molecules Article QuinoxalineTacrine QT78, a Cholinesterase Inhibitor as a Potential Ligand for Alzheimer’s Disease Therapy Eva Ramos 1 , Alejandra Palomino-Antolín 2,3,4 , Manuela Bartolini 5 , Isabel Iriepa 6 , Ignacio Moraleda 6 , Daniel Diez-Iriepa 6,7 , Abdelouahid Samadi 7,8 , Carol V. Cortina 7 , Mourad Chioua 7 , Javier Egea 2,3,4, * , Alejandro Romero 1, * and José Marco-Contelles 7, * 1 Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Complutense University of Madrid, 28040 Madrid, Spain; eva.ramos@ucm.es 2 Molecular Neuroinflammation and Neuronal Plasticity Laboratory, Research Unit, Hospital Universitario Santa Cristina, 28009 Madrid, Spain; apantolin@gmail.com 3 Instituto de Investigación Sanitaria, Hospital Universitario de la Princesa, 28006 Madrid, Spain 4 Instituto-Fundación Teófilo Hernando, Departamento de Farmacología y Terapéutica, Universidad Autónoma de Madrid, 28029 Madrid, Spain 5 Department of Pharmacy and Biotechnology, Alma Mater Studiorum University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy; manuela.bartolini3@unibo.it 6 Departamento de Química Orgánica and Química Inorgánica. Ctra. Madrid-Barcelona, Km. 33,6. Universidad de Alcalá, 28871 Madrid, Spain; isabel.iriepa@uah.es (I.I.); ignacio.moraleda@uah.es (I.M.); daniel.diezi@edu.uah.es (D.D.-I.) 7 Laboratory of Medicinal Chemistry (IQOG, CSIC), C/Juan de la Cierva 3, 28006 Madrid, Spain; samadi@uaeu.ac.ae (A.S.); cvcortina@gmail.com (C.V.C.); mchioua@gmail.com (M.C.) 8 Department of Chemistry, College of Science, United Arab Emirates University, 15551 Al Ain, UAE * Correspondence: javier.egea@inv.uam.es (J.E.); manarome@ucm.es (A.R.); iqoc21@iqog.csic.es (J.M.-C.); Tel.: +34915574402 (J.E.); +34913943970 (A.R.); +34915622900 (J.M.-C.) Academic Editor: Ludovic Jean   Received: 19 February 2019; Accepted: 15 April 2019; Published: 17 April 2019 Abstract: We report the synthesis and relevant pharmacological properties of the quinoxalinetacrine (QT) hybrid QT78 in a project targeted to identify new non-hepatotoxic tacrine derivatives for Alzheimer’s disease therapy. We have found that QT78 is less toxic than tacrine at high concentrations (from 100 µM to 1 mM), less potent than tacrine as a ChE inhibitor, but shows selective BuChE inhibition (IC50 (hAChE) = 22.0 ± 1.3 µM; IC50 (hBuChE) = 6.79 ± 0.33 µM). Moreover, QT78 showed effective and strong neuroprotection against diverse toxic stimuli, such as rotenone plus oligomycin-A or okadaic acid, of biological significance for Alzheimer’s disease. Keywords: Alzheimer’s disease; cholinesterase inhibitor; hepatotoxicity; molecular modeling; neuroprotection; quinoxalines; quinoxalinetacrines; tacrine 1. Introduction Alzheimer’s disease (AD) is the most common neurodegenerative disorder. Many biochemical hallmarks are involved in the progress and development of this pathology: extracellular senile plaques that appear by amyloid-beta peptide deposition and aggregation, hyperphosphorylated tau-protein, neuroinflammation, cell death, oxidative stress, and a strong deficit of neurotransmitters such as acetylcholine, dopamine, and serotonin [1]. These facts have resulted in what has been called the “cholinergic hypothesis of AD”, highlighting that most AD patient impairments are due to deficient cholinergic neurotransmission [2]. Consequently, a few acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitors (rivastigmine, donepezil, galantamine) have been identified and are currently being used in clinical treatment for the therapy of AD [3]. The finding that BuChE Molecules 2019, 24, 1503; doi:10.3390/molecules24081503 www.mdpi.com/journal/molecules Molecules 2018, Molecules 2018, 23, 23, xx FOR FOR PEER PEER REVIEW REVIEW 22 of of 12 12 such as such as acetylcholine, acetylcholine, dopamine, dopamine, and and serotonin serotonin [1].[1]. These These facts facts have have resulted resulted in in what what has has been been called called the “cholinergic the “cholinergic hypothesis hypothesis of of AD”, AD”, highlighting highlighting that that most most ADAD patient patient impairments impairments are are due due to to deficient deficient cholinergic cholinergic neurotransmission [2]. Consequently, a few acetylcholinesterase neurotransmission [2]. Consequently, a few acetylcholinesterase (AChE)2 and (AChE) and Molecules 2019, 24, 1503 of 12 butyrylcholinesterase (BuChE) butyrylcholinesterase (BuChE) inhibitors inhibitors (rivastigmine, (rivastigmine, donepezil, donepezil, galantamine) galantamine) have have been been identified identified and are and are currently currently being being used used in in clinical clinical treatment treatment for for the the therapy therapy of of AD AD [3]. [3]. The The finding finding that that BuChE BuChE activity is raised in AD, and that BuChE activity is concentrated in areas activity is raised in AD, and that BuChE activity is concentrated in areas of the brain engaged in of the brain engaged in cognition, suggests the use of selective BuChE inhibitors inhibitors for AD therapy cognition, suggests the use of selective BuChE inhibitors for AD therapy [4]. Unfortunately, to date,[4]. Unfortunately, Unfortunately, to date, no effective no effective treatment treatment has has been been discovered discovered to to cure cure this this pathology. Consequently, there pathology. Consequently, there isis an an urgent urgent need for novel and more efficient drugs to to treat treat AD AD patients. patients. In the search need for novel and more efficient drugs to treat AD patients. In the search for new therapies to deal for new therapies to deal with this complex and multifactorial disease, the multitarget small molecule with this complex and multifactorial disease, the multitarget small molecule (MTSM) approach [5] is (MTSM) approach [5] is considered one considered one ofof the the most most attractive therapeutic strategies. attractive therapeutic therapeutic strategies. strategies. In this context, and pursuing our search for non-hepatotoxic In this context, and pursuing our search for tacrine (Figure non-hepatotoxic tacrine (Figure 1) 1) analogues analogues [6] [6] showing ChE showing inhibition, we ChE inhibition, we designed designed the the quinoxalinetacrines quinoxalinetacrines (QTs) (QTs) (I,(I, Figure Figure 1) 1) as as new, new, simple, simple, and and readily available agents. Quinoxalines Quinoxalines are are aa class class of of molecules molecules endowed endowed readily available agents. Quinoxalines are a class of molecules endowed with a large of array of with with a a large of array of biological biological properties, properties, including including