Subtly Modulating Glycogen Synthase Kinase 3 β ... - ACS Publications

May 26, 2017 - Subtly Modulating Glycogen Synthase Kinase 3 β: Allosteric Inhibitor. Development and Their Potential for the Treatment of Chronic. Di...
0 downloads 0 Views 9MB Size
Article pubs.acs.org/jmc

Subtly Modulating Glycogen Synthase Kinase 3 β: Allosteric Inhibitor Development and Their Potential for the Treatment of Chronic Diseases Valle Palomo,†,∇ Daniel I. Perez,†,∇ Carlos Roca,† Cara Anderson,‡ Natalia Rodríguez-Muela,§ Concepción Perez,∥ Jose A. Morales-Garcia,⊥,# Julio A. Reyes,∥ Nuria E. Campillo,† Ana M. Perez-Castillo,⊥,# Lee L. Rubin,§ Lubov Timchenko,‡ Carmen Gil,† and Ana Martinez*,† †

Centro de Investigaciones Biológicas-CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain Department of Pediatrics, Division of Neurology, Cincinnati Children’s Hospital, Cincinnati, Ohio 45219, United States § Department of Stem Cell and Regenerative Biology and Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts 02138, United States ∥ Instituto de Quimica Médica-CSIC, Juan del Cierva 3, 28006 Madrid, Spain ⊥ Instituto de Investigaciones Biomedicas-CSIC, Arturo Duperier 4, Madrid, Spain # Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Calle de Valderrebollo 5, 28031 Madrid, Spain ‡

S Supporting Information *

ABSTRACT: Glycogen synthase kinase 3 β (GSK-3β) is a central target in several unmet diseases. To increase the specificity of GSK-3β inhibitors in chronic treatments, we developed small molecules allowing subtle modulation of GSK3β activity. Design synthesis, structure−activity relationships, and binding mode of quinoline-3-carbohydrazide derivatives as allosteric modulators of GSK-3β are presented here. Furthermore, we show how allosteric binders may overcome the β-catenin side effects associated with strong GSK-3β inhibition. The therapeutic potential of some of these modulators has been tested in human samples from patients with congenital myotonic dystrophy type 1 (CDM1) and spinal muscular atrophy (SMA) patients. We found that compound 53 improves delayed myogenesis in CDM1 myoblasts, while compounds 1 and 53 have neuroprotective properties in SMA-derived cells. These findings suggest that the allosteric modulators of GSK-3β may be used for future development of drugs for DM1, SMA, and other chronic diseases where GSK-3β inhibition exhibits therapeutic effects.



However, these compounds rarely reach clinical trials,13 and only tideglusib is currently in clinical development. Tideglusib, an ATP noncompetitive GSK-3β inhibitor, is safe for human treatment.14,15 Clinical trial data indicate signs of efficacy such as dose dependent decreased brain atrophy16 or reduction of phospho-tau and BACE-1 levels in cerebrospinal fluid from Alzheimer’s patients.17 These data, in addition, supported the target engagement of tideglusib and its ability to cross the human blood−brain barrier. The involvement of GSK-3β in various essential molecular pathways, one of them being the oncogenic β-catenin signaling,18 suggests that the inhibitors of GSK-3β might cause side effects and may be one of the reasons for the poor clinical translation.19 However, recent data has shown that only a selective subtle modulation of the enzyme is needed to

INTRODUCTION Glycogen synthase kinase 3 β (GSK-3β) is a constitutively active multifunctional serine/threonine kinase involved in different physiological pathways such as metabolism, cell cycle, development, and neuroprotection.1 It is essential for life, playing a crucial role in the development and childhood stages.2 Simultaneously, GSK-3β has been implicated in various diseases such as diabetes, inflammation, cancer, amyotrophic lateral sclerosis (ALS), Alzheimer’s and Parkinson’s diseases, bipolar disorder, and many other unmet severe pathologies.3−6 An increased expression and activity of GSK-3β has been reported in type II diabetes and obese animal models,7 in skeletal muscle of patients with neuromuscular disease, myotonic dystrophy type 1 (DM1),8 and even more important, in the brains of Alzheimer’s patients and spinal cords from ALS patients.9,10 Consequently, GSK-3β inhibitors have demonstrated to be powerful drugs in animal models11 and emerge as promising effective therapies for these severe diseases.12 © 2017 American Chemical Society

Received: March 13, 2017 Published: May 26, 2017 4983

DOI: 10.1021/acs.jmedchem.7b00395 J. Med. Chem. 2017, 60, 4983−5001

Journal of Medicinal Chemistry

Article

potential, and hydrogen bond capabilities of the final compounds are varied to decipher the key interactions with the allosteric pocket in order to confirm this kind of enzymatic inhibition (Figure 2).

provide a safe GSK-3β homeostasis recovery effect without interfering in other cellular signaling.20 This great challenge may be achieved considering allosteric ligands. Today, allosterism is recognized as an inherent property of all dynamic proteins of biological relevance.21 In fact, allosteric inhibitors bring a novel mechanism of GSK-3β modulation, and to date, very few allosteric modulators of GSK-3β have been described. In addition to substrate competitive GSK-3 inhibitors such as some specific peptides, including L803-mts22 and 5-iminothiadiazoles,23 only the natural sesquiterpene palinurin24 and the quinoline-3-carbohydrazide 1 (VP0.7) have been described25 (Figure 1).

Figure 2. Proposed structural modifications in a 3-carboxamidequinoline scaffold to create diversity for a structure−activity relationship study.

General synthesis of these quinoline derivatives was accomplished by a convergent synthesis previously described26 using 4-hydroxy-3-ethoxycarbonyl-2-oxo-dihydro-quinolines 13−25 and different substituted amines or hydrazines (Scheme 1). The synthesis of the dihydroquinolines starts from the corresponding isatoic anhydride that is alkylated in the presence of sodium hydride and the halogenated alkyl chain to introduce the substituent R2 on the heterocyclic nitrogen (compounds 2−12). These alkylated anhydrides, some of them from commercial suppliers, are treated with diethyl malonate and sodium hydride to obtain the quinolines 13−25. Lastly, these quinolines can be transformed into different amides or hydrazides. Thus, compounds 13−25 were subsequently treated with different hydrazides in solution to achieve the final compounds 26−62. Additionally, three compounds were prepared without the carbohydrazide moiety, which were synthesized from the corresponding quinoline carboxylate. This carboxylate was treated with amine or hydrazide in different solvents at reflux temperature to obtain the amides 63 and 64 and the hydrazide 65. All of the compounds prepared here were unequivocally characterized using analytical and spectroscopic data (1H- and 13 C NMR), which are collected in the Experimental Section and Supporting Information, and when possible for some previously described compounds, melting points were compared (see Experimental Section). GSK-3β Inhibition Assays. The GSK-3β enzymatic inhibition of all of the compounds here prepared was analyzed in our laboratories using human recombinant enzyme and a luminescence method (see Experimental Section).27 All of the compounds were tested initially at a fixed concentration of 10 μM, and only in the cases where the compound’s inhibition was more than 50% of the total activity, the dose−response curve was calculated. Data collected in Tables 1 and 2 are the mean of two different experiments. Our results show that most of the 3carboxamide-quinolines prepared here are GSK-3β inhibitors with IC50 values in the low micromolar range, very similar to that of the first compound discovered 1. The IC50 values obtained here for the new synthesized compounds are rather similar, in the same order of magnitude, pointing to a flat structure−activity relationship which is in agreement with an allosteric modulation. However, the lack of activity of some compounds revealed important structural requirements for the enzymatic inhibition. Thus, the carbonyl group at N′-position of the hydrazide is crucial for the

Figure 1. ATP noncompetitive GSK-3 inhibitors.

Compound 1 is a small heterocyclic molecule which was postulated to bind to GSK-3β between Arg209 and Thr235 in the described pocket 7.25 In this work, we describe a facile synthesis for 1 and expand the methodology for the preparation of related molecules in order to explore the structure−activity relationships of this novel family of allosteric compounds. These studies are supported by refined geometry and docking studies that reinforce the hypothesis of the binding to an allosteric site and open new possibilities to specific drug design. Preliminary studies focused on following β-catenin localization after treatments with different GSK-3β inhibitors are performed. Finally, in regard to the potential translation of this type of inhibitor into the clinic, we report the benefits of some of these allosteric inhibitors of GSK-3β in two muscular chronic diseases: congenital myotonic dystrophy (CDM1) and spinal muscular atrophy (SMA). Using human myoblasts from patients with CDM1, we have shown the correction of delayed myogenesis after treatment with these compounds. Moreover, the survival of motor neurons derived from SMA patients’ induced pluripotent stem cells is increased with allosteric GSK3β modulator treatment.



RESULTS AND DISCUSSION Chemistry. Considering our previous allosteric modulator 1 discovered by virtual screening of our in-house chemical library on GSK-3β as a prototype,25 here we proposed the development of a feasible synthesis for this heterocyclic compound together with some structural modifications that would allow us to build a structure−activity relationship and confirm the allosteric modulation. The 3-carboxamide-quinoline scaffold is maintained in all of the synthesized compounds, while different substituents that may modulate the steric volume, electrostatic 4984

DOI: 10.1021/acs.jmedchem.7b00395 J. Med. Chem. 2017, 60, 4983−5001

Journal of Medicinal Chemistry

Article

Scheme 1a

a

(i) NaH, 0 °C, DMF, R2-X; (ii) NaH, DMF, diethyl malonate; (iii) DMF, 160 °C, 3 min, NH2NHCOR3; (iv) Δ, R4-NH2.

Table 1. GSK-3 Inhibition Evaluation of Quinolines 1 and 26−62

compd no.

R1

1 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

H H H H H H H H H H H H H H H H H

42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62

H 6-F 6-F 6-Cl 6-Cl 6-Cl 6-Cl 6-Cl 6-Br 6-Br 6-Br 6-Br 6-Br 7-Cl 7-Cl 7-Cl 7-Cl 7-Cl 7-Cl 7-Cl 7-Cl

R3

GSK-3 IC50 (μM)

C11H23 C11H23 C7H15 C11H23 C7H15 C15H31 Ph Me Bn (CH2)2-indol-3-yl C7H15 (CH2)2-indol-3-yl (CH2)2-(4-OH-Ph) C11H23 C11H23 C7H15 C11H23

2.8 ± 0.4 4.50 ± 0.30 >20 7.3 ± 0.2 8.7 ± 0.4 insoluble >20 >20 >20 >20 >20 >20 >20 >20 7.78 ± 0.15 7.11 ± 0.25 5.25 ± 0.58

C11H23 C11H23 C7H15 C11H23 C11H23 C7H15 C11H23 C7H15 C11H23 C11H23 C7H15 C11H23 C7H15 C11H23 C7H15 C11H23 C7H15 C11H23 C7H15 C11H23 C7H15

5.51 ± 0.27 5.8 ± 0.8 >20 3.81 ± 0.12 5.95 ± 0.49 8.48 ± 0.30 3.18 ± 0.28 9.42 ± 0.23 3.54 ± 0.18 4.28 ± 0.50 6.28 ± 0.33 2.01 ± 0.18 7.34 ± 0.20 3.12 ± 0.03 9.0 ± 0.3 4.03 ± 0.19 6.66 ± 0.29 2.48 ± 0.26 4.83 ± 0.23 5.99 ± 0.22 5.96 ± 0.17

R2 Et H H Me Me Me Me Me Me Me Et Et Et isobutyl isoprenyl isoprenyl CH2cyclopropyl Bn H H H Me Me Et Et H Me Me Et Et H H Me Me Et Et Bn Bn

Table 2. GSK-3 Inhibition Evaluation of Quinolines 63−65

compd no.

R4

GSK-3 IC50 (μM)

63 64 65

C6H13 CH2CONHCH2COOBn NH-(CH2)2−CN

>20 >20 >20

inhibition, leading to inactive compounds 63−65 when omitted from the structure. Moreover, a long lipophilic tail of 7 or 11 carbon atoms joined to the carboxamide moiety is also a prerequisite for active compounds. In fact, when it is substituted by phenyl, alkyl, benzyl, or dimethylene heteroaryl fragments (compounds 31, 32, 33, 34, and 36), the biological activity is lost. Other changes in the nature of the substituent attached to the heterocyclic nitrogen atom are rather insensitive for GSK-3β inhibition, resulting in compounds with similar enzymatic potency. Finally, introduction of halogen atoms in positions 6 or 7 of the quinoline scaffold produces a slight increase in IC50 value (derivatives 53 and 59 versus 1, for example). To corroborate the kind of inhibition of these new compounds, we selected derivatives 53 and 59 with IC50 values lower than that of the reference compound 1 for different kinetic studies. We determined the competition with ATP or with the substrate used in the enzymatic reaction. The small peptide GS-2 similar to skeletal muscle glycogen synthase was used as substrate. The first set of studies were done varying both ATP (from 1 to 50 μM) and quinoline concentrations. Double-reciprocal plotting of the data is depicted in Figure 3. The intercept of the plot in the vertical axis (1/V) rises when the quinoline concentration increases (from 2.5 to 5 μM), whereas the intercept in the horizontal axis (1/[ATP]) does not change, meaning that, while the enzyme maximal rate (Vmax) decreases in the presence of the inhibitor, the Michaelis−Menten constant (Km) remains unaltered. These results would suggest that derivatives 53 and 59 act as noncompetitive inhibitors of ATP binding because an increase in the ATP concentration (from 1 to 50 μM) does not interfere with enzymatic inhibition. Finally, kinetic experiments were also performed varying the concentrations of both GS-2 (from 12.5 to 100 μM) and quinoline inhibitors 53 and 59 (2.5 and 5 μM), while the ATP concentration was kept constant (1 μM). Double-reciprocal plotting of the data (Figure 4) suggests that these compounds act as noncompetitive inhibitors of GS-2 binding. These results 4985

DOI: 10.1021/acs.jmedchem.7b00395 J. Med. Chem. 2017, 60, 4983−5001

Journal of Medicinal Chemistry

Article

Figure 3. Kinetic data determined for the quinoline derivatives 53 and 59. ATP concentrations in the reaction mixture varied from 1 to 50 μM. Compound concentrations used are depicted in the plot, and the concentration of GS-2 was kept constant at 12.5 μM. Each point is the mean of two different experiments, each one analyzed in duplicate.

Figure 4. Kinetic data determined for the quinoline derivatives 53 and 59. GS-2 concentrations in the reaction mixture varied from 12.5 to 100 μM. Compound concentrations used are depicted in the plot, and the concentration of ATP was kept constant at 1 μM. Each point is the mean of two different experiments, each one analyzed in duplicate.

Figure 5. Kinase profiling for compound 1 (blue), 53 (red), and 59 (green) on different human kinases at a fixed compound concentration of 10 μM.

are similar to that observed for compound 125 and confirm that the new 3-carbohydrazide-quinolines described here are also allosteric inhibitors of GSK-3β, binding to a pocket different from the ones to which ATP and substrate bind. One of the main challenges to overcome when a protein kinase is modulated allosterically is selectivity over other protein kinases. To check that fact, we have tested a couple of compounds, 53 and 59, together with the first prototype, quinoline 1, on a panel of 50 different kinases. Kinase profiling plots are shown in Figure 5, showing a consistent and robust selectivity compatible with an ATP noncompetitive inhibition. These results not only support the selective profile of the allosteric modulators but also rule out the possibility to be pan assay interference compounds.

