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The authors report on the synthesis and biological evaluation of new compounds whose structure combines tacrine and indole moieties. Tacrine–indole heterodimers were designed to inhibit cholinesterases and β-amyloid formation, and to cross the blood–brain barrier. The most potent new acetylcholinesterase inhibitors were compounds 3c and 4d (IC50 = 25 and 39 nM, respectively). Compound 3c displayed considerably higher selectivity for acetylcholinesterase relative to human plasma butyrylcholinesterase in comparison to compound 4d (selectivity index: IC50 [butyrylcholinesterase]/IC50 [acetylcholinesterase] = 3 and 0.6, respectively). Furthermore, compound 3c inhibited β-amyloid-dependent amyloid nucleation in the yeast-based prion nucleation assay and displayed no dsDNA destabilizing interactions with DNA. Compounds 3c and 4d displayed a high probability of crossing the blood–brain barrier. The results support the potential of 3c for future development as a dual-acting therapeutic agent in the prevention and/or treatment of Alzheimer's disease.

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., GRAPHICAL ABSTRACT

Insecticides represent the most crucial element in the integrated management approach to malaria and other vector-borne diseases. The evolution of insect resistance to long-used substances and the toxicity of organophosphates (OPs) and carbamates are the main factors contributing to the development of new, environmentally safe pesticides. In our work, fourteen compounds of 7-methoxytacrine-tacrine heterodimers were tested for their insecticidal effect. Compounds were evaluated in vitro on insect acetylcholinesterase from Anopheles gambiae (AgAChE) and Musca domestica (MdAChE). The evaluation was executed in parallel with testing on human erythrocyte acetylcholinesterase (HssAChE) and human butyrylcholinesterase (HssBChE) using a modified Ellman's method. Compound efficacy was determined as IC

23 p.-6 fig.-1 tab., A novel series of aromatic esters (1a-1m) related to the Amaryllidaceae alkaloid (AA) haemanthamine were designed, synthesized and tested in vitro with particular emphasis on the treatment of neurodegenerative diseases. Some of the synthesized compounds revealed promising acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory profile. Significant human AChE (hAChE) inhibition was demonstrated by 11-O-(3-nitrobenzoyl)haemanthamine (1j) with IC50value of 4.0 ± 0.3 µM. The strongest human BuChE (hBuChE) inhibition generated 1-O-(2-methoxybenzoyl)haemanthamine (1g) with IC50 value 3.3 ± 0.4 µM. Moreover, 11-O-(2-chlorbenzoyl)haemanthamine (1m) was able to inhibit both enzymes in dose-dependent manner. The mode of hAChE and hBuChE inhibition was minutely inspected using enzyme kinetic analysis in tandem with in silico experiments, the latter elucidating crucial interaction in 1j-, 1m-hAChE and 1g-, 1m-hBuChE complexes. The blood–brain barrier (BBB) permeability was investigated applying the parallel artificial membrane permeation assay (PAMPA) to predict the CNS availability of the compounds., This project was supported by Charles University grants (GA UK Nr. 178518, SVV UK 260412, 260 401; Progres/UK Q40 and Q42), by MH CZ - DRO (University Hospital Hradec Kralove, Nr. 00179906), by Long-term development plan (Faculty of Military Health Sciences), and by EFSA-CDN (Nr.CZ.02.1.01/0.0/0.0/16_019/0000841) co-funded by ERDF, by MICU (grant Nr. SAF2016-76693-R to A.M.), and by Czech Science Foundation (Nr. 20-29633 J). Computational resources were provided by the CESNET LM2015042 and the CERIT Scientific Cloud LM2015085, provided under the program “Projects of Large Research,Development, and Innovations Infrastructures”., preprint

Alzheimer’s disease (AD) is a multifactorial neurodegenerative condition of the central nervous system (CNS) that is currently treated by cholinesterase inhibitors and the N-methyl-d-aspartate receptor antagonist, memantine. Emerging evidence strongly supports the relevance of targeting butyrylcholinesterase (BuChE) in the more advanced stages of AD. Within this study, we have generated a pilot series of compounds (1–20) structurally inspired from belladine-type Amaryllidaceae alkaloids, namely carltonine A and B, and evaluated their acetylcholinesterase (AChE) and BuChE inhibition properties. Some of the compounds exhibited intriguing inhibition activity for human BuChE (hBuChE), with a preference for BuChE over AChE. Seven compounds were found to possess a hBuChE inhibition profile, with IC50 values below 1 µM. The most potent one, compound 6, showed nanomolar range activity with an IC50 value of 72 nM and an excellent selectivity pattern over AChE, reaching a selectivity index of almost 1400. Compound 6 was further studied by enzyme kinetics, along with in-silico techniques, to reveal the mode of inhibition. The prediction of CNS availability estimates that all the compounds in this survey can pass through the blood-brain barrier (BBB), as disclosed by the BBB score.

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.

Multitarget-directed ligands (MTDLs) are considered a promising therapeutic strategy to address the multifactorial nature of Alzheimer's disease (AD). Novel MTDLs have been designed as inhibitors of human acetylcholinesterases/butyrylcholinesterases, monoamine oxidase A/B, and glycogen synthase kinase 3β and as calcium channel antagonists via the Biginelli multicomponent reaction. Among these MTDLs, (±)-BIGI-3h was identified as a promising new hit compound showing in vitro balanced activities toward the aforementioned recognized AD targets. Additional in vitro studies demonstrated antioxidant effects and brain penetration, along with the ability to inhibit the aggregation of both τ protein and β-amyloid peptide. The in vivo studies have shown that (±)-BIGI-3h (10 mg/kg intraperitoneally) significantly reduces scopolamine-induced cognitive deficits., L.I. thanks the Regional Council of Franche-Comté (2016YC- 04540 and 04560) for financial support, Mrs. M.-J. Henriot (PHV Pharma) for her support in the HPLC analyses, and Vincent Luzet for preliminary results in synthesis. O.S., J.J., and J.K. acknowledge the support from the grant by Czech Science Foundation no. 20-29633J. T.K., D.J., and V.H. acknowledge support from the Long-term Development Plan (Faculty of Military Health Sciences). R.A. and K.M. thank the Ministry of Education, Youth and Sports of the Czech Republic (ERDF no. CZ.02.1.01/0.0/0.0/16_025/0007444).

