, 97 (s, 1H), 8.30 (d, J = 5.2 Hz, 1H), 8.19 (dd, J = 7.8, 1.0 Hz, 1H), 7.46 (m, 2H), 7.39 (dd, J = 7.4, 1.1 Hz, 1H), Purification by flash chromatography on silica gel using (CH 2 Cl 2 /MeOH 98:2, v/v) gave the urea (±)-47 (126 mg) as an off-white solid in 92% yield. 1 H NMR (300 MHz, DMSO) ? 10.67 (s, 1H), vol.9, p.692
, Purification by flash chromatography on silica gel using (CH 2 Cl 2 /MeOH 98:2, v/v) gave the urea (R)-47 (87 mg) as an off-white solid in 73% yield. NMR and MS spectra were in accordance with those reported above
, The organic phase was dried over MgSO 4 , filtered, and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (CH 2 Cl 2 /EtOAc 90/10, v/v) to afford 42 as a pink solid in 48% yield (237 mg). 1 H NMR (300 MHz, Water was added, and the product was extracted with CH 2 Cl 2 (x2)
, 07 (s, 1H), 8.43 (dd, J = 5.0, 0.6 Hz, 1H), 8.01 (dd, J = 1.7, 0.6 Hz, 1H), 7.30 (dd, General procedure A was followed, using methyl ester 42 (237 mg, 1.25 mmol), to give 44 as a beige solid (114 mg) in 99% yield. 1 H NMR (300 MHz
, But-3-yn-1-yl)picolinoyl azide (46)
, General procedure B was followed, using acyl hydrazine 44 (223 mg) to give the desired acyl azide 46 (196 mg) as a white solid in 83% yield. 1 H NMR (300 MHz
, Hz, 1H), 2.91 (t, J = 7.1 Hz, 2H), 2.55 (td, J = 7.1, 2.6 Hz, 2H), 1.98 (t, J = 2.6 Hz, 1H). 13 C NMR (75 MHz, Hz, 1H), 8.03 (d, J = 1.0 Hz, 1H), 7.42 (dd, J = 4, vol.9
, 2.76 (d, J = 10.3 Hz, 1H), 2.61 (t, J = 5.9 Hz, 2H), 1.99 (d, J = 13.8 Hz, 1H), 1.88 -1.79(m, 6H), 1.79 -1.59 (m, 1H), 1.49 -1.37 (m, 1H), 1.04 (dd, J = 22.2, 12.2 Hz, 1H). 13 C NMR (75 MHz, 3.98 -3.93 (m, 2H), 2.98 (m, 7H)
, General procedure D was followed, using the alkyne (±)-47 (97 mg, 0.28 mmol) and azide 14
, 01 (s, 1H), 8.62 (s, 1H), 8.32 (d, J = 5.4 Hz, 1H), 8.25 (d, J = 7.3 Hz, 1H), 8.04 -7.94 (m, 1H), 7.80 (s, 1H), 7.72 (d, Purification by flash chromatography on silica gel (CH 2 Cl 2 /MeOH/NH 4 OH 97:3:1, v/v/v) gave the desired compound (±)-6 (30 mg) as an off-white solid in 18% yield. 1 H NMR (300 MHz, DMSO) ? 11.06 (bs, 1H), vol.10
, General procedure B was followed using acyl hydrazine 55 (572 mg, 3.55 mmol) to give the desired acyl azide 57 (588 mg) as an off-white solid in 96% yield. 1 H NMR (300 MHz, vol.3
, 1H), 8.35 (t, J = 2.0 Hz, 1H), 3.30 (s, 1H). 13 C NMR (75 MHz
, General procedure C was followed, using the acyl azide 57 (68 mg, 0.44 mmol) and tetrahydropyrido
, Purification by flash chromatography on silica gel using (CH 2 Cl 2 /MeOH/NH 4 OH 96:4:1, v/v/v) gave the urea (±)-59 (81 mg) as a white solid in 65% yield. 1 H NMR (300 MHz, vol.3
, J = 12.0, 1H), 2.59 (d, J = 10.4 Hz, 1H), 1.84 (dd, J = 26.3, 13.0 Hz, 3H), 1.50 (m, 1H), 1.34 (m, 1H), 1.00 (m, 1H). 13 C NMR (75 MHz, p.347
, Hz, 1H), 7.84 (t, J = 7.8 Hz, 1H), vol.7, p.61
