Development of a Potent and Selective G2A (GPR132) Agonist.

G protein-coupled receptor G2A was postulated to be a promising target for the development of new therapeutics in neuropathic pain, acute myeloid leukemia, and inflammation. However, there is still a lack of potent, selective, and drug-like G2A agonists to be used as a chemical tool or as the starting matter for the development of drugs. In this work, we present the discovery and structure-activity relationship elucidation of a new potent and selective G2A agonist scaffold. Systematic optimization resulted in (3-(pyridin-3-ylmethoxy)benzoyl)-d-phenylalanine (T-10418) exhibiting higher potency than the reference and natural ligand 9-HODE and high selectivity among G protein-coupled receptors. With its favorable activity, a clean selectivity profile, excellent solubility, and high metabolic stability, T-10418 qualifies as a pharmacological tool to investigate the effects of G2A activation.

Identification of ligands binding to MB327-PAM-1, a binding pocket relevant for resensitization of nAChRs.

Desensitization of nicotinic acetylcholine receptors (nAChRs) can be induced by overstimulation with acetylcholine (ACh) caused by an insufficient degradation of ACh after poisoning with organophosphorus compounds (OPCs). Currently, there is no generally applicable treatment for OPC poisoning that directly targets the desensitized nAChR. The bispyridinium compound MB327, an allosteric modulator of nAChR, has been shown to act as a resensitizer of nAChRs, indicating that drugs binding directly to nAChRs can have beneficial effects after OPC poisoning. However, MB327 also acts as an inhibitor of nAChRs at higher concentrations and can thus not be used for OPC poisoning treatment. Consequently, novel, more potent resensitizers are required. To successfully design novel ligands, the knowledge of the binding site is of utmost importance. Recently, we performed in silico studies to identify a new potential binding site of MB327, MB327-PAM-1, for which a more affine ligand, UNC0646, has been described. In this work, we performed ligand-based screening approaches to identify novel analogs of UNC0646 to help further understand the structure-affinity relationship of this compound class. Furthermore, we used structure-based screenings and identified compounds representing four new chemotypes binding to MB327-PAM-1. One of these compounds, cycloguanil, is the active metabolite of the antimalaria drug proguanil and shows a higher affinity towards MB327-PAM-1 than MB327. Furthermore, cycloguanil can reestablish the muscle force in soman-inhibited rat muscles. These results can act as a starting point to develop more potent resensitizers of nAChR and to close the gap in the treatment after OPC poisoning.

Synthesis and biological evaluation of novel MB327 analogs as resensitizers for desensitized nicotinic acetylcholine receptors after intoxication with nerve agents.

Poisoning with organophosphorus compounds, which can lead to a cholinergic crisis due to the inhibition of acetylcholinesterase and the subsequent accumulation of acetylcholine (ACh) in the synaptic cleft, is a serious problem for which treatment options are currently insufficient. Our approach to broadening the therapeutic spectrum is to use agents that interact directly with desensitized nicotinic acetylcholine receptors (nAChRs) in order to induce functional recovery after ACh overstimulation. Although MB327, one of the most prominent compounds investigated in this context, has already shown positive properties in terms of muscle force recovery, this compound is not suitable for use as a therapeutic agent due to its insufficient potency. By means of in silico studies based on our recently presented allosteric binding pocket at the nAChR, i.e. the MB327-PAM-1 binding site, three promising MB327 analogs with a 4-aminopyridinium ion partial structure (PTM0056, PTM0062, and PTM0063) were identified. In this study, we present the synthesis and biological evaluation of a series of new analogs of the aforementioned compounds with a 4-aminopyridinium ion partial structure (PTM0064-PTM0072), as well as hydroxy-substituted analogs of MB327 (PTMD90-0012 and PTMD90-0015) designed to substitute entropically unfavorable water clusters identified during molecular dynamics simulations. The compounds were characterized in terms of their binding affinity towards the aforementioned binding site by applying the UNC0642 MS Binding Assays and in terms of their muscle force reactivation in rat diaphragm myography. More potent compounds were identified compared to MB327, as some of them showed a higher affinity towards MB327-PAM-1 and also a higher recovery of neuromuscular transmission at lower compound concentrations. To improve the treatment of organophosphate poisoning, direct targeting of nAChRs with appropriate compounds is a key step, and this study is an important contribution to this research.