Alzheimer’s disease (AD) is a complex disorder with unknown etiology. Currently, only symptomatic therapy of AD is available, comprising cholinesterase inhibitors and N-methyl-d-aspartate (NMDA) receptor antagonists. Drugs targeting only one pathological condition have generated only limited efficacy. Thus, combining two or more therapeutic interventions into one molecule is believed to provide higher benefit for the treatment of AD. In the presented study, we designed, synthesized, and biologically evaluated 15 novel fluoren-9-amine derivatives. The in silico prediction suggested both the oral availability and permeation through the blood–brain barrier (BBB). An initial assessment of the biological profile included determination of the cholinesterase inhibition and NMDA receptor antagonism at the GluN1/GluN2A and GluN1/GluN2B subunits, along with a low cytotoxicity profile in the CHO-K1 cell line. Interestingly, compounds revealed a selective butyrylcholinesterase (BChE) inhibition pattern with antagonistic activity on the NMDARs. Their interaction with butyrylcholinesterase was elucidated by studying enzyme kinetics for compound 3c in tandem with the in silico docking simulation. The docking study showed the interaction of the tricyclic core of new derivatives with Trp82 within the anionic site of the enzyme in a similar way as the template drug tacrine. From the kinetic analysis, it is apparent that 3c is a competitive inhibitor of BChE.

BACKGROUND In the last decade, the concept of uncharged reactivators potentially able to penetrate the CNS has been introduced as an alternative to the classic charged oxime reactivators. However, this concept brings with it several associated drawbacks such as higher lipophilicity, difficulty in administration, lower affinity to cholinesterases, and higher toxicity risk. OBJECTIVE In this study, we compare data obtained for a set of five classic charged reactivators and a set of three recently published uncharged oximes supplemented by two novel ones. METHODS This time, we used only in silico prediction and in vitro approaches. RESULTS Our data showed that tested uncharged oximes have low affinity for cholinesterases, do not possess high reactivation potency, and certainly represent a greater toxicity risk due to higher lipophilicity. We assume that balanced physicochemical properties will be required for the successful treatment of OP poisoning. Nevertheless, the compound meeting such criteria and pinpointed in silico (K1280) failed in this particular case. CONCLUSION From the presented data, it seems that the concept of uncharged reactivators will have to be modified, at least to improve the bioavailability and to satisfy requirements for in vivo administration.

Novel tacrine-benzyl quinolone carboxylic acid (tacrine-BQCA) hybrids were designed based on multi-target directed ligands (MTLDs) paradigm, synthesized and evaluated in vitro as inhibitors of human acetylcholinesterase (hAChE) and human butyrylcholinesterase (hBChE). Tacrine moiety is represented herein as 7-methoxytacrine, 6-chlorotacrine or unsubstituted tacrine forming three different families of seven members, i.e. 21 compounds in overall. Introducing BQCA, a positive modulator of M1 muscarinic acetylcholine receptors (mAChRs), the action of novel compounds on M1 mAChRs was evaluated via Fluo-4 NW assay on the Chinese hamster ovarian (CHO-M1WT2) cell line. All the novel tacrine-BQCA hybrids were able to block the action of hAChE and hBChE in micromolar to nanomolar range. The hAChE kinetic profile of 5p was found to be mixed-type which is consistent with our docking experiments. Moreover, selected ligands were assessed for their potential hepatotoxicity on HepG2 cell line and presumable permeation through the blood-brain barrier by PAMPA assay. Expected agonistic profile towards M1 mAChRs delivered by BQCA moiety was not confirmed. From all the hybrids, 5o can be highlighted as non-selective cholinesterase inhibitor (hAChE IC50 = 74.5 nM; hBChE IC50 = 83.3 nM) with micromolar antagonistic activity towards M1 mAChR (IC50 = 4.23 μM). A non-selective pattern of cholinesterase inhibition is likely to be valuable during the onset as well as later stages of AD.

Kamil Kuca,1,2 Jana Zdarova Karasova,2,3 Ondrej Soukup,2 Jiri Kassa,3 Eva Novotna,2 Vendula Sepsova,2,3 Anna Horova,2 Jaroslav Pejchal,3 Martina Hrabinova,2,3 Eva Vodakova,2 Daniel Jun,2,3 Eugenie Nepovimova,1,2 Martin Valis,4 Kamil Musilek1,2 1Department of Chemistry, Faculty of Science, University of Hradec Kralove, 2Biomedical Research Center, University Hospital Hradec Kralove, 3Department of Toxicology and Military Pharmacy, Faculty of Military Health Sciences, University of Defence, 4Department of Neurology, University Hospital Hradec Kralove, Hradec Kralove, Czech Republic Background: Intoxication by nerve agents could be prevented by using small acetylcholinesterase inhibitors (eg, pyridostigmine) for potentially exposed personnel. However, the serious side effects of currently used drugs led to research of novel potent molecules for prophylaxis of organophosphorus intoxication. Methods: The molecular design, molecular docking, chemical synthesis, in vitro methods (enzyme inhibition, cytotoxicity, and nicotinic receptors modulation), and in vivo methods (acute toxicity and prophylactic effect) were used to study bispyridinium, bisquinolinium, bisisoquinolinium, and pyridinium-quinolinium/isoquinolinium molecules presented in this study. Results: The studied molecules showed non-competitive inhibitory ability towards human acetylcholinesterase in vitro that was further confirmed by molecular modelling studies. Several compounds were selected for further studies. First, their cytotoxicity, nicotinic receptors modulation, and acute toxicity (lethal dose for 50% of laboratory animals [LD50]; mice and rats) were tested to evaluate their safety with promising results. Furthermore, their blood levels were measured to select the appropriate time for prophylactic administration. Finally, the protective ratio of selected compounds against soman-induced toxicity was determined when selected compounds were found similarly potent or only slightly better to standard pyridostigmine. Conclusion: The presented small bisquaternary molecules did not show overall benefit in prophylaxis of soman-induced in vivo toxicity. Keywords: AChE inhibitors, prophylaxis, pre-treatment, nerve agents, toxicity, soman

Background Based on in vitro and in vivo rat experiments, the newly developed acetylcholinesterase (AChE) reactivator, K203, appears to be much more effective in the treatment of tabun poisonings than currently fielded oximes. Methods To determine if this reactivating efficacy would extend to humans, studies were conducted in vitro using human brain homogenate as the source of AChE. The efficacy of K203 was compared with commercially available oximes; pralidoxime, obidoxime and asoxime (HI-6). Results Reactivation studies showed that K203 was the most effective reactivator with a second order kinetic constant (k r) of 2142 min− 1. M− 1, which was 51 times higher than that obtained for obidoxime (k r = 42 min− 1. M− 1). Both pralidoxime and asoxime (HI-6) failed to significantly reactivate tabun-inhibited human AChE. Discussion According to these results and previous studies, using K203, it appears that oxime K203 is the most effective reactivator of tabun-inhibited cholinesterase in several species including humans and should be considered as a possible medical countermeasure to tabun exposure.