, General procedure B was followed, using acyl hydrazine 56 (550 mg, 3.41 mmol), to give the desired acyl azide 58 (534 mg) as an off-white solid in 91% yield. 1 H NMR (300 MHz, vol.3
, 0.9 Hz, 1H), vol.7, p.11
, 44 mmol) and tetrahydropyrido[2,1-a]isoindolone (±)-24 (80 mg, 0.44 mmol). Purification by flash chromatography using (CH 2 Cl 2 /MeOH/NH 4 OH 96:4:1, v/v/v) gave the urea (±)-60 (81 mg) as a white solid in 50% yield
, = 7.5 Hz, 1H), 7.80 (t, J = 7.8 Hz, 1H)
,
,
, The purification by flash chromatography (CH 2 Cl 2 /MeOH/NH 4 OH 97:3:1, v/v/v) gave the desired compound (±)-7 as white off solid (65 mg) in 81% yield. 1 H NMR (300 MHz, DMSO) ? 9
, 2H), 2.64 -2.57 (m, 3H), 1.90 (d, J = 13.2 Hz, 1H)
,
, HRMS (ESI+) calcd for C 35 H 36 N 9 O 2 614.2914, found 614.2991. HPLC: t R = 21
, The general procedure D was followed by using the alkyne (±)-60 (45 mg, 0.13 mmol) and azide 14 (34 mg, 0.13 mmol). The purification by flash chromatography on silica gel (CH 2 Cl 2 /MeOH/NH 4 OH 95:5:1, v/v/v) gave the desired compound (±)-8 as yellow solid
Cells were grown at 37 °C, 5% CO 2 in ECACC recommended media: DMEM for HuH7 and MDA-MB-231, EMEM for MCF7 and CaCo-2, McCoy's for HCT116, and RPMI for PC3 and NCI-H727. All culture media were supplemented by 10% of FBS, 1% of penicillin-streptomycin, and 2 mM glutamine. The human neuroblastoma cell line SH-SY5Y and the Madin-Darby Canine Kidney (MDCK) cells retrovirally transfected with the human MDR1 cDNA (MDCKII-MDR1) were cultured in DMEM supplemented with 100 U/ml penicillin, MDA-MB-231, HCT116, PC3, MCF7 and NCI-H727 cancer cell lines were obtained from the ECACC collection ,
, Cytotoxicity assay Chemicals were solubilized in DMSO at a concentration of 10 mM (stock solution) and diluted in culture medium to the desired final concentrations. The dose-effect cytotoxic assays (IC 50 determination) were performed by increasing the concentration of each chemical (final well concentrations: 0.1 -0.3 -0.9 -3.0 -9.0 -25 µM). The cells were plated in 96-well plates (4000 cells/well). Twenty-four h after seeding, the cells were exposed to chemicals, All cells were grown at 37 ºC in a humidified atmosphere with 5% CO 2 . All materials for cell culturing were purchased from EuroClone, vol.72
, Vina generated the estimated total Gibbs free energy of binding in kcal/mol, which could be converted to the apparent constant, K i , using the relationship ?G=RT.ln (K i ). Docking poses were visualized using the PyMOL molecular graphics system
, The crystal structure of GSK-3? in complex with PF-04802367 (PBD code 5K5N) was used for molecular docking
Crystals were flash-frozen and stored in liquid nitrogen until used for data collection. Diffraction images were indexed and integrated using XDS,[73] and intensities were further scaled and merged with XSCALE, Purified TcAChE (12 mg mL -1 ) was purified as described by, vol.72 ,
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