4-Oxypiperidine Ethers as Multiple Targeting Ligands at Histamine H3 Receptors and Cholinesterases.

This study examines the properties of a novel series of 4-oxypiperidines designed and synthesized as histamine H3R antagonists/inverse agonists based on the structural modification of two lead compounds, viz., ADS003 and ADS009. The products are intended to maintain a high affinity for H3R while simultaneously inhibiting AChE or/and BuChE enzymes. Selected compounds were subjected to hH3R radioligand displacement and gpH3R functional assays. Some of the compounds showed nanomolar affinity. The most promising compound in the naphthalene series was ADS031, which contained a benzyl moiety at position 1 of the piperidine ring and displayed 12.5 nM affinity at the hH3R and the highest inhibitory activity against AChE (IC50 = 1.537 µM). Eight compounds showed over 60% eqBuChE inhibition and hence were qualified for the determination of the IC50 value at eqBuChE; their values ranged from 0.559 to 2.655 µM. Therapy based on a multitarget-directed ligand combining H3R antagonism with additional AChE/BuChE inhibitory properties might improve cognitive functions in multifactorial Alzheimer's disease.

Multifunctional agents against Alzheimer's disease based on oxidative stress: Polysubstituted pyrazine derivatives synthesized by multicomponent reactions.

As Alzheimer's disease (AD) is a neurodegenerative disease with a complex pathogenesis, the exploration of multi-target drugs may be an effective strategy for AD treatment. Multifunctional small molecular agents can be obtained by connecting two or more active drugs or privileged pharmacophores by multicomponent reactions (MCRs). In this paper, two series of polysubstituted pyrazine derivatives with multifunctional moieties were designed as anti-AD agents and synthesized by Passerini-3CR and Ugi-4CR. Since the oxidative stress plays an important role in the pathological process of AD, the antioxidant activities of the newly synthesized compounds were first evaluated. Subsequently, selected active compounds were further screened in a series of AD-related bioassays, including Aß1-42 self-aggregation and deaggregation, BACE-1 inhibition, metal chelation, and protection of SH-SY5Y cells from H2O2-induced oxidative damage. Compound A3B3C1 represented the best one with multifunctional potencies. Mechanism study showed that A3B3C1 acted on Nrf2/ARE signaling pathway, thus increasing the expression of related antioxidant proteins NQO1 and HO-1 to normal cell level. Furthermore, A3B3C1 showed good in vitro human plasma and liver microsome stability, indicating a potential for further development as multifunctional anti-AD agent.

Discovery of novel multifunctional ligands targeting GABA transporters, butyrylcholinesterase, ß-secretase, and amyloid ß aggregation as potential treatment of Alzheimer's disease.

Alzheimer's disease (AD) is a global health problem in the medical sector that will increase over time. The limited treatment of AD leads to the search for a new clinical candidate. Considering the multifactorial nature of AD, a strategy targeting number of regulatory proteins involved in the development of the disease is an effective approach. Here, we present a discovery of new multi-target-directed ligands (MTDLs), purposely designed as GABA transporter (GAT) inhibitors, that successfully provide the inhibitory activity against butyrylcholinesterase (BuChE), ß-secretase (BACE1), amyloid ß aggregation and calcium channel blockade activity. The selected GAT inhibitors, 19c and 22a - N-benzylamide derivatives of 4-aminobutyric acid, displayed the most prominent multifunctional profile. Compound 19c (mGAT1 IC50 = 10 µM, mGAT4 IC50 = 12 µM and BuChE IC50 = 559 nM) possessed the highest hBACE1 and Aß40 aggregation inhibitory activity (IC50 = 1.57 µM and 99 % at 10 µM, respectively). Additionally, it showed a decrease in both the elongation and nucleation constants of the amyloid aggregation process. In contrast compound 22a represented the highest activity and a mixed-type of eqBuChE inhibition (IC50 = 173 nM) with hBACE1 (IC50 = 9.42 µM), Aß aggregation (79 % at 10 µM) and mGATs (mGAT1 IC50 = 30 µM, mGAT4 IC50 = 25 µM) inhibitory activity. Performed molecular docking studies described the mode of interactions with GATs and enzymatic targets. In ADMET in vitro studies both compounds showed acceptable metabolic stability and low neurotoxicity. Successfully, compounds 19c and 22a at the dose of 30 mg/kg possessed statistically significant antiamnesic properties in a mouse model of amnesia caused by scopolamine and assessed in the novel object recognition (NOR) task or the passive avoidance (PA) task.

