Pyrimido[1,2-a]benzimidazoles as inhibitors of oncoproteins ubiquitin specific protease 5 and MYCN in the childhood cancer neuroblastoma

Abstract

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.

Keywords: Neuroblastoma; Pyrimido[1,2-a]benzimidazoles; Structure activity relationship; Ubiquitin-specific protease 5.