Screening for new ligands of the MB327-PAM-1 binding site of the nicotinic acetylcholine receptor.

Intoxications with organophosphorus compounds (OPCs) effect a severe impairment of cholinergic neurotransmission that, as a result of overstimulation may lead to desensitization of nicotinic acetylcholine receptors (nAChRs) and finally to death due to respiratory paralysis. So far, therapeutics, that are capable to address and revert desensitized neuromuscular nAChRs into their resting, i.e. functional state are still missing. Still, among a class of compounds termed bispyridinium salts, which are characterized by the presence of two pyridinium subunits, constituents have been identified, that can counteract organophosphate poisoning by resensitizing desensitized nAChRs. According to comprehensive modeling studies this effect is mediated by an allosteric binding site at the nAChR termed MB327-PAM-1 site. For MB327, the most prominent representative of the bispyridinium salts and all other analogues studied so far, the affinity for the aforementioned binding site and the intrinsic activity measured in ex vivo and in in vivo experiments are distinctly too low, to meet the criteria to be fulfilled for therapeutic use. Hence, in order to identify new compounds with higher affinities for the MB327-PAM-1 binding site, as a basic requirement for an enhanced potency, two compound libraries, the ChemDiv library with 60 constituents and the Tocriscreen Plus library with 1280 members have been screened for hit compounds addressing the MB327-PAM-1 binding site, utilizing the [2H6]MB327 MS Binding Assay recently developed by us. This led to the identification of a set of 10 chemically diverse compounds, all of which exhibit an IC50 value of ≤ 10 µM (in the [2H6]MB327 MS Binding Assay), which had been defined as selection criteria. The three most affine ligands, which besides a quinazoline scaffold share similarities with regard to the substitution pattern and the nature of the substituents, are UNC0638, UNC0642 and UNC0646. With binding affinities expressed as pKi values of 6.01 ± 0.10, 5.97 ± 0.05 and 6.23 ± 0.02, respectively, these compounds exceed the binding affinity of MB327 by more than one log unit. This renders them promising starting points for the development of drugs for the treatment of organophosphorus poisoning by addressing the MB327-PAM-1 binding site of the nAChR.

MS Binding Assays with UNC0642 as reporter ligand for the MB327 binding site of the nicotinic acetylcholine receptor.

Intoxications with organophosphorus compounds (OPCs) based chemical warfare agents and insecticides may result in a detrimental overstimulation of muscarinic and nicotinic acetylcholine receptors evolving into a cholinergic crisis leading to death due to respiratory failure. In the case of the nicotinic acetylcholine receptor (nAChR), overstimulation leads to a desensitization of the receptor, which cannot be pharmacologically treated so far. Still, compounds interacting with the MB327 binding site of the nAChR like the bispyridinium salt MB327 have been found to re-establish the functional activity of the desensitized receptor. Only recently, a series of quinazoline derivatives with UNC0642 as one of the most prominent representatives has been identified to address the MB327 binding site of the nAChR, as well. In this study, UNC0642 has been utilized as a reporter ligand to establish new Binding Assays for this target. These assays follow the concept of MS Binding Assays for which by assessing the amount of bound reporter ligand by mass spectrometry no radiolabeled material is required. According to the results of the performed MS Binding Assays comprising saturation and competition experiments it can be concluded, that UNC0642 used as a reporter ligand addresses the MB327 binding site of the Torpedo-nAChR. This is further supported by the outcome of ex vivo studies carried out with poisoned rat diaphragm muscles as well as by in silico studies predicting the binding mode of UNC0646, an analog of UNC0642 with the highest binding affinity, in the recently proposed binding site of MB327 (MB327-PAM-1). With UNC0642 addressing the MB327 binding site of the Torpedo-nAChR, this and related quinazoline derivatives represent a promising starting point for the development of novel ligands of the nAChR as antidotes for the treatment of intoxications with organophosphorus compounds. Further, the new MS Binding Assays are a potent alternative to established assays and of particular value, as they do not require the use of radiolabeled material and are based on a commercially available compound as reporter ligand, UNC0642, exhibiting one of the highest binding affinities for the MB327 binding site known so far.

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.

Structural Modification of the Natural Product Valerenic Acid Tunes RXR Homodimer Agonism.

