Re-evolution of the 2-Phenylquinolines: Ligand-Based Design

May 27, 2013 - To test whether this discrepancy was due to differences in lipophilicity, the software QikProp(52) was used to calculate the descriptor...
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Re-evolution of the 2‑Phenylquinolines: Ligand-Based Design, Synthesis, and Biological Evaluation of a Potent New Class of Staphylococcus aureus NorA Efflux Pump Inhibitors to Combat Antimicrobial Resistance Stefano Sabatini,*,† Francesca Gosetto,† Nunzio Iraci,† Maria Letizia Barreca,† Serena Massari,† Luca Sancineto,† Giuseppe Manfroni,† Oriana Tabarrini,† Mirjana Dimovska,‡ Glenn W. Kaatz,‡ and Violetta Cecchetti† †

Dipartimento di Chimica e Tecnologia del Farmaco, Università degli Studi di Perugia, 06123 Perugia, Italy John D. Dingell Department of Veterans Affairs Medical Center and the Department of Internal Medicine, Division of Infectious Diseases, School of Medicine, Wayne State University, Detroit, Michigan 48201, United States



S Supporting Information *

ABSTRACT: Overexpression of efflux pumps is an important mechanism by which bacteria evade the effects of antimicrobial agents that are substrates. NorA is a Staphylococcus aureus efflux pump that confers reduced susceptibility to many structurally unrelated agents, including fluoroquinolones, biocides, and dyes, resulting in a multidrug resistant (MDR) phenotype. In this work, a series of 2-phenylquinoline derivatives was designed by means of ligand-based pharmacophore modeling in an attempt to identify improved S. aureus NorA efflux pump inhibitors (EPIs). Most of the 2phenylquinoline derivatives displayed potent EPI activity against the norA overexpressing strain SA-1199B. The antibacterial activity of ciprofloxacin, when used in combination with some of the synthesized compounds, was completely restored in SA1199B and SA-K2378, a strain overexpressing norA from a multicopy plasmid. Compounds 3m and 3q also showed potent synergistic activity with the ethidium bromide dye in a strain overexpressing the MepA MDR efflux pump.



INTRODUCTION In the past few decades, the rate of antimicrobial resistance has increased dramatically and, in recent years, bacterial infections have become increasingly difficult to treat.1,2 The rise of bacterial strains that are developing resistance to more than one first line drug, i.e., multidrug resistant (MDR) strains, is one of the major worldwide public health emergencies.3,4 Epidemic antibiotic resistance has been described for numerous pathogens in varying contexts, including, but not limited to, a global pandemic of methicillin-resistant Staphylococcus aureus (MRSA) infection.5,6 S. aureus is a bacterium responsible for a wide range of infections from simple skin and soft tissue infections to more serious illnesses like pneumonia, endocarditis, and sepsis.7 Among the resistant strains, MRSA is one of the most challenging to treat.8 About 19000 deaths yearly are attributable to MRSA in the United States.9 This exceeds the total number of deaths related to human immunodeficiency virus/AIDS in the U.S. in 2005.10 In the EU in 2008, MRSA accounted for 44% (n = 171200) of healthcare-associated staphylococcal infections and 22% (n = 5400) of attributable extra deaths.11 Since the mid-1990s, there has been an explosion in the number of MRSA infections in the U.S. among populations © XXXX American Chemical Society

lacking risk factors such as recent exposure to the health care system. This increase has been associated with the recognition of new MRSA strains, often called community-associated MRSA (CA-MRSA), that have been responsible for a large proportion of the increased disease burden. CA-MRSA infections, far from being the clinical curiosity they were in the mid-1990s, have become commonplace and have created a public health crisis in U.S. emergency departments and other clinical settings.12 MRSA often express concomitant resistance to many antibacterials such as tetracyclines, aminoglycosides, and fluoroquinolones. Moreover, since 2002, vancomycin resistant strains have also been identified.13 Such strains amplify the difficulty in treating clinical infections related to them. Among the different mechanisms by which bacteria may acquire resistance to antimicrobial agents, one of the most important involves overexpression of efflux pumps.14 Efflux systems are responsible for the extrusion of toxic compounds, including antibacterials, encountered in the bacterial environment.15 In a recent work, the increased expression of one or more MDR efflux pump genes was identified in 151/309 S. aureus Received: February 20, 2013

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phenyl-4H-chromen-4-one moiety, a common feature of natural flavone and flavonolignane EPIs,35−37 to obtain the 2-phenylquinolone nucleus first, and then, after suitable substitutions, the 2-(4′-propoxyphenyl)quinoline derivatives.41 In particular, the C-4 O-ethyl-2-diethylamine and O-ethyl-2-piperidine 2-phenylquinoline derivatives (compounds 141 and 2,41 respectively; Figure 1) resulted in very potent NorA inhibitors, showing 93.4% and 88.5% inhibition of ethidium bromide (EtBr) efflux, respectively, when tested at a 50 μM concentration against SA1199B (norA+/A116E GrlA). Taking these interesting results into account and in an effort to obtain more potent NorA EPIs, we have decided to expand the SAR of this class of EPIs by introducing an O-alkyl or different Oalkylamino chains at the C-4 position of the 2-(4′propoxyphenyl)quinoline nucleus.

Figure 1. Re-evolution of the 2-(4′-propoxyphenyl)quinolines EPIs.

clinical strains (49%) by quantitative reverse transcription-PCR. Among those overexpressing a single gene, norA was the most common (43%), followed by norB (23.2%) and mepA (9.9%).16 NorA is the most studied S. aureus efflux pump. It is a MDR pump belonging to the major facilitator superfamily (MFS)17 and is capable of extruding multiple structurally dissimilar substrates such as hydrophilic fluoroquinolones (norfloxacin and ciprofloxacin) and various biocides and dyes through an antiport mechanism driven by the proton-motive force.18,19 To date, the structural biology of NorA has not been determined. Nevertheless, the sequence homology and the sharing of several substrates with other MDR pumps have led to the hypothesis that NorA may have a large hydrophobic binding site. This structural peculiarity could explain the broad substrate specificity of MDR pumps.20 Efflux pumps are thus viable antibacterial targets and identification and development of potent EPIs is a promising and valid strategy.21 There are a number of reasons to pursue this area, including: (i) this is a nature-inspired approach, as there are examples in which plants produce both antibacterials and EPIs that improve the antibacterial activity (e.g., berberine and the methoxyhydnocarpin, isoflavones and α-linolenic acid, and dalversinol A and a methoxychalcone),22−24 (ii) EPIs used in combination with antibiotics may not only increase antibacterial potency25 but also may expand the antibacterial spectrum and reduce the frequency of the emergence of target-based resistance,26 (iii) EPIs have been shown to reduce biofilm formation and block the antibacterial tolerance of bacterial biofilms.27 Many EPIs capable of potentiating the activity of antimicrobial pump substrates have been identified. Early EPIs included reserpine and verapamil, but concentrations required for pump inhibition activity are too high for them to be clinically relevant.28,29 Several other nonantibiotic compounds, such as omeprazole, paroxetine, chlorpromazine, and respective derivatives, and the synthetic acridone derivative GG918, have been shown to increase the antibacterial potency of pump substrates by inhibiting NorA.30−34 Great efforts to identify EPIs have been made by phytochemists who have reported series of natural EPI compounds, extremely varied in terms of their chemical class and shape, including flavones, isoflavones, porphyrin phaeophorbide A, and acylated glycosides.35,36 To date, there are just a few examples of rationally designed NorA inhibitors, and very little work has been done with respect to their structure−activity relationships (SAR).37−46 Although the therapeutic utility of EPIs has yet to be validated in the clinical setting, this approach holds promise for improving the efficacy and/or extending the clinical utility of existing antibacterials, giving new life to old drugs with secure social and economic benefit.25 In an attempt to obtain new chemotypes endowed with good inhibitory activity against NorA, we have recently modified the 2-



SYNTHESIS OF A FIRST SET OF QUINOLINE DERIVATIVES To confirm the role of the protonable nitrogen of the Oalkylamino chain at the C-4 position of 141 and 2,41 we designed and synthesized a first set of seven compounds bearing an O-alkyl (3a) or suitable O-alkylamines (3b, 3c, 3j, 3t, 3w, and 3y) differing in size and degree of basicity. To assess the NorA EPI function, compounds 3a−c, 3j, 3t, 3w, and 3y were screened for their EtBr efflux inhibition activity at a concentration of 50 μM against SA-1199B. If the screening EPI activity was >80%, minimum inhibitory concentrations (MICs) were determined and a series of concentrations was tested to evaluate the 50% inhibitory concentration (IC50). 141 and reserpine were included as reference compounds. The results of these assays are provided in Table 1 and Figure 2. Table 1. EtBr Efflux Inhibition Activity (%) at 50 μM Concentration, IC50, and MICs of the First Set of 2Phenylquinoline Derivatives versus SA-1199B (norA+/A116E GrlA)

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LIGAND-BASED PHARMACOPHORE MODEL FOR NORA EPIS Because of the lack of structural biology information for NorA and its interaction with inhibitors and/or substrates, we decided to develop a common feature three-dimensional (3D) pharmacophore model for highly active NorA inhibitors by means of the software Phase.47 The main goal of this study was to determine the essential 3D structural requirements that are relevant for interaction with NorA. The data set for the pharmacophore development was built by considering single or chemical classes of compounds tested as NorA inhibitors synthesized by us or reported in the literature by other research groups.39−44,48,49 Our EPI library included the first set of seven quinoline derivatives described herein (i.e., 3a−c, 3j, 3t, 3w, and 3y), 3-phenyl-1,4-benzothiazine derivatives,39 the 6-amino-7thiopyranopyridinyl quinolone ethyl esters, 40 2-(4′propoxyphenyl)quinoline derivatives,41 pyrazolo[4,3-c][1,2]benzothiazines 5,5 dioxide,42 and other compounds recently published.43,44 Examination of the literature revealed a large number of compounds having the biological behavior appropriate for our purposes. However, to ensure high homogeneity in the biological data within the data set, we selected only compounds whose NorA inhibitory activity was evaluated using protocols comparable to the ones used to test our compounds. In particular, three constraints were applied in such selection: (i)

Figure 2. Dose−response EtBr efflux inhibition assays for 3b, 3y, 1,41 and reserpine against SA-1199B (norA+/A116E GrlA).

