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Development of Small Bisquaternary Cholinesterase Inhibitors As Drugs for Pre-Treatment of Nerve Agent Poisonings

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Development of Small Bisquaternary Cholinesterase Inhibitors As Drugs for Pre-Treatment of Nerve Agent Poisonings Journal name: Drug Design, Development and Therapy Article Designation: Original Research Year: 2018 Volume: 12 Drug Design, Development and Therapy Dovepress Running head verso: Kuca et al Running head recto: Small bisquaternary cholinesterase inhibitors open access to scientific and medical research DOI: 133038 Open Access Full Text Article ORIGINAL RESEARCH Development of small bisquaternary cholinesterase inhibitors as drugs for pre-treatment of nerve agent poisonings Kamil Kuca1,2 Background: Intoxication by nerve agents could be prevented by using small acetylcholin- Jana Zdarova Karasova2,3 esterase inhibitors (eg, pyridostigmine) for potentially exposed personnel. However, the serious Ondrej Soukup2 side effects of currently used drugs led to research of novel potent molecules for prophylaxis Jiri Kassa3 of organophosphorus intoxication. Eva Novotna2 Methods: The molecular design, molecular docking, chemical synthesis, in vitro methods Vendula Sepsova2,3 (enzyme inhibition, cytotoxicity, and nicotinic receptors modulation), and in vivo methods (acute toxicity and prophylactic effect) were used to study bispyridinium, bisquinolinium, bisisoqui- Anna Horova2 nolinium, and pyridinium-quinolinium/isoquinolinium molecules presented in this study. Jaroslav Pejchal3 Results: The studied molecules showed non-competitive inhibitory ability towards human 2,3 Martina Hrabinova acetylcholinesterase in vitro that was further confirmed by molecular modelling studies. Several 2 Eva Vodakova compounds were selected for further studies. First, their cytotoxicity, nicotinic receptors modula- Daniel Jun2,3 tion, and acute toxicity (lethal dose for 50% of laboratory animals [LD50]; mice and rats) were 1,2 Eugenie Nepovimova tested to evaluate their safety with promising results. Furthermore, their blood levels were Martin Valis4 measured to select the appropriate time for prophylactic administration. Finally, the protective Kamil Musilek1,2 ratio of selected compounds against soman-induced toxicity was determined when selected 1Department of Chemistry, Faculty compounds were found similarly potent or only slightly better to standard pyridostigmine. of Science, University of Hradec Conclusion: The presented small bisquaternary molecules did not show overall benefit in 2 Kralove, Biomedical Research prophylaxis of soman-induced in vivo toxicity. Center, University Hospital Hradec Kralove, 3Department of Toxicology Keywords: AChE inhibitors, prophylaxis, pre-treatment, nerve agents, toxicity, soman and Military Pharmacy, Faculty of Military Health Sciences, University of Defence, 4Department of Neurology, Introduction University Hospital Hradec Kralove, Organophosphorus compounds (OP) are widely used (eg, in agriculture as pesticides, Hradec Kralove, Czech Republic in industry as softening agents). Some of them were, unfortunately, developed for military purposes as chemical warfare agents. Sarin, soman or VX belong to the most known nerve agents (NA), especially among OP.1 NA mechanism of action is based on its ability to inhibit the enzyme acetylcholinesterase (AChE; EC 3.1.1.7) at peripheral and central cholinergic synapses. Afterward, generalized cholinergic crisis occurs and in severe cases may lead to death.2 To prevent the deleterious effect of NA, several prophylactic approaches were formerly developed.3 AChE protection against the OP using reversible cholinesterase (ChE) inhibitors is the oldest approach for ChE pre-treatment. In this case, the non-competitive or competitive Correspondence: Kamil Kuca reversible inhibitors of AChE are used to protect the irreversible binding of NA, and thus Biomedical Research Center, University Hospital Hradec Kralove, Sokolska 581, protect AChE function until the most of OP is biodegraded. This effect cannot be expected 500 05 Hradec Kralove, Czech Republic for administration of AChE reactivators (oximes) that are known to be very poor AChE Tel +420 603 289 166 Email [email protected] inhibitors.4 Recently, enzymes scavenging or hydrolyzing OP are highly investigated.5,6 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2018:12 505–512 505 Dovepress © 2018 Kuca et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you http://dx.doi.org/10.2147/DDDT.S133038 hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Kuca et al Dovepress 1 2 1 1 1 1 2 1 +21 12+ +21 