The investigation into human butyrylcholinesterase (hBChE) inhibitors as therapeutic agents for Alzheimer's disease (AD) holds significant promise, addressing both symptomatic relief and disease progression. In the pursuit of novel drug candidates with a selective BChE inhibition pattern, we focused on naturally occurring template structures, specifically Amaryllidaceae alkaloids of the carltonine-type. Herein, we explored a series of compounds implementing an innovative chemical scaffold built on the 3- and 4-benzyloxy-benzylamino chemotype. Notably, compounds 28 (hBChE IC50 = 0.171 ± 0.063 μM) and 33 (hBChE IC50 = 0.167 ± 0.018 μM) emerged as top-ranked hBChE inhibitors. In silico simulations elucidated the binding modes of these compounds within hBChE. CNS availability was predicted using the BBB score algorithm, corroborated by in vitro permeability assessments with the most potent derivatives. Compound 33 was also inspected for aqueous solubility, microsomal and plasma stability. Chemoinformatics analysis validated these hBChE inhibitors for oral administration, indicating favorable gastrointestinal absorption in compliance with Lipinski's and Veber's rules. Safety assessments, crucial for the chronic administration typical in AD treatment, were conducted through cytotoxicity testing on human neuroblastoma (SH-SY5Y) and hepatocellular carcinoma (HepG2) cell lines.

• Publication type
• Journal Article MeSH

Oxime reactivators of acetylcholinesterase (AChE) are used as causal antidotes for intended and unintended poisoning by organophosphate nerve agents and pesticides. Despite all efforts to develop new AChE reactivators, none of these drug candidates replaced conventional clinically used oximes. In addition to the therapeutic efficacy, determining the safety profile is crucial in preclinical drug evaluation. The exact mechanism of oxime toxicity and the structure-toxicity relationship are subjects of ongoing research, with oxidative stress proposed as a possible mechanism. In the present study, we investigated four promising bispyridinium oxime AChE reactivators, K048, K074, K075, and K203, and their ability to induce oxidative stress in vitro. Cultured human hepatoma cells were exposed to oximes at concentrations corresponding to their IC50 values determined by the MTT assay after 24 h. Their potency to generate reactive oxygen species, interfere with the thiol antioxidant system, and induce lipid peroxidation was evaluated at 1, 4, and 24 h of exposure. Reactivators without a double bond in the four-carbon linker, K048 and K074, showed a greater potential to induce oxidative stress compared with K075 and K203, which contain a double bond. Unlike oximes with a three-carbon-long linker, the number of aldoxime groups attached to the pyridinium moieties does not determine the oxidative stress induction for K048, K074, K075, and K203 oximes. In conclusion, our results emphasize that the structure of oximes plays a critical role in inducing oxidative stress, and this relationship does not correlate with their cytotoxicity expressed as the IC50 value. However, it is important to note that oxidative stress cannot be disregarded as a potential contributor to the side effects associated with oximes.

• MeSH
• Acetylcholinesterase metabolism   MeSH
• Antidotes pharmacology   MeSH
• Hep G2 Cells MeSH
• Cholinesterase Inhibitors toxicity   MeSH
• Humans MeSH
• Organophosphates toxicity   MeSH
• Oxidative Stress MeSH
• Oximes pharmacology   chemistry   MeSH
• Pyridinium Compounds pharmacology   chemistry   MeSH
• Cholinesterase Reactivators * pharmacology   chemistry   MeSH
• Carbon MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Acetylcholinesterase MeSH
• Antidotes MeSH
• Cholinesterase Inhibitors MeSH
• Organophosphates MeSH
• Oximes MeSH
• Pyridinium Compounds MeSH
• Cholinesterase Reactivators * MeSH
• Carbon MeSH

Alzheimer's disease (AD) is a complex disease with an unknown etiology. Available treatments, limited to cholinesterase inhibitors and N-methyl-d-aspartate receptor (NMDAR) antagonists, provide symptomatic relief only. As single-target therapies have not proven effective, rational specific-targeted combination into a single molecule represents a more promising approach for treating AD, and is expected to yield greater benefits in alleviating symptoms and slowing disease progression. In the present study, we designed, synthesized, and biologically evaluated 24 novel N-methylpropargylamino-quinazoline derivatives. Initially, compounds were thoroughly inspected by in silico techniques determining their oral and CNS availabilities. We tested, in vitro, the compounds' effects on cholinesterases and monoamine oxidase A/B (MAO-A/B), as well as their impacts on NMDAR antagonism, dehydrogenase activity, and glutathione levels. In addition, we inspected selected compounds for their cytotoxicity on undifferentiated and differentiated neuroblastoma SH-SY5Y cells. We collectively highlighted II-6h as the best candidate endowed with a selective MAO-B inhibition profile, NMDAR antagonism, an acceptable cytotoxicity profile, and the potential to permeate through BBB. The structure-guided drug design strategy applied in this study imposed a novel concept for rational drug discovery and enhances our understanding on the development of novel therapeutic agents for treating AD.