anti-neuroinflammation, anti-neuroinflammation, antitumoral, antitumoral, biological properties, including anti-neuroinflammation, antitumoral, antimycobacterial, and antimycobacterial, antimycobacterial, and and antifungal antifungal[7]. activities antifungal activities [7]. activities [7]. In this article, we describe the In this article, we describe the synthesis, synthesis, toxicological toxicological andand neuroprotective neuroprotective properties, properties, asas well well asas the computational computational analysis analysis of of QT78 QT78 (1,2,3,4-tetrahydroquinolino[2,3-b]quinoxalin-12-amine) (1,2,3,4-tetrahydroquinolino[2,3-b]quinoxalin-12-amine) the computational analysis of QT78 (1,2,3,4-tetrahydroquinolino[2,3-b]quinoxalin-12-amine) (Figure (Figure (Figure 1), 1), the as 1), as the as the first firstfirst member member member of this of this of this new new new family family familyof QTof QT of QT derivatives. derivatives. derivatives. Figure 1. Figure 1. Figure General structure General structure 1. General for structure for quinoxalinetacrines for quinoxalinetacrines (QTs) (I), quinoxalinetacrines (QTs) (I), tacrine, tacrine, and and QT78. QT78. 2. Results and 2. Results 2. Results and Discussion and Discussion Discussion The synthesis The synthesis The synthesis ofof QT78 of QT78 has QT78 has been has been carried carried out been carried out as out shown as shown as in shown in Scheme in scheme scheme 1,1, with 1, with aaa good with good yield, good yield, by yield, by reacting by reacting reacting 3-amino-2-quinoxalinecarbonitrile 3-amino-2-quinoxalinecarbonitrile [8] 3-amino-2-quinoxalinecarbonitrile [8] and [8] and cyclohexanone, and cyclohexanone, cyclohexanone, underunder typical under typical Friedländer-type typical Friedländer-type reaction Friedländer-type reaction reaction conditions conditions [9,10]. [9,10]. QT78 QT78 showed showed spectroscopic spectroscopic data data in in good good agreement agreement with with its its structure structure conditions [9,10]. QT78 showed spectroscopic data in good agreement with its structure and excellent and and excellent excellent analytical data analytical data analytical (see (see Supporting data (see Information). Supporting Information). Supporting It is Information). It It is interesting is interesting to interesting to note note that to note that Hamama that Hamama and Hamama and co-workers and co-workers co-workers have have recently recently reported reported the the reaction reaction of of 2-amino-3-cyano-6-methylquinoxaline-1,4-dioxide 2-amino-3-cyano-6-methylquinoxaline-1,4-dioxide have recently reported the reaction of 2-amino-3-cyano-6-methylquinoxaline-1,4-dioxide with several with with several several cycloalkanones, cycloalkanones, and and described described their their antibacterial, antibacterial, antifungal, antifungal, and and antitumor antitumor activities activities cycloalkanones, and described their antibacterial, antifungal, and antitumor activities [11]. [11]. [11]. Scheme 1. Scheme 1. Synthesis of QT78. Synthesis of QT78. First, we First, we First, started we started with started with the with the in in silico the in silico toxicological silico toxicological analysis toxicological [12]. analysis [12]. analysis In order [12]. In In order to order to predict to predict the predict the hepatotoxic the hepatotoxic hepatotoxic potential potential of of QT78, QT78, and and its its main main Phase Phase I I metabolites, metabolites, we we performed performed the the in in silico potential of QT78, and its main Phase I metabolites, we performed the in silico analysis of QT78. silico analysis analysis of of QT78. QT78. The The The result result for result for hepatotoxicity for hepatotoxicity hepatotoxicity was was “nothing was “nothing “nothing to to report”, to report”, showing report”, showing showing that that there that there there is is is notnot enough not enough evidence enough evidence evidence to to to predict predict a a positive/negative positive/negative result. result.This This means means that, structurally, that, structurally,the novel the novel compound compound predict a positive/negative result. This means that, structurally, the novel compound is quite different is quite is different quite to different tacrine, to tacrine, to andand tacrine, consequently, and QT78QT78 consequently, consequently, does not QT78 does does provide any positive not provide not provide prediction any positive any positive of hepatotoxicity. prediction prediction Likewise, of hepatotoxicity. of hepatotoxicity. its ten Phase Likewise, its Likewise, I metabolites its ten ten Phase displayed Phase II metabolites the same metabolites displayed message, displayed the indicating the same same message,that there message, indicating was no indicating that associated that there there wastacrine was no no metabolites associated risk tacrine (Figure 2). metabolites risk (Figure associated tacrine metabolites risk (Figure 2). 2). Molecules 2019, 24, 1503 3 of 12 Molecules 2018, 23, x FOR PEER REVIEW 3 of 12 Figure2.2. Summary Figure Summary metabolic metabolic tree tree after after processing QT78 (query processing QT78 (query compound) compound) for for human human Phase Phase II biotransformation biotransformationreactions reactionsand andtwo twosequences sequencesofofbiotransformation. biotransformation. Thus, Thus,we wechose choseHepG2 HepG2 cells,cells, a well-established in vitroinmodel a well-established vitro and widely model andused human widely hepatoma used human cell line [13], hepatoma to line cell test [13], the hepatotoxicity exerted by QT78, to test the hepatotoxicity exertedusing tacrine by QT78, as the using reference tacrine as thedrug. Cells were reference drug. incubated with QT78 and tacrine in a wide range of doses (1–1000 µM) in a 24 h Cells were incubated with QT78 and tacrine in a wide range of doses (1–1000 μM) in a 24 h period. period. The HepG2 viability The HepG2 was significantly viability was decreased when QT78 significantly was incubated decreased when QT78 at the was highest concentrations incubated at the(300 and highest 1000 µM) (Table(300 concentrations 1). However, and 1000 the μM) toxicity (Tableprofile was significantly 1). However, the toxicitybetter thanwas profile thatsignificantly of tacrine, which better showed a significant viability reduction at lower concentrations (from 30 µM). than that of tacrine, which showed a significant viability reduction at lower concentrations These results (from suggest30 that μM). the hepatotoxic These safety profile results suggest that theof QT78 is better hepatotoxic