Only the compounds carrying a small protonable O-aminoalkyl chain at the C-4 position of the quinolone nucleus (3b and 3y) possessed good NorA EPI activity. In fact, the presence of an O-alkyl chain (3a), a less protonable (3c, 3j, and 3w) or bulky Oalkylamino chain (3t) at the C-4 position resulted in weak or absent NorA EPI activity. The good EtBr efflux inhibition activities of compounds 3b and 3y (92.0 and 96.1%, respectively) were also confirmed by their IC50 values, which were comparable to those of the reference compounds in a dose−response assay (Figure 2). These data highlighted the key role of the protonable Oalkylamino chain at the C-4 position of the 2-(4′propoxyphenyl)quinoline nucleus to obtain potent NorAmediated EtBr efflux inhibition.



Article

COMPUTER-AIDED DESIGN OF NEW QUINOLINE NORA EPIS

On the basis of these data, we elected to synthesize additional quinoline derivatives bearing suitable protonable side chains at the C-4 position, with the aim of expanding our SAR knowledge and improving NorA EPI activity for this class of EPIs. For this purpose, a variety of computational approaches were combined into a multistep procedure (Figure 3) to rationally support the chemical synthesis of new quinoline NorA EPIs, thus minimizing efforts and costs.

Figure 3. Workflow for the identification of the virtual hits and new EPIs.

Figure 4. Geometry of the pharmacophore: (A) distance and (B) angles between pharmacophoric features of APRR hypothesis. C

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Table 2. The Active Set of Compounds Used to Build the Pharmacophore Model and Their Corresponding EPI Activities at 50 μM against SA-1199B (norA+/A116E GrlA)

the NorA inhibitory activity had to be evaluated at a concentration of 50 μM and reported as the percentage of reduction of EtBr efflux,50,51 (ii) the bacterial strain used for these experiments had to be SA-1199B, which overexpress the NorA efflux pump and is characterized by a mutation of topoisomerase IV that reduces its affinity to ciprofloxacin (CPX), and (iii) the compounds should have well-defined chirality. On the basis of these requirements, a total of 65 compounds constituted our data set. The pharmacophore model was developed by means of the Phase47 software. The computational method we used required classification of molecules according to activity. As such, arbitrary activity ranges were established. Molecules possessing EtBr efflux inhibition of >70% at a 50 μM concentration were considered “active”, and those demonstrating 100 >100 >100 >100 >100 nd 100 >100 0.31

nd nd 100 25 50 25 >100 nd 50 >100 0.63

nd nd >100 25 50 50 >100 nd 50 >100 2.50

>100 >100 >100 >100 >100 >100 >100 >100 >100 >100 0.63

>100 >100 >100 >100 >100 >100 >100 >100 >100 >100 10

H

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Figure 9. Effect of compounds 3h, 3i, 3m, 3q, and 3s on the MIC of EtBr against S. aureus SA-K2885 (mepA−) and SA-K2886 (mepA++).

In an attempt to delineate a preliminary SAR for this emerging class of EPIs, it is possible to state that: (i) the highest activities were generally displayed by compounds having the 2-ethylamino alkyl chains linked to the C-4 hydroxyl group (compounds 3d, 3e, and 3h) and (ii) the activity was maintained when the side chain nitrogen atom was included in an aliphatic ring (compounds 3f, 3m, 3q, 3s, and 3x). The influence of the second heteroatom on the aliphatic ring moiety has not yet been completely delineated. Inhibitory activity tended to be lost when the second heteroatom was an oxygen or a sulfur atom. This was confirmed by the fact that the piperazine derivative 3q displayed EtBr efflux inhibitory activity of 93.4% in comparison to morpholine 3k and thiomorpholine 3p, which inhibited the efflux of 86.9% and 57.7%, respectively. Inhibitory activity was completely lost when the side chain nitrogen was included in an aromatic ring (compounds 3c and 3j), in a phthalimide moiety (3w) or disubstituted with benzyl groups (3t). This behavior may be related to the reduction of the nitrogen nucleophilicity, as supported by the low inhibitory activity of the compound 3a, which is missing a protonable nitrogen, or to steric hindrance. The monosubstitution with a benzyl group was, on the other hand, well tolerated. In fact, the compound 3v, in which the side chain nitrogen atom is substituted with an ethyl and a benzyl group, possessed an EtBr inhibitory activity higher than that of the compound 3t, having two benzyl groups as substituents. In conclusion, chemical modifications proposed by in silico analyses led us to confirm the 2-(4′-propoxyphenyl)quinoline nucleus as a very good scaffold to obtain new and potent EPIs. Some of the 2-{[2-(4′-propoxyphenyl)-4-quinolinyl]-oxy}ethanamine derivatives, showing modest or no intrinsic antistaphylococcal activity up to the highest concentration tested (100 μg/mL), were able to restore, in a concentration-dependent manner, the antibacterial activity of CPX against norA-overexpressing S. aureus strains. Both the EPI activity of these derivatives in the EtBr efflux inhibition assay and their synergistic activity with CPX were superior to that shown by the reference compounds against the norA-overexpressing strain SA-1199B. Compounds 3m and 3q also possessed potent synergistic activity with EtBr against a MepA overexpressing strain.

3u, and 3x) (data not shown). These studies identified eight compounds (3e, 3f, 3h, 3i, 3m, 3q, 3s, and 3x) having IC50 values lower than 9 μM, lower than those of both reserpine49 and 141 (Table 4). The MICs of the eight most active compounds (3e, 3f, 3h, 3i, 3m, 3q, 3s, and 3x) were determined using five S. aureus strains with different norA genotypes. Data shown in Table 5 illustrate how these selected compounds had little or no intrinsic antibacterial activity, with MICs ranging from 25 to >100 μg/mL. The compounds were then assayed for their ability to reduce the MIC of CPX (synergistic activity) against the abovementioned S. aureus strains (Figure 8). For compounds 3e, 3f, and 3x, synergistic activity data are available only for SA-1199 (norA wt) and SA-1199B (norA+/A116E GrlA). The isobolograms illustrated in Figure 8 revealed that neither the tested compounds nor the reference compound reserpine49 showed any significant synergistic activity with CPX against S. aureus ATCC 25923 (wt), SA-1199 (norA wt), and SA-K1902 (norA−) strains. These data are in agreement with the low expression of the NorA pump in these strains. On the contrary, a good synergistic effect with CPX was demonstrated by all the tested compounds against SA-K2378 (norA++), with a CPX MIC reduction of 4−32-fold (2.5 → 0.08 μg/mL). In particular, 0.78 μg/mL of compound 3m reduced the CPX MIC 16-fold. When tested against SA-1199B (norA+/A116E GrlA), 3.13 μg/ mL of compounds 3m and 3q showed the best synergistic activity by reducing the CPX MIC 16-fold (10 → 0.63 μg/mL). Given their potent NorA inhibitory activity, the inhibitory activities of compounds 3h, 3i, 3m, 3q, and 3s were evaluated against the MepA (MATE superfamily) efflux pump. This was accomplished using S. aureus strains SA-K2885 (mepA−) and SA-K2886 (mepA++, overexpressed from a plasmid) (Figure 9). In both cases, synergistic activity studies were carried out using EtBr instead of CPX, as it is a better MepA substrate and thus permits a clearer picture of MepA inhibition. As expected, the tested compounds did not show any synergistic activity with EtBr against SA-K2885 (mepA−). However, all were able to reduce the EtBr MIC against SAK2886 (mepA++). The most active compounds were 3m and 3q, which were able to reduce the EtBr MIC 16-fold at concentrations of 0.78 and 1.56 μg/mL, respectively. These data indicate that 2-(4′-propoxyphenyl)]quinolines 3h, 3i, 3m, 3q, and 3s are inhibitors of both the NorA and MepA MDR efflux pumps and are able to completely restore the activity of CPX and EtBr against strains overexpressing such pumps.



EXPERIMENTAL SECTION

Preparation of the Data Set Compounds. Molecules were built using the fragment library tools of the Maestro GUI56 and then submitted to conformational search using the software MacroModel.54 I

