European Journal of Medicinal Chemistry 79 (2014) 413e421

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Original article

4-Oxo-1,4-dihydro-quinoline-3-carboxamides as BACE-1 inhibitors: Synthesis, biological evaluation and docking studies Peng Liu, Yan Niu*, Chao Wang, Qi Sun, Yaya Zhai, Jiapei Yu, Jing Sun, Fengrong Xu, Gang Yan, Wenjie Huang, Lei Liang, Ping Xu* Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 November 2013 Received in revised form 7 April 2014 Accepted 7 April 2014 Available online 12 April 2014

In this work, we report a series of new 4-oxo-1,4-dihydro-quinoline-3-carboxamide derivatives as bsecretase (BACE-1) inhibitors. Supported by docking study, a small library of derivatives were designed, synthesized and biologically evaluated in vitro. The studies revealed that the most potent analog 14e (IC50 ¼ 1.89 mM) with low cellular cytotoxicity and high predicted blood brain barrier permeability, could serve as a good structure for further modification. Ó 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Alzheimer’s disease BACE-1 inhibitors 4-Oxo-1,4-dihydro-quinoline-3carboxamide Docking study

1. Introduction Alzheimer’s disease (AD) is a progressive neurodegenerative disease of brain that leads to irreversible loss of neurons and dementia. This disease is characterized in clinic by loss of brain function, affecting memory, language and learning skills, which inevitably leads to incapacitation and death. The pathological hallmarks of AD pathology include the deposition of b-amyloid peptides (Ab) in the neural parenchyma and the formation of intracellular neurofibrillary tangles [1,2]. The amyloid hypothesis states that Ab peptides are derived from the b-amyloid precursor protein (APP). The cleavage of APP by b-secretase (BACE) produces a soluble amyloid precursor protein-b (sAPPb) and a membrane bound C-terminal fragment called C99. Then, g-secretase cleaves C99 to release Ab40 and Ab42 peptides. Ab42 is prone to selfassemble into fibrils and is the major Ab component in amyloid plagues. An increased production of Ab could induce cell death and ultimately lead to dementia [3]. Considering the role of BACE in APP metabolism, inhibition of BACE is considered a very promising approach for AD treatment [4]. BACE is a transmembrane aspartyl protease that exists in two isoforms with BACE-1 located in the central nervous system and

* Corresponding authors. E-mail addresses: [email protected] (Y. Niu), [email protected] (P. Xu). http://dx.doi.org/10.1016/j.ejmech.2014.04.025 0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

BACE-2 expressed mainly in the periphery [5]. Therefore, BACE-1 is believed to be the most promising therapeutic target for the treatment of AD [6]. Over a decade ago, medicinal chemists in industry and academia have developed many inhibitors of BACE-1, but only few of these compounds have entered into clinic research [7e9]. The initial work in the design of BACE-1 inhibitors was concentrated on peptidomimetic inhibitors [10,11]. Although some of them were very potent in vitro, the majority of the inhibitors were not able to demonstrate significant activity in CNS. In recent years, more research was focused on developing nonpeptidomimetic BACE-1 inhibitors, which were much smaller in size and more BBB permeable, and therefore, more potent for antiAlzheimer’s disease agent development [12,13]. In this paper, we report the design and synthesis of a variety of non-peptidic BACE-1 inhibitors bearing the scaffold of 4-oxo-1,4dihydro-quinoline-3-carboxamide, which was generated from our previous in silico virtual screening [14]. Their potencies to inhibit BACE-1 in vitro were evaluated using Fluorescence Resonance Energy Transfer (FRET) assay. 2. Design Using in silico screening and biological test, we had identified a series of compounds as weak inhibitors against BACE-1 in our previous research [14]. Among them, compound 1 was selected as starting structure in this work for further optimization for the

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following reasons: 1) its simple chemical structure allows for exploiting a series of novel compounds; 2) docking results (Fig. 1B) revealed the quinolin-4-one moiety occupied S1 pocket and the NH group interacted with the catalytic residues of Asp32 through hydrogen bonds (H-bonds); 3) the 4-oxo-1,4-dihydro-quinoline-3carboxamide scaffold was considered as a privileged structure for drug discovery [15], which is found in many biologically active natural products and synthetic therapeutic agents [16,17]. Docking result suggested there were several key interactions between 1 and BACE-1, among which two key H-bonds were formed between the quinolin-4-one ring and the catalytic Asp32 and Thr72, respectively. The other two H-bonds were formed by the 3-substitution with Asp228 in the S1 pocket and Arg235 in the S20 pocket. The above observations indicated that increased potency might be achieved by the introduction of proper substituents, which were expected to extend into S1/S3 pockets, at C-6 position of the quinolin-4-one core. Considering the S3 pocket could accommodate large hydrophobic ligands [18,19] and the S1 pocket was also highly hydrophobic and approximately spherical in shape comprising the aromatic residues like Phe108 and Trp115 [20], a series of the 6-N(3-(trifluoromethyl)phenyl)sulfamoyl or 6-o-tolyl 4-oxo-1,4dihydro-quinoline-3-carboxamides were designed and prepared [21e23], supposing that 6-o-tolyl substitution could orient in the S1 pocket properly forming pep stacking interaction with the aromatic residues, and the 6-sulfamoyl group could extend deeper into the S3 pocket to improve potency [20,24,25]. 3. Chemistry The structures of designed compounds were shown in Table 1 and they were synthesized following the route shown in Scheme 1 and 2. As shown in Scheme 1, 4-oxo-6-(N-(3-(trifluoromethyl)phenyl) sulfamoyl)-1,4-dihydro-quinoline-3-carboxamide analogs 8aeg were prepared in six steps [26]. Starting from commercially available 3-(trifluoromethyl)aniline and 4-nitrobenzensulfonyl chloride 2, sulfonamide 3 was yielded, followed by reduction of eNO2 under H2 atmosphere to afford the aniline 4. Condensation of 4 with diethyl ethoxymethylene malonate gave the diethyl 2-((phenylamino)methylene)malonate derivative 5, which was converted to ethyl quinolone-3-carboxylate 6 by treatment with polyphosphoric acid (PPA) and phosphorus oxychloride (POCl3) [27]. After hydrolysis of the ester with NaOH, the resulting carboxylic acid 7 was then treated with N,N,N0 ,N0 -tetramethyl-o-(1H-benzotriazol-1-yl) uronium hexafluorophosphate (HBTU), N,N-diisopropylethylamine (DIPEA) and various amine to give the desired amides 8aeg. In Scheme 2, 4-bromoaniline was transformed to the diethyl 2(phenylamino)methylene malonate derivative 10 by heating with

