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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.

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.

Summary Searching for the universal oxime, which could be able to reactivate acetylcholinesterase inhibited by various nerve agents is still topic of high interest. In this contribution, oxime HLo-7, that was thoroughly discussed in the last decade, is evaluated . Its universality was tested in vitro using the rat brain homogenate as a source of the cholinesterases. The main members of the nerve agent family (tabun, sarin, soman, cyclosarin and VX) were used for this purpose. As shown, oxime HLo-7 was able to reactivate cholinesterases inhibited by all tested nerve agents with the exception of tabun. Hence, it could not be designated as the broad-spectrum reactivator.

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.

Cholinesterase activity in blood of laboratory rats was monitored. Rats were intoxicated with paraoxon at dosis of 0 - 65 - 125 - 170 - 250 - 500 nmol. The 250 nmol dose was found to be the LD(50). An electrochemical sensor was found useful to provide information about cholinesterase activity. The decrease of cholinesterase activity was correlated to intoxication symptoms and mortality level. It was found that the symptoms of intoxication are not observed while at least 50% of cholinesterase activity in blood remains. The minimal cholinesterase activity essential to survival is around 10%, when compared with the initial state. No changes in levels of low moleculary weight antioxidants were observed.

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.

Because of the threat of misuse of nerve agents as terroristic weapons by the terrorists, animmediate need is felt for the preparation of antidotes on large-scale basis. HI-6 (dichloride anddimethanesulphonate) salt are the most promising acetylcholinesterase reactivators used ascausal antidotes in nerve agents intoxication. In this study, rapid and large-scale preparationof oxime HI-6, the most promising reactivator has been described.

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.

Coupling of two distinct pharmacophores, tacrine and trolox, endowed with different biological properties, afforded 21 hybrid compounds as novel multifunctional candidates against Alzheimer's disease. Several of them showed improved inhibitory properties toward acetylcholinesterase (AChE) in relation to tacrine. These hybrids also scavenged free radicals. Molecular modeling studies in tandem with kinetic analysis exhibited that these hybrids target both catalytic active site as well as peripheral anionic site of AChE. In addition, incorporation of the moiety bearing antioxidant abilities displayed negligible toxicity on human hepatic cells. This striking effect was explained by formation of nontoxic metabolites after 1 h incubation in human liver microsomes system. Finally, tacrine-trolox hybrids exhibited low in vivo toxicity after im administration in rats and potential to penetrate across blood-brain barrier. All of these outstanding in vitro results in combination with promising in vivo outcomes highlighted derivative 7u as the lead structure worthy of further investigation.

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.

1. The efficacy of the oxime HLö-7 and currently used oximes (pralidoxime, obidoxime, HI-6) to reactivate acetylcholinesterase inhibited by various nerve agents (sarin, tabun, cyclosarin, VX) was tested by in vitro methods. 2. Both H oximes (HLö-7, HI-6) were found to be more efficacious reactivators of sarin and VX-inhibited acetylcholinesterase than pralidoxime and obidoxime. On the other hand, their potency to reactivate tabun-inhibited acetylcholinesterase is very low and does not reach the reactivating efficacy of obidoxime. In the case of cyclosarin, the oxime HI-6 was only found to be able to sufficiently reactivate cyclosarin-inhibited acetylcholinesterase in vitro. 3. Thus, the oxime HLö-7 does not seem to be more efficacious reactivator of nerve agent-inhibited acetylcholinesterase than HI-6 according to in vitro evaluation of their reactivation potency and, therefore, it is not more suitable to be introduced for antidotal treatment of nerve agent-exposed people than HI-6.

Tacrine (THA), the first clinically effective acetylcholinesterase (AChE) inhibitor and the first approved drug for the treatment of Alzheimer’s disease (AD), was withdrawn from the market due to its side effects, particularly its hepatotoxicity. Nowadays, THA serves as a valuable scaffold for the design of novel agents potentially applicable for AD treatment. One such compound, namely 7-methoxytacrine (7-MEOTA), exhibits an intriguing profile, having suppressed hepatotoxicity and concomitantly retaining AChE inhibition properties. Another interesting class of AChE inhibitors represents Huprines, designed by merging two fragments of the known AChE inhibitors—THA and (−)-huperzine A. Several members of this compound family are more potent human AChE inhibitors than the parent compounds. The most promising are so-called huprines X and Y. Here, we report the design, synthesis, biological evaluation, and in silico studies of 2-methoxyhuprine that amalgamates structural features of 7-MEOTA and huprine Y in one molecule.

