All chemicals and solvents used were commercially available and were of reagent grade. Where measurements are listed, melting points were determined in open glass capillaries on a Veego digital melting point apparatus and were uncorrected. The infrared (IR) spectra of compounds were recorded on Schimadzu FT-IR 8400S infrared spectrophotometer using an ATR accessory. 1H NMR spectra were recorded on a Bruker Avance II 400 spectrometer, using DMSO-d6 as solvent and tetramethylsilane (TMS) as internal standard. Mass spectral analysis was carried out using Applied Biosystem QTRAP 3200 MS/MS or Waters Xevo G2-XS Quadrupole Time-of-Flight mass spectrometer system in ESI mode. Reactions were monitored by TLC using pre-coated silica gel aluminum plates (Kieselgel 60, 254, E. Merck, Germany); zones were detected visually under ultraviolet irradiation.
General synthesis of 2 chloro-3-formyl quinolines 12 . Commercially available Acetanilide /substituted acetanilides (0.05 mol) were dissolved in 9.6 mL of dimethyl formamide (DMF, 0.125 mol), to which 32 mL of phosphorus oxychloride (0.35 mol) was added gradually at 0° C. The reaction mixture was taken in a round bottom flask (RBF) equipped with a reflux condenser fitted with a drying tube and was heated for 4–16 hours on oil bath at 75–80° C. The solution was then cooled to room temperature and subsequently poured onto 100 mL of icy water. The precipitate formed was collected by filtration and recrystallized from ethyl acetate.
General Synthesis of 2-chloroquinolinyl chalcones 13,14. A mixture of 2-chloro-3-formyl quinolines (1 mmol), the respective acetophenones (1 mmol) and a base (either sodium methoxide (catalytic) or sodium hydroxide (one pellet)) in methanol (4 mL) was stirred at room temperature for 6–24 hours. The resulting precipitate was collected by filtration, washed with water, and recrystallized from DMF-H2O or EtOH-H2O.
General Synthesis of 3-(3-oxo-3-phenylprop-1-enyl)quinolin-2(1H)-ones of Formula I (Fig. 1). A suspension of the 3-(2-chloroquinolin-3-yl)-1-phenylprop-2-en-1-ones (0.001 mol) in 70% acetic acid (10 mL) was heated under reflux for 4–6 hours. Upon completion of the reaction (as indicated by a single spot in a TLC), the reaction mixture was cooled to ambient temperature and the solid product precipitated out was filtered. The filtered product was washed with water, dried and recrystallized in methanol or DMF/water.
(E)-3-(3-(2-Methoxyphenyl)-3-oxoprop-1-enyl)quinolin-2(1H)-one (CTR-17). Yield 81%; M.P. 256–258° C.; FT-IR (KBr) υ (cm− 1): 3153 (NH), 1656 (C═O), 1586, 1557 (C═C), 1240, 1020 (C—O—C); 1H NMR (400 MHz, DMSO-d6): (12.05 (s, 1H), 8.47 (s, 1H), 7.86 (d, J = 16.0 Hz, 1H), 7.73 (d, J = 7.9 Hz, 1H), 7.60–7.44 (m, 4H), 7.34 (d, J = 8.3 Hz, 1H), 7.26–7.19 (m, 2H), 7.08 (td, J = 7.4, 0.9 Hz, 1H), 3.87 (s, 3H); MS-API: [M + H] + 306.1 (calculated 305.1).
