2-Phenylquinoline S. aureus NorA Efflux

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2-Phenylquinoline S. aureus NorA Efflux Pump Inhibitors: Evaluation of the Importance of Methoxy Groups Introduction Tommaso Felicetti, Rolando Cannalire, Donatella Pietrella, Gniewomir Latacz, Annamaria Lubelska, Giuseppe Manfroni, Maria Letizia Barreca, Serena Massari, Oriana Tabarrini, Katarzyna J. Kiec-Kononowicz, Bryan D. Schindler, Glenn W Kaatz, Violetta Cecchetti, and Stefano Sabatini J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00791 • Publication Date (Web): 01 Aug 2018 Downloaded from http://pubs.acs.org on August 1, 2018

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Journal of Medicinal Chemistry

2-Phenylquinoline S. aureus NorA Efflux Pump Inhibitors: Evaluation of the Importance of Methoxy Groups Introduction Tommaso Felicetti,a Rolando Cannalire,a Donatella Pietrella,a Gniewomir Latacz,b Annamaria Lubelska,b Giuseppe Manfroni,a Maria Letizia Barreca,a Serena Massari,a Oriana Tabarrini,a Katarzyna Kieć-Kononowicz,b Bryan D. Schindler,c Glenn W. Kaatz,c Violetta Cecchetti,a and Stefano Sabatinia,* a

Department of Pharmaceutical Sciences, University of Perugia, via del Liceo 1, 06123 Perugia,

Italy. b

Department of Technology and Biotechnology of Drugs, Faculty of Pharmacy, Jagiellonian

University-Medical College, ul. Medyczna 9, 31-688 Cracow, Poland. c

John D. Dingell Department of Veterans Affairs Medical Centre and the Department of Internal

Medicine, Division of Infectious Diseases, School of Medicine, Wayne State University, Detroit, MI 48201, United States. KEYWORDS Efflux pump inhibitors, NorA efflux pump, Staphylococcus aureus, Antimicrobial Resistance Breakers, methoxy-2-phenylquinoline derivatives.

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Abstract

Antimicrobial resistance (AMR) represents a hot topic in drug discovery. Besides the identification of new antibiotics, the use of non-antibiotic molecules to block resistance mechanisms is a powerful alternative. Bacterial efflux pumps exert an early step in AMR development by allowing bacteria to grow at sub-inhibitorial drug concentrations. Thus, efflux pump inhibitors (EPIs) offer a great opportunity to fight AMR. Given our experience in developing Staphylococcus aureus NorA EPIs, in this work, starting from the 2-phenylquinoline hit 1, we planned the introduction of methoxy groups on the basis of their presence in known NorA EPIs. Among the 35 different synthesized derivatives, compounds 3b and 7d exhibited the best NorA inhibition activity by restoring at very low concentrations ciprofloxacin MICs against resistant S. aureus strains. Interestingly, both compounds displayed EPI activities at non-toxic concentrations for human cells as well as highlighted promising results by preliminary pharmacokinetic studies.

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Introduction Antimicrobial resistance (AMR) is a complex global health issue increasingly accelerated by selective pressure resulting from the use and misuse of antimicrobial agents in humans and animals.1 The magnitude of the problem worldwide and the impact of AMR on human health, as well as on costs for the health-care sector and the wider societal impact are still largely underestimated. However, the yearly cost to the US health system alone has been estimated to range between $21 and $34 billions, accompanied by more than 8 million additional days in hospital.1 Microorganisms can acquire resistance by four main mechanisms: i) alteration of the target site, ii) enzymatic drug inactivation/modification, iii) decreased uptake or enhanced efflux of the drug, and iv) biofilm formation.2,3 The use of non-antibiotic adjuvant molecules to target resistance mechanisms for blocking resistance and recovering drug sensibility in resistant strains is a valid approach to overcome the issue of a poor antibiotic availability.4,5 Besides the famous example of the β-lactamase inhibitors commonly used to overcome resistance, various nonantibiotic adjuvant molecules targeting different resistance mechanisms have been developed over the years (see references 4,6 for a more detailed analysis). Among known resistance mechanisms, the extrusion of antimicrobial agents mediated by efflux pumps plays a central role. Indeed, for some drugs, microorganisms can only acquire resistance in the presence of efflux pump activity as this activity reduces intracellular drug concentrations to sub-inhibitory levels and allows the microorganism to develop specific resistance mechanisms.7–11 Of course, overexpression of efflux pumps increases non-specific drug extrusion thereby generating superbugs impossible to treat by common therapies. Therefore, the identification of a nonbactericidal adjuvant compound targeting efflux pumps holds promise over the development of

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new antimicrobial agents that are destined to create new mechanisms of resistance. Indeed, an efflux pump inhibitor (EPI) prevents the evolutionary pressure on bacteria that evolve resistance only for compounds exerting bactericidal or bacteriostatic effects; thus the mild impact on the development of resistance appears a strength for the EPI strategy.12 To date, little has been done in terms of EPI development and no inhibitors have ever reached clinical use; the few known examples of EPIs failed in in-vivo studies owing to poor pharmacokinetic (PK) properties and high toxicity.6,8 Nonetheless, breaking resistance by an adjuvant molecule appears advantageous over the strategy of antibiotic discovery; indeed, a great number of both undiscovered targets and unidentified chemical scaffolds have still to be explored in the panorama of the adjuvant molecules.13 Recently, six bacterial species termed ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) have been identified as a great threat for human health.14,15 Among them, S. aureus and its methicillin-resistant strain (MRSA) are a problem in the hospital setting as well as in the community resulting in a 64% increase of likelihood to die.1 The S. aureus trans-membrane protein NorA, belonging to the Major Facilitator Superfamily, is encoded from norA gene, commonly overexpressed in S. aureus resistant strains16 and upregulated in response to fluoroquinolone treatment.17,18 Through an antiporter mechanism by using the proton motive force, NorA extrudes unrelated toxic compounds including the fluoroquinolone ciprofloxacin (CPX) and the dye ethidium bromide (EtBr).16,19,20 Furthermore, different teams showed that inhibition of efflux in S. aureus, by different EPIs, reduced biofilm production thus indirectly linking NorA and other EPs to the mechanisms involved in biofilm formation.21–23

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To date, few known NorA EPIs have been reported and their main issue is the toxicity at the active concentrations, making these EPIs just tool compounds rather than useful clinical candidates.6 The time has come to deal with the challenge of discovering new NorA EPIs as preclinical candidates for in vivo studies. The efforts of our laboratory have been focused on the design and synthesis of new NorA EPIs able to synergize with CPX against different S. aureus efflux pump overexpressing strains.24–27 In particular, by different studies, the 2-phenylquinoline scaffold was identified as the best core to design and synthesize new NorA EPIs.24–26 In parallel, the selected core was further improved in terms of NorA EPI activity by i) p-OPr functionalization of the C-2 phenyl ring and ii) insertion of an O-alkylamino chain at C-4.24,25 As a result of these efforts, the 2-phenylquinoline 124 (Figure 1) is a representative hit containing both chemical requirements. More recently, 124 and the most potent NorA EPIs reported in literature were used to build a common-feature pharmacophore model with the aim to find common requirements to inhibit NorA.27 Actually, two pharmacophore models were hyphotesized sharing three out of four chemical features: one hydrogen-bond acceptor, one positive charge, one aromatic ring and a forth site that slightly differs between the two models, providing for one aromatic ring or one hydrophobic region, respectively (Figure S1).27 In this work, retaining all key requirements matched by 1,24 we decided to investigate substitutions around the unexplored benzene ring of the quinoline core by chemical functionalization of the C-5, C-6, C-7, and C-8 positions with –OMe group/s, given their large presence on different known NorA EPIs (Figure 1).28 Indeed, many NorA EPIs having natural or synthetic origins show embedded in their backbone at least a –OMe group; examples are: the antipsycotic and antihypertensive reserpine,29 the eucariotyc P-gp inhibitors biricodar, timcodar

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and elacridar,30,31 the calcium channel blocker verapamil,32 the proton pump inhibitor omeprazole33 and the natural compounds capsaicin34 and chrysosplenol D.35 Thus, we designed, synthesized, and biologically evaluated seven new series (2-8) of monoand di-methoxy quinoline derivatives by maintaining p–OPr substitution on the C-2 phenyl and different O-alkylamino chains in quinoline C-4, selected on the basis of the previous acquired SAR information (Figure 1).25 The synthesis of the new derivatives and series was driven by biological data interpretation according to a step-by-step iterative design. Thus, we initially synthesized the four series of mono-methoxy derivatives (2-5 – Figure 1) bearing three different O-alkylamino chains (R1 = ethyl-N,N-diethylamino (a), ethylpiperidine (b), and ethylazepane (c), see Figure 1) with the aim to understand the best position for the –OMe group on the quinoline core. The good EPI activity (Table 1) obtained for the series 3-5 (with -OMe in C-6, C-7, and C-8, respectively, see Figure 1 and Table 1) encouraged us to enlarge these series introducing four more O-alkylamino chains (ethyl-6,7-dimethoxy-tetrahydroisoquinoline

(d),

propyl-N,N-dimethyl

(e),

ethyl-4-

benzylpiperazine (f) and ethylpiperazine (g) – Figure 1) at the C-4 quinoline core. The excellent results (Table 1) obtained for three out of four mono-methoxy series (3-5) led us to explore diOMe functionalization affording three more new series (6-8) (Figure 1).

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Figure 1. Structural modifications of the 2-phenylquinoline core to obtain seven new series (2-8) of NorA EPIs. Chemistry Four different synthetic routes were applied to afford 5-methoxy (series 2 - Scheme 1), 6methoxy and 8-methoxy (series 3 and 5 - Scheme 2), 7-methoxy, 5,7-dimethoxy and 6,7dimethoxy (series 4, 6 and 7 - Scheme 3) and 6,8-dimethoxy (series 8 - Scheme 4) 2phenylquinoline derivatives. 5-Methoxy-2-phenyl-4-hydroxyquinoline derivatives (series 2): by an analogue procedure to that reported for the synthesis of the flavone nucleus,36 in a single step the intermediate 10 was obtained by base-mediated acylation of the 6-hydroxyacetophenone 937 with 4-propoxybenzoyl chloride. Then, derivative 10 was cyclized in H2SO4 at reflux to obtain the flavone derivative 11 that was reacted with HClO4 in HC(OEt)3 to give the corresponding flavylium perchlorate salt

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12. Then, compound 12 was converted into the 5-methoxyquinoline 13 by following the procedure described by Sato and co-workers using 25% NH4OH at room temperature.38 Finally, the subsequent alkylation of 13 with appropriate (chloroalkyl)amines using NaH in dry DMF at 80 °C afforded the desired 5-methoxy derivatives 2a and 2b, while target compound 2c was obtained even from 13 but by MW irradiation using K2CO3 in dry DMF (Scheme 1 and Figure 1). Scheme 1a

a

Reagents and conditions: i) t-BuOK, 4-propoxybenzoyl chloride, dry THF, 0 °C → rt → reflux, 12 h, 47%; ii) AcOH, conc H2SO4, reflux, 40 min, 79%; iii) 70% HClO4, HC(OEt)3, rt → 60 °C, 20 h, 73%; iv) 25% NH4OH, rt, 5 h, 81%; v) (2-chloroethyl)amines, NaH, dry DMF, 90 °C, 20 h, 18-20%; vi) K2CO3, 1-(2-chloroethyl)azepane hydrochloride, dry DMF, MW, 80 °C, 40 min, 16%.

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6-Methoxy and 8-methoxy-2-phenyl-4-hydroxyquinoline derivatives (series 3 and 5): acrylate intermediate 14,24 obtained as previously reported by us, was reacted with the appropriate o-methoxy- or p-methoxy-aniline in benzene with a catalytic amount of p-TsOH to give the aminoacrylate derivatives 15 and 16, respectively. Cyclization of the aminoacrylates 15 and 16 in dowtherm A at 240 °C gave the 6-methoxy-quinoline 17 and 8-methoxy-quinoline 18 that were then functionalized with the appropriate (chloroalkyl)amines, using K2CO3 as base, in dry DMF to obtain the target compounds 6-methoxy-2-phenylquinolines 3a-c, 3e, and 3f and the 8-methoxy-2-phenylquinolines 5a, 5b, and 5e. By using the same procedure, derivatives 17 and 18 were alkylated with 1-bromo-2-chloroethane to give intermediates 19 and 20, respectively. During the reaction aimed to obtain 20, we noted the formation of the vinyl derivative 21. Then, using the same conditions but starting from 19, compound 3d was obtained by reaction with 6,7dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride. In parallel, by reacting derivative 20 with

6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride under MW

conditions

compound 5d was obtained. Differently, starting from 20 and in neat conditions, compound 5c was obtained by reacting with azepane in presence of K2CO3, whereas by reaction with piperazine under MW irradiation compound 5g was achieved. Finally, catalytic reduction of 3f using Pd/C and ammonium formate gave 3g (Scheme 2 and Figure 1). Scheme 2a

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a

Reagents and conditions: i) p-TsOH, benzene, reflux, 19 h, 28-67%; ii) Dowtherm A, 240 °C, 2 h, 88-90%; iii) (2-chloroethyl)amines, or 1-bromo-2-chloroethane, or 6,7-dimethoxy-1,2,3,4tetrahydroisoquinoline, K2CO3, dry DMF, 80-100 °C, 3-20 h, 12-86% or MW, 100 °C, 15-90 min 19-24%; iv) dry MeOH, 10% Pd/C, ammonium formate, 60 °C, 6 h, 20%; v) azepane, K2CO3, 100 °C, 18 h, 58%; vi) piperazine, MW, 120 °C, 15 min, 55%. 7-Methoxy-2-phenyl-4-hydroxyquinoline,

5,7-dimethoxy-2-phenyl-4-hydroxyquinoline

and 6,7-dimethoxy-2-phenyl-4-hydroxyquinoline derivatives (series 4, 6 and 7): by reacting 3-methoxyaniline 22 or 3,5-dimethoxyaniline 23 with 4-propoxybenzoyl chloride, benzamides 2424 and 25 were obtained, respectively. Next, Friedel-Crafts acylation with acetyl chloride using SnCl4 afforded compounds 27 and 28, respectively. In parallel, benzoylation of the commercially available amino acetophenone 26 gave amide intermediate 29. Then, cyclization of 27 with NaH in dry DMF afforded the 2-phenyl-7-methoxy-quinoline derivative 30, whereas with a procedure previously reported by us39 reaction of 28 and 29 with t-BuOK in t-BuOH gave the 5,7-

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dimethoxy and 6,7-dimethoxy quinoline derivatives 31 and 32, respectively. O-alkylation of 3032 with appropriate (chloroalkyl)amines using K2CO3 in dry DMF gave the desired compounds 4a-c, 4e, 4f, 6a, 6b, 7a and 7b; similarly, chloroethyl derivatives 33-35 were obtained. Then, using the same conditions but starting from 33-35, compounds 4d, 6d and 7d were obtained by reaction with 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride. In parallel, by reacting 34 or 35 with azepane in neat conditions, target compounds 6c and 7c were afforded. Finally, catalytic reduction of 4f using Pd/C and ammonium formate in EtOH gave 4g (Scheme 3 and Figure 1). Scheme 3a

a

Reagents and conditions: i) 4-propoxybenzoyl chloride, Et3N, dry THF, 0 °C → rt, 1 h, 7398%; ii) acetyl chloride, SnCl4, dry CH2Cl2, 0 °C → rt, 19 h, 43-49%; iii) NaH, dry DMF, 90 °C, 12 h, 65%; iv) t-BuOK, t-BuOH, 70 °C, 24 h, 41-72%; v) (2-chloroethyl)amines, or 1-bromo-2chloroethane, or 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline, K2CO3, dry DMF, 80-90 °C, 2-

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48 h, 21-75%; vi) dry EtOH, 10% Pd/C, ammonium formate, rt, 1 h, 31%; vii) K2CO3, azepane, reflux, 3-9 h, 10-27%. 6,8-Dimethoxy-2-phenyl-4-hydroxyquinoline derivatives (series 8): 2,4-dimethoxyaniline 36 was acylated with 4-propoxybenzoyl chloride to give intermediate 37 that was chlorinated with PCl5 to afford the imidoyl chloride 38. Nucleophilic substitution of 38 with diethyl malonate, in presence of NaH, gave the poor stable imine intermediate 39 that was immediately cyclized in neat conditions at 170 °C to afford compound 40. After hydrolysis of 40 with KOH in EtOH under MW irradiation at 150 °C, we observed spontaneous decarboxylation thereby obtaining the key synthon 41. Alkylation of 41 with properly selected (chloroalkyl)amines using K2CO3 in dry DMF gave the desired compounds 8a and 8b. Similarly, by using 1-bromo-2chloroethane intermediate 42 was obtained and then reacted with 6,7-dimethoxy-1,2,3,4tetrahydroisoquinoline hydrochloride by using the same conditions to afford compound 8d. Finally, compound 8c was achieved by the reaction between derivative 42 and azepane in neat conditions (Scheme 4 and Figure 1). Scheme 4a

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a

Reagents and conditions: i) 4-propoxybenzoyl chloride, Et3N, dry THF, 0 °C → rt, 1 h, 22%; ii) PCl5, dry benzene, 0 °C, 4 h, 100%; iii) diethyl malonate, NaH, dry DMF, rt → 80 °C, 16 h; iv) neat conditions, 170 °C, 7 h, 25%; v) 20% KOH, EtOH, MW, 150 °C, 30 min, 68%; vi) (2chloroethyl)amines, or 1-bromo-2-chloroethane, or 6,7-dimethoxy-1,2,3,4tetrahydroisoquinoline, K2CO3, dry DMF, 80-90 °C, 3-8 h, 5-56%; vii) azepane, K2CO3, reflux, 3 h, 65%. Results and discussion EtBr efflux assays. All synthesized target compounds (2a-c, 3a-g, 4a-g, 5a-e, 5g, 6a-d, 7a-d, and 8a-d) as well as some intermediates (13, 19-21, 30, 32 and 33) were assayed at 50 µM for their ability to inhibit EtBr efflux in SA-1199B, a S. aureus strain overexpressing the norA gene and also possessing an A116E GrlA substitution.40 Reserpine,41 the first identified NorA EPI, commonly used as standard in EtBr efflux assays, and the starting hit 124 were used as reference compounds. Intermediates 17, 18, 31 and 41 were not tested owing to solubility issues. In this assay, EtBr is used because it is a known efflux pump substrate that emits fluorescence when inside the bacterial cells while it loses fluorescence once extruded.20,42 Thus, by a fluorometric

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method it is possible to determine the efflux inhibition by calculating the difference in fluorescence between treated and untreated cells over five minutes. Since in SA-1199B strain NorA prevails on other efflux pumps due to the norA gene overexpression,40 EtBr efflux inhibition is mainly attributable to NorA inhibition. For all the compounds having an EtBr efflux inhibition higher than 80%, i) dose-response curves at scalar concentrations were built and IC50 values were calculated (Figure 2, Table 1), ii) MIC assays against SA-1199B were performed to exclude any antibacterial effect as interference on the NorA efflux inhibition activity (Table 1).

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Table 1. EtBr efflux inhibition (%) at 50 µM, IC50 and MICs of the seven series (2-8) of 2-phenylquinoline derivatives and intermediates 13, 19-21, 30, 32 and 33 against SA-1199B (norA+/A116E GrlA). LogD at pH = 7.4 predictions. Minimal potentiating concentration 8-fold CPX MIC (MPC8).

