4.1 Synthesis of benzoyl chloride derivatives (1a-i)
To the benzoic acid derivatives (1 mmol), thionyl chloride (4 mmol, 0.28 mL) was added dropwise at room temperature and the resulting mixture was heated under reflux for 2 hours. The extra thionyl chloride was evaporated under vacuum and benzene was added 3 times to remove the remaining of thionyl chloride.
4.2 Synthesis of 7-hydroxy-2-phenyl-4H-chromen-4-one derivatives (2a-i)
Initially, 2,4-dihydroxyacetophenone (152 mg, 1 mmol) and potassium carbonate (553 mg, 4 mmol) were dissolved in dry acetone. Then the appropriate benzoyl chloride derivative (2 mmol) was added dropwise to the reflux solution for 5 minutes, and the resulting mixture was refluxed for 8 hours. After the completion of the reaction, the solvent was evaporated under vacuum. Next, 5 ml of water and methanol (1:1) were added and the mixture was refluxed for 2 hours. The progression of the reaction was monitored by TLC. Then, the mixture was cooled to room temperature and was poured into ice and then neutralized with 5% HCl solution. The precipitates were filtered and purified by preparative TLC (hexane: EtOAc, 1:1).
7-Hydroxy-2-phenyl-4H-chromen-4-one (2a)
White crystal, yield: 23%, mp: >250˚C, IR (KBr, cm-1): 3178 (OH), 1630; 1H NMR (500 MHz, DMSO-d6): δ 10.81 (s, 1H, OH), 8.07 (d, J=8.7 Hz ,2H, H2',6'), 7.89 (d, J=8.7 Hz, 1H, H5), 7.55-7.62 (m, 3H, H3',4',5'), 7.01 (d, J=2.1 Hz, 1H, H8), 6.93 (dd, J=8.7, 2.1 Hz, 1H, H6), 6.91 (s, 1H, H3(. Anal. calcd. for C15H10O3: C, 76.62; H, 4.23; O, 20.15. Found: C, 75.98; H, 4.54; O, 20.01.
7-Hydroxy-2-(4-methylphenyl)-4H-chromen-4-one (2b)
White crystal, yield: 21%, mp: >250˚C, IR (KBr, cm-1): 3069 (OH), 1628; 1H NMR (500 MHz, DMSO-d6): δ 10.78 (s, 1H, OH), 7.96 (d, J=8.7 Hz, 2H, H2',6'), 7.88 (d, J=8.7 Hz, 1H, H5), 7.38 (d, J=8.7 Hz, 2H, H3',5'), 7.0 (d, J=2.1 Hz, 1H, H8), 6.92 (dd, J=8.7, 2.1 Hz, 1H, H6), 6.85 (s, 1H, H3), 2.36 (s, 3H, CH3). Anal. calcd. for C16H12O3: C, 76.18; H, 4.79; O, 19.03. Found: C, 76.33; H, 4.52; O, 18.79.
7-Hydroxy-2-(4-methoxyphenyl)-4H-chromen-4-one (2c)
White crystal, yield: 32%, mp: >250˚C, IR (KBr, cm-1): 2981 (OH), 1685; 1H NMR (500 MHz, DMSO-d6): δ 10.93 (s, 1H, OH), 8.03 (d, J=8.7 Hz ,2H, H2',6'), 7.87 (d, J=8.7 Hz, 1H, H5), 7.11 (d, J=8.7 Hz, 2H, H3',5'), 7.0 (d, J=2.1 Hz, 1H, H8), 6.92 (dd, J=8.7, 2.1 Hz, 1H, H6), 6.80 (s, 1H, H3), 3.86 (s, 3H, OCH3). Anal. calcd. for C16H12O4: C, 71.64; H, 4.51; O, 23.86. Found: C, 71.22; H, 4.82; O, 24.01.
