MYCN癌基因和组蛋白去乙酰化酶（HDACs）是儿童癌症神经母细胞瘤中的关键驱动基因。我们最近描述了一种新型吡啶并苯并咪唑类似物SE486-11，通过对去泛素化酶——泛素特异性蛋白酶5（USP5）的影响增加MYCN泛素化来增强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，以及它们对非恶性和人类神经母细胞瘤细胞系的细胞病学效应的评估。在测试的类似物中，4-（4-甲氧基苯基）苯并[4,5]咪唑[1,2-a]嘧啶（3a）是对神经母细胞瘤细胞最活跃的化合物（IC50≤2 µM），对正常细胞的细胞毒性低（IC50≥15 µM）。我们展示3a化合物在体外结合到USP5蛋白（Kd=0.47 µM），并协同增强HDAC抑制剂对神经母细胞瘤细胞的疗效。此外，对受治疗的神经母细胞瘤细胞USP5和MYCN进行敲除，表明化合物3a的细胞病活性需要USP5和MYCN表达，因此为进一步开发这种嘧啶并[1,2-a]苯并咪唑提供了临床相关的理论基础。版权所有© 2023年Elsevier Inc.。保留所有权利。

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 (Kd = 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.Copyright © 2023 Elsevier Inc. All rights reserved.