The MYCN oncogene and histone deacetylases (HDACs) are key driver genes in the childhood cancer, neuroblastoma. We recently described a novel pyridobenzimidazole analogue, SE486-11, which enhanced the therapeutic effectiveness of HDAC inhibitors by increasing MYCN ubiquitination through effects on the deubiquitinase, ubiquitin-specific protease 5 (USP5). Here we describe the synthesis of a novel series of pyrimido[1,2-a]benzimidazole derivatives, and an evaluation of their cytopathic effects against non-malignant and human neuroblastoma cell lines. Among the tested analogues, 4-(4-methoxyphenyl)benzo[4,5]imidazo[1,2-a]pyrimidine (3a) was the most active compound against neuroblastoma cells (IC₅₀ ≤ 2 µM), with low cytotoxicity (IC₅₀ ≥ 15 µM) to normal cells. We show compound 3a bound to USP5 protein (K_d = 0.47 µM) in vitro and synergistically enhanced the efficacy of HDAC inhibitors against neuroblastoma cells. Moreover, knockdown of USP5 and MYCN in treated neuroblastoma cells showed that both USP5 and MYCN expression was necessary for the cytopathic activity of compound 3a, thus providing a clinically relevant rationale for further development of this of pyrimido[1,2-a]benzimidazole.

MYCN 癌基因和组蛋白脱乙酰酶 (HDAC) 是儿童癌症、神经母细胞瘤的关键驱动基因。我们最近描述了一种新型吡啶并苯并咪唑类似物 SE486-11，它通过影响去泛素酶、泛素特异性蛋白酶 5 (USP5) 来增加 MYCN 泛素化，从而增强 HDAC 抑制剂的治疗效果。在这里，我们描述了一系列新型嘧啶并[1,2- a ]苯并咪唑衍生物的合成，并评估了它们对非恶性和人神经母细胞瘤细胞系的细胞病变作用。在测试的类似物中，4-(4-甲氧基苯基)苯并[4,5]咪唑并[1,2-a]嘧啶(3a)是对抗神经母细胞瘤细胞最有效的化合物(IC ₅₀ ≤ 2 µM)，具有低细胞毒性(对正常细胞的 IC ₅₀ ≥ 15 µM)。我们在体外证明化合物 3a 与 USP5 蛋白 (K _d = 0.47 µM) 结合，并协同增强 HDAC 抑制剂对抗神经母细胞瘤细胞的功效。此外，在治疗的神经母细胞瘤细胞中敲低USP5和MYCN表明，USP5和MYCN表达对于化合物3a的细胞病变活性是必需的，从而为进一步开发嘧啶并[1,2- a ]苯并咪唑提供了临床相关的原理。