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.

A series of novel tacrine derivatives and tacrine–coumarin heterodimers were designed, synthesized, and biologically evaluated for their potential inhibitory effect on both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Of these compounds, tacrine–coumarin heterodimer 7c and tacrine derivative 6b were found to be the most potent inhibitors of human AChE (hAChE), demonstrating IC50 values of 0.0154 and 0.0263 μM. Ligands 6b, 6c, and 7c exhibited the highest levels of inhibitory activity against human BuChE (hBuChE), demonstrating IC50 values that range from 0.228 to 0.328 μM. Docking studies were performed in order to predict the binding modes of compounds 6b and 7c with hAChE/hBuChE.

Organophosphorus compounds represent nerve agents, pesticides and several industrial compounds. Treatment after exposure to organophosphates involves the use of parasympatolytics, acetylcholinesterase (AChE) reactivators/modulators and anticonvulsive drugs. Wider clinical use of several AChE reactivators/modulators might be limited because of possible side effects, including gastrointestinal toxicity. In this study we evaluated the effect of paraoxon and an AChE reactivator (HI-6) on the gastric myoelectric activity in experimental pigs.Six female experimental pigs (mean weight 33 kg) entered the study. Intramuscular paraoxon (1.5 g) was administrated after the baseline gastric electrogastrography (EGG) recording, followed by HI-6 dimethansulphonate (1.5 g i.m.) 10 min. later. A further ten 15-minute-interval EGG recordings were performed. Running spectral analysis was used for the elemental evaluation of the EGG. The results were expressed as dominant frequency of slow waves at all intervals of EGG recordings. EGG power analysis was performed in all animals.Paraoxon induced a non-significant decrease of dominant frequency (2.8±0.6 vs. 2.6±0.5 cycles per min.; p=0.092). Subsequent administration of HI-6 normalised dominant frequency to basal values and increased it significantly within the subsequent 30 minutes (3.0±0.4; p0.001). Paraoxon administration did not influence the power (within a 10-minute exposure). However, the amplitudes increased significantly 90 minutes after administration of HI-6 (819±109 vs. 5054±732 μV2; p0.001).AChE reactivator HI-6 blocked the gastric effect of paraoxon significantly. Subsequent myoelectric changes in the dominant frequency and power were executed by HI-6. The effect of paraoxon was non-significant.

Among all the tacrine congeners synthesized, derivative (III) shows the best activity and selectivity against hAChE and derivative (Id) against hBChE.

New tacrine derivatives 5a – d , 6a – d with piperazino-ethyl spacer linked with corresponding secondary amines and tacrine homodimer 8 were synthesized and tested as cholinesterase inhibitors on human acetylcholinesterase ( h AChE) and human plasmatic butyrylcholinesterase ( h BChE). In most cases the majority of synthesized derivatives exhibit a high AChE and BChE inhibitory activity with IC 50 values in the low-nanomolar range, being clearly more potent than the reference standard tacrine (9-amino-1,2,3,4-tetrahydroacridine, 1 ) and 7-MEOTA (7-methoxy-9-amino-1,2,3,4-tetrahydroacridine). Among them, inhibitors 8 and 5c , showed a strong inhibitory activity against h AChE, with an IC 50 value of 4.49 nM and 4.97, nM resp., and a high selectivity to h AChE. The compound 5d acted as the most potent inhibitor against h BChE with an IC 50 value of 33.7 nM and exhibited also a good selectivity towards h BChE. The dissociation constants K i of the selected inhibitors were compared with their IC 50 values. Molecular modeling studies were performed to predict the binding modes between individual derivatives and h AChE/ h BChE.