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The search was carried on in vacuum using the OPLS 2005 force field. The “Mixed torsional/Low mode sampling method” was employed, using the automatic setup of the search variables; the maximum numbers of steps were set to 10000 and 100 per molecule and rotatable bond, respectively. Conformers in an energy window of 10 kcal/mol were saved, discarding the ones redundant on the basis of their RMSD. Minimization of conformers was performed using the Polak−Ribiere conjugate gradient method, using a maximum of 500 minimization step and 0.0005 kJ/(Å mol) gradient convergence threshold. Pharmacophore Model Generation. Phase 3.347 was used to carry on the pharmacophore modeling studies. First, we established an arbitrary range of activities for all the molecules in the data set (activity is expressed as percentage of inhibition of EtBr efflux). Molecules showing a percentage of EtBr efflux inhibition greater than 70% were considered “active”, while molecules showing a percentage of less than 30% were considered “inactive”. The “active set” was then composed of 14 inhibitors (Table 2), whereas the “inactive” one was composed of 51 molecules (Table S-1 in Supporting Information). Pharmacophoric sites were generated using the default feature definitions as implemented in Phase. Pharmacophore features common to at least seven of 14 active compounds and composed by three, four or five sites were generated, obtaining several hypotheses. The hypotheses were scored by the goodness of the alignment of each active ligand onto hypotheses (“active score”). The scoring algorithm took into account contributions from the alignment of site points and vectors, volume overlap, selectivity, number of active ligands matched, relative conformational energy, and ligand activity. A second score (“inactive score”) was then calculated using the alignment of each inactive molecule. A satisfying hypothesis should show high active scores as well as low inactive scores, thus proving to be able to discriminate between active and inactive molecules. Following the active/inactive scoring ratio as guideline, the best identified hypothesis (APRR: active score = 5.038, inactive score = 1.224) was composed by four pharmacophoric features: one hydrogenbond acceptor (A3), one positive ionizable group (P7), and two aromatic rings (R9 and R10). Focused Library Generation. The Combinatorial Library Enumeration utility in CombiGlide57 was used to build a focused library of quinoline derivatives. Following the a priori decided two synthetic pathways (Figure 7), cores were sketched using the Maestro GUI, the appropriate reagents were searched through the Sigma-Aldrich Web site, downloaded as an sdf file, and prepared with the Reagent Preparation utility in CombiGlide. Cores were then reacted in silico with the reactants to obtain all the possible combinations for each reaction, and structures were untangled and minimized. Output structures with a molecular weight greater than 500 Da and more than two chiral centers were ruled out, and the filtered structures were then submitted to a conformational sampling using MacroModel with the same settings used for the data set. Finally, the conformations were used as input to create a phase database (5060 molecules). Synthesis. All reactions were routinely checked by thin-layer chromatography (TLC) on silica gel 60F254 (Merck) and visualized using UV illumination. Flash chromatography was performed on Merck silica gel 60 (mesh 230−400) using the indicated solvents. Yields were of purified product and were not optimized. Melting points were determined in capillary tubes (Mettler PF62 apparatus) and are uncorrected. Elemental analyses were performed by a Fisons elemental analyzer (model EA1108CHN), and the data for C, H, and N are within 0.4% of the theoretical values (≥95% purity). 1H NMR spectra were recorded at 400 MHz with a Bruker Advance-DRX 400 instrument and with Me4Si as the internal standard. The chemical shift (δ) values are reported in ppm, and the coupling constants (J) are given in Hz. The abbreviations used are as follows: s, singlet; bs, broad singlet; d, doublet; dd, double doublet; t, triplet; m, multiplet. The spectral data are consistent with the assigned structures. Reagents and solvents were purchased from common commercial suppliers and were used as received. For routine aqueous workup, the reaction mixture was extracted with CH2Cl2 or EtOAc. The organic layer was washed with brine, dried over anhydrous Na2SO4, and concentrated with a Büchi

rotary evaporator under reduced pressure. All starting materials were commercially available, unless otherwise indicated. General Procedure for the Synthesis of the 4-[(2Chloroethyl)oxy]-2-[4′-(propyloxy)phenyl]quinolone (5), 4-[(3Methylbutyl)oxy]-2-[4′-(propyloxy)phenyl]quinoline (3a), and the [2-({2-[4′-(Propyloxy)phenyl]quinolin-4-yl}oxy)alkyl]amines (3b−k). To a solution of 2-[4′-(propyloxy)phenyl]quinolin4-ol 441(1.0 equiv) in dry DMF (10 mL) was added K2CO3 (4.0 equiv) and a solution of 1-bromo-2-chloroethane or 1-iodo-3-methylbutane or halo/mesylalkylamines (4.0 equiv) in dry DMF (3 mL) was added dropwise. The reaction mixture was warmed to 70 °C under nitrogen atmosphere with stirring until 2-[4′-(propyloxy)phenyl]quinolin-4-ol 441 disappeared (TLC). The mixture was then poured in water and extracted with EtOAc (3 × 100 mL), and the combined organic extracts were dried over Na2SO4. The organic solvent was then removed under reduced pressure to obtain a residue that was purified by flash column chromatography to give the target compounds. For compounds 3b, 3d, 3e, and 3i, the obtained oil was dissolved in a mixture of Et2O/EtOH and treated with HClg until the target compound precipitated as its HCl salt. 4-[(2-Chloroethyl)oxy]-2-[4′-(propyloxy)phenyl]quinolone (5). Obtained from 1-bromo-2-chloroethane as a white solid after purification by flash chromatography (silica gel/PE/EtOAc 95/5) (65% yield, mp 100.3−101.8 °C). 1H NMR (CDCl3): δ 1.11 (3H, t, J = 7.35 Hz, OCH2CH2CH3), 1.84−1.95 (2H, m, OCH2CH2CH3), 4.02− 4.08 (4H, m, OCH2CH2CH3,OCH2CH2Cl), 4.59 (2H, t, J = 5.67 Hz, OCH2CH2Cl), 7.07 (2H, d, J = 8.87 Hz, H-3′ and H-5′), 7.16 (1H, s, H3), 7.52 (1H, t, J = 7.53 Hz, H-6), 7.75 (1H, t, J = 5.24 Hz, H-7), 8.10− 8.15 (3H, m, H-8, H-2′ and H-6′), 8.26 (1H, d, J = 8.26 Hz, H-5). 4-(3-Methylbutoxy)-2-(4′-propoxyphenyl)quinoline (3a). Obtained from 1-iodo-3-methylbutane as a yellowish solid after purification by flash chromatography (silica gel/PE/EtOAc 97/3) (40% yield, mp 78.2−83.1 °C). 1H NMR (CDCl3): δ 0.98−1.18 (9H, m, CH3), 1.98− 2.07 (5H, m, OCH2CH2CH, OCH2CH2CH and OCH2CH2CH3), 4.04 (2H, t, J = 6.62 Hz, OCH2CH2CH3), 4.34 (2H, t, J = 6.21 Hz, OCH2CH2CH), 7.08 (2H, d, J = 8.35 Hz, H-3′ and H-5′), 7.17 (1H, s, H-3), 7.50 (1H, t, J = 7.08 Hz, H-6), 7.73 (1H, t, J = 7.05 Hz, H-7), 8.10−8.14 (3H, m, H-2′, H-6′ and H-8), 8.23 (1H, d, J = 8.21 Hz, H-5). Anal. (C23H27NO2) C, H, N. N,N-Dimethyl-2-{[2-(4′-propoxyphenyl)-4-quinolinyl]oxy}ethanamine Hydrochloride (3b). Obtained from (2-chloroethyl)dimethylamine hydrochloride as a white solid after purification by flash chromatography (silica gel/CH2Cl2/MeOH 99/1) and precipitation as hydrochloride (56% yield, mp 145.1−146.9 °C). 1H NMR (DMSO-d6): δ 0.95 (3H, t, J = 7.44 Hz, OCH2CH2CH3), 1.68−1.78 (2H, m, OCH2CH2CH3), 2.85 (6H, d, J = 4.72 Hz, NCH3), 3.65−3.80 (2H, m, OCH2CH2N), 4.04 (2H, t, J = 6.49 Hz, OCH2CH2CH3), 4.90−5.20 (2H, m, OCH2CH2N), 7.20 (2H, d, J = 8.74 Hz, H-3′ and H-5′), 7.71− 7.79 (2H, m, H-3 and H-6), 8.02 (1H, t, J = 8.11 Hz, H-7), 8.86 (2H, d, J = 8.86 Hz, H-2′and H-6′), 8.50 (1H,d, J = 8.32 Hz, H-8), 8.59 (1H, d, J = 8.30 Hz, H-5), 11.16 (1H, bs, NH). Anal. (C22H26N2O2·HCl) C, H, N. 2-(4′-Propoxyphenyl)-4-[2-(1H-pyrrol-1-yl)ethoxy]quinoline (3c). Obtained from 1-(2-chloroethyl)-1H-pyrrole55 as a white solid after purification by flash chromatography (silica gel/PE/EtOAc 95/5) (30% yield, mp 127.7−129.0 °C). 1H NMR (CDCl3): δ 1.11 (3H, t, J = 7.35 Hz, OCH2CH2CH3), 1.84−1.94 (2H, m, OCH2CH2CH3), 4.04 (2H, t, J = 6.61 Hz, OCH 2 CH 2 N), 4.53 (4H, m, OCH 2 CH 2 N and OCH2CH2CH3);6.25 (2H, d, J = 2.11 Hz, pyrrole CH), 6.89 (2H, t, J = 2.10 Hz, pyrrole NCH), 7.04−7.09 (3H, m, H-3, H-3′ and H-5′), 7.52 (1H, t, J = 2.55 Hz, H-6), 7.73 (1H, t. J = 2.57 Hz, H-7), 8.05−8.13 (3H, m, H-8, H-2′ and H-6′), 8.20 (1H, d, J = 7.20 Hz, H-5). Anal. (C24H24N2O2) C, H, N. N,N-Dimethyl-2-{[2-(4′-propoxyphenyl)quinolin-4-yl]oxy}propan1-amine Hydrochloride (3d) and N,N-Dimethyl-1-{[2-(4′-propoxyphenyl)-4-quinolinyl]oxy}-2-propanamine Hydrochloride (3e). Obtained from (2-chloropropyl)dimethylamine hydrochloride after purification by flash chromatography (silica gel/CH2Cl2/EtOH 97/3) and precipitation as its HCl salt. 3d: 18% yield; mp 189.7−190.5 °C. 1H NMR (DMSO-d6): δ 1.04 (3H, t, J = 7.33 Hz, OCH2CH2CH3), 1.47 (3H, d, J = 5.89 Hz, J

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Journal of Medicinal Chemistry