than profile safety that of tacrine of QT78 (Supporting Information). is better than that of tacrine (Supporting Information). Table 1. In vitro hepatotoxicity of tacrine and QT78 in HepG2 cells. Table 1. In vitro hepatotoxicity Viability (%)ofHepG2 tacrine and QT78 in HepG2 cells. Cells Compounds 1 µM 3 µM 10 µM 30 µM 100 µM 300 µM 1000 µM Viability (%) HepG2nsCells QT78 96.7 ± 2.14 ns 97.6 ± 2.20 ns 97.1 ± 1.64 ns 95.1 ± 1.50 90.5 ± 1.86 ns 78.1 ± 0.94 *** 68.3 ± 2.92 *** Compounds 93.4 ± 4.69 ns Tacrine 90 ± 2.95 ns 88.7 ± 3.42 ns 81.6 ± 4.88 * 64.3 ± 4.54 *** 40 ± 2.20 *** 34.4 ± 2.73 *** Cell viability was measured as MTT reduction and data were normalized as % control. Data are expressed as 1 µM 3 µM 10 µM 30 µM 100 µM 300 µM 1000 µM the means ± S.E.M. in triplicate of at least three different cell batches. All compounds were assayed at increasing concentrations QT78 (1–1000 96.7  2.14 P < 0.001, µM). ***97.6  2.20* P < 0.05 97.1and ns not significant,  1.64 95.1  1.50with respect to control group. Comparisons 78.1  0.94 68.3  2.92 between drugs and control group were performed bynsone-way ANOVA followed by the Newman–Keuls post-hoc test. ns ns ns 90.5  1.86 ns *** *** Tacrine Next, the 93.4 innsvitro 4.69 90  2.95  3.42 of88.7human 81.6  4.88 64.3  4.54 40  2.20 *** and 34.4  2.73 ns inhibition acetylcholinesterase (hAChE) human ns * *** *** butyrylcholinesterase (hBuChE) was assessed according to the method of Ellman [14] (Supporting Cell viability Information), using was measured tacrine as theas reference MTT reduction and data compound. were normalized Comparison of theasIC%50control. values Data are highlighted expressed as the means ± S.E.M. in triplicate of at least three different cell that QT78 acts as a modest but selective hBuChE inhibitor [IC50 (hAChE) = 22.0 ± 1.3 µM; IC50 batches. All compounds were assayed at increasing concentrations (1–1000 μM). *** P < 0.001, * P < 0.05 and ns not significant, (hBuChE) = 6.79 ± 0.33 µM]. Thus, it is obvious that the enlargement of the three-membered with respect to control group. Comparisons between drugs and control group were performed by tetrahydroacridine scaffold to the four-membered tetrahydroquinolino[2,3-b] quinoxaline nucleus was one-way ANOVA followed by the Newman–Keuls post-hoc test. Next, the in vitro inhibition of human acetylcholinesterase (hAChE) and human butyrylcholinesterase (hBuChE) was assessed according to the method of Ellman [14] (Supporting Information), using tacrine as the reference compound. Comparison of the IC50 values highlighted that QT78 acts as a modest but selective hBuChE inhibitor [IC50 (hAChE) = 22.0 ± 1.3 M; IC50 Molecules 2019, 24, 1503 4 of 12 (hBuChE) = 6.79 ± 0.33 M]. Thus, it is obvious that the enlargement of the three-membered tetrahydroacridine scaffold to the four-membered tetrahydroquinolino[2,3-b] quinoxaline nucleus detrimental was detrimental regarding its activity regarding towardstowards its activity both ChE bothenzymes, leading to ChE enzymes, a decrease leading to a of the inhibitory decrease of the potency inhibitoryof two ordersofoftwo potency magnitude orders (tacrine: of magnitude (tacrine:= IC IC50 (hAChE) 0.374 ± 0.053 µM; 50 (hAChE)  0.053 M; IC50 (hBuChE) = 0.374 = 0.044 IC50 ±(hBuChE) 0.002 µM)= [10]. 0.044Based ± 0.002 onM)these[10]. results, Based weon next carried these out we results, docking studies out next carried to explain docking thestudies binding to modes explainofthe compound binding modes QT78 in of hAChE compound and QT78 hBuChE. in hAChE and hBuChE. The The structures structures of of hAChE hAChE crystallized crystallized withwith fasciculin fasciculin IIII (PDB (PDB ID: ID: 1B41) 1B41) and and hBuChE hBuChE crystallized crystallized with with tacrine tacrine (PDB (PDB ID: ID: 4BDS) 4BDS) werewere used. used. TheThe docking docking of of compound compound QT78 QT78 with with the the target target enzymes enzymes was was performed performed usingusing AutoDock AutoDock Vina Vina [15] [15] software software and and the the results results were were analyzed analyzed withwith Discovery Discovery Studio. Studio. As we have previously described [10], the conformational conformational flexibility flexibility of of AChE AChE waswas considered considered by by allowing allowing eight eight side side chains chains to to be be flexible flexibleduring duringthe thedocking dockingprocess. process. Regarding hAChE, compound Regarding compoundQT78 QT78isisaccommodated accommodatedinin thethe middle middleof the active-site of the active-sitegorge and gorge and no interaction no interaction withwith the catalytic the catalytic triadtriad aminoacids aminoacids (His447, (His447, Ser203 Ser203 and and Glu334) Glu334) is established. is established. The The cyclohexane cyclohexane ringring is oriented is oriented towards towards the bottom the bottom of the ofgorge, the gorge, -alkyl establishing establishing interactions π-alkyl interactions with with Trp86,Trp86, a residue a residue involved involved in thein the interaction interaction with with thethe quaternaryamine quaternary amineofofthe thenatural natural ligand ligand ACh. The complex The complex AChE-QT78 AChE-QT78 was was also also stabilized stabilized by by molecular molecular interactions interactions withwith several several key key residues residues atat the peripheral the peripheral anionic anionic sitesite (PAS). (PAS). There There isis aahydrogen hydrogen bond bond network network in in which which thethe amino amino group group and and the pyrazine the pyrazinenitrogen nitrogenwith withTyr124 Tyr124and andAsp74 Asp74are areinvolved. involved.Additionally, Additionally,the thephenyl phenylringringforms forms a - a π-π  stacking stacking interaction interaction with with Tyr341 Tyr341 andthe and thepyridine pyridinering ringforms formsaaπ-π ‐T-shaped T-shapedinteraction interaction with with Tyr337 Tyr337 (Figure 3a,b). (Figure 3a,b). ToTo sum sum up, up, wewe propose propose thatthat compound compound QT78 QT78 interacts interacts with with the the PAS PAS of of AChE AChE without without catalytic triad catalytic triad residue residue involvement. involvement. b a Figure 3. (a) Figure 3. (a) Three-dimensional Three-dimensional representation representation ofof interactions interactions established established by QT78 with by QT78 with hAChE. hAChE. (b) (b) Two-dimensional schematic view of the interactions between QT78 and Two-dimensional schematic view of the interactions between QT78 and hAChE. hAChE. Docking Docking within within hBuChE hBuChE shows shows that that compound compound QT78QT78 is is deeply deeply inserted inserted into