Guanidines: Synthesis of Novel Histamine H3R Antagonists with Additional Breast Anticancer Activity and Cholinesterases Inhibitory Effect.

This study examines the properties of novel guanidines, designed and synthesized as histamine H3R antagonists/inverse agonists with additional pharmacological targets. We evaluated their potential against two targets viz., inhibition of MDA-MB-231, and MCF-7 breast cancer cells viability and inhibition of AChE/BuChE. ADS10310 showed micromolar cytotoxicity against breast cancer cells, combined with nanomolar affinity at hH3R, and may represent a promising target for the development of an alternative method of cancer therapy. Some of the newly synthesized compounds showed moderate inhibition of BuChE in the single-digit micromolar concentration ranges. H3R antagonist with additional AChE/BuChE inhibitory effect might improve cognitive functions in Alzheimer's disease. For ADS10310, several in vitro ADME-Tox parameters were evaluated and indicated that it is a metabolically stable compound with weak hepatotoxic activity and can be accepted for further studies.

Multitargeting Histamine H3 Receptor Ligands among Acetyl- and Propionyl-Phenoxyalkyl Derivatives.

Alzheimer's disease (AD) is a neurodegenerative disorder, for which there is no effective cure. Current drugs only slow down the course of the disease, and, therefore, there is an urgent need to find effective therapies that not only treat, but also prevent it. Acetylcholinesterase inhibitors (AChEIs), among others, have been used for years to treat AD. Histamine H3 receptors (H3Rs) antagonists/inverse agonists are indicated for CNS diseases. Combining AChEIs with H3R antagonism in one structure could bring a beneficial therapeutic effect. The aim of this study was to find new multitargetting ligands. Thus, continuing our previous research, acetyl- and propionyl-phenoxy-pentyl(-hexyl) derivatives were designed. These compounds were tested for their affinity to human H3Rs, as well as their ability to inhibit cholinesterases (acetyl- and butyrylcholinesterases) and, additionally, human monoamine oxidase B (MAO B). Furthermore, for the selected active compounds, their toxicity towards HepG2 or SH-SY5Y cells was evaluated. The results showed that compounds 16 (1-(4-((5-(azepan-1-yl)pentyl)oxy)phenyl)propan-1-one) and 17 (1-(4-((6-(azepan-1-yl)hexyl)oxy)phenyl)propan-1-one) are the most promising, with a high affinity for human H3Rs (Ki: 30 nM and 42 nM, respectively), a good ability to inhibit cholinesterases (16: AChE IC50 = 3.60 µM, BuChE IC50 = 0.55 µM; 17: AChE IC50 = 1.06 µM, BuChE IC50 = 2.86 µM), and lack of cell toxicity up to 50 µM.

Discovery of new, highly potent and selective inhibitors of BuChE - design, synthesis, in vitro and in vivo evaluation and crystallography studies.

The symptomatic and disease-modifying effects of butyrylcholinesterase (BuChE) inhibitors provide an encouraging premise for researching effective treatments for Alzheimer's disease. Here, we examined a series of compounds with a new chemical scaffold based on 3-(cyclohexylmethyl)amino-2-hydroxypropyl, and we identified a highly selective hBuChE inhibitor (29). Based on extensive in vitro and in vivo evaluations of the compound and its enantiomers, (R)-29 was identified as a promising candidate for further development. Compound (R)-29 is a potent hBuChE inhibitor (IC50 = 40 nM) with selectivity over AChE and relevant off-targets, including H1, M1, α1A and ß1 receptors. The compound displays high metabolic stability on human liver microsomes (90% of the parent compound after 2 h of incubation), and its safety was confirmed through examining the cytotoxicity on the HepG2 cell line (LC50 = 2.85 µM) and hERG inhibition (less than 50% at 10 µM). While (rac)-29 lacked an effect in vivo and showed limited penetration to the CNS in pharmacokinetics studies, compound (R)-29 exhibited a procognitive effect at 15 mg/kg in the passive avoidance task in scopolamine-treated mice.