diethyl ethoxymethylene malonate, and the quinolone product 11 was obtained from 10 with the treatment of PPA and POCl3 in a yield of 77.27%. Then, Suzuki coupling of 11 with o-tolyl boronic acid was employed to give 12 [28]. Finally, the carboxylic acid 13, which was generated from the hydrolysis of ester 12 in presence of NaOH, was condensated with various amines to give 14aeh. 4. Biological evaluations The BACE-1 inhibitory evaluation was performed using a Fluorescence Resonance Energy Transfer (FRET) assay kit supplied by Pan Vera (kit P2985, Madison, WI, USA). The kit protocol was followed by minor modification that the total assay volume was reduced to 15 or 20 mL. Compounds 8aeg and 14aeh were tested at a concentration of 10 mM and their percentages of BACE-1 inhibition were given in Table 1. The most active compounds (% inhibition > 50) were selected to test at more concentrations so that IC50s were determined by using the linear regression parameters. IC50 values were calculated using Graph Pad Prism Software. The cytotoxicity of the discovered active compounds (Table 2) was also evaluated using Human Embryonic Kidney 293 cells (HEK293), which were purchased from the cell bank of the Shanghai Institute of Cell Biology. The cell viability was determined by the CellTiter GloÔ luminescent cell viability assay (Promega). The method to determine the number of viable cells in culture was based on quantitation of ATP present, which signals the presence of metabolically active cells. Staurosporine (SigmaeAldrich) was used as a positive control. The IC50 value was calculated from the dosee response curves generated by plotting the percentage of the viable cells versus test concentrations on a logarithmic scale using Sigma Plot 10.0 software. To evaluate the capabilities of the acquired compounds to penetrate BBB (blood brain barrier), the ADME/T module within Discovery Studio 2.5 software [29] was utilized. The log BB (the logarithm value of brain to plasma concentration ratio) values for each compound were calculated based on a quantitative linear regression model derived from over 800 compounds database that are known to enter the CNS after oral administration. According to the log BB values, the compounds can be classified into four levels, which include very high penetrants (log BB  0.7), high penetrants (0  log BB < 0.7), medium penetrants (0.52 < log BB < 0) and low penetrants (log BB  0.52). 5. Results and discussion All synthesized 6-substituted 4-oxo-1,4-dihydro-quinoline-3carboxamide derivatives were tested for BACE-1 inhibitory potencies with data given in Table 1.

Fig. 1. Predicted binding pose of compound 1 in the active site of BACE-1 (PDB ID: 1TQF).

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Table 1 BACE-1 inhibitory activities of 6-substituted 4-oxo-1,4-dihydro-quinoline-3-carboxamide derivatives.

NO.

1 8a 8b 8c 8d 8e 8f 8g 14a 14b 14c 14d 14e 14f 14g 14h a b c

R

H N-(3-(trifluoromethyl)-phenyl)sulfamoyl N-(3-(trifluoromethyl)-phenyl)sulfamoyl N-(3-(trifluoromethyl)-phenyl)sulfamoyl N-(3-(trifluoromethyl)-phenyl)sulfamoyl N-(3-(trifluoromethyl)-phenyl)sulfamoyl N-(3-(trifluoromethyl)-phenyl)sulfamoyl N-(3-(trifluoromethyl)-phenyl)sulfamoyl o-tolyl o-tolyl o-tolyl o-tolyl o-tolyl o-tolyl o-tolyl o-tolyl

R1

Benzo[d][1,3]dioxol- 5-ylmethyl 4-Fluorobenzyl 3-Fluorobenzyl 4-Methoxybenzyl 3-Methoxybenzyl Furan-2-ylmethyl Pyridine-3ylmethyl Cyclohexyl 4-Fluorobenzyl 3-Fluorobenzyl 4-(Trifluoromethyl)-benzyl 3-(Trifluoromethyl)-benzyl 3-Bromobenzyl Furan-2-ylmethyl Pyridine-3ylmethyl Cyclohexyl

MW

322 519 519 531 531 491 502 493 386 386 436 436 446 358 369 360

BACE-1

BACE-1

Inhibition (%)a,b

IC50 (mM)a

11.3 30.4 84.6 50.4 73.5 71.1 35.7 65.6 13.9 12.3 38.1 67.4 77.6 32.9 10.2 77.8

               

0.2 0.1 3.2 1.9 0.7 3.9 1.6 1.8 0.6 0.6 1.8 2.9 4.9 0.1 0.5 0.7

n.dc n.d 1.85  0.13 7.04  0.48 2.20  0.14 1.61  0.09 n.d 7.18  0.36 n.dc n.d n.d 6.18  0.23 1.89  0.09 n.d n.d 11.53  0.57

values are mean  S.D. of two independent experiments for BACE-1 inhibition. % inhibition of BACE-1 activity at concentration of 10 mM of the tested compound. n.d. ¼ not determined.

Scheme 1. Synthesis of 4-oxo-6-(N-(3-(trifluoromethyl)phenyl)sulfamoyl)-1,4-dihydro-quinoline-3-carboxamide derivatives. (Reagents and conditions: (i) pyridine, 0  C, 3e6 h; (ii) H2, Pd/C, 6 h, rt; (iii) diethyl ethoxymethylene malonate, 130  C, 2 h; (iv) PPA, POCl3, 70  C, 12 h; (v) NaOH, 100  C, 2 h; (vi) HBTU, DIPEA, CH2Cl2, ReNH2, rt, 10 h.)

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Scheme 2. Synthesis of 6-o-tolyl-4-oxo-1,4-dihydro-quinoline-3-carboxamide derivatives. (Reagents and conditions: (i) diethyl ethoxymethylene malonate, 130  C, 2 h; (ii) PPA, POCl3, 70  C, 12 h; (iii) o-tolyl boronic acid, Pd(PPh3)4, K2CO3, H2O, THF, 80  C, 10 h; (iv) NaOH, 100  C, 2 h; (v) HBTU, DIPEA, CH2Cl2, ReNH2, rt, 10 h.)

Compared with the starting compound 1, all acquired compounds with sulfamoyl substitution at the C-6 position (8ae8g) showed significantly improved potencies. As for the various R1, phenyl ring with a fluorine atom substituted at meta- position (8b) exhibited better potency than para- position (8a) did. The metasubstituted phenyl ring with a fluorine atom (8b) or methoxy group (8d) seemed to contribute concurrently to potency. Different from the other analogs, 8g with non-aromatic cyclohexyl substitution at R1 also demonstrated good enzymatic potency (IC50 ¼ 7.18 mM). To explore the binding modes of the 6-sulfamoyl1,4-dihydro-quinoline-3-carboxamides, one of the most active inhibitors 8d was selected as representation and docked to the crystal structure of BACE-1 (PDB: 1TQF). Seven H-bonds were observed in the binding mode as shown in Fig. 2A, among which two were formed between the carbonyl oxygen on the quinolin-4-one moiety and Gln73 in the S10 pocket, and another two were formed by the carbonyl group substituted at 3-position of quinolin-4-one ring Table 2 Cytotoxic profiles and predicted BBB permeabilities of selected compounds. NO.