Molecular Modeling Studies. All of these experimental results prompted us to perform a computational molecular docking study to understand the ligand−protein interaction in detail and the allosteric mechanism of action of these heterocyclic derivatives. The quinoline compounds studied here have at least 3 or 4 protons that can be moved between 6 and 7 different positions marked by nitrogen and oxygen atoms present in the heterocycle and the substituent attached in position 3 leading to different tautomeric forms (Figure 2). Thus, a preliminary computational study was performed to determine the different tautomeric microspecies using the Marvin Sketch software and compound 1 as prototype of this heterocyclic family. Results showed that the more stable tautomeric forms were those with 4986

DOI: 10.1021/acs.jmedchem.7b00395 J. Med. Chem. 2017, 60, 4983−5001

Journal of Medicinal Chemistry

Article

Figure 6. pKa profile of tautomeric form A (top) and tautomeric form B (below) of compound 1.

allosteric cavity previously identified as pocket 725 but considering the favorable protonation state at physiological conditions. The calculated docking scores together with the predicted Ki values were used to evaluate the binding mode of these allosteric modulators. The results obtained showed that these GSK-3β inhibitors need to maintain three different key interactions simultaneously in the allosteric site for enzymatic inhibition. The first one faced the quinoline moiety with Arg209 located in the activation loop of this protein kinase. The nature of this interaction is different for the compounds studied; thus, electrostatic, cation-π, or hydrogen bond types are found. The second one is a crucial hydrogen bond formed between the carbohydrazide group of the inhibitor and Ser236, and the last one is produced by hydrophobic interactions between the aliphatic chain of the inhibitor with the hydrophobic region of the pocket (Leu169, Arg328, Thr330, and Pro331) (Figure 7). These three different interactions seem to be essential for enzyme inhibition. The nonexistence of some of the three key interactions above-mentioned is translated in a decrease or loss of the activity. As an example, inactive compounds 32, 63, and 64 were analyzed. Compounds 32 and 63 bind to the cavity by interactions with Arg209, although these are not present with Ser236 and neither is the hydrophobic region. These facts explain the low affinity of these quinolines and their lack of

a hydroxyl group in positions 2 or 4 of the quinoline heterocycle, forms A and B, respectively (Figure 6). Moreover, using the same software, the pKa profile for A and B tautomeric forms of compound 1, were calculated. Deprotonated microspecies distributions at different pH values are depicted in Figure 6. At physiological pH, the tautomeric form A will have two microspecies coexisting, one with the protonated hydroxyl group (microspecies A) and another deprotonated (microspecies A1). However, the high acidic nature of the 4-hydroxyl group in tautomer B, leads to a unique anionic form at physiological environment (microspecies B1) (Figure 6). Ab initio geometry optimizations and frequency calculations were performed for both anionic species (A1 and B1) showing a difference in Gibbs free energy between them of 0.818 kcal· mol−1 (Figure S1). Both optimized structures maintain the intramolecular H-bond between the hydroxyl group of the quinoline ring and the amine of carbohydrazide, which is the tautomer with the hydroxyl moiety in position 4 (anionic form B1) slightly more favored energetically. Docking experiments were carried out in order to understand the allosteric mechanism of action of 3-carboxamide-quinolines and the differences in the behavior of active and inactive compounds. These studies were performed using AutoDock software (see Experimental Section), centering the grid in the 4987

DOI: 10.1021/acs.jmedchem.7b00395 J. Med. Chem. 2017, 60, 4983−5001

Journal of Medicinal Chemistry

Article

maintained, and therefore, the inhibitory activity is maintained (Figure S3). Finally, docking studies show that bigger aliphatic chains in R3 (C11H23) score better than the smaller ones (C7H15) in agreement with the experimental data (see Table 1). Hydrophobic interactions between the aliphatic chain and Leu169, Val240, Arg328, Thr330, and Pro331 residues appear to be very important to improve the ligand affinity by the allosteric pocket, whereas compounds with the shorter C7H15 chain are less efficient (Figure S4). In order to provide a reasonable hypothesis for the allosteric mechanism of inhibition of this quinoline heterocyclic family, a structural comparative study was performed between the apo form of GSK-3β (1PYX) and the complexes with compound 1 and compound 63, after molecular dynamics experiments. Trajectories for each ligand−GSK-3β complex and GSK-3β without ligand were calculated along 25 ns. Simulations for all systems maintain the stability along the trajectories. Compound 1 is able to restrict the movement of the protein backbone, while compound 63 is not able to restrict this movement (Figure S5), which is in correspondence with the lack of activity of this last compound. More in detail, the comparative analysis of the root-mean-square fluctuations (RMSF) for each residue between the trajectory for apo-GSK-3β and GSK-3β complexed with compound 1 shows the movement of different residues present in the allosteric pocket, such as Arg209 (Figure S6). The major changes are produced in the activation loop region (residues 200−226) inducing an important rearrangement of the residues of the catalytic site. Because of the electrostatic interactions of 1 with Arg209, there is a decrease in the flexibility of the activation loop. The main change is the drastic reduction of the distance between Glu211 and Lys205 (Figures 8 and S7). The interaction of Arg209 with compound 1 does

Figure 7. Suggested binding mode for compound 1. An ionic interaction between the hydroxyl group and Arg209 fixes the ligand to the allosteric cavity, allowing the interactions with Ser236 and the hydrophobic region.

biological activity (see Figure S2). Another interesting example is compound 65 which interacts with Arg209 and Ser236, but the less hydrophobic nature of the chain could prevent its binding to the allosteric site (Figure S2). Among active compounds, the docking study has allowed us to rationalize the relationship between chemical structure and biological activity. Regarding the substituent R1, different halogen atoms in positions 6 and 7 of the quinoline ring, docking results show that the substituent is exposed to solvent without any interaction with target residues. This fact is reflected in the slight variation of IC50 values of the halogen derivatives 1, 53, and 59. Considering R2, the substituent attached to the heterocyclic nitrogen atom of the quinoline ring, small moieties such as H, Me, and Et allow the ionic interaction between the 4-hydroxyl moiety and Arg209 (Figure 7). In the case of bigger substituents such as isoprenyl, CH2-cyclopropyl, and benzyl groups (compounds 39, 40, 41, 42, 61, and 62), the steric hindrance makes the quinoline ring to flip, allowing the formation of an hydrogen bond between the hydroxyl attached to the quinoline and Asn213’s backbone−NH (see Figure S3). This alternative binding mode favors H-bonds between the carbonyl group of the quinoline ring and Arg209 and in addition hydrophobic interactions among R2 substituents and the Arg209’s side chain (Figure S3). In the case of aromatic substituents (compounds 42, 61, and 62), the cation−π interaction between the benzyl group and Arg209 has been found, which would explain that in spite of their different binding modes, the interactions with the Arg209 are still

Figure 8. Local distortion of GSK-3 upon binding compound 1 (yellow). GSK-3 (1PYX) is depicted in green, before binding, and in gray after MD simulation. In the complex 1-GSK-3, the distance between Glu211 and Lys205 is shorter (magenta lines) than that in apo-GSK-3 (blue lines). The hydrogen bond interaction between Glu211 and Lys 205 is present in the complex quinoline 1-GSK-3.

not allow the movement of Glu211, stimulating a reorientation of Glu211 to Lys205 favoring the formation of a strong hydrogen bond. This new bond with Lys205, one of the different basic residues involved in the catalytic recognition site network (Asn213, Arg180, and Arg96), produces different changes of more than 0.5 Å. These conformational changes restrict the movement of the GSK-3β activation loop, modifying the substrate binding. 4988

DOI: 10.1021/acs.jmedchem.7b00395 J. Med. Chem. 2017, 60, 4983−5001

Journal of Medicinal Chemistry

Article

Figure 9. Effect of GSK3 inhibitors 1 and 53 on the expression and cellular location of β-catenin in vitro. LN18 and SH-SY5Y human cell lines were cultured for 72 h in the presence of different GSK-3 inhibitors at a concentration of 10 μM, as indicated in the Experimental Section. 66, 67, and 68, commercial inhibitors of GSK-3, were used as controls. Subcellular distribution of β-catenin (green) was evaluated using immunofluorescence techniques. Nuclei were stained with DAPI (blue). Representative images are shown. A clear cellular membrane distribution of β-catenin is observed in those cells treated with ATP-no competitive compounds, in comparison with the cells cultured in the presence of ATP-competitive compounds. Scale bar, 20 μm.

Effects of Different GSK-3β Inhibitors on β-Catenin Localization. Levels of β-catenin, a multifunctional protein with a pivotal role in embryonic patterning, organogenesis, and adult homeostasis, are normally kept low by a phosphorylation event mediated by GSK-3 (α- and β-isoforms), which targets βcatenin for ubiquitylation and proteosomal degradation.28 When this phosphorylation event is blocked, β-catenin is stabilized and accumulated in the cytosol. Stabilized β-catenin translocates to the nucleus, where it coactivates transcription of different oncogenes.29 The great challenge for a small molecule targeting GSK-3 (α- or β-) is to reduce its aberrant activity found in several diseases without altering its role on β-catenin signaling. By using different techniques (X-ray, molecular dynamics, or NMR),30−32 several allosteric sites that may allow the recognition of one substrate or other have been described in GSK-3β. Moreover, the structural basis for the recruitment of GSK-3β to the Axin-APC scaffold crucial for β-catenin

phosphorylation was described,33 and structural biology studies have shown that it is possible to target the three-dimensional structure of GSK-3β to select for specific substrate-bound forms of the enzyme, enabling selective targeting of one pathway controlled by GSK-3β.34 Considering this background, we decided to explore if the use of several inhibitors of GSK-3β that bind in different enzymatic sites may alter β-catenin localization. We selected two widely used ATP-competitive inhibitors, 66 (SB216763)35 and 67 (alsterpaullone),36 and two allosteric modulators described here, specifically compounds 1 and 53. In addition, we also included 68 (TDZD-8),37 a well-known ATP noncompetitive inhibitor that modulates the enzyme by covalent modification of Cys199. We have used two different human cell lines of glioblastoma and neuroblastoma, LN-18 and SH-SY5Y, respectively, and treated them long-term (72 h) with the different GSK-3β inhibitors. Localization of β-catenin was analyzed by immunohistochemistry. Our results show that 4989

DOI: 10.1021/acs.jmedchem.7b00395 J. Med. Chem. 2017, 60, 4983−5001

Journal of Medicinal Chemistry

Article

Figure 10. Compound 53 improves delayed myogenesis of CDM1 myoblasts. (A) Bright field images of healthy myotubes and CDM1 myotubes differentiated in fusion medium for 4 days. (B) Bright field images of CDM1 myotubes with different doses of compound 53 in fusion medium for 4 days. (C) Western blot analysis of protein extracts from normal (healthy Mt), untreated (CDM1Mt), and treated (CDM1Mt+53) with compound 53 with antibodies to desmin (a differentiation marker) and actin as control.

Figure 11. Compounds 1 and 53 increase the survival of human SMA MNs. (A) Quantification of Islet1+ MNs differentiated from iPSCs derived from SMA type I and type II patients treated with DMSO or increasing concentrations of the indicated compounds for 7 days, fixed, and subjected to immunocytochemistry. (B) Representative images of SMA type I and II Islet1+ MNs treated with the indicated compounds. Scale bar = 50 μm.

whereas in the cell cultures treated with the two ATPcompetitive inhibitors, β-catenin is translocated to the nucleus, in the three cases where the inhibitor targets the enzyme allosterically, localization of β-catenin remains in the cytosol (Figure 9). We here corroborate that allosteric modulators of GSK-3β not only induce a conformational change in the active site by modification of GSK-3β activation loop flexibility but also point to overcoming an important challenge in GSK-3β targeting: reducing the aberrant GSK-3β activity found in several pathologies while not promoting oncogenesis through aberrant β-catenin signaling. On the basis of these results, this

kind of inhibitor should be of great benefit in chronic disease treatments. Effect of GSK-3β Allosteric Modulator on Chronic Diseases. Until now, few data have been obtained from the clinic regarding the safety and the efficacy of GSK-3β inhibitors in patients, and some issues remain unclear. However, it is important to consider that lithium, a weak inhibitor of GSK-3β, is the standard pharmacological therapy for chronic bipolar disorder treatment which may discard mechanism-based toxicities associated with GSK-3β inhibition.38 Moreover, tideglusib, a TDZD-related compound that targets GSK-3β in 4990

DOI: 10.1021/acs.jmedchem.7b00395 J. Med. Chem. 2017, 60, 4983−5001

Journal of Medicinal Chemistry



Article

CONCLUSIONS GSK-3β represents an important drug target for many severe and unmet human diseases. Here, we describe a new family of selective allosteric modulators of GSK-3β45 and provide a careful structural study of their binding mode. The compounds reported here are able to modify the flexibility of the GSK-3β activation loop which not only induces a smooth conformational change in the active site, decreasing the activity, but also points to overcoming an important challenge in GSK-3β targeting: not interfering in the β-catenin signaling. We have shown in this article that modulation of GSK-3β activity by allosteric quinoline 53 in myoblasts from skeletal muscle of patients with congenital DM1 shows that this compound clearly improves CDM1 myogenesis. Moreover, we have shown that allosteric modulation of GSK-3β with quinolines 1 and 53 promotes the survival of SMA motor neurons. Thus, modulation of GSK-3β activity by these allosteric quinoline derivatives opens a new avenue for future chronic treatments of GSK-3β associated diseases, including CDM1 and SMA.

an ATP noncompetitive manner, has no safety concerns in patients of chronic diseases such as progressive supranuclear palsy and Alzheimer’s disease after long-term treatments of 12 and 6 months, respectively.14,15 Until now, the quinoline allosteric modulators reported here have not reached clinical trials, but compound 1 has shown safety and great efficacy in preclinical models of multiple sclerosis39 and fragile X syndrome,40 pointing to a potential use in the chronic treatment of such neurological diseases. Among the untreated diseases where GSK-3β recently emerges as a potential pharmacological target are some rare neuromuscular degenerative diseases. We recently found that the levels of active GSK-3β are increased in skeletal muscle biopsies from patients with myotonic dystrophy type 1 (DM1).8,41 The inhibitors of GSK-3β, lithium and 68, correct the levels of GSK-3β and the GSK-3β-cyclin D3 pathway in a DM1 mouse model (HSALR mice) significantly reducing myotonia, muscle weakness, and myopathy.8,41 Furthermore, although lithium did not improve the neuromuscular phenotype in a mouse model of SMA,42 maleimide-based GSK-3β inhibitors prolonged the median survival time of these mice.43 Given the specific effect of the allosteric modulators here described on GSK-3β activity, we tested if these modulators could be of therapeutic interest for GSK-3β associated neuromuscular diseases, mainly DM1 and SMA. To examine if the allosteric modulators have a beneficial effect in DM1, we determined whether compound 53 improves delayed myogenesis in primary myoblasts from skeletal muscle of patients with congenital DM1 (CDM1). Figure 10 shows that untreated CDM1 myoblasts fail to fuse and form myotubes in a cell culture dish. However, treatment of CDM1 myoblasts during differentiation with compound 53 improved the differentiation of CDM1 myoblasts in a dose-dependent manner. Moreover, an analysis of protein extracts of the different cultures showed that desmin, a differentiation marker, is present in healthy myotubes and is deficient in the CDM1 ones. Treatment with the GSK-3β inhibitor, quinoline 53, corrects expression levels of desmin. These data suggest the therapeutic potential of the allosteric GSK-3β modulator 53 for future therapy of CDM1. Furthermore, we have used human motor neurons (MNs) derived from induced pluripotent stem cells (iPSCs) obtained from a healthy subject (1016A) and SMA patients affected by different disease severities, a severe type II (I-51C) line and a very severe type I (I-38G) line, that have been previously described,44 to evaluate their survival with or without different GSK-3β inhibitors. We have selected two allosteric modulators described here, compounds 1 and 53, and 67, a well-known GSK-3β ATP-competitive inhibitor, and treated the MN cultures from 7 days. Importantly, both compounds 1 and 53 produced a dose-dependent MN survival increase in type I SMA MN cultures (Figure 11). Given that the rate of MN death is notably higher in the most severe type I than in the type II and healthy control cultures, a higher rescue was also expected. The results also showed that these compounds do not display toxicity, even at the highest concentrations tested, whereas 67 was toxic at concentrations higher than 1.25 μM and produced some basal toxicity in the healthy control MNs. These data demonstrate that these non-ATP competitive GSK3β inhibitors prevent the death of iPSC-derived SMA MNs in chronic treatments (7 days).



EXPERIMENTAL SECTION

Chemistry. Substrates were either purchased from commercial sources or used without further purification. Melting points were determined with a Mettler Toledo MP70 apparatus and are uncorrected. Flash column chromatography was carried out at medium pressure using silica gel (E. Merck, grade 60, particle size 0.040−0.063 mm, 230−240 mesh ASTM) with the indicated solvent as eluent. Compounds were detected with UV light (254 nm). 1H NMR spectra were obtained on a Bruker AVANCE-300 spectrometer working at 300 MHz and on the Varian INNOVA-300 operating at 300 MHz. Typical spectral parameters: spectral width 10 ppm, pulse width 9 μs (57°), data size 32 K. Description of signals: s = singlet, bs = broad singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublet, ddd = doublet of dd, and dt = doublet of triplets. 13 C NMR experiments were carried out on the Bruker AVANCE-300 spectrometer operating at 75 MHz. The acquisition parameters: spectral width 16 kHz, acquisition time 0.99 s, pulse width 9 μs (57°), and data size 32 K. J values are reported in hertz. Elemental analyses were performed by the analytical department at CENQUIOR (CSIC) and are reported in the Supporting Information, and the results obtained were within ±0.4% of the theoretical values. HPLC analyses were performed on an Alliance Waters 2690 equipment with a UV detector photodiode array Waters 2996 with MS detector MicromassZQ (Waters) positive electrospray, using the Sunfire column C18, 3.5 μm (50 mm × 4.6 mm) and acetonitrile and Milli-Q water (with 0.1% formic acid) as mobile phase. The standard gradient consisted of a 20 min run (unless otherwise stated) from 10% to 100% acetonitrile gradient at a flow rate of 1.0 mL/min. All of the final compounds have a purity >95% tested by HPLC General Synthesis Procedure for Compounds 2−12. Following the procedure described by Ukrainteset al.,26 the corresponding isatoic anhydride (1 equiv) was dissolved in dry DMF (15 mL), and sodium hydride (1.5 equiv) was added slowly at 0 °C. After 30 min, the corresponding halogenated compound (1.5−3 equiv) was added, and the reaction mixture was stirred at room temperature overnight. Dichloromethane (15 mL) and water (15 mL) were added. The organic phases were washed with brine (15 mL), dried over magnesium sulfate, filtered, and the solvent evaporated. The obtained solid was recrystallized in dichloromethane/hexane to afford the desired compound. N-Isobutylisatoic Anhydride (2). Reagents: isatoic anhydride (1 g, 6.1 mmol); sodium hydride (0.23 g, 9.2 mmol); and isobutyl iodide (3.4 g, 18 mmol). White solid (600 mg, 46%). Mp = 85−86 °C. HPLC: purity >99%, R.t. = 8.6 min. ESI MS (m/z): 220 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 8.10 (dd, J = 1.7, 7.9 Hz, 1H), 7.68 (ddd, J = 1.6, 7.3, 8.4 Hz, 1H), 7.25−7.18 (m, 1H), 7.10 (d, J = 4991

