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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.

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

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

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.

Prolyl oligopeptidase is a cytosolic serine peptidase that hydrolyses proline-containing peptides at the carboxy terminus of proline residues. It has been associated with schizophrenia, bipolar affective disorder, and related neuropsychiatric disorders and therefore may have important clinical implications. Thirty-one isoquinoline alkaloids of various structural types, previously isolated in our laboratory, were screened for their ability to inhibit prolyl oligopeptidase. Promising results have been showed by alkaloids californidine (IC50=55.6±3.5 μM), dihydrosanquinarine (IC50=99.1±7.6 μM), corypalmine (IC50=128.0±10.5 μM) and N-methyllaurotetanine (IC50=135.0±11.7 μM).

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.

Human 17&beta, hydroxysteroid dehydrogenase type 10 is a multifunctional protein involved in many enzymatic and structural processes within mitochondria. This enzyme was suggested to be involved in several neurological diseases, e.g., mental retardation, Parkinson&rsquo, s disease, or Alzheimer&rsquo, s disease, in which it was shown to interact with the amyloid-beta peptide. We prepared approximately 60 new compounds based on a benzothiazolyl scaffold and evaluated their inhibitory ability and mechanism of action. The most potent inhibitors contained 3-chloro and 4-hydroxy substitution on the phenyl ring moiety, a small substituent at position 6 on the benzothiazole moiety, and the two moieties were connected via a urea linker (4at, 4bb, and 4bg). These compounds exhibited IC50 values of 1&ndash, 2 &mu, M and showed an uncompetitive mechanism of action with respect to the substrate, acetoacetyl-CoA. These uncompetitive benzothiazolyl inhibitors of 17&beta, hydroxysteroid dehydrogenase type 10 are promising compounds for potential drugs for neurodegenerative diseases that warrant further research and development.

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.

Asoxime (HI-6) is a well known oxime reactivator used for counteracting intoxication by nerve agents. It is able to reactivate acetylcholinesterase (AChE) inhibited even by sarin or soman. The present experiment was aimed to determine markers of oxidative stress represented by thiobarbituric acid reactive substances and antioxidants represented by ferric reducing antioxidant power, reduced and oxidized glutathione in a Beagle dog model. Two groups of dogs were intramuscularly exposed to single (11.4 mg/kg.b.wt.) or tenfold (114 mg/kg.b.wt.) human therapeutically doses of HI-6. HI-6 affinity for AChE in vitro was evaluated in a separate experiment. Complete serum biochemistry and pharmacokinetics were also performed with significant alteration in blood urea nitrogen, creatine phosphokinase, glucose and triglycerides. Blood samples were collected before HI-6 application and after 30, 60, and 120 min. The overall HI-6 impact on organism is discussed.

Assay of acetylcholinesterase (AChE) activity plays an important role in diagnostic, detection of pesticides and nerve agents, in vitro characterization of toxins and drugs including potential treatments for Alzheimer's disease. These experiments were done in order to determine whether indoxylacetate could be an adequate chromogenic reactant for AChE assay evaluation. Moreover, the results were compared to the standard Ellman's method. We calculated Michaelis constant Km (2.06 × 10(-4) mol/L for acetylthiocholine and 3.21 × 10(-3) mol/L for indoxylacetate) maximum reaction velocity V(max) (4.97 × 10(-7) kat for acetylcholine and 7.71 × 10(-8) kat for indoxylacetate) for electric eel AChE. In a second part, inhibition values were plotted for paraoxon, and reactivation efficacy was measured for some standard oxime reactivators: obidoxime, pralidoxime (2-PAM) and HI-6. Though indoxylacetate is split with lower turnover rate, this compound appears as a very attractive reactant since it does not show any chemical reactivity with oxime antidots and thiol used for the Ellman's method. Thus it can be advantageously used for accurate measurement of AChE activity. Suitability of assay for butyrylcholinesterase activity assessment is also discussed.

Inhibitors of acetylcholinesterase are compounds widely used in the treatment of various diseases, such as Alzheimer’s disease, glaucoma and Myasthenia gravis (MG). Compounds used in the therapy of MG posses a positive charge in the molecule to ensure peripheral effect of action and minimal blood–brain barrier penetration. The most prescribed carbamate inhibitors are however known for many severe side effects related to the carbamylation of AChE. This paper describes preparation and in vitro evaluation of 20 newly prepared bis-isoquinolinium inhibitors of potential concern for MG. The newly prepared compounds were evaluated in vitro on human recombinant AChE and human plasmatic butyrylcholinesterase (BChE). Their inhibitory ability was expressed as IC50 and compared to chosen standards ambenonium dichloride, edrophonium chloride, BW284c51 and ethopropazine hydrochloride. Three novel compounds presented promising inhibition (in nM range) of both enzymes in vitro better or similar to edrophonium and BW284c51, but worse to ambenonium. The novel inhibitors did not present higher selectivity toward AChE or BChE. The kinetic assay confirmed non-competitive inhibition of hAChE by two selected promising novel compounds. Two newly prepared compounds were also chosen for docking studies that confirmed apparent π–π or π–cationic interactions aside the cholinesterases catalytic sites. The SAR findings were discussed.