Molecular weight

IC50 (mM)a,b

Predicted log BB

8b 8c 8d 8e 8g 14d 14e 14h Staurosporine

519.09 531.11 531.11 491.08 493.13 436.14 446.06 360.18 e

>50 >50 >50 >50 >50 >50 >50 >50 0.056

Undefinedc Undefined Undefined Undefined Undefined 0.411 0.351 0.205 n.dd

a b c d

IC50 values are the means of at least two experiments. Cytotoxicity of tested compound at concentration of 50 mM. Undefined ¼ no predicted result. n.d. ¼ not determined.

with Thr72. Additionally, the two NH groups within the quinolin-4one ring and 3-carboxamide moiety respectively formed another two critical H-bonds with the catalytic Asp32 and Asp228. Just as expected, the phenyl ring substituted at the 6-position oriented properly in the S1 pocket and established favorable CeH/p contacts with Ile110. Meanwhile, meta-trifluoromethyl group on the phenyl ring was observed to extend to the S3 pocket and assisted to reinforce the ligand’s binding by forming multiple Van der Waal’s interactions which can well explain the great improvement in potency compared to the 6- non-substituted compound 1. As for the R1 substitutions on 4-oxo-6-(o-tolyl)-1,4-dihydroquinoline-3-carboxamide, the effect of p- or m- substitution on the phenyl ring appeared to follow the same rule for the 6-sulfamoyl analogs that m- substituted 14a and 14c were more favorable than the p- substituted 14b and 14d, although less potent than the corresponding 6-sulfamoyl substituted analogs (8ae8d). Meanwhile, the more bulky 3-trifluoromethyl substituted 14d (IC50 ¼ 6.18 mM) and bromine substituted 14e (IC50 ¼ 1.89 mM) displayed much better activities than the 3-fluorine substituted 14b, suggesting there was large room in the corresponding pocket and bulky substitutions at m- position might be more favorable. Docking result of 14e (Fig. 2B) suggested the 6-tolyl substitution could position well in the S1 pocket and form edge-face pep stacking interactions with the adjacent Phe108 and Trp115. However, compared with 8d, fewer H-bonds were formed and the lower binding score suggested a lower docking affinity, which was consistent with the higher IC50 value in Table 1. The cytotoxicities of the eight active compounds were listed in Table 2 and all the IC50 values were higher than 50 mM, suggesting low distinct cytotoxic effects, and these BACE-1 inhibitors should be safe for normal cells. The predicted log BB values for the eight active compounds in Table 2 suggested that some of these compounds were high

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Fig. 2. Predicted binding pose of 8d (A) and 14e (B) in the active site of BACE-1 (PDB ID: 1TQF).

penetrants of BBB (log BB > 0.2), among which 14d and 14e, were not only high permeable, but also low in molecular weight (MW < 450). As for compounds 8aeg, since these novel structures were outside the 95% and 99% confidence area of database, no reliable prediction could presently be made by the software. 6. Conclusions Aided by docking study, a series of 6-substituted 4-oxo-1,4dihydro-quinoline-3-carboxamides were designed, synthesized and evaluated as BACE-1 inhibitors in an enzymatic assay. Eight compounds were found to have IC50s at mM level, and 14e exhibited optimized drug-like profiles with the relatively low IC50 of 1.89 mM, low molecular weight of 446, high BBB penetrability and high safety to HEK293 cells, making it a good structure for further modification. 7. Experimental section 7.1. General chemical methods Melting points were measured with an X4 apparatus and were uncorrected. 1H NMR spectra were recorded on a Bruker (Bruker BioSpin AG, Fällanden, Switzerland) Avance III 400 MHz system. Chemical shifts were reported in parts per million (ppm) relative to tetramethylsilane (TMS) as internal standard. The spin multiplicities were given as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) or br (broad). MS and high-resolution mass spectra (HRMS) were obtained using Electrospray Ionization (ESI) technique on a Bruker’s Fourier Transform Ion Cyclotron resonance Mass Spectrometer. Thin layer chromatography (TLC) analysis was performed on silica gel GF254 purchased from Qingdao Haiyang Chemical Co. (Qingdao, Shandong Province, China) or Merck (Darmstadt, Germany). 7.1.1. 4-Nitro-N-(3-(trifluoromethyl)phenyl)benzenesulfonamide (3) To a solution of 3-(trifluoromethyl)aniline (0.8 g, 5 mmol) in pyridine (0.8 g, 10 mmol), 4-nitrobenzenesulfonyl chloride (1.1 g, 5 mmol) dissolved in CH2Cl2 (20 mL) was added dropwise in an ice bath. After stirring for 1 h, the ice bath was removed and the mixture was stirred overnight before concentration in vacuum. The residue was extracted with ethyl acetate, washed with 0.5 N HCl, brine in sequence, and dried over anhydrous Na2SO4 to give the crude product, which was purified by column chromatography on silica gel (CH2Cl2/MeOH ¼ 500/1) to give 1.56 g of 3 in a yield of 90.17% as a white solid. m.p. 135e136  C; 1H NMR (400 MHz, CDCl3): d 8.32 (d, 2H, J ¼ 8.8 Hz), 7.98 (d, 2H, J ¼ 8.8 Hz), 7.40e7.48 (m, 2H), 7.36 (s, 1H), 7.30 (d, 1H, J ¼ 6.8 Hz), 6.94 (s, 1H); ESI-MS (m/ z): 345 (MH)