DOI: 10.1021/acs.jmedchem.7b00395 J. Med. Chem. 2017, 60, 4983−5001

Journal of Medicinal Chemistry

Article

8.5 Hz, 1H) 3.93 (d, J = 7.6 Hz, 2H), 2.25−2.10 (m,1H), 1.03 (d, J = 6.7 Hz, 6H). 13C NMR (75 MHz, CDCl3) δ (ppm): 162.4, 158.7, 148.3, 141.8, 137.2, 131.1, 124.0, 114.4, 112.0, 51.7, 26.9, 20.1, 19.9. Anal. (C12H13NO3). N-Isoprenylisatoic Anhydride (3). Reagents: isatoic anhydride (1 g, 6.1 mmol); sodium hydride (0.23 g, 9.2 mmol); and isoprenyl bromide (1.9 g, 12 mmol). White solid (950 mg, 67%). Mp = 119−120 °C (lit.46 119 °C). N-Cyclopropylmethylisatoic Anhydride (4). Reagents: isatoic anhydride (0.8 g, 4.9 mmol); sodium hydride (0.14 g, 5.9 mmol); and cyclopropyl methyl bromide (3.4 g, 18 mmol). Brown solid (530 mg, 50%). Mp = 122−123 °C (lit.47 118−121 °C). N-Benzylisatoic Anhydride (5). Reagents: isatoic anhydride (1 g, 6.1 mmol); sodium hydride (0.22 g, 9.2 mmol); and benzyl bromide (1.4 g, 12.3 mmol). White solid (200 mg, 15%). Mp = 139−140 °C (lit.47 140−141 °C). 5-Chloro-N-methylisatoic Anhydride (6). Reagents: 5-chloroisatoic anhydride (0.8 g, 3.9 mmol); sodium hydride (0.14 g, 5.9 mmol); and methyl iodide (1.6 g, 11.4 mmol). White solid (500 mg, 60%). Mp = 194−195 °C (lit.47 192−195 °C). 5-Chloro-N-ethylisatoic Anhydride (7). Reagents: 5-chloroisatoic anhydride (0.5 g, 2.5 mmol); sodium hydride (0.09 g, 3.8 mmol); and ethyl iodide (0.6 g, 3.8 mmol). White solid (400 mg, 70%). Mp = 145−146 °C (lit.47 143−145 °C). 5-Bromo-N-methylisatoic Anhydride (8). Reagents: 5-bromoisatoic anhydride (1 g, 4.1 mmol); sodium hydride (0.15 g, 6.2 mmol); and methyl iodide (0.9 g, 6.2 mmol). White solid (690 mg, 66%). Mp = 201−202 °C (lit.48 204 °C). 5-Bromo-N-ethylisatoic Anhydride (9). Reagents: 5-bromoisatoic anhydride (2 g, 8.2 mmol); sodium hydride (0.3 g, 12.5 mmol); and ethyl iodide (1.9 g, 12.5 mmol). White solid (1.4 g, 63%). Mp = 177− 178 °C. HPLC: purity >97%, R.t. = 8.4 min. ESI MS (m/z): 272 [M + 2H]+, 270 [M + H]+. 1H NMR (300 MHz, CDCl3) δ (ppm): 8.21 (d, J = 2.4 Hz, 1H), 7.78 (dd, J = 2.4, 8.9 Hz, 1H), 7.02 (d, J = 8.9 Hz, 1H), 4.05 (q, J = 7.2 Hz, 2H), 1.31 (t, J = 7.2 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ (ppm): 157.3, 146.9, 140.0, 139.0, 133.2, 116.6, 115.5, 113.4, 40.3, 12.0. Anal. (C10H8BrNO3). 4-Chloro-N-methylisatoic Anhydride (10). Reagents: 4-chloroisatoic anhydride (0.9 g, 4.5 mmol); sodium hydride (0.12 g, 5.4 mmol); and methyl iodide (1.9 g, 13.7 mmol). Yellow solid (400 mg, 42%). Mp = 215−216 °C (lit.47 216−218 °C). 4-Chloro-N-ethylisatoic Anhydride (11). Reagents: 4-chloroisatoic anhydride (0.9 g, 4.5 mmol); sodium hydride (0.12 g, 5.0 mmol); and ethyl iodide (1.3 g, 8.3 mmol). White solid (650 mg, 63%). Mp = 156−157 °C. HPLC: purity >97%, R.t. = 8.1 min. ESI MS (m/z): 228 [M + 2H]+, 226 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 8.00 (d, J = 8.4 Hz, 1H), 7.64 (d, J = 1.7 Hz, 1H), 7.37 (dd, J = 1.7, 8.4 Hz, 1H), 4.06 (q, J = 7.0 Hz, 2H), 1.20 (t, J = 7.0 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 158.8, 147.6, 142.8, 142.5, 131.8, 124.1, 114.9, 111.4, 40.2, 12.3. Anal. (C10H8ClNO3). N-Benzyl-4-chloroisatoic Anhydride (12). Reagents: 4-chloroisatoic anhydride (1.0 g, 5.0 mmol); sodium hydride (0.19 g, 7.8 mmol); and benzyl bromide (1.7 g, 10.1 mmol). White solid (650 mg, 63%). Mp = 167−168 °C (lit.47 165−170 °C). General Synthesis Procedure for Compounds 13−25. Following the procedure described by Coppola et al.49 diethylmalonate was added dropwise to a sodium hydride solution in dry DMF (15 mL) under argon atmosphere. The reaction was stirred for 30 min at room temperature. Then, the mixture was added to a round-bottomed flask containing the corresponding isatoic anhydride derivative dissolved in DMF (15 mL) under argon atmosphere. The mixture was heated at 50 °C during 5 h. To the mixture, dichlorometane (30 mL), water (50 mL), and HCl (1 N) were added to reach pH 5. The organic layer was isolated and washed with brine (30 mL). Then, the organic phase was dried over dry magnesium sulfate and filtered, and the solvent was evaporated. The solid was recrystallized in dichloromethane/hexane to obtain the desired compound. Ethyl 4-Hydroxy-1-isobutyl-2-oxo-1,2-dihydroquinoline-3-carboxylate (13). Reagents: N-isobutylisatoic anhydride (1) (0.3 g, 1.3 mmol); sodium hydride (0.138 g, 5.7 mmol); and diethyl malonate

(1.1 g, 6.8 mmol). Yellow oil (150 mg, 38%). HPLC: purity >95%, R.t. = 10.0 min. ESI MS (m/z): 290 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 13.02 (s, 1H), 8.06 (dd, J = 1.55, 8.3 Hz, 1H), 7.72 (ddd, J = 1.60, 7.0, 8.7 Hz, 1H), 7.55 (d, J = 8.6 Hz, 1H), 7.29 (ddd, J = 0.86, 7.0, 7.9 Hz, 1H), 4.33 (q, J = 8.6 Hz, 2H), 4.07 (d, J = 7.4 Hz, 2H), 2.10 (sept, J = 7.0 Hz, 1H), 1.31 (t, J = 7.4 Hz, 3H), 0.89 (d, J = 7.4 Hz, 6H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 169.2, 165.5, 158.9, 140.0, 133.6, 124.6, 121.7, 115.3, 114.5, 101.0, 61.4, 47.5, 26.6, 19.8, 13.9. Anal. (C16H19NO4). Ethyl 4-Hydroxy-1-isoprenyl-2-oxo-1,2-dihydroquinoline-3-carboxylate (14). Reagents: N-isopropenylisatoic anhydride (2) (0.3 g, 1.3 mmol); sodium hydride (0.065 g, 1.3 mmol); and diethyl malonate (1.0 g, 6.5 mmol). White solid (80 mg, 21%). Mp = 123−124 °C. HPLC: purity >96%, R.t. = 10.2 min. ESI MS (m/z): 302 [M + H]+. 1 H NMR (300 MHz, DMSO-d6) δ (ppm): 14.21 (s, 1H), 8.18 (dd, J = 1.6, 8.3 Hz, 1H), 7.65 (td, J = 1.6, 7.1, 7.9 Hz, 1H), 7.24 (m, 2H), 5.10 (m, 1H), 4.88 (d, J = 6.0 Hz, 2H), 4.51 (q, J = 7.1 Hz, 2H), 1.80 (s, 3H), 1.63 (s, 3H), 1.41 (t, J = 7.1 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ (ppm): 172.9, 171.8, 159.5, 140.9, 135.8, 134.3, 125.9, 121.8, 119.7, 115.2, 114.7, 98.0, 62.5, 40.9, 25.7, 18.5, 14.4. Anal. (C13H13NO3). Ethyl 1-Benzyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylate (16). Reagents: N-benzylisatoic anhydride (4) (0.85 g, 3.4 mmol); sodium hydride (0.18 g, 7.37 mmol); and diethyl malonate (2.7 g, 16.8 mmol). White solid (100 mg, 9%), Mp = 117−118 °C. HPLC: purity >99%, R. t. = 9.9 min. ESI MS (m/z): 324 [M + H]+. 1H NMR (300 MHz, CDCl3) δ (ppm): 14.29 (s, 1H), 8.13 (dd, J = 1.5, 8.0 Hz, 1H), 7.48−7.43 (m, 1H), 7.23−7.18 (m, 2H), 7.15−7.11 (m, 5H), 5.44 (s, 2H), 4.45 (q, J = 7.1 Hz, 2H), 1.42 (t, J = 7.1 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ (ppm): 171.7, 171.0, 158.7, 139.8, 135.4, 133.3, 127.7, 126.2, 125.4, 124.7, 121.0, 113.9, 96.8, 61.4, 44.6, 13.21. Anal. (C19H17NO4). Ethyl 6-Chloro-4-hydroxy-2-oxo-1,2-dihydroquiniline-3-carboxylate (17). Reagents: 5-chloroisatoicanhydride (1.0 g, 5.0 mmol); sodium hydride (0.25 g, 10.4 mmol); and diethyl malonate (4.0 g, 25 mmol). White solid (0.06 g, 9%). Mp = 251 °C (lit.50 250 °C decomp). Ethyl 6-Chloro-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline3-carboxylate (18). Reagents: 5-chloro-N-methylisatoicanhydride (5) (0.8 g, 3.5 mmol); sodium hydride (0.18 g, 7.1 mmol); and diethyl malonate (2.8 g, 18 mmol). White solid (300 mg, 30%). Mp = 136− 137 °C (lit.49 132−135 °C). Ethyl 6-Chloro-1-ethyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3carboxylate (19). Reagents: 5-chloro-N-ethylisatoicanhydride (6) (0.8 g, 3.5 mmol); sodium hydride (0.18 g, 7.1 mmol); and diethyl malonate (2.8 g, 18 mmol). White solid (500 mg, 48%). Mp = 140− 141 °C. HPLC: purity >99%, R.t. = 9.9 min. ESI MS (m/z): 298 [M + 2H]+, 296 [M + H]+. 1H NMR (300 MHz, CDCl3) δ (ppm): 14.14 (s, 1H), 8.09 (d, J = 2.5 Hz, 1H), 7.54 (dd, J = 2.5, 9.1 Hz 1H), 7.19 (d, J = 2.0 Hz, 1H), 4.44 (q, J = 7.1 Hz, 2H), 4.21 (q, J = 7.1 Hz, 2H), 1.42 (t, J = 7.1 Hz, 3H), 1.25 (t, J = 7.1 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ (ppm): 171.4, 169.5, 157.7, 137.8, 133.3, 126.4, 124.2, 115.1, 114.5, 97.5, 61.2, 36.4,14.3, 13.2. Anal. (C14H14ClNO4). Ethyl 6-Bromo-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline3-carboxylate (21). Reagents: 5-bromo-N-methylisatoicanhydride (7) (0.6 g, 2.3 mmol); sodium hydride (0.12 g, 4.8 mmol); and diethyl malonate (1.8 g, 11.5 mmol). White solid (0.3 g, 40%). Mp = 139− 140 °C. HPLC: purity >96%, R.t. = 9.6 min. ESI MS (m/z): 330 [M + 2H], 328 [M + H]+. 1H NMR (300 MHz, CDCl3) δ (ppm): 14.14 (s, 1H), 8.28 (d, J = 2.4 Hz, 1H), 7.74 (dd, J = 2.4, 9.0 Hz, 1H), 7.18 (d, J = 9.0 Hz, 1H), 4.50 (q, J = 7.1 Hz, 2H), 3.62 (s, 3H), 1.48 (t, J = 7.1 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ (ppm): 172.5, 170.6, 159.3, 140.3, 137.2, 128.2, 116.5, 116.0, 115.0, 98.7, 62.7, 29.4, 14.3. Anal. (C13H12BrNO4). Ethyl 6-Bromo-1-ethyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3carboxylate (22). Reagents: 5-bromo-N-ethylisatoicanhydride (8) (0.5 g, 1.8 mmol); sodium hydride (0.09 g, 3.7 mmol); and diethyl malonate (1.5 g, 4.25 mmol). White solid (0.3 g, 48%). Mp = 129− 130 °C. HPLC: purity >99%, R.t. = 10.6 min. ESI MS (m/z): 342 [M + 2H]+, 340 [M + H]. 1H NMR (300 MHz, CDCl3) δ (ppm): 14.14 4992

DOI: 10.1021/acs.jmedchem.7b00395 J. Med. Chem. 2017, 60, 4983−5001

Journal of Medicinal Chemistry

Article

(s, 1H), 8.23 (d, J = 2.4 Hz, 1H), 7.67 (dd, J = 2.4, 9.0 Hz 1H), 7.13 (d, J = 9.0 Hz, 1H), 4.44 (q, J = 7.1 Hz, 2H), 4.20 (q, J = 7.1 Hz, 2H), 1.42 (t, J = 0.5,7.1 Hz, 3H), 1.24 (t, J = 1.5,7.2 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ (ppm): 172.4, 170.4, 158.7, 139.2, 137.0, 128.7, 116.6, 115.7, 114.6, 98.5, 62.5, 37.4, 14.2, 12.7. Anal. (C14H14BrNO4). Ethyl 7-Chloro-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline3-carboxylate (23). Reagents: 4-chloro-N-methylisatoicanhydride (9) (0.4 g, 2.1 mmol); sodium hydride (0.1 g, 4.2 mmol); and diethyl malonate (1.7 g, 10.2 mmol). White solid (280 mg, 49%). Mp = 153− 154 °C. HPLC: purity >99%, R.t. = 5.1 min. ESI MS (m/z): 284 [M + 2H]+, 282 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 12.95 (s, 1H), 8.02 (d, J = 8.6 Hz, 1H), 7.59 (d, J = 1.8 Hz, 1H), 7.33 (dd, J = 1.9, 8.7 Hz, 1H), 4.32 (q, J = 7.1 Hz, 2H), 3.52 (s, 3H), 1.30 (t, J = 7.1 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 168.9, 164.6, 159.2, 141.7, 138.9, 126.7, 122.5, 115.1, 113.7, 102.3, 61.8, 49.1, 29.4, 14.4. Anal. (C13H12ClNO4). Ethyl 7-Chloro-1-ethyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3carboxylate (24). Reagents: 4-chloro-N-ethylisatoic anhydride (10) (0.6 g, 2.6 mmol); sodium hydride (0.1 g, 4.6 mmol); and diethyl malonate (1.9 g, 11.5 mmol). White solid (460 mg, 62%). Mp = 141− 142 °C. HPLC: purity >99%, R.t. = 9.9 min. ESI MS (m/z): 298 [M + 2H]+, 296 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 13.00 (s, 1H), 8.03 (d, J = 8.6 Hz, 1H), 7.64 (d, J = 1.8 Hz, 1H), 7.33 (dd, J = 1.9, 8.6 Hz, 1H), 4.32 (q, J = 7.1 Hz, 2H), 4.19 (q, J = 7.1 Hz, 2H), 1.30 (t, J = 7.1 Hz, 3H), 1.15 (t, J = 7.1 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 169.0, 164.8, 158.6, 140.7, 139.0, 127.0, 122.3, 114.6, 113.9, 102.0, 61.8, 37.1,14.4, 13.0. Anal. (C14H14ClNO4). Ethyl 1-Benzyl-7-chloro-4-hydroxy-2-oxo-1,2-dihydroquinoline3-carboxylate (25). Reagents: N-benzyl-4-chloroisatoic anhydride (11) (0.5 g, 1.9 mmol); sodium hydride (0.09 g, 3.9 mmol); and diethyl malonate (1.6 g, 9.6 mmol). White solid (900 mg, 94%). Mp = 172−173 °C. HPLC: purity >99%, R.t. = 10.8 min. ESI MS (m/z): 360 [M + 2H]+, 358 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 13.17 (s, 1H), 8.07 (d, J = 8.6 Hz, 1H), 7.43 (d, J = 1.8 Hz, 1H), 7.35−7.32 (m, 3H), 7.26−7.23 (m, 1H), 7.18 (dd, J = 1.3, 8.6 Hz, 2H), 5.47 (s, 2H), 4.34 (q, J = 7.1 Hz, 2H), 1.31 (t, J = 7.1 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 168.5, 164.8, 158.9, 140.6, 138.7, 136.5, 128.7, 127.1, 126.6, 126.4, 122.3, 114.9, 113.8, 101.6, 61.5, 44.5, 13.9. Anal. (C19H16ClNO4). General Procedure for the Synthesis of Compounds 1 and 26− 62. Following the procedure described by Jonsson et al.,51 the corresponding ethyl carboxylates were mixed with the corresponding hydrazide, and the mixture was heated in DMF at 160 °C for 3 min. The mixture was cooled down to room temperature, MeOH (5 mL) was added, and a white precipitate was formed. The precipitate was filtered and washed with MeOH to afford the products. N′-Dodecanoyl-1-ethyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3carbohydrazide (1). Reagents: ethyl 1-ethyl-4-hydroxy-2-oxo-1,2dihydroquinoline-3-carboxylate (0.17 g, 0.67 mmol) and dodecanoylhydrazide (0.14 g, 0.67 mmol). White solid (142 mg, 91%). Mp = 133−134 °C. HPLC (10 min run): purity >99%, R.t. = 3.82 min. ESI MS (m/z): 430 [M + H]. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 16.22 (s, 1H), 12.05 (d, J = 3.7 Hz, 1H), 10.80 (d, J = 3.7 Hz, 1H), 8.09 (d, J = 8.0 Hz, 1H), 7.82 (t, J = 7.1 Hz, 1H), 7.68 (d, J = 8.6 Hz, 1H), 7.38 (t, J = 7.4 Hz, 1H), 4.31 (d, J = 6.7 Hz, 2H), 2.24 (m, 2H), 1.55 (m, 2H), 1.23 (m, 19H), 0.84 (m, 3H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 161.5, 171.4, 170.3, 168.0, 139.6, 135.3, 125.4, 123.2, 115.8, 96.4, 37.6, 33.7, 31.9, 29.6, 29.6, 29.4, 29.2, 25.7, 22.7, 14.5, 13.4. Anal. (C24H35N3O4). N′-Dodecanoyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carbohydrazide (26). Reagents: ethyl 4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylate (0.030 g, 0.15 mmol) and dodecanoylhydrazide (0.030 g, 0.15 mmol). White solid (34 mg, 98%). Mp = 256−257 °C. HPLC (10 min run): purity >99%, R.t. = 2.19 min. ESI MS (m/z): 402 [M + H]. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 16.11 (s, 1H), 11.98 (s, 1H), 11.92 (s, 1H), 10.63 (s, 1H), 7.98 (d, J = 8.1 Hz, 1H), 7.71 (t, J = 7.7 Hz, 1H), 7.39 (d, J = 8.3 Hz, 1H), 7.31 (t, J = 7.6 Hz, 1H), 2.23 (t, J = 7.2 Hz, 2H), 1.57 (d, J = 6.6 Hz, 2H), 1.25 (s, 16H), 0.85 (t, J = 6.2 Hz); 13C NMR (75 MHz, DMSO-d6) δ (ppm): 162.4, 172.2, 170.0, 167.8, 139.2, 134.6, 124.3, 122.9, 116.3, 114.5,

