Reagents and chemicals
Melting points were determined by using an XT-4 binocular microscope (Beijing Tech Instrument Co., China) without correction. IR spectra were recorded on a Bruker VECTOR 22 spectrometer. NMR spectra were recorded in a CDCl3 solvent using a JEOL-ECX 500 NMR spectrometer operating at 500 MHz for 1H, and at 125 MHz for 13C by using TMS as internal standard. Elemental analysis was performed on an Elementar Vario-III CHN analyzer. Silica gel (200–300 mesh) and TLC plates (Qingdao Marine Chemistry Co., Qingdao, China) were used for chromatography. All solvents (Yuda Chemistry Co., Guiyang, China) were analytical grade, and used without further purification unless otherwise noted.
Synthetic procedures
6-methyl-quinazolin-4(3H)-one, 8-methyl-quinazolin-4(3H)-one, 6-methyl-4-chloroquiazoline, and 8-methyl-4-chloroquiazoline were prepared according to a previously described method [33]. Intermediate (E)-4-(2-hydroxyphenyl)-3-butylene-2-one was prepared according to a previously reported [34].
General synthetic procedures for the preparation of compounds 5a–5n
Compounds 2 (10 mmol), 3 (10 mmol) and K2CO3 (70 mmol) in 20 mL of acetonitrile was stirred at 30–40 °C for 3.5 h. The reaction mixture was concentrated and allowed to cool. The solid product obtained was filtered, and recrystallized with ethanol to afford the desired solid compound 4a or 4b, respectively. To the mixture of compound 4a or 4b (0.5 mmol) and sodium hydroxide (1%) in 20 mL of 75 vol% ethanol/water solution was added substituted aldehydes (0.5 mmol). The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated and suspended in water (20 mL), adjusted with 5% HCl to pH 7, and filtered. Recrystallization with ethanol afforded the desired solid compounds 5a–5n.
(1E,4E)-1-(2-fluorophenyl)-5-(2-((6-methylquinazolin-4-yl)oxy)phenyl)penta-1,4-dien-3-one (5a)
Yield: 52.6%; yellow powder; mp: 121–123 °C; IR (KBr, cm−1) ν: 3442, 1657, 1622, 1596, 1465, 1398, 1356, 1221, 983; 1H NMR (CDCl3, 500 MHz) δ: 8.70 (s, 1H, Qu-2-H), 8.23 (d, J = 12.00 Hz, 1H, F–Ar–CH=), 7.93 (d, J = 8.6 Hz, 1H, Ar–CH=), 7.78–7.85 (m, 3H, Qu-5,7,8-H), 7.47–7.50 (m, 3H, F–Ar-4,6-H, Ar-3-H), 7.30–7.39 (m, 5H, F–Ar-3,5-H, Ar-4,5-H, F–Ar–C=CH), 7.10 (d, J = 16.0 Hz, 1H, Ar–C=CH), 6.81 (d, J = 14.8 Hz, 1H, Ar-6-H), 2.61 (s, 3H, CH3); 13C NMR (CDCl3, 125 MHz) δ: 188.8, 166.4, 153.4, 153.4, 151.7, 150.4, 136.9, 136.7, 136.5, 131.7, 129.4, 128.2, 127.9, 127.7, 127.2, 127.1, 126.6, 126.5, 123.6, 123.5, 122.3, 116.4, 21.9; Anal. Calcd for C25H19FN2O2: C 76.08; H 4.67; N 6.83; Found: C 76.42; H 4.78; N 6.80.
