Abstract

Background Sorafenib is a tyrosine kinase inhibitor (TKI) used to treat hepatocellular carcinoma (HCC), but this drug causes clinically significant toxicities in approximately 50% of patients. Given the high frequency and severity of these side effects, it is necessary to develop new, safer drugs to treat this cancer.Purpose Novel 2,6,9-trisubstituted pyrimidine derivatives were synthesised and evaluated as potential antitumour agents for HCC.Materials and Methods Twelve compounds (6a-l) were obtained by a four-step synthetic procedure using a simple and efficient methodology in which two key reactions were promoted by microwave irradiation. Subsequently, compounds 6a-l were evaluated in vitro for cytotoxic activity against the HCC cell line HepG2 and other cell lines; in vivo in the HepG2 xenograft tumour model; and in silico (docking and dynamic simulations).Results and discussion Compound 6e proved to be the most promising of this series (IC50 = 5.6 mu M), as well as being more index selective than sorafenib and with lower cytotoxicity in Vero cells (18.92 mu M). In addition, 6e was further evaluated in Huh-7 cells and demonstrated selectivity for HCC. Docking studies on the proposed targets, VEGFR-2 and B-raf, indicated that 6e could bind to them with binding energies and interaction patterns similar to those of sorafenib. The 6e interaction pattern at the VEGFR-2 binding site was corroborated by dynamic studies over 100 ns. A possible mechanism of 6e-induced HepG2 cell death was investigated. Experiments on caspases-3, -7, -8, -9, Apaf-1, Cyt-c, ERK1/2, and p53 showed that they were all activated, whereas Bcl-2 was inhibited by 6e in HepG2 cells. Furthermore, 6e induced the accumulation of reactive oxygen species (ROS) in HepG2 cells. These results suggest that apoptosis in HepG2 was caused by: (i) a caspase-dependent pathway and (ii) changes in the cellular levels of Bcl-2 family proteins and ROS. In addition, 6e attenuated the growth of HepG2 xenograft tumours in mice at a dose of 1 mg/kg for 3 weeks.Conclusion Based on these results, this pyrimidine derivative could be an interesting compound for the design of new agents against HCC.