Alzheimer’s disease is debilitating neurodegenerative disorder in the elderly. Current therapy relies on administration of acetylcholinesterase inhibitors (AChEIs) -donepezil, rivastigmine, galantamine, and N-methyl-d-aspartate receptor antagonist memantine. However, their therapeutic effect is only short-term and stabilizes cognitive functions for up to 2 years. Given this drawback together with other pathological hallmarks of the disease taken into consideration, novel approaches have recently emerged to better cope with AD onset or its progression. One such strategy implies broadening the biological profile of AChEIs into so-called multi-target directed ligands (MTDLs). In this review article, we made comprehensive literature survey emphasising on donepezil template which was structurally converted into plethora of MTLDs preserving anti-cholinesterase effect and, at the same time, escalating the anti-oxidant potential, which was reported as a crucial role in the pathogenesis of the Alzheimer’s disease.

Fifteen Amaryllidaceae alkaloids (1–15) of various structural types were isolated by standard chromatographic methods from fresh bulbs of Narcissus poeticus cv. Pink Parasol. The chemical structures were elucidated by MS, and 1D and 2D NMR spectroscopic analyses, and by comparison with literature data. Narcipavline (5) and narcikachnine (6) are reported here for the first time. In their structure are combined two basic structural types of Amaryllidaceae alkaloids (galanthamine- and galanthindole-structural types), which represent a new structural type of these compounds. Alkaloids isolated in sufficient amounts were evaluated for their human erythrocytic acetylcholinesterase, and human serum butyrylcholinesterase (HuBuChE) inhibition activity using Ellman’s method. Z-Gly-Pro-p-nitroanilide was used as substrate in the prolyl oligopeptidase (POP) assay. Untested alkaloids were also screened for their cytotoxic activity against a small panel of human cancer cells, which spanned cell lines from different tissue types. In parallel, MRC-5 human fibroblasts were employed to determine overall toxicity against noncancerous cells. Some compounds were evaluated for their antiprotozoal activity. The newly isolated alkaloid narcipavline (5) showed interesting HuBuChE inhibition activity (IC50 = 24.4 ± 1.2 µM), and norlycoramine (11) demonstrated promising POP inhibition (IC50 = 0.21 ± 0.01 mM).

The search for novel and effective therapeutics for Alzheimer's disease (AD) is the main quest that remains to be resolved. The goal is to find a disease-modifying agent able to confront the multifactorial nature of the disease positively. Herewith, a family of huprineY-tryptophan heterodimers was prepared, resulting in inhibition of cholinesterase and neuronal nitric oxide synthase enzymes, with effect against amyloid-beta (Aβ) and potential ability to cross the blood-brain barrier. Their cholinesterase pattern of behavior was inspected using kinetic analysis in tandem with docking studies. These heterodimers exhibited a promising pharmacological profile with strong implication in AD.

Background Pharmaceuticals with targets in the cholinergic transmission have been used for decades and are still fundamental treatments in many diseases and conditions today. Both the transmission and the effects of the somatomotoric and the parasympathetic nervous systems may be targeted by such treatments. Irrespective of the knowledge that the effects of neuronal signalling in the nervous systems may include a number of different receptor subtypes of both the nicotinic and the muscarinic receptors, this complexity is generally overlooked when assessing the mechanisms of action of pharmaceuticals. Methods We have search of bibliographic databases for peer-reviewed research literature focused on the cholinergic system. Also, we have taken advantage of our expertise in this field to deduce the conclusions of this study. Results Presently, the life cycle of acetylcholine, muscarinic receptors and their effects are reviewed in the major organ systems of the body. Neuronal and non-neuronal sources of acetylcholine are elucidated. Examples of pharmaceuticals, in particular cholinesterase inhibitors, affecting these systems are discussed. The review focuses on salivary glands, the respiratory tract and the lower urinary tract, since the complexity of the interplay of different muscarinic receptor subtypes is of significance for physiological, pharmacological and toxicological effects in these organs. Conclusion Most pharmaceuticals targeting muscarinic receptors are employed at such large doses that no selectivity can be expected. However, some differences in the adverse effect profile of muscarinic antagonists may still be explained by the variation of expression of muscarinic receptor subtypes in different organs. However, a complex pattern of interactions between muscarinic receptor subtypes occurs and needs to be considered when searching for selective pharmaceuticals. In the development of new entities for the treatment of for instance pesticide intoxication, the muscarinic receptor selectivity needs to be considered. Reactivators generally have a muscarinic M2 receptor acting profile. Such a blockade may engrave the situation since it may enlarge the effect of the muscarinic M3 receptor effect. This may explain why respiratory arrest is the major cause for deaths by esterase blocking.

Tacrine was the first drug to be approved for Alzheimer’s disease (AD) treatment, acting as a cholinesterase inhibitor. The neuropathological hallmarks of AD are amyloid-rich senile plaques, neurofibrillary tangles, and neuronal degeneration. The portfolio of currently approved drugs for AD includes acetylcholinesterase inhibitors (AChEIs) and N-methyl-d-aspartate (NMDA) receptor antagonist. Squaric acid is a versatile structural scaffold capable to be easily transformed into amide-bearing compounds that feature both hydrogen bond donor and acceptor groups with the possibility to create multiple interactions with complementary sites. Considering the relatively simple synthesis approach and other interesting properties (rigidity, aromatic character, H-bond formation) of squaramide motif, we combined this scaffold with different tacrine-based derivatives. In this study, we developed 21 novel dimers amalgamating squaric acid with either tacrine, 6-chlorotacrine or 7-methoxytacrine representing various AChEIs. All new derivatives were evaluated for their anti-cholinesterase activities, cytotoxicity using HepG2 cell line and screened to predict their ability to cross the blood-brain barrier. In this contribution, we also report in silico studies of the most potent AChE and BChE inhibitors in the active site of these enzymes., This project was conceived by JMC (IQOG, CSIC, Madrid, Spain), initially carried out experimentally by EM, in Madrid, thanks to a Short-Term Scientific Mission (from 30 January until the 28 April 2017) granted by EU COST Action (CA15135: “Multi-target paradigm for innovative ligand identification in the drug discovery process (MuTaLig)”), and continued in Hradec Kralove (Czech Republic) by EM in collaboration with BS, under the supervision of JK, who organized and planned all the biological analysis and wrote the manuscript. JMC is very thankful to EM, BS, and JK for their collaboration, and to the COST Action CA15135 for supporting this project. The study was supported by a grant of Ministry of Defence “Long Term Development Plan” Medical Aspects of Weapons of Mass Destruction of the Faculty of Military Health Sciences, University of Defence, by the Ministry of Education, Youth and Sports of Czech Republic (project ERDF no. CZ.02.1.01/0.0/0.0/18_069/0010054) and by MH CZ - DRO (University Hospital Hradec Kralove, No. 00179906). JMC thanks MINECO (Government of Spain) (SAF2015-65586-R) for support.

