Antitumor activity of a pexidartinib bioisostere inhibiting CSF1 production and CSF1R kinase activity in human hepatocellular carcinoma

Macrophage colony-stimulating factor (M-CSF, also known as CSF1) in tumor tissues stimulates tumor growth and tumor-induced angiogenesis through an autocrine and paracrine action on CSF1 receptor (CSF1R). In the present study, novel bioisosteres of pexidartinib (1) were synthesized and evaluated their inhibitory activities against CSF1R kinase and tumor growth. Among newly synthesized bioisosteres, compound 3 showed the highest inhibition (95.1%) against CSF1R tyrosine kinase at a fixed concentration (1 μM). The half maximal inhibitory concentration (IC50) of pexidartinib (1) and compound 3 was 2.7 and 57.8 nM, respectively. Unlike pexidartinib (1), which cross-reacts to three targets with structural homology, such as CSF1R, c-KIT, and FLT3, compound 3 inhibited CSF1R, c-KIT, but not FLT3, indicating compound 3 may be a more selective CSF1R inhibitor than pexidartinib (1). The inhibitory effect of compound 3 on the proliferation of various cancer cell lines was the strongest in U937 cells followed by THP-1 cells. In the case of cancer cell lines derived from solid tumors, the anti-proliferative activity of compound 3 was weaker than pexidartinib (1), except for Hep3B. However, compound 3 was safer than pexidartinib (1) in terminally differentiated normal cells such as macrophages. Pexidartinib (1) and compound 3 suppressed the production of CSF1 in Hep3B liver cancer cells as well as in the co-culture of Hep3B cells and macrophages. Also, pexidartinib (1) and compound 3 decreased the population ratio of the M2/M1 phenotype and inhibited their migration. Importantly, compound 3 preferentially inhibited M2 phenotype over M1, and the effect was about 4 times greater than that of pexidartinib (1). In addition, compound 3 inhibited maintenance of cancer stem cell population. In a chick chorioallantoic membrane (CAM) tumor model implanted with Hep3B cells, tumor growth and tumor-induced angiogenesis were significantly blocked by compound 3 to a similar extent as pexidartinib (1). Overall, compound 3, a bioisostere of pexidartinib, is an effective dual inhibitor to block CSF1R kinase and CSF1 production, resulting in significant inhibition of tumor growth.