99.4, 33.3, 31.7, 31.0, 29.4, 29.1, 28.9, 25.3, 22.5, 14.3. Anal. (C22H31N3O4). 4-Hydroxy-N′-octanoyl-2-oxo-1,2-dihydroquinoline-3-carbohydrazide (27). Reagents: ethyl 4-hydroxy-2-oxo-1,2-dihydroquinoline-3carboxylate (0.050 g, 0.2 mmol) and octanoylhydrazide (0.030 g, 0.2 mmol). White solid (48 mg, 83%). Mp = 254−255 °C. HPLC (10 min run): purity 95%, R.t. = 2.14 min. ESI MS (m/z): 346 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 16.10 (s, 1H), 11.97 (s, 2H), 10.67 (s, 1H), 7.99 (d, J = 8.1 Hz, 1H), 7.69 (d, J = 7.5 Hz, 1H), 7.49− 6.98 (m, 2H), 2.23 (t, J = 7.2 Hz, 2H), 1.55 (s, 2H), 1.27 (s, 8H), 0.86 (d, J = 6.6 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 162.8, 172.5, 170.0, 167.9, 139.2, 134.4, 124.4, 122. 9, 116.3, 96.1, 33.3, 31.5, 28.9, 28.8, 25.3, 22.4, 14.3. Anal. (C18H23N3O4). N′-Dodecanoyl-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline3-carbohydrazide (28). Reagents: ethyl 4-hydroxy-1-methyl-2-oxo1,2-dihydroquinoline-3-carboxylate (0.15 g, 0.61 mmol) and dodecanoylhydrazide (0.13 g, 0.61 mmol). White solid (137 mg, 93%). Mp = 219−220 °C. HPLC (10 min run): purity >99%, R.t. = 3.02 min. ESI MS (m/z): 416 [M + H]. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 16.15 (s, 1H), 11.91 (s, 1H), 10.38 (s, 1H), 8.15 (d, J = 8.0 Hz, 1H), 7.85 (ddd, J = 8.7, 7.1, 1.6 Hz, 1H), 7.66 (d, J = 8.6 Hz, 1H), 7.49− 7.25 (m, 1H), 3.69 (s, 3H), 2.28 (t, J = 7.3 Hz, 2H), 1.64 (s, 2H), 1.64−1.11 (m, 16H), 0.89 (t, J = 6.7 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ (ppm): 161.9, 171.1, 169.1, 166.6, 140.1, 134.1, 125.4, 122.5, 115.6, 114.4, 96.3, 34.4, 31.9, 29.6, 29.4, 29.3, 29.2, 25.4, 22.7, 14.1. Anal. (C23H33N3O4). 4-Hydroxy-1-methyl-N′-octanoil-2-oxo-1,2-dihydroquinoline-3carbohydrazide (29). Reagents: ethyl 4-hydroxy-1-methyl-2-oxo-1,2dihydroquinoline-3-carboxylate (0.30 g, 1.2 mmol) and octanoylhydrazide (0.19 g, 1.2 mmol). White solid (330 mg, 99%). Mp = 157− 158 °C. HPLC (10 min run): purity >99%, R.t. = 5.53 min. ESI MS (m/z): 360 [M + H]). 1H NMR (300 MHz, DMSO-d6) δ (ppm): 16.24 (s, 1H), 11.99 (d, J = 3.5 Hz, 1H), 10.75 (d, J = 3.4 Hz, 1H), 8.08 (d, J = 8.0 Hz, 1H), 7.92−7.74 (m, 1H), 7.64 (d, J = 8.7 Hz, 1H), 7.39 (t, J = 7.6 Hz, 1H), 3.64 (t, J = 4.9 Hz, 3H), 2.22 (t, J = 7.3 Hz, 2H), 1.79−1.34 (m, 2H), 1.25 (s, 8H), 0.85 (t, J = 6.1 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 161.5, 171.0, 169.9, 167.6, 140.2, 134.9, 124.8, 123.1, 115.9, 115.1, 96.1, 33.3, 31.5, 29.6, 28.9, 28.7, 25.3, 22.4, 14.3. Anal. (C19H25N3O4). 4-Hydroxy-1-methyl-N′-palmitoyl-2-oxo-1,2-dihydroquinoline-3carbohydrazide (30). Reagents: ethyl 4-hydroxy-1-methyl-2-oxo-1,2dihydroquinoline-3-carboxylate(0.30 g, 1.2 mmol) and palmitoylhydrazide (0.33 mg, 1.2 mmol). White solid (532 mg, 94%). Mp = 149− 150 °C. HPLC (10 min run): purity 95%, R.t. = 7.24 min. ESI MS (m/ z): 472 [M + H]. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 16.25 (s, 1H), 11.99 (s, 1H), 10.76 (s, 1H), 8.09 (d, J = 7.8 Hz, 1H), 7.83 (m, 1H), 7.64 (d, J = 8.3 Hz, 1H), 7.39 (m, 1H), 3.63 (m, J = 2.7 Hz, 3H), 2.21 (m, 2H), 1.52 (s, 2H), 1.20 (s, 24H), 0.81 (d, J = 3.0 Hz, 3H). Anal. (C27H41N3O4). N′-Benzoyl-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3carbohydrazide (31). Reagents: ethyl 4-hydroxy-1-methyl-2-oxo-1,2dihydroquinoline-3-carboxylate (0.50 g, 2.02 mmol) and phenylhydrazide (0.27 g, 2.02 mmol). White solid (585 mg, 86%). Mp = 138−139 °C (lit.26 137−139 °C). N′-Acetyl-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carbohydrazide (32). Reagents: ethyl 4-hydroxy-1-methyl-2-oxo-1,2dihydroquinoline-3-carboxylate (0.40 g, 1.62 mmol) and methylhydrazide (0.12 g, 1.62 mmol). White solid (524 mg, 89%). Mp = 252− 253 °C. HPLC (10 min run): purity >99%, R.t. = 3.83 min. ESI MS (m/z): 276 [M + H]. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 16.21 (s, 1H), 11.99 (d, J = 4.1 Hz, 1H), 10.79 (d, J = 4.1 Hz, 1H), 8.08 (dd, J = 8.0, 1.3 Hz, 1H), 7.82 (t, J = 7.9 Hz, 1H), 7.63 (d, J = 8.6 Hz, 1H), 7.38 (t, J = 7.6 Hz, 1H) 3.63 (s, 3H), 1.96 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 161.5, 171.0, 167.5, 166.8, 140.2, 135.0, 124.8, 123.1, 115.9, 115.1, 96.0, 29.6, 20.7. Anal. (C13H13N3O4). 4-Hydroxy-1-methyl-2-oxo-N′-(2-phenylacetyl)-1,2-dihydroquinoline-3-carbohydrazide (33). Reagents: ethyl 4-hydroxy-1-methyl-2oxo-1,2-dihydroquinoline-3-carboxylate (0.30 g, 1.2 mmol) and benzylhydrazide (0.18 g, 1.2 mmol). White solid (273 mg, 80%). Mp = 227−228 °C. HPLC (10 min run): purity >99%, R.t. = 4.90 min. 4993

DOI: 10.1021/acs.jmedchem.7b00395 J. Med. Chem. 2017, 60, 4983−5001

Journal of Medicinal Chemistry

Article

δ (ppm): 16.32 (s, 1H), 12.05 (d, J = 4.1 Hz, 1H), 10.79 (d, J = 4.1 Hz, 1H), 8.12 (d, J = 1.6 Hz, 1H), 7.9−7.75 (m, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.35 (t, J = 7.6 Hz, 1H), 4.18 (d, J = 7.5 Hz, 2H), 2.30−2.05 (m, 3H), 1.52−1.35 (m, 2H), 1.25−1.05 (m, 16H), 0.95−0.80 (m, 9H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 170.6, 169.4, 167.0, 161.5, 139.4, 134.5, 124.6, 122.6, 115.9, 114.9, 95.5, 47.85, 31.3, 29.0, 28.9, 28.7, 28.6, 28.5, 26.7, 24.9, 22.0, 19.8, 13.9. Anal. (C26H39N3O4). N′-Dodecanoyl-4-hydroxy-1-isoprenyl-2-oxo-1,2-dihydroquinoline-3-carbohydrazide (39). Reagents: ethyl carboxylate 13, (0.2 g, 0.66 mmol) and dodecanoylhydrazide (0.16 g, 0.73 mmol). White solid (125 mg, 40%). Mp = 107−108 °C. HPLC: purity >99%, R.t. = 4.9 min. ESI MS (m/z): 470 [M + 2H]+. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 16.28 (s, 1H), 11.97 (d, J = 3.9 Hz, 1H), 10.77 (d, J = 3.9 Hz, 1H), 8.11 (dd, J = 7.9, 1.5 Hz, 1H), 7.83 (td, J = 9.18, 7.2, 1.6 Hz, 1H), 7.51 (d, J = 8.6 Hz, 1H), 7.39 (t, J = 8.6 Hz, 1H), 5.51−5.05 (m, 1H), 4.95−4.85 (m, 2H), 2.23 (t, J = 7.3 Hz, 2H), 1.85 (s, 3H), 1.67 (s, 3H), 1.6−1.40 (m, 2H), 1.35−1.1 (m, 16H), 0.84 (t, J = 6.5 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 170.7, 169.6, 167.3, 160.9, 139.0, 135.8, 134.7, 124.7, 122.7, 119.2, 115.5, 114.9, 95.7, 32.9, 31.3, 29.0, 28.9, 28.8, 28.7, 28.5, 25.3, 24.9, 22.1, 18.2, 13.9. Anal. (C27H39N3O4). 4-Hydroxy-1-isoprenyl-N′-octanoil-2-oxo-1,2-dihydroquinoline3-carbohydrazide (40). Reagents: ethyl carboxylate 13 (0.2 g, 0.66 mmol), octanoylhydrazide (0.12 g, 0.73 mmol). White solid (125 mg, 22%). Mp = 114−115 °C. HPLC: purity >99%, R.t. = 13.7 min. ESI MS (m/z): 414 [M + H]+. 1H NMR (300 MHz, CDCl3) δ (ppm): 15.37 (s, 1H), 12.38 (d, J = 5.3 Hz, 1H), 8.67−8.55 (m, 1H), 8.06 (d, J = 7.9 Hz, 1H), 7.57 (t, J = 7.9 Hz, 1H), 7.22−7.14 (m, 2H), 5.1−5.02 (m, 1H), 4.81−4.76 (m, 2H), 2.26 (t, J = 7.5 Hz, 2H), 1.80 (s, 3H), 1.7−1.52 (m, 5H), 1.38−1.1 (m, 8H), 0.79 (t, J = 6.4 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ (ppm): 170.1, 169.6, 166.9, 161.7, 139.7, 136.5, 134.09, 125.1, 122.4, 119.1, 115.8, 114.9, 96.4, 40.9, 34.4, 31.8, 29.3, 29.1, 25.7, 26.6, 22.7, 18.5, 14.2. Anal. (C23H31N3O4). 1-(Cyclopropylmethyl)-N′-dodecanoyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carbohydrazide (41). Following the general synthesis procedure for compounds 13−25, ethyl 1-(cyclopropylmethyl)-4hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylate (15) was synthesized and used here without further purification. Diethyl malonate (1.1 g, 6.8 mmol) was added dropwise to a sodium hydride (0.007 g, 2.9 mmol) solution in dry DMF (15 mL) under argon atmosphere. The reaction was stirred for 30 min at room temperature. Then, the mixture was added to a round-bottomed flask containing the Ncyclopropylmethylisatoic anhydride 4 (0.3 g, 1.3 mmol) dissolved in DMF (15 mL) under argon atmosphere. The mixture was heated at 50 °C during 5 h. To the mixture, dichloromethane (30 mL), water (50 mL), and HCL (1N) were added to reach pH 5. The organic layer was isolated and washed with brine (30 mL). The organic phase was dried over magnesium sulfate and filtered, and the solvent was evaporated. The solid (0.13 g, 0.45 mmol) was used in the next step without further purification after reaction with dodecanoylhydrazide (0.15 g, 0.68 mmol) to yield hydrazide 41. White solid (90 mg, 49%). Mp = 121−122 °C. HPLC: purity >99%, R.t. = 15.8 min. ESI MS (m/z): 456 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 16.30 (s, 1H), 12.01 (d, J = 4.1 Hz, 1H), 10.76 (d, J = 4.1 Hz, 1H), 8.12 (d, J = 7.9 Hz, 1H), 7.80−7.61 (m, 2H), 7.35 (t, J = 7.6 Hz, 1H), 4.23 (d, J = 6.9 Hz, 2H), 2.23 (t, J = 7.6 Hz, 2H), 1.6−1.42 (m, 2H), 1.32−1.17 (m, 16H), 0.84 (t, J = 6.9 Hz, 3H), 0.52−0.42 (m, 5H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 170.7, 169.5, 167.0, 161.3, 139.3, 134.6, 124.6, 122.7, 115.8, 114.9, 95.6, 45.2, 32.9, 31.3, 29.0, 28.9, 28.8, 28.7, 28.5, 25.0, 22.1, 13.9, 9.8, 3.7. Anal. (C26H37N3O4). 1-Benzyl-N′-dodecanoyl-4-hydroxy-2-oxo-1,2-dihydroquinoline3-carbohydrazide (42). Reagents: ethyl carboxylate 16 (0.04 g, 0.12 mmol) and dodecanoylhydrazide (0.033 g, 0.15 mmol). White solid (45 mg, 75%). Mp = 167−168 °C. HPLC: purity >99%, R.t. = 9.6 min. ESI MS (m/z): 516 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 15.63 (s, 1H), 12.13 (d, J = 4.1 Hz, 1H), 8.25−8.08 (m, 2H), 7.3−7.1 (m, 7H), 5.46 (s, 2H), 2.35−2.10 (m, 2H), 1.75−1.50 (m, 2H), 1.5−1.1 (m, 16H), 0.9−0.75 (m, 3H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 170.5, 168.2, 165.8, 161.1, 138.7, 134.9, 133.2,

