Discovery of novel pyridone-benzamide derivatives possessing a 1-methyl-2-benzimidazolinone moiety as potent EZH2 inhibitors for the treatment of B-cell lymphomas

Enhancer of zeste homolog 2 (EZH2) is a promising therapeutic target for diffuse large B-cell lymphoma. In this study, based on the binding model of 1 (tazemetostat) with polycomb repressive complex 2 (PRC2), we designed and synthesized a series of tazemetostat analogs bearing a 1-methyl-2-benzimidazolinone moiety to improve the inhibitory activity of EZH2 wild-type (WT) and Y641 mutants and enhance metabolic stability. After the assessment of the structure-activity relationship at enzymatic and cellular levels, compound N40 was identified. Biochemical assays showed that compound N40 (IC₅₀ = 0.32 nM) exhibited superior inhibitory activity against EZH2 WT, compared with 1 (IC₅₀ = 1.20 nM), and high potency against EZH2 Y641 mutants (EZH2 Y641F, IC₅₀ = 0.03 nM; EZH2 Y641N, IC₅₀ = 0.08 nM), which were approximately 10-fold more active than those of 1 (EZH2 Y641F, IC₅₀ = 0.37 nM; EZH2 Y641N, IC₅₀ = 0.85 nM). Furthermore, compound N40 (IC₅₀ = 3.52 ± 1.23 nM) effectively inhibited the proliferation of Karpas-422 cells and was more potent than 1 (IC₅₀ = 35.01 ± 1.28 nM). Further cellular experiments showed that N40 arrested Karpas-422 cells in the G1 phase and induced Apoptosis in a dose-dependent manner. Moreover, N40 inhibited the trimethylation of lysine 27 on histone H3 (H3K27Me3) in Karpas-422 cells bearing the EZH2 Y641N mutant. Additionally, N40 (T_1/2 = 177.69 min) showed improved metabolic stability in human liver microsomes compared with 1 (T_1/2 = 7.97 min). Our findings suggest N40 as a promising EZH2 inhibitor; further investigation remains warranted to confirm our findings and further develop N40.

EZH2 inhibitor; Improved in vitro activity; Polycomb repressive complex 2 (PRC2); Tazemetostat analogs; Trimethylation of lysine 27 on histone H3 (H3K27Me3).