Connecting GSK-3ß Inhibitory Activity with IKK-ß or ROCK-1 Inhibition to Target Tau Aggregation and Neuroinflammation in Alzheimer's Disease-Discovery, In Vitro and In Cellulo Activity of Thiazole-Based Inhibitors.

GSK-3ß, IKK-ß, and ROCK-1 kinases are implicated in the pathomechanism of Alzheimer's disease due to their involvement in the misfolding and accumulation of amyloid ß (Aß) and tau proteins, as well as inflammatory processes. Among these kinases, GSK-3ß plays the most crucial role. In this study, we present compound 62, a novel, remarkably potent, competitive GSK-3ß inhibitor (IC50 = 8 nM, Ki = 2 nM) that also exhibits additional ROCK-1 inhibitory activity (IC50 = 2.3 µM) and demonstrates anti-inflammatory and neuroprotective properties. Compound 62 effectively suppresses the production of nitric oxide (NO) and pro-inflammatory cytokines in the lipopolysaccharide-induced model of inflammation in the microglial BV-2 cell line. Furthermore, it shows neuroprotective effects in an okadaic-acid-induced tau hyperphosphorylation cell model of neurodegeneration. The compound also demonstrates the potential for further development, characterized by its chemical and metabolic stability in mouse microsomes and fair solubility.

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.

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.

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.

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.

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.

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.

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 multifunctional agents against Alzheimer's disease among non-imidazole histamine H3 receptor ligands. In vitro and in vivo pharmacological evaluation and computational studies of piperazine derivatives.

In the search for new treatments for complex disorders such as Alzheimer's disease the Multi-Target-Directed Ligands represent a very promising approach. The aim of the present study was to identify multifunctional compounds among several series of non-imidazole histamine H3 receptor ligands, derivatives of 1-[2-thiazol-5-yl-(2-aminoethyl)]-4-n-propylpiperazine, 1-[2-thiazol-4-yl-(2-aminoethyl)]-4-n-propylpiperazine and 1-phenoxyalkyl-4-(amino)alkylopiperazine using in vitro and in vivo pharmacological evaluation and computational studies. Performed in vitro assays showed moderate potency of tested compounds against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Molecular modeling studies have revealed possible interactions between the active compounds and both AChE and BuChE as well as the human H3 histamine receptor. Computational studies showed the high drug-likeness of selected compounds with very good physicochemical profiles. The parallel artificial membrane permeation assay proved outstanding blood-brain barrier penetration in test conditions. The most promising compound, A12, chemically methyl(4-phenylbutyl){2-[2-(4-propylpiperazin-1-yl)-1,3-thiazol-5-yl]ethyl}amine, possesses good balanced multifunctional profile with potency toward studied targets - H3 antagonist activity (pA2 = 8.27), inhibitory activity against both AChE (IC50 = 13.96 µM), and BuChE (IC50 = 14.62 µM). The in vivo pharmacological studies revealed the anti-amnestic properties of compound A12 in the passive avoidance test on mice.

Anti-Alzheimer's multitarget-directed ligands with serotonin 5-HT6 antagonist, butyrylcholinesterase inhibitory, and antioxidant activity.

Serotonin 5-HT6 receptors, butyrylcholinesterase (BuChE) and oxidative stress are related to the pathophysiology of Alzheimer's disease. Inhibition of BuChE provides symptomatic treatment of the disease and the same effect was demonstrated for 5-HT 6 antagonists in clinical trials. Oxidative stress is regarded as a major and primary factor contributing to the development of Alzheimer's disease; therefore, antioxidant agents may provide a disease-modifying effect. Combining BuChE inhibition, 5-HT 6 antagonism, and antioxidant properties may result in multitarget-directed ligands providing cognition-enhancing properties with neuroprotective activity. On the basis of the screening of the library of 5-HT 6 antagonists against BuChE, we selected two compounds and designed their structural modifications that could lead to improved BuChE inhibitory activity. We synthesized two series of compounds and tested their affinity and functional activity at 5-HT 6 receptors, BuChE inhibitory activity and antioxidant properties. Compound 12 with K i and K b values against 5-HT 6 receptors of 41.8 and 74 nM, respectively, an IC 50 value of 5 µM against BuChE and antioxidant properties exceeding the activity of ascorbic acid is a promising lead structure for further development of anti-Alzheimer's agents.

Design, Synthesis, and Biological Evaluation of 2-(Benzylamino-2-Hydroxyalkyl)Isoindoline-1,3-Diones Derivatives as Potential Disease-Modifying Multifunctional Anti-Alzheimer Agents.