2-(4-chlorophenyl)-7-Hydroxy-4H-chromen-4-one (2d)
White crystal, yield:12%, mp: >250˚C, IR (KBr, cm-1): 3278 (OH), 1645; 1H NMR (500 MHz, DMSO-d6): δ 10.79 (s, 1H, OH), 8.10 (d, J=8.7 Hz, 2H, H2',6'), 7.89 (d, J=8.7 Hz, 1H, H5), 7.64 (d, J=8.7 Hz, 2H, H3',5'), 7.01 (d, J=2.1 Hz, 1H, H8), 6.95 (s, 1H, H3), 6.93 (d, J=2.1 Hz, 1H, H6). Anal. calcd. for C15H9ClO3: C, 66.07; H, 3.33; O, 17.60. Found: C, 65.89; H, 3.62; O, 17.33.
2-(4-bromophenyl)-7-Hydroxy-4H-chromen-4-one (2e)
White crystal, yield:16%, mp: >250˚C, IR (KBr, cm-1): 3301 (OH), 1607; 1H NMR (500 MHz, DMSO-d6): δ 10.69 (s, 1H, OH), 8.02 (d, J=8.7 Hz, 2H, H2',6'), 7.88 (d, J=8.7 Hz, 1H, H5), 7.78 (d, J=8.7 Hz, 2H, H3',5'), 6.99 (d, J=2.1 Hz, 1H, H8), 6.94 (s, 1H, H3), 6.92 (dd, J=8.7, 2.1 Hz, 1H, H6). Anal. calcd. for C15H9BrO3: C, 56.81; H, 2.86; O, 15.13. Found: C, 56.63; H, 3.07; O, 15.43.
2-(2,3-dimethoxyphenyl)-7-Hydroxy-4H-chromen-4-one (2f)
White crystal, yield:13%, mp: >250˚C, IR (KBr, cm-1): 3005 (OH), 1627; 1H NMR (500 MHz, DMSO-d6): δ 10.42 (s, 1H, OH), 7.90 (d, J=8.7 Hz ,2H, H5), 7.32 (dd, J=8.7, 2.1 Hz, 1H, H6), 7.19 (dd, J=8.7, 2.1 Hz, 1H, H4'), 6.94 (d, J=2.1 Hz, 1H, H8), 6.91-6.93 (m, 2H, H5',6'), 6.61 (s, 1H, H3), 3.88 (s, 1H, OCH3), 3.82 (s, 1H, OCH3). Anal. calcd. for C17H14O5: C, 68.45; H, 4.73; O, 26.82. Found: C, 68.18; H, 5.01; O, 26.65.
2-(2,4-dimethoxyphenyl)-7-Hydroxy-4H-chromen-4-one (2g)
White crystal, yield:15%, mp: >250˚C, IR (KBr, cm-1): 2998 (OH), 1638; 1H NMR (500 MHz, DMSO-d6): δ 10.35 (s, 1H, OH), 8.11 (d, J=8.7 Hz ,1H, H5), 7.84 (d, J=8.7 Hz, 1H, H6'), 7.08 (s, 1H, H3), 7.01 (d, J=8.7 Hz, 1H, H6), 6.97 (s, 2H, H3'), 6.60 (d, J=8.7 Hz, 1H, H5'), 6.52 (s, 1H, H8), 3.89 (s, 1H, OCH3), 3.86 (s, 1H, OCH3). Anal. calcd. for C17H14O5: C, 68.45; H, 4.73; O, 26.82. Found: C, 68.76; H, 4.41; O, 27.12.
2-(3,4-dimethoxyphenyl)-7-Hydroxy-4H-chromen-4-one (2h)
White crystal, yield:17%, mp: >250˚C, IR (KBr, cm-1): 3013 (OH), 1653; 1H NMR (500 MHz, DMSO-d6): δ 10.38 (s, 1H, OH), 7.94 (d, J=8.7 Hz ,1H, H5), 7.70 (dd, J=8.7, 2.1 Hz, 1H, H6'), 7.59 (s, 1H, H3), 7.37 (dd, J=8.7, 2.1 Hz, 1H, H6), 7.11-7.14 (m, 2H, H2',5'), 6.97 (s, 1H, H8), 3.89 (s, 1H, OCH3), 3.85 (s, 1H, OCH3). Anal. calcd. for C17H14O5: C, 68.45; H, 4.73; O, 26.82. Found: C, 68.09; H, 4.98; O, 26.86.
