Discovery and biological evaluation of N-(3-(7-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-4-methyl-2-oxo-2H-pyrimido[4,5-d][1,3]oxazin-1(4H)-yl)phenyl)acrylamide as potent Bruton’s tyrosine kinase inhibitors


Bruton’s tyrosine kinase (BTK) is a key component of the B cell receptor (BCR) signaling pathway and plays a crucial role in B cell malignancies and autoimmune disorders; thus, it is an attractive target for the treatment of B cell related diseases. Here, we evaluated the BTK inhibitory activity of a series of pyrimido[4,5-d][1,3]oxazin-2-one derivatives. Combining this evaluation with structure-activity relationship (SAR) analysis, we found that compound 2 exhibited potent BTK kinase inhibitory activity, with an IC50 of 7 nM. This derivative markedly inhibited BTK activation in TMD8 B cell lymphoma cells and thus inhibited the in vitro growth of the cells. Further studies revealed that compound 2 dose dependently arrested TMD8 cells at G1 phase, accompanied by decreased levels of Rb, phosphorylated Rb, and cyclin D1. Moreover, following treatment with compound 2, TMD8 cells underwent apoptosis associated with PARP and caspase 3 cleavage. Interestingly, the results of the kinase activity assay on a small panel of 35 kinases showed that the kinase selectivity of compound 2 was superior to that of the first-generation inhibitor ibrutinib, suggesting that compound 2 could be a second-generation inhibitor of BTK. In conclusion, we identified a potent and highly selective BTK inhibitor worthy of further development.

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The research is supported in part by the National Key Research and Development Program (Grant 2016YFA0502304), the Special Program for Applied Research on Super Computation of the NSFC-Guangdong Joint Fund (the second phase) under grant No. U1501501, and the Fundamental Research Funds for the Central Universities.

Author information

MZL, PRS, DD, YYD, JD, and HIL designed the study; MZL, PRS, LJT, and TZ performed the research; DD and YYD  contributed the compound; MZL, PRS, DD, and YYD  analyzed the data; and MZL, PRS, DD, HIL, and HX  wrote the paper.

Correspondence to Hua Xie or Hong-lin Li or Jian Ding.

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  • B cell receptor
  • Bruton’s tyrosine kinase
  • ibrutinib
  • small-molecule inhibitor
  • B cell malignancies