R2

R3

EtBr efflux inib. (%)

IC50 (µM)

MIC (µM)

LogD (pH 7.4)a

MPC8 (µg/mL)b

2a

5-OMe

H

96.5 ± 1.6

9.4 ± 0.4

30.6

3.09

3.13

2b

5-OMe

H

83.4 ± 1.6

19.1 ± 0.2

59.4

3.37

NTc

2c

5-OMe

H

74.8 ± 3.1

NDd

NTc

3.31

NTc

3a

6-OMe

H

99.0 ± 1.0

4.7 ± 0.4

122

3.05

6.25

3b

6-OMe

H

97.6 ± 1.3

4.2 ± 0.5

238

3.34

0.78

3c

6-OMe

H

92.2 ± 0.4

8.1 ± 0.2

28.7

3.28

245

3.06

6.25

4b

7-OMe

H

100.0 ± 1.5

4.0 ± 0.3

238

3.31

3.13

4c

7-OMe

H

91.3 ± 1.9

4.9 ± 1.1

212.3

3.25

3.13

4d

7-OMe

H

91.0 ± 1.2

4.7 ± 0.3

>189

4.95

3.13

4e

7-OMe

H

100.0 ± 3.6

4.3 ± 0.1

232

2.44

6.25

4f

7-OMe

H

92.1 ± 2.7

7.3 ± 0.4

>195

5.22

>100

4g

7-OMe

H

100.0 ± 2.3

5.5 ± 0.6

237

1.85

6.25

5a

8-OMe

H

83.1 ± 1.6

13.7 ± 1.1

245

3.05

NTc

5b

8-OMe

H

88.7 ± 2.0

11.9 ± 0.4

238

3.31

NTc

5c

8-OMe

H

72.8 ± 5.5

NDd

NTc

3.24

NTc

5d

8-OMe

H

65.5 ± 0.5

NDd

NTc

4.95

NTc

5e

8-OMe

H

64.7 ± 0.9

NDd

NTc

2.45

NTc

5g

8-OMe

H

100.0 ± 3.1

9.9 ± 0.7

>237

1.85

25

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6a

5-OMe

7-OMe

93.1 ± 1.6

2.8 ± 0.2

>228

2.81

1.56

6b

5-OMe

7-OMe

49.7 ± 2.1

NDd

NTc

3.10

NTc

6c

5-OMe

7-OMe

74.1 ± 0.9

NDd

NTc

3.04

NTc

6d

5-OMe

7-OMe

86.5 ± 3.1

1.6 ± 0.3

179

4.71

228

2.79

50

7b

6-OMe

7-OMe

92.1 ± 2.0

3.9 ± 0.2

>222

3.08

3.13

7c

6-OMe

7-OMe

67.9 ± 0.7

NDd

NTc

3.01

NTc

7d

6-OMe

7-OMe

94.1 ± 1.5

1.1 ± 0.1

>179

4.71

0.78

8a

6-OMe

8-OMe

92.4 ± 1.1

4.6 ± 0.4

>228

2.79

6.25

8b

6-OMe

8-OMe

93.8 ± 1.5

3.8 ± 0.1

>222

3.08

6.25

8c

6-OMe

8-OMe

80.3 ± 2.0

1.8 ± 0.2

>215

3.02

1.56

8d

6-OMe

8-OMe

86.7 ± 1.8

3.8 ± 1.0

45

4.72

3.13

5-OMe

H

91.4 ± 1.5

4.1 ± 0.3

40.4

4.24

NTc

19

6-OMe

H

10.5 ± 1.5

NDd

NTc

NDc

NTc

20

8-OMe

H

6.8 ± 2.3

NDd

NTc

NDc

NTc

13

H

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8-OMe

H

13.3 ± 0.8

NDd

NTc

NDc

NTc

30

H

7-OMe

H

55.7 ± 2.1

NDd

NTc

NDc

NTc

32

H

6-OMe

7-OMe

79.2 ± 0.6

NDd

NTc

NDc

NTc

33

7-OMe

H

57.5 ± 0.9

NDd

NTc

NDc

NTc

124

H

H

93.4 ± 1.2

8.9 ± 0.1

>241

3.32

12.5

84.8 ± 0.7

9.2 ± 0.9

>164

2.71

>100

reserpine41

Predicted by ChemAxon.43 bMinimal potentiating concentration (MPC) able to reduce 8-fold the CPX MIC (MPC8) reported in µg/mL. cNot tested. dNot determined for such compounds that did not reach the 80% EtBr efflux inhibition. Values in bold highlight EtBr efflux inhibition higher than 80% and their corresponding IC50 values. a

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Journal of Medicinal Chemistry

Overall, all the compounds bearing a protonable alkylamino chain showed modest to excellent EtBr efflux inhibition with the exception of 6-OMe derivatives 3d and 3f. In contrast, intermediates of the synthesis (19-21, 30, 32 and 33) exhibited a poor efflux inhibition with the exception of 13. However, this compound also had antibacterial activity at the tested concentration, coupled with a poor solubility and for these reasons it was discarded. Series 2 having C-5 –OMe group showed good EtBr efflux inhibition at 50 µM comparable to the reference compounds 124 and reserpine.41 However, the two best derivatives 2a and 2b exhibited high IC50 coupled with low MICs values, thus, we decided to discard this series for further chemical optimizations. On the contrary, series 3 and 4 bearing C-6 or C-7 –OMe group, respectively, yielded the best derivatives among the mono-methoxy series (2-5). Indeed, compounds 3a, 3b, and 4a-g showed high percentages of EtBr efflux inhibition coupled with low IC50 and high MIC values, resulting in a slight improvement over reference compounds 124 and reserpine.41 In the C-6 –OMe derivatives 3d and 3f, the presence of a bulky and aromatic portion at the C-4 O-alkylamino chain was detrimental to EtBr efflux inhibition activity. A propyl-N,Ndimethyl chain in the same position (compound 3e) produced moderate EtBr efflux inhibition, almost reaching the 80% threshold. However, this trend was not observed in series 4 with C-7 OMe substituent where compounds 4d-f showed excellent percentages of EtBr inhibition. Also, in the series 3, a greater heterocycle (compound 3c), or a double protonable nitrogen (compound 3g) highlighted good percentage of EtBr efflux inhibition but coupled with high IC50 values and especially low MICs. Once again, this trend was not observed in the series 4, where compounds 4c and 4g retained low IC50 and high MIC values. Thinking in terms of chemical physical properties, it is of note as the drastic increase in lipophilicity (see predicted logD in Table 1) for compound 3f with respect to the parent compounds (3a-e and 3g) having more polar alkylamino

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chains decreased the activity differently from 4f when compared to the polar derivatives of series (4a-e and 4g). Shifting attention towards C-8 –OMe series 5, compounds 5a, 5b and 5g showed good EtBr efflux inhibition (> 80%) while 5c-e did not reach the established threshold. Once again, ethylN,N-diethyl 5a and ethylpiperidine 5b derivatives proved to be the best compounds of this series exceeded only by 5g; however, they showed results in the same range of the reference compounds, thereby displaying as –OMe in C-8 position does not significantly contribute in inhibiting EtBr efflux. As mentioned above, the good results obtained from mono-methoxy series (3-5) guided us to introduce a further –OMe group. Thus, we designed and synthesized three new series (6-8) bearing four representative O-alkylamino chains (a-d) at C-4 of the quinoline nucleus (Figure 1). Since C-5 –OMe series 2 and C-8 –OMe series 5 yielded less interesting compounds, the synthesis of compounds carrying –OMe groups both in C-5 and C-8 positions was not planned. Different combinations of –OMe groups (5,7-dimethoxy (6), 6,7-dimethoxy (7), and 6,8dimethoxy (8)) produced excellent results and worthy of note led to an overall reduction in terms of logD values with all the dimethoxy derivatives having predicted values below 5 (Table 1). In particular, compounds 6a, 6d, 7a, 7b, 7d, and 8a-d exhibited high percentages of EtBr efflux inhibition coupled with high MIC values, except for 8d, and especially most of them displayed an interesting decrease in IC50 values respect to 1,24 reserpine,41 and all the monomethoxy compounds. In particular, compounds 6d, 7d, and 8c showed IC50 values lower than 2 µM, highlighting that in two series out of three the bulky lipophilic group 6,7-dimethoxy-1,2,3,4tetrahydroisoquinoline (chain d – Figure 1) resulted as the best substituent.

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Journal of Medicinal Chemistry

Overall, good results were observed for dimethoxy derivatives, often exhibiting low IC50 coupled with high MIC values. On the other hand, when attention is shifted at C-4 position it was impossible to delineate an O-alkylamino chain as the best substituent. This is because its influence on activity depends on the –OMe position, thereby strengthening our choice to test for each series a set of chains.

Figure 2. Dose−response EtBr efflux inhibition assays for 5-OMe compounds (13, 2a, 2b -panel A), 6-OMe compounds (3a-c, 3g -panel B), 7-OMe compounds (4a-g -panel C), 8-OMe compounds (5a, 5b, 5g -panel D), and dimethoxy compounds (6a, 6d, 7a, 7b, 7d, 8a-d -panel E) against SA-1199B (norA+/A116E GrlA); 124 and reserpine41 were included in all panels as comparison.

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MIC evaluation. The most potent compounds able to inhibit EtBr efflux with IC50 < 10 µM were evaluated for their antibacterial effect against two different S. aureus strains: ATCC 25923 (norA wild-type) and SA-1199 (norA wild-type) (Table 2). Compounds showing low MIC values (ranging from 28.7 and 45 µM – Table 1) against SA-1199B but with EtBr efflux IC50 values lower than MICs (2a, 3c, and 8d) were still taken forward, since the efflux inhibition is obtained at non-antibacterial concentrations. Since ideal EPIs should not possess antibacterial activity, MIC assessment is instrumental in order to know the concentrations to be used in the EPI activity evaluation. Thus, high MIC values were well-accepted because an antibacterial effect at low concentrations can produce false positives in the next step where the synergistic effect of our compounds is evaluated in combination with CPX (i.e. EPIs with low MIC values synergize with CPX by their antibacterial effect rather than/or in addition to the NorA inhibition). As shown in Table 2, all the compounds except 2a, 3c, and 8d showed no significant antibacterial activity against all S. aureus test strains.

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Journal of Medicinal Chemistry

Table 2. Intrinsic antibacterial activity (MICs - µg/mL) of compounds 2a, 3a-c, 4a-g, 5g, 6a, 6d, 7a, 7b, 7d, 8a-d, 1,24 reserpine,41 CPX and EtBr against S. aureus strains used in checkerboard assays. CC50 (µg/mL) values on HepG2 and THP-1 cells. MICs µg/mL

Compd.

S. aureus ATCC 25923 (wt)

CC50 µg/mL

SA-1199B SA-1199 SA-K1902 SA-K2378 SA-K2885 SA-K2886 (norA+/A116E (norA wt) (norA-) (norA++) (mepA-) (mepA+) GrlA)

HepG2 cells

THP-1 cells

2a

12.5

12.5

12.5

NTa

NTa

NTa

NTa

NTa

NTa

3a

50

50

50

NTa

NTa

NTa

NTa

5.0 ± 3.1

NTa

3b

50

>100

100

25

25

12.5

12.5

42.0 ± 21.6

>100 ± 0.0

3c

12.5

25

12.5

NTa

NTa

NTa

NTa

NTa

NTa

4a

50

>100

>100

25

25

NTa

NTa

7.7 ± 2.3

NTa

4b

50

>100

100

NTa

NTa

NTa

NTa

71.5 ± 15.9

NTa

4c

>100

100

100

NTa

NTa

NTa

NTa

NTa

NTa

4d

>100

>100

>100

NTa

NTa

NTa

NTa

NTa

NTa

4e

100

100

100

NTa

NTa

NTa

NTa

NTa

NTa

4f

>100

>100

>100

NTa

NTa

NTa

NTa

NTa

NTa

4g

100

100

100

NTa

NTa

NTa

NTa

NTa

NTa

5g

>100

>100

>100

NTa

NTa

NTa

NTa

NTa

NTa

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6a

100

>100

>100

25

25

NTa

NTa

NTa

NTa

6d

100

100

100

NTa

NTa

NTa

NTa

NTa

NTa

7a

100

>100

>100

NTa

NTa

NTa

NTa

NTa

NTa

7b

100

>100

>100

NTa

NTa

NTa

NTa

NTa

NTa

7d

>100

>100

>100

100

100

100

100

8a

100

>100

>100

NTa

NTa

NTa

NTa

NTa

NTa

8b

100

>100

>100

NTa

NTa

NTa

NTa

NTa

NTa

8c

>100

>100

>100

NTa

NTa

NTa

NTa

NTa

NTa

8d

25

25

25

25

25

NTa

NTa

NTa

NTa

74.0 ± 18.9

37.0 ± 0.4

124

100

>100

>100

50

50

NTa

NTa

21.6 ± 4.6

NTa

reserpine41

>100

>100

>100

>100

>100

NTa

NTa

NTa

NTa

CPX

0.31

0.63

10

0.31

2.50

NTa

NTa

138.6b

NTa

EtBr

NTa

NTa

NTa

NTa

NTa

0.63

25

NTa

NTa

a

Not tested. bCC50 values previously reported by us.23

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Journal of Medicinal Chemistry

Synergistic activity. Checkerboard assays were performed with the aim to determine if a synergistic effect between our EPIs and CPX existed (Figure 3). At first glance, it is evident that against wild-type S. aureus strains (ATCC 25923 and SA-1199) CPX possesses a low MIC value and therefore the synergistic effect is almost absent. On the other hand, as expected, in the S. aureus strain overexpressing norA (SA-1199B), when CPX MIC value is high (10 µg/mL), a synergistic effect can be readily appreciated for all the compounds tested. Indeed, scalar dilutions of them led to a high reduction of CPX MIC. For our compounds, the synergistic effect was expressed as the minimal potentiating concentration (MPC) able to reduce the CPX MIC n-fold (MPCn) and for comparative purposes the MPC8 values were provided in Table 1. Firstly, at a concentration of 1.56 µg/mL (a value significantly below of the MIC of all EPIs) 15 out of 21 tested EPIs showed at least an MPC4 against SA-1199B. Focusing our attention on the most potent EPIs, at 0.78 µg/mL derivatives 2a, 4a, 4c, 4d, 6d, 8c, and 8d showed an MPC4 while best compounds 3b and 7d exhibited an MPC8 (Figure 3). Therefore, both 3b and 7d at non-antibacterial concentrations (128-fold lower than MICs) were able to reduce the CPX MIC to 1.25 µg/mL (an 8-fold reduction). Analysing data in terms of SAR, it is evident as a bulky group that increases logD, such as a O-ethyltetrahydroisoquinoline linked at the C-4 position, coupled with –OMe at C-7 (compound 4d), or at both C-5 and C-7 (compound 6d), or at C-6 and C-7 (compound 7d), or at C-6 and C-8 (compound 8d) showed excellent synergism with CPX. Of note, O-ethylazepane at the C-4 position is suitable when a –OMe group is present at the C-6 or C-7 positions (compounds 3c and 4c). On the contrary, the presence of –OMe group at C-6 and C-7 together resulted in a detrimental effect on inhibitory activity (compound 7c). When an O-ethyl-N,N-diethyl amino group is linked at the C-4 position, -OMe group was preferred at C-5 or C-7 (compounds 2a and 4a). Interestingly, when –OMe is present in both positions together

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(compound 7a) EPI activity is still damaged. The C-4 O-ethylpiperidine chain in combination with C-6 –OMe produced the best EPI (compound 3b) but when it was combined with –OMe in other positions a reduced activity was observed. Finally, a O-propyl-N,N-dimethyl chain (e), a Oethyl-benzylpiperazine (f) or a O-ethylpiperazine (g) linked at the C-4 position showed a drastic decrease in the activity regardless of the position of the –OMe group. Worthy of note, the best compounds 3b and 7d showed an evident improvement over the starting hit 124 and reference compound reserpine,41 resulting in an MPC8 of 0.78 µg/mL (3b and 7d) instead of 12.5 and >100 µg/mL (124 and reserpine,41 respectively - Figure 3 and Table 2). Focusing attention on the central point of our rational design, we observed that -OMe introduction on the quinoline scaffold yielded more potent NorA EPIs with respect to the starting des-methoxy hit 1.24 Overall, C-6 mono-methoxy or C-6/C-7 di-methoxy introductions were preferred over other pattern of methoxy substituents. In addition, we proved that 2phenylquinoline scaffold benefits from -OMe group presence thereby indicating, by an extensive SAR analysis, the C-6 position as the best to obtain potent EPIs. Given the random although significant presence of methoxy groups on the known NorA EPIs, it appears hard trying a direct comparison of their activities with our compounds. However, our findings clearly suggest that the presence of -OMe groups may be a strength to improve EPI activity of known NorA desmethoxy inhibitors.

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Journal of Medicinal Chemistry

Figure 3. Effect on the MIC of CPX of mono-methoxy compounds (2a, 3a-c, 4a-g, 5g) against ATCC 25923 (panel A), SA-1199 (panel C), and SA-1199B (panel E); and dimethoxy compounds (6a, 6d, 7a, 7b, 7d, 8a-d) against ATCC 25923 (panel B), SA-1199 (panel D), SA1199B (panel F). 124 and reserpine41 were included as reference compounds. To further support our hypothesis that the synergistic effect with CPX of the synthesized EPIs was mainly due to NorA inhibition, MIC evaluation (Table 2) and checkerboard assays (Figure 4) were performed for compounds 3b, 4a, 6d, 7d, and 8d, selected as representatives from each

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series, against two engineered S. aureus strains: SA-K1902 (norA deleted) and SA-K2378 (overexpressing norA);44 derivative 124 and reserpine41 were included in the test as reference compounds (Figure 4).