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8.81 Hz, H-3′and H-5′), 7.22−7.35 (2H, m, H-3 and pyridine CH), 7.54 (1H, t, J = 6.99 Hz, H-6), 7.66−7.85 (3H, m, H-7 and pyridine CH), 8.05−8.15 (3H, m, H-8, H-2′ and H-6′), 8.34 (1H, d, J = 7.52 Hz, H-8), 8.70 (1H, d, J = 4.75 Hz, pyridine CH). Anal. (C24H22N2O2) C, H, N. 4-[2-(4-Morpholinyl)ethoxy]-2-(4′-propoxyphenyl)quinoline (3k). Obtained from 4-(2-chloroethyl)morpholine hydrochloride as a white solid after purification by flash chromatography (silica gel/CH2Cl2/ MeOH 99/1) (24% yield, mp 141.5−142.5 °C). 1H NMR (CDCl3): δ 1.02 (3H, t, J = 7.44 Hz, OCH2CH2CH3), 1.71−1.89 (2H, m, OCH2CH2CH3), 2.61−2.73 (4H, m, morpholine NCH2), 3.05 (2H, t, J = 4.99 Hz, OCH2CH2N), 3.72−3.83 (4H, m, morpholine OCH2), 3.95 (2H, t, J = 6.57 Hz, OCH2CH2CH3), 4.48 (2H, t, J = 5.33 Hz, OCH2CH2N), 6.98 (2H, d, J = 8.75 Hz, H-3′ and H-5′), 7.12 (1H, s, H3), 7.44 (1H, t, J = 7.98 Hz, H-6), 7.68 (1H, t, J = 7.73 Hz, H-7), 8.03− 8.10 (3H, m, H-8, H-2′ and H-6′), 8.21 (1H, d, J = 8.16 Hz, H-5). Anal. (C24H28N2O3) C, H, N. General Procedure for the Synthesis of the [2-({2-[4′(Propyloxy)phenyl]quinolin-4-yl}oxy)alkyl]amines (3l−x). To a solution of 4-[(2-chloroethyl)oxy]-2-[4′-(propyloxy)phenyl]quinolone (5) (1.0 equiv) in dry DMF (10 mL) was added K2CO3 (4.0 equiv) followed by the dropwise addition of a solution of the suitable cyclic/ dialkylamine (4.0 equiv) in dry DMF (3 mL). The reaction mixture was warmed at 80 °C under a nitrogen atmosphere with stirring until 4-[(2chloroethyl)oxy]-2-[4′-(propyloxy)phenyl]quinolone (5) disappeared (TLC). The mixture was then poured into water and extracted with EtOAc (3 × 100 mL). The organic solvent was removed under reduced pressure to obtain a residue that was purified by flash chromatography or crystallized from cyclohexane to give the target compounds. For compound 3o, the obtained oil was dissolved in a mixture of Et2O/ EtOH and treated with HClg until the target compound precipitated as its HCl salt. 2-(4′-Propoxyphenyl)-4-[2-(1-pyrrolidinyl)ethoxy]quinoline (3l). Obtained from pyrrolidine as a white solid after crystallization from cyclohexane (18% yield, mp 99.7−101.2 °C). 1H NMR (CDCl3): δ 1.01 (3H, t, J = 7.47 Hz OCH2CH2CH3), 1.70−1.95 (6H, m, OCH2CH2CH3 and pyrrolidine CH2), 2.60−2.80 (4H, m, pyrrolidine NCH2), 3.06 (2H, t, J = 5.86 Hz, OCH2CH2N), 3.96 (2H, t, J = 6.62 Hz, OCH2CH2CH3), 4.39 (2H, t, J = 5.85 Hz, OCH2CH2N), 6.97 (2H, d, J = 9.55 Hz, H-3′and H-5′), 7.10 (1H, s, H-3), 7.39 (1H, t, J = 6.88 Hz, H-6), 7.63 (1H, t, J = 6.92, H-7), 7.95−8.05 (3H, m, H-8, H-2′and H-6′), 8.15 (1H, d, J = 8.26 Hz, H-5). Anal. (C24H28N2O2) C, H, N. 4-[2-(1-Azepanyl)ethoxy]-2-(4′-propoxyphenyl)quinoline (3m). Obtained from azepane as a white solid after purification by flash chromatography (silica gel/CH2Cl2/MeOH 99/1) (21% yield, mp 74.9−75.8 °C). 1H NMR (CDCl3): δ 1.11 (3H, t, J = 7.43 Hz, OCH2CH2CH3), 1.55−2.10 (10H, m, OCH2CH2CH3 and azepane CH2), 2.92−3.08 (4H, m, azepane NCH2), 3.26 (2H, t, J = 5.74 Hz, OCH2CH2N), 4.05 (2H, t, J = 6.60 Hz, OCH2CH2CH3), 4.47 (2H, t, J = 5,90 Hz, OCH2CH2N), 7.08 (2H, d, J = 8.82 Hz, H-3′and H-5′), 7.21 (1H, s, H-3), 7.49 (1H, t, J = 7.03 Hz, H-6), 7.73 (1H, t, J = 8.35 Hz, H7), 8.05−8.15 (3H, m, H-8, H-2′, and H-6′), 8.19 (1H, d, J = 8.39 Hz, H5). Anal. (C26H32N2O2) C, H, N. 4-[2-(3,4-Dihydroisoquinolin-2(1H)-yl)ethoxy]-2-(4′propoxyphenyl)quinoline (3n). Obtained from 1,2,3,4-tetrahydroisoquinoline as a white solid after purification by flash chromatography (silica gel/PE/EtOAc 90/10) (23% yield, mp 74.9−76.3 °C). 1H NMR (CDCl3): δ 1.08 (3H, t, J = 7.41 Hz, OCH2CH2CH3) 1.82−1.95 (2H, m, OCH2CH2CH3), 2.99 (4H, s, dihydroisoquinoline CH2), 3.20 (2H, t, J = 5.77 Hz, OCH2CH2N), 3.89 (2H, s, dihydroisoquinoline CH2), 4.01 (2H, t, J = 6.59 Hz, OCH2CH2CH3), 4.52 (2H, t, J = 5.80 Hz, OCH2CH2N), 7.03−7.07 (3H, m, dihydroisoquinoline CH, H-3′ and H-5′), 7.13−7.21 (3H, m, dihydroisoquinoline CH), 7.27 (1H, s, H-3), 7.49 (1H, t, J = 7.95 Hz, H-6), 7.71 (1H, t, J = 7.01 Hz, H-7), 8.07−8.10 (3H, m, H-8, H-2′ and H-6′), 8.21 (1H, d, J = 8.15 Hz, H-5). Anal. (C29H30N2O2) C, H, N. 4-[2-(6,7-Dimethoxy-3,4-dihydro-2(1H)-isoquinolinyl)ethoxy]-2(4′-propoxyphenyl)quinoline Hydrochloride (3o). Obtained from 6,7bis(methyloxy)-1,2,3,4-tetrahydroisoquinoline hydrochloride as a white solid after purification by flash chromatography (silica gel/EtOAc/ EtOH 98/2) (68% yield, mp 138.7−140.2 °C). 1H NMR (CDCl3): δ