7-Hydroxy-2-(2,3,4-trimethoxy phenyl)-4H-chromen-4-one (2i)
White crystal, yield:26%, mp: >250˚C, IR (KBr, cm-1): 3014 (OH), 1629; 1H NMR (500 MHz, DMSO-d6): δ 10.80 (s, 1H, OH), 8.21 (d, J=8.7 Hz ,1H, H5), 7.87 (d, J=8.7 Hz, 1H, H6'), 7.56 (d, J=8.7 Hz, 1H, H5'), 7.23 (s, 1H, H3), 7.12-7.16 (m, 1H, H6), 6.78 (s, 1H, H8), 4.19 (s, 1H, OCH3), 4.11 (s, 1H, OCH3), 3.91 (s, 1H, OCH3). Anal. calcd. for C18H16O6: C, 65.85; H, 4.91; O, 29.24. Found: C, 65.30; H, 5.12; O, 29.40.
Synthesis of 4-oxo-2-phenyl-4H-chromen-7-yl methanesulfonate (3a-i)
To a solution of 2a-i (1 mmol) in dry tetrahydrofuran (5 mL) methanesulfonyl chloride (1.5 mmol) and triethylamine (2 mmol) was added dropwise in an ice bath and stirred at room temperature overnight. Then the volatiles were evaporated under vacuum. To this residue water was added and it was extracted with ethyl acetate. The organic phase was dried with sodium sulfate and the solvent was evaporated under vacuum. The product was purified by preparative TLC (hexane: EtOAc, 3:2).
4-oxo-2-phenyl-4H-chromen-7-yl methanesulfonate (3a)
White crystal, yield:67%, mp:120-121 ˚C, IR (KBr, cm-1): 1638, 1335 (S=O), 1199; 1H NMR (500 MHz, DMSO-d6): δ 8.17 (d, J=8.4 Hz, 1H, H5), 8.15 (d, J=8.4 Hz, 2H, H2',6'), 7.91 (d, J=1.9 Hz, 1H, H8), 7.67-7.59 (m, 3H, H3',4',5'), 7.50 (dd, J=8.4, 1.9 Hz, 1H, H6), 7.13 (s, 1H, H3), 3.55 (s, 3H, CH3SO2). Anal. calcd. for C16H12O5S: C, 60.75; H, 3.82; O, 25.29. Found: C, 60.81; H, 4.02; O, 25.53.
2-(4-methyl phenyl)-4-oxo-4H-chromen-7-yl methanesulfonate (3b)
White crystal, yield:69%, mp:184-185 ˚C, IR (KBr, cm-1): 1642, 1342 (S=O), 1177; 1H NMR (500 MHz, DMSO-d6): δ 8.15 (d, J=8.7 Hz, 1H, H5), 8.06 (d, J=8.7 Hz, 2H, H2',6'), 7.91 (d, J=2.2 Hz, 1H, H8), 7.48 (dd, J=8.7, 2.2 Hz, 1H, H6), 7.42 (dd, J=8.7, 2.2 Hz, 1H, H3',5'), 7.08 (s, 1H, H3), 3.55 (s, 3H, CH3SO2), 2.83 (s, 3H, CH3), 13C NMR (125 MHz, DMSO-d6): 177.0 C4, 163.9 C2, 156.6 C8a, 152.9 C7, 142.88 C4', 130.2 C3',5', 128.1 C5, 127.5 C1', 126.8 C2',6', 122.5 C4a, 120.4 C6, 112.6 C8, 106.7 C3, 38.2 C-S, 21.4 CH3. Anal. calcd. for C17H14O5S: C, 61.81; H, 4.27; O, 24.22. Found: C, 61.48; H, 4.43; O, 24.18.