7.1.2. 4-Amino-N-(3-(trifluoromethyl)phenyl)benzenesulfonamide (4) To a stirred solution of 3 (1.06 g, 3 mmol) in ethanol (20 mL) was added a catalytic amount of Pd/C (100 mg). The mixture was stirred under a hydrogen atmosphere (60 psi) for 6 h at room temperature before the catalyst was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel. (CH2Cl2/CH3OH ¼ 200/1), and 4 was afforded as a white solid (405 mg, 41.83% yield). m.p. 105e 106  C; 1H NMR (400 MHz, CDCl3): d 7.58 (d, 2H, J ¼ 8.8 Hz), 7.30e 7.36 (m, 3H), 7.25e7.29 (m, 1H), 7.20 (s, 1H), 6.60 (d, 2H, J ¼ 8.8 Hz), 4.15 (s, br, 2H); ESI-MS (m/z): 315 (MH) 7.1.3. Diethyl 2-(((4-(N-(3-(trifluoromethyl)phenyl)sulfamoyl) phenyl)amino)methylene)-malonate (5) A mixture of diethyl ethoxymethylene malonate (130 mg, 0.63 mmol) and 4 (200 mg, 0.63 mmol) was heated to 120  C for 2 h. When cooled to room temperature, the mixture was diluted with ethyl acetate (30 mL) and washed with water (20 mL  3). The organic layers were combined, dried with Na2SO4 and concentrated under reduced pressure. The residue was purified using chromatography on silica gel (CH2Cl2/MeOH ¼ 100:1) to afford 5 (281 mg, 91.53% yield) as a white solid. m.p. 143e144  C; 1H NMR (400 MHz, CDCl3): d 11.08 (d, 1H, J ¼ 13.2 Hz), 8.46 (d, 1H, J ¼ 13.2 Hz), 7.80 (d, 2H, J ¼ 8.8 Hz), 7.38 (m, 4H), 7.32 (m, 1H), 7.15 (d, 2H, J ¼ 8.8 Hz), 4.23e4.34 (m, 4H), 1.34 (m, 6H); ESI-MS (m/z): 487 (M þ H)þ 7.1.4. Ethyl 4-oxo-6-(N-(3-(trifluoromethyl)phenyl)sulfamoyl)-1,4dihydro-quinoline-3-carboxylate (6) To a three-necked flask, 5 (300 mg, 0.61 mmol), PPA (1.5 g) and POCl3 (2.5 g) were added at room temperature. The mixture was heated to 70  C for 12 h and then cooled to room temperature. The mixture was treated with aqueous Na2CO3 solution to remove the remanent acid and filtered, and the solid was washed with water and dried to afford the product, which could be used in next step. m.p. 211e212  C; 1H NMR (400 MHz, DMSO-d6): d 12.80 (s, 1H, br), 10.92 (s, 1H), 8.60 (s, 1H), 8.56 (d, 1H, J ¼ 2.0 Hz), 8.02 (dd, 1H, J1 ¼ 8.7 Hz, J2 ¼ 2.0 Hz), 7.80 (d, 1H, J ¼ 8.7 Hz), 7.50 (t, 1H, J ¼ 7.7 Hz), 7.36e7.45 (m, 3H), 4.24 (q, 2H, J ¼ 7.2 Hz), 1.29 (t, 3H, J ¼ 7.2 Hz); ESI-MS (m/z): 441 (M þ H)þ. 7.1.5. 4-Oxo-6-(N-(3-(trifluoromethyl)phenyl)sulfamoyl)-1,4dihydro-quinoline-3-carboxylic acid (7) A mixture of 6 and NaOH (25 mg, 0.61 mmol) in H2O (20 mL) was stirred under reflux for 2 h. The mixture was filtered and then cooled to room temperature. The solution was acidified with 2 N HCl until pH ¼ 3 to afford solid, which was filtered, washed with water and dried to get product 7 (60 mg, two steps yield: 24%). m.p. 137e138  C; 1H NMR (400 MHz, DMSO-d6): d 14.47 (br, 1H), 8.95 (s, 1H), 8.67 (d, 1H, J ¼ 2.1 Hz), 8.14 (dd, 1H, J1 ¼ 8.8 Hz, J2 ¼ 2.1 Hz), 7.96 (d, 1H, J ¼ 8.8 Hz), 7.50 (t, 1H, J ¼ 8.0 Hz), 7.39e7.42 (m, 3H); HRMS (ESI) for C17H11F3N2O5S: calcd. 413.04135 (M þ H)þ, found: 413.04116 (M þ H)þ.