Figure 4. Effect of compounds 3b, 4a, 6d, 7d, 8d, 1,24 and reserpine41 on the MIC of CPX against SA-K1902 (norA−) and SA-K2378 (norA++). Overall, as expected all the tested compounds were able to synergize with CPX against SAK2378 (norA++) while no significant synergism was observed against SA-K1902 (norA-). In accordance with the findings obtained using SA-1199B, all compounds widely reduced the CPX MIC to low concentrations against SA-K2378 (norA++). In particular, at 0.78 µg/mL compounds 3b and 8d showed a CPX MIC reduction of 8-fold and even derivative 4a by 16fold. Cytotoxicity evaluation. To demonstrate that our EPIs inhibit NorA efflux at nontoxic concentrations, CC50 values on human liver epithelial (HepG2) cells were measured for compounds 3b, 4a, and 7d (Table 2). These compounds were selected on the basis of having good EPI activity against both SA-1199B and SA-K2378 S. aureus strains. Although compound 4a showed a low CC50 (7.7 µg/mL), the others (3b and 7d) showed higher values (42.0 and 74.0 µg/mL, respectively). This indicated that both inhibited NorA efflux and completely restored the

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Journal of Medicinal Chemistry

CPX MIC at concentrations significantly below their CC50. In particular, 3b and 7d showed an MPC8 about 50- and 95-fold lower than their CC50, respectively. Therefore, when compared with the starting hit 1,24 already better than reserpine, the novel NorA EPIs 3b and 7d showed a 16fold improved MPC8 (Table 1) against SA-1199B coupled with a lower toxicity. Indeed, parent compound 124 exhibited a MPC8 of 12.5 µg/mL which was very close to its CC50 value (21.6 µg/mL). However, intrigued from the large difference between the CC50 of 3b and 4a, which differ by an adjacent position of the –OMe group and for a N,N-diethyl group instead of a piperidine ring, we also assessed the CC50 values on HepG2 cells for the parental compounds 3a and 4b (Table 2). CC50 values suggest that –OMe group position is not significant in terms of cytotoxicity while the piperidine ring plays an important role with respect to N,N-diethyl group, making compounds 3b and 4b safer than the parental 3a and 4a. In order to confirm the good toxic profiles for best compounds 3b and 7d, we performed cytotoxic evaluations against the human monocytic cell line (THP-1). Worthing to note, both compounds still exhibited CC50 values significantly higher than their MPC8 (Table 2), with compound 3b having a CC50 >100 µg/mL and showing a CC50/MPC8 ratio >128-fold. Membrane depolarization. To ensure that our two hit compounds (3b and 7d) did not inhibit NorA in a nonspecific way by destroying proton motive force needed for NorA function, membrane polarization was assessed by cytofluorimeter analysis using the fluorescent probe 3,3’-diethyloxacarbocyanine iodide (DiOC2(3)) as its distribution is proton gradient-sensitive. DiOC2(3) in the presence of a bacterial membrane potential exhibits red fluorescence that shifts to green emission as the membrane potential is lost, thereby allowing for calculation of the percentage of membrane polarization by a red/green fluorescence ratio.45 SA-1199B strain was

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treated with three different concentrations (1, 5 and 10 µg/mL) of compounds 3b and 7d for 30 minutes and the proton ionophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) at a fixed concentration of 1.02 µg/mL (5 µM), used as positive control (Figure S2). Both compounds 3b and 7d showed a modest concentration-dependent membrane depolarization, slightly more evident for compound 7d than 3b. Interestingly, both of them at 1 µg/mL exhibited a poor depolarizing effect (< 20% for 7d and < 10% for 3b), thereby highlighting that at a concentration as low as 0.78 µg/mL at which CPX MIC is reduced by 8-fold (MPC8) in checkerboard assays both compounds inhibit NorA in a specific manner. MepA inhibition activity. Since the selective inhibition of a pump in bacteria may result in turn in an overexpression of different efflux pumps belonging to other families, EPI-mediated NorA inhibition could lead to a restored resistant S. aureus strain due to overexpression of other pumps. As a key example, the development of pyridopyrimidine derivatives reported as specific inhibitors of the MexB efflux pump in P. aeruginosa was stopped because it was not able to inhibit other kinds of efflux pumps such as MexY.46 However, a large degree of efflux pump inhibition can be reached owing to a conserved hydrophobic binding pocket present in several bacterial efflux pumps belonging to different families.47 Therefore, we selected our two hit compounds 3b and 7d with the aim of investigating their effect on the EtBr MIC against a S. aureus strain (SA-K2886) overexpressing MepA (mepA++), an efflux pump different from NorA and belonging to the Multidrug And Toxic Compound Extrusion family.48 In this assay, we used as a comparison the MepA knock-out SA-K2885 strain (mepA-) (Figure 5).48

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Journal of Medicinal Chemistry

Figure 5. Effect of compounds 3b and 7d on the MIC of EtBr against SA-K2885 (mepA-) and SA-K2886 (mepA++). Compounds 3b and 7d at concentrations of 1.56 and 3.13 µg/mL, respectively, showed a high synergistic effect against SA-K2886 lowering EtBr MIC from 25 to ≤ 1.56 µg/mL. On the contrary, as expected the two compounds did not show any significant synergistic effect with EtBr against SA-K2885 (mepA-). Worthy of note, both compounds showed this potentiating activity at values much lower than their MICs against the involved strains (Table 2). We have demonstrated that the new hit EPIs (3b and 7d), at concentrations that are nontoxic for human cells, are able to inhibit MepA-mediated efflux in addition to that mediated by NorA. The inhibition of pumps belonging to different families is an advantage for the development of new EPIs, thereby bypassing the overexpression of other kind of pumps. PK studies. To establish a preliminary PK profile for the new hit compounds 3b and 7d, we performed in vitro metabolic stability assays on mouse liver microsomes (MLMs) and evaluated their effect on two different cytochromes: CYP3A4 and 2D6. Initially, the metabolic stability of compounds 3b and 7d was examined in silico using the MetaSite49 computational tool which suggested demethylation as the main metabolic pathway (100% of probability) (Figures S3 and S4). In particular, for derivative 3b the main demethylation may occur on the –OMe group at C-6

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of the 2-phenylquinoline scaffold while for compound 7d, the demethylation prediction mainly indicates the –OMe group at the C-6 of the tetrahydroisoquinoline substituent. Subsequently, the in vitro metabolic stability of compounds 3b and 7d was evaluated by using MLMs. The UPLC spectrum of 3b after 2 hours of incubation showed it to be metabolically stable as more than 90% remained as such in the reaction mixture. Three additional peaks related to three metabolites M1-M3 (Figure S5) were observed. MS spectra (Figure S6) combined with in silico data allowed us to identify the possible structures of the M1 metabolite as the demethylated (Figure S3) and M2 metabolite either as the oxided at the open piperidine ring or the hydroxylated (in Figures S7 the four main metabolites are proposed), respectively. Unfortunately, due to the problems with MS spectra identification, the molecular mass of M3 was not determined (Figure S8). In parallel, the UPLC analysis of the reaction mixture after 2 hours incubation of 7d with MLMs led to the identification of a 62% of unchanged compound 7d and eight different metabolites M1-M8 (Figure S9). Then, by combining in silico predictions with spectral data of identified MS/MS fragments of metabolites derived from in vitro reactions (Figure S10 and S11), we identified reactions of hydroxylation and oxidation over the ethyl tetrahydroisoquinoline substituent as the most common events occurring in the presence of MLMs (Figures S12-14). Interestingly, in vitro data that indicated hydroxylation and oxidation-mediated ring opening as main clues differed from results by MetaSite49 that predicted mainly demethylation. In addition, compounds 3b and 7d were evaluated for their effect on cytochromes P450 (CYPs) 3A4 and 2D6. The luminescence CYP3A4 and CYP2D6 P450-Glo™ assays50 based on the conversion of the beetle D-luciferin derivative into D-luciferin by recombinant human CYPs 3A4 or 2D6 isoenzymes were used. As reference compounds, the CYP3A4 inhibitor

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Journal of Medicinal Chemistry

ketoconazole and the CYP2D6 inhibitor quinidine were used. The CYP3A4 assay showed around 60% inhibition by 7d at 10 µM whereas no effect was observed for 3b. Both compounds demonstrated no significant CYP3A4 inhibition at 1 µM (Figure S15), a concentration close to their MPC8. Similarly, both 3b and 7d at 1 µM did not inhibit CYP2D6 that, on the other hand, was significantly inhibited when these compounds were tested at 10 µM (Figure S16). Therefore, from these preliminary PK studies, mono-methoxy derivative 3b exhibited a better profile than the dimethoxy derivative 7d, highlighting a metabolically stable behavior when submitted to the action of MLMs. In addition, at 1 µM, a concentration at which it shows excellent EPI activity, the lead compound 3b did not show any significant inhibition of the main cytochromes CYP3A4 and 2D6. Conclusions In this work, we report the design, synthesis and biological evaluation of a new large series of methoxy-2-phenylquinoline derivaties as S. aureus NorA EPIs. Two novel methoxy-2phenylquinoline derivatives (3b and 7d) were devoid of any significant antibacterial effect and showed an MPC8 of 0.78 µg/mL in reducing CPX MIC against SA-1199B, a norA overexpressing strain harbouring a grlA mutation. Thus, methoxy introduction on the 2phenylquinoline core allowed us to obtain potent S. aureus EPIs and indicated the C-6 position as a hot spot for chemical optimization, consistently to our recent studies.51 The ability of compounds 3b and 7d to inhibit NorA efflux has been indirectly proved by EtBr efflux assays against SA-1199B and confirmed by the lack of any significant synergistic activity against SA-K1902 (norA-). Both compounds exhibited no (3b) or poor (7d) depolarazing effect at their MPC8 values on the S. aureus membrane, thereby excluding a NorA efflux inhibition due to the proton motive force disruption. Taken together, these data suggest that compounds 3b and

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7d may inhibit NorA efflux mechanism in a specific manner because the absence of any significant depolarizing effect on the bacterial membrane indirectly rules out any increase in bacterial membrane permeability, as also demonstrated by the lack of any significant synergistic effect against SA-K1902 that does not posses the NorA pump due to norA gene deletion. In addition, since a substantial difference in MIC values is expected by efflux pump substrates being tested against wild-type and norA overexpressing strains, as it appears evident for CPX against SA-K1902 and SA-K2378 (8-fold difference), the similar MICs against these strains observed for 3b and 7d suggest that both compounds are not NorA substrates. Thus, although the information on a direct binding of 3b and 7d to NorA is not yet available, data point toward the important finding that NorA-mediated efflux inhibition does not occur in a nonspecific mechanism such as proton motive force disruption, increase in membrane permeability or competitive extrusion with effluxed drugs. Furthermore, both compounds inhibited the S. aureus MepA efflux pump, revealing methoxy-2-phenylquinoline scaffold as suitable to obtain broad spectrum EPIs strongly needed to fully overcome a pump-mediated resistance. Interestingly, cytotoxicity evaluation against different cell lines (HepG2 and THP-1 cells) for compounds 3b and 7d showed CC50 values significantly higher than the concentrations able to exert the synergistic effect (MPC8 of 0.78 µg/mL). Finally, preliminary PK studies displayed a good metabolic stability for compound 3b coupled with no effect in inhibiting cytochromes CYP3A4 and 2D6 at the useful concentrations to show synergistic activity. Therefore, by combining all information obtained from this extensive work around the 2phenylquinoline core, compound 3b emerged as a promising lead compound that deserves to be further characterized as a potential candidate in view of a possible in vivo evaluation to give the proof-of-concept for a therapeutic potential of NorA EPIs.

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Supporting information Figures S1-S16 mentionated in the manuscript. Data used to build Figure 3-5 are furnished as Tables S1-S7. Molecular formula strings and some data (CSV) Experimental section PAINS filters All the tested compounds were examined for known classes of pan-assay interference compounds by using the PAINS remover filter at http://zinc15.docking.org/patterns/home/52 and none of the compounds was found as potential PAINS. The most active compounds (2a, 3a-c, 4a-g, 5g, 6a, 6d, 7a, 7b, 7d, 8a-d) were examined for known classes of molecular aggregators by using http://zinc15.docking.org/patterns/home/52 and none of them was found as a potential aggregator. Furthermore, none of the compounds emitted fluorescence at the same wavelength as EtBr, the dye employed to evaluate the EPI activity in the real-time fluorimetric assays. Bacterial Strains The strains of S. aureus employed were ATCC 25923 (wild-type), SA-K1902 (norA-deleted),44 SA-1199 (wt), and SA-1199B (overexpressing norA and also possessessing an A116E GrlA substitution).40 In addition, SA-K2378, which overexpresses norA from a multicopy plasmid, was also used.44 This strain was produced by cloning norA and its promoter into plasmid pCU1 and then introducing the construct into SA-K1902.44 SA-K2885 and SA-K2886 are norA-deleted strains containing the empty expression vector pALC2073 and pALC2073-mepA, respectively.48 Genes cloned into pALC2073 are under control of a xyl/tetO promoter, which is inducible by

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0.05 µg/mL tetracycline. This concentration of tetracycline was included in all experiments utilizing these strains.53 EtBr Efflux. Cells were grown overnight in cation-supplemented Mueller-Hinton broth (SMHB) containing no additive at 35 °C with. Organisms were diluted into the same medium as used for overnight growth until an optical density at 600 nm (OD600) of 0.7−0.8 was achieved. Cells were then pelleted and resuspended at OD600 = 0.8 in 0.5 mL aliquots of SMHB containing EtBr plus carbonyl cyanide m-chlorophenylhydrazone (CCCP) to “load” cells with EtBr (final concentrations, 25 µM for EtBr and 100 µM for CCCP). After 20 min at room temperature, cells were pelleted then resuspended in 1 mL of fresh SMHB, and 200 µL aliquots were immediately transferred into the wells of opaque 96-well plates containing or lacking a 50 µM concentration of each test compound. Fluorescence was monitored continuously using a BioTek FLx800 microplate reader (BioTek Instruments Inc., Winooski, VT) at excitation and emission wavelengths of 485 and 645 nm, respectively, for 5 min. Experiments were performed in triplicate with two technical replicates per biological replicate. Efflux activity of SA-1199B was expressed as percent fluorescence decrease over a 5 min time course. Inhibition of this efflux by test compounds was determined using the equation [efflux in the absence] − [efflux in the presence of test [compound]/[efflux in the absence of test compound] × 100, giving the percent efflux inhibition observed. If a 50 µM concentration of test compound achieved at least 80% efflux inhibition, a series of concentrations were tested to quantify the potency by determining the 50% inhibitory concentration (IC50). IC50 determinations also were performed for selected compounds having less than 80% efflux inhibition.54,55 Microbiologic Procedures. MICs were determined by microdilution techniques according to CLSI guidelines.56 The effect of combining reserpine, 1,24 or scalar dilutions of freshly prepared

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solutions of each selected compound on the MICs of CPX was also determined. Checkerboard combination studies using CPX and EtBr and the tested compounds were performed as described previously.57 Cell viability assay. Compounds 1, 3a, 3b, 4a, 4b, 7d and CPX were tested on human liver epithelial cell line (HepG2) and compounds 3b and 7d against human leukemic monocyte cell line (THP-1). HepG2 or THP-1 cells were grown in RPMI 1640 supplemented with 10% heatinactivated fetal calf serum, 10,000 units penicillin and 10 µg streptomycin/mL overnight to confluence. Monolayers were treated for 24 h at 37 °C with scalar concentration of tested compounds (0-250 µg/mL). Cell viability was then evaluated using an ATP bioluminescence kit (Via Light kit; Cambrex). Results are expressed as 50% cytotoxic concentration (CC50). The CC50 was defined as the concentration required to reduce the cell number by 50% compared to that for the untreated controls. Each concentration was tested in triplicate. Membrane potential assay. The effect of 3b and 7d on the membrane potential was measured using the BacLight Bacterial Membrane Potential Kit (Molecular Probes, Life Technologies) according to the manufacturer’s instructions. Briefly, SA-1199B was grown in Mueller Hinton Broth (MHB) at 37 °C until reaching an OD600 of 0.6. Bacterial cells were then washed in PBS and diluted to 1×106 CFU/mL with filtered PBS (filter 0.22 µm) in flow cytometry tubes. 10 µL of 3 mM of 3,3'-diethyloxacarbocyanine iodide (DiOC2(3) in DMSO) were added to each tube (final concentration 30 µM) and mixed. Then, 3b or 7d from a stock solution in DMSO (10 mg/mL) was added to reach final concentrations of 1, 5 and 10 µg/mL. As positive control, 10 µL of 500 µM carbonyl cyanide m-chlorophenyl hydrazone (CCCP, final concentration 5 µM) was used to eradicate the proton gradient by eliminating the membrane potential. The samples were analyzed after 30 minutes by measuring the fluorescence using a cytometer Attune NxT

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(ThermoFisher Scientific) with a laser emitting at 488 nm and collecting in the green and red channels. The red to green fluorescence ratio was determined and normalized against the emission from the DiOC2(3) blank tube having 1 mL of the bacterial suspension and a final concentration of DiOC2(3) of 30 µM. The results are presented as the percentage of depolarized membranes compared with the drug-free control. Metabolic stability. The stock solutions of 3b and 7d (10 mM) were prepared in DMSO. Commercial mouse liver microsomes were purchased from Sigma-Aldrich (St. Louis, USA). The biotransformations were carried out using 1 mg/mL of MLMs in 200 µL of reaction buffer containing 0.1 M Tris-HCl (pH 7.4) and the test compound with final volume 50 µM. Due to the problems with 3b and 7d solubility the total volume of DMSO in the reaction mixture was exceed up to ~3%. The reaction mixture was preincubated at 37 °C for 5 min and then, the reaction was started by adding 50 µL of NADPH Regeneration System (Promega, Madison, WI, USA). The reaction was terminated after 120 min by the addition of 200 µL of cold methanol. The mixture was next centrifuged at 14000 rpm for 15 min and the UPLC/MS analysis of the supernatant was performed. Mass spectra were recorded on UPLC/MS system consisted of a Waters Acquity UPLC (Waters, Milford, USA), coupled to a Waters TQD mass spectrometer (electrospray ionization mode ESI-tandem quadrupole). The in silico study was performed by MetaSite 4.1.1 provided by Molecular Discovery Ltd.49 The highest metabolism probability sites were analyzed during this study by liver computational model. Chemistry

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All starting materials, reagents, and solvents were purchased from common commercial suppliers and were used as such, unless otherwise indicated. Organic solutions were dried over anhydrous Na2SO4 and concentrated with a rotary evaporator at low pressure. The reactions carried out under MW irradiation were performed employing a microwave reactor BIOTAGE INITIATORTM 2.0 version 2.3, build 6250. All reactions were routinely checked by thin-layer chromatography (TLC) on silica gel 60F254 (Merck) and visualized by using UV or iodine. Chromatography separations were carried out using Sigma Aldrich aluminium oxide activated, basic, Brockmann I; flash chromatography separations were carried out on Merck silica gel 60 (mesh 230-400). Melting points were determined in capillary tubes (Electrotermal model 9100) and are uncorrected. Yields were of purified products and were not optimized. 1H NMR spectra were recorded at 200 or 400 MHz (Bruker Avance DRX-200 or 400, respectively) while

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C

NMR spectra were recorded at 101 MHz (Bruker Avance DRX-400). Chemical shifts are given in ppm (δ) relative to TMS. Spectra were acquired at 298 K. Data processing was performed with standard Bruker software XwinNMR and the spectral data are consistent with the assigned structures. The purity of the tested compounds was evaluated by combustion analysis using a Fisons elemental analyzer, model EA1108CHN, and data for C, H, and N are within 0.4% of the theoretical values (≥95% sample purity). General procedure (A) for the synthesis of compounds 3a-e, 4a-c, 4e, 4f, 5a, 5b, 5e, 6a, 6b, 6d, 7a, 7b, 7d, 8a, 8b, 8d, 19-21, 33-35, 42. To a suspension of derivative 17-19, 30-35, 41 or 42 (1.0 equiv) and K2CO3 (3.0 equiv) in dry DMF (15 mL per mmol), chloroalkylamine (2.0 equiv) was added. The reaction mixture was stirred at 80-100 °C for 3-48 h under nitrogen atmosphere. The mixture was then poured in water and extracted with EtOAc (3 x 100 mL) and the organic layers were dried over Na2SO4 and

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evaporated to dryness under reduced pressure to obtain a residue that was purified by column chromatography or crystallization to give the target compounds 3a-e, 4a-c, 4e, 4f, 5a, 5b, 5e, 6a, 6b, 6d, 7a, 7b, 7d, 8a, 8b, 8d, 19-21, 33-35, 42. N,N-diethyl-2-{[5-methoxy-2-(4-propoxyphenyl)quinolin-4-yl]oxy}ethanamine (2a). To a suspension of NaH (0.24 g, 5.81 mmol) in dry DMF (5 mL), a solution of derivative 13 (0.60 g, 1.93 mmol) in dry DMF (5 mL) was added. After 5 min, a solution of (2-chloroethyl) diethylamino hydrochloride (0.67 g, 3.87 mmol) in dry DMF (5 mL) was added dropwise. The reaction mixture was stirred at 90 °C for 20 h and then poured in water and extracted with EtOAc. The organic layers were dried over Na2SO4 and evaporated to dryness under reduced pressure. After crystallization by EtOH, compound 2a was obtained as a yellowish solid (20% yield, mp 216.0-217.0 °C). 1H NMR (DMSO-d6, 400 MHz): δH 1.01 (3H, t, J = 7.5 Hz, OCH2CH2CH3), 1.06 (6H, t, J = 8.0 Hz, NCH2CH3 x 2), 1.76-1.87 (2H, m, OCH2CH2CH3), 2.632.75 (4H, m, NCH2CH3 x 2), 2.79-2.97 (2H, m, OCH2CH2N), 3.91-4.04 (4H, m, OCH2CH2N and OCH2CH2CH3), 4.12 (3H, m, OCH3), 6.97-7.09 (3H, m, H6, H3’, and H5’), 7.32 (1H, d, J = 8.6 Hz, H8), 7.37 (1H, s, H3), 7.79 (1H, t, J = 8.4 Hz, H7), 8.20 (2H, d, J = 8.6 Hz, H2’ and H6’). 13

C NMR (DMSO-d6, 101 MHz): δC 10.41, 11.32, 22.39, 41.77, 46.22, 49.69, 57.26, 70.04,