into the the active active site. site. Nevertheless, it can be seen that the structure of the docked compound does not fit in the Nevertheless, it can be seen that the structure of the docked compound does not fit in the space space occupied by tacrine (Figure 4a), which could account for its weaker BuChE inhibitory activity. As shown in Figure 4b, the binding mode of compound QT78 is characterized by the following features: the NH2 group forms two hydrogen bonds with the catalytic triad residues His438 and Ser198. Moreover, the pyridine and cyclohexane rings interact with the residues in the acyl-binding pocket (ABP) (Phe329 Molecules 2018, 23, x FOR PEER REVIEW 5 of 12 occupied by tacrine (Figure 4a), which could account for its weaker BuChE inhibitory activity. As shown Molecules in 24, 2019, Figure 1503 4b, the binding mode of compound QT78 is characterized by the following features: 5 of 12 the NH2 group forms two hydrogen bonds with the catalytic triad residues His438 and Ser198. Moreover, the pyridine and cyclohexane rings interact with the residues in the acyl-binding pocket and Leu286). (ABP) Also, (Phe329 andtheLeu286). pyridine ringthe Also, allowed an amide-π pyridine stacked ring allowed interaction an amide- with Gly116 stacked in the interaction with oxyanion hole (OH). Gly116 in the oxyanion hole (OH). a b Figure Figure 4. (a) 4. (a) Molecular Molecular docking docking model model for compound for compound QT78QT78 and tacrine and tacrine withinwithin the hBuChE the hBuChE binding- binding-site. site. Compound Compound QT78 isQT78 coloredis colored orange, orange, and tacrine, and tacrine, in Different in cherry. cherry. Different subsites subsites of thesite of the active active weresite were colored: colored: catalytic catalytic anionic anionic site (CAS) site in(CAS) green,in green, oxyanion oxyanion hole (OH)hole (OH) in red, in red,binding choline cholinesite binding site in in violet violet (CBS), (CBS), acyl acylpocket binding binding pocket (ABP) (ABP) and in yellow, in yellow, and peripheral peripheral anionic siteanionic (PAS) insite (PAS) blue. in blue.bonding Hydrogen Hydrogen interactions bonding are shown in dashed interactions are shown blackinlines. (b) Two-dimensional dashed black lines. (b) interaction map of compound Two-dimensional interactionQT78, map of which is boundQT78, compound to thewhich activeissite of hBuChE. bound to the active site of hBuChE. TheThe molecular molecular docking dockingresults show results thatthat show QT78 is located QT78 in the is located inactive site of the active both site enzymes, of both but inbut enzymes, hBuChE, the ligand in hBuChE, interacts the ligand with the interacts catalytic with triad residues, the catalytic whereas whereas triad residues, in hAChEinthe ligandthe hAChE is further ligand is away. These results agree with the experimental results, which showed that QT78 further away. These results agree with the experimental results, which showed that QT78 is a modest is a modest but selective hBuChE but selective inhibitor. hBuChE inhibitor. In In addition, for essential addition, for essential biological biological activity, activity,drug drug candidates candidates should should alsoalsohavehave anan ideal ideal pharmacokinetic profile. In this context, we have computed Lipinski’s pharmacokinetic profile. In this context, we have computed Lipinski's rule of five and other rule of five and other pharmacokinetic pharmacokinetic properties properties of of QT78, QT78,using usingthethe QikProp QikProp module module of of Schrödinger Schrödinger software software (QikProp, (QikProp, version 3.8, Schrodinger, LLC, New York, NY, USA, version 3.8, Schrodinger, LLC, New York, NY, USA, 2013). 2013). Compounds Compounds violating moremore violating than than one ofone these ofrules thesemay have rules maybioavailability limitations. have bioavailability The ADME limitations. The predictions of compoundof ADME predictions QT78 showed satisfactory compound QT78 showed results satisfactory highlighting drug-like characteristicsdrug-like results highlighting based on characteristics Lipinski’s rulebased of fiveon (see Table S1,rule Lipinski's Supporting Information) of five (see [16]. Table S1, Supporting Information) [16]. TheThedrugs drugsused forfor used neurological neurological disorder disorder treatment treatment actact ononthethe central nervous central nervous system system (CNS). (CNS). CNS drugs show values of MW < 450, hydrogen-bond donors < 3, CNS drugs show values of MW < 450, hydrogen-bond donors < 3, hydrogen-bond acceptorshydrogen-bond acceptors < 7, < 7, number of hydrogen bond donor < 5, van der Waals surface area of polar nitrogen number of hydrogen bond donor < 5, van der Waals surface area of polar nitrogen and oxygen atoms and oxygen atoms < 90, 0.30 cross BBB readily, while compounds with a log BB < −1.00 are poorly distributed into the brain [17]. The QPlogBB value for QT78 is −0.332, indicating it has a good potential for BBB penetration. The literature data suggest that polar surface area (PSA) is a measure of the hydrogen bonding capacity of a molecule, and its value should not exceed a certain limit if the compound is intended Molecules 2018, 23, x FOR PEER REVIEW 6 of 12 to be CNS active. The most active CNS drugs have a PSA lower than 70 A2 . The value of PSA for QT78 is 59.549 Å2 , suggesting a good penetration of the BBB. Similarly, the percentage of human oral Further, the predicted value for the partition coefficient (QPlogPo/w = 2.534), critical for the absorption is very high (100%), while other physicochemical descriptors obtained by QikProp (Table S1, estimation of absorption within the body, is in the optimum range (−2.0–6.5). Supporting Information) are within the acceptable range for human use, thereby indicating that QT78 Drugs must cross the blood–brain barrier (BBB) to exert their activity on the CNS; thus, one of is a potential drug-like molecule and possible CNS drug. the most used descriptors for CNS penetration is logBB. Experimental values of log BB cover a span Finally, following the protocols described in the Supporting Information, we evaluated the from about −2.00 to +1.04. Within this range, compounds with log BB > 0.30 cross BBB readily, while neuroprotective profile of QT78 against two toxic insults related to neurodegeneration [18,19], using compounds with a log BB < −1.00 are poorly distributed into the brain [17]. The QPlogBB value for melatonin (10 nM) as a reference compound: (a) a model of reactive oxygen species (ROS) generation, QT78 is −0.332, indicating it has a good potential for BBB penetration. based