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7.1.6. N-(4-fluorobenzyl)-4-oxo-6-(N-(3-(trifluoromethyl)phenyl) sulfamoyl)-1,4-dihydro-quinoline-3-carboxamide (8a) A mixture of 7 (100 mg, 0.24 mmol), HBTU (92 mg, 0.24 mmol) and DIPEA (63 mg, 0.48 mmol) was dissolved in CH2Cl2 (30 mL) under N2 atmosphere. The solution was stirred at room temperature for 15 min before 4-fluorobenzylamine (30 mg, 0.24 mmol) was added. Then the mixture was stirred for additional 12 h. The solution was concentrated under reduced pressure and the residue was extracted with ethyl acetate (30 mL  3) and washed with water (20 mL  3). The combined organic layers were concentrated and purified by chromatography on silica gel (CH2Cl2/ MeOH ¼ 100:1) to afford 8a as a white solid (35 mg, 28.2% yield). m.p. 243e244  C; 1H NMR (400 MHz, DMSO-d6): d 13.05 (s, 1H), 10.93 (s, 1H), 10.14 (t, 1H, J ¼ 6.0 Hz), 8.82 (s, 1H), 8.63 (d, 1H, J ¼ 2.0 Hz), 8.05 (dd, 1H, J1 ¼ 8.8 Hz, J2 ¼ 2.0 Hz), 7.86 (d, 1H, J ¼ 8.8 Hz), 7.46e7.52 (m, 1H), 7.35e7.42 (m, 5H), 7.16 (t, 2H, J ¼ 8.8 Hz), 4.54 (d, 2H, J ¼ 5.6 Hz); 13C NMR (100 MHz, DMSO-d6): d 176.02, 164.29, 162.86, 160.47, 145.58, 142.03, 138.84, 136.01, 135.49, 131.19, 130.10, 129.80, 125.98, 125.73, 123.80, 121.43, 121.13, 116.30, 115.68, 115.47, 112.55, 41.89; HRMS (ESI) for C24H17F4N3O4S: calcd. 520.09487 (M þ H)þ, found: 520.09421 (M þ H)þ. 7.1.7. N-(3-fluorobenzyl)-4-oxo-6-(N-(3-(trifluoromethyl)phenyl) sulfamoyl)-1,4-dihydro-quinoline-3-carboxamide (8b) The procedure described for 8a was used, starting from 7 (100 mg, 0.24 mmol), HBTU (92 mg, 0.24 mmol), DIPEA (63 mg, 0.48 mmol) and 3-fluorobenzylamine (30 mg, 0.24 mmol), and 8b was obtained as a white solid (32 mg, 25.8% yield). m.p. 222e223  C; 1 H NMR (400 MHz, DMSO-d6): d 13.04 (s, 1H), 10.91 (s, 1H), 10.18 (s, 1H), 8.83 (s, 1H), 8.54 (s, 1H), 8.07 (d, 1H, J ¼ 8.8 Hz), 7.87 (d, 1H, J ¼ 8.8 Hz), 7.46e7.54 (m, 1H), 7.33e7.44 (m, 4H), 6.97e7.18 (m, 3H), 4.59 (d, 2H, J ¼ 5.2 Hz); 13C NMR (100 MHz, DMSO-d6): d 176.05, 164.43, 163.93, 161.51, 145.62, 143.01, 142.04, 138.84, 135.50, 131.18, 130.79, 130.12, 126.00, 125.76, 123.78, 121.42, 121.15, 116.32, 114.44, 114.05, 112.51, 110.00, 42.14; HRMS (ESI) for C24H17F4N3O4S: calcd. 520.09487 (M þ H)þ, found: 520.09428 (M þ H)þ 7.1.8. N-(4-methoxybenzyl)-4-oxo-6-(N-(3-(trifluoromethyl) phenyl)sulfamoyl)-1,4-dihydro-quinoline-3-carboxamide (8c) According to procedure described for 8a, starting from 7 (100 mg, 0.24 mmol), HBTU (92 mg, 0.24 mmol), DIPEA (63 mg, 0.48 mmol) and 4-methoxybenzylamine (33 mg, 0.24 mmol), 8c was obtained as a white solid (26 mg, 20.4% yield). m.p. 250e251  C; 1H NMR (400 MHz, DMSO-d6): d 12.99 (s, 1H), 10.91 (s, 1H), 10.07 (s, 1H), 8.83 (s, 1H), 8.65 (s, 1H), 8.06 (d, 1H, J ¼ 8.0 Hz), 7.86 (d, 1H, J ¼ 8.4 Hz), 7.48 (d, 1H, J ¼ 7.2 Hz), 7.35e7.45 (m, 3H), 7.28 (d, 2H, J ¼ 7.6 Hz), 6.91 (d, 2H, J ¼ 7.6 Hz), 4.50 (d, 2H, J ¼ 4.4 Hz), 3.64 (s, 3H); 13C NMR (100 MHz, DMSO-d6): d 181.02, 169.09, 163.77, 150.52, 147.01, 143.83, 140.45, 136.71, 136.18, 135.52, 135.18, 134.17, 130.96, 128.77, 127.75, 126.12, 121.28, 119.31, 117.63, 60.48, 47.08; HRMS (ESI) for C25H20F3N3O5S: calcd. 532.11485 (M þ H)þ, found: 532.11419 (M þ H)þ. 7.1.9. N-(3-methoxybenzyl)-4-oxo-6-(N-(3-(trifluoromethyl)phenyl) sulfamoyl)-1,4-dihydro-quinoline-3-carboxamide (8d) The procedure described for 8a was used, starting from 7 (100 mg, 0.24 mmol), HBTU (92 mg, 0.24 mmol), DIPEA (63 mg, 0.48 mmol) and 3-methoxybenzylamine (33 mg, 0.24 mmol), 8d was obtained as a white solid (27 mg, 21.2% yield). m.p. 181e182  C; 1 H NMR (400 MHz, DMSO-d6): d 10.13 (s, 1H), 8.82 (s, 1H), 8.64 (d, 1H, J ¼ 2.0 Hz), 8.05 (dd, 1H, J1 ¼ 8.8 Hz, J2 ¼ 2.0 Hz), 7.85 (d, 1H, J ¼ 8.8 Hz), 7.48 (d, 1H, J ¼ 7.2 Hz), 7.36e7.42 (m, 3H), 7.26 (t, 1H, J ¼ 8.0 Hz), 6.91 (m, 2H), 6.84 (d, 1H, J ¼ 8.0 Hz), 4.55 (d, 2H, J ¼ 5.6 Hz), 3.74 (s, 3H); 13C NMR (100 MHz, DMSO-d6): d 176.05, 164.24, 159.84, 145.58, 142.02, 141.41, 138.82, 135.44, 131.20, 130.09, 129.98, 125.97, 125.76, 123.78, 121.43, 121.10, 119.89, 116.26, 113.15, 112.62, 55.44, 42.57; HRMS (ESI) for C25H20F3N3O5S: calcd. 532.11485 (M þ H)þ, found: 532.11411 (M þ H)þ.