Twenty-one known Amaryllidaceae alkaloids of various structural types and one undescribed alkaloid, named narcimatuline, have been isolated from fresh bulbs of Narcissus pseudonarcissus L. cv. Dutch Master. The chemical structures were elucidated by combination of MS, HRMS, 1D and 2D NMR spectroscopic techniques, and by comparison with literature data. Narcimatuline amalgamates two basic scaffolds of Amaryllidaceae alkaloids in its core, namely galanthamine and galanthindole. All isolated compounds were evaluated for their in vitro acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), prolyl oligopeptidase (POP), and glycogen synthase kinase-3β (GSK-3β) inhibitory activities. The most interesting biological profile was demonstrated by newly isolated alkaloid narcimatuline.

A combination of tacrine and tryptophan led to the development of a new family of heterodimers as multi-target agents with potential to treat Alzheimer's disease. Based on the in vitro biological profile, compound S-K1035 was found to be the most potent inhibitor of human acetylcholinesterase (hAChE) and human butyrylcholinesterase (hBChE), demonstrating balanced IC50 values of 6.3 and 9.1 nM, respectively. For all the tacrine-tryptophan heterodimers, favorable inhibitory effect on hAChE as well as on hBChE was coined to the optimal spacer length ranging from five to eight carbon atoms between these two pharmacophores. S-K1035 also showed good ability to inhibit Aβ42 self-aggregation (58.6 ± 5.1% at 50 μM) as well as hAChE-induced Aβ40 aggregation (48.3 ± 6.3% at 100 μM). The X-ray crystallographic analysis of TcAChE in complex with S-K1035 pinpointed the utility of the hybridization strategy applied and the structures determined with the two K1035 enantiomers in complex with hBChE could explain the higher inhibition potency of S-K1035. Other in vitro evaluations predicted the ability of S-K1035 to cross blood-brain barrier and to exert a moderate inhibition potency against neuronal nitric oxide synthase. Based on the initial promising biochemical data and a safer in vivo toxicity compared to tacrine, S-K1035 was administered to scopolamine-treated rats being able to dose-dependently revert amnesia.

Three new alkaloids, bersavine (3), muraricine (4), and berbostrejdine (8), together with seven known isoquinoline alkaloids (1-2, 5-7, 9, and 10) were isolated from an alkaloidal extract of the root bark of Berberis vulgaris. The structures of the isolated compounds were determined by spectroscopic methods, including 1D and 2D NMR techniques, HRMS, and optical rotation, and by comparison of the obtained data with those in the literature. The NMR data of berbamine (5), aromoline (6), and obamegine (7) were completely assigned employing 2D NMR experiments. Alkaloids isolated in sufficient amounts were evaluated for their in vitro acetylcholinesterase, butyrylcholinesterase (BuChE), prolyl oligopeptidase, and glycogen synthase kinase-3β inhibitory activities. Selected compounds were studied for their ability to permeate through the blood-brain barrier. Significant human BuChE ( hBuChE) inhibitory activity was demonstrated by 6 (IC50 = 0.82 ± 0.10 μM). The in vitro data were further supported by computational analysis that showed the accommodation of 6 in the active site of hBuChE.

Twenty known Amaryllidaceae alkaloids of various structural types, and one undescribed alkaloid of narcikachnine-type, named narcieliine (3), have been isolated from fresh bulbs of Zephyranthes citrina. The chemical structures of the isolated alkaloids were elucidated by a combination of MS, HRMS, 1D and 2D NMR, and CD spectroscopic techniques, and by comparison with literature data. The absolute configuration of narcieliine (3) has also been determined. Compounds isolated in a sufficient quantity were evaluated for their in vitro acetylcholinesterase (AChE; E.C. 3.1.1.7), butyrylcholinesterase (BuChE; E.C. 3.1.1.8), and prolyl oligopeptidase (POP; E.C. 3.4.21.26) inhibition activities. Significant human AChE/BuChE (hAChE/hBuChE) inhibitory activity was demonstrated by the newly described alkaloid narcieliine (3), with IC50 values of 18.7 ± 2.3 µM and 1.34 ± 0.31 µM, respectively. This compound is also predicted to cross the blood–brain barrier (BBB) through passive diffusion. The in vitro data were further supported by in silico studies of 3 in the active site of hAChE/hBuChE.

Irreversible inhibition of acetylcholinesterase (AChE) by organophosphates leads to many failures in living organism and ultimately in death. Organophosphorus compounds developed as nerve agents such as tabun, sarin, soman, VX and others belong to the most toxic chemical warfare agents and are one of the biggest threats to the modern civilization. Moreover, misuse of nerve agents together with organophosphorus pesticides (e.g. malathion, paraoxon, chlorpyrifos, etc.) which are annually implicated in millions of intoxications and hundreds of thousand deaths reminds us of insufficient protection against these compounds. Basic treatments for these intoxications are based on immediate administration of atropine and acetylcholinesterase reactivators which are currently represented by mono- or bis-pyridinium aldoximes. However, these antidotes are not sufficient to ensure 100 % treatment efficacy even they are administered immediately after intoxication, and in general, they possess several drawbacks. Herein, we have reviewed new efforts leading to the development of novel reactivators and proposition of new promising strategies to design novel and effective antidotes. Structure-activity relationships and biological activities of recently proposed acetylcholinesterase reactivators are discussed and summarized. Among further modifications of known oximes, the main attention has been paid to dual binding site ligands of AChE as the current mainstream strategy. We have also discussed new chemical entities as potential replacement of oxime functional group.