OCHCH3CH2N), 1.76- 1.87 (2H, m, OCH2CH2CH3), 2.80−2.95 (8H, m, OCHCH3CH2N and NCH3), 3.75−3.85 (1H, m, OCHCH3CH2N), 4.07 (2H, t, J = 5.03 Hz, OCH2CH2CH3), 7.17 (2H, d, J = 9.09 Hz, H-3′ and H-5′), 7.63 (1H, t, J = 8.43 Hz, H-6), 7.73 (1H, s, H-3), 7.87 (1H, t, J = 8.51 Hz, H-7), 8.25 (1H, d, J = 8.39 Hz, H-8), 8.50−8.75 (3H, m, H-5, H-2′ and H-6′), 10.40 (1H, bs, NH). Anal. (C23H28N2O2·HCl) C, H, N. 3e: 34% yield; mp 80.7−81.5 °C. 1H NMR (DMSO-d6): δ 1.05 (3H, t, J = 7.21 Hz, OCH2CH2CH3), 1.54 (3H, d, J = 6.66 Hz, OCH2CHCH3N), 1.77−1.79 (2H, m, OCH2CH2CH3), 2.90 (6H, d, J = 4.66 Hz, NCH 3 ), 4.05−4.20 (3H, m,OCH 2 CH 2 CH 3 and OCH2CHCH3N), 4.95 (2H, d, J = 4.61 Hz, OCH2CHCH3N), 7.29 (2H, d, J = 8.84 Hz, H-3′ and H-5′), 7.79−7.87 (2H, m, H-3 and H-6), 8.05 (1H, t, J = 8.70 Hz, H-7), 8.32 (2H, d, J = 8.93 Hz, H-2′ and H-6′), 8.51 (1H, d, J = 8.51 Hz, H-8), 8.63 (1H, d, J = 8.77 Hz, H-5), 11.10 (1H, bs, NH). Anal. (C23H28N2O2·HCl) C, H, N. 4-[(1-Methylpiperidin-3-yl)oxy]-2-(4′-propoxyphenyl)quinoline (3f). Obtained from 1-methylpiperidin-3-yl methanesulfonate58 as a white solid after purification by flash chromatography (silica gel/ CH2Cl2/MeOH 99/1) (16% yield, mp 72.5−73.8 °C). 1H NMR (CDCl3): δ 1.11 (3H, t, J = 7.34 Hz, OCH2CH2CH3), 1.84−1.94 (6H, m, OCH2CH2CH3 and piperidine CH2), 2.08−2.36 (2H, m, piperidine CH2), 2.63 (3H, s, NCH3), 2.90−3.30 (2H, m, piperidine CH2), 4.05 (2H, t, J = 6.60 Hz, OCH2CH2CH3), 4.05 (2H, t, J = 6.60 Hz, OCH2CH2CH3), 4.30−4.35 (1H, m, piperidine CH), 7.07 (2H, d, J = 8.89 Hz, H-3′ and H-5′), 7.31 (1H, s, H-3), 7.52 (1H, t, J = 8.15 Hz, H6), 7.72 (1H, t, J = 8.08 Hz, H-7), 8.09−8.13 (3H, m, H-8, H-2′ and H6′), 8.22 (1H, d, J = 8.29 Hz, H-5). Anal. (C25H30N2O2) C, H, N. 4-(1-Azabicyclo[2.2.2]oct-3-yloxy)-2-(4′-propoxyphenyl)quinoline (3g). Obtained from 3-chloro-1-azabicyclo[2.2.2]octane hydrochloride as a colorless oil after purification by flash chromatography (silica gel/ CH2Cl2/EtOH 98/2) (11% yield). 1H NMR (CDCl3): δ 1.08 (3H, t, J = 7.36 Hz, OCH2CH2CH3), 1.63−1.92 (6H, m, OCH2CH2CH3 and 2 × CH2 azabicyclo), 2.41−2.53 (1H, m, CH azabicyclo)), 2.88−3.51 (6H, m, 3 × CH2 azabicyclo), 4.02 (2H, t, J = 6.63 Hz, OCH2CH2CH3), 4.74−4.78 (1H, m, CH azabicyclo), 6.95−7.08 (3H, m, H-3, H-3′ and H-5′), 7.47 (1H, t, J = 6.70 Hz, H-6), 7.70 (1H, t, J = 7.05 Hz, H-7), 8.03−8.10 (3H, m, H-8, H-2′ and H-6′), 8.21 (1H, d, J = 7.05 Hz, H-5). Anal. (C25H28N2O2) C, H, N. N,N-Diisopropyl-N-(2-{[2-(4′-propoxyphenyl)-4-quinolinyl]oxy}ethyl)amine (3h). Obtained from N-(2-chloroethyl)-N-(1methylethyl)propan-2-amine hydrochloride as a white solid after purification by flash chromatography (silica gel/PE/EtOAc 80/20) and precipitated as its HCl salt (25% yield, mp 75.5−78.8 °C). 1H NMR (CDCl3): δ 1.10−1.13 (15H, m, OCH2CH2CH3 and isopropylic CH3), 1.81−1.91 (2H, m, OCH2CH2CH3), 3.00−3.16 (4H, m, OCH2CH2N and isopropylic CH), 4.01 (2H, t, J = 6.61 Hz, OCH2CH2CH3), 4.20 (2H, t, J = 7.17 Hz, OCH2CH2N), 7.04 (2H, d, J = 8.80 Hz, H-3′ and H5′), 7.17 (1H, s, H-3), 7.46 (1H, t, J = 8.09 Hz, H-6), 7.69 (1H, d, J = 8.05 Hz, H-7), 8.07−8.11 (3H, m, H-8, H-2′ and H-6′), 8.20 (1H, d, J = 8.26 Hz, H-5). Anal. (C26H34N2O2) C, H, N. N,N-Dimethyl-3-{[2-(4′-propoxyphenyl)-4-quinolinyl]oxy}-1-propanamine Hydrochloride (3i). Obtained from (3-chloropropyl)dimethylamine hydrochloride as a white solid after purification by flash column chromatography (silica gel/CHCl3/MeOH 99/1) and precipitated as its HCl salt (57% yield, mp 145.1−146.2 °C). 1H NMR (DMSO-d6): δ 1.04 (3H, t, J = 7.44 Hz, OCH2CH2CH3), 1.80−2.00 (2H, m, OCH2CH2CH3), 2.40−2.50 (2H, m, OCH2CH2CH2NCH3), 2.38 (6H, d, J = 4.85 Hz,OCH2CH2CH2N(CH3)2), 3.35−3.45 (2H, m, OCH2CH2CH2N(CH3)2), 4.13 (2H, t, J = 6.57 Hz, OCH2CH2CH3), 4.81 (2H, t, J = 6.58 Hz, OCH2CH2CH2N(CH3)2), 7.28 (2H, d, J = 8.60 Hz, H-3′and H-5′), 7.79 (1H, s, H-3), 7.86 (1H, t, J = 8.16 Hz, H-6), 8.13 (1H, t, J = 7.00 Hz, H-7), 8.35 (2H, d, J = 8.61 Hz, H-2′ and H-6′), 8.45 (1H, d, J = 8.19 Hz, H-8), 8.68 (1H, d, J = 9.06 Hz, H-5). Anal. (C23H28N2O2·HCl) C, H, N. 2-(4′-Propoxyphenyl)-4-(2-pyridinylmethoxy)quinoline (3j). Obtained from 2-(chloromethyl)pyridine hydrochloride as a white solid after purification by flash chromatography (silica gel/PE/EtOAc 90/10) (62% yield, mp 138.0−138.8 °C). 1H NMR (CDCl3): δ 1.10 (3H, t, J = 7.30 Hz, OCH2CH2CH3), 1.84−1.94 (2H, m, OCH2CH2CH3), 4.03 (2H, t, J = 6.56 Hz, OCH2CH2N), 5.57 (2H, s, OCH2), 7.06 (2H, d, J = K

dx.doi.org/10.1021/jm400262a | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

1.60 (8H, m, 2 × NCH2 CH 2 CH 2 CH 3 ), 1.84−1.95 (2H, m, OCH2CH2CH3), 2.64 (4H, t, J = 6.91 Hz, 2 × NCH2CH2CH2CH3), 3.10 (2H, t, J = 5.44 Hz, OCH2CH2N), 4.05 (2H, t, J = 6.07 Hz, OCH2CH2CH3), 4.37 (2H, t, J = 6.11 Hz, OCH2CH2N), 7.07 (2H, d, J = 8.81 Hz, H-3′ and H-5′), 7.19 (1H, s, H-3), 7.48 (1H, t, J = 7.57 Hz, H6), 7.72 (1H, t, J = 7.79 Hz, H-7), 8.08−8.15 (3H, m, H-8, H-2′ and H6′), 8.21 (1H, d, J = 8.28 Hz, H-5). Anal. (C28H38N2O2) C, H, N. N-Benzyl-N-ethyl-2-{[2-(4′-propoxyphenyl)quinolin-4-yl]oxy}ethanamine (3v). Obtained from ethyl(phenylmethyl)amine as a white semisolid after purification by flash chromatography (silica gel/CH2Cl2/ Et2O 97/3) (19% yield). 1H NMR (CDCl3): δ 1.11 (3H, t, J = 7.46 Hz, OCH2CH2CH3), 1.20 (3H, t, J = 6.99 Hz, NCH2CH3), 1.85−1.95 (2H, m, OCH2CH2CH3), 2.79 (2H, q, J = 7.00 Hz, NCH2CH3), 3.13 (2H, t, J = 5.89 Hz, OCH2CH2N), 3.81 (2H, s, CH2Ph), 4.06 (2H, t, J = 6.59 Hz, OCH2CH2CH3), 4.34 (2H, t, J = 6.05 Hz, OCH2CH2N), 7.05−7.11 (3H, m, H-3, H-3′ and H-5′), 7.30−7.47 (6H, m, H-6 and phenyl CH), 7.71 (1H, t, J = 7.77 Hz, H-7), 8.07−8.16 (4H, m, H-5, H-8, H-2′ and H6′). Anal. (C29H32N2O2) C, H, N. 2-(2-{[2-(4′-Propoxyphenyl)-4-quinolinyl]oxy}ethyl)-1H-isoindole1,3(2H)-dione (3w). Obtained from phthalimide potassium salt as a white solid after purification by flash chromatography (silica gel/ cyclohexane/EtOAc 95/5) (54% yield, mp 195.5−196.3 °C). 1H NMR (CDCl3): δ 1.11 (3H, t, J = 7.48 Hz, OCH2CH2CH3), 1.80−2.00 (2H, m, OCH2CH2CH3), 4.07 (2H, t, J = 5.51 Hz, OCH2CH2N), 4.38 (2H, t, J = 4.97 Hz, OCH2CH2CH3), 4.67 (2H, t, J = 5.97 Hz, OCH2CH2N), 7.07 (2H, d, J = 8.85 Hz, H-3′and H-5′), 7.18 (1H, s, H-3), 7.57 (1H, t, J = 7.06 Hz, H-6), 7.70−7.87 (3H, m, H-8, H-5″ and H-6″), 7.90−8.05 (2H, m, H-4″ and H-7″), 8.10−8.25 (4H, m, H-5, H-7, H-2′ and H-6′). Anal. (C28H24N2O4) C, H, N. 4-[2-(1,4-Dioxa-8-azaspiro[4.5]dec-8-yl)ethoxy]-2-(4′propoxyphenyl)quinoline (3x). Obtained from 1,4-dioxa-8azaspiro[4.5]decane as a white solid after purification by flash chromatography (silica gel/PE/EtOAc 70/30) (45% yield, mp 125.0− 126.4 °C). 1H NMR (CDCl3): δ 1.10 (3H, t, J = 7.34 Hz, OCH2CH2CH3), 1.81−1.94 (6H, m, OCH2CH2CH3, and piperidine CH2), 2.76−2.85 (4H, m, piperidine CH2N), 3.09 (2H, t, J = 5.69 Hz, OCH2CH2N), 4.00−4.08 (6H, m, OCH2CH2CH3 and dioxolane CH2), 4.45 (2H, t, J = 5.79 Hz, OCH2CH2N), 7.07 (2H, d, J = 8.83 Hz, H-3′ and H-5′), 7.18 (1H, s, H-3), 7.48 (1H, t, J = 6.98 Hz, H-6), 7.72 (1H, t, J = 7.71 Hz, H-7), 8.08−8.22 (4H, m, H-5, H-8, H-2′ and H-6′). Anal. (C27H32N2O4) C, H, N. 2-{[2-(4′-Propoxyphenyl)-4-quinolinyl]oxy}ethylamine (3y). To a solution of 2-(2-{[2-(4′-propoxyphenyl)-4-quinolinyl]oxy}ethyl)-1Hisoindole-1,3(2H)-dione (3w) (0.10 g, 0.22 mmol) in EtOH (10 mL) was added 98% hydrazine hydrate solution (0.33 g, 0.66 mmol), and the reaction was refluxed for 8 h with stirring. The reaction mixture was cooled and filtered. The filtrate was evaporated under reduced pressure to obtain a residue that was purified by flash chromatography (silica gel/ CHCl3/MeOH 99/1) to give compound 3y (0.01 g, 14% yield) as a yellowish oil. 1H NMR (DMSO-d6): δ 1.03 (3H, t, J = 7.41 Hz, OCH2CH2CH3), 1.68−1.80 (2H, m, OCH2CH2CH3), 3.50−3.65 (2H, m, OCH2CH2NH2), 3.70−3.85 (2H, m, OCH2CH2NH2), 4.04 (2H, t, J = 6.48 Hz, OCH2CH2CH3), 7.01 (1H, s, H-3), 7.10 (2H, d, J = 8.77 Hz, H-3′and H-5′), 7.40−7.48 (3H, m, H-6 and NH2), 7.69 (1H, t, J = 7.69 Hz, H-7), 7.89 (1H, d, J = 8.45 Hz, H-8), 8.15 (2H, d, J = 8.84 Hz, H2′and H-6′), 8.28 (1H, d, J = 8.46 Hz, H-5). Anal. (C20H22N2O2) C, H, N. 1-(2-{[2-(4′-Propoxyphenyl)quinolin-4-yl]oxy}ethyl)piperidin-4one (3z). To a solution of 4-[2-(1,4-dioxa-8-azaspiro[4.5]dec-8yl)ethoxy]-2-(4′-propoxyphenyl)quinolone 3x (0.07 g, 0.16 mmol) in MeOH (2 mL) was added a HCl solution (9% in H2O, 2 mL), and the reaction was refluxed for 6 h with stirring. The reaction mixture was cooled and filtered. The solid obtained was then dissolved in CHCl3 and dried over Na2SO4. The solvent was removed in vacuo to yield compound 3z as a white solid (0.025g, 40% yield, mp 204.4−206.0 °C). 1 H NMR (MeOD-d4): δ 1.10 (3H, t, J = 7.33 Hz, OCH2CH2CH3), 1.832.02 (2H, m, OCH2CH2CH3), 2.08−2.14 (4H, m, piperidinone CH2N), 3.13−3.32 (4H, m, piperidinone CH2CO), 3.90−3.98 (2H, m, OCH2CH2N), 4.27 (2H, t, J = 6.42 Hz, OCH2CH2 CH3), 5.00−5.07 (2H, m, OCH2CH2N), 7.26 (2H, d, J = 8.97 Hz, H-3′ and H-5′), 7.74