on the cocktail of the mitochondrial respiratory chain blockers rotenone and oligomycin A The literature data suggest that polar surface area (PSA) is a measure of the hydrogen bonding (R/O), and (b) hyperphosphorylation of tau protein using okadaic acid (OA), a well-known protein capacity of a molecule, and its value should not exceed a certain limit if the compound is intended to phosphatase inhibitor. As shown in Figure 5, QT78 revealed an interesting neuroprotective activity. be CNS active. The most active CNS drugs have a PSA lower than 70 A2. The value of PSA for QT78 At the concentrations of 0.1 and 0.3 µM, QT78 produced a significant increase in cell viability against is 59.549 Å2, suggesting a good penetration of the BBB. Similarly, the percentage of human oral R/O (16.7% and 21.4%, respectively). Similarly, QT78 at 0.1 µM showed a potent increase in cell absorption is very high (100%), while other physicochemical descriptors obtained by QikProp (Table viability against OA (18.9%). In all cases, when we increased the concentration of the compound to 1 S1, Supporting Information) are within the acceptable range for human use, thereby indicating that µM, the protective capacity decreased against all toxic stimuli tested. QT78 is a potential drug-like molecule and possible CNS drug. A Figure 5. Cont. Molecules 2019, 24, 1503 7 of 12 7 of 12 B Figure 5. Neuroprotective properties of QT78 (0.1, 0.3 and 1 µM) in SH-SY5Y cells against (A) rotenone Figure 5. Neuroprotective (30 µM)/oligomycin A (10 µM) or (B)properties of QT78 okadaic acid (0.1, (20 nM) 0.3 and toxicity. Data1 are in SH-SY5Y M)expressed cells±against as means SEM (A) rotenone (30 M)/oligomycin A (10 M) or (B) okadaic acid (20 nM) toxicity. Data are expressed of five different cultures performed in triplicate (untreated control set to 100%). *** p < 0.001, compared as means to control; ###± SEM of five different cultures performed in triplicate p < 0.001, p < 0.01 and p < 0.05 compared to toxic stimuli. ## # (untreated control set to 100%). *** p < 0.001, compared to control; ### p < 0.001, ## p < 0.01 and # p < 0.05 compared to toxic stimuli. 3. Materials and Methods Finally, following the protocols described in the Supporting Information, we evaluated the 3.1. Chemistry Methodsprofile of QT78 against two toxic insults related to neurodegeneration [18,19], using neuroprotective melatoninwere Reactions (10 nM) as a reference monitored compound: by TLC (a) a model using precoated of reactive silica oxygen species gel aluminum (ROS) generation, plates containing a based indicator fluorescent on the cocktail (Merck,of the Kenilworth, 5539, mitochondrial NJ, respiratory United States).chain blockers Detection rotenone was done byandUV oligomycin (254 nm) A (R/O), followed byand (b) hyperphosphorylation charring of tau with sulfuric–acetic acid protein spray, using okadaic 1% aqueous acid permanganate potassium (OA), a well-known protein solution phosphatase or 0.5% inhibitor.acid phosphomolybdic As in shown in Figure 95% EtOH. 5, QT78 revealed Anhydrous Na2 SO4 was an interesting used to dryneuroprotective organic solutionsactivity. At the concentrations of 0.1 and 0.3 μM, QT78 produced a significant increase in during work-ups and the removal of solvents was carried out under vacuum with a rotary evaporator.cell viability against Flash column chromatography was performed using silica gel 60 (230–400 mesh, Merck). Melting pointsin cell R/O (16.7% and 21.4%, respectively). Similarly, QT78 at 0.1 μM showed a potent increase were viability against determined on aOA (18.9%). Kofler blockIn allare and cases, when we increased uncorrected. IR spectrathe concentration were obtained onofa the compound to 1 Perkin-Elmer μM, the protective capacity decreased against all toxic stimuli 1 tested. Spectrum One spectrophotometer (Waltham, MA, United States). H-NMR spectra were recorded with a Varian VXR-200S spectrometer (Palo Alto, CA, United States), using tetramethylsilane as internal 3. Materials standard and Methods and 13 C-NMR spectra were recorded with a Bruker WP-200-SY (Billerica, MA, United States). All the assignments for protons and carbons were in agreement with 2D COSY, HSQC, HMBC, and 1D 3.1. Chemistry Methods NOESY spectra. Values (*) can be interchanged. The purity of compounds was checked by elemental Reactions on analyses, conducted were monitored a Carlo Erba EAby1108 TLCapparatus using precoated silica (Sabadell, gel and Spain), aluminum confirmed plates to becontaining >95%. a fluorescent indicator (Merck, 5539, Kenilworth, NJ, United States). Detection was done by UV (254 3.2. Synthesis of QT78by charring with sulfuric–acetic acid spray, 1% aqueous potassium permanganate nm) followed solution A mixtureorof0.5% phosphomolybdic acid in 95%[8] 3-amino-2-quinoxalinecarbonitrile EtOH. Anhydrous (160 mg, Na2SO 0.94 mmol), 4 was used to(146 cyclohexanone dryµL, organic 1.41 mmol) and AlCl3 (186 mg, 1.41 mmol) in 1,2-dichloroethane (6 mL) was irradiated in a MWrotary solutions during work-ups and the removal of solvents was carried out under vacuum with a evaporator. apparatus at 95 ◦ Flash C, 250column W and chromatography was performed 20 atm, for 2 h. Then, the mixtureusing wassilica gel at cooled 60 0(230–400 mesh, ◦ C, diluted Merck). with Melting a mixture of Hpoints 2 O/THFwere determined (1:1), treated on with a Kofler NaOH block 15% and until are uncorrected. basic, and IR extracted spectra several were timesobtained with on 1 a Perkin-Elmer methylene Spectrum chloride. The organic One layerspectrophotometer (Waltham, was washed with brine MA, United until neutral, States). dried with Na2 SO H-NMR spectra 4 , filtered were recorded and evaporated. with a was The residue Varian VXR-200S purified spectrometer by column (Palo Alto, chromatography (CH2 ClCA, 2 /MeOH United States), (from 1% tousing 5%)), tetramethylsilane 13 affording 1,2,3,4-tetrahydroquinolino[2,3-b]quinoxalin-12-amine (QT78, Figure 6) (172 mg, 88%): as internal standard and C-NMR spectra were recorded with a Bruker WP-200- SY (Billerica, MA, United States). All the assignments for protons and carbons were in agreement with 2D COSY, HSQC, HMBC, and 1D NOESY spectra. Values (*) can be interchanged. The purity of A mixture of 3-amino-2-quinoxalinecarbonitrile [8] (160 mg, 0.94 mmol), cyclohexanone (146 µ L, 1.41 mmol) and AlCl3 (186 mg, 1.41 mmol) in 1,2-dichloroethane (6 mL) was irradiated in a MW apparatus at 95 °C, 250 W and 20 atm, for 2 h. Then, the mixture was cooled at 0 °C, diluted with a mixture of H2O/THF (1:1), treated with NaOH 15% until basic, and extracted several times