• Keywords
• Alzheimer’s disease, N-methyl-d-aspartate receptor, acetylcholinesterase, enzyme inhibition, monoamine oxidase A/B, multi-target directed ligands,
• MeSH
• Acetylcholinesterase metabolism   MeSH
• Alzheimer Disease * drug therapy   MeSH
• Cholinesterase Inhibitors therapeutic use   MeSH
• Monoamine Oxidase Inhibitors therapeutic use   MeSH
• Humans MeSH
• Monoamine Oxidase metabolism   MeSH
• Neuroblastoma * drug therapy   MeSH
• Drug Design MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Acetylcholinesterase MeSH
• Cholinesterase Inhibitors MeSH
• Monoamine Oxidase Inhibitors MeSH
• Monoamine Oxidase MeSH

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.

• Keywords
• 7-methoxytacrine, Alzheimer's disease, cholinesterases, in vitro, indole, tacrine,
• MeSH
• Acetylcholinesterase metabolism   MeSH
• Alzheimer Disease drug therapy   MeSH
• Amyloid beta-Peptides metabolism   MeSH
• Cholinesterase Inhibitors chemistry   pharmacology   MeSH
• Molecular Targeted Therapy MeSH
• Dimerization MeSH
• DNA chemistry   MeSH
• Blood-Brain Barrier MeSH
• Indoles chemistry   pharmacology   MeSH
• Inhibitory Concentration 50 MeSH
• Humans MeSH
• Ligands MeSH
• Neuroprotective Agents chemistry   pharmacology   MeSH
• Drug Evaluation, Preclinical MeSH
• Molecular Dynamics Simulation MeSH
• Molecular Docking Simulation MeSH
• Tacrine chemistry   pharmacology   MeSH
• Protein Binding MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Acetylcholinesterase MeSH
• Amyloid beta-Peptides MeSH
• Cholinesterase Inhibitors MeSH
• DNA MeSH
• Indoles MeSH
• Ligands MeSH
• Neuroprotective Agents MeSH
• Tacrine MeSH

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.

• Keywords
• Alzheimer’s disease, N-methyl-d-aspartate receptor, acetylcholinesterase, butyrylcholinesterase, fluorene, in silico, in vitro, multi-target directed ligands,
• MeSH
• Alzheimer Disease drug therapy   enzymology   genetics   pathology   MeSH
• Butyrylcholinesterase chemistry   drug effects   genetics   MeSH
• CHO Cells MeSH
• Cholinesterase Inhibitors chemistry   pharmacology   MeSH
• Cricetulus MeSH
• Fluorenes chemistry   pharmacology   MeSH
• Blood-Brain Barrier drug effects   MeSH
• Enzyme Inhibitors pharmacology   MeSH
• Humans MeSH
• Computer Simulation MeSH
• Receptors, N-Methyl-D-Aspartate antagonists & inhibitors   genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Butyrylcholinesterase MeSH
• Cholinesterase Inhibitors MeSH
• fluorene MeSH Browser
• Fluorenes MeSH
• Enzyme Inhibitors MeSH
• N-methyl D-aspartate receptor subtype 2A MeSH Browser
• NR2B NMDA receptor MeSH Browser
• Receptors, N-Methyl-D-Aspartate MeSH

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.

• Keywords
• 6-chlorotacrine, 7-methoxytacrine, Alzheimer’s disease, bis(7)-tacrine, cholinesterases, in silico, in vitro, squaramides, tacrine,
• MeSH
• Acetylcholinesterase metabolism   MeSH
• Butyrylcholinesterase metabolism   MeSH
• Quinine analogs & derivatives   chemistry   pharmacology   MeSH
• Cholinesterase Inhibitors chemical synthesis   chemistry   pharmacology   MeSH
• Kinetics MeSH
• Humans MeSH
• Models, Molecular MeSH
• Molecular Structure MeSH
• Tacrine chemistry   pharmacology   MeSH
• Dose-Response Relationship, Drug MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Acetylcholinesterase MeSH
• Butyrylcholinesterase MeSH
• Quinine MeSH
• Cholinesterase Inhibitors MeSH
• squaramide MeSH Browser
• Tacrine MeSH

Therapeutic application of newly developed oximes is limited due to their adverse effects on different tissues. Within this article, it has been investigated which morphological changes could be observed in Wistar rats after the treatment with increasing doses of selected acetyl cholinesterase reactivators - asoxime, obidoxime, K027, K048, and K075. Subsequently, heart, diaphragm and musculus popliteus were obtained for pathohistological and semiquantitative analysis 24 hrs and 7 days after im administration of a single dose of 0.1 LD50, 0.5 LD50, and 1.0 LD50 of each oxime. Different muscle damage score was based on an estimation scale from 0 (no damage) to 5 (strong damage). In rats treated with 0.1 LD50 of each oxime, muscle fibres did not show any change. The intensive degeneration was found in all muscles after treatment with 0.5 LD50 of asoxime and obidoxime, respectively. Acute toxic muscle injury was developed within 7 days following treatment with 0.5 LD50 and 1.0 LD50 of each oxime, with the highest values in K048 and K075 group (P < 0.001 vs. control and asoxime), respectively. The early muscle alterations observed in our study seem to contribute to the pathogenesis of the oxime-induced toxic muscle injury, which probably manifests as necrosis and/or inflammation.