Alzheimer's disease is a multifactorial disease that is characterized mainly by Amyloid-β (A-β) deposits, cholinergic deficit and extensive metal (copper, iron)-induced oxidative stress. In this work we present details of the synthesis, antioxidant and copper-chelating properties, DNA protection study, cholinergic activity and amyloid-antiaggregation properties of new multifunctional tacrine-7-hydroxycoumarin hybrids. The mode of interaction between copper(II) and hybrids and interestingly, the reduction of Cu(II) to Cu(I) species (for complexes Cu-5e-g) were confirmed by EPR measurements. EPR spin trapping on the model Fenton reaction, using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap, demonstrated a significantly suppressed formation of hydroxyl radicals for the Cu-5e complex in comparison with free copper(II). This suggests that compound 5e upon coordination to free copper ion prevents the Cu(II)-catalyzed decomposition of hydrogen peroxide, which in turn may alleviate oxidative stress-induced damage. Protective activity of hybrids 5c and 5e against DNA damage in a Fenton system (copper catalyzed) was found to be in excellent agreement with the EPR spin trapping study. Compound 5g was the most effective in the inhibition of acetylcholinesterase (hAChE, IC50=38nM) and compound 5b was the most potent inhibitor of butyrylcholinesterase (hBuChE, IC50=63nM). Compound 5c was the strongest inhibitor of A-β1-40 aggregation, although a significant inhibition (>50%) was detected for compounds 5b, 5d, 5e and 5g. Collectively, these results suggest that the design and investigation of multifunctional agents containing along with the acetylcholinesterase inhibitory segment also an antioxidant moiety capable of alleviating metal (copper)-induced oxidative stress, may be of importance in the treatment of Alzheimer's disease.

Berberine, a naturally occurring compound, possesses an interesting multipotent pharmacological profile potentially applicable for Alzheimer’s disease (AD) treatment. In this study, a series of novel 22 berberine derivatives was developed and tested in vitro. Berberine core was substituted at position 9-O of its aromatic ring region. All the hybrids under the study revealed multi-targeted profile inhibiting prolyl oligopeptidase, acetylcholinesterase and butyrylcholinesterase highlighting 4a, 4g, 4j, 4l and 4s possessing balanced activities in the micromolar range. The top-ranked candidates in terms of the most pronounced potency against POP, AChE and BChE can be classified as 4d, 4u and 4v, bearing 4-methylbenzyl, (naphthalen-2-yl)methylene and 1-phenoxyethyl moieties, respectively. In vitro data were corroborated by detailed kinetic analysis of the selected lead molecules. 4d, 4u and 4v were also inspected for their potential to inhibit aggregation of two abberant proteins in AD, namely amyloid beta and tau, indicating their potential disease-modifying properties. To explain the results of our study, we carried out docking simulation to the active sites of the respective enzyme with the best berberine derivatives, along with QSAR study. We also investigated compounds’ potential permeability through blood-brain barrier by applying parallel artificial membrane permeation assay and addressed their cytotoxicity profile.

The acetylcholinesterase (AChE) reactivators (e.g., obidoxime, asoxime) became an essential part of organophosphorus (OP) poisoning treatment, together with atropine and diazepam. They are referred to as a causal treatment of OP poisoning, because they are able to split the OP moiety from AChE active site and thus renew its function. In this approach, fifteen novel AChE reactivators were determined. Their molecular design originated from former K-oxime compounds K048 and K074 with remaining oxime part of the molecule and modified part with heteroarenium moiety. The novel compounds were prepared, evaluated in vitro on human AChE (HssAChE) inhibited by tabun, paraoxon, methylparaoxon or DFP and compared to commercial HssAChE reactivators (pralidoxime, methoxime, trimedoxime, obidoxime, asoxime) or previously prepared compounds (K048, K074, K075, K203). Some of presented oxime reactivators showed promising ability to reactivate HssAChE comparable or higher than the used standards. The molecular modelling study was performed with one compound that presented the ability to reactivate GA-inhibited HssAChE. The SAR features concerning the heteroarenium part of the reactivator&rsquo, s molecule are described.

Background: Based on the prevalence studies, the number of people suffering from dementia will almost double every 20 years, to 65.7 million in 2030 and 115.4 million in 2050, assuming no changes in mortality, effective preventative measures, definitive diagnostic guidelines or curative treatment. From the abovementioned epidemiological data, it is obvious that dementia constitutes a major public health problem not only at present, but unfortunately also in the future. Objectives and Methods: Several N-alkylated tacrine (THA) derivatives have already been synthesized by Pomponi et al., in 1997. However, these compounds were tested for their anti-AChE activity using enzyme isolated from Electrophorus electricus. For this reason, we have decided to extend the previously reported series of THA derivatives and consequently test them in the battery of experiments, the results of which have served to more relevant evaluation of these compounds from the perspective of Alzeimer´s disease compared to that published by Pomponi. Results and Conclusion: In summary, all compounds of interest effectively inhibited ChEs in vitro. One of the most promising derivatives 8 bearing an N-octyl chain showed 2.5-fold higher AChE inhibitory activity in relation to tacrine. With respect to blood-brain barrier (BBB) penetration, it can be claimed that synthesized analogues are presumably able to cross the BBB. From the point of view of hepatotoxicity, selected Nalkylated tacrine derivatives exerted worse results compared to tacrine. However, in vitro results are only illustrative, therefore, only in vivo experiments could determine the real value of selected N-alkylated THA derivatives.

Nerve agents and oxon forms of organophosphorus pesticides act as strong irreversible inhibitors of two cholinesterases in the human body: acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BChE; EC 3.1.1.8), and are therefore highly toxic compounds. For the recovery of inhibited AChE, antidotes from the group of pyridinium or bispyridinium aldoxime reactivators (pralidoxime, obidoxime, HI-6) are used in combination with anticholinergics and anticonvulsives. Therapeutic efficacy of reactivators (called “oximes”) depends on their chemical structure and also the type of organophosphorus inhibitor. Three novel oximes (K131, K142, K153) with an oxime group in position four of the pyridinium ring were designed and then tested for their potency to reactivate human (Homo sapiens sapiens) AChE (HssACHE) and BChE (HssBChE) inhibited by the pesticide paraoxon (diethyl 4-nitrophenyl phosphate). According to the obtained results, none of the prepared oximes were able to satisfactorily reactivate paraoxon-inhibited cholinesterases. On the contrary, extraordinary activity of obidoxime in the case of paraoxon-inhibited HssAChE reactivation was confirmed. Additional docking studies pointed to possible explanations for these results.