Reversible inhibitors (e.g., pyridostigmine bromide, neostigmine bromide) of carbamate origin are used in the early treatment of Myasthenia gravis (MG) to block acetylcholinesterase (AChE) native function and conserve efficient amount of acetylcholine for decreasing number of nicotinic receptors. Carbamate inhibitors are known for many undesirable side effects related to the reversible inhibition of AChE. In contrast, this paper describes 20 newly prepared bispyridinium inhibitors of potential concern for MG. Although some compounds from this series have been known before, they were not assayed for cholinesterase inhibition yet. The newly prepared compounds were evaluated in vitro on human erythrocyte AChE and human plasmatic butyrylcholinesterase (BChE). Their inhibitory ability was expressed as IC(50) and compared to standard carbamate drugs. Three compounds presented promising inhibition (in muM range) of both enzymes in vitro similar to the used standards. The novel inhibitors did not present selectivity between AChE and BChE. Two newly prepared compounds were chosen for docking studies and confirmed apparent pi-pi or pi-cationic interactions aside enzyme's catalytic sites. The kinetics assay confirmed non-competitive inhibition of AChE by two best newly prepared compounds.

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.

An electrochemical sensor is introduced as a tool applicable for diagnosis of intoxication by cholinesterase inhibitors caused by the well-known nerve agent VX. The traditional Ellman method was chosen for comparison with the sensor's analytical parameters. Both methods are based on estimation of blood cholinesterase inhibition as a marker of intoxication. While Ellman´s method provided a limit of detection of 5.2´10-7 M for blood containing VX, the electrochemical sensor was able to detect 4.0´10-7 M. Good correlation between both methods was observed (R = 0.92). The electrochemical sensor could be considered a convenient tool for a fast yet accurate method, easily available for field as well as laboratory use. Time and cost savings are key features of the sensor-based assay.

Acetylcholinesterase (AChE; EC 3.1.1.7) reactivators are used as a part of the antidotal therapy of organophosphorus pesticide and nerve agent intoxications. Cyclosarin is one member of the nerve agent family. In this article, we compared the reactivation potency of five structurally different AChE reactivators (pralidoxime, trimedoxime, methoxime, HS-6, and BI-6) to reactivate cyclosarin-inhibited cholinesterases of human brain. The results demonstrate that the bisquaternary monooxime reactivator BI-6 seems to be the most potent reactivator of cyclosarin-inhibited cholinesterases. Moreover, according to the results, we can describe basic structural requirements, which are necessary for the efficacious reactivation process.

Organophosphorus compounds such as nerve agents inhibit, practically irreversibly, cholinesterases by their phosphorylation in the active site of these enzymes. Current antidotal treatment used in the case of acute nerve agent intoxications consists of combined administration of anticholinergic drug (usually atropine) and acetylcholinesterase (AChE, EC 3.1.1.7) reactivator (HI-6, obidoxime, pralidoxime), which from a chemical view is a derivative from the group of pyridinium or bispyridinium aldoximes (commonly called "oxime"). Oximes counteract acetylcholine increase, resulting from AChE inhibition. In the human body environment these compounds are powerful nucleophiles and are able to break down the bond between AChE and nerve agent molecule. This process leads to renewal of enzyme functionality -- to its reactivation. The usefulness of oxime in the reactivation process depends on its chemical structure and on the nerve agent whereby AChE is inhibited. Due to this fact, selection of suitable reactivator in the treatment of intoxications is very important. In our work, we have compared differences in the in vitro inhibition potency of VX and Russian VX on rat, pig and human brain, and subsequently we have tested reactivation of rat brain cholinesterase inhibited by these agents using oxime HI-6, obidoxime, pralidoxime, trimedoxime and methoxime. The results showed that no major differences in the reactivation process of both VX and Russian VX-inhibited cholinesterase. The similarity in reactivation was caused by analogous chemical structure of either nerve agent; and that oxime HI-6 seems to be the most effective reactivator tested, which confirms that HI-6 is currently the most potent reactivator of AChE inhibited by nerve agents. The results obtained in our study should be considered in the future development of new AChE reactivators.