93.02, 105.57, 108.03, 111.36, 115.49, 120.97, 130.12, 136.17, 155.09, 159.71, 158.61, 164.09, 165.78. Anal calcd for C25H32N2O3: C, 73.50; H, 7.90; N, 6.86; found: C, 73.41; H, 7.92; N, 6.84. 5-methoxy-4-(2-(piperidin-1-yl)ethoxy)-2-(4-propoxyphenyl)quinoline (2b). Following the same procedure reported for compound 2a, starting from derivative 13 and using (2-chloroethyl)piperidine hydrochloride, compound 2b, after purification by flash column

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chromatography (CHCl3/MeOH 99/1), was obtained as a yellowish solid (18% yield, mp 121.5123.0 °C). 1H NMR (DMSO-d6, 400 MHz): δ H 0.99 (3H, t, J = 7.4 Hz, OCH2CH2CH3), 1.401.49 (2H, m, piperidine CH2), 1.52-1.57 (4H, m, piperidine CH2 x 2), 1.74-1.82 (2H, m, OCH2CH2CH3), 2.41-2.58 (4H, m, piperidine NCH2 x 2), 2.68 (2H, t, J = 5.9 Hz, OCH2CH2N), 3.91 (2H, t, J = 5.9 Hz, OCH2CH2N), 4.08 (2H, t, J = 6.5 Hz, OCH2CH2CH3), 4.14 (3H, s, OCH3), 7.19 (2H, d, J = 8.8 Hz, H3’, and H5’), 7.33 (1H, d, J = 8.4 Hz, H6), 7.46 (1H, s, H3), 7.58 (1H, d, J = 8.0 Hz, H8), 7.97 (1H, t, J = 8.5 Hz, H7), 8.29 (2H, d, J = 9.0 Hz, H2’ and H6’). 13

C NMR (DMSO-d6, 101 MHz): δC 10.32, 21.36, 23.42, 24.57, 54.83, 55.05, 57.11, 67.35,

71.81, 104.12, 108.11, 114.01, 115.07, 122.65, 128.61, 130.39, 131.45, 148.33, 153.75, 161.36, 162.22, 170.21. Anal calcd for C26H32N2O3: C, 74.26; H, 7.67; N, 6.66; found: C, 74.48; H, 7.66; N, 6.85. 4-(2-(azepan-1-yl)ethoxy)-5-methoxy-2-(4-propoxyphenyl)quinoline (2c). In a MW vial, derivative 13 (0.50 g, 1.62 mmol), 1-(2-chloroethyl)azepane hydrochloride (0.63 g, 3.20 mmol), and K2CO3 (1.10 g, 8.00 mmol) were suspended in dry DMF (6 mL). The mixture was irradiated by MW according to the following conditions: time = 40 min, temperature = 80 °C, pressure = 5 bar, cooling = ON. The reaction mixture was poured in ice/water and then filtered. After crystallization by EtOH, compound 2c was obtained as a yellow solid (16% yield, mp 204.0-204.5 °C). 1H NMR (CDCl3, 400 MHz): δH 1.01 (3H, t, J = 7.4 Hz, OCH2CH2CH3), 1.63-1.69 (8H, m, azepane CH2 x 4), 1.76-1.85 (2H, m, OCH2CH2CH3), 2.71-2.81 (4H, m, azepane NCH2 x 2), 2.96-3.04 (2H, m, OCH2CH2N), 3.91-4.03 (4H, m, OCH2CH2N and OCH2CH2CH3), 4.15 (3H, s, OCH3), 7.03-7.10 (3H, m, H6, H3’, and H5’), 7.31 (1H, s, H3), 7.34 (1H, d, J = 8.5 Hz, H8), 7.78 (1H, t, J = 8.5 Hz, H7), 8.18 (2H, d, J = 8.9 Hz, H2’ and H6’). 13C

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NMR (CDCl3, 101 MHz): δC 10.41, 22.39, 26.92, 27.45, 42.22, 53.48, 54.93, 57.42, 70.03, 94.55, 105.18, 108.14, 111.39, 115.47, 120.95, 129.72, 130.09, 136.21, 153.96, 155.02, 164.06, 167.07. Anal calcd for C27H34N2O3: C, 74.62; H, 7.89; N, 6.45; found: C, 74.55; H, 7.86; N, 6.46. N,N-diethyl-2-{[6-methoxy-2-(4-propoxyphenyl)quinolin-4-yl]oxy}ethanamine (3a). General procedure A (time = 3 h, T = 90 °C): starting from derivative 17 (0.30 g, 0.97 mmol) and using (2-chloroethyl)diethylamine hydrochloride, compound 3a was obtained after crystallization by cyclohexane as a white solid (53% yield, mp 97.0-99.0 °C). 1H NMR (CDCl3, 400 MHz): δH 1.03 (3H, t, J = 8.7 Hz, OCH2CH2CH3), 1.07-1.23 (6H, m, NCH2CH3 x 2), 1.781.87 (2H, m, OCH2CH2CH3), 2.77 (4H, q, J = 7.2 Hz, NCH2CH3 x 2), 3.13 (2H, t, J = 6.3 Hz, OCH2CH2N), 3.91 (3H, s, OCH3), 3.98 (2H, t, J = 6.6 Hz, OCH2CH2CH3), 4.38 (2H, t, J = 6.5 Hz, OCH2CH2N), 7.01 (2H, d, J = 8.8 Hz, H3’ and H5’), 7.12 (1H, s, H3), 7.31 (1H, dd, J = 2.9 and 9.1 Hz, H7), 7.40 (1H, d, J = 2.7 Hz, H5), 7.95 (1H, d, J = 9.1 Hz, H8), 8.05 (2H, d, J = 8.8 Hz, H2’ and H6’). 13C NMR (CDCl3, 101 MHz): δC 10.55, 12.01, 18.09, 22.59, 51.77, 55.49, 67.06, 69.59, 98.34, 99.78, 114.64, 120.68, 122.01, 128.52, 130.58, 132.69, 145.12, 156.05, 156.92, 160.04, 160.89. Anal calcd for C25H32N2O3: C, 73.50; H, 7.90; N, 6.86; found: C, 73.57; H, 7.88; N, 6.85. 6-methoxy-4-(2-piperidin-1-ylethoxy)-2-(4-propoxyphenyl)quinoline (3b). General procedure A (time = 3 h, T = 80 °C): starting from derivative 17 (0.30 g, 0.97 mmol) and using 1-(2-chloroethyl)piperidine hydrochloride, compound 3b was obtained after crystallization by cyclohexane as a white solid (83% yield, mp 98.0-100.0 °C). 1H NMR (CDCl3, 400 MHz): δH 1.10 (3H, t, J = 7.4 Hz, OCH2CH2CH3), 1.38-1.49 (2H, m, piperidine CH2), 1.63-

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Journal of Medicinal Chemistry

1.66 (4H, m, piperidine CH2 x 2), 1.78-1.87 (2H, m, OCH2CH2CH3), 2.58-2.73 (4H, m, piperidine NCH2 x 2), 2.99 (2H, t, J = 6.0 Hz, OCH2CH2N), 3.91 (3H, s, OCH3), 3.97 (2H, t, J = 6.6 Hz, OCH2CH2CH3), 4.46 (2H, t, J = 6.0 Hz, OCH2CH2N), 7.06 (2H, d, J = 8.8 Hz, H3’ and H5’), 7.15 (1H, s, H3), 7.31 (1H, dd, J = 2.8 and 8.9 Hz, H7), 7.47 (1H, d, J = 2.9 Hz, H5), 7.95 (1H, d, J = 9.0 Hz, H8), 8.01 (2H, d, J = 8.9 Hz, H2’ and H6’). 13C NMR (CDCl3, 101 MHz): δC 10.52, 22.58, 23.97, 25.87, 55.11, 55.51, 57.49, 66.47, 69.60, 98.41, 99.94, 114.66, 120.72, 121.87, 128.51, 130.60, 132.70, 141.14, 156.06, 156.94, 160.06, 160.78. Anal calcd for C26H32N2O3: C, 74.26; H, 7.67; N, 6.66; found: C, 73.15; H, 7.64; N, 6.68. 4-(2-azepan-1-ylethoxy)-6-methoxy-2-(4-propoxyphenyl)quinoline (3c). General procedure A (time = 3 h, T = 100 °C): starting from derivative 17 (0.40 g, 1.35 mmol) and using 1-(2-chloroethyl)azepane hydrochloride, compound 3c was obtained after purification by flash column chromatography (CHCl3/MeOH 99/1) as a white solid (12% yield, mp 89.0-92.5 °C). 1H NMR (CDCl3, 400 MHz): δH 1.02 (3H, t, J = 7.4 Hz, OCH2CH2CH3), 1.58-1.63 (4H, m, azepane CH2 x 2), 1.65-1.74 (4H, m, azepane CH2 x 2), 1.80-1.90 (2H, m, OCH2CH2CH3), 2.86 (4H, t, J = 5.5 Hz, azepane NCH2 x 2), 3.16 (2H, t, J = 5.9 Hz, OCH2CH2N), 3.89 (3H, s, OCH3), 3.95 (2H, t, J = 6.7 Hz, OCH2CH2CH3), 4.35 (2H, t, J = 5.9 Hz OCH2CH2N), 6.98 (2H, d, J = 8.7 Hz, H3’ and H5’), 7.11 (1H, s, H3), 7.28 (1H, dd, J = 2.8 and 9.2 Hz, H7), 7.39 (1H, d, J = 2.8 Hz, H5), 7.93 (1H, d, J = 9.2 Hz, H8), 7.98 (2H, d, J = 8.9 Hz, H2’ and H6’).

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(CDCl3, 101 MHz): δC 10.54, 22.59, 27.00, 27.88, 55.51, 55.96, 56.17, 66.83, 69.59, 98.39, 99.84, 114.64, 120.71, 121.95, 128.53, 130.57, 132.69, 145.12, 156.08, 156.93, 160.04, 160.85. Anal calcd for C27H34N2O3: C, 74.62; H, 7.89; N, 6.45; found: C, 74.55; H, 7.93; N, 6.42.

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4-[2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethoxy]-6-methoxy-2-(4propoxyphenyl)quinoline hydrochloride (3d). General procedure A (time = 16 h, T = 80 °C): starting from derivative 19 (0.50 g, 1.35 mmol) and using 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride, compound 3d was obtained after purification by flash column chromatography (CH2Cl2/EtOAc 98/2) as a white oil (15% yield); then, compound 3d was dissolved into Et2O and HClgas was bubbled until obtaining compound 3d as a white hydrochloride solid that was collected by filtration (83% yield, 133.0134.0 °C). 1H NMR (DMSO-d6, 400 MHz): δH 1.00 (3H, t, J = 6.6 Hz, OCH2CH2CH3), 1.711.80 (2H, m, OCH2CH2CH3), 3.45-3.75 (11H, m, OCH3 x 3 and tetrahydroisoquinoline CH2), 3.78-3.81 (2H, m, tetrahydroisoquinoline NCH2), 4.05 (2H, t, J = 6.4 Hz, OCH2CH2CH3), 4.42 (2H, m, tetrahydroisoquinoline NCH2), 4.69 (2H, m, OCH2CH2N), 4.87 (2H, m, OCH2CH2N), 6.40 (1H, s, H5’’), 6.50 (1H, s H8’’), 7.18 (2H, d, J = 8.7 Hz, H3’ and H5’), 7.50-7.54 (1H, m, H7), 7.70-7.77 (1H, m, H5), 7.75 (1H, s, H3), 8.15 (2H, d, J = 8.8 Hz, H2’ and H6’), 8.40 (1H, d, J = 8.9 Hz, H8), 15.12 (1H, bs, NH).

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C NMR (DMSO-d6, 101 MHz): δC 10.81, 22.39, 27.90,

42.05, 45.39, 45.66, 55.84, 56.28, 63.06, 69.95, 100.81, 101.15, 101.65, 109.70, 109.99, 112.05, 112.21, 115.49, 120.93, 122.10, 124.80, 125.28, 126.09, 131.47, 147.68, 153.18, 155.18, 158.76, 162.56, 166.05. Anal calcd for C32H37ClN2O5: C, 68.01; H, 6.60; N, 4.96; found: C, 68.15; H, 6.57; N, 4.95. (3-{[6-Methoxy-2-(4-propoxyphenyl)quinolin-4-yl]oxy-N,N-dimethylpropan-1-amine (3e). General procedure A (time = 14 h, T = 80 °C): starting from derivative 17 (0.50 g, 1.62 mmol) and using (3-chloropropyl)dimethylamine hydrochloride, compound 3e was obtained after purification by flash column chromatography (CH2Cl2/MeOH 95/5) as a pale brown solid (28%

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yield, mp 72.0-74.0 °C). 1H NMR (CDCl3, 400 MHz): δH 1.03 (3H, t, J = 7.5 Hz, OCH2CH2CH3), 1.78-1.87 (2H, m, OCH2CH2CH3), 2.10-2.25 (2H, m, OCH2CH2CH2N), 2.35 (6H, s, N(CH3)2), 2.65 (2H, t, J = 8.0 Hz, OCH2CH2CH2N), 3.92 (3H, s, OCH3), 3.95 (2H, t, J = 6.5 Hz, OCH2CH2CH3), 4.30 (2H, t, J = 7.8 Hz, OCH2CH2CH2N), 6.98 (2H, d, J = 8.7 Hz, H3’ and H5’), 7.11 (1H, s, H3), 7.32 (1H, dd, J = 2.7 and 8.9 Hz, H7), 7.41 (1H, d, J = 2.8 Hz, H5), 7.93 (1H, d, J =8.9 Hz, H8), 8.15 (2H, d, J = 8.8 Hz, H2’and H6’).

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C NMR (CDCl3, 101

MHz): δC 10.54, 22.59, 27.08, 45.37, 55.57, 56.32, 66.38, 69.58, 98.38, 99.87, 114.62, 120.72, 121.83, 128.54, 130.57, 132.71, 145.08, 156.09, 156.91, 160.02, 160.94. Anal calcd for C24H30N2O3: C, 73.07; H, 7.67; N, 7.10; found: C, 72.98; H, 7.68; N, 7.09. 4-[2-(4-benzylpiperazin-1(1H)-yl)ethoxy]-6-methoxy-2-(4-propoxyphenyl)quinoline (3f). In a MW vial, derivative 17 (0.81 mmol, 0.30 g), K2CO3 (3.23 mmol, 0.45 g), and 1-benzyl-4-(2chloroethyl)piperazine (3.23 mmol, 0.57 g) were suspended in dry DMF (5 mL). The mixture was irradiated by MW according the following conditions: time = 15 min, temperature = 100 °C, pressure = 5 bar, cooling = OFF. The reaction mixture was poured in ice/water and extracted with EtOAc. The organic layers were washed with brine, dried over Na2SO4, and evaporated under reduced pressure to afford a yellow oil. After purification by flash column chromatography (CHCl3/MeOH 99/1), compound 3f was obtained as a white solid (24% yield, mp 118.0-119.0 °C). 1H NMR (CDCl3, 400 MHz): δH 1.01 (3H, t, J = 7.3 Hz, OCH2CH2CH3), 1.69-1.83 (2H, m, OCH2CH2CH3), 2.43-2.57 (4H, m, piperazine NCH2 x 2), 2.61-2.75 (4H, m, piperazine NCH2 x 2), 2.99-3.03 (2H, m, OCH2CH2N), 3.50 (2H, s, benzylic CH2), 3.91 (3H, s, OCH3), 3.97 (2H, t, J = 7.3 Hz, OCH2CH2CH3), 4.38-4.46 (2H, m, OCH2CH2N), 6.91 (2H, d, J = 8.7 Hz, H3’ and H5’), 7.21-7.43 (8H, m, H3, H5, H7, and benzylic Ar-H), 7.91-8.03 (3H, m, H8, H2’, and H6’).

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13

C NMR (CDCl3, 101 MHz): δC 10.52, 22.58, 52.98, 53.64, 55.53, 56.80, 62.94, 66.53, 69.61,

98.37, 99.89, 114.67, 120.69, 121.94, 127.22, 128.30, 128.51, 129.33, 130.59, 132.67, 145.11, 156.02, 156.98, 160.07, 160.75. Anal calcd for C32H37N3O3: C, 75.12; H, 7.29; N, 8.21; found: C, 75.32; H, 7.31; N, 8.19. 6-methoxy-4-[2’-piperazin-2’(1H)-ylethoxy]-2-(4’-propoxyphenyl)quinoline (3g). To a solution of compound 3f (0.34 g, 0.66 mmol) in dry MeOH (4 mL), ammonium formate (0.39 g, 3.27 mmol) and 10% Pd/C (0.30 g, 1:1 w/w) were added. The reaction mixture was stirred at 60 °C for 6 h. After cooling, Pd/C was filtered over Celite and to the filtrate was added cyclohexane (10 mL) to precipitate the exceeding ammonium formate. After the filtration and evaporation of the organic solvents, the obtained oil was purified by flash column chromatography (CH2Cl2/MeOH 98/2) to give compound 3g as a yellow solid (20% yield, mp 89.5-91.0 °C). 1H NMR (CDCl3, 400 MHz): δH 1.05 (3H, t, J = 7.6 Hz, OCH2CH2CH3), 1.711.89 (2H, m, OCH2CH2CH3), 2.58-2.77 (8H, m, piperazine CH2 x 4), 3.03 (2H, t, J = 5.6 Hz, OCH2CH2N), 3.89 (3H, s, OCH3), 3.95 (2H, t, J = 6.6 Hz, OCH2CH2CH3), 4.39 (2H, t, J = 5.7 Hz, OCH2CH2N), 6.95 (2H, d, J = 8.7 Hz, H3’ and H5’), 7.08 (1H, s, H3), 7.31 (1H, dd, J = 2.8 and 8.4 Hz, H7), 7.37 (1H, d, J = 2.8 Hz, H5), 7.90 (1H, d, J = 8.3 Hz, H8), 8.01 (2H, d, J = 8.9 Hz, H2’ and H6’).

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C NMR (CDCl3, 101 MHz): δC 10.54, 22.59, 48.59, 56.67, 55.55, 56.80,

66.72, 69.60, 98.37, 99.79, 114.65, 120.67, 122.01, 128.51, 130.59, 132.68, 145.12, 156.03, 156.97, 160.05, 160.77. Anal calcd for C25H31N3O3: C, 71.23; H, 7.41; N, 9.97; found: C, 71.02; H, 7.32; H, 9.99. N,N-diethyl-2-{[7-methoxy-2-(4-propoxyphenyl)quinolin-4-yl]oxy}ethanamine (4a).

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General procedure A (time = 14 h, T = 90 °C): starting from derivative 30 (0.30 g, 0.97 mmol) and using (2-chloroethyl)diethylamine hydrochloride, compound 4a was obtained after purification by flash column chromatography (CH2Cl2/MeOH 99/1) as a white solid (68% yield, mp 65.0-67.5 °C). 1H NMR (CDCl3, 400 MHz): δH 1.04 (3H, t, J = 7.5 Hz, OCH2CH2CH3), 1.14 (6H, t, J = 7.1 Hz, NCH2CH3 x 2), 1.81-1.88 (2H, m, OCH2CH2CH3), 2.73 (4H, q, J = 7.2 Hz, NCH2CH3 x 2), 3.07 (2H, t, J = 6.0 Hz, OCH2CH2N), 3.93 (3H, s, OCH3), 3.98 (2H, t, J = 6.6 Hz, OCH2CH2CH3), 4.34 (2H, t, J = 6.1 Hz, OCH2CH2N), 6.99 (2H, d, J = 8.8 Hz, H3’ and H5’), 7.02 (1H, s, H3,), 7.06 (1H, dd, J = 2.5 and 9.2 Hz, H6), 7.39 (1H, d, J = 2.4 Hz, H8), 7.948.06 (3H, m, H5, H2’, and H6’).

