3.7.1. Cell Culture

Liver cancer Huh7.5 cell line used was obtained from the American Type Culture Collection (ATCC). Human normal liver LO2 cell line used was purchased from China Center for Type Culture Collection (CCTCC). Huh7.5 cells and LO2 cells were cultured at 37 ◦C in RPMI-1640 medium and DMEM medium, respectively, supplemented with 10% fetal bovine serum (FBS, PAN Biotech, Aidenbach, Germany), 100 U/mL penicillin, and 100 mg/mL streptomycin. All experiments were carried out with the same batch of cell line between passages 2 and 5 [21].

#### 3.7.2. Cell Proliferation Assay

The cytotoxic activities of compounds **1**–**10** against Huh7.5 liver tumor cells and human normal liver LO2 cell line were determined by the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, 6 <sup>×</sup> <sup>10</sup><sup>3</sup> of logarithmically growing Huh7.5 cells and human normal liver LO2 cell line were plated in the 96-well plate at 37 ◦C for 24 h. Then, cells were treated with DMSO (as the vehicle control) and increasing concentrations of test compounds (with the final concentration of 1, 2, 4, 5, 8, 10, 15, 20 μM) for 48 h, respectively. MTT solution (5 mg/mL, 20 μL per well) was added and incubated for another 4 h. After supernate from the wells were removed, DMSO was added to

each well to dissolve purple crystals of formazan with gentle shaking for 10 min, and optical density at 490 nm was read by a multi-detection microplate reader (Infinite M1000 Pro, Tecan, Switzerland). Sorafenib was used as positive control. All the compounds and positive control were dissolved and diluted in DMSO. All tests were performed in triplicate. The values of relative cell viability were calculated as percentages of absorbance from the treated samples to absorbance from the vehicle control [21].