with methylene chloride. The organic layer was washed with brine until neutral, dried with Na82SO Molecules 2019, 24, 1503 4, of 12 filtered and evaporated. The residue was purified by column chromatography (CH2Cl2/MeOH (from 1% to 5%)), affording 1,2,3,4-tetrahydroquinolino[2,3-b]quinoxalin-12-amine (QT78, Figure 6) (172 mp ◦ C; IR (KBr) 3292, 2938, 1622, 1605, 1501, 1375, 1347, 1326, cm−1 ; 1 H-NMR mg, 238–240 88%): mp 238–240 °C; IR (KBr) 3292, 2938, 1622, 1605, 1501, 1375, 1347, 1326, cm −1; 1H-NMR (300 MHz, (300 DMSO) δ 8.17 (dd, J= 2.1, 8.8 Hz, 2H, H7, H10), 8.01–7.70 (m, 2H, H8, H9), MHz, DMSO) δ 8.17 (dd, J= 2.1, 8.8 Hz, 2H, H7, H10), 8.01–7.70 (m, 2H, H8, H9), 7.10 (bs, 7.10 (bs, 2H, NH ), 2.98–2.96 2 2H, NH 2), (m, 2H, H4), 13 C-NMR 2.98–2.96 (m, 2.69–2.56 (m, 2H, H1), 2H, H4), 2.69–2.56 1.88–1.85 (m, 2H, (m, 4H, (m, H1), 1.88–1.85 H2, 4H, H3);H2, (75 MHz, H3); 13C-NMR DMSO) (75 MHz, δ 165.8δ DMSO) (2C, 165.8C12,(2C,C4a), C12, 148.9 C4a), (C5a), 147.5 (C11a), 148.9 (C5a), 144.2 (C10a)*, 147.5 (C11a), 140.0 (C6a)*, 144.2 (C10a)*, 131.0 (C9)**, 140.0 (C6a)*, 130.6 (C8)**, 131.0 (C9)**, 129.3 130.6 (C8)**, (C7)***, 129.0 (C10)***, 110.3 (C12a), 34.5 (C4), 23.9 (C1), 22.3 (C2)*, 21.9 (C3)*; MS (EI) 129.3 (C7)***, 129.0 (C10)***, 110.3 (C12a), 34.5 (C4), 23.9 (C1), 22.3 (C2)*, 21.9 (C3)*; MS (EI) m/z (%): m/z (%): 250 [M + ] (100), 235 235 (18).(18). HRMS. Calc. for for C15C H1514HN144N(M + +H) + 250 [M +] (100), HRMS. Calc. 4 (M H)+: :251.12912. 251.12912.Found: Found: 251.12989. 251.12989. Anal. Cald. for C15 H 15H14 N : C, 71.98; H, 5.64; N, 22.38. Found: C, 71.75; H, 5.61; 14N44: C, 71.98; H, 5.64; N, 22.38. Found: C, 71.75; H, 5.61; N, 22.15. N, 22.15. Figure 6. Structure Figure 6. Structure of of QT78. QT78. 3.3. In Silico Prediction Systems for Toxicology and Metabolism 3.3. In Silico Prediction Systems for Toxicology and Metabolism Meteor Nexus (v.3.1.0, Lhassa Limited, Leeds, UK) and Derek Nexus (v.6.0.1, Lhassa Limited, Meteor Nexus (v.3.1.0, Lhassa Limited, Leeds, UK) and Derek Nexus (v.6.0.1, Lhassa Limited, Leeds, UK) knowledge-based expert systems (Knowledge Bases: Meteor KB 2015 1.0.0, Derek KB Leeds, UK) knowledge-based expert systems (Knowledge Bases: Meteor KB 2015 1.0.0, Derek KB 2015 2015 1.0) were employed for metabolism and toxicity predictions [20,21]. Hepatotoxicity was selected 1.0) were employed for metabolism and toxicity predictions [20,21]. Hepatotoxicity was selected as as an “end point”. Derek Nexus assesses predictions based on a summary of evidences, evaluating an “end point”. Derek Nexus assesses predictions based on a summary of evidences, evaluating alerts alerts and estimating the likelihood of occurrence [21]. The compound was considered to have a and estimating the likelihood of occurrence [21]. The compound was considered to have a structural structural alert for the selected endpoint (hepatotoxicity) if the prediction in Derek Nexus™ was alert for the selected endpoint (hepatotoxicity) if the prediction in Derek Nexus™ was ‘certain’, ‘certain’, ‘probable’, ‘plausible’ or ‘equivocal’. The predictions ‘doubted’, ‘improbable’, ‘impossible’, ‘probable’, ‘plausible’ or ‘equivocal’. The predictions ‘doubted’, ‘improbable’, ‘impossible’, ‘inactive’ ‘inactive’ or ‘no alert’ were regarded as negative. When Derek has no knowledge on which to base a or ‘no alert’ were regarded as negative. When Derek has no knowledge on which to base a prediction, prediction, the message “nothing to report” is displayed; this absence of evidence is not synonymous the message “nothing to report” is displayed; this absence of evidence is not synonymous with a with a prediction of inactivity. prediction of inactivity. The metabolism was predicted by Meteor Nexus version 3.1.0, (Lhasa ltd., Leeds, UK), a SAR The metabolism was predicted by Meteor Nexus version 3.1.0, (Lhasa ltd., Leeds, UK), a SAR (structure-activity relationship) tool used to predict the likely metabolic fate of a chemical structure (structure-activity relationship) tool used to predict the likely metabolic fate of a chemical structure through a knowledge-base composed of a biotransformation dictionary, rules and example metabolic through a knowledge-base composed of a biotransformation dictionary, rules and example metabolic pathways. Meteor was setup for human Phase I biotransformation reactions, since in most cases the pathways. Meteor was setup for human Phase I biotransformation reactions, since in most cases the reactive metabolites are generated particularly through Phase I metabolic reactions [22]. An absolute reactive metabolites are generated particularly through Phase I metabolic reactions [22]. An absolute reasoning biotransformation ranking method was applied as a qualitative rule-based approach to reasoning biotransformation ranking method was applied as a qualitative rule-based approach to evaluate the likelihood level (probable, plausible, equivocal, doubted, and improbable). The minimal evaluate the likelihood level (probable, plausible, equivocal, doubted, and improbable). The minimal likelihood level selected was “plausible”, meaning that the weight of evidence supports the proposition. likelihood level selected was “plausible”, meaning that the weight of evidence supports the All possible first and second-generation metabolites have been generated and afterwards processed for proposition. All possible first and second-generation metabolites have been generated and hepatotoxicity predictions. afterwards processed for hepatotoxicity predictions. 3.4. In Vitro Toxicity of QT78 and Tacrine in HepG2 Cells 3.4. In Vitro Toxicity of QT78 and Tacrine in HepG2 Cells The human hepatoma HepG2 cell line was kindly provided by the IdiPAZ Institute for Health Research (Madrid, Spain). They were cultured in Eagle´s minimum essential medium (EMEM) supplemented with 15 nonessential amino acids, 1 mM sodium pyruvate, 10 % heat-inactivated FBS, 100 units/mL penicillin, and 100 µg/mL streptomycin (reagents from Invitrogen, Madrid, Spain). Cultures were seeded into flasks containing supplemented medium and maintained at 37 ◦ C in a humidified atmosphere of 5% CO2 and 95% air. Culture media were changed every 2 days. Cells were sub-cultured after partial digestion with 0.25% trypsin-EDTA. For assays, cells were subcultured in 96-well plates at a seeding density of 1 × 105 cells per well. When the cells reached 80% confluence, the medium was replaced with fresh medium containing 1–1000 µM QT78 and tacrine, or 0.1% DMSO as a vehicle control. Molecules 2019, 24, 1503 9 of 12 3.5. Inhibition of Human AChE and BuChE The ability of QT78 to inhibit