(E)-3-(3-(2-Methoxyphenyl)-3-oxoprop-1-enyl)-6-methylquinolin-2(1H)-one (CTR-18). Yield 86%; M.P. 222–224° C.; FT-IR (KBr) υ (cm− 1): 3145 (NH), 1654 (C═O), 1584, 1558 (C═C), 1241, 1019 (C—O—C); 1H NMR (400 MHz, DMSO-d6): δ 11.88 (s, 1H), 8.15 (s, 1H), 7.89 (d, J = 15.9 Hz, 1H), 7.58 (d, J = 15.9 Hz, 1H), 7.50 (t, J = 7.5 Hz, 2H), 7.44 (s, 1H), 7.32 (d, J = 8.5 Hz, 1H), 7.26 (d, J = 8.4 Hz, 1H), 7.10 (d, J = 8.3 Hz, 1H), 7.04 (t, J = 7.4 Hz, 1H), 3.90 (s, 3H), 2.39 (s, 3H), MS-API: [M + H] + 320.1 (calculated 319.12).
(E)-7-Methoxy-3-(3-(2-methoxyphenyl)-3-oxoprop-1-enyl) quinolin-2(1H)-one (CTR-19). Yield 83%; M.P. 227–229° C.; FT-IR (KBr) υ (cm− 1): 3144 (NH), 1656 (C═O), 1559 (C═C), 1167, 1021 (C—O—C); 1H NMR (400 MHz, DMSO-d6): δ 11.96 (s, 1H), 8.40 (s, 1H), 7.85 (d, J = 16.0 Hz, 1H), 7.60–7.42 (m, 3H), 7.32–7.18 (m, 4H), 7.08 (t, J = 7.4 Hz, 1H), 3.87 (s, 3H), 3.81 (s, 3H); MS-API: [M + H] + 336.1 (calculated 335.12).
(E)-6-Methoxy-3-(3-(2-methoxyphenyl)-3-oxoprop-1-enyl) quinolin-2(1H)-one (CTR-20). Yield 83%; M.P. 227–229° C.; FT-IR (KBr) υ (cm− 1); 3155 (NH), 1652 (C═O), 1597, 1558 (C═C), 1164, 1022 (C—O—C); 1H NMR (400 MHz, DMSO-d6): δ 11.91 (s, 1H), 8.37 (s, 1H), 7.78 (d, J = 15.9 Hz, 1H), 7.64 (d, J = 8.8 Hz, 1H), 7.57–7.49 (m, 2H), 7.48–7.40 (m, 1H), 7.20 (d, J = 8.5 Hz, 1H), 7.07 (t, J = 7.7 Hz, 1H), 6.89–6.81 (m, 2H), 3.85 (s, 3H), 3.84 (s, 3H); MS-API: [M + H] + 336.1 (calculated 335.12).
(E)-3-(3-(2,6-dimethoxyphenyl)-3-oxoprop-1-enyl)quinolin-2(1H)-one (CTR-25).
Yield 65%; M.P. 236–238° C.; FT-IR (ATR) υ (cm− 1): 3149 (NH), 1667 (C═O), 1591, 1558 (C═C), 1252, 1058 (C—O—C); 1H NMR (400 MHz, DMSO-d6): δ 12.00 (s, 1H), 8.41 (s, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.47–7.55 (m, 1H), 7.32–7.41 (m, 2H), 7.21–7.31 (m, 2H), 7.18 (t, J = 7.6 Hz, 1H), 6.73 (d, J = 8.5 Hz, 2H), 3.69 (s, 6H); MS-API: [M + H] + 336.2 (calculated 335.12).
(E)-3-(3-(2-ethoxyphenyl)-3-oxoprop-1-enyl)quinolin-2(1H)-one (CTR-32)
Yield 77%; M.P. 199–201° C.; FT-IR (ATR) υ (cm− 1): 3128 (NH), 1651 (C═O), 1597, 1555 (C═C), 1169, 1023 (C—O—C); 1H NMR (400 MHz, DMSO-d6): δ 12.02 (s, 1H), 8.39 (s, 1H), 8.00 (d, J = 15.8 Hz, 1H), 7.68 (dd, J = 8.0, 1.3 Hz, 1H), 7.44–7.56 (m, 4H), 7.26–7.32 (m, 1H), 7.11–7.23 (m, 2H), 7.02 (td, J = 7.5, 1.0 Hz, 1H), 4.12 (q, J = 7.0 Hz, 2H), 1.31 (t, J = 6.9 Hz, 3H). MS-API: [M + H] + 320.2 (calculated 319.12).