1.11 (3H, t, J = 7.31 Hz, OCH2CH2CH3), 1.80−2.00 (2H, m, OCH2CH2CH3), 2.85−3.10 (4H, m, dihydroisoquinoline CH2), 3.23 (2H, t, J = 5.47 Hz, OCH2CH2N), 3.84 (2H, s, dihydroisoquinoline CH2), 3.88 (3H, s, OCH3), 3.90 (3H, s, OCH3), 4.05 (2H, t, J = 6.62 Hz, OCH2CH2CH3), 4.56 (2H, t, J = 5.74 Hz, OCH2CH2N), 6.58 (1H, s, dihydroisoquinoline CH), 6.66 (1H, s, dihydroisoquinoline CH), 7.06 (2H, d, J = 8.85 Hz, H-3′and H-5′), 7.24 (1H, s, H-3), 7.51 (1H, t, J = 8.09 Hz, H-6), 7.74 (1H, t, J = 6.76 Hz, H-7), 8.03−8.18 (3H, m, H-8, H2′ and H-6′), 8.24 (1H, d, J = 8.26 Hz, H-5). Anal. (C31H34N2O4·HCl) C, H, N. 2-[4′-(Propyloxy)phenyl]-4-[(2-thiomorpholin-4-ylethyl)oxy]quinoline (3p). Obtained from thiomorpholine as yellow a solid after purification by flash chromatography (silica gel/CH2Cl2/MeOH 98/2) (30% yield, mp 0.151.2−152.0 °C). 1H NMR (CDCl3): δ 1.11 (3H, t, J = 7.34 Hz, OCH2CH2CH3), 1.83−1.95 (2H, m, OCH2CH2CH3), 2.70− 2.80 (4H, m, thiomorpholine NCH2), 2.97−3.05 (4H, m, thiomorpholine SCH2), 3.10 (2H, t, J = 5.74 Hz, OCH2CH2N), 4.05 (2H, t, J = 6.63 Hz, OCH2CH2CH3), 4.43 (2H, t, J = 5.56 Hz, OCH2CH2N), 7.07 (2H, d, J = 8.84 Hz, H-3′ and H-5′), 7.18 (1H, s, H-3), 7.50 (1H, t, J = 8.29 Hz, H-6), 7.74 (1H, t, J = 8.18 Hz, H-7), 8.09−8.25 (4H, m, H-5, H-8, H-2′ and H-6′). Anal. (C24H28N2O2S) C, H, N. 4-[2-(1-Piperazinyl)ethoxy]-2-(4′-propoxyphenyl)quinoline (3q). Obtained from piperazine as a brownish solid after purification by flash chromatography (silica gel/CH2Cl2/MeOH 98/2) (30% yield, mp 121.8−123.1 °C). 1H NMR (CDCl3): δ.1.15 (3H, t, J = 7.34 Hz, OCH2CH2CH3), 1.80−2.00 (2H, m, OCH2CH2CH3), 2.03 (1H, s, NH), 2.75−2.85 (2H, m, OCH2CH2N), 3.07 (8H, t, J = 4.08 Hz, piperazine CH2), 4.05 (2H, t, J = 6.62 Hz, OCH2CH2CH3), 4.46 (2H, t, J = 5.63 Hz, OCH2CH2N), 7.07 (2H, d, J = 8.85 Hz, H-3′ and H-5′), 7.31 (1H, s, H-3), 7.50 (1H, t, J = 7.00 Hz, H-6), 7.74 (1H, t, J = 5.41 Hz, H7), 8.10 (3H, m, H-8, H-2′ and H-6′), 8.18 (1H, d, J = 8.70 Hz, H-5). Anal. (C24H29N3O2) C, H, N. 4-[2-(4-Methyl-1-piperazinyl)ethoxy]-2-(4′-propoxyphenyl)quinoline (3r). Obtained from 1-methylpiperazine as white a solid after crystallization from cyclohexane (28% yield, mp 108.7−110.1 °C). 1H NMR (CDCl3): δ 1.11 (3H, t, J = 7.40 Hz, OCH2CH2CH3), 1.78−2.00 (2H, m, OCH2CH2CH3), 2.35 (3H, s, NCH3), 2.45−2.65 (4H, m, piperazine CH2), 2.70−2.85 (4H, m, piperazine CH2), 3.07 (2H, t, J = 5.74 Hz, OCH2CH2N), 4.05 (2H, t, J = 6.59 Hz, OCH2CH2CH3), 4.47 (2H, t, J = 5.67 Hz, OCH2CH2N), 7.07 (2H, d, J = 8.86 Hz, H-3′and H5′), 7.19 (1H, s, H-3), 7.49 (1H, t, J = 6.93 Hz, H-6), 7.72 (1H, t, J = 6.95 Hz, H-7), 8.05−8.15 (3H, m, H-8, H-2′and H-6′), 8.20 (1H, d, J = 8.32 Hz, H-5). Anal. (C25H31N3O2) C, H, N. 4-[2-(4-Benzylpiperazin-1-yl)ethoxy]-2-(4′-propoxyphenyl)quinoline (3s). Obtained from 1-(phenylmethyl)piperazine as a yellow solid after purification by flash chromatography (silica gel/PE/EtOAc 80/20) (43% yield, mp 102.5−103.3 °C). 1H NMR (CDCl3): δ 1.08 (3H, t, J = 7.26 Hz, OCH 2 CH 2 CH 3 ), 1.81−1.92 (2H, m, OCH2CH2CH3), 2.54−2.57 (4H, m, piperazine CH2), 2.72−2.74 (4H, m, piperazine CH2), 3.03 (2H, t, J = 5.85 Hz, OCH2CH2N), 3.55 (2H, s, CH2Ph), 4.02 (2H, t, J = 6.55 Hz, OCH2CH2CH3), 4.43 (2H, t, J = 5.92 Hz, OCH2CH2N), 7.04 (2H, d, J = 8.83 Hz, H-3′ and H-5′), 7.15 (1H, s, H-3), 7.27−7.33 (5H, m, phenyl CH), 7.45 (1H, t, J = 8.80 Hz, H-6), 7.68 (1H, t, J = 6.89 Hz, H-7), 8.05−8.17 (4H, m, H-5, H-8, H-2′ and H-6′). Anal. (C31H35N3O2) C, H, N. N,N-Dibenzyl-2-{[2-(4′-propoxyphenyl)quinolin-4-yl]oxy}ethanamine (3t). Obtained from bis(phenylmethyl)amine as a white solid after purification by flash chromatography (silica gel/PE/EtOAc 95/5) (30% yield, mp 76.6−78.4 °C). 1H NMR (CDCl3): δ 1.11 (3H, t, J = 7.43 Hz, OCH2CH2CH3), 1.85−1.95 (2H, m, OCH2CH2CH3), 4.05 (2H, t, J = 6.59 Hz, OCH2CH2CH3), 4.40−4.56 (6H, m, OCH2CH2N and CH2Ph), 4.80 (2H, t, J = 3.95 Hz, OCH2CH2N), 7.02−7.46 (14H, m, H-3, H-6,H-3′, H-5′ and phenyl CH), 7.73 (1H, t, J = 7.63 Hz, H-7), 8.09−8.23 (4H, m, H-5, H-8, H-2′ and H-6′). Anal. (C34H34N2O2) C, H, N. Dibutyl(2-{[2-(4′-propoxyphenyl)quinolin-4-yl]oxy}ethyl)amine (3u). Obtained from dibutylamine as a white solid after purification by flash chromatography (silica gel/CH2Cl2/Acetone 97/3) (35% yield, mp 64.0−65.7 °C). 1H NMR (CDCl3): δ 0.97 (6H, t, J = 7.17 Hz, 2 × NCH2CH2CH2CH3), 1.11 (3H, t, J = 7.31 Hz,OCH2CH2CH3,), 1.26− L