ESI MS (m/z): 352 [M + H]. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 16.13 (s, 1H), 12.07 (d, J = 4.1 Hz, 1H), 11.13 (d, J = 4.2 Hz, 1H), 8.07 (dd, J = 8.0, 1.2 Hz, 1H), 7.81 (t, J = 7.8 Hz, 1H), 7.61 (d, J = 8.6 Hz, 1H), 7.42−7.19 (m, 6H), 3.61 (s, 3H), 3.60 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 161.1, 170.6, 167.3, 167.1, 139.8, 135.4, 134.6, 129.1, 128.3, 126.6, 124.4, 122.7, 115.5, 114.6, 95.6, 39.7, 29.2. Anal. (C19H17N3O4). N′-2-(Indol-3-yl)propanoyl-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carbohydrazide (34). Reagents: ethyl 4-hydroxy-1methyl-2-oxo-1,2-dihydroquinoline-3-carboxylate (0.20 g, 0.81 mmol) and 3-(indol-3-yl)-propanohydrazide (0.16 g, 0.81 mmol). White solid (0.90 mg, 45%). Mp = 235−236 °C. HPLC (10 min run): purity 96%, R.t. = 4.72 min. ESI MS (m/z): 405 [M + H]. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 16.22 (s, 1H), 12.08 (d, J = 4.1 Hz, 1H), 10.92 (d, J = 4.1 Hz, 1H), 10.79 (s, 1H), 8.09 (d, J = 7.9 Hz, 1H), 7.82 (t, J = 7.8 Hz, 1H), 7.63 (d, J = 8.6 Hz, 1H), 7.56 (d, J = 7.7 Hz, 1H), 7.39 (t, J = 7.6 Hz, 1H), 7.33 (d, J = 8.1 Hz, 1H), 7.16 (s, 1H), 7.06 (t, J = 7.4 Hz, 1H), 6.98 (t, J = 7.4 Hz, 1H), 3.64 (s, 3H), 2.99 (t, J = 7.5 Hz, 2H), 2.63 (t, J = 7.6 Hz, 2H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 161.1, 170.6, 169.1, 166.9, 139.8, 136.2, 134.5, 126.9, 124.4, 122.7, 122.3, 120.9, 118.3, 118.1, 115.5, 114.7, 113.3, 111.3, 95.6, 33.8, 29.2, 20.7. Anal. (C22H20N4O4). 1-Ethyl-4-hydroxy-N′-octanoil-2-oxo-1,2-dihydroquinoline-3-carbohydrazide (35). Reagents: ethyl 1-ethyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylate (0.10 g, 0.38 mmol) and octanoylhydrazide (0.06 g, 0.38 mmol). White solid (100 mg, 99%). Mp = 146−147 °C. HPLC (10 min run): purity >99%, R.t. = 5.80 min. ESI MS (m/z): 374 [M + H]. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 16.24 (s, 1H), 12.03 (d, J = 3.9 Hz, 1H), 10.79 (d, J = 3.9 Hz, 1H), 8.11 (d, J = 7.8 Hz, 1H), 7.84 (t, J = 7.1 Hz, 1H), 7.70 (d, J = 8.6 Hz, 1H), 7.40 (t, J = 7.3 Hz, 1H), 4.32 (q, J = 6.8 Hz, 2H), 2.24 (t, J = 7.1 Hz, 2H), 1.56 (m, 2H), 1.33−1.21 (m, 11H), 0.86 (t, J = 5.9 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 160.7, 170.6, 169.5, 167.1, 138.7, 134.7, 124.6, 122.6, 115.2, 114.9, 95.6, 36.9, 32.8, 31.1, 28.4, 28.3, 25.0, 22.0, 13.9, 12.7. Anal. (C20H27N3O4). 1-Ethyl-N′-2-(indol-3-yl)propanoyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carbohydrazide (36). Reagents: ethyl 1-ethyl-4-hydroxy2-oxo-1,2-dihydroquinoline-3-carboxylate (0.10 g, 0.38 mmol) and 3(indol-3-il)-propanohydrazide (0.08 g, 0.38 mmol). White solid (65 mg, 72%). Mp = 258−259 °C. HPLC (10 min run): purity 97%, R.t. = 3.72 min. ESI MS (m/z): 419 [M + H]. 1H NMR (300 MHz, DMSOd6) δ (ppm): 16.23 (s, 1H), 12.13 (d, J = 4.0 Hz, 1H), 10.96 (d, J = 3.9 Hz, 1H), 10.82 (s, 1H), 8.12 (d, J = 8.0 Hz, 1H), 7.84 (t, J = 7.8 Hz, 1H), 7.70 (d, J = 8.7 Hz, 1H), 7.57 (d, J = 7.7 Hz, 1H), 7.53−7.29 (m, 2H), 7.17 (s, 1H), 7.08 (t, J = 7.1 Hz, 1H), 6.99 (t, J = 7.1 Hz, 1H), 4.33 (q, J = 6.6 Hz, 2H), 3.01 (t, J = 7.5 Hz, 2H), 2.64 (t, J = 7.6 Hz, 2H), 1.24 (t, J = 6.9 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 161.6, 171.4, 170.0, 167.8, 139.6, 137.0, 135.3, 127.8, 125.4, 123.3, 123.0, 121.6, 119.0, 118.9, 115.9, 115.8, 114.2, 112.0, 96.5, 37.6, 34.7, 21.4, 13.4. Anal. (C23H22N4O4). 1-Ethyl-N′-2-(4-hydroxyphenyl)propanoyl-4-hydroxy-2-oxo-1,2dihydroquinoline-3-carbohydrazide (37). Reagents: ethyl 1-ethyl-4hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylate (0.10 g, 0.38 mmol) and 3-(4-hydroxyphenyl)propanhydrazide (0.08 g, 0.38 mmol). White solid (70 mg, 73%). Mp = 165−166 °C. HPLC (10 min run): purity 99%, R.t. = 4.45 min. ESI MS (m/z): 396 [M + H]. 1 H NMR (300 MHz, DMSO-d6) δ (ppm): 16.21 (s, 1H), 12.09 (d, J = 4.2 Hz, 1H), 10.89 (d, J = 4.2 Hz, 1H), 9.18 (s, 1H), 8.12 (d, J = 8.0 Hz, 1H), 7.84 (t, J = 7.1 Hz, 1H), 7.71 (d, J = 8.6 Hz, 1H), 7.40 (t, J = 7.5 Hz, 1H), 7.03 (d, J = 8.3 Hz, 2H), 6.68 (d, J = 8.4 Hz, 2H), 4.33 (q, J = 6.9 Hz, 2H), 2.77 (t, J = 7.5 Hz, 2H), 2.50 (t, J = 7.5 Hz, 2H), 1.24 (t, J = 6.9 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 161.1, 171.0, 169.1, 167.3, 155.9, 139.2, 135.1, 131.3, 129.5, 125.1, 123.0, 115.6, 115.4, 115.3, 96.0, 37.3, 35.4, 30.3, 13.1. Anal. (C21H21N3O5). N′-Dodecanoyl-4-hydroxy-1-isobutyl-2-oxo-1,2-dihydroquinoline-3-carbohydrazide (38). Reagents: ethyl carboxylate 12, (0.2 g, 0.66 mmol) and dodecanoylhydrazide (0.17 g, 0.78 mmol). White solid (50 mg, 21%). Mp = 112−113 °C. HPLC: purity 97%, R.t. = 6.6 min. ESI MS (m/z): 458 [M + H]+. 1H NMR (300 MHz, DMSO-d6) 4994

DOI: 10.1021/acs.jmedchem.7b00395 J. Med. Chem. 2017, 60, 4983−5001

Journal of Medicinal Chemistry

Article

133.6, 126.6, 123.1, 117.0, 116.0, 96.1, 36.8, 32.6, 30.8, 28.5, 28.5, 28.4, 28.2, 28.1, 28.0, 24.5, 21.5, 13.2, 12.1. Anal. (C24H34ClN3O4). 6-Chloro-1-ethyl-4-hydroxy-N′-octanoyl-2-oxo-1,2-dihydroquinoline-3-carbohydrazide (49). Reagents: ethyl carboxylate 19 (0.2 g, 0.68 mmol) and octanoylhydrazine (0.119 g, 0.74 mmol). White solid (190 mg, 70%), Mp = 160−161 °C. HPLC: purity >98%, R.t. = 13.1 min. ESI MS (m/z): 410 [M + 2H]+, 408 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 16.33 (s, 1H), 11.98 (d, J = 4.1 Hz, 1H), 10.81 (d, J = 4.1 Hz, 1H), 8.03 (d, J = 2.3 Hz, 1H), 7.86 (dd, J = 9.2, 2.6 Hz, 1H), 7.75 (d, J = 9.2 Hz, 1H), 4.3 (q, J = 6.9 Hz, 2H), 2.24 (t, J = 7.3 Hz, 2H), 1.60−1.53 (m, 2H), 1.35−1.18 (m, 11H), 0.87 (t, J = 6.4 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ (ppm): 170.3, 166.6, 161.3, 138.2, 134.8, 127.7, 124.2, 118.2, 117.0, 97.1, 39.8, 37.8, 33.6, 31.7, 29.2, 28.9, 25.6, 22.6, 14.5, 13.3. Anal. (C20H26ClN3O4). 6-Bromo-N′-dodecanoyl-4-hydroxy-2-oxo-2-hydroquinoline-3carbohydrazide (50). Following the general synthesis procedure for compounds 13−25, ethyl 6-bromo-4-hydroxy-2-oxo-2-hydroquinoline-3-carboxylate (20) was synthesized and used here without further purification. Diethyl malonate (3.3 g, 20.8 mmol) was added dropwise to a sodium hydride (0.2 g, 8.3 mmol) solution in dry DMF (15 mL) under argon atmosphere. The reaction was stirred for 30 min at room temperature. Then, the mixture was added to a round-bottomed flask containing the 5-bromoisatoic anhydride (1.0 g, 4.1 mmol) dissolved in DMF (15 mL) under argon atmosphere. The mixture was heated at 50 °C during 5 h. To the mixture, dichloromethane (30 mL), water (50 mL), and HCL (1 N) were added to reach pH 5. The organic layer was isolated and washed with brine (30 mL). The organic phase was dried over magnesium sulfate and filtered, and the solvent was evaporated. The solid (0.07 g, 0.25 mmol) was used in the next step without further purification after reaction with dodecanoylhydrazide (0.06 g, 0.27 mmol) to yield hydrazide 50. White solid (103 mg, 85%). Mp = 286−287 °C. ESI MS (m/z): 482 [M + 2H]+, 480 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 16.20 (s, 1H), 12.17 (bs, 1H), 11.91 (d, J = 4.0 Hz, 1H), 10.76 (d, J = 4.1 Hz, 1H), 8.12−8.05 (m, 1H), 7.86 (dd, J = 8.7 Hz, 1H), 7.33 (d, J = 8.9 Hz, 1H), 2.21 (t, J = 7.3 Hz, 2H), 1.60−1.42 (m, 2H), 1.20 (s, 16H), 0.84 (t, J = 6.6 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 170.4, 169.4, 167.0, 161.5, 137.6, 136.0, 134.9, 132.3, 126.0, 125.4, 117.9, 113.8, 32.6, 30.7, 28.4, 28.3, 28.2, 28.1, 28.0, 24.4, 21.4, 13.2. Anal. (C22H30BrN3O4). 6-Bromo-N′-dodecanoyl-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carbohydrazide (51). Reagents: ethyl carboxylate 21 (0.12 g, 0.36 mmol) and dodecanoylhydrazine (0.08 g, 0.4 mmol). White solid (100 mg, 55%). Mp = 160−161 °C. HPLC: purity >98%, R.t. = 14.3 min. ESI MS (m/z): 496 [M + 2H]+, 494 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 16.23 (s, 1H), 11.85 (bs, J = 4.3 Hz, 1H), 10.51 (bs, J = 5.5 Hz, 1H), 8.14 (d, J = 2.2 Hz, 1H), 7.91 (dd, J = 9.1, 1.4 Hz, 1H), 7.58 (d, J = 9.1 Hz, 1H), 3.6 (s,3H), 2.26 (t, J = 7.4 Hz,2H), 1.60−1.45 (m, 2H), 1.27 (s, 16H), 0.87 (t, J = 6.4 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 170.4, 167.8, 161.8, 139.7, 137.4, 127.0, 118.6, 117.3, 115.4, 97.2, 32.6, 30.7, 28.9, 28.5, 28.4, 28.2, 28.0, 24.4, 21.5, 13.2. Anal. (C23H32BrN3O4). 6-Bromo-4-hydroxy-1-methyl-N′-octanoyl-2-oxo-1,2-dihydroquinoline-3-carbohydrazide (52). Reagents: ethyl carboxylate 21 (0.15 g, 0.46 mmol); dodecanoylhydrazine (0.08 g, 0.5 mmol). White solid (130 mg, 64%). Mp = 185−186 °C. HPLC: purity >98%, R.t. = 9.8 min. ESI MS (m/z): 440 [M + 2H]+, 438 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 16.30 (s, 1H), 11.94 (d, J = 4.2 Hz, 1H), 10.81 (d, J = 4.2 Hz, 1H), 8.12 (s, 1H), 7.95 (d, J = 9.3 Hz, 1H), 7.61 (d, J = 9.0 Hz, 1H), 3.60 (s, 3H), 2.23 (t, J = 7.4 Hz, 2H), 1.60−1.42 (m, 2H), 1.27 (s, 8H), 0.86 (t, J = 6.2 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 169.4, 169.3, 166.8, 160.8, 138.7, 136.4, 126.0, 117.7, 116.2, 114.5, 96.3, 32.7, 30.8, 29.2, 28.2, 27.9, 24.6, 21.6, 13.5. Anal. (C19H24BrN3O4). 6-Bromo-N′-dodecanoyl-1-ethyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carbohydrazide (53). Reagents: ethyl carboxylate 22 (0.12 g, 0.36 mmol) and dodecanoylhydrazine (0.08 g, 0.4 mmol). White solid (150 mg, 80%). Mp = 146−147 °C. HPLC: purity >98%, R.t. = 6.2 min. ESI MS (m/z): 510 [M + 2H]+, 508 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 16.28 (s, 1H), 11.97 (s, 1H), 10.81 (s, 1H), 8.16 (s, 1H), 7.97 (dd, J = 9.6, 2.5 Hz, 1H), 7.62 (d, J =

127.9, 126.4, 125.4, 121.6, 114.9, 114.2, 95.1, 44.9, 33.4, 30.9, 28.6, 28.4, 28.3, 28.2, 24.4, 21.7, 13.1. Anal. (C29H37N3O4). N′-Dodecanoyl-6-fluoro-4-hydroxy-2-oxo-1,2-dihydroquinoline3-carbohydrazide (43). Reagents: ethyl 6-fluoro-4-hydroxy-2-oxo-1,2dihydroquinoline-3-carboxylate (20 mg, 0.07 mmol) and dodecanoylhydrazide (17 mg, 0.07 mmol). White solid (15 mg, 93%). Mp = 178−179 °C. HPLC (10 min run): purity 95%, R.t. = 2.35 min. ESI MS (m/z): 419 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 16.16 (s, 1H), 12.11 (s, 1H), 11.94 (d, J = 3.9 Hz, 1H), 10.72 (s, 1H), 7.72−7.56 (m, 2H), 7.42 (m, 1H), 2.21 (t, J = 7.3 Hz, 2H), 1.52 (m, 2H), 1.23 (s, 16H), 0.83 (t, J = 6.7 Hz, 3H). Anal. (C22H30FN3O4). 6-Fluoro-4-hydroxy-N′-octanoyl-2-oxo-1,2-dihydroquinoline-3carbohydrazide (44). Reagents: ethyl 6-fluoro-4-hydroxy-2-oxo-1,2dihydroquinoline-3-carboxylate (20 mg, 0.08 mmol) and octanoylhydrazide (13 mg, 0.08 mmol). White solid (24 mg, 83%). Mp = 290− 291 °C. HPLC (10 min run): purity 98%, R.t. = 2.37 min. ESI MS (m/ z): 364 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 16.15 (s, 1H), 11.95 (d, J = 3.6 Hz, 1H), 12.13 (s, 1H), 10.75 (d, J = 3.7 Hz, 1H), 7.66 (m, 2H), 7.43 (m, 1H), 2.23 (t, J = 7.3 Hz, 2H), 1.55 (s, 2H), 1.40 (m, 8H), 0.86 (d, J = 6.6 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 162.2, 171.4, 170.0, 167.5, 157.6, 136.0, 123.2, 118.7, 118.6, 114.9, 108.8, 96.8, 33.3, 31.5, 28.8, 28.7, 25.3, 22.4, 14.3. Anal. (C18H22FN3O4). 6-Chloro-N′-dodecanoyl-4-hydroxy-2-oxo-2-hydroquinoline-3carbohydrazide (45). Reagents: ethyl carboxylate 17 (0.037 g, 0.14 mmol) and dodecanoylhydrazine (0.034 g, 0.15 mmol). White solid (45 mg, 73%). Mp = 285−286 °C. HPLC (10 min run): purity >99%, R.t. = 6.2 min. ESI MS (m/z): 438 [M + 2H]+, 436 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 16.22 (s, 1H), 12.18 (s, 1H), 11.9 (d, J = 4.2 Hz, 1H), 10.75 (bs, 1H), 7.97−7.90 (m, 1H), 7.76 (dd, J = 2.46, 8.8 Hz, 1H), 7.40 (d, J = 8.8 Hz, 1H), 2.2 (t, J = 7.3 Hz, 2H), 1.60−1.45 (m, 2H), 1.2 (s, 16H), 0.85 (t, J = 6.4 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 169.3, 169.1, 167.1, 164.0, 134.7, 133.4, 132.0, 129.4, 125.8, 117.7, 99.0, 32.6, 30.7, 28.4, 28.3, 28.2, 28.1, 28.0, 24.4, 21.5, 13.2. Anal. (C22H30ClN3O4). 6-Chloro-N′-dodecanoyl-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carbohydrazide (46). Reagents: ethyl carboxylate 18 (0.10 g, 0.36 mmol) and dodecanoylhydrazine (0.08 g, 0.39 mmol). White solid (143 mg, 90%). Mp = 157−158 °C. HPLC: purity >99%, R.t. = 13.9 min. ESI MS (m/z): 452 [M + 2H]+, 450 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 16.33 (s, 1H), 11.96 (d, J = 4.2 Hz, 1H), 10.81 (d, J = 4.2 Hz, 1H), 8.00 (d, J = 2.5 Hz, 1H), 7.86 (dd, J = 9.1, 2.5 Hz, 1H), 7.69 (d, J = 9.1 Hz, 1H), 3.6 (s,3H), 2.23 (t, J = 7.3 Hz, 2H),1.60−1.47 (m, 2H), 1.25 (s, 16H), 0.85 (t, J = 6.4 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 169.6, 169.4, 166.8, 160.8, 138.4, 133.7, 126.9, 122.9, 117.5, 115.8, 96.2, 32.9, 29.2, 28.6, 28.5, 28.3, 28.3, 28.2, 24.6, 21.7,13.5. Anal. (C23H32ClN3O4). 6-Chloro-4-hydroxy-1-methyl-N′-octanoyl-2-oxo-1,2-dihydroquinoline-3-carbohydrazide (47). Reagents: ethyl carboxylate 18 (0.1 g, 0.36 mmol) and octanoylhydrazine (0.062 g, 0.39 mmol). White solid (120 mg, 86%). Mp = 187−188 °C. HPLC: purity >97%, R.t. = 12.3 min. ESI MS (m/z): 396 [M + 2H]+, 394 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 16.33 (s, 1H), 11.96 (d, J = 4.2 Hz, 1H), 10.8 (d, J = 4.2 Hz, 1H), 8.06 (d, J = 2.6 Hz, 1H), 7.86 (dd, J = 9.0, 2.5 Hz, 1H), 7.68 (d, J = 9.1 Hz, 1H), 3.6 (s, 3H), 2.24 (t, J = 7.4 Hz, 2H), 1.6−1.45 (m, 2H), 1.26 (s, 16H), 0.87 (t, J = 6.2 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 169.4, 166.8, 161.1, 160.8, 138.4, 133.7, 126.9, 122.9, 117.5, 115.9, 96.2, 32.7, 30.8, 29.2, 28.2, 28.0, 24.6, 21.6, 13.5. Anal. (C19H24ClN3O4). 6-Chloro-N′-dodecanoyl-1-ethyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carbohydrazide (48). Reagents: ethyl carboxylate 19 (0.2 g, 0.68 mmol) and dodecanoylhydrazine (0.16 g, 0.75 mmol). White solid (234 mg, 74%). Mp = 149−150 °C. HPLC: purity >98%, R.t. = 14.9 min. ESI MS (m/z): 466 [M + 2H]+, 464 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 16.31 (s, 1H), 11.90 (d, J = 4.2 Hz, 1H), 10.81 (d, J = 4.2 Hz, 1H), 8.05 (d, J = 2.6 Hz, 1H), 7.97 (dd, J = 9.1, 2.6 Hz, 1H), 7.62 (d, J = 9.2 Hz, 1H), 4.3 (q, J = 6.9 Hz, 2H), 2.23 (t, J = 7.3 Hz, 2H), 1.35−1.17 (m, 19H), 0.86 (t, J = 7.4 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 169.2, 166.6, 160.3, 137.2, 4995

