*4.8. Cytotoxicity Bioassay*

Cell viability was analyzed by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliu mromide (MTT) assay as previously described [33]. In brief, cells were seeded in a 96-well plate at a density of 5 × 10<sup>3</sup> per well overnight and treated with compounds for demand time. OD570 values were detected using a Hybrid Multi-Mode Reader (Synergy H1, BioTek, Santa Clara, CA, USA). The experiment was independently repeated three times.

#### *4.9. Plate Clone Formation Assay*

PC-3 cells were seeded in the six-well plate at a density of 1000 cells per well overnight, then cells were treated with DMSO (0.1 %, *v*/*v*), docetaxel (0.1 μM), compound **4** (0.16 μM, 0.32 μM, and 0.64 μM), respectively, for demand time. The cell clone colony formation was observed after two weeks of treatment. Cells were fixed with 4% formaldehyde for 30 min

and washed with PBS buffer, then stained with crystal violet stain solution for 30 min. The dye solution was removed and washed the cells with PBS buffer again. Cell colonies were recorded and analyzed by the colony count analysis system (GelCount, Oxford Optronix, Oxford, UK). The experiment was repeated three times independently.

#### *4.10. Apoptosis and Cell Cycle Assay*

PC-3 cells were seeded in the six-well plate at a density of 2.0 × 10<sup>5</sup> cells/well and incubated overnight and treated with DMSO (0.1 %, *v*/*v*), docetaxel (1.0 μM), compound **4** (2.5 μM, 5.0 μM, 10 μM), respectively, for 48 h. Then, cells were collected and stained with annexin V-FITC and PI solution, following the manufacturer's manual (BMS500FI-300, Thermo Fisher Scientific, Waltham, MA, USA). Apoptotic rates and cell cycle distribution of PC-3 cells were examined and analyzed by flow cytometer (NovoCyte, Agilent, Santa Clara, CA, USA). Each experiment was repeated three times independently.