Alzheimer's disease (AD) is a neurodegenerative disorder. Symptomatic treatment is available by inhibitors of acetylcholinesterase (AChE) such as rivastigmine, galantamine and donepezil. As huperzine is a promising compound for AD treatment, our study was aimed at evaluating its pertinent implications in oxidative stress.Laboratory guinea pigs were exposed to huperzine A at doses of 0, 5, 25, 125 and 625 µg/kg. The animals were observed for cognitive disorders and sacrificed one hour after exposure. Tonic-clonic seizures were noticed, but only in highly dosed animals. Ferric reducing antioxidant power (FRAP), thiobarbituric acid reactive substances (TBARS), glutathione reductase and glutathione S-transferase were assessed in frontal, temporal and parietal lobes, the cerebellum, liver, spleen and kidney.Only minimal changes in enzymatic markers were recognized. Huperzine was not implicated in oxidative stress enhancement as the TBARS values remained quite stable. Surprisingly, antioxidants accumulated in the examined brain compartments as the FRAP value was significantly elevated following all doses of huperzine.We discuss the potency of huperzine in enhancing the antioxidant capacity of the central nervous system. Huperzine is probably implicated in more processes than cholinesterase inhibition only.

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

Carbamate inhibitors (e.g., pyridostimine bromide) are used as a pre-exposure treatment for the prevention of organophosphorus poisoning. They work by blocking acetylcholinesterase's (AChE) native function and thus protect AChE against irreversible inhibition by organophosphorus compounds. However, carbamate inhibitors are known for many undesirable side-effects related to the carbamylation of AChE. In this Letter, 19 analogues of SAD-128 were prepared and evaluated as cholinesterase inhibitors. The screening results showed promising inhibitory ability of four compounds better to used standards (pralidoxime, obidoxime, BW284c51, ethopropazine, SAD-128). Four most promising compounds were selected for further molecular docking studies. The SAR was stated from obtained data. The former receptor studies were reported and discussed. The further in vivo studies were recommended in the view of OP pre-exposure treatment.

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.

Four novel bisquaternary aldoxime cholinesterase reactivators differing in their chemical structure were prepared. Afterwards, their biological activity was evaluated for their ability to reactivate acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BuChE, EC 3.1.1.8) inhibited by paraoxon. Their reactivation activity was compared with standard reactivators—pralidoxime, obidoxime and HI-6—which are clinically used at present. As it resulted, none of the prepared compounds surpassed obidoxime, which is considered to be the most potent compound if used for reactivation of AChE inhibited by paraoxon. In case of BuChE reactivation, two compounds (K053 and K068) achieved similar results as obidoxime.

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).

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.

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

Eleven isoquinoline alkaloids (1–11) were isolated from dried leaves of Peumus boldus Mol. by standard chromatographic methods. The chemical structures were elucidated by MS, and 1D and 2D NMR spectroscopic analysis, and by comparison with literature data. Compounds isolated in sufficient amount were evaluated for their acetylcholinesterase, and butyrylcholinesterase inhibition activity using Ellman's method. In the prolyl oligopeptidase assay, Z-Gly-Pro- p-nitroanilide was used as substrate. Promising butyrylcholinesterase inhibition activities were demonstrated by two benzylisoquinoline alkaloids, reticuline (8) and N-methylcoclaurine (9), with IC50 values of 33.6 ± 3.0 μM and 15.0 ± 1.4 μM, respectively. Important prolyl oligopeptidase inhibition activities were shown by N-methyllaurotetanine (6) and sinoacutine (4) with IC50 values of 135.4 ± 23.2 μM and 143.1 ± 25.4 μM, respectively. Other tested compounds were considered inactive.

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.

Searching for new potent acetylcholinesterase (AChE; E.C. 3.1.1.7) reactivators (oximes) is a very time-consuming process. At our department, we are able to synthesize more than 50 new AChE reactivators per year. Owing to this fact, we have to select promising reactivators using our in vitro method (potentiometric titration, pH 8 and temperature 25 degrees C; source of cholinesterases, rat brain homogenate; time of inhibition by nerve agents, 30 min; time of reactivation, 10 min) prior to in vivo experiments. For this purpose, we are using two-phase in vitro evaluation of reactivator potency. In the first phase, reactivation potency of all newly synthesized AChE reactivators is tested at two concentrations: 10(-3) M and 10(-5) M. Afterwards, all reactivators achieving reactivation potency over 15% (especially at the concentration 10(-5) M) are tested. The second phase consists of the measurement of the relationship between concentration of the oxime and its reactivation ability. In most cases, the reactivation bell-shaped curve is obtained. The most potent AChE reactivators are selected and provided for further experiments during our development process.

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.