2-(4-methoxy phenyl)-4-oxo-4H-chromen-7-yl methanesulfonate (3c)
White crystal, yield:78%, mp:208-212 ˚C, IR (KBr, cm-1): 1637, 1363 (S=O), 1179; 1H NMR (500 MHz, DMSO-d6): δ 8.13 (d, J=8.7 Hz, 1H, H5), 8.09 (d, J=8.9 Hz, 2H, H3',5'), 7.85 (d, J=2.2 Hz, 1H, H8), 7.46 (d, J=8.7 Hz, 1H, H6), 7.13 (d, J=8.9 Hz, 2H, H2',6'), 6.98 (s, 1H, H3), 3.87 (s, 3H, OCH3), 3.52 (s, 3H, CH3SO2), 13C NMR (125 MHz, DMSO-d6): 176.7 C4, 163.6 C2, 163 C4', 156.6 C8a, 153 C7, 128.7 C2',6', 127.5 C5, 123.3 C1', 122.8 C4a, 120.5 C6, 115.1 C3',5', 112.7 C8, 106.0 C3, 56.0 C-O, 37.4 C-S. Anal. calcd. for C17H14O6S: C, 58.95; H, 4.07; O, 27.72. Found: C, 59.22; H, 4.33; O, 28.10.
2-(4-chloro phenyl)-4-oxo-4H-chromen-7-yl methanesulfonate (3d)
White crystal, yield:71%, mp:201-203 ˚C, IR (KBr, cm-1): 1636, 1340 (S=O), 1179; 1H NMR (500 MHz, DMSO-d6): δ 8.19 (d, J=8.7 Hz, 2H, H2',6'), 8.16 (d, J=8.7 Hz, 1H, H5), 7.92 (d, J=2.2 Hz, 1H, H8), 7.69 (d, J=8.7 Hz, 2H, H3',5'), 7.50 (dd, J=8.7, 2.2 Hz, 1H, H6), 7.16 (s, 1H, H3), 3.55 (s, 3H, CH3SO2); MS: m/z 350 [M+1]. Anal. calcd. for C16H11ClO5S: C, 54.78; H, 3.16; O, 22.81. Found: C, 54.42; H, 3.33; O, 23.01.
2-(4-bromo phenyl)-4-oxo-4H-chromen-7-yl methanesulfonate (3e)
White crystal, yield:65%, mp:229-230 ˚C, IR (KBr, cm-1): 1640, 1340 (S=O), 1178; 1H NMR (500 MHz, DMSO-d6): δ 8.16 (d, J=8.7 Hz, 1H, H5), 8.11 (d, J=8.7 Hz, 1H, H2',6'), 7.92 (d, J=2.2 Hz, 1H, H8), 7.83 (d, J=8.7 Hz, 2H, H3',5'), 7.50 (dd, J=8.7, 2.2 Hz, 1H, H6), 7.17 (s, 1H, H3), 3.55 (s, 3H, CH3SO2); MS: m/z 395 [M+1]. Anal. calcd. for C16H11BrO5S: C, 48.62; H, 2.81; O, 22.24. Found: C, 48.43; H, 3.13; O, 22.56.
