In vitro activity: compound 11 (HJC0152 hydrochloride) significantly inhibits cell proliferation and induces apoptosis accompanying cellular morphological changes at concentrations of 1, 5, and 10 μM. Results show that treatment with 10 μM HJC0152 hydrochloride decreases the STAT3 promoter activity in MDA-MB-231 cells by approximately 32%, and increasing the dose of HJC0152 hydrochloride to 20 μM further decreases STAT3 promoter activity by 62% as compare with control. Total STAT3 is reduced after treatment with HJC0152 hydrochloride. HJC0152 hydrochloride induces cleaved caspase-3 and down regulates cyclin D1 in MDA-MB-231 cells.

Kinase Assay: HJC0152 hydrochloride is a novel, orally bioavailable and potent inhibitor of the signal transducers and activators of transcription 3 (STAT3). It has potential application for treating human head and neck squamous cell carcinoma (HNSCC).

Cell Assay: Breast cancer MDA-MB-231 cells are incubated in 6-well plates (2.5×105/well). Cells are then treated with DMSO, or HJC0152 hydrochloride (compound 11) at different concentrations for 48 h, and then both adherent and floating cells are collected, washed once with PBS. Resuspended cells are incubated with 100 μL PBS containing 1% BSA and 100 μL Annexin V and dead cell detection reagent at room temperature for 20 min. Apoptosis is measured immediately using the Muse Cell Analyzer with the MuseTM Apoptosis Kit.