Retinoid X receptors (RXR) are ligand-sensing transcription factors with a unique role in nuclear receptor signaling as universal heterodimer partners. RXR modulation holds potential in cancer, neurodegeneration and metabolic diseases but adverse effects of RXR activation and lack of selective modulators prevent further exploration as therapeutic target. The natural product valerenic acid has been discovered as RXR agonist with unprecedented preference for RXR subtype and homodimer activation. To capture structural determinants of this activity profile and identify potential for optimization, we have studied effects of structural modification of the natural product on RXR modulation and identified an analogue with enhanced RXR homodimer agonism.

Structure-Guided Design of Nurr1 Agonists Derived from the Natural Ligand Dihydroxyindole.

The neuroprotective transcription factor Nurr1 was recently found to bind the dopamine metabolite 5,6-dihydroxyindole (DHI) providing access to Nurr1 ligand design from a natural template. We screened a custom set of 14 k extended DHI analogues in silico for optimized descendants to select 24 candidates for microscale synthesis and in vitro testing. Three out of six primary hits were validated as novel Nurr1 agonists with up to sub-micromolar binding affinity, highlighting the druggability of the Nurr1 surface region lining helix 12. In vitro profiling confirmed cellular target engagement of DHI descendants and demonstrated remarkable additive effects of combined Nurr1 agonist treatment, indicating diverse binding sites mediating Nurr1 activation, which may open new avenues in Nurr1 modulation.

Cryo-EM structure of GABA transporter 1 reveals substrate recognition and transport mechanism.

The inhibitory neurotransmitter γ-aminobutyric acid (GABA) is cleared from the synaptic cleft by the sodium- and chloride-coupled GABA transporter GAT1. Inhibition of GAT1 prolongs the GABAergic signaling at the synapse and is a strategy to treat certain forms of epilepsy. In this study, we present the cryo-electron microscopy structure of Rattus norvegicus GABA transporter 1 (rGAT1) at a resolution of 3.1 Å. The structure elucidation was facilitated by epitope transfer of a fragment-antigen binding (Fab) interaction site from the Drosophila dopamine transporter (dDAT) to rGAT1. The structure reveals rGAT1 in a cytosol-facing conformation, with a linear density in the primary binding site that accommodates a molecule of GABA, a displaced ion density proximal to Na site 1 and a bound chloride ion. A unique insertion in TM10 aids the formation of a compact, closed extracellular gate. Besides yielding mechanistic insights into ion and substrate recognition, our study will enable the rational design of specific antiepileptics.

Targeting the Alternative Vitamin E Metabolite Binding Site Enables Noncanonical PPARγ Modulation.

The lipid-sensing transcription factor PPARγ is the target of antidiabetic thiazolidinediones (TZD). At two sites within its ligand binding domain, it also binds oxidized vitamin E metabolites and the vitamin E mimetic garcinoic acid. While the canonical interaction within the TZD binding site mediates classical PPARγ activation, the effects of the second binding on PPARγ activity remain elusive. Here, we identified an agonist mimicking dual binding of vitamin E metabolites and developed a selective ligand of the second site, unveiling potential noncanonical regulation of PPARγ activities. We found that this alternative binding event can simultaneously occur with orthosteric ligands and it exerted different effects on PPARγ-cofactor interactions compared to both orthosteric PPARγ agonists and antagonists, indicating the diverse roles of the two binding sites. Alternative site binding lacked the pro-adipogenic effect of TZD and mediated no classical PPAR signaling in differential gene expression analysis but markedly diminished FOXO signaling, suggesting potential therapeutic applications.

Development of a Potent Nurr1 Agonist Tool for In Vivo Applications.

Nuclear receptor related 1 (Nurr1) is a neuroprotective transcription factor and an emerging target in neurodegenerative diseases. Despite strong evidence for a role in Parkinson's and Alzheimer's disease, pharmacological control and validation of Nurr1 are hindered by a lack of suitable ligands. We have discovered considerable Nurr1 activation by the clinically studied dihydroorotate dehydrogenase (DHODH) inhibitor vidofludimus calcium and systematically optimized this scaffold to a Nurr1 agonist with nanomolar potency, strong activation efficacy, and pronounced preference over the highly related receptors Nur77 and NOR1. The optimized compound induced Nurr1-regulated gene expression in astrocytes and exhibited favorable pharmacokinetics in rats, thus emerging as a superior chemical tool to study Nurr1 activation in vitro and in vivo.