human ChE activity was assessed using Ellman’s assay [14]. A Jasco V-530 double beam spectrophotometer connected to a HAAKE DC30 thermostating system (Thermo Haake, Vreden, Germany) was used. Stock solutions of the tested compound and reference compound tacrine (1–2 mM) were prepared in methanol and water, respectively, and diluted in the same solvent used to prepare stock solution. The assay solution consisted of a 0.1 M phosphate buffer, pH 8.0, with the addition of 340 µM 5,50 -dithiobis (2-nitrobenzoic acid), 0.02 unit/mL human recombinant AChE or BuChE from human serum (Sigma-Aldrich, Milan, Italy), and 550 µM substrate, i.e., acetylthiocholine iodide or butyrylthiocholine iodide, respectively (Sigma-Aldrich, Milan, Italy). Either 50 µL of a solution of the tested compound at increasing concentrations or 50 µL of solvent (methanol or water) were added to the assay solution and preincubated at 37 ◦ C with the enzyme for 20 min before the addition of substrate. The rate of absorbance increase at 412 nm was followed for 3 min. In parallel, blanks containing all components except the enzyme were prepared to account for the non-enzymatic hydrolysis of the substrate. The reaction rates were compared and the percentage of inhibition due to the presence of test compound was calculated. Each concentration was analyzed in duplicate/triplicate. The percentage of inhibition of the enzyme activity due to the presence of inhibitor was calculated. Inhibition plots were obtained for each compound by plotting the percentage of inhibition versus the logarithm of inhibitor concentration in the assay solution. The linear regression parameters were determined for each curve and the IC50 extrapolated. 3.6. Molecular Modeling The structure of compound QT78 was built using Discovery Studio 2.1 [15]. The molecular geometry of QT78 was energy-minimized using the adopted-based Newton–Raphson algorithm with the CHARMm force field [23] with a convergence criterion for the energy gradient 0.01 kcal (mol-Å)−1 . The ligand was set up for docking with the help of AutoDockTools (ADT; version 1.5.4) to define the torsional degrees of freedom to be considered during the docking process. All the rotatable bonds of the ligand were allowed to rotate freely. The enzyme structure used for the calculations was the human AChE (Protein Data Band code 1B41). To give flexibility to the binding site, residues lining the site were allowed to move. These residues were Trp286, Tyr124, Tyr337, Tyr341, Tyr72, Asp74, Thr75, Trp86. Docking calculations for hBuChE were performed following a similar protocol to that described before for hAChE. The coordinates of hBuChE were obtained from the Protein Data Bank (PDB ID: 4BDS). For docking purposes, initial proteins were prepared by removing all water molecules, heteroatoms, any co-crystallized solvent and the ligand. Proper bonds, bond orders, hybridization and charges were assigned using protein model tool in the Discovery Studio, version 2.1, software package. A CHARMm force field was applied using the receptor ligand interactions tool in the Discovery Studio software package. ADT was used to add hydrogens and partial charges for proteins using Gasteiger charges and to generate the docking input files. AutoDock Vina software was employed for all the protein-ligand calculations and they were applied to the whole protein target (blind docking). For hAChE, the grid box was built with a resolution of 1 Å and 60 × 60 × 72 Å and it was positioned at the middle of the protein (x = 116.546; y = 110.33; z = −134.181). For hBuChE, a grid box of 66 × 60 × 74 with grid points separated 1 Å was positioned in the middle of the protein (x = 136.0; y = 123.59; z = 38.56). The resultant structure files were analyzed using Discovery Studio. 3.7. Neuroprotection Studies in Human Neuroblastoma Cells Human SH-SY5Y cells were maintained in a 1:1 mixture of Nutrient Mixture F-12 and Eagle´s minimum essential medium (EMEM) supplemented with 15 nonessential amino acids, 1 mM sodium pyruvate, 10% heat-inactivated FBS, 100 units/mL penicillin, and 100 µg/mL streptomycin. Cultures were seeded into flasks containing supplemented medium and maintained at 37 ◦ C in a humidified atmosphere of 5% CO2 and 95% air. For assays, SH-SY5Y cells were sub-cultured in 96-well plates at a Molecules 2019, 24, 1503 10 of 12 seeding density of 5 × 104 cells per well for 2 days. The cells were then incubated with 30 µM rotenone and 10 µM oligomycin-A (R/O) or okadaic acid (20 nM) (Sigma-Aldrich, Madrid, Spain) according to previous studies [18,19] with or without QT78 at different concentrations for 24 h. In this study, all cells were used at a low passage number (<14). 3.8. Measurement of Cell Viability MTT reduction was performed as described [24] for both HepG2 and SH-SY5Y human cell lines. Briefly, 50 µL of the MTT labeling reagent, at a final concentration of 0.5 mg/mL, was added. After incubation for 2 h, in a humidified incubator at 37 ◦ C with 5% CO2 and 95% air (v/v), the supernatant was removed, the obtained purple formazan product was re-suspended in 100 µL of DMSO. Colorimetric determination of MTT reduction was measured in an ELISA microplate reader at 540 nm. Non-treated cells were considered controls and were taken as 100% viability. 3.9. Statistical Analysis Statistical analyses were performed using GraphPad Prism software (version 6, San Diego, CA, USA). The data were analyzed by t-test or ANOVA with Tukey’s multiple comparison test, as indicated. P values less than 0.05 were considered significant. 4. Conclusions In the search for a new MTSM [5] for AD [25–27], the most common neurodegenerative disorder [28], the design, synthesis and pharmacological properties of QT78, the parent compound of a new family of tacrine derivatives bearing the quinoxaline heterocyclic ring core, was investigated. As a result, we have identified QT78 as a promising hit compound, which is less toxic than tacrine at high concentrations (from 100 µM to 1 mM), but less potent than tacrine as an inhibitor of both ChEs, with a modest selectivity towards BuChE. At 0.1 and 0.3 µM, QT78 exhibited a significant neuroprotective effect against different toxic stimuli, including ROS generation and hyperphosphorylation of tau protein. In addition, computational analyses shed light into the toxicological profile, binding and ADME properties of QT78. Taken together, the reported data are relevant to the search for new anti-AD agents. Our work is now in progress to prepare and evaluate new, related QTs based on QT78. The results will be reported elsewhere in due course. Supplementary Materials: Supporting Information is available free of charge on the website and includes Materials and Methods, NMR and HRMS spectra, and the ADME analysis. Author Contributions: Conceptualization, J.M.