(1E,4E)-1-(2-chlorophenyl)-5-(2-((6-methylquinazolin-4-yl)oxy)phenyl)penta-1,4-dien-3-one (5b)
Yield: 46.3%; yellow powder; mp: 152–154 °C; IR (KBr, cm−1) ν: 3445, 1653, 1618, 1584, 1481, 1400, 1359, 1223, 986; 1H NMR (CDCl3, 500 MHz) δ: 8.69 (s, 1H, Qu-2-H), 8.22 (d, J = 8.0 Hz, 1H, Cl–Ar–CH=), 7.76–7.95 (m, 4H, Ar–CH=, Qu-5,7,8-H), 7.38–7.53 (m, 3H, Cl–Ar-3,6-H, Ar-3-H), 7.23–7.31 (m, 5H, Cl–Ar-4,5-H, Ar-5-H, Ar–C=CH, Cl–Ar–C=CH), 7.21 (m, 1H, Ar-4-H), 6.82 (d, J = 14.8 Hz, 1H, Ar-6-H), 2.62 (s, 3H, CH3); 13C NMR (CDCl3, 125 MHz) δ: 188.6, 167.1, 154.3, 153.1, 151.0, 142.6, 142.1, 136.5, 134.5, 133.3, 132.6, 130.0, 129.6, 129.4, 127.4, 125.8, 125.6, 122,9, 122.7, 121.2, 116.3, 17.7; Anal. Calcd for C26H19ClN2O2: C 73.2; H 4.50; N 6.56; Found: C 73.27; H 4.56; N 6.42.
(1E,4E)-1-(4-chlorophenyl)-5-(2-((6-methylquinazolin-4-yl)oxy)phenyl)penta-1,4-dien-3-one (5c)
Yield: 55.8%; yellow powder; mp: 173–176 °C; IR (KBr, cm−1) ν: 3445, 1653, 1622, 1558, 1489, 1373, 1229, 986; 1H NMR (CDCl3, 500 MHz) δ: 8.70 (s, 1H, Qu-2-H), 8.23 (d, J = 12.0 Hz, 1H, Cl–Ar–CH=), 7.93 (d, J = 8.6 Hz, 1H, Ar–CH=), 7.78–7.85 (m, 3H, Qu-5,7,8-H), 7.47–7.50 (m, 3H, Cl–Ar-2,6-H, Ar-3-H), 7.30–7.39 (m, 5H, Cl–Ar-3,5-H, Ar-4,5-H, Cl–Ar–C=CH), 7.10 (d, J = 16.0 Hz, 1H, Ar–C=CH), 6.81 (d, J = 14.8 Hz, 1H, Ar-6-H), 2.61 (s, 3H, CH3); 13C NMR (CDCl3, 125 MHz) δ: 185.7, 167.4, 154.3, 153.1, 148.1, 147.4, 134.5, 134.1, 133.5, 132.3,131.3, 130.0, 129.8, 129.2, 128.9, 127.3, 127.1, 122.8, 122.6, 121.1, 116.3, 17.8; Anal. Calcd for C26H19ClN2O2: C 73.15; H 4.49; N 6.56; Found: C 72.43; H 4.12; N 6.79.
(1E,4E)-1-(2-chloro-5-nitrophenyl)-5-(2-((6-methylquinazolin-4-yl)oxy)phenyl)penta-1,4-dien-3-one (5d)
Yield: 58.2%; yellow powder; mp: 176–178 °C; IR (KBr, cm−1) ν: 3445, 1653, 1622, 1576, 1522, 1458, 1348, 1277, 1221, 983; 1H NMR (CDCl3, 500 MHz) δ: 8.68 (s, 1H, Qu-2-H), 8.40 (s, 1H, Cl–Ar-6-H), 8.21 (d, J = 15.0 Hz, 1H, Cl–Ar-4-H), 8.10–8.12 (d, J = 10.0 Hz, 1H, Qu-8-H), 7.73–7.92 (m, 5H, Cl–Ar–CH=, Qu-5,7-H, Cl–Ar-3-H, Ar–CH=), 7.54–7.57 (m, 2H, Cl–Ar–C=CH, Ar-3-H), 6.91–7.41 (m, 4H, Ar-4,5,6-H, Ar–C=CH), 2.61 (s, 3H, CH3); 13C NMR (CDCl3, 125 MHz) δ: 187.8, 179.6, 158.5, 153.3, 151.9, 146.8, 138.1, 136.7, 136.4, 134.6, 132.1, 131.3, 130.7, 130.2, 128.4, 127.8, 126.7, 125.1, 123.6, 122.9, 122.5, 122.2, 116.7, 21.9; Anal. Calcd for C26H18N3O4: C 66.18; H 3.84; N 8.90; Found: C 65.81; H 3.66; N 9.30.