Serotonin 5-HT6 Receptor Ligands and Butyrylcholinesterase Inhibitors Displaying Antioxidant Activity-Design, Synthesis and Biological Evaluation of Multifunctional Agents against Alzheimer's Disease.

Neurodegeneration leading to Alzheimer's disease results from a complex interplay of a variety of processes including misfolding and aggregation of amyloid beta and tau proteins, neuroinflammation or oxidative stress. Therefore, to address more than one of these, drug discovery programmes focus on the development of multifunctional ligands, preferably with disease-modifying and symptoms-reducing potential. Following this idea, herein we present the design and synthesis of multifunctional ligands and biological evaluation of their 5-HT6 receptor affinity (radioligand binding assay), cholinesterase inhibitory activity (spectroscopic Ellman's assay), antioxidant activity (ABTS assay) and metal-chelating properties, as well as a preliminary ADMET properties evaluation. Based on the results we selected compound 14 as a well-balanced and potent 5-HT6 receptor ligand (Ki = 22 nM) and human BuChE inhibitor (IC50 = 16 nM) with antioxidant potential expressed as a reduction of ABTS radicals by 35% (150 µM). The study also revealed additional metal-chelating properties of compounds 15 and 18. The presented compounds modulating Alzheimer's disease-related processes might be further developed as multifunctional ligands against the disease.

Benzophenone Derivatives with Histamine H3 Receptor Affinity and Cholinesterase Inhibitory Potency as Multitarget-Directed Ligands for Possible Therapy of Alzheimer's Disease.

The multitarget-directed ligands demonstrating affinity to histamine H3 receptor and additional cholinesterase inhibitory potency represent a promising strategy for research into the effective treatment of Alzheimer's disease. In this study, a novel series of benzophenone derivatives was designed and synthesized. Among these derivatives, we identified compound 6 with a high affinity for H3R (Ki = 8 nM) and significant inhibitory activity toward BuChE (IC50 = 172 nM and 1.16 µM for eqBuChE and hBuChE, respectively). Further in vitro studies revealed that compound 6 (4-fluorophenyl) (4-((5-(piperidin-1-yl)pentyl)oxy)phenyl)methanone) displays moderate metabolic stability in mouse liver microsomes, good permeability with a permeability coefficient value (Pe) of 6.3 × 10-6 cm/s, and its safety was confirmed in terms of hepatotoxicity in the HepG2 cell line. Therefore, we investigated the in vivo activity of compound 6 in the Passive Avoidance Test and the Formalin Test. While compound 6 did not show a statistically significant influence on memory and learning, it showed analgesic properties in both acute (ED50 = 20.9 mg/kg) and inflammatory (ED50 = 17.5 mg/kg) pain.

Development and crystallography-aided SAR studies of multifunctional BuChE inhibitors and 5-HT6R antagonists with ß-amyloid anti-aggregation properties.

The lack of an effective treatment makes Alzheimer's disease a serious healthcare problem and a challenge for medicinal chemists. Herein we report interdisciplinary research on novel multifunctional ligands targeting proteins and processes involved in the development of the disease: BuChE, 5-HT6 receptors and ß-amyloid aggregation. Structure-activity relationship analyses supported by crystallography and docking studies led to the identification of a fused-type multifunctional ligand 50, with remarkable and balanced potencies against BuChE (IC50 = 90 nM) and 5-HT6R (Ki = 4.8 nM), and inhibitory activity against Aß aggregation (53% at 10 µM). In in vitro ADME-Tox and in vivo pharmacokinetic studies compound 50 showed good stability in the mouse liver microsomes, favourable safety profile and brain permeability with the brain to plasma ratio of 6.79 after p.o. administration in mice, thus being a promising candidate for in vivo pharmacology studies and a solid foundation for further research on effective anti-AD therapies.