Background: The design of new heterodimeric dual binding site acetylcholinesterase inhibitors constitutes the main goal-directed to the development of new anticholinesterase agents with the expanded pharmacological profile. Multi-target compounds are usually designed by combining in a hybrid molecule with two or more pharmacophoric moieties that are known to enable interaction with the selected molecular targets. Methods: All compounds were tested for their inhibitory activity on human AChE/BChE. The Ellman´s method was used to determine inhibition kinetics and IC50 values. In order to predict passive bloodbrain penetration of novel compounds, modification of the parallel artificial membrane permeation assay has been used. Docking studies were performed in order to predict the binding modes of new hybrids with hAChE/ hBChE respectively. Results: In this study, we described the design, synthesis, and evaluation of series tacrine-coumarin and tacrine-quinoline compounds which were found to show potential inhibition of ChEs and penetration of the blood-brain barrier. Conclusion: Tacrine-quinoline hybrids 7a exhibited the highest activity towards hBChE (IC50 = 0.97 µmol) and 7d towards hAChE (IC50 = 0.32 µmol). Kinetic and molecular modelling studies revealed that 7d was a mixed-type AChE inhibitor (Ki = 1.69 µmol) and 7a was a mixed-type BChE inhibitor (Ki = 1.09 µmol). Moreover, hybrid 5d and 7c could penetrate the CNS.

This publication was supported by the project Czech National Institute of Mental Health (NIMH – CZ; no. ED2.1.00/03.0078), Ministry of Education, Youth and Sports of the Czech Republic (no. LD14009), MH CZ - DRO (UHHK, 00179906), the University of Defence of the Czech Republic (long term development plan), MINECO (Government of Spain; Grant SAF2012-33304), the School of Biology at the University of St Andrews, and by COST Action CM1103. In this study, novel indolotacrine analogues have been designed, synthesized and evaluated as potential drugs for the treatment of Alzheimer’s disease. Based on a multi-target-directed ligand approach, novel compounds have been designed to act simultaneously as cholinesterase and monoamine oxidase (MAO) inhibitors. Prepared compounds were also evaluated for their antioxidant, cytotoxic, hepatotoxic and permeability (Blood-Brain Barrier penetration) properties. Indolotacrine 9b (9-methoxy-2,3,4,6-tetrahydro-1H-indolo[2,3-b]quinolin-11-amine) showed the most promising results in the in vitro assessment being a potent inhibitor of acetylcholinesterase (IC50 = 1.5 µM), butyrylcholinesterase (IC50 = 2.4 µM) and monoamine oxidase A (IC50 = 0.49 µM) and a weak inhibitor of monoamine oxidase B (IC50 = 53.9 µM). Although its cytotoxic (IC50 = 5.5 ± 0.4 µM) and hepatotoxic (IC50 = 1.22 ± 0.11 µM) profile is not as good as the standard 7-methoxytacrine (IC50 = 63 ± 4 µM and IC50 = 11.50 ± 0.77 µM respectively), the overall improvement in the inhibitory activities and potential to cross blood-brain barrier make indolotacrine 9b a promising lead compound for further development and investigation. Postprint

A novel series of 6-chlorotacrine-scutellarin hybrids was designed, synthesized and the biological activity as potential anti-Alzheimer’s agents was assessed. Their inhibitory activity towards human acetylcholinesterase (hAChE) and human butyrylcholinesterase (hBChE), antioxidant activity, ability to cross the blood-brain barrier (BBB) and hepatotoxic profile were evaluated in vitro. Among these compounds, hybrid K1383, bearing two methylene tether between two basic scaffolds, was found to be very potent hAChE inhibitor (IC50 = 1.63 nM). Unfortunately, none of the hybrids displayed any antioxidant activity (EC50 ≥ 500 μM). Preliminary data also suggests a comparable hepatotoxic profile with 6-Cl-THA (established on a HepG2 cell line). Kinetic studies performed on hAChE with the most active compound in the study, K1383, pointed out to a mixed, non-competitive enzyme inhibition. These findings were further corroborated by docking studies.

organophosphorus compounds act as irreversible inhibitors of the vital enzyme acetylcholinesterase (AChE). this leads in the accumulation of acetylcholine (ACh) leading to cholinergic crisis and death. The main therapeutic approach is based on immediate administration of an ache reactivator as an antidote enabling recovery of the ache function. Areas covered: This review covers the development of AChE reactivators in order to introduce a new efficient drug that will overcome significant failures of common antidotes. Further options together with methods of detection are also discussed in order to assure a complete insight into the treatment of intoxication. Expert opinion: Since organophosphates belong to the most toxic chemical warfare agents, efficient antidotes are a matter of importance. The solution of how to limit the basic drawbacks of clinically used reactivators remained a spotlight for many researches worldwide. Recent strategies of the treatment of OP exposure bring us new possibilities which may overcome classic antidotes. The importance of detection of OP also has to be taken into consideration. Especially, with the fast spreading toxic effect when death can occur within minutes.

A novel series of acridine-coumarin hybrids was synthesized and biologically evaluated for their potential inhibitory effect on both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The newly synthesized derivatives 9a-d have shown higher activity against human AChE (hAChE) compared with 7-MEOTA as the standard drug. Among them derivative 9b exhibited the most potent acetylcholinesterase inhibitory activity, with an IC50 value of 5.85μM compared with 7-MEOTA (IC50=15μM). Molecular modelling studies were performed to predict the binding modes of compounds 9b, 9c and 9f with hAChE/hBuChE.

Two series of non-symmetrical bisquaternary pyridinium–quinolinium and pyridinium–isoquinolinium compounds were prepared as molecules potentially applicable in myasthenia gravis treatment. Their inhibitory ability towards human recombinant acetylcholinesterase and human plasmatic butyrylcholinesterase was determined and the results were compared to the known effective inhibitors such as ambenonium dichloride, edrophonium bromide and experimental compound BW284C51. Two compounds, 1-(10-(pyridinium-1-yl)decyl)quinolinium dibromide and 1-(12-(pyridinium-1-yl)dodecyl)quinolinium dibromide, showed very promising affinity for acetylcholinesterase with their IC50 values reaching nM inhibition of acetylcholinesterase. These most active compounds also showed satisfactory selectivity towards acetylcholinesterase and they seem to be very promising as leading structures for further modifications and optimization. Two of the most promising compounds were examined in the molecular modelling study in order to find the possible interactions between the ligand and tested enzyme.