(1E,4E)-1-(2,4-dichlorophenyl)-5-(2-((6-methylquinazolin-4-yl)oxy)phenyl)penta-1,4-dien-3-one (5e)
Yield: 60.5%; yellow powder; mp: 211–214 °C; IR (KBr, cm−1) ν: 3443, 1655, 1618, 1582, 1499, 1371, 1225, 986; 1H NMR (CDCl3, 500 MHz) δ: 8.68 (s, 1H, Qu-2-H), 8.21 (s, 1H, Qu-5-H), 7.60–7.93 (m, 4H, Qu-7,8-H, Cl–Ar–CH=, Ar–CH=), 7.38–7.43 (m, 4H, Cl–Ar-3-H, Ar-3-H, Cl–Ar-5,6-H), 7.26–7.31 (m, 3H, Ar-4,5-H, Cl–Ar–C=CH), 7.12 (d, J = 16.5 Hz, 1H, Ar–C=CH), 6.80 (d, J = 16.1 Hz, 1H, Ar-6-H), 2.61 (s, 3H, CH3); 13C NMR (CDCl3, 125 MHz) δ: 188.5, 167.1, 153.4, 153.1, 151.4, 142.7, 137.9, 136.6, 136.5, 134.5, 132.3, 131.6, 130.2, 130.1, 128.7, 128.3, 127.6, 127.4, 125.0, 122.7, 121.2, 116.2, 17.7; Anal. Calcd for C26H18Cl2N2O2: C 67.69; H 3.93; N 6.07; N 7.07; Found: C 67.56; H 3.45; N 5.65.
(1E,4E)-1-(2,6-dichlorophenyl)-5-(2-((6-methylquinazolin-4-yl)oxy)phenyl)penta-1,4-dien-3-one (5f)
Yield: 55.2%; yellow powder; mp: 187–189 °C; IR (KBr, cm−1) ν: 3443, 1655, 1618, 1582, 1499, 1333, 1225, 986; 1H NMR (CDCl3, 500 MHz) δ: 8.68 (s, 1H, Qu-2-H), 8.20 (s, 1H, Qu-5-H), 7.89 (d, J = 8.5 Hz, 1H, Qu-8-H), 7.80–7.85 (m, 2H, Ar–CH=, Cl–Ar–CH=), 7.73 (d, J = 8.8 Hz, 1H, Qu-7-H), 7.52–7.61 (m, 2H, Cl–Ar-3,5-H), 7.39 (m, 1H, Cl–Ar-4-H), 7.24–7.30 (m, 3H, Ar-3,5-H, Ar–C=CH), 7.15 (m, 1H, Ar-4-H), 7.06 (d, J = 16.0 Hz, 1H, Cl–Ar–C=CH), 7.00 (d, J = 16.5 Hz, 1H, Ar-6-H), 2.60 (s, 3H, CH3); 13C NMR (CDCl3, 125 MHz) δ: 188.9, 166.4, 153.4, 151.7, 150.4, 138.4, 137.5, 136.7, 136.5, 135.2, 132.9, 132.3, 131.8, 129.9, 128.9, 128.2, 128.0, 127.9, 127.5, 126.7, 123.6, 122.2, 116.0, 21.9; Anal. Calcd for C26H18Cl2N2O2: C 67.69; H 3.93; N 6.07; Found: C 68.06; H 4.14; N 6.11.