Benocyclidine (BTCP) as Non-labelled Reporter Ligand for MS Binding Assays for the PCP Ion Channel Binding Site of the Desensitized Torpedo Nicotinic Acetylcholine Receptor (nAChR).

In this study we present MS Binding Assays for the PCP ion channel binding site of Torpedo californica nicotinic acetylcholine receptor (nAChR) as an alternative to radioligand binding assays. As MS Marker Benocyclidine (BTCP) was employed, found to be more affine (Kd of 84.2 nM) than the radioligands, e. g. [3 H]PCP, used so far in respective binding assays. Based on a highly sensitive and fast LC-ESI-MS/MS method for quantification of BTCP samples, BTCP MS Binding Assays for the PCP ion channel binding site of Torpedo nAChR could be established comprising saturation, kinetic and competition experiments. The affinities obtained in competitive BTCP MS Binding Assays for ligands addressing the PCP ion channel binding site of Torpedo nAChR were in excellent accord with those reported from radioligand experiments. Thus, the new BTCP MS Binding Assays represent a potent and reliable alternative to radioligand binding assays used so far for the characterization of ligand binding to the PCP ion channel binding site of the nAChR.

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.

A novel binding site in the nicotinic acetylcholine receptor for MB327 can explain its allosteric modulation relevant for organophosphorus-poisoning treatment.

Organophosphorus compounds (OPCs) are highly toxic compounds that can block acetylcholine esterase (AChE) and thereby indirectly lead to an overstimulation of muscarinic and nicotinic acetylcholine receptors (nAChRs). The current treatment with atropine and AChE reactivators (oximes) is insufficient to prevent toxic effects, such as respiratory paralysis, after poisonings with various OPCs. Thus, alternative treatment options are required to increase treatment efficacy. Novel therapeutics, such as the bispyridinium non-oxime MB327, have been found to reestablish neuromuscular transmission by interacting directly with nAChR, probably via allosteric mechanisms. To rationally design new, more potent drugs addressing nAChR, knowledge of the binding mode of MB327 is fundamental. However, the binding pocket of MB327 has remained elusive. Here, we identify a new potential allosteric binding pocket (MB327-PAM-1) of MB327 located at the transition of the extracellular to the transmembrane region using blind docking experiments and molecular dynamics simulations. MB327 forms striking interactions with the receptor at this site. The interacting amino acids are highly conserved among different subunits and different species. Correspondingly, MB327 can interact with several nAChR subtypes from different species. We predict by rigidity analysis that MB327 exerts an allosteric effect on the orthosteric binding pocket and the transmembrane domain after binding to MB327-PAM-1. Furthermore, free ligand diffusion MD simulations reveal that MB327 also has an affinity to the orthosteric binding pocket, which agrees with recently published results that related bispyridinium compounds show inhibitory effects via the orthosteric binding site. The newly identified binding site allowed us to predict structural modifications of MB327, resulting in the more potent resensitizers PTM0062 and PTM0063.

Simultaneous LC-ESI-MS/MS Quantification of Levosimendan and Its Metabolites for Therapeutic Drug Monitoring of Cardiac Surgery Patients.

Levosimendan is used in severe chronic cardiac insufficiency, also within the peri-operative setting. Real-life pharmacokinetic data in surgical patients is lacking, making therapeutic drug monitoring (TDM) of levosimendan, its pharmacologically active metabolite OR-1896, and its intermediate OR-1855 important. A simultaneous highly sensitive quantification of levosimendan and its metabolites in small-volume samples has not yet been described. Here, levosimendan (LLOQ 0.450 nM), OR-1896, and OR-1855 (LLOQ both 1.0 nM) were successfully quantified by LC-ESI-MS/MS after liquid-liquid extraction in 300 µL of blood. A short C8 column under reversed-phase conditions enabled simultaneous and fast quantification of levosimendan in the negative and the metabolites in the positive ionization mode in a single run within 2 min. Interestingly and unexpectedly, constitutional isomers of levosimendan metabolites with identical mass transitions and similar retention times were observed in surgical patients' samples, which we identified as the metamizole metabolites 4-aminoantipyrine and 4-acetamidoantipyrine. A longer C8 column and a modified mobile phase enabled selective quantification of all analytes in a single run within 7 min. We developed, validated, and applied highly sensitive LC-ESI-MS/MS methods for simultaneous quantification of levosimendan and its metabolites, enabling efficient TDM of cardiac surgery patients even with additional metamizole administration.