Discovery of 1-(phenylsulfonyl)-1H-indole-based multifunctional ligands targeting cholinesterases and 5-HT6 receptor with anti-aggregation properties against amyloid-beta and tau.

Multifunctional ligands as an essential variant of polypharmacology are promising candidates for the treatment of multi-factorial diseases like Alzheimer's disease. Based on clinical evidence and following the paradigm of multifunctional ligands we have rationally designed and synthesized a series of compounds targeting processes involved in the development of the disease. The biological evaluation led to the discovery of two compounds with favorable pharmacological characteristics and ADMET profile. Compounds 17 and 35 are 5-HT6R antagonists (Ki = 13 nM and Ki = 15 nM respectively) and cholinesterase inhibitors with distinct mechanisms of enzyme inhibition. Compound 17, a tacrine derivative is a reversible inhibitor of acetyl- and butyrylcholinesterase (IC50 = 8 nM and IC50 = 24 nM respectively), while compound 35 with rivastigmine-derived phenyl N-ethyl-N-methylcarbamate fragment is a selective, pseudo-irreversible inhibitor of butyrylcholinesterase (IC50 = 455 nM). Both compounds inhibit aggregation of amyloid ß in vitro (75% for compound 17 and 68% for 35 at 10 µM) moreover, compound 35 is a potent tau aggregation inhibitor in cellulo (79%). In ADMET in vitro studies both compounds showed acceptable metabolic stability on mouse liver microsomes (28% and 60% for compound 17 and 35 respectively), no or little effect on CYP3A4 and 2D6 up to a concentration of 10 µM and lack of toxicity on HepG2 cell line (IC50 values of 80 and 21 µM, for 17 and 35 respectively). Based on the pharmacological characteristics and favorable pharmacokinetic properties, we propose compounds 17 and 35 as an excellent starting point for further optimization and in-depth biological studies.

Biphenylalkoxyamine Derivatives-Histamine H3 Receptor Ligands with Butyrylcholinesterase Inhibitory Activity.

Neurodegenerative diseases, e.g., Alzheimer's disease (AD), are a key health problem in the aging population. The lack of effective therapy and diagnostics does not help to improve this situation. It is thought that ligands influencing multiple but interconnected targets can contribute to a desired pharmacological effect in these complex illnesses. Histamine H3 receptors (H3Rs) play an important role in the brain, influencing the release of important neurotransmitters, such as acetylcholine. Compounds blocking their activity can increase the level of these neurotransmitters. Cholinesterases (acetyl- and butyrylcholinesterase) are responsible for the hydrolysis of acetylcholine and inactivation of the neurotransmitter. Increased activity of these enzymes, especially butyrylcholinesterase (BuChE), is observed in neurodegenerative diseases. Currently, cholinesterase inhibitors: donepezil, rivastigmine and galantamine are used in the symptomatic treatment of AD. Thus, compounds simultaneously blocking H3R and inhibiting cholinesterases could be a promising treatment for AD. Herein, we describe the BuChE inhibitory activity of H3R ligands. Most of these compounds show high affinity for human H3R (Ki < 150 nM) and submicromolar inhibition of BuChE (IC50 < 1 µM). Among all the tested compounds, 19 (E153, 1-(5-([1,1'-biphenyl]-4-yloxy)pentyl)azepane) exhibited the most promising in vitro affinity for human H3R, with a Ki value of 33.9 nM, and for equine serum BuChE, with an IC50 of 590 nM. Moreover, 19 (E153) showed inhibitory activity towards human MAO B with an IC50 of 243 nM. Furthermore, in vivo studies using the Passive Avoidance Task showed that compound 19 (E153) effectively alleviated memory deficits caused by scopolamine. Taken together, these findings suggest that compound 19 can be a lead structure for developing new anti-AD agents.