7.1.10. N-(furan-2-ylmethyl)-4-oxo-6-(N-(3-(trifluoromethyl) phenyl)sulfamoyl)-1,4-dihydro-quinoline-3-carboxamide (8e) The procedure described for 8a was used, starting from 7 (100 mg, 0.24 mmol), HBTU (92 mg, 0.24 mmol), DIPEA (63 mg, 0.48 mmol) and furfurylamine (23 mg, 0.24 mmol), 8e was obtained as a white solid (28 mg, 23.76% yield). m.p. 209e210  C; 1H NMR (400 MHz, DMSO-d6): d 13.01 (s, 1H), 10.92 (s, 1H), 10.06 (t, 1H, J ¼ 5.6 Hz), 8.81 (s, 1H), 8.64 (d, 1H, J ¼ 2.0 Hz), 8.05 (dd, 1H, J1 ¼ 8.8 Hz, J2 ¼ 2.4 Hz), 7.86 (d, 1H, J ¼ 8.8 Hz), 7.61 (s, 1H), 7.49 (t, 1H, J ¼ 8.0 Hz), 7.34e7.44 (m, 3H), 6.42 (m, 1H), 6.31 (t, 1H, J ¼ 2.8 Hz), 4.56 (d, 2H, J ¼ 5.6 Hz); 13C NMR (100 MHz, DMSO-d6): d 176.01, 164.09, 152.55, 145.63, 142.79, 142.03, 138.82, 135.53, 131.19, 130.11, 125.94, 125.74, 123.80, 121.46, 121.14, 116.33, 112.39, 110.95, 107.38, 35.86; HRMS (ESI) for C22H16F3N3O5S: calcd. 492.08355 (M þ H)þ, found: 492.08316 (M þ H)þ. 7.1.11. 4-Oxo-N-(pyridin-3-ylmethyl)-6-(N-(3-(trifluoromethyl) phenyl)sulfamoyl)-1,4-dihydro-quinoline-3-carboxamide (8f) The procedure described for 8a was used, starting from 7 (100 mg, 0.24 mmol), HBTU (92 mg, 0.24 mmol), DIPEA (63 mg, 0.48 mmol) and 3-(aminomethyl)pyridine (26 mg, 0.24 mmol), 8f was obtained as a white solid (20 mg, 16.6% yield). m.p. 248e 249  C; 1H NMR (400 MHz, DMSO-d6): d 13.05 (s, 1H), 10.95 (s, 1H), 10.19 (t, 1H, J ¼ 4.0 Hz), 8.83 (s, 1H), 8.65 (d, 1H, J ¼ 2.0 Hz), 8.58 (s, 1H), 8.47 (d, 1H, J ¼ 3.6 Hz), 8.07 (dd, 1H, J1 ¼ 8.8 Hz, J2 ¼ 2.0 Hz), 7.87 (d, 1H, J ¼ 8.8 Hz), 7.75 (d, 1H, J ¼ 7.6 Hz), 7.50 (t, 1H, J ¼ 7.6 Hz), 7.34e7.44 (m, 4H), 4.60 (d, 2H, J ¼ 4.0 Hz); 13C NMR (100 MHz, DMSO-d6): d 175.99, 164.49, 149.36, 148.57, 145.63, 142.03, 138.82, 135.66, 135.50, 131.20, 130.12, 125.97, 125.71, 123.99, 123.78, 121.45, 121.13, 116.26, 112.46, 40.29; HRMS (ESI) for C23H17F3N4O4S: calcd. 503.09954 (M þ H)þ, found: 503.09929 (M þ H)þ. 7.1.12. N-cyclohexyl-4-oxo-6-(N-(3-(trifluoromethyl)phenyl) sulfamoyl)-1,4-dihydro-quinoline-3-carboxamide (8g) The procedure described for 8a was used, starting from 7 (100 mg, 0.24 mmol), HBTU (92 mg, 0.24 mmol), DIPEA (63 mg, 0.48 mmol) and cyclohexylamine (24 mg, 0.24 mmol), 8g was obtained as a white solid (42 mg, 35.6% yield). m.p. 245e246  C; 1H NMR (400 MHz, DMSO-d6): d 12.97 (s, 1H), 10.90 (s, 1H), 9.80 (s, 1H), 8.78 (s, 1H), 8.67 (s, 1H), 8.05 (s, 1H), 7.87 (s, 1H), 7.30e7.60 (m, 4H), 3.85 (s, 1H), 1.11e1.86 (m, 11H); 13C NMR (100 MHz, DMSO-d6): d 181.12, 168.07, 150.34, 146.97, 143.84, 140.42, 136.17, 135.23, 134.99, 130.93, 130.78, 128.73, 127.79, 126.37, 126.09, 121.31, 117.79, 52.26, 37.87, 30.67, 29.54; HRMS (ESI) for C23H22F3N3O4S: calcd. 494.13559 (M þ H)þ, found: 494.13505 (M þ H)þ. 7.1.13. Diethyl 2-(((4-bromophenyl)amino)methylene)malonate (10) Following the same synthetic procedure as described to obtain 5, starting from the 4-bromoaniline 9 (6 g, 34.88 mmol) and diethyl ethoxymethylene malonate (7.53 g, 34.88 mmol), 10 was obtained as a white solid (5.1 g, 42.9% yield). m.p. 94e95  C; 1H NMR (400 MHz, CDCl3): d 11.00 (d, 1H, J ¼ 13.2 Hz), 8.46 (d, 1H, J ¼ 13.6 Hz), 7.48 (d, 2H, J ¼ 8.8 Hz), 7.02 (d, 2H, J ¼ 8.8 Hz), 4.17e 4.38 (m, 4H), 1.25e1.45 (m, 6H); ESI-MS (m/z): 342 (M þ H)þ. 7.1.14. Ethyl 6-bromo-4-oxo-1,4-dihydro-quinoline-3-carboxylate (11) The procedure described for 6 was used, starting from 10 (5 g, 14.9 mmol), PPA (10 g) and POCl3 (6.8 g), 11 was obtained as a white solid (3.4 g, 77.44% yield). m.p. 247e248  C; 1H NMR (400 MHz, DMSO-d6): d 8.60 (s, 1H), 8.17 (d, 1H, J ¼ 8.0 Hz), 7.61e7.71 (m, 2H), 7.39 (t, 1H, J ¼ 7.2 Hz), 4.21 (q, 2H, J ¼ 7.2 Hz), 1.28 (t, 3H, J ¼ 7.2 Hz); ESI-MS (m/z): 296 (M þ H)þ. 7.1.15. Ethyl 4-oxo-6-(o-tolyl)-1,4-dihydro-quinoline-3-carboxylate (12) A mixture of o-tolyl boronic acid (760 mg, 5.58 mmol), 11 (1.5 g, 5.08 mmol), K2CO3 (1.05 g, 7.66 mmol), Pd(PPh3)4 (295 mg) and