(E)-3-(3-(2-ethoxyphenyl)-3-oxoprop-1-enyl)-6-methoxyquinolin-2(1H)-one (CTR-40)
Yield 86%; M.P. 233–235° C.; FT-IR (KBr) υ (cm− 1): 3166 (NH), 1652 (C═O), 1598, 1560 (C═C), 1245, 1023 (C—O—C); 1H NMR (400 MHz, DMSO-d6): δ 11.94 (s, 1H), 8.33 (s, 1H), 8.00 (d, J = 15.8 Hz, 1H), 7.43–7.53 (m, 3H), 7.17–7.26 (m, 3H), 7.15 (d, J = 8.3 Hz, 1H), 7.02 (t, J = 7.5 Hz, 1H), 4.12 (q, J = 6.8 Hz, 2H), 3.77 (s, 3H), 1.31 (t, J = 7.0 Hz, 3H); MS-API: [M + H] + 350.2 (calculated 349.13).
(E)-8-Methoxy-3-(3-(2-methoxyphenyl)-3-oxoprop-1-enyl) quinolin-2(1H)-one (CTR-21). Yield 71%. 1H NMR (400 MHz, DMSO-d6): 3.86 (s, 3H), 3.91 (s, 3H), 7.06 (t, 1H), 7.17 (t, 3H), 7.29 (d, 1H), 7.47 (d, 1H), 7.52 (m, 2H), 7.86 (d, 1H), 8.45 (s 1H), 11.16 (1H). MS-API: [M + H] + 336.1 (Calculated: 335.12).
(E)-6,7-dimethoxy-3-(3-(2-methoxyphenyl)-3-oxoprop-1-enyl)quinolin-2(1H)-one (CTR-24)
1H NMR (400 MHz, DMSO-d6) δ 11.83 (s, 1H), 8.27 (s, 1H), 7.74 (d, J = 16.01 Hz, 1H), 7.38–7.56 (m, 3H), 7.14–7.19 (m, 2H), 7.03 (dt, J = 0.88, 7.44 Hz,1H), 6.83 (s, 1H), 3.82 (s, 3H), 3.81 (s, 3H), 3.77 (s, 3H)
(E)-3-(3-(2,5-dimethoxyphenyl)-3-oxoprop-1-enyl)quinolin-2(1H)-one (CTR-26)
1H NMR (400 MHz, DMSO-d6) δ 12.00 (s, 1H), 8.42 (s, 1H), 7.83 (d, J = 15.76 Hz, 1H), 7.69 (d, J = 8.00 Hz, 1H), 7.45–7.57 (m, 2H), 7.30 (d, J = 8.25 Hz,1H), 7.19 (t, J = 7.50 Hz, 1H), 7.04–7.14 (m, 2H), 6.98 (d, J = 2.75 Hz, 1H), 3.78 (s, 3H), 3.72 (s, 3H)
(E)-3-(3-(2,4-dimethoxyphenyl)-3-oxoprop-1-enyl)quinolin-2(1H)-one (CTR-27)
1H NMR (400 MHz, DMSO-d6) δ 11.98 (s, 1H), 8.38 (s, 1H), 7.99 (d, J = 16.01 Hz, 1H), 7.70 (d, J = 7.75 Hz, 1H), 7.49–7.58 (m, 3H), 7.30 (d, J = 8.25 Hz,1H), 7.19 (t, J = 7.50 Hz, 1H), 3.86 (s, 3H), 3.83 (s, 3H)
(E)-3-(3-(5-fluoro-2-methoxyphenyl)-3-oxoprop-1-enyl)quinolin-2(1H)-one (CTR-29)
1H NMR (400 MHz, DMSO-d6) δ 12.02 (s, 1H), 8.44 (s, 1H), 7.82 (d, J = 16.01 Hz, 1H), 7.69 (d, J = 7.75 Hz, 1H), 7.47–7.59 (m, 2H), 7.37 (dt, J = 3.25,8.63 Hz, 1H), 7.30 (d, J = 8.25 Hz, 1H), 7.26 (dd, J = 3.25, 8.76 Hz, 1H), 7.17–7.22 (m, 2H), 3.29 (s, 3H)
(E)-3-(3-(4-fluoro-2-methoxyphenyl)-3-oxo prop-1-enyl)quinolin-2(1H)-one (CTR-30)
1H NMR (400 MHz, DMSO-d6) δ 12.01 (s, 1H), 8.42 (s, 1H), 7.87 (d, J = 16.01 Hz, 1H), 7.69 (d, J = 7.75 Hz, 1H), 7.48–7.58 (m, 3H), 7.30 (d, J = 8.25 Hz,1H), 7.19 (t, J = 7.50 Hz, 1H), 7.09 (dd, J = 2.25, 11.51 Hz, 1H), 6.87 (dt, J = 2.50, 8.38 Hz, 1H), 3.86 (s, 3H)
(E)-3-(3-(2,5-dimethoxyphenyl)-3-oxoprop-1-enyl)-6-methoxyquinolin-2(1H)-one (CTR-34)