13

C NMR (CDCl3, 101 MHz): δC 10.54, 11.79, 22.58, 47.93,

51.29, 55.49, 66.79, 69.60, 96.84, 107.30, 114.63, 117.64, 122.80, 128.75, 132.63, 151.12, 158.96, 160.26, 161.11, 161.84. Anal calcd for C25H32N2O3: C, 73.50; H, 7.90; N, 6.86; found: C, 73.27; H, 7.92; N, 6.86. 7-methoxy-4-(2-piperidin-1-ylethoxy)-2-(4-propoxyphenyl)quinoline (4b). General procedure A (time = 14 h, T = 90 °C): starting from derivative 30 (0.30 g, 0.97 mmol) and using 1-(2-chloroethyl)piperidine hydrochloride, compound 4b, after purification by flash column chromatography (CH2Cl2/MeOH 98/2) was obtained as a white solid (41% yield, mp 92.0-94.0 °C). 1H NMR (CDCl3, 400 MHz): δH 1.04 (3H, t, J = 7.3 Hz, OCH2CH2CH3), 1.421.51 (2H, m, piperidine CH2), 1.61-1.69 (4H, m, piperidine CH2 x 2), 1.78-1.87 (2H, m, OCH2CH2CH3), 2.57-2.72 (4H, m, piperidine NCH2 x 2), 2.98 (2H, t, J = 6.0 Hz, OCH2CH2N), 3.93 (3H, s, OCH3), 3.96 (2H, t, J = 6.6 Hz, OCH2CH2CH3), 4.40 (2H, t, J = 6.0 Hz, OCH2CH2N), 6.99 (2H, d, J = 8.8 Hz, H3’ and H5’), 7.01 (1H, s, H3), 7.06 (1H, dd, J = 2.5 and 9.1 Hz, H6), 7.39 (1H, d, J = 2.5 Hz, H8), 7.99-8.02 (3H, m, H5, H2’, and H6’).

13

C NMR

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(CDCl3, 101 MHz): δC 10.54, 22.58, 23.93, 25.80, 55.07, 55.50, 57.52, 66.36, 69.59, 96.86, 107.29, 114.64, 117.64, 122.85, 129.74, 132.66, 151.12, 158.98, 160.26, 161.12, 161.77. Anal calcd for C26H32N2O3: C, 74.26; H, 7.67; N, 6.66; found: C, 74.05; H, 7.69; N, 6.68. 4-(2-azepan-1-ylethoxy)-7-methoxy-2-(4-propoxyphenyl)quinoline hydrochloride (4c). General procedure A (time = 14 h, T = 90 °C): starting from derivative 30 (0.50 g, 1.62 mmol) and using 1-(2-chloroethyl)azepane hydrochloride, compound 4c, after purification by flash column chromatography (CH2Cl2/MeOH 98:2) was obtained as a yellow oil (21% yield); then, compound 4c was dissolved into Et2O and HClgas was bubbled until obtaining compound 4c as a white hydrochloride solid that was collected by filtration (94% yield, mp 192.5-194.5 °C). 1H NMR (DMSO-d6, 400 MHz): δH 0.98 (3H, t, J = 7.4 Hz, OCH2CH2CH3), 1.57-2.03 (10H, m, OCH2CH2CH3 and azepane CH2 x 4), 3.20-3.30 (4H, m, azepane NCH2 x 2), 3.50-3.60 (2H, m, OCH2CH2N), 3.94 (3H, s, OCH3), 4.07 (2H, t, J = 6.6 Hz, OCH2CH2CH3), 5.05 (2H, m, OCH2CH2N), 7.20 (2H, d, J = 8.6 Hz, H3' and H5'), 7.38 (1H, dd, J = 2.0 and 9.0 Hz, H6), 7.64 (1H, s, H3), 8.05 (1H, m, H8), 8.28 (2H, d, J = 8.5 Hz, H2' and H6'), 8.43 (1H, d, J = 9.1 Hz, H5), 11.20 (1H, bs, NH). 13C NMR (DMSO-d6, 101 MHz): δC 10.80, 22.37, 23.02, 26.57, 54.39, 54.62, 56.61, 65.73, 69.99, 100.34, 101.21, 113.95, 115.55, 119.92, 123.78, 125.60, 131.59, 140.01, 155.81, 162.82, 163.86, 166.45. Anal calcd for C27H35ClN2O3: C, 68.85; H, 7.49; N, 5.95; found: C, 68.74; H, 7.51; N, 5.96. 4-[2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethoxy]-7-methoxy-2-(4propoxyphenyl)quinoline (4d). General procedure A (time = 20 h, T = 80 °C): starting from derivative 33 (0.40 g, 1.08 mmol) and using 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride, compound 4d, after

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Journal of Medicinal Chemistry

purification by flash column chromatography (CH2Cl2/EtOAc 90/10) was obtained as a white solid (21% yield, mp 108.5-110.0 °C. 1H NMR (CDCl3, 400 MHz): δH 1.03 (3H, t, J = 7.4 Hz, OCH2CH2CH3),

1.75-1.92

(2H,

m,

OCH2CH2CH3),

2.86

(2H,

t,

J

=

5.3

Hz,

tetrahydroisoquinoline CH2), 2.94 (2H, t, J = 5.8 Hz, tetrahydroisoquinoline NCH2), 3.15 (2H, t, J = 5.3 Hz, OCH2CH2N), 3.70-3.80 (8H, m, OCH3 x 2 and tetrahydroisoquinoline NCH2), 3.854.00 (5H, m, OCH3 and OCH2CH2CH3), 4.50 (2H, t, J = 4.1 Hz, OCH2CH2N), 6.48 (1H, s, H8''), 6.52 (1H, s, H5''), 6.98 (2H, d, J = 8.8 Hz, H3' and H5'), 7.06 (1H, s, H3), 7.08 (1H, dd, J = 2.4 and 9.1 Hz, H6), 7.43 (1H, s, H8), 8.02 (2H, d, J = 8.8 Hz, H2' and H6'), 8.05 (1H, d, J = 9.2 Hz, H5).

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C NMR (CDCl3, 101 MHz): δC 10.51, 22.57, 28.43, 51.63, 55.51, 55.93, 56.42, 66.95,

69.62, 96.91, 107.23, 109.43, 111.39, 114.67, 117.71, 122.88, 125.77, 125.96, 128.78, 132.45, 147.33, 147.69, 151.00, 158.91, 160.35, 161.23, 161.94. Anal calcd for C32H36N2O5: C, 72.70; H, 6.86; N, 5.30; found: C, 72.61; H, 6.89; N, 5.31. (3-{[7-methoxy-2-(4-propoxyphenyl)quinolin-4-yl]oxy}propyl)dimethylamine hydrochloride (4e). General procedure A (time = 15 h, T = 80 °C): starting from derivative 30 (0.50 g, 1.62 mmol) and using (3-chloropropyl)dimethylamine hydrochloride, compound 4e was obtained after purification by flash column chromatography (CH2Cl2/MeOH 98/2) as a yellow oil (33% yield); HClgas was bubbled into a solution in Et2O of the obtained oil to afford compound 4e as a white hydrochloride solid that was collected by filtration (yield 89%, mp 187.5-191.0° C). 1H NMR (DMSO-d6, 400 MHz): δH 0.98 (3H, t, J = 7.3 Hz, OCH2CH2CH3), 1.68-1.80 (2H, m, OCH2CH2CH3), 2.33-2.41 (2H, m, OCH2CH2CH2N), 2.75 (6H, s, N(CH3)2), 3.21-3.34 (2H, m, OCH2CH2CH2N), 3.95 (3H, s, OCH3), 4.05 (2H, t, J = 5.6 Hz, OCH2CH2CH3), 4.60 (2H, t, J = 5.9 Hz, OCH2CH2CH2N), 7.15 (2H, d, J = 8.7 Hz, H3' and H5'), 7.28 (1H, dd, J = 2.4 and 8.9

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Hz, H6), 7.58 (1H, s, H3), 8.08 (1H, m, H8), 8.18-8.30 (3H, m, H5, H2', and H6'), 11.14 (1H, bs, NH). 13C NMR (DMSO-d6, 101 MHz): δC 10.78, 22.37, 23.84, 42.40, 53.88, 56.62, 68.67, 70.01, 100.23, 101.04, 113.93, 115.58, 119.94, 124.81, 125.11, 131.62, 141.05, 155.78, 162.86, 163.89, 166.86. Anal calcd for C24H31ClN2O3: C, 66.89; H, 7.25; N, 6.50; found: C, 66.95; H, 7.24; N, 6.50. 4-[2-(4-benzylpiperazin-1-yl)ethoxy]-7-methoxy-2-(4-propoxyphenyl)quinoline (4f). General procedure A (time = 2 h, T = 80 °C): starting from derivative 30 (1.00 g, 3.23 mmol) and using 1-benzyl-4-(2-chloroethyl)piperazine, compound 4f was obtained after crystallization by Et2O as a white solid (75% yield, mp 123.5-125.0 °C). 1H NMR (CDCl3, 400 MHz): δH 1.02 (3H, t, J = 7.40 Hz, OCH2CH2CH3), 1.73-1.87 (2H, m, OCH2CH2CH3), 2.43-2.57 (4H, m, piperazine NCH2 x 2), 2.59-2.81 (4H, m, piperazine NCH2 x 2), 2.98 (2H, t, J = 5.9 Hz, OCH2CH2N), 3.44 (2H, s, benzylic CH2), 3.92 (3H, s, OCH3), 3.99 (2H, t, J = 6.6 Hz, OCH2CH2CH3), 4.38 (2H, t, J = 6.1 Hz, OCH2CH2N), 6.85-7.04 (3H, m, H3, H3', and H5'), 7.06 (1H, dd, J = 2.4 and 9.0 Hz, H6), 7.24-7.33 (5H, m, ArH x 5), 7.39-7.44 (1H, m, H8), 7.87-8.06 (3H, m, H5, H2', and H6').

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C NMR (CDCl3, 101 MHz): δC 10.52, 22.58, 52.93, 53.56, 55.48,

56.84, 62.92, 66.58, 69.62, 96.79, 107.29, 114.66, 117.68, 122.84, 127.23, 128.28, 128.75, 129.32, 132.63, 134.86, 154.35, 159.32, 160.29, 161.17, 161.82. Anal calcd for C32H37N3O3: C, 75.15; H, 7.29; N, 8.21; found: C, 75.27; H, 7.26; N, 8.19. 7-methoxy-4-(2-piperazin-1-ylethoxy)-2-(4-propoxyphenyl)quinoline (4g). To a solution of compound 4f (1.00 g, 1.95 mmol) in EtOH (150 mL), ammonium formate (0.62 g, 9.78 mmol) and Pd/C (1.00 g, 1/1:w/w) were added. The reaction mixture was stirred at rt for 1 h and then Pd/C was filtered over CeliteTM and the filtrate was evaporated to dryness under

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Journal of Medicinal Chemistry

reduced pressure to give a yellow oil. After purification by flash column chromatography (CH2Cl2/MeOH 98/2) compound 4g was obtained as a white solid (31% yield, mp 183.5-185.0 °C). 1H NMR (DMSO-d6, 400 MHz): δH 0.97 (3H, t, J = 7.3 Hz, OCH2CH2CH3), 1.71-1.76 (2H, m, OCH2CH2CH3), 2.78-2.83 (4H, m, piperazine NCH2 x 2), 2.87-2.93 (2H, m, OCH2CH2N), 3.01-3.06 (4H, m, piperazine NCH2 x 2), 3.84 (3H, s, OCH3), 3.98 (2H, t, J = 6.4 Hz, OCH2CH2CH3), 4.45-4.48 (2H, m, OCH2CH2N), 7.00 (2H, d, J = 8.5 Hz, H3' and H5'), 7.15 (1H, dd, J = 2.5 and 9.0 Hz, H6), 7.28-7.35 (2H, m, H3 and H8), 8.00 (1H, d, J = 9.1 Hz, H5), 8.17 (2H, d, J = 8.5 Hz, H2' and H6'), 8.85-9.00 (1H, bs, NH).

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C NMR (DMSO-d6, 101 MHz): δC

10.87, 22.51, 43.41, 50.00, 55.84, 56.42, 66.79, 69.52, 97.23, 107.86, 114.60, 114.84, 117.76, 123.16, 129.14, 131.96, 150.91, 158.07, 160.43, 161.13, 161.85. Anal calcd for C25H31N3O3: C, 71.23; H, 7.41; N, 9.97; found: C, 71.38; H, 7.39; N, 9.96. N,N-diethyl-2-{[8-methoxy-2-(4-propoxyphenyl)quinolin-4-yl]oxy}ethanamine (5a). General procedure A (time = 3 h, T = 90 °C): starting from derivative 18 (0.30 g, 0.97 mmol) and (2-chloroethyl)diethylamine hydrochloride, compound 5a was obtained after crystallization by cyclohexane as a white solid (76% yield, mp 111.0-111.5 °C). 1H NMR (DMSO-d6, 400 MHz): δH 1.04 (3H, t, J = 7.5 Hz, OCH2CH2CH3), 1.14 (6H, t, J = 6.3 Hz, NCH2CH3 x 2), 1.711.85 (2H, m, OCH2CH2CH3), 2.64-2.71 (4H, m, NCH2CH3 x 2), 3.01-3.11 (2H, m, OCH2CH2N), 3.97 (2H, t, J = 6.6 Hz, OCH2CH2CH3), 4.05 (3H, s, OCH3), 4.32-4.41 (2H, m, OCH2CH2N), 6.95-7.07 (3H, m, H7, H3’, and H5’), 7.19 (1H, s, H3), 7.34 (1H, t, J = 8.1 Hz, H6), 7.68 (1H, dd, J = 2.1 and 7.9 Hz, H5), 8.33 (2H, d, J = 8.9 Hz, H2’ and H6’).

13

C NMR (CDCl3, 101

MHz): δC 10.51, 11.80, 22.58, 47.95, 51.30, 56.09, 66.97, 69.58, 98.58, 108.43, 113.34, 114.57,

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121.20, 124.99, 128.86, 132.63, 141.09, 155.24, 157.16, 160.27, 161.77. Anal calcd for C25H32N2O3: C, 73.50; H, 7.90; N, 6.86; found: C, 73.34; H, 7.88; N, 6.87. 8-methoxy-4-(2-piperidin-1-ylethoxy)-2-(4-propoxyphenyl)quinoline (5b). General procedure A (time = 3 h, T = 90 °C): starting from derivative 18 (0.30 g, 0.97 mmol) and 1-(2-chloroethyl)piperidine hydrochloride, compound 5b was obtained after crystallization by cyclohexane as a white solid (86% yield, mp 107.0-108.5 °C). 1H NMR (CDCl3, 400 MHz): δH 1.04 (3H, t, J = 7.3 Hz, OCH2CH2CH3), 1.44-1.51 (2H, m, piperidine CH2), 1.63-1.72 (4H, m, piperidine CH2 x 2), 1.77-1.85 (2H, m, OCH2CH2CH3), 2.63-2.71 (4H, m, piperidine NCH2 x 2), 2.99 (2H, t, J = 5.5 Hz, OCH2CH2N), 3.97 (2H, t, J = 6.6 Hz, OCH2CH2CH3), 4.05 (3H, s, OCH3), 4.43 (2H, t, J = 5.8 Hz, OCH2CH2N), 6.97 (2H, d, J = 8.8 Hz, H3’ and H5’), 7.08 (1H, dd, J = 1.2 and 7.9 Hz, H7), 7.18 (1H, s, H3), 7.34 (1H, t, J = 7.8 Hz, H6), 7.69 (1H, d, J = 8.3 Hz, H5), 8.09 (2H, d, J = 8.9 Hz, H2’ and H6’). 13C NMR (CDCl3, 101 MHz): δC 10.54, 22.58, 23.85, 25.69, 55.03, 56.10, 57.45, 66.33, 69.56, 98.62, 108.41, 113.34, 114.56, 121.17, 125.04, 128.87, 132.57, 141.04, 155.20, 157.20, 160.26, 161.64. Anal calcd for C26H32N2O3: C, 74.26; H, 7.67; N, 6.66; found: C, 74.22; H, 7.68; N, 6.65. 4-(2-(azepan-1-yl)ethoxy)-8-methoxy-2-(4-propoxyphenyl)quinoline (5c). To a solution of derivative 20 (0.40 g, 1.08 mmol) in azepane (3 mL), K2CO3 (0.74 g, 5.38 mmol) was added. The reaction was stirred at 100 °C for 18 h. After cooling, Et2O was added to the mixture and the obtained solid was filtered. After crystallization by cyclohexane, compound 5c was obtained as a white solid (58% yield, mp 109.0-110.0 °C). 1H NMR (CDCl3, 400 MHz): δH 1.00 (3H, t, J = 7.0 Hz, OCH2CH2CH3), 1.57-1.64 (4H, m, azepane CH2 x 2), 1.67-1.79 (4H, m, azepane CH2 x 2), 1.85-1.91 (2H, m, OCH2CH2CH3), 2.75-2.88 (4H, m, azepane NCH2 x 2),

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Journal of Medicinal Chemistry

3.16 (2H, t, J = 6.0 Hz, OCH2CH2N), 3.98 (2H, t, J = 7.0 Hz, OCH2CH2CH3), 4.05 (3H, s, OCH3), 4.37 (2H, t, J = 6.0 Hz, OCH2CH2N), 6.96-7.07 (3H, m, H7, H3’ and H5’), 7.18 (1H, s, H3), 7.34 (1H, t, J = 8.2 Hz, H6), 7.69 (1H, d, J = 8.5 Hz, H5), 8.09 (2H, d, J = 8.7 Hz, H2’ and H6’).

13

C NMR (CDCl3, 101 MHz): δC 10.54, 22.58, 26.99, 27.79, 55.93, 56.09, 56.22, 66.85,

69.59, 98.59, 108.39, 113.38, 114.55, 121.22, 125.00, 128.88, 132.62, 141.04, 155.18, 157.19, 160.24, 161.81. Anal calcd for C27H34N2O3: C, 74.62; H, 7.89; N, 6.45; found: C, 74.59; H, 7.90; N, 6.45. 4-[2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethoxy]-8-methoxy-2-(4propoxyphenyl)quinoline (5d). In a MW vial, derivative 20 (0.10 g, 0.27 mmol), K2CO3 (0.19 g, 1.34 mmol), and 6,7dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride (0.19 g, 0.81 mmol) were suspended in dry DMF (5 mL). The mixture was irradiated by MW according the following conditions: time = 90 min, temperature = 100 °C, pressure = 5 bar, cooling = OFF. The reaction mixture was poured in ice/water and the obtained precipitate was filtered. After purification by flash column chromatography (CH2Cl2/Acetone 85/15), compound 5d was obtained as a yellow solid (19% yield, mp 112.0-113.0 °C). 1H NMR (CDCl3, 400 MHz): δH 1.00 (3H, t, J = 7.3 Hz, OCH2CH2CH3), 1.78-1.86 (2H, m, OCH2CH2CH3), 2.78-2.93 (2H, m, tetrahydroisoquinoline CH2), 2.98-3.03 (2H, m, tetrahydroisoquinoline NCH2), 3.18-2.27 (2H, m, tetrahydroisoquinoline NCH2), 3.78-3.91 (8H, m, OCH2CH2N and OCH3 x 2), 3.97 (2H, t, J = 6.6 Hz OCH2CH2CH3), 4.07 (3H, s, OCH3), 4.48-4.53 (2H, m, OCH2CH2N), 6.51 (1H, s, H5’’), 6.60 (1H, s , H8’’), 6.94 (2H, d, J = 8.7 Hz, H3’ and H5’), 7.07 (1H, d, J = 8.0 Hz, H7), 7.15 (1H, s, H3), 7.35 (1H, t, J = 7.8 Hz, H6), 7.70 (1H, d, J = 8.5 Hz, H5), 8.10 (2H, d, J = 8.7 Hz, H2’ and H6’).