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H2O (3.8 mL) in THF (30 mL) was heated to reflux under N2 atmosphere for 12 h. After the mixture was cooled to room temperature, filtered, the crude solid product 12 was purified by recrystallization with ethyl acetate. (1.2 g, 76.92% yield). m.p. 208e 209  C; 1H NMR (400 MHz, DMSO-d6): d 12.59 (s, 1H), 8.60 (s, 1H), 8.06 (s, 1H), 7.71 (s, 2H), 7.22e7.37 (m, 4H), 4.23 (q, 2H, J ¼ 7.2 Hz), 2.26 (s, 3H), 1.29 (t, 3H, J ¼ 7.2 Hz); HRMS (ESI) for C19H17NO3: calcd. 308.12812 (M þ H)þ, found: 308.12776 (M þ H)þ. 7.1.16. 4-oxo-6-(o-tolyl)-1,4-dihydro-quinoline-3-carboxylic acid (13) The procedure described for 7 was used, starting from 12 (4.5 g, 14.65 mmol) and NaOH (1.3 g, 32.50 mmol), 13 was obtained as a white solid (3.12 g, 76.47% yield). m.p. 252e253  C; 1H NMR (400 MHz, DMSO-d6): d 13.87 (br, 1H), 8.92 (d, 1H, J ¼ 6.4 Hz), 8.18 (s, 1H), 7.90e8.00 (m, 2H), 7.27e7.39 (m, 4H), 2.26 (s, 3H); HRMS (ESI) for C17H13NO3: calcd. 280.09682 (M þ H)þ, found: 280.09642 (M þ H)þ. 7.1.17. N-(4-fluorobenzyl)-4-oxo-6-(o-tolyl)-1,4-dihydro-quinoline3-carboxamide (14a) The procedure described for 8a was used, starting from 13 (100 mg, 0.35 mmol), HBTU (135.6 mg, 0.35 mmol), DIPEA (93 mg) and 4-fluorobenzylamine (45 mg, 0.35 mmol), 14a was obtained as a white solid (91 mg, 67.4% yield). m.p. 180e181  C; 1H NMR (400 MHz, DMSO-d6): d 12.82 (s, 1H), 10.42 (t, 1H, J ¼ 5.6 Hz), 8.82 (s, 1H), 8.15 (s, 1H), 7.78 (s, 2H), 7.20e7.50 (m, 6H), 7.17 (t, 2H, J ¼ 8.8 Hz), 4.56 (d, 2H, J ¼ 5.6 Hz), 2.24 (s, 3H); 13C NMR (100 MHz, DMSO-d6): d 176.55, 164.99, 162.71, 160.48, 144.17, 140.65, 138.51, 138.33, 136.31, 135.28, 134.21, 130.99, 130.12, 129.83, 128.21, 126.64, 126.43, 125.55, 119.50, 115.47, 115.68, 111.36, 41.86, 20.58; HRMS (ESI) for C24H19FN2O2: calcd. 387.15033 (M þ H)þ, found: 387.15003 (M þ H)þ. 7.1.18. N-(3-fluorobenzyl)-4-oxo-6-(o-tolyl)-1,4-dihydro-quinoline3-carboxamide (14b) The procedure described for 8a was used, starting from 13 (100 mg, 0.35 mmol), HBTU (135.6 mg, 0.35 mmol), DIPEA (93 mg, 0.72 mmol) and 3-fluorobenzylamine (45 mg, 0.35 mmol), 14b was obtained as a white solid. (93 mg, 68.8% yield). m.p. 193e194  C; 1H NMR (400 MHz, DMSO-d6): d 12.86 (s, 1H), 10.47 (s, 1H), 8.82 (s, 1H), 8.15 (s, 1H), 7.79 (s, 2H), 7.01e7.44 (m, 8H), 4.59 (d, 2H, J ¼ 4.0 Hz), 2.25 (s, 3H); 13C NMR (100 MHz, DMSO-d6): d 176.55, 165.12, 163.85, 161.50, 144.25, 143.14, 140.63, 138.52, 138.33, 135.29, 134.24, 130.99, 130.81, 130.13, 128.22, 126.65, 126.41, 125.54, 123.76, 119.52, 114.35, 111.25, 40.28, 20.60; HRMS (ESI) for C24H19FN2O2: calcd. 387.15033 (M þ H)þ, found: 387.14985 (M þ H)þ. 7.1.19. 4-Oxo-6-(o-tolyl)-N-(4-(trifluoromethyl)benzyl)-1,4dihydro-quinoline-3-carboxamide (14c) The procedure described for 8a was used, starting from 13 (100 mg, 0.35 mmol), HBTU (135.6 mg, 0.35 mmol), DIPEA (93 mg, 0.72 mmol) and p-trifluoromethylbenzylamine (63 mg, 0.35 mmol), 14c was obtained as a white solid. (99 mg, 65.13% yield) m.p. 252e253  C; 1H NMR (400 MHz, DMSO-d6): d 12.86 (s, 1H), 10.52 (t, 1H, J ¼ 5.6 Hz), 8.82 (s, 1H), 8.16 (s, 1H), 7.79 (s, 2H), 7.71 (d, 2H, J ¼ 8.0 Hz), 7.56 (d, 2H, J ¼ 8.0 Hz), 7.24e7.38 (m, 4H), 4.67 (d, 2H, J ¼ 5.6 Hz), 2.25 (s, 3H); 13C NMR (100 MHz, DMSO-d6): d 176.55, 165.22, 145.13, 144.26, 140.62, 138.52, 138.35, 135.29, 134.26, 131.00, 130.13, 128.33, 128.23, 126.66, 126.41, 125.73, 125.53, 119.53, 111.22, 42.18, 20.60; HRMS (ESI) for C25H19F3N2O2: calcd. 437.14714 (M þ H)þ, found: 437.14772 (M þ H)þ. 7.1.20. 4-Oxo-6-(o-tolyl)-N-(3-(trifluoromethyl)benzyl)-1,4dihydro-quinoline-3-carboxamide (14d) The procedure described for 8a was used, starting from 13 (100 mg, 0.35 mmol), HBTU (135.6 mg, 0.35 mmol), DIPEA (93 mg, 0.72 mmol) and m-trifluoromethylbenzylamine (63 mg,