1E,4E)-1-(2,5-dimethoxyphenyl)-5-(2-((6-methylquinazolin-4-yl)oxy)phenyl)penta-1,4-dien-3-one (5g)
Yield: 49.6%; yellow powder; mp: 122–123 °C; IR (KBr, cm−1) ν: 3443, 1653, 1618, 1576, 1497, 1458, 1360, 1223, 1114, 1045; 1H NMR (CDCl3, 500 MHz) δ: 8.68 (s, 1H, Qu-2-H), 8.22 (s, 1H, Qu-5-H), 7.81–7.92 (m, 5H, Qu-7,8-H, Ar–CH=, CH3O–Ar–CH=, Ar-3-H), 7.75 (d, J = 8.6 Hz, 1H, CH3O–Ar–C=CH), 7.51 (m, 1H, Ar-5-H), 7.38 (m, 1H, Ar-4-H), 7.17 (d, J = 16.0 Hz, 1H, Ar–C=CH), 6.99 (d, J = 2.8 Hz, 1H, Ar-6-H), 6.89–6.94 (m, 2H, CH3O–Ar-3,6-H), 6.81 (d, J = 2.8 Hz, 1H, CH3O–Ar-4-H), 3.76 (s, 6H, 2-OCH3), 2.57 (s, 3H, CH3); 13C NMR (CDCl3, 125 MHz) δ: 189.3, 166.5, 153.5, 153.4, 153.2, 151.6, 150.4, 138.8, 138.4, 136.6, 136.2, 131.5, 128.4, 128.1, 127.8, 127.1, 126.7, 126.6, 123.5, 122.4, 117.6, 113.2, 112.5, 56.1, 55.8, 21.9; Anal. Calcd for C28H24N2O4: C 74.3; H 5.35; N 6.19; Found: C 74.3; H 5.48; N 5.95.
(1E,4E)-1-(2-fluorophenyl)-5-(2-((8-methylquinazolin-4-yl)oxy)phenyl)penta-1,4-dien-3-one (5h)
Yield: 50.4%; yellow powder; mp: 155–157 °C; IR (KBr, cm−1) ν: 3445, 1653, 1620, 1582, 1506, 1481, 1398, 1223, 984; 1H NMR (CDCl3, 500 MHz) δ: 8.79 (s, 1H, Qu-2-H), 8.31 (d, J = 8.0 Hz, 1H, F–Ar–CH=), 7.77–7.85 (m, 3H, Qu-5,7-H, Ar–CH=), 7.67 (d, J = 16.5 Hz, 1H, F–Ar-6-H), 7.59 (m, 1H, Qu-6-H), 7.53 (m, 1H, F–Ar-4-H), 7.29–7.43 (m, 4H, Ar-3,5-H, F–Ar-3,5-H), 7.05–7.14 (m, 3H, Ar-4-H, F–Ar–C=CH, Ar–C=CH), 6.95 (d, J = 16.5 Hz, 1H, Ar-6-H), 2.76 (s, 3H, CH3); 13C NMR (CDCl3, 125 MHz) δ: 188.8, 167.1, 153.2, 151.7, 151.1, 136.9, 136.6, 136.0, 134.5, 131.9, 131.9, 129.3, 128.4, 128.1, 127.8, 127.8, 127.6, 126.6, 124.5, 123.6, 121.1, 116.4, 17.8; Anal. Calcd for C25H19FN2O2: C 76.08; H 4.67; N 6.83; Found: C 75.81; H 4.53; N 7.04.
(1E,4E)-1-(2-chlorophenyl)-5-(2-((8-methylquinazolin-4-yl)oxy)phenyl)penta-1,4-dien-3-one (5i)
Yield: 41.8%; yellow powder; mp: 152–154 °C; IR (KBr, cm−1) ν: 3443, 1655, 1616, 1595, 1481, 1406, 1358, 1229, 979; 1H NMR (CDCl3, 500 MHz) δ: 8.79 (s, 1H, Qu-2-H), 8.30 (d, J = 8.5 Hz, 1H, Cl–Ar–CH=), 7.96 (d, J = 16.5 Hz, 1H, Ar–CH=), 7.76–7.85 (m, 3H, Qu-5,6,7-H), 7.50-7.59 (m, 3H, Ar-3-H, Cl–Ar-3,6-H), 7.38-7.40 (m, 2H, Cl–Ar-4,5-H), 7.29–7.39 (m, 2H, Cl–Ar–C=CH, Ar–C=CH), 7.14–7.25 (m, 2H, Ar-4,5-H), 6.81 (d, J = 16.0 Hz, 1H, Ar-6-H), 2.77 (s, 3H, CH3); 13C NMR (CDCl3, 125 MHz) δ: 188.7, 167.1, 153.2, 151.7, 151.1, 139.2, 137.2, 136.6, 135.4, 134.6, 131.7, 131.3, 130.3, 128.4, 128.3, 128.1, 127.7, 127.6, 127.1, 126.6, 123.6, 121.1, 116.1, 17.8; Anal. Calcd for C26H19ClN2O2: C 73.15; H 4.49; N 6.56; Found: C 73.04; H 4.74; N 6.76%.