1H NMR (400 MHz, DMSO-d6) δ 11.92 (s, 1H), 8.36 (s, 1H), 7.82 (d, J = 16.01 Hz, 1H), 7.50 (d, J = 16.01 Hz, 1H), 7.18–7.26 (m, 3H), 7.07–7.12 (m, 2H), 6.98 (d, J = 2.50 Hz, 1H), 3.78 (s, 3H), 3.77 (s, 3H), 3.72 (s, 3H)
(E)-3-(3-(2,4-dimethoxyphenyl)-3-oxoprop-1-enyl)-6-methoxyquinolin-2(1H)-one (CTR-35)
1H NMR (400 MHz, DMSO-d6) δ 11.89 (s, 1H), 8.31 (s, 1H), 7.99 (d, J = 15.76 Hz, 1H), 7.57 (d, J = 2.75 Hz, 1H), 7.54 (d, J = 9.76 Hz, 1H), 7.16–7.26 (m,3H), 6.67 (d, J = 2.25 Hz, 1H), 6.62 (dd, J = 2.25, 8.51 Hz, 1H), 3.87 (s, 3H), 3.83 (s, 3H), 3.77 (s, 3H)
(E)-6-methoxy-3-(3-(2-methoxy-4-(trifluoro methoxy)phenyl)-3-oxoprop-1-enyl)quinolin-(1H)-one (CTR-36)
1H NMR (400 MHz, DMSO-d6) δ 11.94 (s, 1H), 8.37 (s, 1H), 7.83 (d, J = 15.76 Hz, 1H), 7.58 (d, J = 8.50 Hz, 1H), 7.51 (d, J = 15.76 Hz, 1H), 7.15–7.28 (m,4H), 7.04 (d, J = 8.50 Hz, 1H), 3.87 (s, 3H), 3.77 (s, 3H)
(E)-3-(3-(5-fluoro-2-methoxyphenyl)-3-oxoprop-1-enyl)-6-methoxyquinolin-2(1H)-one (CTR-37)
1H NMR (400 MHz, DMSO-d6) δ 11.94 (s, 1H), 8.37 (s, 1H), 7.82 (d, J = 16.01 Hz, 1H), 7.50 (d, J = 16.01 Hz, 1H), 7.37 (dt, J = 3.25, 8.63 Hz, 1H), 7.23–7.29 (m, 2H), 7.17–7.22 (m, 3H), 3.82 (s, 3H), 3.77 (s, 3H)
(E)-3-(3-(4-fluoro-2-methoxyphenyl)-3-oxoprop-1-enyl)-6-methoxyquinolin-2(1H)-one (CTR-38)
1H NMR (400 MHz, DMSO-d6) δ 11.91 (br. s., 1H), 8.35 (s, 1H), 7.86 (d, J = 16.01 Hz, 1H), 7.48–7.58 (m, 2H), 7.17–7.29 (m, 3H), 7.09 (dd, J = 2.50,11.51 Hz, 1H), 6.88 (dt, J = 2.38, 8.44 Hz, 1H), 3.86 (s, 3H), 3.77 (s, 3H)
Cell lines. The human MDA-MB-231, MCF7, MDA-MB-468, RPMI-8226 and HeLa cell lines were purchased from ATCC and maintained in Dulbecco's Modified Eagle Medium (DMEM) - high glucose supplemented with 10% fetal bovine serum (FBS) and antibiotics/antimycotics. Cell line authentication was carried out by Genetica DNA Laboratories (Burlington, NC) using a short tandem repeat (STR) profiling method (March 2015; July 2015; September 2016). Melanoma cells E-055 (MZ-Mel-3), E-097 (Mel-SOE), E-112 (UKRV-Mel-38), E-157 (UKRV-Mel-17) were obtained from the European Searchable Tumour Line Database (ESTDAB) cell bank (a kind gift of Dr. Graham Pawelec, Tubingen University, Germany). MDA-MB-435 was purchased from the Division of Cancer Treatment and Diagnosis Tumor Repository. All melanoma cells were cultured in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% FBS and P/S/antimycotics. Primary human epidermal melanocytes from normal adult abdominal skin were purchased from ATCC (cat # PCS-200-013) and Cell Applications (cat#104-05a). The melanocytes were cultured in the media supplied by the companies.