2-(2,3-dimethoxy phenyl)-4-oxo-4H-chromen-7-yl methanesulfonate (3f)
White crystal, yield:78%, mp:208-212 ˚C, IR (KBr, cm-1): 1648, 1368 (S=O), 1149;1H NMR (500 MHz, DMSO-d6): δ 8.17 (d, J=8.7 Hz, 1H, H5), 7.82 (d, J=2.2 Hz, 1H, H8), 7.50 (dd, J=8.9, 2.2 Hz, 1H, H6), 7.42 (d, J=2.2 Hz, 1H, H4'), 7.26-7.33 (m, 2H, H5',6'), 6.83 (s, 1H, H3), 3.89 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.53 (s, 3H, CH3SO2); ); 13C NMR (125 MHz, DMSO-d6): 177.2 C4, 163.9 C2, 161.9 C8a, 161.0 C7, 157.0 C3', 155.9 C2', 131.1 C5, 120.8 C1', 120.6 C5', 117.7 C6', 114.9 C4a, 112.5 C4', 110.9 C3, 110.0 C6, 105.1 C8, 59.5 C-O2', 58.5 C-O3', 42.0 C-S. Anal. calcd. for C18H16O7S: C, 57.44; H, 4.28; O, 29.76. Found: C, 57.73; H, 4.56; O, 30.03.
2-(2,4-dimethoxy phenyl)-4-oxo-4H-chromen-7-yl methanesulfonate (3g)
White crystal, yield:78%, mp:208-212 ˚C, IR (KBr, cm-1): 1659, 1349 (S=O), 1133; 1H NMR (500 MHz, DMSO-d6): δ 8.06 (d, J=8.7 Hz, 1H, H5), 7.94 (dd, J=8.9, 2.2 Hz, 1H, H6'), 7.62 (d, J=2.2, 1H, H8), 7.56 (d, J=2.2 Hz, 1H, H3'), 7.51 (dd, J=8.9, 2.2 Hz, 1H, H6), 7.27 (dd, J=8.9, 2.2 Hz, 1H, H 5'), 4.02 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 3.50 (s, 3H, CH3SO2); ); 13C NMR (125 MHz, DMSO-d6): 178.3 C4, 163.0 C2, 162.8 C4', 160.4 C2', 159.4 C8a, 157.9 C7, 130.2 C6', 128.7 C5, 117.7 C4a, 114.4 C1', 113.4 C3, 111.0 C6, 106.1 C5', 105.1 C8, 101.2 C3', 55.5 C-O2', 55.4 C-O4', 40.3 C-S. Anal. calcd. for C18H16O7S: C, 57.44; H, 4.28; O, 29.76. Found: C, 57.61; H, 4.55; O, 29.98.
2-(3,4-dimethoxy phenyl)-4-oxo-4H-chromen-7-yl methanesulfonate (3h)
White crystal, yield:78%, mp:208-212 ˚C, IR (KBr, cm-1): 1655, 1329 (S=O), 1169; 1H NMR (500 MHz, DMSO-d6): δ 7.94 (d, J=8.7 Hz, 1H, H5), 7.70 (d, J=8.9 Hz, 1H, H6'), 7.59 (s, 1H, H8), 7.37 (d, J=8.7 Hz, 1H, H6), 7.11-7.15 (m, 2H, H2',5'), 6.98 (s, 1H, H3), 4.01 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 3.85 (s, 3H, CH3SO2); 13C NMR (125 MHz, DMSO-d6): 178.9 C4, 165 C2, 160.0 C8a, 157.6 C7, 154.2 C3', 149.3 C4', 130.0 C5, 128.0 C1', 127.5 C6', 126.8 C4a, 112.5 C5', 108 C6, 105.2 C2', 104.5 C8, 103.9 C3, 56.9 C-O3', 57.4 C-O4', 42.4 C-S. Anal. calcd. for C18H16O7S: C, 57.44; H, 4.28; O, 29.76. Found: C, 57.09; H, 4.46; O, 29.88.