(1E,4E)-1-(4-chlorophenyl)-5-(2-((8-methylquinazolin-4-yl)oxy)phenyl)penta-1,4-dien-3-one (5j)
Yield: 58.6%; yellow powder; mp: 161–163 °C; IR (KBr, cm−1) ν: 3445, 1647, 1616, 1576, 1481, 1406, 1358, 1227, 937; 1H NMR (CDCl3, 500 MHz) δ: 8.79 (s, 1H, Qu-2-H), 8.20–8.34 (m, 3H, Qu-5,6,7-H), 7.72–7.86 (m, 4H, Ar–CH=, Ar-3-H, Cl–Ar–C=CH, Cl–Ar=CH), 7.52–7.64 (m, 4H, Cl–Ar-2,3,5,6-H), 7.41-7.42 (m, 1H, Ar-5-H), 7.30–7.32 (m, 1H, Ar-4-H), 7.11–7.14 (d, J = 15.0 Hz, 1H, Ar-6-H), 6.93–6.96 (d, J = 15.0 Hz, 1H, Ar–C=CH), 2.77 (s, 3H, CH3); 13C NMR (CDCl3, 125 MHz) δ: 188.6, 167.1, 153.2, 153.1, 151.7, 151.1, 141.9, 136.9, 136.7, 134.6, 131.7, 129.5, 129.2, 128.4, 128.1, 127.6, 127.2, 126.7, 125.8, 123.6, 121.1, 17.8; Anal. Calcd for C26H19ClN2O2: C 73.15; H 4.49; N 6.56; Found: C 73.36; H 4.65; N 6.86.
(1E,4E)-1-(2-chloro-5-nitrophenyl)-5-(2-((8-methylquinazolin-4-yl)oxy)phenyl)penta-1,4-dien-3-one (5k)
Yield: 54.5%; yellow powder; mp: 198–200 °C; IR (KBr, cm−1) ν: 3420, 1676, 1626, 1560, 1522, 1479, 1402, 1348, 1221, 980; 1H NMR (CDCl3, 500 MHz) δ: 8.78 (s, 1H, Qu-2-H), 8.39 (s, 1H, Cl–Ar-6-H), 8.32 (d, J = 8.0 Hz, 1H, Cl–Ar-4-H), 8.13 (d, J = 8.3 Hz, 1H, Qu-5-H), 7.76-7.88 (m, 4H, Ar–CH=, Cl–Ar–CH=, Qu-6, 7-H), 7.45–7.59 (m, 3H, Ar-3-H, Cl–Ar-3-H, Cl–Ar–C=CH), 7.23–7.40 (m, 2H, Ar-4,5-H), 7.10 (d, J = 12.5 Hz, 1H, Ar–C=CH), 6.93 (d, J = 16.0 Hz, 1H, Ar-6-H), 2.75 (s, 3H, CH3);13C NMR (CDCl3, 125 MHz) δ: 187.8, 167.1, 153.1, 151.8, 151.0, 146.7, 141.6, 138.2, 136.7, 136.6, 134.6, 132.0, 131.3, 130.1, 128.6, 127.7, 126.9, 126.7, 125.1, 123.7, 122.5, 120.9, 116.1, 17.7; Anal. Calcd for C26H18ClN3O4: C 66.18; H 3.84; N 8.90; Found: C 66.30; H 3.84; N 8.86.