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(1H, s, H-3), 7.87 (1H, t, J = 7.98 Hz, H-6), 8.07−8.18 (3H, m, H-7, H2′ and H-6′), 8.24 (1H, d, J = 7.91 Hz, H-8), 8.58 (1H, d, J = 8.67 Hz, H5). Anal. (C25H28N2O3) C, H, N. Bacterial Strains. The strains of S. aureus employed included ATCC 25923 (wild-type), SA-K2378, which overexpresses norA from a multicopy plasmid and was produced by cloning norA and its promoter into plasmid pCU1 and then introducing the construct into SA-K1902 (norA−).59 In addition, SA-1199B (overexpressing norA and also possessing an A116E GrlA substitution) and its isogenic parent SA-1199 (norA wt) were also used.19,51 SA-K2885 and K2886 are norA-deleted strains containing the empty expression vector pALC2073 and pALC2073-mepA, respectively.60 Genes cloned into pALC2073 are under control of a xyl/tetO promoter, which is inducible by 0.05 μg/mL tetracycline.61 This concentration of tetracycline was included in all experiments utilizing these strains. EtBr Efflux. The loss of EtBr from S. aureus SA-1199B (norA +/A116EGrlA) was determined fluorometrically as previously described.50 With the exception of reserpine, which was replicated numerous times, experiments were usually performed in duplicate, and the results were expressed as mean total efflux over a 5 min time course. The effect of various concentrations of reserpine and tested compounds on the EtBr efflux of SA-1199B (norA+/A116E GrlA) was compared to that in their absence, allowing the calculation of the percentage reduction in efflux. Dose−response experiments were performed by testing increasing concentrations of inhibitors, and the 50% inhibitory concentration (IC50) was determined by inspection of the resultant plots (see Supporting Information). Microbiologic Procedures. MICs were determined by microdilution techniques according to CLSI guidelines.62 The effect of combining reserpine or various test compounds, with scalar dilutions of freshly prepared solutions of each selected compound, on the MICs of CPX was also determined. Checkerboard combination studies using CPX and tested compounds were performed as described previously.63



REFERENCES

(1) Spellberg, B.; Guidos, R.; Gilbert, D.; Bradley, J.; Boucher, H. W.; Scheld, W. M.; Bartlett, J. G.; Edwards, J., Jr. The epidemic of antibioticresistant infections: a call to action for the medical community from the Infectious Diseases Society of America. Clin. Infect. Dis. 2008, 46, 155− 164. (2) Boucher, H. W.; Talbot, G. H.; Bradley, J. S.; Edwards, J. E.; Gilbert, D.; Rice, L. B.; Scheld, M.; Spellberg, B.; Bartlett, J. Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America. Clin. Infect. Dis. 2009, 48, 1−12. (3) Standing up to antimicrobial resistance. Nature. Rev. Microbiol. 2010, 8, 836. (4) Taubes, G. The bacteria fight back. Science 2008, 356−361. (5) Chambers, H. F. Community-associated MRSAresistance and virulence converge. N. Engl. J. Med. 2005, 352, 1485−1487. (6) Moran, G. J.; Krishnadasan, A.; Gorwitz, R. J.; Fosheim, G. E.; McDougal, L. K.; Carey, R. B.; Talan, D. A. Methicillin-resistant S. aureus infections among patients in the emergency department. N. Engl. J. Med. 2006, 355, 666−674. (7) Lowy, F. D. Staphylococcus aureus infections. N. Engl. J. Med. 1998, 339, 520−532. (8) Carbon, C. MRSA and MRSE: is there an answer? Clin. Microbiol. Infect. 2000, 6 (Suppl 2), 17−22. (9) Klein, E.; Smith, D. L.; Laxminarayan, R. Hospitalizations and deaths caused by methicillin-resistant Staphylococcus aureus, United States, 1999−2005. Emerg. Infect. Dis. 2007, 13, 1840−1846. (10) Bancroft, E. A. Antimicrobial resistance: it’s not just for hospitals. JAMA, J. Am. Med. Assoc. 2007, 298, 1803−1804. (11) The Bacterial Challenge: Time to React; ECDC/EMEA Joint Working Group, 2009; http://www.ecdc.europa.eu/en/publications/ Publications/0909_TER_The_Bacterial_Challenge_Time_to_React. pdf (accessed June 7, 2013). (12) David, M. Z.; Daum, R. S. Community-associated methicillinresistant Staphylococcus aureus: epidemiology and clinical consequences of an emerging epidemic. Clin. Microbiol. Rev. 2010, 23, 616−687. (13) Centers for Disease Control and Prevention. Public Health Dispatch: Vancomycin-Resistant Staphylococcus aureusPennsylvania. Morb. Mortal. Wkly. Rep. 2002, 51, 902. (14) Lynch, A. S. Efflux systems in bacterial pathogens: an opportunity for therapeutic intervention? An industry view. Biochem. Pharmacol. 2006, 71, 949−956. (15) Marquez, B. Bacterial efflux systems and efflux pumps inhibitors. Biochimie 2005, 87, 1137−1147. (16) Patel, D.; Kosmidis, C.; Seo, S. M.; Kaatz, G. W. Ethidium bromide MIC screening for enhanced efflux pump gene expression or efflux activity in Staphylococcus aureus. Antimicrob. Agents Chemother. 2010, 54, 5070−5073. (17) Pao, S. S.; Paulsen, I. T.; Saier, M. H., Jr. Major facilitator superfamily. Microbiol. Mol. Biol. Rev. 1998, 62, 1−34. (18) Yoshida, H.; Bogaki, M.; Nakamura, S.; Ubukata, K.; Konno, M. Nucleotide sequence and characterization of the Staphylococcus aureus norA gene, which confers resistance to quinolones. J. Bacteriol. 1990, 172, 6942−6949. (19) Kaatz, G. W.; Seo, S. M. Mechanisms of fluoroquinolone resistance in genetically related strains of Staphylococcus aureus. Antimicrob. Agents Chemother. 1997, 41, 2733−2737. (20) Neyfakh, A. A. Mystery of multidrug transporters: the answer can be simple. Mol. Microbiol. 2002, 44, 1123−1130. (21) Mahamoud, A.; Chevalier, J.; Alibert-Franco, S.; Kern, W. V.; Pages, J. M. Antibiotic efflux pumps in Gram-negative bacteria: the inhibitor response strategy. J. Antimicrob. Chemother. 2007, 59, 1223− 1229. (22) Stermitz, F. R.; Lorenz, P.; Tawara, J. N.; Zenewicz, L. A.; Lewis, K. Synergy in a medicinal plant: antimicrobial action of berberine potentiated by 5′-methoxyhydnocarpin, a multidrug pump inhibitor. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 1433−1437. (23) Morel, C.; Stermitz, F. R.; Tegos, G.; Lewis, K. Isoflavones as potentiators of antibacterial activity. J. Agric. Food Chem. 2003, 51, 5677−5679.

ASSOCIATED CONTENT

* Supporting Information S

Structure of the “inactive set” of EPIs used to build the pharmacophore model, fitness values of the “active set” of compounds perfectly matching the APRR hypothesis, EtBr efflux inhibition dose−response experiment plots used to determine the 50% inhibitory concentration (IC50), and elemental analysis data for target compounds. This material is available free of charge via the Internet at http://pubs.acs.org.



Article

AUTHOR INFORMATION

Corresponding Author

*Phone: +39 075 5855130. Fax: +39 075 5855115. E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We thank Dr. C. Guercini for helpful discussions and R. Bianconi and C. Moriconi for their technical support.



ABBREVIATIONS USED MDR, multidrug resistance; MRSA, methicillin-resistant Staphylococcus aureus; MFS, major facilitator superfamily; EPI, efflux pump inhibitor; EtBr, ethidium bromide; CPX, ciprofloxacin; LBDD, ligand-based drug design M