Cyanobiphenyls: Novel H3 receptor ligands with cholinesterase and MAO B inhibitory activity as multitarget compounds for potential treatment of Alzheimer's disease.

Alzheimer's disease (AD) is a complex and incurable illness that requires the urgent approval of new effective drugs. However, since 2003, no new molecules have shown successful results in clinical trials, thereby making the common "one compound - one target" paradigm questionable. Recently, the multitarget-directed ligand (MTDL) approach has gained popularity, as compounds targeting at least two biological targets may be potentially more effective in treating AD. On the basis of these findings, we designed, synthesized, and evaluated through biological assays a series of derivatives of alicyclic amines linked by an alkoxy bridge to an aromatic lipophilic moiety of [1,1'-biphenyl]-4-carbonitrile. The research results revealed promising biological activity of the obtained compounds toward the chosen targets involved in AD pathophysiology; the compounds showed high affinity (mostly low nanomolar range of Ki values) for human histamine H3 receptors (hH3R) and good nonselective inhibitory potency (micromolar range of IC50 values) against acetylcholinesterase from electric eel (eeAChE) and equine serum butyrylcholinesterase (eqBuChE). Moreover, micromolar/submicromolar potency against human monoamine oxidase B (hMAO B) was detected for some compounds. The study identified compound 5 as a multiple hH3R/eeAChE/eqBuChE/hMAO B ligand (5: hH3R Ki = 9.2 nM; eeAChE IC50 = 2.63 µM; eqBuChE IC50 = 1.30 µM; hMAO B IC50 = 0.60 µM). Further in vitro studies revealed that compound 5 exhibits a mixed type of eeAChE and eqBuChE inhibition, good metabolic stability, and moderate hepatotoxicity effect on HepG2 cells. Finally, compound 5 showed a beneficial effect on scopolamine-induced memory impairments, as assessed by the passive avoidance test, thus revealing the potential of this compound as a promising agent for further optimization for AD treatment.

Discovery of multifunctional anti-Alzheimer's agents with a unique mechanism of action including inhibition of the enzyme butyrylcholinesterase and γ-aminobutyric acid transporters.

Looking for an effective anti-Alzheimer's agent is very challenging; however, a multifunctional ligand strategy may be a promising solution for the treatment of this complex disease. We herein present the design, synthesis and biological evaluation of novel hydroxyethylamine derivatives displaying unique, multiple properties that have not been previously reported. The original mechanism of action combines inhibitory activity against disease-modifying targets: ß-secretase enzyme (BACE1) and amyloid ß (Aß) aggregation, along with an effect on targets associated with symptom relief - inhibition of butyrylcholinesterase (BuChE) and γ-aminobutyric acid transporters (GATs). Among the obtained molecules, compound 36 exhibited the most balanced and broad activity profile (eeAChE IC50 = 2.86 µM; eqBuChE IC50 = 60 nM; hBuChE IC50 = 20 nM; hBACE1 IC50 = 5.9 µM; inhibition of Aß aggregation = 57.9% at 10 µM; mGAT1 IC50 = 10.96 µM; and mGAT2 IC50 = 19.05 µM). Moreover, we also identified 31 as the most potent mGAT4 and hGAT3 inhibitor (IC50 = 5.01 µM and IC50 = 2.95 µM, respectively), with high selectivity over other subtypes. Compounds 36 and 31 represent new anti-Alzheimer agents that can ameliorate cognitive decline and modify the progress of disease.

Multidirectional in vitro and in cellulo studies as a tool for identification of multi-target-directed ligands aiming at symptoms and causes of Alzheimer's disease.

Effective therapy of Alzheimer's disease (AD) requires treatment with a combination of drugs that modulate various pathomechanisms contributing to the disease. In our research, we have focused on the development of multi-target-directed ligands - 5-HT6 receptor antagonists and cholinesterase inhibitors - with disease-modifying properties. We have performed extended in vitro (FRET assay) and in cellulo (Escherichia coli model of protein aggregation) studies on their ß-secretase, tau, and amyloid ß aggregation inhibitory activity. Within these multifunctional ligands, we have identified compound 17 with inhibitory potency against tau and amyloid ß aggregation in in cellulo assay of 59% and 56% at 10 µM, respectively, hBACE IC50=4 µM, h5TH6 K i=94 nM, hAChE IC50=26 nM, and eqBuChE IC50=5 nM. This study led to the development of multifunctional ligands with a broad range of biological activities crucial not only for the symptomatic but also for the disease-modifying treatment of AD.