The potency of currently used oximes to reactivate sarin-inhibited acetylcholinesterase (AChE) in various parts of pig brain and whole pig brain was evaluated using in vitro methods. Significant differences in reactivation potency among all tested oximes were observed. At concentrations (10−4 M) corresponding to recommended doses in vivo, the oxime HI-6 seems to be a more efficacious reactivator of sarin-inhibited AChE in whole pig brain as well as in cerebral hemispheres and cerebellum compared with the other oximes studied. Nevertheless, there are not any differences in the potency of oximes tested to reactivate sarin-inhibited AChE in medulla oblongata. Thus, the oxime HI-6 appears to be the most promising oxime among currently available oximes for the antidotal treatment of acute sarin poisoning, although it is not more efficacious than other currently used oximes in medulla oblongata, whose function is necessary for the vital functions of respiration and circulation. Copyright © 2005 John Wiley & Sons. Ltd.

A known alkaloid (+)-chenabinol (1) and two new secobisbenzylisoquinoline alkaloids were isolated by standard chromatographic methods from the root bark of Berberis vulgaris L. The structures of the new alkaloids, named berkristine (2) and verfilline (3), were established by spectroscopic (including 2D NMR), and HRMS (ESI) methods. The alkaloids were tested for their inhibition activity of human cholinesterases and prolyl oligopeptidase. Compound 1 inhibited human butyrylcholinesterase with an IC50 value of 44.8 ± 5.4 μM.

Ongoing studies of Zephyranthes robusta resulted in the isolation of the lycorine-type alkaloid previously called carinatine and 10- O-demethylgalanthine. The NMR data given previously for this compound were revised and completed by two-dimensional 1H-1H and 1H-13C chemical shift correlation experiments. The name of the isolated compound was corrected to 9- O-demethylgalanthine in accordance with the currently used system of numbering of lycorine-type alkaloids. 9- O-Demethylgalanthine and galanthine, a previously isolated alkaloid from Z. robusta, were inactive in acetylcholinesterase/butyrylcholinesterase assays (IC50 > 500 μM), but showed important prolyl oligopeptidase inhibition activity.

Alkaloid extracts of eight Narcissus (Amaryllidaceae) species and varieties were studied with respect to their acetylcholinesterase (HuAChE) and butyrylcholinesterase (HuBuChE) inhibitory activity and alkaloid patterns. Thirty alkaloids were determined by GC/MS, and twenty-five of them identified from their mass spectra, retention times and retention indexes. Promising HuAChE inhibition activity was demonstrated by six Narcissus taxa and HuBuChE inhibition by N. jonquila cv. Double Campernelle and N. nanus cv. Elka with IC50 values of 24.1 ± 1.9 μg/mL and 25.1 ± 1.8 μg/mL, respectively. Two alkaloids were isolated in pure form using preparative TLC and identified as the galanthamine type alkaloid narwedine and the lycorine type alkaloid incartine. Both compounds were tested for their biological activity. They were considered inactive in HuAChE/HuBuChE assays, but showed promising prolyl oligopeptidase inhibition activities with IC50 values of 0.95 ± 0.12 mM and 0.91 ± 0.09 mM, respectively.

Oxime K203 seems to be the most promising oxime in case of reactivation of tabun-inhibited acetylcholinesterase (AChE). Although it was originally developed for treatment of tabun intoxications, it is able to reactivate cholinesterases inhibited by other nerve agents. This study is aimed at the evaluation of its potency in vitro against other nerve agents. For this purpose, sarin, tabun, cyclosarin, soman, VX, Russian VX and DFP were selected as members of the nerve agent family to check its universality. At high concentrations (10(-3) M), oxime K203 reached promising reactivation activity. At low concentrations, relevant for human use (10(-5) M), promising reactivation potency was obtained only with tabun. In conclusion, oxime K203 reactivates other nerve agents-inhibited cholinesterases, however its broad-spectrum reactivation is limited at high, for human not attainable, concentrations only.

Nerve agents such as sarin, VX and tabun are organophosphorus compounds able to inhibit an enzyme acetylcholinesterase (AChE). AChE reactivators and anticholinergics are generally used as antidotes in the case of intoxication with these agents. None from the known AChE reactivators is able to reactivate AChE inhibited by all kinds of nerve agents. In this work, reactivation potency of seventeen structurally different AChE reactivators was tested in vitro and subsequently, relationship between their chemical structure and biological activity was outlined. VX was chosen as appropriate member of the nerve agent family.