DOI: 10.1021/acs.jmedchem.7b00395 J. Med. Chem. 2017, 60, 4983−5001

Journal of Medicinal Chemistry

Article

8.8 Hz, 1H), 4.30 (q, J = 6.9 Hz, 2H), 2.24 (t, J = 7.2 Hz, 2H), 1.60− 1.45 (m, 2H), 1.26 (s, 19H), 0.85 (t, J = 8.6 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 169.3, 166.7, 160.3, 137.6, 136.4, 126.2, 117.2, 116.5, 114.2, 96.0, 36.7, 32.6, 30.8, 28.5, 28.4, 28.2, 28.1, 28.0, 24.4, 21.5, 13.2, 12.1. Anal. (C24H34BrN3O4). 6-Bromo-1-ethyl-4-hydroxy-N′-octanoyl-2-oxo-1,2-dihydroquinoline-3-carbohydrazide (54). Reagents: ethyl carboxylate 22 (0.12 g, 0.36 mmol) and octanoylhydrazine (0.4 mmol, 0.06 g). White solid (130 mg, 80%). M.p.= 171−172 °C. HPLC: purity >98%, R.t. = 13.5 min. ESI MS (m/z): 454 [M + 2H]+, 452 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 16.24 (s, 1H), 11.88 (d, J = 4.0 Hz, 1H), 10.61 (d, J = 4.1 Hz, 1H), 8.12 (d, J = 2.5 Hz, 1H), 7.90 (dd, J = 9.1, 2.5 Hz, 1H), 7.61 (d, J = 9.1 Hz, 1H), 4.30 (q, J = 7.0 Hz, 2H), 2.22 (t, J = 7.4 Hz,2H), 1.57−1.50 (m, 2H), 1.30−1.17 (m, 11H), 0.84 (t, J = 6.7 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 169.4, 166.7, 160.4, 137.8, 136.7, 126.3, 117.5, 116.6, 114.4, 96.2, 36.9, 32.8, 30.9, 28.3, 28.1, 24.7, 21.7, 13.6, 12.4. Anal. (C20H26BrN3O4). 7-Chloro-N′-dodecanoyl-4-hydroxy-2-oxo-1,2-dihydroquinoline3-carbohydrazide (55). Reagents: ethyl 7-chloro-4-hydroxy-2-oxo-1,2dihydroquinoline-3-carboxylate (17 mg, 0.06 mmol) and dodecanoylhydrazide (14 mg, 0.06 mmol). White solid (13 mg, 92%). Mp = 291−292 °C. HPLC (10 min run): purity 97%, R.t. = 2.91 min. ESI MS (m/z): 436 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 16.21 (s, 1H), 12.10 (s, 1H), 11.80 (s, 1H), 10.71 (s, 1H), 7.98 (d, J = 8.6 Hz, 1H), 7.42 (d, J = 8.8 Hz, 1H), 7.31 (dd, J = 8.7, 2.0 Hz, 1H), 2.20 (t, J = 7.4 Hz, 2H), 1.53 (s, 2H), 1.23 (s, 16H), 0.82 (d, J = 6.7 Hz, 3H). Anal. (C22H30ClN3O4). 7-Chloro-4-hydroxy-N′-octanoyl-2-oxo-1,2-dihydroquinoline-3carbohydrazide (56). Reagents: ethyl 7-chloro-4-hydroxy-2-oxo-1,2dihydroquinoline-3-carboxylate(20 mg, 0.07 mmol) and octanoylhydrazide (12 mg, 0.07 mmol). White solid (16 mg, 86%). Mp = 290− 291 °C. HPLC (10 min run): purity 95%, R.t. = 5.45 min. ESI MS (m/ z): 379 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 16.12 (s, 1H), 12.09 (s, 1H), 11.79 (s, 1H), 10.71 (s, 1H), 7.94 (d, J = 5.0 Hz, 1H), 7.48−7.16 (m, 2H), 2.21 (t, J = 7.3 Hz, 2H), 1.61−1.42 (m, 2H), 1.25 (s, 8H), 0.85 (t, J = 6.6 Hz, 3H). Anal. (C18H22ClN3O4). 7-Chloro-N′-dodecanoyl-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carbohydrazide (57). Reagents: ethyl carboxylate 23 (0.10 g, 0.36 mmol) and dodecanoylhydrazine (0.08 g, 0.39 mmol). White solid (105 mg, 65%). Mp = 165−166 °C. HPLC: purity >98%, R.t. = 13.8 min. ESI MS (m/z): 452 [M + 2H]+, 450 [M + H]+.1H NMR (300 MHz, DMSO-d6) δ (ppm): 16.30 (s, 1H), 11.89 (bs, 1H), 10.81 (bs, 1H), 8.06 (d, J = 8.6 Hz, 1H), 7.74 (d, J = 1.6, Hz, 1H), 7.69 (dd, J = 8.6, 1.7 Hz, 1H), 3.60 (s,3H), 2.23 (t, J = 7.2 Hz, 2H), 1.53− 1.27 (m, 2H), 1.23 (s, 16H), 0.84 (t, J = 6.5 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 170.0, 169.4, 166.9, 161.0, 140.6, 139.1, 126.0, 122.6, 114.9, 113.5, 113.4, 32.7, 30.9, 29.2, 29.1, 28.6, 28.5, 28.3, 28.3, 28.2, 24.6, 21.7,13.5. Anal. (C23H32ClN3O4). 7-Chloro-4-hydroxy-1-methyl-N′-octanoyl-2-oxo-1,2-dihydroquinoline-3-carbohydrazide (58). Reagents: ethyl carboxylate 23 (0.1 g, 0.36 mmol) and octanoilhydrazine (0.063 g, 0.39 mmol). White solid (70 mg, 50%). Mp = 164−165 °C. HPLC: purity >99%, R.t. = 7.5 min. ESI MS (m/z): 396 [M + 2H]+, 394 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 16.31 (s, 1H), 11.89 (bs, 1H), 10.5 (bs, 1H), 8.09 (dd, J = 8.6, 2.5 Hz, 1H), 7.72−7.70 (m,1H), 7.43−7.40 (m, 1H), 3.64 (s, 3H), 2.25 (t, J = 7.4 Hz,2H), 1.6−1.42 (m, 2H), 1.29 (s, 8H), 0.88 (t, J = 6.6 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 170.1, 169.5, 167.0, 161.1, 140.6, 139.2, 126.0, 122.6, 115.0, 113.5, 95.7, 32.7, 30.8, 29.2, 29.1, 28.2, 28.0, 24.6, 21.7, 13.5. Anal. (C19H24ClN3O4). 7-Chloro-N′-dodecanoyl-1-ethyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carbohydrazide (59). Reagents: ethyl carboxylate 24 (0.15 g, 0.5 mmol) and dodecanoylhydrazine (0.124 g, 0.56 mmol). White solid (221 mg, 95%). Mp = 138−139 °C. HPLC: purity >98%, R.t. = 14.7 min. ESI MS (m/z): 466 [M + 2H]+, 464 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 16.17 (s, 1H), 11.91 (bs, 1H), 10.81 (bs, 1H), 8.11 (d, J = 8.6 Hz, 1H), 7.75 (d, J = 1.8 Hz, 1H), 7.42 (dd, J = 8.6, 1.8 Hz, 1H), 4.30 (q, J = 6.9 Hz, 2H), 2.23 (t, J = 7.2 Hz, 2H), 1.54−1.40 (m, 2H), 1.37−1.10 (m, 19H), 0.84 (t, J = 6.3 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 169.9, 169.3, 166.7,

160.6, 139.4, 139.2, 126.2, 122.4, 114.3, 113.7, 95.5, 36.7, 32.6, 30.8, 28.5, 28.4, 28.3, 28.2, 24.5, 21.6, 13.3, 12.1. Anal. (C24H34ClN3O4). 7-Chloro-1-ethyl-4-hydroxy-N′-octanoyl-2-oxo-1,2-dihydroquinoline-3-carbohydrazide (60). Reagents: ethyl carboxylate 24 (0.15 g, 0.5 mmol) and octanoylhydrazine (0.09 g, 0.55 mmol). White solid (60 mg, 29%). Mp = 161−162 °C. HPLC: purity >99%, R.t. = 8.2 min. ESI MS (m/z): 410 [M + 2H]+, 408 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 16.31 (s, 1H), 11.91 (d, J = 4.1 Hz,1H), 10.81 (d, J = 4.1 Hz, 1H), 8.09 (d, J = 8.6 Hz,1H), 7.90 (d, J = 1.9 Hz, 1H), 7.61 (dd, J = 8.6, 1.8 Hz, 1H), 4.30 (q, J = 7.0 Hz, 2H), 2.23 (t, J = 7.3 Hz, 2H), 1.60−1.45 (m, 2H), 1.35−1.12 (m, 11H), 0.86 (t, J = 6.6 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 170.0, 169.4, 166.9, 160.7, 139.5, 139.3, 126.3, 122.6, 114.6, 113.7, 95.6, 36.9, 32.7, 30.8, 28.2, 28.0, 24.6, 21.6, 13.5, 12.3. Anal. (C20H26ClN3O4). 1-Benzyl-7-chloro-N′-dodecanoyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carbohydrazide (61). Reagents: ethyl carboxylate 25 (0.20 g, 0.56 mmol) and dodecanoylhydrazine (0.133 g, 0.62 mmol). White solid (250 g, 85%). Mp = 167−168 °C. HPLC: purity >99%, R.t. = 5.1 min. ESI MS (m/z): 528 [M + 2H]+, 526 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 16.49 (s, 1H), 11.87 (d, J = 4.2 Hz, 1H), 10.87 (d, J = 4.2 Hz, 1H), 8.11 (d, J = 8.6 Hz, 1H), 7.57 (d, J = 1.8 Hz, 1H), 7.41 (dd, J = 8.6, 1.8 Hz, 1H), 7.36−7.30 (m, 2H), 7.28−7.20 (m, 3H), 5.61 (s, 2H), 2.23 (t, J = 7.3 Hz, 2H), 1.60−1.50 (m, 2H), 1.30−1.10 (m, 16H), 0.84 (t, J = 6.8 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 170.7, 169.5, 166.9, 161.5, 140.1, 139.3, 136.1, 128.7, 127.2, 126.6, 126.4, 123.2, 115.5, 114.1, 95.8, 44.8, 32.9, 31.3, 29.0, 28.9, 28.7, 28.7, 28.5, 24.9, 22.1, 13.9. Anal. (C29H36ClN3O4). 1-Benzyl-7-chloro-4-hydroxy-N′-octanoyl-2-oxo-1,2-dihydroquinoline-3-carbohydrazide (62). Reagents: ethyl carboxylate 25 (0.20 g, 0.5 mmol) and octanoylhydrazine (0.100 g, 0.62 mmol). White solid (196 mg, 74%). Mp = 182−183 °C. HPLC: purity >98%, R.t. = 2.4 min. ESI MS (m/z): 472 [M + 2H]+, 470 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 16.51 (s, 1H), 11.86 (d, J = 4.2 Hz, 1H), 10.8 (d, J = 4.2 Hz, 1H), 8.11 (d, J = 8.6 Hz, 1H), 7.57 (d, J = 1.8 Hz, 1H), 7.42 (dd, J = 8.6, 1.8 Hz, 1H), 7.36−7.30 (m, 2H), 7.28−7.20 (m, 3H), 5.6 (s, 2H), 2.24 (t, J = 7.4 Hz, 2H), 1.60−1.50 (m, 2H), 1.35−1.15 (m, 8H), 0.86 (t, J = 6.8 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 170.7, 169.6, 166.9, 161.5, 140.1, 139.3, 136.1, 128.7, 127.3, 126.7, 126.4, 123.2, 115.5, 114.1, 95.8, 44.8, 32.9, 31.2, 28.5, 28.4, 24.9, 22.1, 13.9. Anal. (C25H28ClN3O4). N-Hexyl-4-hydroxy-1-methyl-2-oxo-1,2-dihydro-quinoline-3-carboxamide (63). Over ethyl 4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylate (0.300 g, 1.27 mmol) in EtOH (3 mL), hexylamine (0.77 mL, 1.27 mmol) was added. The mixture was heated under reflux for 5 h. After that time, the mixture was cooled down to room temperature, and water (6 mL) was added. The mixture was acidified with a solution of HCl [1 M] and was extracted with water and dichlorometane. The organic phase was dried with anhydrous MgSO4, and the solvent was evaporated under reduced pressure. The final product was purified by flash chromatography using a 1:1 mixture of AcOEt/hexane as eluent to afford a white solid (214 mg, 51%). Mp = 29−30 °C. HPLC (10 min run): purity >99%, R.t. = 2.99 min. ESI MS (m/z): 303 [M + H]. 1H NMR (300 MHz, CDCl3) δ (ppm): 10.34 (s, 1H), 8.06 (dd, J = 7.9, 1.2 Hz, 1H), 7.93−7.63 (m, 1H), 7.58 (d, J = 8.5 Hz, 1H), 7.35 (t, J = 7.5 Hz, 1H), 3.60 (s, 3H), 3.42−3.34 (m, 2H), 1.67−1.42 (m, 3H), 1.29 (m, 6H), 0.86 (t, J = 6.6 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ (ppm): 161.6, 171.2, 170.4, 139.6, 134.1, 124.4, 122.4, 115.3, 115.2, 95.8, 30.8, 28.9, 28.6, 26.0, 22.0, 13.8. Anal. (C17H22N2O3). N-(2-((2-(Benzyloxy)-2-oxoethyl)amino)-2-oxoethyl)-4-hydroxy1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamide (64). Over ethyl 4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylate (0.200 g, 0.8 mmol), in 3 mL of EtOH, Gly-Gly benzyl ester ptoluenesulfonate salt (0.320 g, 0.8 mmol) was added. The mixture was heated under reflux for 5 h. After that time, the mixture was cooled down to room temperature, and water (6 mL) was added. The mixture was acidified with a solution of HCl [1 M] and was extracted with water and dichlorometane. The organic phase was dried with anhydrous MgSO4, and the solvent was evaporated under reduced 4996

DOI: 10.1021/acs.jmedchem.7b00395 J. Med. Chem. 2017, 60, 4983−5001

Journal of Medicinal Chemistry

Article

ions. Solvated systems were first minimized for 8000 steps with the initial 4500 steps using the steepest descent algorithm. The final 3500 steps used the conjugate gradient energy minimization with constraints applied to the protein residues. This was followed by two minimization stages of 8000 steps, with the last 3500 using the conjugate gradient decreasing the restrains to the system. The system was equilibrated to 300 K and 1 atm, using a 9 steps protocol, applying energetic restraints of 15 kcal mol−1 Å−1 from the initial step and gradually decreasing them until their disappearance. The production trajectories of 25 ns were obtained in an isothermal−isobaric ensemble. All bonds involving hydrogen atoms were constrained with the SHAKE algorithm.64 A cutoff of 10 Å was used for the Lennard-Jones interaction and the short-range electrostatic interactions. A Berendsen barostat65 and a Langevin thermostat were used to regulate the system pressure and temperature, respectively. Trajectories of 25 ns were calculated, analyzing them using the CPPTRAJ module66 and Xmgrace software67 to obtain the graphics and visual molecular dynamics (VMD)68 for visual inspection.