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0.35 mmol), 14d was obtained as a white solid (110 mg, 72.36% yield); m.p. 216e217  C; 1H NMR (400 MHz, DMSO-d6): d 12.82 (s, 1H), 10.50 (s, 1H), 8.81 (s, 1H), 8.15 (s, 1H), 7.78 (s, 2H), 7.51e7.74 (m, 4H), 7.24e7.37 (m, 4H), 4.66 (d, 2H, J ¼ 5.6 Hz), 2.25 (s, 3H); 13C NMR (100 MHz, DMSO-d6): d 176.55, 165.22, 144.26, 141.82, 140.66, 138.54, 138.37, 135.29, 134.26, 132.01, 130.99, 130.02, 128.22, 126.41, 125.55, 124.33, 123.99, 119.53, 111.22, 42.16, 20.58; HRMS (ESI) for C25H19F3N2O2: calcd. 437.14714 (M þ H)þ, found: 437.14763 (M þ H)þ . 7.1.21. N-(3-bromobenzyl)-4-oxo-6-(o-tolyl)-1,4-dihydroquinoline-3-carboxamide (14e) The procedure described for 8a was used, starting from 13 (100 mg, 0.35 mmol), HBTU (135.6 mg, 0.35 mmol), DIPEA (93 mg, 0.72 mmol) and 3-bromobenzylamine (66 mg, 0.35 mmol), 14e was obtained as a white solid (89 mg, 57.05% yield); m.p. 184e185  C; 1H NMR (400 MHz, DMSO-d6): d 12.81 (s, 1H), 10.44 (t, 1H, J ¼ 5.6 Hz), 8.81 (s, 1H), 8.16 (s, 1H), 7.78 (s, 2H), 7.54 (s, 1H), 7.45 (d, 1H, J ¼ 7.6 Hz), 7.23e7.40 (m, 6H), 4.57 (d, 2H, J ¼ 5.6 Hz), 2.25 (s, 3H); 13 C NMR (100 MHz, DMSO-d6): d 176.55, 165.14, 144.24, 143.13, 140.66, 138.45, 138.37, 135.30, 134.23, 131.02, 130.55, 130.13, 128.21, 126.92, 126.64, 126.44, 125.57, 122.12, 119.51, 112.25, 42.00, 20.58; HRMS (ESI) for C24H19BrN2O2: calcd. 447.07027 (M þ H)þ, found: 447.07010 (M þ H)þ. 7.1.22. N-(furan-2-ylmethyl)-4-oxo-6-(o-tolyl)-1,4-dihydro-quinoline-3-carboxamide (14f) The procedure described for 8a was used, starting from 13 (100 mg, 0.35 mmol), HBTU (135.6 mg, 0.35 mmol), DIPEA (93 mg, 0.72 mmol) and furfurylamine (35 mg, 0.35 mmol), 14f was obtained as a white solid. (46 mg, 36.8% yield); m.p. 208e209  C; 1H NMR (400 MHz, CDCl3): d 12.86 (s, 1H), 11.05 (s, 1H), 8.80 (d, 1H, J ¼ 5.6 Hz), 8.50 (s, 1H), 8.21 (s, 1H), 7.63 (t, 1H, J ¼ 7.6 Hz), 7.50 (q, 2H, J ¼ 8.4 Hz), 7.35 (d, 1H, J ¼ 7.6 Hz), 7.23e7.26 (m, 1H), 7.08e7.19 (m, 2H), 6.77e6.86 (m, 1H), 4.89 (d, 2H, J ¼ 5.2 Hz), 2.18 (s, 3H); 13C NMR (100 MHz, CDCl3): d 177.27, 166.65, 157.91, 148.77, 143.69, 140.29, 139.06, 138.11, 137.37, 135.26, 134.03, 130.50, 129.95, 127.71, 125.99, 122.55, 121,89, 118.52, 115.51, 110.94, 44.71, 20.42; HRMS (ESI) for C22H18N2O3: calcd. 359.13902 (M þ H)þ, found: 359.13880 (M þ H)þ. 7.1.23. 4-Oxo-N-(pyridin-3-ylmethyl)-6-(o-tolyl)-1,4-dihydroquinoline-3-carboxamide (14g) The procedure described for 8a was used, starting from 13 (100 mg, 0.35 mmol), HBTU (135.6 mg, 0.35 mmol), DIPEA (93 mg, 0.72 mmol) and 3-(aminomethyl)pyridine (39 mg, 0.35 mmol), 14g was obtained as a white solid (93 mg, 72.09% yield); m.p. 198e 199  C; 1H NMR (400 MHz, DMSO-d6): d 12.83 (s, 1H), 10.46 (s, 1H), 8.81 (s, 1H), 8.59 (s, 1H), 8.47 (s, 1H), 8.15 (s, 1H), 7.76 (m, 3H), 7.18e 7.42 (m, 5H), 4.60 (s, 2H), 2.24 (s, 3H); 13C NMR (100 MHz, DMSOd6): d 176.52, 165.18, 149.40, 148.58, 144.20, 140.63, 138.51, 138.34, 135.65, 135.28, 134.23, 130.99, 130.12, 128.21, 126.64, 126.42, 125.54, 123.99, 119.51, 111.27, 40.23, 20.58; HRMS (ESI) for C23H19N3O2: calcd. 370.15500 (M þ H)þ, found: 370.15507 (M þ H)þ. 7.1.24. N-cyclohexyl-4-oxo-6-(o-tolyl)-1,4-dihydro-quinoline-3carboxamide (14h) The procedure described for 8a was used, starting from 13 (100 mg, 0.35 mmol), HBTU (135.6 mg, 0.35 mmol), DIPEA (93 mg, 0.72 mmol) and cyclohexylamine (36 mg, 0.35 mmol), 14h was obtained as a white solid (83 mg, 65.8% yield); m.p. 253e254  C; 1H NMR (400 MHz, DMSO-d6): d 12.79 (s, 1H), 10.09 (d, 1H, J ¼ 7.6 Hz), 8.77 (s, 1H), 8.16 (s, 1H), 7.77 (s, 2H), 7.18e7.44 (m, 4H), 3.85 (d, 1H, J ¼ 7.6 Hz), 2.26 (s, 3H), 1.86 (d, 2H, J ¼ 10.8 Hz), 1.67 (s, 2H), 1.54 (s, 1H), 1.20e1.44 (m, 5H); 13C NMR (100 MHz, DMSO-d6): d 176.60, 163.79, 143.95, 140.64, 138.48, 138.19, 135.28, 134.15, 131.01, 130.15, 128.20, 126.66, 126.39, 125.56, 119.46, 111.57, 47.18, 32.99, 25.71,

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24.62, 20.61; HRMS (ESI) for C23H24N2O2: calcd. 361.19105 (M þ H)þ, found: 361.19119 (M þ H)þ. 7.2. Molecular docking The model of BACE-1 was constructed by removing all water molecules from the X-ray structure of human BACE-1 “apo” form (PDB ID: 1TQF). The protein was prepared in Discovery Studio 2.5, with the amino acids ionized at pH 7.4, hydrogens added and charges calculated. The 3D models of ligands were built using modified CHARMm force field-based structure minimization implemented in Discovery Studio’s ’prepare ligands’ protocol. Docking simulations were carried out by means of GOLD 3.0.1 version. The host ligand was redocked into the binding pocket and the RMSD between the docked pose and the active conformation was 1.031, confirming that the docking protocol was reliable. 7.3. Cytotoxicity assay The assay was performed with the Human Embryonic Kidney 293 cells (HEK293) which were purchased from the cell bank of the Shanghai Institute of Cell Biology. The HEK293 cell line was cultured in the DMEM medium. The media was supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 units/mL streptomycin (Invitrogen). The cells were maintained at 37  C in a humidified environment with 5% CO2. The cell viability was determined by using the CellTiter GloÔ luminescent cell viability assay (Promega). Briefly, the cells were seeded into 384-well plates at an initial density of 1000 cells/well in 45 mL of medium. Then the cells were treated with compounds at varying concentrations. The first compound work concentration is 100 mM, and then three fold dilution ten more times. Staurosporine (SigmaeAldrich, catalog No.S4400) was used as a positive control. After incubation for 72 h, 10% of CellTiter GloÔ reagent was added and luminescent signals were read on a VeriScan reader (Thermo Fisher Scientific). The IC50 value was calculated from the curves generated by plotting the percentage of the viable cells versus test concentrations on a logarithmic scale using Sigma Plot 10.0 software. Acknowledgments This work was supported by the National Natural Science Foundation of China (21002002, 21172012), the National Basic Research Program of China (2012CB518000) and the Specialized Research Fund for the Doctoral Program of Higher Education of China (20120001110010). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2014.04.025. References [1] Jeffrey L. Cummings, Alzheimer’s disease, The New England Journal of Medicine 351 (2004) 56e57. [2] Roland Jakob-Roetne, Helmut Jacobsen, Alzheimer’s disease: from pathology to therapeutic approaches, Angewandte Chemie International Edition 17 (2009) 3030e3059. [3] Linnea R. Freeman, Le Zhang, Kalavathi Dasuri, Sun-Ok Fernandez-Kim, Annadora J. Bruce-Keller, Jrffrey N. Keller, Mutant amyloid precursor protein differentially alters adipose biology under obesogenic and non-obesogenic conditions, Current Alzheimer Research 2 (2012) 217e226. [4] M.L. Bolognesi, R. Matera, A. Minarini, M. Rosini, C. Melchiorre, Alzheimer’s disease: new approaches to drug discovery, Current Opinion in Chemical Biology 13 (2009) 303e308.

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