Novel Functionalized Amino Acids as Inhibitors of GABA Transporters with Analgesic Activity.

Neuropathic pain resistance to pharmacotherapy has encouraged researchers to develop effective therapies for its treatment. γ-Aminobutyric acid (GABA) transporters 1 and 4 (mGAT1 and mGAT4) have been increasingly recognized as promising drug targets for neuropathic pain (NP) associated with imbalances in inhibitory neurotransmission. In this context, we designed and synthesized new functionalized amino acids as inhibitors of GABA uptake and assessed their activities toward all four mouse GAT subtypes (mGAT1-4). According to the obtained results, compounds 2RS,4RS-39c (pIC50 (mGAT4) = 5.36), 50a (pIC50 (mGAT2) = 5.43), and 56a (with moderate subtype selectivity that favored mGAT4, pIC50 (mGAT4) = 5.04) were of particular interest and were therefore evaluated for their cytotoxic and hepatotoxic effects. In a set of in vivo experiments, both compounds 50a and 56a showed antinociceptive properties in three rodent models of NP, namely, chemotherapy-induced neuropathic pain models (the oxaliplatin model and the paclitaxel model) and the diabetic neuropathic pain model induced by streptozotocin; however compound 56a demonstrated predominant activity. Since impaired motor coordination is also observed in neuropathic pain conditions, we have pointed out that none of the test compounds induced motor deficits in the rotarod test.

Screening for New Inhibitors of Glycine Transporter 1 and 2 by Means of MS Binding Assays.

A straightforward screening of a compound library comprising 2439 substances for the identification of new inhibitors for the neurotransmitter transporters GlyT1 and GlyT2 is described. Screening and full-scale competition experiments were performed using recently developed GlyT1 and GlyT2 MS Binding Assays. That way for both targets, GlyT1 and GlyT2, ligands were identified, which exhibited affinities (pKi values) in the low micromolar to sub-micromolar range. The majority of these binders exhibit new chemical scaffolds in the class of GlyT1 and GlyT2 inhibitors, which could be of interest for the development of new ligands with improved affinities for the target proteins. Additionally, compounds with excellent fluorescent properties were found for GlyT2, which renders them promising compounds for future fluorescence-based techniques. All in all, this study demonstrates that MS Binding Assays represent a powerful technology platform also well suited for the screening of compound libraries in a highly reliable and effective manner.

Development of tricyclic N-benzyl-4-hydroxybutanamide derivatives as inhibitors of GABA transporters mGAT1-4 with anticonvulsant, antinociceptive, and antidepressant activity.

γ-Aminobutyric acid (GABA) neurotransmission has a significant impact on the proper functioning of the central nervous system. Numerous studies have indicated that inhibitors of the GABA transporters mGAT1-4 offer a promising strategy for the treatment of several neurological disorders, including epilepsy, neuropathic pain, and depression. Following our previous results, herein, we report the synthesis, biological evaluation, and structure-activity relationship studies supported by molecular docking and molecular dynamics of a new series of N-benzyl-4-hydroxybutanamide derivatives regarding their inhibitory potency toward mGAT1-4. This study allowed us to identify compound 23a (N-benzyl-4-hydroxybutanamide bearing a dibenzocycloheptatriene moiety), a nonselective GAT inhibitor with a slight preference toward mGAT4 (pIC50 = 5.02 ± 0.11), and compound 24e (4-hydroxy-N-[(4-methylphenyl)-methyl]butanamide bearing a dibenzocycloheptadiene moiety) with relatively high inhibitory activity toward mGAT2 (pIC50 = 5.34 ± 0.09). In a set of in vivo experiments, compound 24e successively showed predominant anticonvulsant activity and antinociception in the formalin model of tonic pain. In contrast, compound 23a showed significant antidepressant-like properties in mice. These results were consistent with the available literature data, which indicates that, apart from seizure control, GABAergic neurotransmission is also involved in the pathophysiology of several psychiatric diseases, however alternative mechanisms underlying this action cannot be excluded. Finally, it is worth noting that the selected compounds showed unimpaired locomotor skills that have been indicated to give reliable results in behavioral assays.

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.