13

C NMR

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(CDCl3, 101 MHz): δC 10.54, 22.58, 28.00, 51.47, 55.12, 55.92, 56.12, 66.70, 69.57, 98.69, 108.45, 109.34, 111.28, 113.32, 114.57, 121.15, 125.08, 125.91, 128.88, 132.52, 141.08, 147.39, 147.78, 155.24, 157.21, 160.29, 161.62. Anal calcd for C32H36N2O5: C, 72.70; H, 6.86; N, 5.30; found: C, 72.61; H, 6.87; N, 5.32. (3-{[8-methoxy-2-(4-propoxyphenyl)quinolin-4-yl]oxy}propyl)dimethylamine (5e). General procedure A (time = 20 h, T = 90 °C): starting from derivative 18 (0.40 g, 1.29 mmol) and using (3-chloropropyl)dimethylamine hydrochloride, compound 5e was obtained after purification by flash column chromatography (CH2Cl2/MeOH 95/5) as a white solid (25% yield, mp 86.0-87.0 °C). 1H NMR (CDCl3, 400 MHz): δH 1.00 (3H, t, J = 7.3 Hz, OCH2CH2CH3), 1.77-1.88 (2H, m, OCH2CH2CH3), 2.11-2.20 (2H, m, OCH2CH2CH2N), 2.31 (6H, s, N(CH3)2), 2.60 (2H, t, J = 7.2 Hz, OCH2CH2CH2N), 3.94 (2H, t, J = 6.4 Hz, OCH2CH2CH3), 4.07 (3H, s, OCH3), 4.37 (2H, t, J = 6.1 Hz, OCH2CH2CH2N), 6.94-7.05 (3H, m, H7, H3’ and H5’), 7.18 (1H, s, H3), 7.34 (1H, t, J = 8.1 Hz, H6), 7.72 (1H, d, J = 7.6 Hz, H5), 8.09 (2H, d, J = 8.2 Hz, H2’ and H6’). 13C NMR (CDCl3, 101 MHz): δC 10.51, 22.58, 27.18, 45.42, 56.09, 56.31, 66.46, 69.57, 98.54, 108.40, 113.36, 114.54, 121.26, 124.92, 128.88, 132.71, 141.06, 155.23, 157.20, 160.23, 161.93. Anal calcd for C24H30N2O3: C, 73.07; H, 7.67; N, 7.10; found: C, 74.21; H, 7.65; N, 7.08. 8-methoxy-4-(2-piperazin-1-ylethoxy)-2-(4-propoxyphenyl)quinoline (5g). In a MW vial, derivative 20 (0.29 g, 0.77 mmol) and piperazine (1.11 g, 15.38 mmol) were irradiated by MW according the following conditions: time = 15 min, temperature = 120 °C, pressure = 5 bar, cooling = OFF. The reaction mixture was poured in ice/water and 2N HCl was added to neutralize pH. The obtained precipitated was filtered and after purification by flash

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Journal of Medicinal Chemistry

column chromatography (Al2O3, CH2Cl2/MeOH 97/3) compound 5g was obtained as a white solid (55% yield, 167.0-168.0 °C). 1H NMR (CDCl3, 400 MHz): δH 1.05 (3H, t, J = 7.8 Hz, OCH2CH2CH3), 1.77-1.88 (2H, m, OCH2CH2CH3), 2.57-2.78 (4H, m, piperazine NCH2 x 2), 2.93 (2H, t, J = 4.9 Hz, piperazine NCH2), 3.02 (2H, t, J = 4.9 Hz, piperazine NCH2), 3.19 (2H, t, J = 4.5 Hz, OCH2CH2N), 3.97 (2H, t, J = 6.6 Hz, OCH2CH2CH3), 4.05 (3H, s, OCH3), 4.34-4.41 (2H, m, OCH2CH2N), 6.98 (2H, d, J = 7.1 Hz, H3' and H5'), 7.04 (1H, dd, J = 2.4 and 7.9 Hz, H7), 7.16 (1H, s, H3), 7.33 (1H, t, J = 7.7 Hz, H6), 7.67 (1H, dd, J = 2.4 and 8.4 Hz, H5), 8.06 (2H, d, J = 8.8 Hz, H2’ and H6’).

13

C NMR (CDCl3, 101 MHz): δC 10.49, 22.57, 43.83, 51.32,

53.51, 56.12, 66.74, 69.60, 98.53, 108.54, 113.39, 114.62, 121.06, 125.07, 130.79, 132.57, 154.59, 155.26, 157.14, 157.76, 160.31. Anal calcd for C25H31N3O3: C, 71.23; H, 7.41; N, 9.97; found: C, 71.35; H, 7.40; N, 9.96. (2-{[5,7-dimethoxy-2-(4-propoxyphenyl)quinolin-4-yl]oxy}ethyl)diethylamine (6a). General procedure A (time = 27 h, T = 90 °C): starting from derivative 31 (0.20 g, 0.59 mmol) and using (2-chloroethyl)diethylamine hydrochloride, compound 6a was obtained after purification by flash column chromatography (CH2Cl2/MeOH 98/2) as a white solid (25% yield, 90.0-92.0 °C). 1H NMR (CDCl3, 400 MHz): δH 1.04 (3H, t, J = 7.4 Hz, OCH2CH2CH3), 1.11 (6H, t, J = 7.4 Hz, NCH2CH3 x 2), 1.78-1.85 (2H, m, OCH2CH2CH3), 2.74 (4H, q, J = 6.9 Hz, NCH2CH3 x 2), 3.07 (2H, t, J = 5.5 Hz, OCH2CH2N), 3.88 (3H, s, OCH3), 3.91 (3H, s, OCH3), 3.97 (2H, t, J = 6.6 Hz, OCH2CH2CH3), 4.29 (2H, t, J = 5.7 Hz, OCH2CH2N), 6.42 (1H, d, J = 2.0 Hz, H6), 6.98-7.01 (3H, m, H3, H3’, and H5’), 7.03 (1H, d, J = 1.9 Hz, H8), 8.01 (2H, d, J = 8.7 Hz, H2’ and H6’). 13C NMR (CDCl3, 101 MHz): δC 10.52, 11.86, 22.57, 47.89, 51.55, 55.50, 55.90, 67.54, 69.60, 97.59, 98.05, 100.63, 114.60, 128.66, 132.25, 153.21, 157.83, 158.61,

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159.86, 160.29, 160.90, 163.79. Anal calcd for C26H34N2O4: C, 71.21; H, 7.81; N, 6.39; found: C, 71.49; H, 7.80; N, 6.38. 5,7-dimethoxy-4-(2-piperidin-1-ylethoxy)-2-(4-propoxyphenyl)quinoline (6b). General procedure A (time = 24 h, T = 90 °C): starting from derivative 31 (0.20 g, 0.59 mmol) and using 1-(2-chloroethyl)piperidine hydrochloride, compound 6b, after purification by flash column chromatography (CH2Cl2/MeOH 99/1), was obtained as a white solid (34% yield, 91.093.0 °C). 1H NMR (CDCl3, 400 MHz): 1.04 (3H. t, J = 7.2 Hz, OCH2CH2CH3), 1.38-1.47 (2H, m, piperidine CH2), 1.51-1.63 (4H, m, piperidine CH2 x 2), 1.79-1.85 (2H, m, OCH2CH2CH3), 2.51-2.69 (4H, m, piperidine NCH2 x 2), 2.93 (2H, t, J = 5.1 Hz, OCH2CH2N), 3.88 (3H, s, OCH3), 3.91 (3H, s, OCH3), 3.97 (2H, t, J = 6.3 Hz, OCH2CH2CH3), 4.32 (2H, t, J = 5.2 Hz, OCH2CH2N), 6.42 (1H, s, H6), 6.91-7.04 (4H, m, H3, H8, H3’, and H5’), 7.99 (2H, d, J = 8.2 Hz, H2’ and H6’).

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C NMR (CDCl3, 101 MHz): δC 10.51, 22.57, 24.14, 26.11, 55.27, 55.48,

55.92, 57.65, 67.32, 69.59, 97.55, 98.08, 100.63, 107.32, 114.60, 128.64, 132.33, 153.21, 157.86, 158.62, 160.26, 160.89, 163.78. Anal calcd for C27H34N2O4: C, 71.97; H, 7.61; N, 6.22; found: C, 72.04; H, 7.60; N, 6.22. 4-(2-azepan-1-ylethoxy)-5,7-dimethoxy-2-(4-propoxyphenyl)quinoline (6c). Under N2 atmosphere, to a solution of derivative 34 (0.15 g, 0.39 mmol) in azepane (1.50 mL, 12.47 mmol), K2CO3 (0.27 g, 1.93 mmol) was added portionwise and the reaction mixture was stirred at reflux for 3 h. Reaction mixture was poured in water and extracted with EtOAc. The organic layers were washed with brine, dried over Na2SO4, and evaporated to dryness under vacuum to give a brown oil. After purification by flash column chromatography (CH2Cl2/MeOH 99/1), compound 6c was obtained as a white solid (10% yield, 121.0-122.5 °C). 1H NMR

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Journal of Medicinal Chemistry

(CDCl3, 400 MHz): 1.04 (3H, t, J = 7.3 Hz, OCH2CH2CH3), 1.52-1.63 (4H, m, azepane CH2 x 2), 1.67-1.88 (6H, m, azepane CH2 x 2 and OCH2CH2CH3), 7.89-3.01 (4H, m, azepane NCH2 x 2), 3.19-3.27 (2H, m, OCH2CH2N), 3.78-4.02 (8H, m, OCH3 x 2 and OCH2CH2CH3), 4.25-4.31 (2H, m, OCH2CH2N), 6.43 (1H, d, J = 2.0 Hz, H6), 6.93-7.04 (4H, H3, H8, H3’, and H5’), 8.01 13

(2H, d, J = 8.6 Hz, H2’ and H6’).

C NMR (CDCl3, 101 MHz): δC 10.51, 22.57, 24.14, 27.05,

28.91, 55.64, 55.72, 56.25, 56.97, 69.60, 97.55, 98.14, 100.70, 108.36, 114.62, 128.68, 132.04, 141.65, 153.20, 156.31, 158.65, 160.34, 160.94. Anal calcd for C28H36N2O4: C, 72.39; H, 7.81; N, 6.03; found: C, 72.15; H, 7.83; N, 6.03. 4-[2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethoxy]-5,7-dimethoxy-2-(4propoxyphenyl)quinoline (6d). General procedure A (time = 36 h, T = 80 °C): starting from derivative 34 (0.20 g, 0.52 mmol) and using 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride, compound 6d, after purification by flash column chromatography (CH2Cl2/MeOH 99/1), was obtained as a white solid (26% yield, 128.0-130.0 °C). 1H NMR (CDCl3, 400 MHz): 1.04 (3H, t, J = 7.4 Hz, OCH2CH2CH3),

1.77-1.86

(2H,

m,

OCH2CH2CH3),

2.85

(2H,

t,

J

=

5.6

Hz,

tetrahydroisoquinoline CH2), 2.96 (2H, t, J = 6.4 Hz, tetrahydroisoquinoline NCH2), 3.12 (2H, t, J = 5.5 Hz, OCH2CH2N), 3.78-3.82 (8H, m, OCH3 x 2 and tetrahydroisoquinoline NCH2), 3.91 (3H, s, OCH3), 3.92 (3H, s, OCH3), 4.01 (2H, t, J = 5.9 Hz, OCH2CH2CH3), 4.42 (2H, t, J = 5.6 Hz, OCH2CH2N), 6.44 (1H, d, J = 2.0 Hz, H6), 6.50 (1H, s, H8’’), 6.59 (1H, s, H5’’), 6.97 (2H, d, J = 8.7 Hz, H3’ and H5’), 7.02 (1H, s, H3), 7.04 (1H, d, J = 2.1 Hz, H8), 7.99 (2H, d, J = 8.7 Hz, H2’ and H6’).

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C NMR (CDCl3, 101 MHz): δC 10.50, 22.57, 28.72, 51.78, 54.89, 55.51,

55.77, 55.94, 56.24, 56.64, 67.81, 69.60, 97.63, 98.11, 100.68, 107.31, 109.42, 111.38, 114.61,

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125.93, 126.46, 128.65, 132.27, 147.24, 147.58, 153.24, 157.84, 158.65, 160.29, 160.93, 163.72. Anal calcd for C33H38N2O6: C, 70.95; H, 6.86; N, 5.01; found: C, 71.11; H, 6.85; N, 5.00. (2-{[6,7-dimethoxy-2-(4-propoxyphenyl)quinolin-4-yl]oxy}ethyl)diethylamine (7a). General procedure A (time = 3 h, T = 90 °C): starting from derivative 32 (0.20 g, 0.61 mmol) and using (2-chloroethyl)diethylamine hydrochloride, compound 7a was obtained after purification by flash column chromatography (CHCl3/MeOH 99/1) as a yellow solid (30% yield, mp 116.0-118.0 °C). 1H NMR (CDCl3, 400 MHz): δH 1.05 (3H, t, J = 7.4 Hz, OCH2CH2CH3), 1.19 (6H, t, J = 6.3 Hz, NCH2CH3 x 2), 1.79-1.87 (2H, m, OCH2CH2CH3), 2.63-2.71 (4H, m, NCH2CH3 x 2), 3.11-3.23 (2H, m, OCH2CH2N), 3.96-4.05 (8H, m, OCH2CH2CH3 and OCH3 x 2), 4.37-4.45 (2H, m, OCH2CH2N), 7.00 (2H, d, J = 8.7 Hz, H3’ and H5’), 7.06 (1H, s, H8), 7.37 (1H, s, H3), 7.50 (1H, s, H5), 8.00 (2H, d, J = H2’ and H6’).

13

C NMR (CDCl3, 101 MHz): δC

10.55, 11.70, 22.59, 48.01, 51.09, 55.98, 56.13, 66.60, 69.60, 97.24, 99.74, 108.14, 114.41, 114.65, 128.50, 132.70, 146.11, 148.62, 152.50, 156.65, 160.04, 160.66. Anal calcd for C26H34N2O4: C, 71.21; H, 7.81; N, 6.39; found: C, 71.42; H, 7.79; N, 6.39. 6,7-dimethoxy-4-(2-piperidin-1-ylethoxy)-2-(4-propoxyphenyl)quinoline (7b). General procedure A (time = 3 h, T = 90 °C): starting from derivative 32 (0.28 g, 0.87 mmol) and using 1-(2-chloroethyl)piperidine hydrochloride, compound 7b was obtained after purification by flash column chromatography (CH2Cl2/MeOH 98/2) as a pink solid (27% yield, 75.0-77.0 °C). 1H NMR (CDCl3, 400 MHz): δH 1.04 (3H, t, J = 7.4 Hz, OCH2CH2CH3), 1.441.53 (2H, m, piperidine CH2), 1.63-1.71 (4H, m, piperidine CH2 x 2), 1.77-1.87 (2H, m, OCH2CH2CH3), 2.59-2.74 (4H, m, piperidine NCH2 x 2), 2.97-3.05 (2H, m, OCH2CH2N), 3.964.02 (8H, m, OCH2CH2CH3 and OCH3 x 2), 4.39-4.48 (2H, m, OCH2CH2N), 6.99 (2H, d, J = 8.7

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HZ, H3’ and H5’), 7.06 (1H, s, H8), 7.37 (1H, s, H3), 7.42 (1H, s, H5), 7.99 (2H, d, J = 8.7 Hz, H2’ and H6’). 13C NMR (CDCl3, 101 MHz): δC 10.55, 22.60, 23.89, 25.73, 26.91, 55.09, 56.01, 57.43, 66.22, 69.59, 97.33, 99.77, 108.14, 114.47, 114.65, 128.50, 132.74, 146.14, 148.62, 152.51, 156.57, 160.02, 160.60. Anal calcd for C27H34N2O4: C, 71.97; H, 7.61; N, 6.22; found: C, 71.85; H, 7.63; N, 6.23. 4-(2-azepan-1-ylethoxy)-6,7-dimethoxy-2-(4-propoxyphenyl)quinoline (7c). To a solution of derivative 35 (0.16 g, 0.41 mmol) in azepane (4 mL, 48.89 mmol), K2CO3 (0.29 g, 2.07 mmol) was added portionwise. The reaction mixture was stirred at reflux for 9 h, then was poured in water and the obtained precipitate was filtered. After purification by flash chromatography column (CH2Cl2/MeOH 98/2), derivative 7c was obtained as a pink solid (27% yield, 110.5-112.5 °C). 1H NMR (CDCl3, 400 MHz): δH 1.04 (3H, t, J = 7.4 Hz, OCH2CH2CH3), 1.58-1.62 (4H, m, azepane CH2 x 2), 1.67-1.74 (4H, m, azepane CH2 x 2), 1.78-1.87 (2H, m, OCH2CH2CH3), 2.65-2.74 (4H, m, azepane NCH2 x 2), 3.18 (2H, t, J = 5.6 Hz, OCH2CH2N), 3.96-4.02 (8H, m, OCH2CH2CH3 and OCH3 x 2), 4.39 (2H, t, J = 5.5 Hz, OCH2CH2N), 6.99 (2H, d, J = 8.7 Hz, H3’ and H5’), 7.05 (1H, s, H8), 7.37 (1H, s, H3), 7.41 (1H, s, H5), 7.99 (2H, d, J = 8.7 Hz, H2’ and H6’). 13C NMR (CDCl3, 101 MHz): δC 10.52, 22.59, 27.00, 27.76, 28.02, 55.92, 55.99, 56.15, 68.23, 70.17, 97.27, 99.80, 108.17, 114.15, 114.64, 128.49, 132.82, 146.17, 148.61, 152.50, 156.68, 160.01, 160.70. Anal calcd for C28H36N2O4: C, 72.39; H, 7.81; N, 6.03; found: C, 72.50; H, 7.79; N, 6.02. 4-[2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethoxy]-6,7-dimethoxy-2-(4propoxyphenyl)quinoline (7d).