(1E,4E)-1-(2,4-dichlorophenyl)-5-(2-((8-methylquinazolin-4-yl)oxy)phenyl)penta-1,4-dien-3-one (5l)
Yield: 58.6%; yellow powder; mp: 175–178 °C; IR (KBr, cm−1) ν: 3445, 1653, 1618, 1576, 1481, 1408, 1358, 1229, 984; 1H NMR (CDCl3, 500 MHz) δ: 8.79 (s, 1H, Qu-2-H), 8.30 (d, J = 8.0 Hz, 1H, Cl–Ar–CH=), 7.76–7.94 (m, 3H, Ar–CH=, Qu-5,7-H), 7.53–7.57 (m, 2H, Qu-6-H, Cl–Ar-3-H), 7.38–7.47 (m, 3H, Ar-3-H, Cl–Ar-5,6-H), 7.29–7.31 (m, 2H, Cl–Ar-4-H), 7.38–7.41 (m, 2H, Cl–Ar–C=CH, Ar-5-H), 7.15–7.17 (m, 2H, Ar–C=CH, Ar-4H), 6.78 (d, J = 16.5 Hz, 1H, Ar-6-H), 2.77 (s, 3H, CH3); 13C NMR (CDCl3, 125 MHz) δ: 188.5, 167.1, 153.2, 151.8, 151.1, 139.1, 137.5, 136.7, 135.4, 134.6, 134.1, 133.3, 131.8, 131.7, 129.2, 128.4, 128.0, 127.6, 127.4, 126.7, 125.8, 123.6, 121.0, 116.1, 17.7; Anal. Calcd for C26H18Cl2N2O2: C 67.69; H 3.93; N 6.07; Found: C 67.27; H 4.03; N 5.96%.
(1E,4E)-1-(2,6-dichlorophenyl)-5-(2-((8-methylquinazolin-4-yl)oxy)phenyl)penta-1,4-dien-3-one (5m)
Yield: 56.1%; yellow powder; mp: 161–163 °C; IR (KBr, cm−1) ν: 3421, 1676, 1620, 1587, 1481, 1400, 1359, 1225, 984; 1H NMR (CDCl3, 500 MHz) δ: 8.76 (s, 1H, Qu-2-H), 8.28 (d, J = 8.5 Hz, 1H, Ar–CH=), 7.73–7.85 (m, 3H, Cl–Ar–CH=, Qu-5,7-H), 7.52–7.59 (m, 3H, Cl–Ar-3,5-H, Qu- 6-H), 7.29–7.41 (m, 4H, Ar-3, 5-H, Cl–Ar-4-H, Cl–Ar–C=CH), 7.16 (m, 1H, Ar-4-H), 7.07 (d, J = 16.0 Hz, 1H, Ar–C=CH), 6.98 (d, J = 17.0 Hz, 1H, Ar-6-H), 2.75 (s, 3H, CH3); 13C NMR (CDCl3, 125 MHz) δ: 188.96, 167.10, 153.15, 151.70, 150.99, 137.59, 136.75, 135.68, 135.17, 134.57, 132.97, 131.85, 129.88, 128.85, 128.36, 127.88, 127.65,127.45, 126.72, 123.60, 121.04, 116.02, 17.79; Anal. Calcd for C26H18Cl2N2O2 (461): C, 67.69; H, 3.93; N, 6.07; N, 7.07%. Found: 67.36; H, 3.96; N, 5.84%.
(1E,4E)-1-(2,5-dimethoxyphenyl)-5-(2-((8-methylquinazolin-4-yl)oxy)phenyl)penta-1,4-dien-3-one (5n)
Yield: 43.6%; yellow powder; mp: 176–178 °C; IR (KBr, cm−1) ν: 3445, 1647, 1616, 1570, 1491, 1373, 1211, 984; 1H NMR (CDCl3, 500 MHz) δ: 8.79 (s, 1H, Qu-2-H), 8.30 (d, J = 8.6 Hz, 1H, CH3O–Ar–CH=), 7.75–7.92 (m, 4H, Ar–CH=, Qu-5,6,7-H), 7.50–7.59 (m, 2H, Ar-3,5-H), 7.39 (m, 1H, Ar-4-H), 7.15–7.29 (m, 2H, CH3O–Ar–C=CH, Ar–C=CH), 6.98 (s, 1H, CH3O–Ar-6-H), 6.89–6.93 (m, 2H, Ar-6-H, CH3O–Ar-3-H), 6.81 (d, J = 8.6 Hz, 1H, CH3O–Ar-4-H), 3.77 (s, 6H, 2CH3O), 2.76 (s, 3H, CH3); 13C NMR (CDCl3, 125 MHz) δ: 189.25, 167.14, 153.57, 153.18, 151.66, 151.03, 138.78, 136.58, 136.25, 134.51, 131.45, 128.41, 128.23, 127.55, 127.24, 126.58, 124.21, 123.54, 121.16, 120.94, 117.61, 116.16, 113.22, 112.47, 56.08, 55.85, 17.75. Anal. Calcd for C28H24N2O4 (453): C, 74.32; H, 5.35; N, 6.19; %. Found: C, 74.55; H, 5.68; N, 5.95%.