The complex nature of Alzheimer's disease calls for multidirectional treatment. Consequently, the search for multi-target-directed ligands may lead to potential drug candidates. The aim of the present study is to seek multifunctional compounds with expected activity against disease-modifying and symptomatic targets. A series of 15 drug-like various substituted derivatives of 2-(benzylamino-2-hydroxyalkyl)isoindoline-1,3-diones was designed by modification of cholinesterase inhibitors toward ß-secretase inhibition. All target compounds have been synthesized and tested against eel acetylcholinesterase (eeAChE), equine serum butyrylcholinesterase (eqBuChE), human ß-secretase (hBACE-1), and ß-amyloid (Aß-aggregation). The most promising compound, 12 (2-(5-(benzylamino)-4-hydroxypentyl)isoindoline-1,3-dione), displayed inhibitory potency against eeAChE (IC50 = 3.33 µM), hBACE-1 (43.7% at 50 µM), and Aß-aggregation (24.9% at 10 µM). Molecular modeling studies have revealed possible interaction of compound 12 with the active sites of both enzymes-acetylcholinesterase and ß-secretase. IN CONCLUSION: modifications of acetylcholinesterase inhibitors led to the discovery of a multipotent anti-Alzheimer's agent, with moderate and balanced potency, capable of inhibiting acetylcholinesterase, a symptomatic target, and disease-modifying targets: ß-secretase and Aß-aggregation.

Lipophilic properties of anti-Alzheimer's agents determined by micellar electrokinetic chromatography and reversed-phase thin-layer chromatography.

Lipophilicity as one of the most important physicochemical properties of the biologically active compounds is closely related to their pharmacokinetic parameters and therefore, it is taken into account at the design stage of new drugs. Among the novel, fast, and reliable methods for determination of the lipophilicity of compounds micellar electrokinetic chromatography (MEKC) is considered to be an appropriate one for bioactive molecules, as it closely mimics the physiological conditions. In this paper MEKC was used for the estimation of log P values for 49 derivatives of phthalimide, tetrahydroisochinoline and indole, designed and synthesized as potential anti-Alzheimer's agents with cholinesterase inhibitory activity. RP-TLC method was applied for determination of another lipophilicity descriptor - RM0 . The results of both experimental methods were compared with each other giving satisfactory correlation (R = 0.784), and with computational methods (Marvin, ChemOffice Software) resulting in weaker correlation (R = 0.466-0.687). The lipophilicity-activity relationship was finally established, showing significant influence of lipophilicity on cholinesterase inhibition in some subgroups of phthalimide derivatives.

Synthesis, Molecular Modelling and Biological Evaluation of Novel Heterodimeric, Multiple Ligands Targeting Cholinesterases and Amyloid Beta.

Cholinesterases and amyloid beta are one of the major biological targets in the search for a new and efficacious treatment of Alzheimer's disease. The study describes synthesis and pharmacological evaluation of new compounds designed as dual binding site acetylcholinesterase inhibitors. Among the synthesized compounds, two deserve special attention--compounds 42 and 13. The former is a saccharin derivative and the most potent and selective acetylcholinesterase inhibitor (EeAChE IC50 = 70 nM). Isoindoline-1,3-dione derivative 13 displays balanced inhibitory potency against acetyl- and butyrylcholinesterase (BuChE) (EeAChE IC50 = 0.76 µM, EqBuChE IC50 = 0.618 µM), and it inhibits amyloid beta aggregation (35.8% at 10 µM). Kinetic studies show that the developed compounds act as mixed or non-competitive acetylcholinesterase inhibitors. According to molecular modelling studies, they are able to interact with both catalytic and peripheral active sites of the acetylcholinesterase. Their ability to cross the blood-brain barrier (BBB) was confirmed in vitro in the parallel artificial membrane permeability BBB assay. These compounds can be used as a solid starting point for further development of novel multifunctional ligands as potential anti-Alzheimer's agents.

Synthesis and Biological Evaluation of Benzochromenopyrimidinones as Cholinesterase Inhibitors and Potent Antioxidant, Non-Hepatotoxic Agents for Alzheimer's Disease.

We report herein the straightforward two-step synthesis and biological assessment of novel racemic benzochromenopyrimidinones as non-hepatotoxic, acetylcholinesterase inhibitors with antioxidative properties. Among them, compound 3Bb displayed a mixed-type inhibition of human acetylcholinesterase (IC50 = 1.28 ± 0.03 µM), good antioxidant activity, and also proved to be non-hepatotoxic on human HepG2 cell line.

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.

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.

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.

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.

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.