Sulforhodamine B (SRB) assay. The SRB assay was used to assess drug-induced cytotoxicity and cell proliferation as previously described20–22 with a few modifications. Briefly, cells were plated 10,000 to 25,000 cells/cm2 in 96-well plates (100 µl medium) and allowed to adhere overnight. The cell counts were different for each cell line to ensure optimal cell growth for the duration of the assay. Eight different concentrations of compound solutions using 2–4 times serial dilutions (100 µl). Cells were then incubated for an additional 48 hours and then fixed with 100 µl ice-cold 10% (w/v) trichloroacetic acid (TCA) (without removing the cell medium, final concentration, 3.3% of TCA) and stained with 0.4% SRB (w/v) in 1% acetic acid (v/v). The relative growth rate (%) was calculated for each of the compound concentrations according to the following formula: 100 * (T – T0)/(C – T0), in which T is the optical density (OD) after exposure to a certain concentration of the compound after 48 hours, T0 is the OD at the start of drug exposure (time zero) and C is the control growth. The GI50 for each compound was obtained from a non-linear sigmoidal dose-response (variable slope) curve fitted by GraphPad Prism v.4.03 software.
PBMCs isolation. Peripheral blood mononuclear cells from healthy donors were isolated by density gradient centrifugation using Ficoll solution. Briefly, whole blood (collected from Canadian Blood Services under Institutional Research Ethics Board Project # 18–099) was gently layered over an equal volume of Ficoll and centrifuged at 800 ×g for 20 minutes with no brake. The Buffy coat was carefully collected by pipetting in 50 mL tubes and topped off with PBS. The cell isolates were subsequently mixed by inversion and centrifuged at 300 ×g for 12 minutes. The supernatant was then carefully aspirated without disturbing the cell pellet, and the PBMCs were resuspended in ammonium-chloride-potassium (ACK) lysis buffer. The tubes were left to stand for 15 minutes to allow for red blood cell lysis. PBS was again added to the cells and centrifuged at 300 ×g for 12 minutes and pelleted PBMCs were resuspended in RPMI + 10% heat inactivated FBS. Following counting, cells were centrifuged again and cryopreserved with a freezing medium composed of 40% FBS, 10% DMSO and 50% culture medium. Cryovials were placed in a − 80°C for 24 hours prior to transfer to liquid nitrogen for long-term storage.