12+ &O ; ; 6$' WULPHGR[LPH RELGR[LPH Figure 1 Bisquaternary compounds tested or used for organophosphorus protection or reactivation. During last decade, some attention was turned back to 4-positioned pyridinium analogs bridged also by varying the reversible ChE inhibitors including SAD-128, a bisqua- linkage (Figure 3).15,16 ternary compound, which was considered as pre-treatment The design of presented molecules originated from known drug candidate in eighties (Figure 1).7–10 In fact, it was the first compounds (eg, ambenonium dichloride, BW284c51 or bisquaternary compound used for this purpose and lacking SAD-128) that were known for excellent or selective cholinest- oxime moiety that is common for bisquaternary reactivators erase inhibition in vitro (ambenonium dichloride, BW284c51) (eg, trimedoxime, obidoxime). The major reason for use of or in vivo (SAD-128). The heteroaromatic moieties were charged reversible AChE inhibitor was challenge of rapidly used to cover π-cationic interactions with the aromatic amino aging organophosphates (eg, soman), which are very difficult acid residues in the cholinesterase binding pocket (especially to treat after intoxication, but might be prevented prior to AChE). The varying linkage was adapted to possibly connect intoxication by combining peripherally and centrally acting the AChE cation–π binding site with the peripheral aromatic reversible AChE inhibitor. In this case, the charged inhibitor binding site. In this case, the better reversible inhibition of would serve as a peripherally acting one. With this hypoth- AChE was hypothesized for some of the chosen linkers (one esis, several novel series of bisquaternary compounds were to 12 methylene units). Some heteroatoms, double bonds or prepared and evaluated (Figures 2 and 3). In this article, we aromatic rings were also introduced to the linkage to improve would like to summarize results, which were obtained by our the other inhibitor-enzyme interactions within the AChE bind- group within the last seven years for bisquaternary reversible ing pocket including π-π interactions (double bonds, aromatic AChE inhibitors with possible impact on protection against rings) of hydrogen bonding (ether fragment). OP compounds. The whole summary is divided into several The synthesis of presented molecules was fairly simple sections that are presented consecutively step by step such (Figure 4). The heteroaromatic compound was dissolved in as in the development process: synthesis, in vitro evaluation, dimethylformamide (DMF) or acetonitrile (MeCN) upon cytotoxicity, nicotinic receptor evaluation, toxicity, pharma- addition of dihalogenated linking molecule and stirred cokinetics and in vivo evaluation of the prophylactic effect. at about 70°C for several hours/days. The products were obtained in good yield and purity, which was checked by Design and synthesis standard techniques (NMR, ESI-MS).17 The bisquaternary compounds were designed as simple molecules with two pyridinium, quinolinium, isoquinolinium In vitro screening, molecular docking and or mixed pyridinium-quinolinium/isoquinolinium moieties structure–activity relationship connected by variety of linkers (Figure 2).11–14 The latter The in vitro screening of prepared molecules was done on compounds were derived from 4-(tert-butyl)pyridinium or human recombinant AChE and human plasmatic BChE 1 $ 1 1 $ 1 1 $ 1 1 $ 1 ; ; ; ; ; &O%U 1 $ 1 $ &+ ± (= &+&+ &+&+ &+ ±2 &+ ± ; Figure 2 Bispyridinium, bisquinolinium, bisisoquinolinium and pyridinium-quinolinium/isoquinolinium compounds designed as small cholinesterase inhibitors. 506 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2018:12 Dovepress Dovepress Small bisquaternary cholinesterase inhibitors 5 1 1 $ 1 1 ± ± ; ; 5 ; &O%U 5 0H(WWHUW%XW3K%Q &+ 3K $ &+ ± (= &+&+ &+&+ &+ ±2 &+ ± 12%Q2+&+2+ &+ 2+ 1 &+ &+ 12+&2&+&22+ &22&+&21+&1 Figure 3 4-(tert-butyl)pyridinium or 4-positioned pyridinium analogs designed as small cholinesterase inhibitors. (hBChE) to exclude the possible interspecies differences. The non-competitive binding. The symmetrical molecules half maximal inhibitory concentration (IC50) was determined (K298, K344, K474, K524) presented closure of hAChE for all molecules and the selected compounds were further gorge entrance via π-π or π-cationic interactions, where tested in kinetic experiments in vivo. The IC50 data were one heteroarenium moiety was positioned into the gorge compared to pattern bisquaternary molecules with similar and the second one was located outside the gorge close to mechanism of action (ambenonium dichloride, BW284c51 its entrance.11–13 Differently,
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