4-oxo-2-(2,3,4-trimethoxy phenyl)-4H-chromen-7-yl methanesulfonate (3i)
White crystal, yield:78%, mp:208-212 ˚C, IR (KBr, cm-1): 1680, 1328 (S=O), 1145; 1H NMR (500 MHz, DMSO-d6): δ 8.15 (d, J=8.7 Hz, 1H, H5), 7.81 (s, 1H, H8), 7.67 (d, J=8.7 Hz, 1H, H6), 7.47 (d, J=8.9 Hz, 1H, H6'), 7.03 (d, J=8.9 Hz, 1H, H5'), 6.84 (s, 1H, H3), 3.89 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 3.53 (s, 3H, CH3SO2); 13C NMR (125 MHz, DMSO-d6): 178.8 C4, 168.5 C2, 157.5 C8a, 157 C7, 154.6 C4', 152.9 C2', 141.9 C3', 129.0 C5, 122.7 C6', 119.5 C1', 118.9 C4a, 110.3 C3, 109.0 C6, 107.5 C5', 103.0 C8, 56.6 C-O2', 54.9 C-O3', 54.0 C-O4', 38.4 C-S. Anal. calcd. for C19H18O8S: C, 56.15; H, 4.46; O, 31.49. Found: C, 56.55; H, 4.87; O, 31.73.
4.3 Cell culture
A human breast cancer cell line, MCF-7, was obtained from the Iranian Biological Resource Center (Tehran, Iran) and maintained in RPMI 1640 medium (Biowest). The medium was supplemented with 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA) and 1% antibiotics (Penicillin and Streptomycin). The cells were incubated at 37 °C under the standard condition of 95% humidity and 5% CO2 to reach 70% cell confluency [24].
4.4 Cell proliferation assay
The cytotoxicity of compounds was assessed by MTT assay which measures the percentage of viable cells. Cells were seeded in a 96-well cell culture plate at 7 × 103 cells/well and incubated for 48 h. Then, cells were exposed to fresh medium containing different concentrations of compounds. Subsequently, the medium was replaced with tetrazolium salt (5 mg/ml of PBS) (MTT, (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) (Sigma, St.Louis, MO, USA) followed by additional 4 h incubation at 37 °C. The formed formazan crystals were dissolved in 100 µl of Dimethyl sulfoxide (DMSO) (Merck) and measured for absorbance at 570 nm using a plate reader (BioRad, Model 680). Also finally, IC50 values were defined as the half-maximal inhibitory concentration.
4.5 Flow cytometry analysis of cellular apoptosis
The Annexin V-FITC/PI (Propidium iodide) dual staining assay (BD Pharmingen™ kit) was used to determine the induction of apoptosis by the most potent compound 3c, in comparison with commercial anticancer drug docetaxel. The breast cancer cell line MCF-7 (3 × 105 cells/well) were seeded into 6-well plates and incubated overnight at 37 °C under 5% CO2 overnight. After treatment the cells with IC50 concentrations of compound 3c, docetaxel as positive control, and 1% DMSO as negative control, incubated with for 24h. Then, the treated cells were trypsinized, harvested and washed with PBS (pH 7.4). The cell suspension was centrifuged at 1200 and resuspended in 500 µl of 1× annexin V binding buffer containing 1.4 M NaCl, 25 mM CaCl2, 0.1 M HEPES/ NaOH (pH 7.4). The cells were double stained with 5 µL of Annexin V-PE and 5 µL of PI solution and incubated at room temperature in dark for 15 mins. After adding 400 μL of 1× annexin binding buffer to the vials, the cells were analyzed by flow cytometry using FITC signal detector (FL1) and PI staining by the phycoerythrin emission signal detector (FL2) [25].
4.6 Docking analysis
To determine the possible binding modes of the compound, docking analysis was carried out against the STS enzyme using the smina molecular docking The X-ray crystal structures of STS enzyme (PID: 1P49) were extracted from the PBD site and were prepared by removing solvent molecules and the co-crystallized ligands. Polar hydrogen atoms were added to the enzymes and the Kollmann charges were assigned. The compound (3c) was drawn using Marvin Sketch and subjected to energy minimization using the steepest descent algorithm and then Gasteiger charges were calculated by Open Babel. Binding sites were determined automatically using the coordinates of native co-crystallized ligands of enzymes [26].