the human body—acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BuChE; EC 3.1.1.8). Subsequent accumulation of acetylcholine at synaptic clefts can result in cholinergic crisis and possible death of intoxicated organism. For the recovery of inhibited AChE, derivatives from the group of pyridinium or bispyridinium aldoximes (called oximes) are used. Their efficacy depends on their chemical structure and also type of organophosphorus inhibitor. In this study, we have tested potency of selected cholinesterase reactivators (pralidoxime, obidoxime, trimedoxime, methoxime and H-oxime HI-6) to reactivate human erythrocyte AChE and human plasma BuChE inhibited by pesticide paraoxon. For this purpose, modified Ellman's method was used and two different concentrations of oximes (10 and 100 μM), attainable in the plasma within antidotal treatment of pesticide intoxication were tested. Results demonstrated that obidoxime (96.8%) and trimedoxime (86%) only reached sufficient reactivation efficacy in case of paraoxon-inhibited AChE. Other oximes evaluated did not surpassed more than 25% of reactivation. In the case of BuChE reactivation, none of tested oximes surpassed 12.5% of reactivation. The highest reactivation efficacy was achieved for trimedoxime (12.4%) at the concentration 100 μM. From the data obtained, it is clear that only two from currently available oximes (obidoxime and trimedoxime) are good reactivators of paraoxon-inhibited AChE. In the case of BuChE, none of these reactivators could be used for its reactivation.     

Oximes: Reactivators of phosphorylated acetylcholinesterase and antidotes in therapy against tabun poisoning

One of the therapeutic approaches to organophosphate poisoning is to reactivate AChE with site-directed nucleophiles such as oximes. However, pyridinium oximes 2-PAM, HI-6, TMB-4 and obidoxime, found as the most effective reactivators, have limiting reactivating potency in tabun poisoning. We tested oximes varying in the type of ring (pyridinium and/or imidazolium), the length and type of the linker between rings, and in the position of the oxime group on the ring to find more effective oximes to reactivate tabun-inhibited human erythrocyte AChE. Three of our tested pyridinium oximes K027, K048, K074, along with TMB-4, were the most promising for AChE reactivation. Promising oximes were further tested in vivo on tabun poisoned mice not only as antidotes in combination with atropine but also as pretreatment drug. Herein, we showed that a promising treatment in tabun poisoning by selected oximes and atropine could be improved if oximes are also used in pretreatment. Since the reactivating efficacy of the oximes in vitro corresponded to their therapeutic efficacy in vivo, it seems that pharmacological effect of these oximes is indeed primarily related to the reactivation of tabun-phosphorylated AChE.     

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.     

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.     

New series of monoquaternary pyridinium oximes: Synthesis and reactivation potency for paraoxon-inhibited electric eel and recombinant human acetylcholinesterase

The preparation of a series of monoquaternary pyridinium oximes bearing either a heterocyclic side chain or a functionalized aliphatic side chain and the corresponding in vitro evaluation for reactivation of paraoxon-inhibited electric eel acetylcholinesterase (EeAChE) and recombinant human acetylcholinesterase (rHuAChE) are reported. Several newly synthesized compounds efficiently reactivated inhibited EeAChE, but were poor reactivators of inhibited rHuAChE. Compounds bearing a thiophene ring in the side chain (20, 23, 26 and 29) showed better reactivation (24–37% for EeAChE and 5–9% for rHuAChE) compared to compounds with furan and isoxazole heterocycles (0–8% for EeAChE and 2–3% for rHuAChE) at 10^?5 M. The N-pyridyl-CH₂COOH analog 8 reactivated EeAChE (36%) and rHuAChE (15%) at 10^?4 M with a k_r value better than 2-pyridine aldoxime methiodide (2-PAM) for rHuAChE.     

A comparison of the therapeutic and reactivating efficacy of newly developed bispyridinium compounds (K206, K269) with currently available oximes against tabun in rats and mice

The potency of newly developed bispyridinium compounds (K206, K269) in reactivating tabun-inhibited acetylcholinesterase and eliminating tabun-induced lethal toxic effects was compared with commonly used oximes (obidoxime, trimedoxime, the oxime HI-6) using in vivo methods. Studies which determined percentage of reactivation of tabun-inhibited blood and tissue AChE in poisoned rats showed that the reactivating efficacy of both newly developed oximes is comparable with obidoxime and trimedoxime in blood but lower than the reactivating potency of trimedoxime and obidoxime in the diaphragm and brain. Nevertheless, the differences in reactivating efficacy of obidoxime, trimedoxime and K206 was not significant while the potency of K269 to reactivate tabun-inhibited acetylcholinesterase was significantly lower. Both newly developed oximes were also found to be relatively efficacious in elimination of the lethal toxic effects in tabun-poisoned mice. Their therapeutic efficacy corresponds to the therapeutic potency of obidoxime. The oxime HI-6, relatively efficacious against soman, did not seem to be an adequately effective oxime in reactivation of tabun-inhibited AChE and to counteract lethal effects of tabun. Both newly developed oximes (K206, K269) are significantly more efficacious in reactivating tabun-inhibited AChE in rats and to eliminate lethal toxic effects of tabun in mice than the oxime HI-6 but their reactivating and therapeutic potency does not prevail over the effectiveness of currently available obidoxime and trimedoxime and, therefore, they are not suitable for their replacement of commonly used oximes for the treatment of acute tabun poisoning.     

Oxime-induced decarbamylation and atropine/oxime therapy of guinea pigs intoxicated with pyridostigmine

The generally accepted explanation for the effects of oximes in countering organophosphorus (OP) anticholinesterase is reactivation of the inhibited acetylcholinesterase (AChE). With soman, the inhibited AChE rapidly becomes resistant to oxime reactivation due to a phenomenon called aging. Thus, pretreatment with pyridostigmine (Py) or physostigmine (Ph) followed by atropine sulfate therapy is required to achieve significant protection against soman; the effectiveness of a pretreatment/therapy (P/T) regimen can be further increased against certain OPs (e.g. sarin and VX) by including an oxime in the therapy regimen. The P/T regimen is clouded by a controversy concerning the use of oximes in the treatment of carbamate intoxication, because 2-PAM has been reported to exacerbate intoxication by some carbamates and to have no effect on decarbamylation rates. To better understand the role of oxime therapy in the theory of pretreatment of OP intoxication we examined the effects of 2-PAM and HI-6 on the rate of decarbamylation of Py-inhibited erythrocyte AChE in vitro and in vivo, and studied the effects of atropine plus 2-PAM or HI-6 on Py toxicity. In decarbamylation experiments, Py-inhibited guinea pig erythrocytes were washed free of excess Py and incubated with vehicle or oxime (2 X 10(-4) M, pH 7.3 and 37 degrees C). Aliquots were assayed for AChE activity at various times during a 60 min incubation period. Rate constants were calculated and compared to determine whether the presence of oxime affected decarbamylation. The data from in vitro and in vivo experiments revealed that oximes accelerated the decarbamylation (p less than 0.05) of inhibited AChE. Lethality data for Py-treated guinea pigs showed that treatment with atropine (23 mumoles/kg, im) plus 2-PAM or HI-6 (145 mumoles/kg, im) at one min after injection of Py increased the protective ratio from 4.2 (atropine only) to 5.1 and 12.2, respectively. It is suggested that the enhanced therapeutic efficacy of atropine by oximes against Py intoxication is related to oxime-induced reactivation.     

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.