Compound 1 bearing with benzo [cd]indol-2(1H)-one scaffold was identified as an effective BRD4 inhibitor through the AlphaScreen-based high-throughput screening and its high-resolution crystal structure with BRD4_BD1 protein. A series of 48 compounds were designed and synthesized by structural optimization on compound 1. All the compounds have been evaluated for their BRD4 inhibitory activities. The results showed that compounds 23, 24, 28 and 44 are the most potential ones with the IC₅₀ values of 1.02 μM, 1.43 μM, 1.55 μM and 3.02 μM, respectively. According to their co-crystal structures in complex with BRD4_BD1 and the protein thermal shift assays, the binding modes were revealed that the additional indirect hydrogen bonds and hydrophobic interactions make such four compounds more active than 1 against BRD4. Furthermore, compounds 1, 23 and 44 were chosen to evaluate for their antiproliferative activities on the MLL-AF4-expression acute leukemia cell line (MV4-11), other cancer cell lines (MDA-MB-231, A549, 22Rv1) and the non-cancer cell lines (HUV-EC-C, MRC5, RPTEC). The results showed that these compounds exhibited good and selective inhibitory activities against MV4-11 cells with the IC₅₀ values of 11.67 μM, 5.55 μM, and 11.54 μM, respectively, and could act on the cell proliferation by blocking cell cycle at G1 phase. They could markedly down-regulate the expressions of the c-Myc, Bcl-2 and CDK6 oncogenes in MV4-11 in the qRT-PCR and western blot studies, which further demonstrated that compound 1 and its derivatives could serve as a promising therapeutic strategy for MLL leukemia by targeting BRD4_BD1 protein.

通过基于AlphaScreen的高通量筛选及其具有BRD4_BD1蛋白的高分辨率晶体结构，带有苯并[ cd ] indol -2（1 H）-1骨架的化合物1被鉴定为有效的BRD4抑制剂。通过化合物1的结构优化设计和合成了48种化合物。已经评估了所有化合物的BRD4抑制活性。显示的结果表明，化合物23，24，28和44是与IC最有潜力的人₅₀值分别为1.02μM，1.43μM，1.55μM和3.02μM。根据它们与BRD4_BD1配合使用的共晶体结构和蛋白质热位移分析，结合模式显示出额外的间接氢键和疏水相互作用使这四种化合物对BRD4的活性高于1。此外，化合物1，23和44被选择，以评估它们对MLL-AF4-表达急性白血病细胞系（MV4-11），其它癌细胞系（MDA-MB-231，A549，种前列腺）和抗增殖活性非癌细胞系（HUV-EC-C，MRC5，RPTEC）。结果表明，这些化合物对带有IC _50的MV4-11细胞表现出良好的选择性抑制活性值分别为11.67μM，5.55μM和11.54μM，并且可以通过阻止G1期的细胞周期来作用于细胞增殖。在qRT-PCR和Western blot研究中，它们可以显着下调MV4-11中c-Myc，Bcl-2和CDK6癌基因的表达，这进一步证明了化合物1及其衍生物可以作为一种有前途的治疗策略通过靶向BRD4_BD1蛋白治疗MLL白血病。