PBMCs thawing. PBMC cryovials were transferred from liquid nitrogen and placed in a 37°C water bath until almost completely thawed. 1 mL of pre-warmed medium was added drop-wise and the entire contents of the cryovial was transferred to a 15-mL tube. Pre-warmed medium was then added to bring the volume up to 10 mL. The PBMCs were centrifuged at 300 ×g for 10 minutes, and the cell pellet was gently resuspended with 5 mL of warm medium. The tubes were subsequently placed in a 37°C incubator in a 5° angled rack for 4 hours. An additional 5 mL warm medium containing of 30 units/mL benzonase nuclease was then injected into the cell solution and left to stand at room temperature for 5 minutes before centrifugation for 10 minutes at 300 ×g. The pelleted cells were resuspended in the medium with 100 units/mL of IL-2 (Cedarlane Laboratories, Burlington, ON, Canada) and a small aliquot was used for cell counting and viability staining. The cells were plated in 96-well plates at a density of 80,000-120,000 cellules/well/100 µL.
PBMCs culture and MTS assay. Following a 48-hour incubation period, eight different concentrations of compound solutions using two to four-fold serial dilutions were added to the cells (100 µl). Cells were incubated for an additional 48 hours and cytotoxicity was established using the CellTiter 96 cytotoxicity assay (Promega). 40 µl of the tetrazolium dye was added to each well of the plate and then incubated for 2 hours. OD was then read directly at 490 nm using the automated Biotek Synergy H4 plate reader. The GI50 was calculated similarly to the SRB.
Cell cycle analysis by flow cytometry. Cell cycle progression was determined using flow cytometry. UKRV-Mel-38 and HeLa cells were plated onto 10 cm plate at a density of 0.5 million and treated them with or without the compounds the following day for 12, 24 or 48 hours. Cells were then harvested and centrifuged at 1,000 rpm for 5 minutes, washed in PBS and then fixed with ice-cold ethanol (70%) for at least 24 hours at − 20°C. The cells were centrifuged, re-suspended in PBS solution, followed by centrifugation. The cell pellet was then stained with 100 µg/mL propidium iodide (PI) and 100 µg/mL RNase A in distilled water for at least 1 hour. DNA content was measured using a Beckmann Coulter Cytomics FC500 (Beckman Coulter, Fullerton, CA), and the proportion of cell populations in G0/G1, S, and G2/M phases of cell cycle was calculated on the basis of DNA distribution histograms using CXP software (Beckman Coulter, Fullerton, CA)
Cell Cycle Analysis by immunofluorescence and Western blots. Cell cycle analysis, including immunofluorescence and western blot analysis was carried out as described previously10.
Tubulin polymerization assay. Microtubule assembly was assessed using a Tubulin Polymerization assay kit (BK011P; Cytoskeleton Inc., Denver, CO). Purified porcine tubulin proteins (> 99% purity) were suspended in G-PEM buffer containing 80 mM PIPES, 2 mM MgCl2, 0.5 mM EGTA, 1 mM GTP (pH 6.9) and 20% glycerol. Polymerization was started by incubating at 37°C and followed by absorbance readings every 1 minute at 340 nm for 1 hour using a Synergy H4 plate reader.
Microsome-based assay. Microsome metabolism assay was carried out as per manufacturer protocol (ThermoFisher), with 5 µM of starting compound. Analytes were measured on a Waters Xevo G2-XS Quadrupole Time-of-Flight mass spectrometer. Half-life was calculated using exponential one phase decay (GraphPad Prism 5). Hydroxychloroquine was used as an internal standard (500 pg/µL).