-C., J.E. and A.R.; Software, I.I., I.M. and D.D.-I.; Investigation, E.R., A.P.-A., M.B., A.S., C.V.C. and M.C.; Writing—Original Draft Preparation, J.M.-C.; Writing—Review & Editing, J.M.-C., J.E. and A.R; Supervision, J.M.-C., J.E. and A.R; Funding Acquisition, J.M.-C. Funding: J.M.-C is indebted to MICINN (SAF2006-08764-C02-01 and ISCIII [RED RENEVAS (RD06/0026/1002)] for financial support. Also this work was supported by grants from Fondo de Investigaciones Sanitarias (FIS)(ISCIII/FEDER)(Programa MIguel Servet CP14/00008;PI16/00735) and Fundación Mutua Madrileña to J.E. Acknowledgments: J.M.-C. is indebted to Nico Justin for reading and correcting the manuscript. Conflicts of Interest: The authors declare no conflict of interest. References 1. Liu, L.; Luo, S.; Zeng, L.; Wang, W.; Yuan, L.; Jian, X. Degenerative alterations in noradrenergic neurons of the locus coeruleus in Alzheimer’s disease. Neural Regen. Res. 2013, 8, 2249–2255. [PubMed] 2. Mesulam, M.-M. Cholinergic circuitry of the human nucleus basalis and its fate in Alzheimer’s disease. J. Comp. Neurol. 2013, 521, 4124–4144. [CrossRef] [PubMed] 3. Holzgrabe, U.; Kapkova, P.; Alptüzün, V.; Scheiber, J.; Kugelmann, E. Targeting acetylcholinesterase to treat neurodegeneration. Expert Opin. Ther. Targets 2007, 11, 161–179. [CrossRef] [PubMed] 4. Greig, N.H.; Lahiri, D.K.; Sambamurti, K. Butyrylcholinesterase: An important new target in Alzheimer’s disease therapy. Int. Psychogeriatr. 2002, 14, 77–91. [CrossRef] Molecules 2019, 24, 1503 11 of 12 5. Oset-Gasque, M.J.; Marco-Contelles, J. Alzheimer’s disease, the "one-molecule, one-target" paradigm, and the multitarget directed ligand approach. ACS Chem. Neurosci. 2018, 9, 401–403. [CrossRef] [PubMed] 6. Romero, A.; Cacabelos, R.; Oset-Gasque, M.J.; Samadi, A.; Marco-Contelles, J. Novel tacrine-related drugs as potential candidates for the treatment of Alzheimer’s disease. Bioorg. Med. Chem. Lett. 2013, 23, 1916–1922. [CrossRef] 7. Tariq, S.; Somakala, K.; Amir, M. Quinoxaline: An insight into the recent pharmacological advances. Eur. J. Med. Chem. 2018, 143, 542–557. [CrossRef] 8. Monge, A.; Palop, J.A.; Del Castillo, J.C.; Caldero, J.M.; Roca, J.; Romero, G.; Del Rio, J.; Lasheras, B. Novel antagonists of 5-HT3 receptors. Synthesis and biological evaluation of piperazinylquinoxaline derivatives. J. Med. Chem. 1993, 36, 2745–2750. [CrossRef] 9. Marco-Contelles, J.; Pérez-Mayoral, E.; Samadi, A.; Carreiras, M.C.; Soriano, E. Recent advances in the Friedländer reaction. Chem. Rev. 2009, 109, 2652–2671. [CrossRef] 10. Chioua, M.; Buzzi, E.; Moraleda, I.; Iriepa, I.; Maj, M.; Wnorowski, A.; Giovannini, C.; Tramarin, A.; Portali, F.; Ismaili, L.; et al. Tacripyrimidines, the first tacrine-dihydropyrimidine hybrids, as multi-target-directed ligands for Alzheimer’s disease. Eur. J. Med. Chem. 2018, 155, 839–846. [CrossRef] 11. Hamama, W.S.; Waly, S.M.; Said, S.B.; Zoorob, H.H. Annulation of o-aminoquinoxaline-1,4-dioxidenitrile with ketonic compounds under Friedländer-type cyclocondensation and its biological evaluation. J. Heterocyclic Chem. 2018, 55, 1554–1563. [CrossRef] 12. Patocka, J.; Jun, D.; Kuca, K. Possible role of hydroxylated metabolites of tacrine in drug toxicity and therapy of Alzheimer’s disease. Curr. Drug Metab. 2008, 9, 332–335. [CrossRef] 13. Choi, J.M.; Oh, S.J.; Lee, S.Y.; Im, J.H.; Oh, J.M.; Ryu, C.S.; Kwak, H.C.; Lee, J.Y.; Kang, K.W.; Kim, S.K. HepG2 cells as an in vitro model for evaluation of cytochrome P450 induction by xenobiotics. Arch. Pharm. Res. 2015, 38, 691–704. [CrossRef] 14. Ellman, G.L.; Courtney, K.D.; Andres, V., Jr.; Feather-Stone, R.M. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 1961, 7, 88–95. [CrossRef] 15. Trott, O.; Olson, A.J. AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 2010, 31, 455–461. [CrossRef] 16. Lipinski, C.A.; Lombardo, F.; Dominy, B.W.; Feeney, P.J. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev. 2001, 46, 3–26. [CrossRef] 17. Iyer, M.; Mishra, R.; Han, Y.; Hopfinger, A.J. Predicting blood-brain barrier partitioning of organic molecules using membrane-interaction QSAR analysis. Pharm. Res. 2002, 19, 1611–1621. [CrossRef] [PubMed] 18. de los Ríos, C.; Egea, J.; Marco-Contelles, J.; León, R.; Samadi, A.; Iriepa, I.; Moraleda, I.; Gálvez, E.; García, A.G.; López, M.G.; et al. Synthesis, inhibitory activity of cholinesterases, and neuroprotective profile of novel 1,8-naphthyridine derivatives. J. Med. Chem. 2010, 53, 5129–5143. [CrossRef] 19. Egea, J.; Rosa, A.O.; Cuadrado, A.; García, A.G.; López, M.G. Nicotinic receptor activation by epibatidine induces heme oxygenase-1 and protects chromaffin cells against oxidative stress. J. Neurochem. 2007, 102, 1842–1852. [CrossRef] 20. Langowski, J.; Long, A. Computer systems for the prediction of xenobiotic metabolism. Adv. Drug Deliv. Rev. 2002, 54, 407–415. [CrossRef] 21. Marchant, C.A.; Briggs, K.A.; Long, A. In silico tools for sharing data and knowledge on toxicity and metabolism: Derek for windows, meteor, and vitic. Toxicol. Mech. Methods 2008, 18, 177–187. [CrossRef] [PubMed] 22. Njuguna, N.M.; Masimirembwa, C.; Chibale, K. Identification and characterization of reactive metabolites in natural products-driven drug discovery. J. Nat. Prod. 2012, 75, 507–513. [CrossRef] [PubMed] 23. Brooks, B.R.; Bruccoleri, R.E.; Olafson, B.D.; States, D.J.; Swaminathan, S.; Karplus, M. A program for macromolecular energy, minimization, and dynamics calculations. J. Comput. Chem. 1983, 4, 187–217. [CrossRef] 24. Denizot, F.; Lang, R. Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J. Immunol. Methods 1986, 89, 271–277. [CrossRef] 25. Querfurth, H.W.; LaFerla, F.M. Alzheimer’s disease. N. Engl. J. Med. 2010, 362, 329–344. [CrossRef] Molecules 2019, 24, 1503 12 of 12 26. Sloane, P.D.; Zimmerman, S.; Suchindran, C.; Reed, P.; Wang, L.; Boustani, M.; Sudha, S. The public health impact of Alzheimer’s disease, 2000–2050: Potential implication of treatment advances. Annu. Rev. Public Health 2002, 23, 213–231. [CrossRef] 27. Marco-Contelles, J. Facts, results, and perspectives of the current Alzheimer’s disease research. ACS Chem. Neurosci. 2019, 10, 1127–1128. [CrossRef] [PubMed] 28. Scarpini, E.; Scheltens, P.; Feldman, H. Treatment of Alzheimer’s disease: Current status and new perspectives. Lancet Neurol. 2003, 2, 539–547. [CrossRef] Sample Availability: Not available. © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).