Rational design of new multitarget histamine H3 receptor ligands as potential candidates for treatment of Alzheimer's disease.

Design and development of multitarget-directed ligands (MTDLs) has become a very important approach in the search of new therapies for Alzheimer's disease (AD). In our present research, a number of xanthone derivatives were first designed using a pharmacophore model for histamine H3 receptor (H3R) antagonists/inverse agonists, and virtual docking was then performed for the enzyme acetylcholinesterase. Next, 23 compounds were synthesised and evaluated in vitro for human H3R (hH3R) affinity and inhibitory activity on cholinesterases. Most of the target compounds showed hH3R affinities in nanomolar range and exhibited cholinesterase inhibitory activity with IC50 values in submicromolar range. Furthermore, the inhibitory effects of monoamine oxidases (MAO) A and B were investigated. The results showed low micromolar and selective human MAO B (hMAO B) inhibition. Two azepane derivatives, namely 23 (2-(5-(azepan-1-yl)pentyloxy)-9H-xanthen-9-one) and 25 (2-(5-(azepan-1-yl)pentyloxy)-7-chloro-9H-xanthen-9-one), were especially very promising and showed high affinity for hH3R (Ki = 170 nM and 100 nM respectively) and high inhibitory activity for acetylcholinesterase (IC50 = 180 nM and 136 nM respectively). Moreover, these compounds showed moderate inhibitory activity for butyrylcholinesterase (IC50 = 880 nM and 394 nM respectively) and hMAO B (IC50 = 775 nM and 897 nM respectively). Furthermore, molecular docking studies were performed for hH3R, human cholinesterases and hMAO B to describe the mode of interactions with these biological targets. Next, the two most promising compounds 23 and 25 were selected for in vivo studies. The results showed significant memory-enhancing effect of compound 23 in dizocilpine-induced amnesia in rats in two tests: step-through inhibitory avoidance paradigm (SIAP) and transfer latency paradigm time (TLPT). In addition, favourable analgesic effects of compound 23 were observed in neuropathic pain models. Therefore, compound 23 is a particularly promising structure for further design of new MTDLs for AD.

Novel application of capillary electrophoresis with a liposome coated capillary for prediction of blood-brain barrier permeability.

Profiling blood-brain barrier permeability of bioactive molecule is an important issue in early drug development, being a part of the optimization process of a compound's physicochemical properties, and hence pharmacokinetic profile. The study aimed to develop and optimize a new in vitro method for assessment of the compound's brain penetration. The tool is proposed as an alternative to the PAMPA-BBB (Parallel Artificial Membrane Permeability Assay for Blood-Brain Barrier) and based on a capillary electrochromatography (CEC) technique. It utilizes liposomes as structural substitutes of biological membranes, which are used as a capillary inner wall coating material. Following optimization of analysis conditions, migration times for a set of 25 reference drugs (mainly non-ionized in pH 7.4) were examined in a liposome coated capillary. On that basis, the retention factor (log k) was determined for each reference drug. Obtained log k values and experimentally received reference permeability parameters: log BB (in vivo data) and log Pe (PAMPA-BBB data) were compared with one another. Correlation coefficients were calculated, giving comparable results for CEC log k/log BB and analogical PAMPA-BBB log Pe/log BB analyses. Approximate ranges of log k for the central nervous system (CNS) permeable (CNS(+)) and non-permeable (CNS(-)) drugs were established.

Dual Action of Dipyridothiazine and Quinobenzothiazine Derivatives-Anticancer and Cholinesterase-Inhibiting Activity.