Alzheimer´s disease (AD) is one of the most discussed diseases of present time. Because of the aging of our population it is high threat for future. Due to this, new drugs combating AD are still developed. In this study, twelve bisquaternary isoquinolinium de-rivatives differing in the linker between two heteroarenium rings were synthesized and tested for their potency to inhibit brain cho-linesterases. According to the obtained in vitro results, the anticholinesterase activity grew up with the length of the connection chain. Possible binding to the enzyme cavity was described using software Autodock. Moreover, LogP and molecular volume of prepared com-pounds were predicted. As resulted, the highest probability of synthesized compounds to penetrate blood-brain barrier could be expected for the compound with linker having eight methylenes. Keywords: Quinolinium, Quaternary compounds, Inhibitor, Cholinesterase, Alzheimer´s disease, Anticholinergic. INTRODUCTION At present, there is increasing number of people suffering from diseases connecting with the increasing age. Among them, Alz-heimer´s disease (AD) is well discussed by the scientific commu-nity. It belongs along with cancer, AIDS and cardiovascular dis-eases among the highest threats influencing the whole population. Although this disease is known already for more than one hundred years, there is no treatment able to stop its degenerative progress. Currently, there are just few AD treatments (Donepezil, Galan-tamine, Rivastigmine, Tacrine, Memantine) approved by FDA. All of them, except Memantine, are potent inhibitors of acetylcho-linesterase (AChE; EC 3.1.1.7). Although the cholinergic approach is able to have only one- or two-year benefit, economically, one or two more years of the productive life are very important [1,2]. Due to this reason, development of new AChE inhibitors as new AD-drugs candidates is still possible approach how to combat AD [3-5]. During last years, there were published several articles discuss-ing penetration of quaternary compounds into the blood-brain bar-rier (BBB) [6-8]. Thanks to this, also quaternary compounds with increased lipophility could be considered as an alternative for treatment of AD or at least as its supplement. Recently, there were published several articles describing quaternary compounds (expe-cially pyridinium compounds) as potential AD treatment [9-12]. In 2004, we found that more potent inhibitors of AChE than pyridi-nium salts seem to be quaternary compounds with conjugated aro-matic rings (quinolinium, isoquinolinium or acridinium) [13]. Thanks to the added aromatic rings, also their lipophilicity is higher if compared with pyridinium compounds. And with the lipophilic-ity, hand in hand, higher penetration through the BBB can be ex-pected. Because of the fact that affinity of bisquaternary compounds is higher than that of monoquaternary ones, in this article, we turned our attention to bisquaternary isoquinolinium compounds differing in the length of the connection chain between two isoquinolines.

New cholinesterase reactivators are synthesized as potential antidotes for treatment of organophosphorus agent poisonings or as part of pseudo catalytic scavengers for improvement of the nerve agent prophylaxis. In this study, three novel potential cholinesterase reactivators (K064 - (E)-1-(pyridinium)-4-(2-hydroxyiminomethylpyridinium)-but-2-ene dibromide; K065 - (E)-1-(quinolinium)-4-(2-hydroxyiminomethylpyridinium)-but-2-ene dibromide; K066 - (E)-1- (isoquinolinium)-4-(2-hydroxyiminomethylpyridinium)-but-2-ene dibromide) were synthesized and tested for their po- tency to reactivate acetylcholinesterase (AChE, EC 3.1.1.3) and butyrylcholinesterase (BChE, 3.1.1.8) inhibited by pesti- cide paraoxon. As resulted, none from the synthesized compounds surpassed currently clinically used reactivators (prali- doxime, obidoxime and HI-6).

Eleven Amaryllidaceae alkaloids (1–11) were isolated from fresh bulbs of Chlidanthus fragrans Herb. The chemical structures were elucidated by MS, and 1D and 2D NMR spectroscopic experiments. Complete NMR assignments were achieved for deoxypretazzetine (1). All compounds were evaluated for their erythrocytic acetylcholinesterase and serum butyrylcholinesterase inhibition activity using Ellman's method. In the prolyl oligopeptidase assay, Z-Gly-Pro- p-nitroanilide was used as substrate. In biological assays, only the crinine type Amaryllidaceae alkaloid undulatine showed promising acetylcholinesterase and prolyl oligopeptidase inhibition activity with IC50 values of 23.0 ± 1.0 μM and 1.96 ± 0.12 mM, respectively. Other isolated compounds were considered inactive.