pressure. The final product was purified by flash chromatography using a 1:1 mixture of AcOEt/hexane as eluent to afford a white solid (58 mg, 17%). Mp = 181−182 °C. HPLC (10 min run): purity 95%, R.t. = 4.89 min. ESI MS (m/z): 424 [M + H]. 1H NMR (300 MHz, CDCl3) δ (ppm): 17.04 (s, 1H), 10.59 (t, J = 5.3 Hz, 1H), 8.61 (t, J = 5.8 Hz, 1H), 8.07 (dd, J = 8.0, 1.4 Hz, 1H), 7.80 (dd, J = 8.6, 7.2 Hz, 1H), 7.60 (d, J = 8.6 Hz, 1H), 7.44−7.27 (m, 6H), 5.13 (s, 2H), 4.11 (d, J = 5.4 Hz, 2H), 3.96 (d, J = 5.8 Hz, 2H), 3.62 (s, 3H). 13C NMR (75 MHz, CDCl3) δ (ppm): 161.4, 170.9, 170.6, 168.3, 169.6, 139.8, 135.8, 134.3, 128.4, 128.0, 127.9, 124.5, 122.5, 115.3, 115.0, 96.2, 65.9, 41.8, 40.7, 29.0. Anal. (C22H21N2O6). N′-(2-Cyanoethyl)-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carbohydrazide (65). Ethyl 4-hydroxy-1-methyl-2-oxo-1,2dihydroquinoline-3-carboxylate(0.200 g, 0.81 mmol) was mixed with 2-cyanoethylhydrazine (0.070 g, 0.81 mmol) in DMF (2 mL) at 160 °C for 5 min. Then, the mixture was cooled to room temperature, and MeOH (10 mL) was slowly added. The precipitate formed was filtered to afford a white solid (73 mg, 26%). Mp = 115−116 °C. HPLC (10 min run): purity 95%, R.t. = 4.21. ESI MS (m/z): 287 [M + H]. 1H NMR (300 MHz, CDCl3): δ 15.96 (s, 1H), 11.18 (d, J = 5.8 Hz, 1H), 5.96−5.55 (m, 1H), 8.08 (d, J = 8.0 Hz, 1H), 7.82 (t, J = 7.8 Hz, 1H), 7.62 (d, J = 8.6 Hz, 1H), 7.38 (t, J = 7.6 Hz, 1H), 3.62 (s, 3H), 3.12 (m, 2H), 2.67 (t, J = 6.5 Hz, 2H). 13C NMR (75 MHz, CDCl3): δ 161.5, 170.8, 170.2, 140.0, 134.7, 124.7, 123.0, 120.0, 115.8, 115.3, 96.2, 46.7, 29.5, 16.7. Anal. (C14H14N4O3). Molecular Modeling. pKa and Tautomerization Study for Compound 1. Preliminary tautomer search and pKa calculations were developed using Marvin Sketch v.15.12.7 (Marvin 15.12.7, 2015, ChemAxon (http://www.chemaxon.com)).Quantum chemical calculations were carried out using Jaguar v9.0 software.52,53 Full geometry optimizations for each species studied have been carried out using a DFT functional, such as B3LYP using the 6-31+G* basis set.54 The frequency calculations carried out confirm that all the optimized structures correspond to the true minima as no negative vibration frequency was observed. The effect of solvent (water) is taken into consideration using the Poisson−Boltzmann solvation model.55,56 Docking Studies. Before docking calculations, the 1PYX crystal structure was prepared using Maestro Protein Preparation Wizard,57,58 removing ligands, metals, and water molecules, adding hydrogens, ionizing the residues at pH 7.0, and modeling missing loops between residues Ser119 to Glu125 and Pro286 to Phe291 and its side chains using Prime and running a restrained minimization of the protein structure until the heavy atoms converge to 3.0 of RMSD. Ligands were prepared taking into account the previous tautomer studies, keeping fixed the best geometry for the hydrazine group. Docking studies for quinolines were developed using AutoDock v.4.2.59 A grid of points was computed covering the allosteric cavity, using 50 × 50 × 50 grid points with a spacing of 0.375 Å. In docking experiments, a Lamarckian genetic algorithm was chosen, and default parameters were used except “Number of GA runs”, “Population size”, and “Maximum number of evals”, which were set to 200, 200, and 2.5 × 106, respectively. The final best docking clusters (within 2.0 Å RMSD) according to the binding energies and the relative population provided by AutoDock were analyzed by visual inspection. Molecular Dynamics Studies. MD was performed with an Asus 1151 h170LVX-GTX-980Ti workstation, with an Intel Core i7-6500 K Processor (12 M Cache, 3.40 GHz) and 16 GB DDR4 2133 MHz RAM. The workstation had Nvidia GeForce GTX 980Ti available for GPU computations. AMBER1260 with ff14SB61 was used to develop MD studies in order to refine the docking poses for active and inactive compounds (compounds 1 and 63, respectively) and to appreciate conformational changes that could explain the allosteric mechanism of compound 1. Best poses for compounds 1 and 63 were selected as the starting point for this studio. Ligand geometry optimization was calculated using Jaguar,52 using the method HF6-311++ (d,p). Semiempirical charges AM1-BCC for optimized geometries of both ligands were calculated using the Antechamber package.62 Systems were solvated using the TIP3P model63 for water molecules, getting into a cubic box, equilibrating the system charge by adding chloride



BIOLOGY GSK-3β Studies. Reagents. Human recombinant GSK-3β was purchased from Millipore (Millipore, Iberica S.A.U.) The prephosphorylated polypeptide substrate was purchased from Millipore (Millipore, Iberica S.A.U.). Kinase-Glo Luminescent Kinase Assay was obtained from Promega (Promega Biotech, Ibérica, SL). ATP and all other reagents were from SigmaAldrich (St. Louis, MO). The assay buffer contained 50 mM HEPES (pH 7.5), 1 mM EDTA, 1 mM EGTA, and 15 mM magnesium acetate. Enzymatic Inhibition. The method of Bakiet al.27 was followed for the inhibition of GSK-3β. Kinase-Glo assays were performed in assay buffer using black 96-well plates. In a typical assay, 10 μL (10 μM) of test compound (dissolved in dimethyl sulfoxide [DMSO] at 1 mM concentration and diluted in advance in assay buffer to the desired concentration) and 10 μL (20 ng) of enzyme were added to each well followed by 20 μL of assay buffer containing 50 μM substrate and 2 μM ATP. The final DMSO concentration in the reaction mixture did not exceed 1%. After 30 min of incubation at 30 °C, the enzymatic reaction was stopped with 40 μL of Kinase-Glo reagent. Glowtype luminescence was recorded after 10 min using a FLUOstar Optima (BMG Labtechnologies GmbH, Offenburg, Germany) multimode reader. The activity is proportional to the difference of the total and consumed ATP. The inhibitory activities were calculated on the basis of maximal activities measured in the absence of inhibitor. The IC50 was defined as the concentration of each compound that reduces 50% the enzymatic activity with respect to that without inhibitors. Five different concentrations by duplicate were used to determine the dose/response curve. Kinetic Studies. To investigate the inhibitory mechanism of 53 and 59 on GSK-3β, several kinetic experiments were performed. Lineweaver−Burk plots of enzyme kinetics are shown in Figures 3 and 4. Kinetic experiments were performed varying the concentrations of both GS-2 (from 12.5 μM to 100 μM) and inhibitors (5 and 2 μM), while the ATP concentration was kept constant (1 μM). Double-reciprocal plotting of the data, Figure 4, suggests that 53 and 59 act as noncompetitive inhibitors of GS2 binding. Kinetic experiments varying both ATP (from 1 to 50 μM) and inhibitor (5 and 2.5 μM) concentrations, while the GS2 concentration was kept constant (25 μM), were performed using the ADP-Glo Kinase Assay.69 Double-reciprocal plotting of the data is depicted in Figure 3. These results would suggest 4997

DOI: 10.1021/acs.jmedchem.7b00395 J. Med. Chem. 2017, 60, 4983−5001

Journal of Medicinal Chemistry

Article

with papain/DNase solution (Worthington) and plated as single cells on astrocyte-coated 384-well plates (Greiner). The medium used was Neurobasal containing 2% B27 and 1% N2 (Life Technologies) supplemented with 20 ng/mL of BDNF, GDNF, and CNTF (R&D Systems). Cells were fixed in 4% paraformaldehyde at room temperature for 20 min and subsequently immunostained with Islet1 (Abcam) and MAP2 (Lifespan Biosciences) antibodies followed by fluorescently labeled secondary antibodies (Life Technologies). Hoechst 33342 (Life Technologies) was used for nuclear staining. Images of cells were captured using an automated Operetta wide field live-imaging or Opera confocal microscope (PerkinElmer) at 20× magnification. Subsequent image quantification was performed using the Columbus Image Data Storage and Analysis System (PerkinElmer). The number of Islet1 positive MNs was calculated as the ratio of the number of surviving MNs in compound-treated wells to the number of surviving MNs in DMSO-treated wells for each individual cell line.

that the compounds act as noncompetitive inhibitors of ATP binding because an increase in the ATP concentration (from 1 to 50 μM) does not interfere with enzymatic inhibition. β-Catenin Localization Evaluation. Cell Culture and Treatments. LN18 human glioblastoma cells were obtained from Dr. Peinado (Centro Nacional de Investigaciones Oncológicas, Madrid) and maintained in DMEM medium (Sigma) with 10% fetal bovine serum (FBS), 2 mM glutamine, and 100 μg·mL−1 penicillin/streptomycin (Invitrogen). SHSY5Y human neuroblastoma cells were purchased from Sigma and propagated in DMEM supplemented with 10% FBS, 10 mM glutamine, and 5 mg·mL−1 penicillin/streptomycin, at 37 °C and 5% CO2. Cultures grown on glass coverslips were treated with the different compounds for 72 h. The concentration of the different compounds was chosen based on their effectiveness in previously published works.70,71 Vehicle-treated cultures were used as a control. Immunocytochemistry. At the end of the treatment period, cells were processed for immunocytochemistry. Cells were washed with PBS, fixed for 30 min with 4% paraformaldehyde at RT, and permeabilized for 30 min with 0.1% Triton X-100 at 37 °C. After 1 h of incubation with a mouse anti-β-catenin antibody (BD Biosciences), cells were washed with PBS and incubated with an Alexa-488-goat anti mouse antibody (Invitrogen) for 45 min at 37 °C. Nuclei were counterstained with DAPI (blue). Images were acquired using a LSM710 confocal microscope (Zeiss). Confocal microscope settings were adjusted to optimize signal-to-noise ratios. To compare fluorescence signals from different preparations, settings were fixed for all samples within the same analysis. Myoblast Cell Culture. Human primary myoblasts from a control patient with normal skeletal muscle histology and normal metabolism and from a patient with CDM1 containing about 1,000 repeats were grown in myoblast growth medium containing an F10 medium, fetal bovine serum (15%), sodium bicarbonate (1%), defined supplemental calf serum (5%), Lglutamine (1%), and penicillin/streptomycin (1%). Human myoblasts were grown for no more than 15−20 passages. To induce differentiation, the growth medium was replaced with the fusion medium containing DMEM medium, supplemented with horse medium and insulin. The growth medium was replaced every other day, and the fusion medium was replaced daily. Different doses of compound 53, dissolved in DMSO, were added to the fresh fusion medium every day. Whole cell protein extracts were prepared from human myotubes maintained in fusion medium for 4 days using RIPA buffer and analyzed by Western blot assay with antibodies to desmin and actin from Sigma according to the manufacturer’s protocols. Human iPSC-Derived MN Differentiation and Survival Assay. The Harvard iPSC core generated the iPSC lines from the SMA patient fibroblasts, and the Harvard IRB reviewed and determined it to be not human subject research. The information regarding the healthy control and SMA iPSC lines was recently described.44 To induce differentiation of human iPSCs into MNs, iPSCs were cultured as embryoid bodies and were treated with 1 μM LDN (Stemgent) and 10 μM SB431542 (Stemgent) for the first 2 days. Additionally, 1 μM retinoic acid (Sigma-Aldrich) was added at day 3 and 10 ng/mL BDNF and 1 μM smoothened agonist 1.3 (EMD Millipore) 3 days later. At day 10, 2.5 μM DAPT and 2 μM Ara-C were added to eliminate progenitors and dividing cells. After 15 days of differentiation, the cultures were dissociated



ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00395. Elemental analyses of all synthesized compounds; optimized geometries and Gibbs energy values for tautomers A and B of compound 1; binding mode on GSK-3β for derivatives 32, 63, and 65 and 39, 40, 61, and 62; LogP versus pIC50 of the inhibitors; RMSF values of backbone atoms in the three trajectories studied by molecular dynamics; RMSF profile for the trajectory of GSK-3β against the complex GSK-3β//Comp 1 and distance between Lys205 and Glu211 along the three molecular dynamics trajectories (PDF) Molecular formula strings and the associated biochemical and biological data (CSV)



AUTHOR INFORMATION

Corresponding Author

*Phone: +34-91-837-3112. Fax: +34-91-536-0432. E-mail: ana. [email protected]. ORCID

Daniel I. Perez: 0000-0003-1774-4471 Carmen Gil: 0000-0002-3882-6081 Ana Martinez: 0000-0002-2707-8110 Author Contributions ∇

V.P. and D.I.P. contributed equally to this work.

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was financially supported by MINECO (grant no. SAF2012-37979-C03-01 and SAF2016-76693-R to A.M. and IJCI-2014-20767 to V.P.). CCHMC funds L.T. A.M. and C.G. are members of the CIB Intramural Program “Molecular Machines for Better Life” (MACBET).



ABBREVIATIONS USED GSK-3β, glycogen synthase kinase 3 β; DM1, myotonic dystrophy type 1; CDM1, congenital myotonic dystrophy; 4998

DOI: 10.1021/acs.jmedchem.7b00395 J. Med. Chem. 2017, 60, 4983−5001

Journal of Medicinal Chemistry

Article

(16) Hoglinger, G. U.; Huppertz, H. J.; Wagenpfeil, S.; Andres, M. V.; Belloch, V.; Leon, T.; Del Ser, T. Tideglusib reduces progression of brain atrophy in progressive supranuclear palsy in a randomized trial. Mov. Disord. 2014, 29, 479−487. (17) Lovestone, S.; Boada, M.; Dubois, B.; Hull, M.; Rinne, J. O.; Huppertz, H. J.; Calero, M.; Andres, M. V.; Gomez-Carrillo, B.; Leon, T.; del Ser, T. A phase II trial of tideglusib in Alzheimer’s disease. J. Alzheimer’s Dis. 2015, 45, 75−88. (18) Takahashi-Yanaga, F. Activator or inhibitor? GSK-3 as a new drug target. Biochem. Pharmacol. 2013, 86, 191−199. (19) Hall, A. P.; Escott, K. J.; Sanganee, H.; Hickling, K. C. Preclinical toxicity of AZD7969: Effects of GSK3beta inhibition in adult stem cells. Toxicol. Pathol. 2015, 43, 384−399. (20) Martinez, A.; Perez, D. I.; Gil, C. Lessons learnt from glycogen synthase kinase 3 inhibitors development for Alzheimer’s disease. Curr. Top. Med. Chem. 2013, 13, 1808−1819. (21) Doller, D. Allosterism in Drug Discovery; Royal Society of Chemistry: Cambridge, U.K., 2016. (22) Avrahami, L.; Licht-Murava, A.; Eisenstein, M.; EldarFinkelman, H. GSK-3 inhibition: achieving moderate efficacy with high selectivity. Biochim. Biophys. Acta, Proteins Proteomics 2013, 1834, 1410−1414. (23) Palomo, V.; Perez, D. I.; Perez, C.; Morales-Garcia, J. A.; Soteras, I.; Alonso-Gil, S.; Encinas, A.; Castro, A.; Campillo, N. E.; Perez-Castillo, A.; Gil, C.; Martinez, A. 5-imino-1,2,4-thiadiazoles: first small molecules as substrate competitive inhibitors of glycogen synthase kinase 3. J. Med. Chem. 2012, 55, 1645−1661. (24) Bidon-Chanal, A.; Fuertes, A.; Alonso, D.; Perez, D. I.; Martinez, A.; Luque, F. J.; Medina, M. Evidence for a new binding mode to GSK3: allosteric regulation by the marine compound palinurin. Eur. J. Med. Chem. 2013, 60, 479−489. (25) Palomo, V.; Soteras, I.; Perez, D. I.; Perez, C.; Gil, C.; Campillo, N. E.; Martinez, A. Exploring the binding sites of glycogen synthase kinase 3. Identification and characterization of allosteric modulation cavities. J. Med. Chem. 2011, 54, 8461−8470. (26) Ukrainets, I. V.; Tkach, A. A.; Yang, L. Y. 4-hydroxy-2quinolones. 149*. Synthesis, chemical transformations, and biological properties of β-N-acylhydrazides of 1-R-4-hydroxy-2-oxo-1,2-dihydroquinoline-4-carboxylic acids. Chem. Heterocycl. Compd. 2008, 44, 1347−1354. (27) Baki, A.; Bielik, A.; Molnar, L.; Szendrei, G.; Keseru, G. M. A high throughput luminescent assay for glycogen synthase kinase-3beta inhibitors. Assay Drug Dev. Technol. 2007, 5, 75−83. (28) Stamos, J. L.; Weis, W. I. The beta-catenin destruction complex. Cold Spring Harbor Perspect. Biol. 2013, 5, a007898. (29) Sakanaka, C.; Sun, T. Q.; Williams, L. T. New steps in the Wnt/ beta-catenin signal transduction pathway. Recent. Prog. Horm. Res. 2000, 55, 225−236. (30) Doble, B. W.; Woodgett, J. R. GSK-3: tricks of the trade for a multi-tasking kinase. J. Cell Sci. 2003, 116, 1175−1186. (31) Arfeen, M.; Patel, R.; Khan, T.; Bharatam, P. V. Molecular dynamics simulation studies of GSK-3β ATP competitive inhibitors: understanding the factors contributing to selectivity. J. Biomol. Struct. Dyn. 2015, 33, 2578−2593. (32) Leroy, A.; Landrieu, I.; Huvent, I.; Legrand, D.; Codeville, B.; Wieruszeski, J. M.; Lippens, G. Spectroscopic studies of GSK3{beta} phosphorylation of the neuronal tau protein and its interaction with the N-terminal domain of apolipoprotein E. J. Biol. Chem. 2010, 285, 33435−33444. (33) Dajani, R.; Fraser, E.; Roe, S.; Yeo, M.; Good, V. M.; Thompson, V.; Dale, T. C.; Pearl, L. H. Structural basis for recruitment of glycogen synthase kinase 3β to the axin−APC scaffold complex. EMBO J. 2003, 22, 494−501. (34) Stamos, J. L.; Chu, M. L.; Enos, M. D.; Shah, N.; Weis, W. I. Structural basis of GSK-3 inhibition by N-terminal phosphorylation and by the Wnt receptor LRP6. eLife 2014, 3, e01998. (35) Coghlan, M. P.; Culbert, A. A.; Cross, D. A.; Corcoran, S. L.; Yates, J. W.; Pearce, N. J.; Rausch, O. L.; Murphy, G. J.; Carter, P. S.; Roxbee Cox, L.; Mills, D.; Brown, M. J.; Haigh, D.; Ward, R. W.;