• MeSH
• Diaphragm drug effects   injuries   MeSH
• Muscle, Skeletal drug effects   injuries   MeSH
• Rats MeSH
• Myositis chemically induced   MeSH
• Necrosis MeSH
• Oximes toxicity   MeSH
• Rats, Wistar MeSH
• Pyridinium Compounds toxicity   MeSH
• Heart drug effects   MeSH
• Muscles drug effects   pathology   MeSH
• Toxicity Tests, Acute MeSH
• Dose-Response Relationship, Drug MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 1-(4-hydroxyiminomethylpyridinium)-3-(carbamoylpyridinium) propane dibromide MeSH Browser
• 1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium)butane MeSH Browser
• K075 compound MeSH Browser
• Oximes MeSH
• Pyridinium Compounds MeSH

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, nerve agents, pre-treatment, prophylaxis, soman, toxicity,
• MeSH
• Acetylcholinesterase metabolism   MeSH
• Cell Line MeSH
• Cholinesterase Inhibitors chemistry   pharmacology   MeSH
• HeLa Cells MeSH
• Small Molecule Libraries chemistry   pharmacology   MeSH
• Humans MeSH
• Models, Molecular MeSH
• Molecular Structure MeSH
• Nerve Agents adverse effects   MeSH
• Soman adverse effects   MeSH
• Cell Survival drug effects   MeSH
• Dose-Response Relationship, Drug MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Acetylcholinesterase MeSH
• Cholinesterase Inhibitors MeSH
• Small Molecule Libraries MeSH
• Nerve Agents MeSH
• Soman MeSH

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.

• Keywords
• 2-methoxyhuprine, 7-MEOTA, Alzheimer’s disease, acetylcholinesterase, butyrylcholinesterase, huprine Y, tacrine,
• MeSH
• Acetylcholinesterase MeSH
• Enzyme Activation drug effects   MeSH
• Alzheimer Disease drug therapy   MeSH
• Aminoquinolines chemical synthesis   chemistry   pharmacology   MeSH
• Butyrylcholinesterase MeSH
• Cholinesterase Inhibitors chemistry   pharmacology   MeSH
• Blood-Brain Barrier metabolism   MeSH
• Heterocyclic Compounds, 4 or More Rings chemistry   pharmacology   MeSH
• Hydrolysis MeSH
• Inhibitory Concentration 50 MeSH
• Catalytic Domain MeSH
• Humans MeSH
• Molecular Conformation MeSH
• Models, Molecular MeSH
• Molecular Structure MeSH
• Cell Line, Tumor MeSH
• Drug Discovery * MeSH
• Permeability MeSH
• Drug Design MeSH
• Tacrine analogs & derivatives   chemistry   pharmacology   MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Cell Survival drug effects   MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 7-methoxytacrine MeSH Browser
• Acetylcholinesterase MeSH
• Aminoquinolines MeSH
• Butyrylcholinesterase MeSH
• Cholinesterase Inhibitors MeSH
• Heterocyclic Compounds, 4 or More Rings MeSH
• huprine Y MeSH Browser
• Tacrine MeSH

Acetylcholinesterase (AChE) reactivators were developed for the treatment of organophosphate intoxication. Standard care involves the use of anticonvulsants (e.g., diazepam), parasympatolytics (e.g., atropine) and oximes that restore AChE activity. However, oximes also bind to the active site of AChE, simultaneously acting as reversible inhibitors. The goal of the present study is to determine how oxime structure influences the inhibition of human recombinant AChE (hrAChE). Therefore, 24 structurally different oximes were tested and the results compared to the previous eel AChE (EeAChE) experiments. Structural factors that were tested included the number of pyridinium rings, the length and structural features of the linker, and the number and position of the oxime group on the pyridinium ring.

• MeSH
• Acetylcholinesterase chemistry   MeSH
• Cholinesterase Inhibitors chemistry   MeSH
• Catalytic Domain MeSH
• Humans MeSH
• Oximes chemistry   MeSH
• Molecular Docking Simulation MeSH
• Protein Binding MeSH
• Hydrogen Bonding MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Acetylcholinesterase MeSH
• Cholinesterase Inhibitors MeSH
• Oximes MeSH