This study presents the design, synthesis, and characterization of bisindole molecules as anti-cancer agents against Tousled-like kinases (TLKs). We show that compound 2 composed of an indirubin-3′-oxime group linked with a (N-methylpiperidin-2-yl)ethyl moiety possessed inhibitory activity toward both TLK1 and TLK2 in vitro and diminished the phosphorylation level of the downstream substrate anti-silencing function 1 (ASF1) in replicating cells. The treatment of compound 2 impaired DNA replication, slowed S-phase progression, and triggered DNA damage response in replicating cells. Structure optimization further discovered six derivatives exhibiting potent TLK inhibitory activity and revealed the importance of the tertiary amine-containing moiety of the side chain. Moreover, the derivatives 6, 17, 19, and 20 strongly suppressed the growth of triple-negative breast cancer MDA-MB-231 cells, non-small cell lung cancer A549 cells, and colorectal cancer HCT-116 cells, while normal lung fibroblast MRC5 and IMR90 cells showed a lower response to these compounds. Taken together, this study identifies tertiary amine-linked indirubin-3′-oximes as potent anticancer agents that inhibit TLK activity.

本研究介绍了双吲哚分子作为抗 Tousled 样激酶 (TLK) 的抗癌剂的设计、合成和表征。我们表明，由与 ( N-甲基哌啶-2-基) 乙基部分连接的靛玉红-3'-肟基团组成的化合物2在体外对 TLK1 和 TLK2 具有抑制活性，并降低下游底物抗磷酸化水平。在复制细胞中沉默功能 1 (ASF1)。化合物2的处理DNA 复制受损，S 期进程减慢，并在复制细胞中引发 DNA 损伤反应。结构优化进一步发现了六种表现出有效的 TLK 抑制活性的衍生物，并揭示了侧链含叔胺部分的重要性。此外，衍生物6、17、19和20强烈抑制三阴性乳腺癌 MDA-MB-231 细胞、非小细胞肺癌 A549 细胞和结肠直肠癌 HCT-116 细胞的生长，而正常肺成纤维细胞MRC5 和 IMR90 细胞对这些化合物的反应较低。总之，本研究将叔胺连接的靛玉红-3'-肟确定为抑制 TLK 活性的有效抗癌剂。