Development of a novel indirubin derivative with enhanced anticancer properties: synthesis, in Vitro, and in Vivo evaluation

The indirubin ( 2 ) derivative undergoes condensation with hydroxylamine hydrochloride, resulting in the formation of a novel indirubin derivative ( 3 ) that incorporates both the oxime group and the piperidine heterocycle. This compound is subsequently treated with hydrochloric acid in ethanol, leading to the formation of the hydrochloride salt ( 3.HCl ), which exhibits excellent solubility in water. The chemical structures of compounds ( 3 ) and ( 3.HCl ) were determined using 1D and 2D nuclear magnetic resonance (NMR) spectroscopy, as well as high-resolution mass spectrometry with electrospray ionization (ESI). To evaluate their potential as anticancer agents, in vitro assays were conducted using three human cancer cell lines (A549, Hep-G2, SW480) and one murine cancer cell line (B16F10). These compounds, along with indirubin ( 1 ) and Bortezomib (BTZ), were tested. The results revealed that compounds ( 3 ) and ( 3.HCl ) demonstrated significant antitumor activity against all four cancer cell lines, with concentrations ranging from 0.37 ± 0.01 to 0.53 ± 0.06 μM. Importantly, their activity surpassed that of indirubin ( 1 ) and BTZ. Furthermore, an in vivo anticancer activity assay was performed on compound ( 3.HCl ) using the B16F10 cell line. After 18 days, the tumor size in the group treated with compound ( 3.HCl ) was approximately eight times smaller than that of the control group, and nearly three times smaller than the group treated with BTZ.