Design, synthesis, and biological evaluation of indazole-based PLK4 inhibitors

Polo-like kinase 4 (PLK4), a member of the serine/threonine protein kinase family, serves as a central regulator of centriole duplication and plays a critical role in eukaryotic mitosis. Overexpression of PLK4 in several cancer types underscores its potential as a therapeutic target. In our previous studies, compound 28t demonstrated acceptable kinase inhibitory activity but exhibited poor cellular activity. Consequently, 28t was selected as a lead compound for further optimization. Through functional group migration and rational drug design strategies, we conducted structural modifications that ultimately yielded 23 novel indazole-based PLK4 inhibitors. Among these, compound C05 exhibited exceptional kinase inhibitory activity (IC₅₀ < 0.1 nM). At the cellular level, C05 demonstrated potent antiproliferative effects against IMR-32 (neuroblastoma), MCF-7 (breast cancer), and H460 (non-small cell lung cancer) cell lines, with IC₅₀ values of 0.948 μM, 0.979 μM, and 1.679 μM, respectively. Notably, compound C05 demonstrated favorable kinase selectivity towards PLK4 among the 10 kinases tested, achieving an inhibition rate of 87.45%. Further pharmacological experimental studies, including apoptosis induction, cell cycle arrest analysis, and clonogenic formation experiments, revealed that C05 outperformed the positive control LCR-263 in both potency and efficacy. Western blot analysis demonstrated that compound C05 effectively suppressed PLK4 autophosphorylation at 4 μM. Unfortunately, compound C05 demonstrated poor metabolic stability in human liver microsomes (HLMs), exhibiting a short half-life (<i>T</i> _1/2) of 2.69 minutes under standard incubation conditions. Notwithstanding the suboptimal metabolic stability, the compelling biological activity profile of compound C05 warrants further structural refinement.