Cell culture
Human gastric cancer cell line MGC-803, human prostate cancer cell line PC3, and human breast cancer cell line Bcap-37 and one normal cell line NIH3T3 were obtained from Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). NIH3T3 was routinely maintained in a DMEM medium, while all the other cell lines were cultured in a 1640 medium. All the cells were grown in the medium supplemented with 10% FBS at 37 °C with 5% CO2.
MTT assay
The growth-inhibitory effects of the test compounds were determined on MGC-803, PC3, Bcap-37, and NIH3T3 cells. All cell types were seeded into 96-well plates at a density of 2 × 103 cells/well 100 μL of the proper culture medium and incubated with increasing concentrations of the compounds at 37 °C under cell culturing conditions. An MTT assay (Roche Molecular Biochemicals, 1465-007) was performed 72 h later according to the instructions provided by Roche. The precipitated formazan crystals were dissolved in SDS, and the absorbance was read at 595 nm with a microplate reader (BIO-RAD, model 680), which is directly proportional to the number of living cells in culture. The experiment was performed in triplicate. The percentage cytotoxicity was calculated using the formula.
$$\% {\text{Cytotoxicity}} = \left[ {\left( {{\text{Control}}_{\text{abs}} - {\text{Blank}}_{\text{abs}} } \right) - \left( {{\text{Test}}_{\text{abs}} - {\text{Blank}}_{\text{abs}} } \right)} \right]/\left( {{\text{Control}}_{\text{abs}} - {\text{Blank}}_{\text{abs}} } \right)\; \times \; 100$$
AO/EB staining
Cells were seeded in 6-well culture plates at a density of 5 × 104 cells/mL in 0.6 mL of medium and allowed to adhere to the plates overnight. The cells were incubated with different concentrations of compounds or vehicle solution (0.1% DMSO) in a medium containing 10% FBS for 12 h. After the treatment, the cover slip with monolayer cells was inverted on the glass slide with 20 μL of AO/EB stain (100 μg/mL), and finally analyzed for morphological characteristics of cell apoptosis under a fluorescence microscope (Olympus Co., Japan).
Hoechst 33,258 staining
Cells were seeded in 6-well culture plates at a density of 5 × 104 cells/mL in 0.6 mL of medium and allowed to adhere to the plates overnight. The cells were incubated with different concentrations of compounds or vehicle solution (0.1% DMSO) in a medium containing 10% FBS for 12 h. After the treatment, the cells were fixed with 4% paraformaldehyde for 10 min, followed by incubation with Hoechst 33,258 staining solution (Beyotime) for 5 min and finally analyzed for morphological characteristics of cell apoptosis under a fluorescence microscope (Olympus Co., Japan).
Flow cytometry analysis
To further quantitative analysis of apoptosis, the cells were washed with PBS, stained with annexinV-FITC and propidium iodide (PI) using the AnnexinV-FITC kit (KeyGEN BioTECH). The cells were then subjected to flow cytometry according to manufacturer’s instructions and the stained cells were analyzed by FACS can flow cytometer (Becton–Dickinson, CA, USA).
Statistical analysis
All statistical analysis was performed with SPSS Version 19.0. Data was analyzed by one-way ANOVA. Mean separations were performed using the least significant difference method. Each experiment was replicated thrice, and all experiments yielded similar results. Measurements from all the replicates were combined, and treatment effects were analyzed.