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General procedure A (time = 48 h, T = 80 °C): starting from derivative 35 (0.10 g, 0.26 mmol) and using 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride, compound 7d was obtained after purification by flash column chromatography (CH2Cl2/EtOAc 95/5) as a a white solid (32% yield, mp 156.0-158.5 °C). 1H NMR (CDCl3, 400 MHz): δH 1.01 (3H, t, J = 7.2 Hz, OCH2CH2CH3), 1.77-1.84 (2H, m, OCH2CH2CH3), 2.68-2.87 (2H, m, tetrahydroisoquinoline CH2), 2.91-2.97 (2H, m, tetrahydroisoquinoline NCH2), 3.15 (2H, t, J = 5.4 Hz, OCH2CH2N), 3.64-4.01 (16H, m, OCH2CH2CH3, tetrahydroisoquinoline NCH2, and OCH3 x 4), 4.47 (2H, t, J = 5.7 Hz, OCH2CH2N), 6.49 (1H, s, H8’’), 6.58 (1H, s, H5’’), 6.97 (2H, d, J = 8.6 Hz, H3’ and H5’), 7.06 (1H, s, H8), 7.39 (1H, s, H3), 7.41 (1H, s, H5), 7.97 (2H, d, J = 8.5 Hz, H2’ and H6’). 13

C NMR (CDCl3, 101 MHz): δC 10.53, 23.77, 28.52, 51.70, 55.89, 55.98, 56.11, 56.48, 56.79,

61.15, 66.91, 69.01, 97.40, 99.24, 108.09, 109.29, 110.30, 111.33, 114.63, 125.77, 126.02, 128.49, 129.72, 146.11, 147.28, 147.63, 148.63, 152.24, 160.03, 160.72, 165.43. Anal calcd for C33H38N2O6: C, 70.95; H, 6.86; N, 5.01; found: C, 71.03; H, 6.87; N, 4.99. 2-{[6,8-dimethoxy-2-(4-propoxyphenyl)quinolin-4-yl]oxy}-N,N-diethylethanamine (8a). General procedure A (time = 3 h, T = 90 °C): starting from derivative 41 (0.20 g, 0.59 mmol) and using (2-chloroethyl)diethylamine hydrochloride, compound 8a was obtained after crystallization by EtOH as a white solid (52% yield, mp 108.0-109.0° C). 1H NMR (CDCl3, 400 MHz): δH 1.02 (3H, t, J = 7.4 Hz, OCH2CH2CH3), 1.12 (6H, t, J = 7.1 Hz, NCH2CH3 x 2), 1.771.86 (2H, m, OCH2CH2CH3), 2.68 (4H, q, J = 7.1 Hz, NCH2CH3 x 2), 3.06 (2H, t, J = 5.6 Hz, OCH2CH2N), 3.90 (3H, s, OCH3), 3.96 (2H, t, J = 6.4 Hz, OCH2CH2CH3), 4.01 (3H, s, OCH3), 4.30 (2H, t, J = 5.5 Hz, OCH2CH2N), 6.68 (1H, d, J = 2.1 Hz, H7), 6.96-7.00 (3H, m, H5, H3’, and H5’), 7.16 (1H, s, H3), 8.04 (2H, d, J = 8.7 Hz, H2’ and H6’). 13C NMR (CDCl3, 101 MHz): δC 10.52, 12.20, 22.59, 48.15, 51.24, 55.40, 56.13, 67.29, 69.56, 91.39, 98.93, 101.43, 114.53,

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121.43, 128.55, 132.78, 137.52, 154.84, 156.29, 157.25, 159.95, 161.02. Anal calcd for C26H34N2O4: C, 71.21; H, 7.81; N, 6.39; found: C, 71.30; H, 7.81; N, 6.37. 6,8-dimethoxy-4-(2-piperidin-1-ylethoxy)-2-(4-propoxyphenyl)quinoline (8b). General procedure A (time = 3 h, T = 90 °C): starting from derivative 41 (0.20 g, 0.597 mmol) and using 1-(2-chloroethyl)piperidine hydrochloride, compound 8b was obtained after crystallization by cyclohexane as a white solid (56% yield, mp 121.0-121.5 °C). 1H NMR (CDCl3, 400 MHz): δH 1.04 (3H, t, J = 7.4 Hz, OCH2CH2CH3), 1.40-1.46 (2H, m, piperidine CH2), 1.58-1.64 (4H, m, piperidine CH2 x 2), 1.77-1.86 (2H, m, OCH2CH2CH3), 2.53-2.61 (4H, m, piperidine NCH2 x 2), 2.95 (2H, t, J = 5.5 Hz, OCH2CH2N), 3.90 (3H, s, OCH3), 3.95 (2H, t, J = 6.7 Hz, OCH2CH2CH3), 4.01 (3H, s, OCH3), 4.38 (2H, t, J = 6.1 Hz, OCH2CH2N), 6.68 (1H, d, J = 2.5 Hz, H7), 6.96-6.99 (3H, m, H5, H3’, and H5’), 7.17 (1H, s, H3), 8.04 (2H, d, J = 8.7 Hz, H2’ and H6’).

13

C NMR (CDCl3, 101 MHz): δC 10.52, 22.59, 24.12, 26.09, 55.19, 55.43,

56.14, 57.60, 66.69, 69.56, 91.41, 99.03, 101.41, 114.54, 121.45, 128.55, 132.76, 137.52, 154.86, 156.30, 157.27, 159.86, 160.91. Anal calcd for C27H34N2O4: C, 71.97; H, 7.61; N, 6.22; found: C, 72.04; H, 7.60; N, 6.22. 4-(2-azepan-1-ylethoxy)-6,8-dimethoxy-2-(4-propoxyphenyl)quinoline (8c). To a solution of compound 42 (0.25 g, 0.645 mmol) in azepane (3.00 mL), K2CO3 (0.45 g, 3.23 mmol) was added and the reaction mixture was stirred at reflux for 3 h. The reaction mixture was concentrated under vacuum and then poured into ice/water. The obtained solid was filtered and then crystalized by Et2O to afford compound 8c as a white solid (65% yield, mp 107.5-109.0 °C). 1H NMR (CDCl3, 400 MHz): δH 1.04 (3H, t, J = 7.4 Hz, OCH2CH2CH3), 1.53-1.61 (4H, m, azepane CH2 x 2), 1.63-1.71 (4H, m, azepane CH2 x 2), 1.77-1.86 (2H, m, OCH2CH2CH3), 2.83-

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2.86 (4H, m, azepane NCH2 x 2), 3.13 (2H, t, J = 5.5 Hz, OCH2CH2N), 3.90 (3H, s, OCH3), 3.96 (2H, t, J = 6.4 Hz, OCH2CH2CH3), 4.02 (3H, s, OCH3), 4.33 (2H, t, J = 6.0 Hz, OCH2CH2N), 6.68 (1H, d, J = 1.6 Hz, H7), 6.96-7.02 (3H, m, H5, H3’, and H5’), 7.17 (1H, s, H3), 8.04 (2H, d, J = 8.5 Hz, H2’ and H6’).

13

C NMR (CDCl3, 101 MHz): δC 10.52, 22.59, 27.02, 28.33, 55.42,

55.91, 56.03, 56.25, 67.20, 69.56, 91.42, 98.98, 101.42, 114.54, 121.48, 128.56, 132.81, 137.52, 154.86, 156.29, 157.26, 159.95, 161.04. Anal calcd for C28H36N2O4: C, 72.39; H, 7.81; N, 6.03; found: C, 72.41; H, 7.80; N, 6.02. 4-[2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethoxy]-6,8-dimethoxy-2-(4propoxyphenyl)quinoline (8d). General procedure A (time = 8 h, T = 80 °C): starting from derivative 42 (0.25 g, 0.65 mmol) and using 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride, compound 8d was obtained after purification by flash column chromatography (CH2Cl2/MeOH 99/1) as a white solid (5% yield, mp 116.0-118.0 °C). 1H NMR (CDCl3, 400 MHz): δH 1.04 (3H, t, J = 7.2 Hz, OCH2CH2CH3), 1.77-1.84 (2H, m, OCH2CH2CH3), 2.73-2.79 (2H, m, tetrahydroisoquinoline CH2), 2.85-2.91 (2H, m, tetrahydroisoquinoline NCH2), 3.16 (2H, t, OCH2CH2N), 3.78-3.87 (8H, m, tetrahydroisoquinoline NCH2 and OCH3 x 2), 3.91 (3H, s, OCH3), 3.96 (2H, t, J = 6.8 Hz, OCH2CH2CH3), 4.03 (3H, s, OCH3), 4.48 (2H, t, J = 5.6 Hz, OCH2CH2N), 6.50 (1H, s, H8’’), 6.59 (1H, s, H5’’), 6.70 (1H, d, J = 1.8 Hz, H7), 6.94 (2H, d, J = 8.6 Hz, H3’ and H5’), 7.02 (1H, d, J = 1.8 Hz, H5), 7.21 (1H, s, H3), 8.03 (2H, d, J = 7.5 Hz, H2’ and H6’). 13C NMR (CDCl3, 101 MHz): δC 10.52, 22.59, 28.58, 51.72, 55.45, 55.90, 56.14, 56.24, 56.50, 57.02, 67.03, 69.56, 91.40, 99.13, 101.44, 109.36, 111.37, 114.55, 121.44, 125.84, 126.15, 128.55, 132.71, 137.57,

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147.29, 147.64, 154.87, 156.34, 157.33, 159.99, 160.87. Anal calcd for C33H38N2O6: C, 70.95; H, 6.86; N, 5.01; found: C, 71.10; H, 6.85; N, 4.99. (2Z)-3-Hydroxy-3-(2-hydroxy-6-methoxyphenyl)-1-(4-propoxyphenyl)prop-2-en-1-one (10). In dry conditions, 1-(2-hydroxy-6-methoxyphenyl)ethan-1-one 937 (4.00 g, 24.10 mmol) dissolved in dry THF (14 mL) was added dropwise at 0 °C to a suspension of t-BuOK (3.24 g, 29.00 mmol) in dry THF (14 mL). The mixture was stirred for 30 min at rt and then the freshly prepared 4-propoxybenzoyl chloride (4.76 g, 26.40 mmol) was dripped at 0 °C. The mixture was stirred at reflux overnight and then concentrated under reduced pressure, dissolved in CH2Cl2, washed with 5% sodium bicarbonate solution (2 x 200 mL) and brine. The organic layers were dried over Na2SO4 and evaporated to dryness under reduced pressure. After crystallization by EtOH compound 10 was obtained as a yellow solid (47% yield, mp 107.5-109.0 °C). 1H NMR (CDCl3, 400 MHz): δH 1.06 (3H, t, J = 7.4 Hz, OCH2CH2CH3), 1.89-1.81 (2H, m, OCH2CH2CH3), 3.45 (3H, s, OCH3), 4.01 (2H, t, J = 6.5 Hz, OCH2CH2CH3), 4.58 (1H, s, C=CH), 6.61 (1H, d, J = 8.4 Hz, H3’), 6.98 (2H, d, J = 8.8 Hz, H3 and H5), 7.30-7.32 (1H, m, H5’), 7.35-7.38 (1H, m, H4’), 7.95 (2H, d, J = 8.9 Hz, H2 and H6), 12.55 (1H, s, OH), 13.10 (1H, s, OH). 5-Methoxy-2-(4-propoxyphenyl)-4H-chromen-4-one (11). To a solution of derivative 10 (1.00 g, 3.00 mmol) in glacial acetic acid (3 mL), H2SO4 (0.10 mL) was added. The mixture was stirred at reflux for 40 min, poured in ice/water, and then filtered. After purification by flash column chromatography (CHCl3/MeOH 97/3) compound 11 was obtained as a white solid (79% yield, mp 143.5-144.5 °C). 1H NMR (CDCl3, 200 MHz): δH 0.96 (3H, t, J = 7.4 Hz, OCH2CH2CH3), 1.81-1.90 (2H, m, OCH2CH2CH3), 3.84-3.97 (5H, m,

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OCH2CH2CH3 and OCH3), 6.70 (1H, s, H3), 6.91 (1H, d, J = 8.8 Hz, H6), 7.05 (2H, d, J = 8.8 Hz, H3’and H5’), 7.16 (1H, dd, J = 1.1 and 8.5 Hz, H8), 7.51 (1H, t, J = 8.3 Hz, H7), 7.89 (2H, d, J = 8.9 Hz, H2’ and H6’). 5-methoxy-2-(4-propoxyphenyl)quinolin-4-ol (13). To a stirring solution of flavone 11 (2.00 g, 6.40 mmol) in ethyl orthoformate (100 mL), 70% perchloric acid (0.97 g, 9.70 mmol) was slowly added and then the mixture was stirred at 60 °C for 20 h. After filtration, compound 12 was obtained as yellow solid in 73% yield and was dissolved as such in a stirring 25% aqueous ammonia solution (60 mL) and vigorously stirred at rt for 5 h. The mixture was filtered and the solid washed with water. After purification by column chromatography (Al2O3, CHCl3 100%), compound 13 was obtained as a yellow solid (81% yield, mp 163.5-165.5 °C). 1H NMR (DMSO-d6, 400 MHz): δH 0.98 (3H, t, J = 7.4 Hz, OCH2CH2CH3), 1.72-1.77 (2H, m, OCH2CH2CH3), 3.86-4.01 (5H, m, OCH2CH2CH3 and OCH3), 6.61 (1H, s, H3), 6.97 (1H, d, J = 8.4 Hz, H6), 7.04 (2H, d, J = 8.9 Hz, H3’and H5’), 7.11 (1H, dd, J = 1.0 and 8.4 Hz, H8), 7.54 (1H, t, J = 8.4 Hz, H7), 7.87 (2H, d, J = 8.9 Hz, H2’ and H6’), 10.21 (1H, s, OH). Anal calcd for C19H19NO3: C, 73.77; H, 6.19; N, 4.53; found: C, 73.81; H, 6.20; N, 4.51. Ethyl (2E,Z)-3-[(4-methoxyphenyl)amino]-3-(4-propoxyphenyl)acrylate (15). To a solution of ethyl 3-oxo-3-(4-propoxyphenyl)propanoate 1424 (5.00 g, 20 mmol) in benzene (150 mL), 4-methoxyaniline (12.30 g, 100.00 mmol) and p-TsOH (1.00 g, 4.00 mmol) were added. The reaction was refluxed for 19 h by using Dean-Stark trap. After cooling, the solvent was evaporated and water (100 mL) was added and the aqueous phase was acidified with 2 N HCl (pH = 5); then, the mixture was extracted with CH2Cl2 and the organic layers were washed

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with brine, dried over Na2SO4, and evaporated to dryness under reduced pressure. After purification by flash column chromatography (cyclohexane/Et2O 90/10), compound 15 was obtained as a white solid (67% yield, mp 89.0-91.0 °C). 1H NMR (CDCl3, 200 MHz): δH 1.06 (3H, t, J = 7.4 Hz, OCH2CH2CH3), 1.35 (3H, t, J = 7.1 Hz, OCH2CH3), 1.78-1.85 (2H, m, OCH2CH2CH3), 3.76 (3H, s, OCH3), 3.93 (2H, t, J = 7.1 Hz, OCH2CH2CH3), 4.23 (2H, q, J = 7.1 Hz, OCH2CH3), 4.95 (1H, s, C=CH), 6.79-6.84 (4H, m, H2’, H3’, H5’, and H6’), 6.87 (2H, d, J = 8.8 Hz, H3 and H5), 7.29 (2H, d, J = 7.9 Hz, H2 and H6/8), 10.23 (1H, bs, NH). Ethyl (2E,Z)-3-[(2-methoxyphenyl)amino]-3-[4-(1-methylbutyl)phenyl]acrylate (16). Following the same procedure reported for compound 15, starting from 1424 and using 2methoxyaniline, compound 16, after purification by flash column chromatography (petroleum ether/EtOAc 95/5), was obtained as a white solid (28% yield, mp 84.0-86.0 °C). 1H NMR (CDCl3, 200 MHz): δH 1.07 (3H, t, J = 7.4 Hz, OCH2CH2CH3), 1.34 (3H, t, J = 7.1 Hz, OCH2CH3), 1.75-1.89 (2H, m, OCH2CH2CH3), 3.89-4.00 (5H, m, OCH2CH2CH3 and OCH3), 4.25 (2H, q, J = 7.1 Hz, OCH2CH3), 5.01 (1H, s, C=CH), 6.30 (1H, d, J = 8.0 Hz, H3’), 6.566.63 (1H, m, H4’), 6.81-6.91 (4H, m, H3, H5, H5’, and H6’), 7.32 (2H, d, J = 8.8 Hz, H2 and H6), 10.32 (1H, bs, NH). 6-Methoxy-2-(4-propoxyphenyl)quinolin-4-ol (17). Derivative 15 (1.00 g, 2.80 mmol) was suspended in Dowtherm A (6 mL) and stirred at 240 °C for 2 h. Then, cyclohexane (10 mL) was added to the mixture and the solid obtained was filtered. After purification by flash column chromatography (CH2Cl2/MeOH 98/2) compound 17 was obtained as a white solid (88% yield, mp >300 °C). 1H NMR (DMSO-d6, 200 MHz): δH 1.02 (3H, t, J = 7.4 Hz, OCH2CH2CH3), 1.74-1.84 (2H, m, OCH2CH2CH3), 3.87 (3H, s, OCH3), 4.04

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(2H, t, J = 6.5 Hz, OCH2CH2CH3), 6.30 (1H, s, H3), 7.14 (2H, d, J = 8.9 Hz, H3’ and H5’), 7.32 (1H, dd, J = 2.9 and 9.1 Hz, H7), 7.52 (1H, d, J = 2.7 Hz, H5), 7.73-7.80 (3H, m, H8, H2’, and H6’), 11.60 (1H, s, OH). Anal calcd for C19H19NO3: C, 73.77; H, 6.19; N, 4.53; found: C, 73.80; H, 6.20; N, 4.53. 8-Methoxy-2-(4-propoxyphenyl)quinolin-4-ol (18). Following the same procedure reported for compound 17 and starting from derivative 16, compound 18, after purification by flash column chromatography (CH2Cl2/MeOH 98/2) was obtained as a white solid (90% yield, mp 227.0-228.0 °C). 1H NMR (DMSO-d6, 200 MHz): δH 1.02 (3H, t, J = 7.3 Hz, OCH2CH2CH3), 1.70-1.87 (2H, m, OCH2CH2CH3), 4.00-4.16 (5H, m, OCH2CH2CH3 and OCH3), 6.29 (1H, s, H3), 7.11 (2H, d, J = 6.8 Hz, H3’ and H5’), 7.24-7.35 (2H, m, H6 and H7), 7.60-7.80 (3H, m, H5, H2’, and H6’), 10.61 (1H, s, OH). Anal calcd for C19H19NO3: C, 73.77; H, 6.19; N, 4.53; found: C, 73.74; H, 6.19; N, 4.55. 4-(2’-chloroethoxy)-6-methoxy-2-(-4’-propoxyphenyl)quinoline (19). General procedure A (time = 5 h, T = 80 °C): starting from derivative 17 (3.00 g, 9.69 mmol) and using 1-bromo-2-chloroethane, compound 19 was obtained after crystallization by Et2O as a light brown solid (55% yield, mp 119.0-121.0°C). 1H NMR (CDCl3, 400 MHz): δH 1.00 (3H, t, J =

7.1 Hz, OCH2CH2CH3), 1.80-1.95 (2H, m, OCH2CH2CH3), 3.95-4.15 (7H, m, OCH2CH2Cl,

OCH2CH2CH3, and OCH3), 4.50 (2H, t, J = 6.1 Hz OCH2CH2Cl), 7.05 (2H, d, J = 8.8 Hz, H3’ and H5’), 7.10 (1H, s, H3), 7.40 (1H, dd, J = 2.9 and 9.2 Hz, H7), 7.45 (1H, d, J = 3.1 Hz, H5), 8.00-8.10 (3H, m, H8, H2’, and H6’). Anal calcd for C21H22ClNO3: C, 67.83; H, 5.96; N, 3.77; found: C, 67.80; H, 5.95; N, 3.78.