Four thin-layer chromatographic (TLC) methods for identification of twelve bisquaternary acetylcholinesterase reactivator HI-6 salts (sulfate, chloride, acetate, bromide, phosphate, mesylate, tartrate, iodide, malonate, salicylate, maleinate, and tosylate) are described. By use of TLC, particular E and Z isomers of each salt on the oxime group were also separated. These TLC methods could be of high interest for rapid identification of each salt and for purity control.

Oxime K027 is a low-toxic bisquaternary compound originally developed as a reactivator of acetylcholinesterase (AChE) inhibited by nerve agents. The reactivation potency of K027 has been tested as a potential reactivator of AChE inhibited by tabun, sarin, cyclosarin, soman, VX, Russian VX, paraoxon, methylchlorpyrifos, and DDVP. The results show that oxime K027 reactivated AChE inhibited by almost all tested inhibitors to more than 10%, which is believed to be enough for saving the lives of intoxicated organisms. In the case of cyclosarin- and soman-inhibited AChE, oxime K027 did not reach sufficient reactivation potency.

Preparation of 1-(4-hydroxy-iminomethylpyridinium)-3-pyridiniumpropane dibromide is described. This compound represents a new acetylcholinesterase (AChE) reactivator, which has no substituents on the second pyridinium ring as found in other commonly used AChE reactivators. The reactivation ability of this reactivator was tested on tabun- and cyclosarin-inhibited AChE. According to the results obtained, the new compound (without substitution and with decreased molecule size) showed increased reactivation potency in case of cyclosarin inhibited AChE. A potent oxime for treatment of tabun and cyclosarin-caused intoxications was thus obtained via slight modification of the reactivator structure (compared to trimedoxime and K027).

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

This paper describes an in vitro study of three potential acetylcholinesterase (AChE; EC 3.1.1.7) reactivators derived from a monoquaternary reactivator pralidoxime. Compounds used were pyridinium-2-aldoxime-4- carbamoyl-N-methyl iodide (TO231), pyridinium-2-aldoxime-4-ethoxycarbonyl-N-methyl iodide (TO237), and pyridinium-2-aldoxime-5-ethoxycarbonyl-N-methyl iodide (TO238). Pralidoxime and obidoxime were used for comparison. Nerve agent cyclosarin and pesticide chlorpyrifos were used as organophosphorus cholinesterase inhibitors. The source of AChE was rat brain homogenate. None of the tested oximes was able to reactivate cyclosarin-inhibited AChE (at 1.0 mmol L-1 oxime concentration). In case of chlorpyrifos, TO231 was the most potent AChE reactivator with an 82 % reactivation at 1.0 mmol L-1 oxime concentration. This reactivating potency equals that of pralidoxime and obidoxime. TO238 was less effective, and TO237 did not reactivate chlorpyrifos-inhibited AChE at all. None of the tested AChE reactivators, reference compounds included, could be considered universal for both chlorpyrifos- and cyclosarin-inhibited AChE., U ovome in vitro istraživanju testiran je reaktivacijski potencijal triju reaktivatora acetilkolinesteraze (AChE; EC 3.1.1.7) dobivenih iz monokvaternog reaktivatora pralidoksima. Testirani su sljedeći spojevi: 4-karbamoil-2-aldoksim-1-metilpiridinijev jodid (TO231), 4-etoksikarbonil-2-aldoksim-1-metilpiridinijev jodid (TO237) te 5-etoksikarbonil-2-aldoksim-1-metilpiridinijev jodid (TO238). Za usporedbu su uzeti pralidoksim i obidoksim. Kao inhibitori kolinesteraze rabljeni su živćani otrov ciklosarin i pesticid klorpirifos. Izvor acetilkolinesteraze bio je homogenat mozga štakora. Nijedan od testiranih oksima nije uspio reaktivirati acetilkolinesterazu inhibiranu ciklosarinom pri koncentraciji oksima od 1.0 mmol L-1. Kod inhibicije klorpirifosom TO231 pokazao se najsnažnijim reaktivatorom acetilkolinesteraze s reaktivacijom od 82 % pri koncentraciji oksima od 1.0 mmol L-1. Ovaj reaktivacijski potencijal jednak je onomu pralidoksima i obidoksima. Slabiju je djelotvornost iskazao TO238, a TO237 nije uspio reaktivirati acetilkolinesterazu inhibiranu klorpirifosom. Stoga nijedan od testiranih spojeva (uključujući i referentne oksime) ne može služiti kao univerzalni reaktivator acetilkolinesteraze inhibirane klorpirifosom i ciklosarinom.