Rho相关的螺旋卷曲蛋白激酶（ROCK1和ROCK2）以及肌营养不良相关的Cdc-42结合激酶（MRCKα和MRCKβ）是细胞增殖和细胞可塑性的关键调节因子，这是与癌细胞迁移和浸润密切相关的过程。我们之前报道了一种新颖的小分子化合物（DJ4），是ROCK1/2和MRCKα/β专一性多激酶抑制剂的发现。在这里，我们进一步研究了DJ4在非小细胞肺癌和三阴性乳腺癌细胞中的抗增殖和凋亡效应。为了进一步优化DJ4对ROCK/MRCK的抑制效力，我们生成了一个包含27个类似物的文库。在各种结构修饰中，我们鉴定出了四个附加的活性类似物，其ROCK/MRCK的抑制效力得到了增强。对这些活性类似物在非小细胞肺癌、乳腺癌和黑素瘤细胞系中的抗增殖和细胞周期抑制效应进行了检查。通过使用NCI-60人类癌细胞系列来测试DJ4和活性类似物对多种癌细胞类型的抗增殖效果。最后，对这些新类似物在高度浸润的MDA-MB-231乳腺癌细胞中进行了抗迁移效应的测试。综上所述，我们的结果表明，针对ROCK1/2的专一性抑制剂（DJE4、DJ-Allyl）能够抑制细胞增殖并在G2/M期引起细胞周期阻滞，但相比于同时抑制ROCK1/2和MRCKα/β的剂型（DJ4和DJ110），其对细胞死亡诱导的效果较小。

The Rho associated coiled-coil containing protein kinase (ROCK1 and ROCK2) and myotonic dystrophy-related Cdc-42 binding kinases (MRCKα and MRCKβ) are critical regulators of cell proliferation and cell plasticity, a process intimately involved in cancer cell migration and invasion. Previously, we reported the discovery of a novel small molecule (DJ4) selective multi-kinase inhibitor of ROCK1/2 and MRCKα/β. Herein, we further characterized the anti-proliferative and apoptotic effects of DJ4 in non-small cell lung cancer and triple-negative breast cancer cells. To further optimize the ROCK/MRCK inhibitory potency of DJ4, we generated a library of 27 analogs. Among the various structural modifications, we identified four additional active analogs with enhanced ROCK/MRCK inhibitory potency. The anti-proliferative and cell cycle inhibitory effects of the active analogs were examined in non-small cell lung cancer, breast cancer, and melanoma cell lines. The anti-proliferative effectiveness of DJ4 and the active analogs was further demonstrated against a wide array of cancer cell types using the NCI-60 human cancer cell line panel. Lastly, these new analogs were tested for anti-migratory effects in highly invasive MDA-MB-231 breast cancer cells. Together, our results demonstrate that selective inhibitors of ROCK1/2 (DJE4, DJ-Allyl) inhibited cell proliferation and induced cell cycle arrest at G2/M but were less effective in cell death induction compared with dual ROCK1/2 and MRCKα/β (DJ4 and DJ110).