The inverse correlation observed between Alzheimer's disease (AD) and cancer has prompted us to look for cholinesterase-inhibiting activity in phenothiazine derivatives that possess anticancer properties. With the use of in silico and in vitro screening methods, our study found a new biological activity in anticancer polycyclic, tricyclic, and tetracyclic compounds. The virtual screening of a library of 120 ligands, which are the derivatives of azaphenothiazine, led to the identification of 25 compounds that can act as potential inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Biological assays revealed the presence of selective inhibitors of eeAChE (electric eel AChE) or eqBuChE (equine serum BuChE) and nonselective inhibitors of both enzymes among the tested compounds. Their potencies against eeAChE were in a submicromolar-to-micromolar range with IC50 values from 0.78 to 19.32 µM, while their IC50 values against eqBuChE ranged from 0.46 to 10.38 µM. The most potent among the compounds tested was the tetracyclic derivative, 6-(4-diethylaminobut-2-ynyl)-9-methylthioquinobenzothiazine 24, which was capable of inhibiting both enzymes. 9-Fluoro-6-(1-piperidylethyl)quinobenzothiazine 23 was found to act as a selective inhibitor of eqBuChE with an IC50 value of 0.46 µM. Compounds with such a dual antitumor and cholinesterase-inhibitory activity can be considered as a valuable combination for the treatment of both cancer and AD prevention. The results presented in this study might open new directions of research on the group of tricyclic phenothiazine derivatives.

Search for new multi-target compounds against Alzheimer's disease among histamine H3 receptor ligands.

Multi-target-directed ligands seem to be an interesting approach to the treatment of complex disorders such as Alzheimer's disease. The aim of the present study was to find novel multifunctional compounds in a non-imidazole histamine H3 receptor ligand library. Docking-based virtual screening was applied for selection of twenty-six hits which were subsequently evaluated in Ellman's assay for the inhibitory potency toward acetyl- (AChE) and butyrylcholinesterase (BuChE). The virtual screening with high success ratio enabled to choose multi-target-directed ligands. Based on docking results, all selected ligands were able to bind both catalytic and peripheral sites of AChE and BuChE. The most promising derivatives combined the flavone moiety via a six carbon atom linker with a heterocyclic moiety, such as azepane, piperidine or 3-methylpiperidine. They showed the highest inhibitory activities toward cholinesterases as well as well-balanced potencies against H3R and both enzymes. Two derivatives were chosen - 5 (IC50 = 0.46 µM (AChE); 0.44 µM (BuChE); Ki = 159.8 nM (H3R)) and 17 (IC50 = 0.50 µM (AChE); 0.76 µM (BuChE); Ki = 228.2 nM (H3R)), and their inhibition mechanism was evaluated in kinetic studies. Both compounds displayed non-competitive mode of AChE and BuChE inhibition. Compounds 5 and 17 might serve as good lead structures for further optimization and development of novel multi-target anti-Alzheimer's agents.

1-Benzylpyrrolidine-3-amine-based BuChE inhibitors with anti-aggregating, antioxidant and metal-chelating properties as multifunctional agents against Alzheimer's disease.

Complex pathomechanism of Alzheimer's disease (AD) prompts researchers to develop multifunctional molecules in order to find effective therapy against AD. We designed and synthesized novel multifunctional ligands for which we assessed their activities towards butyrylcholinesterase, beta secretase, amyloid beta (Aß) and tau protein aggregation as well as antioxidant and metal-chelating properties. All compounds showed dual anti-aggregating properties towards Aß and tau protein in the in cellulo assay in Escherichia coli. Of particular interest are compounds 24b and 25b, which efficiently inhibit aggregation of Aß and tau protein at 10 µM (24b: 45% for Aß, 53% for tau; 25b: 49% for Aß, 54% for tau). They display free radical scavenging capacity and antioxidant activity in ABTS and FRAP assays, respectively, and selectively chelate copper ions. Compounds 24b and 25b are also the most potent inhibitors of BuChE with IC50 of 2.39 µM and 1.94 µM, respectively. Promising in vitro activities of the presented multifunctional ligands as well as their original scaffold are a very interesting starting point for further research towards effective anti-AD treatment.