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4-(2-chloroethoxy)-8-methoxy-2-(4-propoxyphenyl)quinoline

(20)

and

8-methoxy-2-(4-

propoxyphenyl)-4-(vinyloxy)quinoline (21). General procedure A (time = 2 h, T = 80 °C): starting from derivative 18 (3.00 g, 9.70 mmol) and 1-bromo-2-chloroethane, compounds 20 and 21 were obtained after purification by flash column chromatography (cyclohexane/EtOAc 80/20) as white solids. 20 (Rf < by TLC): (55% yield, mp 131.0-136.0 °C). 1H NMR (CDCl3, 400 MHz): δH 1.00 (3H, t, J = 7.3 Hz, OCH2CH2CH3), 1.87-1.93 (2H, m, OCH2CH2CH3), 3.90-4.00 (4H, m, OCH2CH2CH3 and OCH2CH2Cl), 4.05 (3H, s, OCH3), 4.50 (2H, t, J = 7.1 Hz, OCH2CH2Cl), 6.90-7.00 (2H, m, H3’ and H5’), 7.01 (1H, dd, J = 1.1 and 8.5 Hz, H7), 7.12 (1H, s, H3), 7.35 (1H, t, J = 9.0 Hz, H6), 7.75 (1H, dd, J = 1.5 and 9.0 Hz, H5), 8.10 (2H, d, J = 7.7 Hz, H2’ and H6’). Anal calcd for C21H22ClNO3: C, 67.83; H, 5.96; N, 3.77; found: C, 67.78; H, 5.95; N, 3.77. 21 (Rf > by TLC): (18% yield, mp 112-114 °C). 1H NMR (CDCl3, 400 MHz): δH 1.00 (3H, t, J = 7.3 Hz, OCH2CH2CH3), 1.83-1.89 (2H, m, OCH2CH2CH3), 3.95 (2H, t, J = 7.3 Hz, OCH2CH2CH3), 4.05 (3H, s, OCH3), 4.75 (1H, dd, J = 1.9 and 6.1 Hz, vinylic-H), 5.10 (1H, dd, J = 1.9 and 14 Hz, vinylic-H), 6.85 (1H, dd, J = 6.0 and 14 Hz, vinylic-H), 6.90-6.95 (2H, m, H3’ and H5’), 7.01 (1H, dd, J = 1.1 and 8.0 Hz, H7), 7.20 (1H, s, H3), 7.35 (1H, t, J = 8.0 Hz, H6), 7.70 (1H, dd, J = 1.2 and 8.0 Hz, H5), 8.00-8.10 (2H, m, H2’ and H6’). Anal calcd for C21H21NO3: C, 75.20; H, 6.31; N, 4.18; found: C, 75.13; H, 6.28; N, 4.19. N-(3-methoxyphenyl)-4-propoxybenzamide (24).24 To a solution of 3-methoxyaniline 22 (5.46 g, 44.40 mmol) and Et3N (11.60 mL, 83.20 mmol) in dry THF (30 mL), 4-propoxybenzoyl chloride (5.50 g, 27.70 mmol) was added dropwise. The reaction mixture was stirred at rt for 14 h, then poured in 2 N HCl solution and the solid obtained

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was filtrated. After crystallization by EtOH, compound 2424 was obtained as a white solid (92% yield, mp 112.0-114.0 °C). N-(3,5-dimethoxyphenyl)-4-propoxybenzamide (25). Following the same procedure reported for compound 2424 but starting from 23, after trituration by a mixture of cyclohexane/EtOAc, compound 25 was obtained as a white solid (98% yield, 126.5-127.5 °C). 1H NMR (CDCl3, 200 MHz): δH 1.10 (3H, t, J = 7.3 Hz, OCH2CH2CH3), 1.841.93 (2H, m, OCH2CH2CH3), 3.86 (6H, s, OCH3 x 2), 4.03 (2H, t, J = 6.6 Hz, OCH2CH2CH3), 6.32 (1H, t, J = 2.21 Hz, H4’), 6.94 (1H, d, J = 2.2 Hz, H2’), 7.01 (2H, d, J = 8.7 Hz, H3 and H5), 7.68-7.74 (1H, m, H6’), 7.86 (2H, d, J = 8.3 Hz, H2 and H6). N-(2-acetyl-5-methoxyphenyl)-4-propoxybenzamide (27). In dry conditions at 0 °C, to a solution of derivative 2424 (6.95 g, 24.38 mmol) and SnCl4 (25.41 g, 97.54 mmol) in dry CH2Cl2 (100 mL), acetyl chloride (7.65 g, 97.54 mmol) was added dropwise and the mixture was stirred to rt for 19 h. The reaction mixture was poured in ice/water, acidified with 2 N HCl, and extracted with CH2Cl2. The organic layers were washed with brine, dried over Na2SO4, and evaporated to dryness under reduced pressure. After purification by flash column chromatography (petroleum ether/EtOAc 90/10), compound 27 was obtained as a white solid (43% yield, mp 120.0-122.5 °C). 1H NMR (CDCl3, 200 MHz): δH 1.06 (3H, t, J = 7.5 Hz, OCH2CH2CH3), 1.76-1.97 (2H, m, OCH2CH2CH3), 2.70 (3H, s, COCH3), 3.97 (3H, s, OCH3), 4.03 (2H, t, J = 6.6 Hz, OCH2CH2CH3), 6.68 (1H, dd, J = 2.6 and 8.9 Hz, H4’), 7.04 (2H, d, J = 8.9 Hz, H3 and H5), 7.90 (1H, d, J = 9.0 Hz, H3’), 8.10 (2H, d, J = 9.8 Hz, H2 and H6), 8.72 (1H, d, J = 2.6 Hz, H6’), 13.06 (1H, s, NH).

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N-(2-acetyl-3,5-dimethoxyphenyl)-4-propoxybenzamide (28). Following the same procedure reported for compound 27 but starting from 25, after crystallization from a mixture of cyclohexane/EtOAc, compound 28 was obtained as a light green solid (49% yield, 133.0-135.0 °C). 1H NMR (CDCl3, 200 MHz): δH 1.07 (3H, t, J = 7.2 Hz, OCH2CH2CH3), 1.79-1.91 (2H, m, OCH2CH2CH3), 2.64 (3H, s, COCH3), 3.90 (3H, s, OCH3), 3.93 (3H, s, OCH3), 4.02 (2H, t, J = 6.6 Hz, OCH2CH2CH3), 6.24 (1H, s, H4’), 6.99 (2H, d, J = 8.8 Hz, H3 and H5), 8.02 (2H, d, J = 8.7 Hz, H2 and H6), 8.27 (1H, s, H6’). N-(2-acetyl-4,5-dimethoxyphenyl)-4-propoxybenzamide (29). Following the same procedure reported for compound 2424 but starting from 26, after trituratation in Et2O/EtOH, compound 29 was obtained as a white solid (73% yield, 122.0-124.0 °C). 1H NMR (CDCl3, 200 MHz): 1.10 (3H, t, J = 7.3 Hz, OCH2CH2CH3), 1.80-1.98 (2H, m, OCH2CH2CH3), 2.71 (3H, s, COCH3), 3.97 (3H, s, OCH3), 4.01-4.08 (5H, m, OCH2CH2CH3 and OCH3), 7.05 (2H, d, J = 6.9 Hz, H3 and H5), 7.37 (1H, s, H6’), 8.08 (2H, d, J = 6.8 Hz, H2 and H6), 8.83 (1H, s, H3’). 7-methoxy-2-(4-propoxyphenyl)quinolin-4-ol (30). To a mixture of NaH (0.83 g, 34.71) in dry DMF (20 mL), a solution of derivative 27 (2.27 g, 6.94 mmol) in dry DMF (15 mL) was added dropwise. The reaction mixture was stirred at 90 °C for 12 h, then poured in ice/water, and acidified with 2 N HCl up to pH = 3. The obtained precipitate was filtered to give a white solid. After purification by flash column chromatography (petroleum ether/EtOAc 70/30) compound 30 was obtained as a white solid (65% yield, mp 258.0-260.0 °C). 1H NMR (DMSO-d6, 200 MHz): δH 1.02 (3H, t, J = 7.3 Hz, OCH2CH2CH3),

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1.67-1.88 (2H, m, OCH2CH2CH3), 3.88 (3H, s, OCH3), 4.04 (2H, t, J = 6.5 Hz, OCH2CH2CH3), 6.26 (1H, s, H3), 6.93 (1H, dd, J = 2.4 and 8.9 Hz, H6), 7.13 (2H, d, J = 8.8 Hz, H3’ and H5’), 7.27 (1H, d, J = 2.5 Hz, H8), 7.81 (2H, d, J = 8.9 Hz, H2’ and H6’), 8.00 (1H, d, J = 8.9 Hz, H5), 11.40 (1H, bs, OH). Anal calcd for C19H19NO3: C, 73.77; H, 6.19; N, 4.53; found: C, 73.71; H, 6.18; N, 4.54. 5,7-dimethoxy-2-(4-propoxyphenyl)quinolin-4-ol (31). To a solution of the derivative 28 (2.45 g, 6.88 mmol) in t-BuOH (40 mL), t-BuOK (2.70 g, 24.08 mmol) was added portionwise and the reaction mixture was stirred at 70 °C for 24 h. The reaction mixture was poured in ice/water and acidified to pH 2 with 2 N HCl to give a precipitate that was filtered. After crystallization by EtOH, compound 31 was obtained as a white solid (72% yield, 113.0-115.0 °C). 1H NMR (DMSO-d6, 400 MHz): δH 0.91 (3H, t, J = 7.2 Hz, OCH2CH2CH3), 1.66-1.77 (2H, m, OCH2CH2CH3), 3.87 (6H, s, OCH3 x 2), 3.99 (2H, t, J = 6.7 Hz, OCH2CH2CH3), 6.60 (1H, d, J = 2.1 Hz, H8), 6.93 (1H, s, H3), 7.13 (2H, d, J = 8.7 Hz, H3’ and H5’), 7.17 (1H, d, J = 2.3 Hz, H6), 7.87 (2H, d, J = 8.9 Hz, H2’ and H6’), 10.15 (1H, s, OH). Anal calcd for C20H21NO4: C, 70.78; H, 6.24; N, 4.13; found: C, 70.83; H, 6.25; N, 4.11. 6,7-dimethoxy-2-(4-propoxyphenyl)quinolin-4-ol (32). Following the same procedure reported for compound 31 but starting from 29, after crystallization from a mixture of Et2O/EtOH, compound 32 was obtained as a white solid (41% yield, mp 169.5-172.0 °C). 1H NMR (DMSO-d6, 400 MHz): δH 1.00 (3H, t, J = 7.2 Hz, OCH2CH2CH3), 1.69-1.74 (2H, m, OCH2CH2CH3), 3.87 (3H, s, OCH3), 3.90 (3H, s, OCH3), 4.04 (2H, t, J = 6.7 Hz, OCH2CH2CH3), 6.73 (1H, s, H8), 7.17 (2H, d, J = 8.9 Hz, H3’ and H5’),

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7.43 (1H, s, H3), 7.48 (1H, s, H5), 7.85 (2H, d, J = 8.9 Hz, H2’ and H6’), 10.17 (1H, s, OH). Anal calcd for C20H21NO4: C, 70.78; H, 6.24; N, 4.13; found: C, 70.81; H, 6.24; N, 4.12. 4-(2-chloroethoxy)-7-methoxy-2-(4-propoxyphenyl)quinoline (33). General procedure A (time = 15 h, T = 80 °C): starting from derivative 30 (1.00 g, 3.23 mmol) and using 1-bromo-2-chloroethane, compound 33 was obtained after purification by flash column chromatography (petroleum ether/EtOAc 90/10) (36% yield, mp 116.0-118.0 °C). 1H NMR (CDCl3, 400 MHz): δH 1.00 (3H, t, J = 7.4 Hz, OCH2CH2CH3), 1.75-1.95 (2H, m, OCH2CH2CH3), 3.95-4.05 (7H, m, OCH3, OCH2CH2Cl, OCH2CH2CH3), 4.50 (2H, t, J = 5.8, Hz, OCH2CH2Cl), 6.87-7.04 (3H, m, H8, H3’, and H5’), 7.05 (1H, dd, J = 3.5 and 7.8 Hz, H6), 7.45 (1H, s, H3), 7.95-8.05 (3H, m, H5, H2’, and H6’). Anal calcd for C21H22ClNO3: C, 67.83; H, 5.96; N, 3.77; found: C, 67.88; H, 5.98; N, 3.76. 4-(2-chloroethoxy)-5,7-dimethoxy-2-(4-propoxyphenyl)quinoline (34). General procedure A (time = 15 h, T = 80 °C): starting from derivative 31 (0.86 g, 2.63 mmol) and using 1-bromo-2-chloroethane, compound 34 was obtained after crystallization by Et2O/EtOH as a white solid (71% yield, 199.0-201.0 °C). 1H NMR (CDCl3, 200 MHz): δH 1.07 (3H, t, J = 7.4 Hz, OCH2CH2CH3), 1.74-1.93 (2H, m, OCH2CH2CH3), 3.81-4.07 (10H, m, OCH3 x 2, OCH2CH2CH3, and OCH2CH2Cl), 4.74 (2H, t, J = 4.9 Hz, OCH2CH2Cl), 6.49 (1H, d, J = 2.0 Hz, H8), 6.92 (1H, s, H6), 6.99 (2H, d, J = 8.3 Hz, H3’ and H5’), 8.19 (2H, d, J = 8.9 Hz, H2’ and H6’), 8.38 (1H, s, H3). 4-(2-chloroethoxy)-6,7-dimethoxy-2-(4-propoxyphenyl)quinoline (35).

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General procedure A (time = 6 h, T = 80 °C): starting from derivative 32 (0.30 g, 0.89 mmol) and using 1-bromo-2-chloroethane, compound 35 was obtained after purification by flash column chromatography (CHCl3/MeOH 99/1) as a yellow solid (45% yield, mp 208.0-210.0 °C). 1

H NMR (DMSO-d6, 200 MHz): δH 1.02 (3H, t, J = 7.0 Hz, OCH2CH2CH3), 1.70-1.88 (2H, m,

OCH2CH2CH3), 3.87 (3H, s, OCH3), 3.99 (3H, s, OCH3), 4.11 (2H, t, J = 6.5 Hz, OCH2CH2CH3), 4.21 (2H, t, J = 5.0 Hz, OCH2CH2Cl), 4.92 (2H, t, J = 4.8 Hz, OCH2CH2Cl), 7.22 (2H, d, J = 8.7 Hz, H3’ and H5’), 7.48 (1H, s, H8), 7.66 (1H, s, H3), 7.97 (1H, s, H5), 8.21 (2H, d, J = 8.7 Hz, H2’ and H6’). N-(2,4-dimethoxyphenyl)-4-propoxybenzamide (37). Following the same procedure reported for compound 2424 but starting from 36, after trituration with Et2O/EtOH, compound 37 was obtained as a brown solid (22% yield, mp 97.0-98.5 °C). 1HNMR (CDCl3, 200 MHz): δH 1.09 (3H, t, J = 7.4 Hz, OCH2CH2CH3), 1.79-1.96 (2H, m, OCH2CH2CH3), 3.85 (3H, s, OCH3), 3.92 (3H, s, OCH3), 4.01 (2H, t, J = 6.5 Hz, OCH2CH2CH3), 6.54-6.61 (2H, m, H3’ and H5’), 6.99 (2H, d, J = 8.6 Hz, H3 and H5), 7.88 (2H, d, J = 8.6 Hz, H2 and H6), 8.31 (1H, s, NH), 8.43 (1H, d, J = 9.6 Hz, H6’). N-(2, 4-dimethoxyphenyl) -4-propoxybenzenecarboximidoyl chloride (38). Under N2 atmosphere, to a solution of compound 37 (2.19 g, 6.95 mmol) in benzene (20 mL) at 0 °C, PCl5 (1.59 g, 7.65 mmol) was added portionwise. The reaction mixture was stirred at 0 °C for 4 h, then benzene and POCl3 were evaporated under vacuum to obtain compound 38 as a brown solid that was used as such for the next step (100% yield). 1H-NMR (CDCl3, 200 MHz): δH 1.10 (3H, t, J = 7.4 Hz, OCH2CH2CH3), 1.80-1.87 (2H, m, OCH2CH2CH3), 3.80 (3H, s, OCH3), 3.90 (3H, s, OCH3), 4.05 (2H, t, J = 6.6 Hz, OCH2CH2CH3), 6.50 (1H, s, H3’), 6.60 (2H, d, J = 8.8

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Hz, H3 and H5), 7.05 (2H, d, J = 8.6 Hz, H2 and H6), 7.20 (1H, d, J = 9.2 Hz, H6’), 8.40 (1H, d, J = 8.8 Hz, H5’). Ethyl-6,8-dimethoxy-4-oxo-2-(4-propoxyphenyl)-1,4-dihydroquinoline-3-carboxylate (40). Under N2 atmosphere, to a suspension of NaH (1.16 g, 29.16 mmol) in dry DMF (12 mL), diethyl malonate (3.10 g, 19.41 mmol) was added dropwise and stirred at rt for 30 min. Then, a solution of compound 38 (2.40 g, 7.19 mmol) in dry DMF (8 mL) was slowly added dropwise. The reaction mixture was stirred at 80 °C for 16 h, then poured in ice/water and extracted with EtOAc. The collected organic layers were washed with brine, dried over Na2SO4 and evaporated under vacuum to obtain intermediate 39 as a brown oil. As such, 39 was heated in neat conditions at 170 °C for 7 h. Then, the reaction mixture was allowed to cool up to rt and the crude product was triturated with Et2O to afford compound 40 as a solid (25% yield. mp 109.0110.0° C). 1H-NMR (CDCl3, 200 MHz): δH 1.01-1.20 (6H, m, OCH2CH2CH3 and OCH2CH3), 1.79-1.97 (2H, m, OCH2CH2CH3), 3.94 (3H, s, OCH3), 3.97-4.07 (5H, m, OCH3 and OCH2CH2CH3), 4.23 (2H, q, J = 7.1 Hz, OCH2CH3), 6.75 (1H, d, J = 2.1 Hz, H7), 7.02 (2H, d, J = 8.6 Hz, H3’ and H5’), 7.38 (1H, d, J = 2.0 Hz, H5), 7.54 (2H, d, J = 8.5 Hz, H2’ and H6’), 10.34 (1H, s, OH). 6,8-dimethoxy-2-(4-propoxyphenyl)quinolin-4-ol (41). In a MW vial, to a suspension of KOH (2.00 g, 35.64 mmol) in EtOH (10 mL), derivative 40 (0.82 g, 1.99 mmol) was added portionwise. The reaction was carried out under MW irradiation employing the following conditions: temperature = 150 °C, time = 30 min, P = 15 bar, Cooling = ON. The reaction mixture was concentrated under vacuum, poured into ice/water, and 2N HCl was added to neutralize pH. After filtration and purification by flash column chromatography (CHCl3/MeOH 95/5), compound 41 was obtained as a white solid (68% yield, mp 191.0-191.5

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°C). 1H-NMR (DMSO-d6, 200 MHz): δH 1.03 (3H, t, J = 7.4 Hz, OCH2CH2CH3), 1.75-1.82 (2H, m, OCH2CH2CH3), 3.94 (3H, s, OCH3), 4.00-4.10 (5H, m, OCH3 and OCH2CH2CH3), 7.05 (1H, s, H3), 7.12-7.19 (4H, m, H5, H7, H3’, and H5’), 7.80 (2H, d, J = 8.8 Hz, H2’ and H6’). ). Anal calcd for C20H21NO4: C, 70.78; H, 6.24; N, 4.13; found: C, 70.75; H, 6.23; N, 4.14. 4-(2-chloroethoxy)-6,8-dimethoxy-2-(4-propoxyphenyl)quinoline (42). General procedure A (time = 6 h, T = 80 °C): starting from derivative 41 (1.25 g, 3.69 mmol) and using 1-bromo-2-chloroethane, compound 42 was obtained after crystallization by Et2O/EtOH as a white solid (52% yield, mp 129.5-131.0 °C). 1H NMR (CDCl3, 200 MHz): δH 1.10 (3H, t, J = 7.3 Hz, OCH2CH2CH3), 1.87-1.91 (2H, m, OCH2CH2CH3), 3.99-4.09 (10H, m, OCH3 x 2, OCH2CH2Cl, and OCH2CH2CH3), 4.57 (2H, t, J = 5.8 Hz, OCH2CH2Cl), 6.77 (1H, d, J = 2.6 Hz, H7), 7.02-7.10 (3H, m, H5, H3’, and H5’), 7.19 (1H, s, H3), 8.10 (2H, d, J = 8.7 Hz, H2’ and H6’). Author contribution Corresponding author *(S.S.) E-mail: [email protected]. Phone: +39 075 585 5130. Fax: +39 075 585 5115 Acknowledgments RC was supported by a grant from the “Società Italiana per la Terapia Antinfettiva (S.I.T.A.)”. Abbreviations

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AMR, antimicrobial resistance; CPX, ciprofloxacin; EPI, efflux pump inhibitor; EtBr, ethidium bromide; MPC, minimum potentiating concentration; MLM, mouse liver microsome; MW, microwave; PAINS, pan-assay interference compounds; PK, pharmacokinetic. Bibliography (1)

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Sabatini, S.; Gosetto, F.; Iraci, N.; Barreca, M. L.; Massari, S.; Sancineto, L.; Manfroni, G.; Tabarrini, O.; Dimovska, M.; Kaatz, G. W.; Cecchetti, V. Re-Evolution of the 2Phenylquinolines: Ligand-Based Design, Synthesis, and Biological Evaluation of a Potent New Class of Staphylococcus aureus NorA Efflux Pump Inhibitors to Combat Antimicrobial Resistance. J. Med. Chem. 2013, 56, 4975–4989.

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