Novel multifunctional tacrines for Alzheimer's disease were obtained by Ugi-reaction between ferulic (or lipoic acid), a melatonin-like isocyanide, formaldehyde, and tacrine derivatives, according to the antioxidant additive approach in order to modulate the oxidative stress as therapeutic strategy. Compound 5c has been identified as a promising permeable agent showing excellent antioxidant properties, strong cholinesterase inhibitory activity, less hepatotoxicity than tacrine, and the best neuroprotective capacity, being able to significantly activate the Nrf2 transcriptional pathway., B.R. and L.I. thank the Regional Council of Franche-Comté (France) for financial support, and M.-J. Henriot (PHV Pharma) for support in the HPLC analyses. M.B. thanks the Regional Council of Franche-Comté(France) for a Ph.D. grant. J.E. has a grant from ISCIII (Programa Miguel Servet, CP14/00008). D.J., J.J., V.S., and O.S. thank Jitka Pichova for a skillful technical assistance. This work was supported by the project MH CZ-DRO (UHHK, 00179906) and by Long Term Development Plan-1011 of Faculty of Military Health Sciences, University of Defence (Czech Republic). R.L. thanks ISCIII (Programa Miguel Servet, CP11/00165; grant PI14/00372), Bayer AG “From targets to novel drugs” (grant 2015-03-1282) and Fundación FIPSE (grant 12-00001344-15). P.M. thanks MECD for a FPU fellowship (FPU13/03737).

Alzheimer’s disease (AD) is a debilitating progressive neurodegenerative disorder that ultimately leads to the patient’s death. Despite the fact that novel pharmacological approaches endeavoring to block the neurodegenerative process are still emerging, none of them have reached use in clinical practice yet. Thus, palliative treatment represented by acetylcholinesterase inhibitors (AChEIs) and memantine are still the only therapeutics used. Following the multi-target directed ligands (MTDLs) strategy, herein we describe the synthesis, biological evaluation and docking studies for novel 7-methoxytacrine-p-anisidine hybrids designed to purposely target both cholinesterases and the amyloid cascade. Indeed, the novel derivatives proved to be effective non-specific cholinesterase inhibitors showing non-competitive AChE inhibition patterns. This compounds’ behavior was confirmed in the subsequent molecular modeling studies.

A new tacrine based cholinesterase inhibitor, N-(bromobut-3-en-2-yl)-7-methoxy-1,2,3,4-tetrahydroacridin-9-amine (1), was designed and synthesized to interact with specific regions of human acetylcholinesterase and human butyrylcholinesterase. Its inhibitory ability towards cholinesterases was determined and compared to tacrine (THA) and 9-amino-7-methoxy-1,2,3,4-tetrahydroacridine (7-MEOTA). The assessment of IC50 values revealed 1 as a weak inhibitor of both tested enzymes.

Existence of alternative entrances in acetylcholinesterase (AChE) could explain the contrast between the very high AChE catalytic efficiency and the narrow and long access path to the active site revealed by X-ray crystallography. Alternative entrances could facilitate diffusion of the reaction products or at least water and ions from the active site. Previous molecular dynamics simulations identified side door and back door as the most probable alternative entrances. The simulations of non-inhibited AChE suggested that the back door opening events occur only rarely (0.8% of the time in the 10ns trajectory). Here we present a molecular dynamics simulation of non-inhibited AChE, where the back door opening appears much more often (14% of the time in the 12ns trajectory) and where the side door opening was observed quite frequently (78% of trajectory time). We also present molecular dynamics, where the back door does not open at all, or where large conformational changes of the AChE omega loop occur together with alternative passage opening events. All these differences in AChE dynamical behavior are caused by different protonation states of two glutamate residues located on bottom of the active site gorge (Glu202 and G450 in Mus musculus AChE). Our results confirm the results of previous molecular dynamics simulations, expand the view and suggest the probable reasons for the overall conformational behavior of AChE omega loop.

Butyrylcholinesterase is considered to be an endogenous stoichiometric bioscavenger of organophosphorus compounds (OPs), but due to limited concentration of BChE in the organism, stoichiometric reduction of OP is not always sufficient. This can be improved by creating a pseudo-catalytic scavenger adding oximes as reactivators of inhibited exogenous BChE. In order to improve the BChE bioscavenging function in tabun or paraoxon poisoning, we tested in vitro reactivation of phosphorylated human plasma BChE by bispyridinium oximes varying in the length and type of the linker between rings, and in the position of the oxime group on the ring. Among the tested oximes, the most potent reactivators of tabun-inhibited BChE were K117 [1,1′-(2,2′-oxybis(ethane-2,1-diyl))bis(4-hydroxyiminomethyl pyridinium) bromide] and K127 [4-carbamoyl-1-(2-(2-(4-(hydroxyiminomethyl) pyridinium-1-yl)ethoxy)ethyl)pyridinium bromide]. Reactivation by these oximes (1 mM) reached about 50% of control activity after only 20 min; however, reactivation stopped at 70%. Reactivation of paraoxon-inhibited BChE by all of the selected oximes was slow. Using molecular mechanics, we performed docking of the oximes to tabun-inhibited BChE in order to discuss possible structural modifications of bispyridinium oximes to improve reactivation of phosphorylated BChE.

Obidoxime, a well-known bis-pyridinium reactivator, is often the preferred antidote of organophosphorus poisoning caused by pesticides and tabun. It is also considered to be an allosteric modulator of muscarinic receptors, preferably M2 sub-type. This study compared the effect of obidoxime and atropine in vivo and in vitro on the cholinergic stimulation of the rat heart (M2) and the urinary bladder (M3). The results showed that obidoxime exerts anti-muscarinic effects, that may play an important role in the treatment of organophosphourus poisoning, and that the muscarinic receptor inhibition profile shows M2 receptor selectivity. This anti-muscarinic effect is much smaller that the effect of atropine and might be due to the allosteric inhibition of the receptors. The results also indicate that the acetylcholinesterase inhibition and the muscarinic receptor antagonism occur at different concentrations and dose levels.