SMA, spinal muscular atrophy; MN, motor neuron; RMSF, root-mean-square fluctuations; iPSCs, induced pluripotent stem cells; DMF, dimethylformamide; EtOH, ethanol; MeOH, methanol; DMSO, dimethyldulfoxide; AcOEt, ethyl acetate



REFERENCES

(1) Rayasam, G. V.; Tulasi, V. K.; Sodhi, R.; Davis, J. A.; Ray, A. Glycogen synthase kinase 3: more than a namesake. Br. J. Pharmacol. 2009, 156, 885−898. (2) Niceta, M.; Stellacci, E.; Gripp, K. W.; Zampino, G.; Kousi, M.; Anselmi, M.; Traversa, A.; Ciolfi, A.; Stabley, D.; Bruselles, A.; Caputo, V.; Cecchetti, S.; Prudente, S.; Fiorenza, M. T.; Boitani, C.; Philip, N.; Niyazov, D.; Leoni, C.; Nakane, T.; Keppler-Noreuil, K.; Braddock, S. R.; Gillessen-Kaesbach, G.; Palleschi, A.; Campeau, P. M.; Lee, B. H.; Pouponnot, C.; Stella, L.; Bocchinfuso, G.; Katsanis, N.; Sol-Church, K.; Tartaglia, M. Mutations impairing GSK3-mediated MAF phosphorylation cause cataract, deafness, intellectual disability, seizures, and a Down syndrome-like facies. Am. J. Hum. Genet. 2015, 96, 816−825. (3) O’Leary, O.; Nolan, Y. Glycogen synthase kinase-3 as a therapeutic target for cognitive dysfunction in neuropsychiatric disorders. CNS Drugs 2015, 29, 1−15. (4) Gao, C.; Holscher, C.; Liu, Y.; Li, L. GSK3: a key target for the development of novel treatments for type 2 diabetes mellitus and Alzheimer disease. Rev. Neurosci. 2012, 23, 1−11. (5) Palomo, V.; Perez, D. I.; Gil, C.; Martinez, A. The potential role of glycogen synthase kinase 3 inhibitors as amyotrophic lateral sclerosis pharmacological therapy. Curr. Med. Chem. 2011, 18, 3028− 3034. (6) Golpich, M.; Amini, E.; Hemmati, F.; Ibrahim, N. M.; Rahmani, B.; Mohamed, Z.; Raymond, A. A.; Dargahi, L.; Ghasemi, R.; Ahmadiani, A. Glycogen synthase kinase-3 beta (GSK-3beta) signaling: Implications for Parkinson’s disease. Pharmacol. Res. 2015, 97, 16−26. (7) Henriksen, E. J. Dysregulation of glycogen synthase kinase-3 in skeletal muscle and the etiology of insulin resistance and type 2 diabetes. Curr. Diabetes Rev. 2010, 6, 285−293. (8) Jones, K.; Wei, C.; Iakova, P.; Bugiardini, E.; Schneider-Gold, C.; Meola, G.; Woodgett, J.; Killian, J.; Timchenko, N. A.; Timchenko, L. T. GSK3beta mediates muscle pathology in myotonic dystrophy. J. Clin. Invest. 2012, 122, 4461−4472. (9) Leroy, K.; Yilmaz, Z.; Brion, J. P. Increased level of active GSK3beta in Alzheimer’s disease and accumulation in argyrophilic grains and in neurones at different stages of neurofibrillary degeneration. Neuropathol. Appl. Neurobiol. 2007, 33, 43−55. (10) Yang, W.; Leystra-Lantz, C.; Strong, M. J. Upregulation of GSK3beta expression in frontal and temporal cortex in ALS with cognitive impairment (ALSci). Brain Res. 2008, 1196, 131−139. (11) Martinez, A. Preclinical efficacy on GSK-3 inhibitors: towards a future generation of powerful drugs. Med. Res. Rev. 2008, 28, 773−796. (12) Palomo, V.; Martinez, A. Glycogen synthase kinase 3 (GSK-3) inhibitors: a patent update (2014−2015). Expert Opin. Ther. Pat. 2017, 27, 657−666. (13) Georgievska, B.; Sandin, J.; Doherty, J.; Mortberg, A.; Neelissen, J.; Andersson, A.; Gruber, S.; Nilsson, Y.; Schott, P.; Arvidsson, P. I.; Hellberg, S.; Osswald, G.; Berg, S.; Falting, J.; Bhat, R. V. AZD1080, a novel GSK3 inhibitor, rescues synaptic plasticity deficits in rodent brain and exhibits peripheral target engagement in humans. J. Neurochem. 2013, 125, 446−456. (14) del Ser, T.; Steinwachs, K. C.; Gertz, H. J.; Andres, M. V.; Gomez-Carrillo, B.; Medina, M.; Vericat, J. A.; Redondo, P.; Fleet, D.; Leon, T. Treatment of Alzheimer’s disease with the GSK-3 inhibitor tideglusib: a pilot study. J. Alzheimer’s Dis. 2013, 33, 205−215. (15) Tolosa, E.; Litvan, I.; Hoglinger, G. U.; Burn, D.; Lees, A.; Andres, M. V.; Gomez-Carrillo, B.; Leon, T.; Del Ser, T. A phase 2 trial of the GSK-3 inhibitor tideglusib in progressive supranuclear palsy. Mov. Disord. 2014, 29, 470−478. 4999

DOI: 10.1021/acs.jmedchem.7b00395 J. Med. Chem. 2017, 60, 4983−5001

Journal of Medicinal Chemistry

Article

Smith, D. G.; Murray, K. J.; Reith, A. D.; Holder, J. C. Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription. Chem. Biol. 2000, 7, 793−803. (36) Leost, M.; Schultz, C.; Link, A.; Wu, Y. Z.; Biernat, J.; Mandelkow, E. M.; Bibb, J. A.; Snyder, G. L.; Greengard, P.; Zaharevitz, D. W.; Gussio, R.; Senderowicz, A. M.; Sausville, E. A.; Kunick, C.; Meijer, L. Paullones are potent inhibitors of glycogen synthase kinase-3beta and cyclin-dependent kinase 5/p25. Eur. J. Biochem. 2000, 267, 5983−5994. (37) Martinez, A.; Alonso, M.; Castro, A.; Perez, C.; Moreno, F. J. First non-ATP competitive glycogen synthase kinase 3 beta (GSK3beta) inhibitors: thiadiazolidinones (TDZD) as potential drugs for the treatment of Alzheimer’s disease. J. Med. Chem. 2002, 45, 1292− 1299. (38) Malhi, G. S.; McAulay, C.; Gershon, S.; Gessler, D.; Fritz, K.; Das, P.; Outhred, T. The Lithium Battery: assessing the neurocognitive profile of lithium in bipolar disorder. Bipolar Disord. 2016, 18, 102−115. (39) Beurel, E.; Kaidanovich-Beilin, O.; Yeh, W. I.; Song, L.; Palomo, V.; Michalek, S. M.; Woodgett, J. R.; Harrington, L. E.; EldarFinkelman, H.; Martinez, A.; Jope, R. S. Regulation of Th1 cells and experimental autoimmune encephalomyelitis by glycogen synthase kinase-3. J. Immunol. 2013, 190, 5000−5011. (40) Franklin, A. V.; King, M. K.; Palomo, V.; Martinez, A.; McMahon, L. L.; Jope, R. S. Glycogen synthase kinase-3 inhibitors reverse deficits in long-term potentiation and cognition in fragile X mice. Biol. Psychiatry 2014, 75, 198−206. (41) Wei, C.; Jones, K.; Timchenko, N. A.; Timchenko, L. GSK3beta is a new therapeutic target for myotonic dystrophy type 1. Rare. Dis. 2013, 1, e26555. (42) Dachs, E.; Piedrafita, L.; Hereu, M.; Esquerda, J. E.; Caldero, J. Chronic treatment with lithium does not improve neuromuscular phenotype in a mouse model of severe spinal muscular atrophy. Neuroscience 2013, 250, 417−433. (43) Chen, P. C.; Gaisina, I. N.; El-Khodor, B. F.; Ramboz, S.; Makhortova, N. R.; Rubin, L. L.; Kozikowski, A. P. Identification of a maleimide-based glycogen synthase kinase-3 (GSK-3) inhibitor, BIP135, that prolongs the median survival time of Δ7 SMA KO mouse model of spinal muscular atrophy. ACS Chem. Neurosci. 2012, 3, 5−11. (44) Rodriguez-Muela, N.; Litterman, N. K.; Norabuena, E. M.; Mull, J. L.; Galazo, M. J.; Sun, C.; Ng, S.-Y.; Makhortova, N. R.; White, A.; Lynes, M. M.; Chung, W. K.; Davidow, L. S.; Macklis, J. D.; Rubin, L. L. Single-cell analysis of SMN reveals its broader role in neuromuscular disease. Cell Rep. 2017, 18, 1484−1498. (45) Martínez, A.; Gil, C.; Gil, C.; Palomo Ruiz, V.; Pérez Martín, M. C.; Pérez Fernández, D. I. Moduladores alostéricos de GSK-3 de ́ naturaleza heterociclica. WO2013045736, Apr 4, 2013. (46) Pirrung, M. C.; Blume, F. Rhodium-mediated dipolar cycloaddition of diazoquinolinediones. J. Org. Chem. 1999, 64, 3642−3649. (47) Hardtmann, G. E.; Koletar, G.; Pfister, O. R. The chemistry of 2H-3,1-benzoxazine-2,4(1H)dione (isatoic anhydrides) 1. The synthesis of N-substituted 2H-3,1-benzoxazine-2,4(1H)diones. J. Heterocycl. Chem. 1975, 12, 565−572. (48) Doleschall, G.; Lempert, K. Ü ber die umlagerung von 2-(2aminophenyl)-1,4-dihydro-1-methylchinazolinon-(4) in 3,4-dihydro-2(2-methylaminophenyl)-chinazolinon-(4). Tetrahedron 1969, 25, 2539−2547. (49) Coppola, G. M.; Hardtmann, G. E. The chemistry of 2H-3,1benzoxazine-2,4(1H)dione (isatoic anhydride). 7. Reactions with anions of active methylenes to form quinolines. J. Heterocycl. Chem. 1979, 16, 1605−1610. (50) Rowley, M.; Leeson, P. D.; Stevenson, G. I.; Moseley, A. M.; Stansfield, I.; Sanderson, I.; Robinson, L.; Baker, R.; Kemp, J. A. 3Acyl-4-hydroxyquinolin-2(1H)-ones. Systemically active anticonvulsants acting by antagonism at the glycine site of the N-methyl-Daspartate receptor complex. J. Med. Chem. 1993, 36, 3386−3396. (51) Jönsson, S.; Andersson, G.; Fex, T.; Fristedt, T.; Hedlund, G.; Jansson, K.; Abramo, L.; Fritzson, I.; Pekarski, O.; Runström, A.; Sandin, H.; Thuvesson, I.; Björk, A. Synthesis and biological evaluation

of new 1,2-dihydro-4-hydroxy-2-oxo-3-quinolinecarboxamides for treatment of autoimmune disorders: Structure−activity relationship. J. Med. Chem. 2004, 47, 2075−2088. (52) Bochevarov, A. D.; Harder, E.; Hughes, T. F.; Greenwood, J. R.; Braden, D. A.; Philipp, D. M.; Rinaldo, D.; Halls, M. D.; Zhang, J.; Friesner, R. A. Jaguar: A high-performance quantum chemistry software program with strengths in life and materials sciences. Int. J. Quantum Chem. 2013, 113, 2110−2142. (53) Schrödinger, Release 2016-4: Jaguar; Schrödinger, LLC: New York, NY, 2016. (54) Becke, A. D. Density-functional thermochemistry. IV. A new dynamical correlation functional and implications for exact-exchange mixing. J. Chem. Phys. 1996, 104, 1040−1046. (55) Tannor, D. J.; Marten, B.; Murphy, R.; Friesner, R. A.; Sitkoff, D.; Nicholls, A.; Honig, B.; Ringnalda, M.; Goddard, W. A. Accurate first principles calculation of molecular charge distributions and solvation energies from ab initio quantum mechanics and continuum dielectric theory. J. Am. Chem. Soc. 1994, 116, 11875−11882. (56) Marten, B.; Kim, K.; Cortis, C.; Friesner, R. A.; Murphy, R. B.; Ringnalda, M. N.; Sitkoff, D.; Honig, B. New model for calculation of solvation free energies: Correction of self-consistent reaction field continuum dielectric theory for short-range hydrogen-bonding effects. J. Phys. Chem. 1996, 100, 11775−11788. (57) Schrödinger Release 2016-1: Schrödinger Suite 2016-1 Protein Preparation Wizard: Epik, Impact, and Prime; Schrödinger, LLC, New York, NY, 2016. (58) Sastry, G. M.; Adzhigirey, M.; Day, T.; Annabhimoju, R.; Sherman, W. Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments. J. Comput.-Aided Mol. Des. 2013, 27, 221−234. (59) Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D. S.; Olson, A. J. AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J. Comput. Chem. 2009, 30, 2785−2791. (60) Case, D. A.; Cheatham, T. E., III, Simmerling, C. L.; Wang, J.; Duke, R. E.; Luo, R.; Walker, R. C.; Zhang, W.; Merz, K. M.; Roberts, B.; Hayik, S.; Roitberg, A.; Seabra, G.; Swails, J.; Götz, A. W.; Kolossváry, I.; Wong, K. F.; Paesani, F.; Vanicek, J.; Wolf, R. M.; Liu, J.; Wu, X.; Brozell, S. R.; Steinbrecher, T.; Gohlke, H.; Cai, Q.; Ye, X.; Wang, J.; Hsieh, M.-J.; Cui, G.; Roe, D. R.; Mathews, D. H.; Seetin, M. G.; Salomon-Ferrer, R.; Sagui, C.; Babin, V.; Luchko, T.; Gusarov, S.; Kovalenko, A.; Kollman, P. A. AMBER 12, University of California: San Francisco, CA, 2012. (61) Maier, J. A.; Martinez, C.; Kasavajhala, K.; Wickstrom, L.; Hauser, K. E.; Simmerling, C. ff14SB: Improving the accuracy of protein side chain and backbone parameters from ff99SB. J. Chem. Theory Comput. 2015, 11, 3696−3713. (62) Wang, J.; Wang, W.; Kollman, P. A.; Case, D. A. Automatic atom type and bond type perception in molecular mechanical calculations. J. Mol. Graphics Modell. 2006, 25, 247−260. (63) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 1983, 79, 926−935. (64) Lippert, R. A.; Bowers, K. J.; Dror, R. O.; Eastwood, M. P.; Gregersen, B. A.; Klepeis, J. L.; Kolossvary, I.; Shaw, D. E. A common, avoidable source of error in molecular dynamics integrators. J. Chem. Phys. 2007, 126, 046101. (65) Berendsen, H. J. C.; Postma, J. P. M.; van Gunsteren, W. F.; DiNola, A.; Haak, J. R. Molecular dynamics with coupling to an external bath. J. Chem. Phys. 1984, 81, 3684−3690. (66) Roe, D. R.; Cheatham, T. E. PTRAJ and CPPTRAJ: Software for processing and analysis of molecular dynamics trajectory data. J. Chem. Theory Comput. 2013, 9, 3084−3095. (67) XMGRACE, version 5.1.19; Center for Coastal and LandMargin Research, Oregon Graduate Institute of Science and Technology: Beaverton, OR,2005. (68) Humphrey, W.; Dalke, A.; Schulten, K. VMD: Visual molecular dynamics. J. Mol. Graphics 1996, 14, 33−38. 5000

DOI: 10.1021/acs.jmedchem.7b00395 J. Med. Chem. 2017, 60, 4983−5001

Journal of Medicinal Chemistry

Article

(69) ADP-Glo Kinase Assay Technical Manual: https://www. promega.es/resources/protocols/technical-manuals/0/adp-glo-kinaseassay-protocol/ (accessed May 8, 2017). (70) Morales-Garcia, J. A.; Palomo, V.; Redondo, M.; Alonso-Gil, S.; Gil, C.; Martinez, A.; Perez-Castillo, A. Crosstalk between phosphodiesterase 7 and glycogen synthase kinase-3: two relevant therapeutic targets for neurological disorders. ACS Chem. Neurosci. 2014, 5, 194−204. (71) Morales-Garcia, J. A.; Susin, C.; Alonso-Gil, S.; Perez, D. I.; Palomo, V.; Perez, C.; Conde, S.; Santos, A.; Gil, C.; Martinez, A.; Perez-Castillo, A. Glycogen synthase kinase-3 inhibitors as potent therapeutic agents for the treatment of Parkinson disease. ACS Chem. Neurosci. 2013, 4, 350−360.

5001

DOI: 10.1021/acs.jmedchem.7b00395 J. Med. Chem. 2017, 60, 4983−5001