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(24) Belofsky, G.; Percivill, D.; Lewis, K.; Tegos, G. P.; Ekart, J. Phenolic metabolites of Dalea versicolor that enhance antibiotic activity against model pathogenic bacteria. J. Nat. Prod. 2004, 67, 481−484. (25) Wright, G. D. Resisting resistance: new chemical strategies for battling superbugs. Chem. Biol. 2000, 7, R127−R132. (26) Lomovskaya, O.; Lee, A.; Hoshino, K.; Ishida, H.; Mistry, A.; Warren, M. S.; Boyer, E.; Chamberland, S.; Lee, V. J. Use of a genetic approach to evaluate the consequences of inhibition of efflux pumps in Pseudomonas Aeruginosa. Antimicrob. Agents Chemother. 1999, 43, 1340− 1346. (27) Kvist, M.; Hancock, V.; Klemm, P. Inactivation of efflux pumps abolishes bacterial biofilm formation. Appl. Environ. Microbiol. 2008, 74, 7376−7382. (28) Schmitz, F. J.; Fluit, A. C.; Luckefahr, M.; Engler, B.; Hofmann, B.; Verhoef, J.; Heinz, H. P.; Hadding, U.; Jones, M. E. The effect of reserpine, an inhibitor of multidrug efflux pumps, on the in vitro activities of ciprofloxacin, sparfloxacin and moxifloxacin against clinical isolates of Staphylococcus aureus. J. Antimicrob. Chemother. 1998, 42, 807−810. (29) Aeschlimann, J. R.; Dresser, L. D.; Kaatz, G. W.; Rybak, M. J. Effects of NorA inhibitors on in vitro antibacterial activities and postantibiotic effects of levofloxacin, ciprofloxacin, and norfloxacin in genetically related strains of Staphylococcus aureus. Antimicrob. Agents Chemother. 1999, 43, 335−340. (30) Vidaillac, C.; Guillon, J.; Arpin, C.; Forfar-Bares, I.; Ba, B. B.; Grellet, J.; Moreau, S.; Caignard, D. H.; Jarry, C.; Quentin, C. Synthesis of omeprazole analogues and evaluation of these as potential inhibitors of the multidrug efflux pump NorA of Staphylococcus aureus. Antimicrob. Agents Chemother. 2007, 51, 831−838. (31) Munoz-Bellido, J. L.; Munoz-Criado, S.; Garcia-Rodriguez, J. A. Antimicrobial activity of psychotropic drugs: selective serotonin reuptake inhibitors. Int. J. Antimicrob. Agents 2000, 14, 177−180. (32) Kaatz, G. W.; Moudgal, V. V.; Seo, S. M.; Hansen, J. B.; Kristiansen, J. E. Phenylpiperidine selective serotonin reuptake inhibitors interfere with multidrug efflux pump activity in Staphylococcus aureus. Int. J. Antimicrob. Agents 2003, 22, 254−261. (33) Kaatz, G. W.; Moudgal, V. V.; Seo, S. M.; Kristiansen, J. E. Phenothiazines and thioxanthenes inhibit multidrug efflux pump activity in Staphylococcus aureus. Antimicrob. Agents Chemother. 2003, 47, 719− 726. (34) Gibbons, S.; Oluwatuyi, M.; Kaatz, G. W. A novel inhibitor of multidrug efflux pumps in Staphylococcus aureus. J. Antimicrob. Chemother. 2003, 51, 13−17. (35) Gibbons, S. Anti-staphylococcal plant natural products. Nat. Prod. Rep. 2004, 21, 263−277. (36) Stavri, M.; Piddock, L. J.; Gibbons, S. Bacterial efflux pump inhibitors from natural sources. J. Antimicrob. Chemother. 2007, 59, 1247−1260. (37) Guz, N. R.; Stermitz, F. R.; Johnson, J. B.; Beeson, T. D.; Willen, S.; Hsiang, J.; Lewis, K. Flavonolignan and flavone inhibitors of a Staphylococcus aureus multidrug resistance pump: structure−activity relationships. J. Med. Chem. 2001, 44, 261−268. (38) Samosorn, S.; Bremner, J. B.; Ball, A.; Lewis, K. Synthesis of functionalized 2-aryl-5-nitro-1H-indoles and their activity as bacterial NorA efflux pump inhibitors. Bioorg. Med. Chem. 2006, 14, 857−865. (39) Sabatini, S.; Kaatz, G. W.; Rossolini, G. M.; Brandini, D.; Fravolini, A. From phenothiazine to 3-phenyl-1,4-benzothiazine derivatives as inhibitors of the Staphylococcus aureus NorA multidrug efflux pump. J. Med. Chem. 2008, 51, 4321−4330. (40) Pieroni, M.; Dimovska, M.; Brincat, J. P.; Sabatini, S.; Carosati, E.; Massari, S.; Kaatz, G. W.; Fravolini, A. From 6-Aminoquinolone Antibacterials to 6-Amino-7-thiopyranopyridinylquinolone Ethyl Esters as Inhibitors of Staphylococcus aureus Multidrug Efflux Pumps. J. Med. Chem. 2010, 53, 4466−4480. (41) Sabatini, S.; Gosetto, F.; Manfroni, G.; Tabarrini, O.; Kaatz, G. W.; Patel, D.; Cecchetti, V. Evolution from a Natural Flavones Nucleus to Obtain 2-(4-Propoxyphenyl)quinoline Derivatives As Potent Inhibitors of the S. aureus NorA Efflux Pump. J. Med. Chem. 2011, 54, 5722−5736.

(42) Sabatini, S.; Gosetto, F.; Serritella, S.; Manfroni, G.; Tabarrini, O.; Iraci, N.; Brincat, J. P.; Carosati, E.; Villarini, M.; Kaatz, G. W.; Cecchetti, V. Pyrazolo[4,3-c][1,2]benzothiazines 5,5-dioxide: a promising new class of Staphylococcus aureus NorA efflux pump inhibitors. J. Med. Chem. 2012, 55, 3568−3572. (43) Brincat, J. P.; Carosati, E.; Sabatini, S.; Manfroni, G.; Fravolini, A.; Raygada, J. L.; Patel, D.; Kaatz, G. W.; Cruciani, G. Discovery of Novel Inhibitors of the NorA Multidrug Transporter of Staphylococcus aureus. J. Med. Chem. 2011, 54, 354−365. (44) Brincat, J. P.; Broccatelli, F.; Sabatini, S.; Frosini, M.; Neri, A.; Kaatz, G. W.; Cruciani, G.; Carosati, E. Ligand Promiscuity between the Efflux Pumps Human P-Glycoprotein and S. aureus NorA. ACS Med. Chem. Lett. 2012, 3, 248−251. (45) Pieroni, M.; Sabatini, S.; Massari, S.; Kaatz, G. W.; Cecchetti, V.; Tabarrini, O. Searching for innovative quinolone-like scaffolds: synthesis and biological evaluation of 2,1-benzothiazine 2,2-dioxide derivatives. Med. Chem. Commun. 2012, 3, 1092−1097. (46) Handzlik, J.; Matys, A.; Kieć-Kononowicz, K. Recent Advances in Multi-Drug Resistance (MDR) Efflux Pump Inhibitors of Gram-Positive Bacteria S. aureus. Antibiotics 2013, 2, 28−45. (47) Phase version 3.3; Schrödinger, LLC: New York, 2011. (48) Kaatz, G. W.; Moudgal, V. V.; Seo, S. M.; Hansen, J. B.; Kristiansen, J. E. Phenylpiperidine selective serotonin reuptake inhibitors interfere with multidrug efflux pump activity in Staphylococcus aureus. Int. J. Antimicrob. Agents 2003, 22, 254−261. (49) Michalet, S.; Cartier, G.; David, B.; Mariotte, A. M.; DijouxFranca, M. G.; Kaatz, G. W.; Stavri, M.; Gibbons, S. NCaffeoylphenalkylamide derivatives as bacterial efflux pump inhibitors. Bioorg. Med. Chem. Lett. 2007, 17, 1755−1758. (50) Kaatz, G. W.; Seo, S. M.; O’Brien, L.; Wahiduzzaman, M.; Foster, T. J. Evidence for the existence of a multidrug efflux transporter distinct from NorA in Staphylococcus aureus. Antimicrob. Agents Chemother. 2000, 44, 1404−1406. (51) Price, C. T.; Kaatz, G. W.; Gustafson, J. E. The multidrug efflux pump NorA is not required for salicylate-induced reduction in drug accumulation by Staphylococcus aureus. Int. J. Antimicrob. Agents 2002, 20, 206−213. (52) QikProp, version 3.4; Schrödinger, LLC: New York, 2011. (53) Neyfakh, A. A. The Multidrug Efflux Transporter of Bacillus subtilis Is a Structural and Functional Homolog of the Staphylococcus NorA Protein. Antimicrob. Agents Chemother. 1992, 36, 484−485. (54) MacroModel, version 9.9; Schrödinger, LLC: New York, 2011. (55) Carpio, H.; Galeazzi, E.; Greenhouse, R.; Guzmán, A.; Velarde, E.; Antonio, Y.; Franco, F.; Leon, A.; Pérez, V.; Salas, R.; Valdés, D.; Ackrell, J.; Cho, D.; Gallegra, P.; Halpern, O.; Koehler, R.; Maddox, M. L.; Muchowski, M. J.; Prince, A.; Tegg, D.; Thurber, T. C.; Van Horn, A. R.; Wren, D. Synthesis of l,2-dihydro-3H-pyrrolo[1,2-a]pyrrole-1-carboxylic acids and homologous pyridine and azepine analogues thereof. Can. J. Chem. 1982, 60, 2295−2312. (56) Maestro, version 9.2; Schrödinger, LLC: New York, 2011. (57) CombiGlide, version 2.7; Schrödinger, LLC: New York, 2011. (58) Spinks, D.; Armer, R. E.; Miller, D. J.; Rankovic, Z.; Spinks, G.; Mestres, J.; Jaap, D. R. Preparation of 1-arylsulfonyl-3-substituted indoles and indolines for the treatment of central nervous system disorders. PCT Int. Appl. WO 2003068220 A1 20030821, 2003. (59) Augustin, J.; Rosenstein, R.; Wieland, B.; Schneider, U.; Schnell, N.; Engelke, G.; Entian, K. D.; Gotz, F. Genetic analysis of epidermin biosynthetic genes and epidermin-negative mutants of Staphylococcus epidermidis. Eur. J. Biochem. 1992, 204, 1149−1154. (60) Kaatz, G. W.; McAleese, F.; Seo, S. M. Multidrug resistance in Staphylococcus aureus due to overexpression of a novel multidrug and toxin extrusion (MATE) transport protein. Antimicrob. Agents Chemother. 2005, 49, 1857−1864. (61) Bateman, B. T.; Donegan, N. P.; Jarry, T. M.; Palma, M.; Cheung, A. L. Evaluation of a tetracycline-inducible promoter in Staphylococcus aureus in vitro and in vivo and its application in demonstrating the role of sigB in microcolony formation. Infect. Immun. 2001, 69, 7851−7857. N

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Journal of Medicinal Chemistry

Article

(62) Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically,7th ed.; Approved Standard M7-A7; Clinical and Laboratory Standards Institute: Wayne PA, 2006. (63) Eliopoulos, G. M.; Moellering, R. C. J. Antimicrobial combinations. In Antibiotics in Laboratory Medicine; Lorian, V., Ed.; Williams and Wilkins: Baltimore, MD. 1991; pp 432−492.

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