Newly developed acetylcholinesterase reactivators K117 [1,5-bis(4-hydroxyiminomethylpyridinium)-3-oxapentane dichloride] and K127 [(1-(4-hydroxyiminomethylpyridinium)-5-(4-carbamoylpyridinium)-3-oxapentane dibromide)] were tested for their potency to reactivate tabun-inhibited human brain cholinesterases. Pralidoxime and trimedoxime were chosen as standard reference reactivators. Human tissue was used, as that was closer on the real treatment of human beings. As a result, oxime K127 was found as the best tested reactivator according to the constant kr, characterizing the overall reactivation process. On the contrary, the maximal reactivation ability expressed as percentage of reactivation was the best for trimedoxime. This differences were caused as a result of using the enzyme from different species. Due to this, experiments on human tissue should be conducted after in vitro and in vivo tests on animals to eliminate such important failures of promising oximes.

Organophosphorus pesticides and nerve agents are highly toxic to humans and other living organisms, primarily because of their interaction with enzyme acetylcholinesterase. The aim of our study was to find suitable reactivators of acetylcholinesterase and butyrylcholinesterase and to recommend the most efficacious compounds for the next evaluation as antidotes for intoxication by pesticides.Eighteen structurally different oxime reactivators were tested for their in vitro ability to reactivate paraoxon-inhibited human erythrocyte acetylcholinesterase and human plasma butyrylcholinesterase to find out structure-activity relationship within this set of compounds. Their reactivation ability was compared with commercially available acetylcholinesterase reactivators (pralidoxime, methoxime, trimedoxime, obidoxime, and HI-6).The best reactivation ability was achieved with obidoxime, trimedoxime, compounds K027, K075, K203, and K048. We have also tested reactivation of butyrylcholinesterase with the aim to recommend an efficient reactivator, able to perform a "pseudo catalytic" bioscavenger with butyrylcholinesterase, which is developed as new antidote of organophosphate poisonings. Such combination could allow an enhancement of prophylactic and therapeutic efficiency of administered enzyme. Compounds K117, K269, K075, and trimedoxime were found to be the most potent reactivators of inhibited butyrylcholinesterase.In this work, we have evaluated only reactivation of paraoxon-inhibited cholinesterases. To get better understanding of this problem, a larger number of organophosphorus inhibitors should be used.

Acetylcholinesterase reactivators are crucial antidotes for the treatment of organophosphate intoxication. Eighteen monoquaternary reactivators of acetylcholinesterase with modified side chain were developed in an effort to extend the properties of pralidoxime. The known reactivators (pralidoxime, HI-6, obidoxime, trimedoxime, methoxime) and the prepared compounds were tested in vitro on a model of tabun- and paraoxon-inhibited AChE. Monoquaternary reactivators were not able to exceed the best known compounds for tabun poisoning, but some of them did show reactivation better or comparable with pralidoxime for paraoxon poisoning. However, extensive differences were found by a SAR study for various side chains on the non-oxime part of the reactivator molecule.

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 microM), 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 microM. 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.

Acetylcholinesterase (AChE, EC 3.1.1.7) is an important enzyme for cholinergic nerve transmission. The action of toxic organophosphates such as nerve agents is based on AChE inhibition. The death following acute nerve agent poisoning is due to central or peripheral respiratory/cardiac failure. Therefore, the changes in AChE activity following nerve agents acting predominantly on the central (sarin, soman) or peripheral (VX) level were studied. It is known that AChE activity in different structures exists in relative excess. Female Wistar rats intoxicated with sarin, soman, and VX in different doses (0.5–2.0 × LD 50 ) were divided into groups of survived and died animals. AChE activities in diaphragm, brain parts (pontomedullar area, frontal cortex, basal ganglia, in some cases other parts of the brain) were determined and the rest of activity (in %) was correlated with survival/death of animals. More precise elucidation of action of nerve agents and the assessment of minimal AChE activity in different organs compatible with the survival of organism poisoned with nerve agents were the aims of this study.

Class of monoquaternary pyridinium oximes was in vitro tested as potential reactivators of acetylcholinesterase (AChE; EC 3.1.1.7) inhibited by nerve agent sarin. Human brain homogenate was used as an appropriate source of cholinesterases. Reactivation potency of novel oximes was compared with currently available reactivators - pralidoxime, obidoxime, and HI-6. According to the obtained results, only five reactivators were able to satisfactorily renew cholinesterase potency (pralidoxime, obidoxime, HI-6, 4-PAM, and K119). Unfortunately, none of the novel tested reactivators surpassed the reactivation potency of the currently most promising reactivator, HI-6. This study shows that monoquaternary reactivators are unable to reactivate nerve agent-inhibited AChE. Due to this, in future, only bisquaternary compounds derived from HI-6 or obidoxime should be designed as new potential cholinesterase reactivators.

Acetylcholinesterase reactivators are crucial antidotes for the treatment of organophosphate intoxication. Fifteen new monooxime reactivators of acetylcholinesterase with a (E)-but-2-ene linker were developed in an effort to extend the properties of K-oxime (E)-1-(4-carbamoylpyridinium)-4-(4-hydroxyiminomethylpyridinium)-but-2-ene dibromide (K203). The known reactivators (pralidoxime, HI-6, obidoxime, K075, K203) and the new compounds were tested in vitro on a model of tabun- and paraoxon-inhibited AChE. Monooxime reactivators were not able to exceed the best known compounds for tabun poisoning, but some of them did show reactivation comparable with known compounds for paraoxon poisoning. However, extensive differences were found by a SAR study for various substitutions on the non-oxime part of the reactivator molecule.

Acetylcholinesterase reactivators are crucial antidotes for the treatment of organophosphate intoxication. Among the organophosphates, with the exception of soman, tabun (GA) intoxications are the least responsive to treatment with commercially available therapeutics. A rational design was used to increase reactivation ability and decrease the toxicity of the novel reactivator. (E)-1-(4-carbamoylpyridinium)-4-(4-hydroxyiminomethylpyridinium)-but-2-ene dibromide (K203) has better properties than previously tested compounds in vitro and, therefore, is a potential candidate for the treatment of GA intoxication in vivo.

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.

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.

Acetylcholinesterase activity in defined brain regions was determined using biochemical and histochemical methods 30 min after treating rats with sarin, soman or VX (0.5 × LD50). Enzyme inhibition was high in the pontomedullar area and frontal cortex, but was low in the basal ganglia. Histochemical and biochemical results correlated well. Determination of the activity in defined brain structures was a more sensitive parameter than determination in whole brain homogenate where the activity was a “mean” of the activities in different structures. The pontomedullar area controls respiration, so that the special sensitivity of acetylcholinesterase to inhibition by nerve agents in this area is important for understanding the mechanism of death caused by nerve agents. Thus, acetylcholinesterase activity is the main parameter investigated in studies searching for target sites following nerve agent poisoning.