3,5,7-Trisubstituted pyrazolo[4,3-d]pyrimidines have been identified as potent inhibitors of cyclin-dependent kinases (CDKs), which are established drug targets. Herein, we describe their further structural modifications leading to novel nanomolar inhibitors with strong antiproliferative activity. We determined the crystal structure of fully active CDK2/A2 with 5-(2-amino-1-ethyl)thio-3-cyclobutyl-7-[4-(pyrazol-1-yl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyrimidine (24) at 1.7 Å resolution, confirming the competitive mode of inhibition. Biochemical and cellular assays in lymphoma cell lines confirmed the expected mechanism of action through dephosphorylation of retinoblastoma protein and RNA polymerase II, leading to induction of apoptosis. Importantly, we also revealed an interesting ability of compound 24 to induce proteasome-dependent degradation of cyclin K both in vitro and in a patient-derived xenograft in vivo. We propose that 24 has a dual mechanism of action, acting as a kinase inhibitor and as a molecular glue inducing an interaction between CDK12 and DDB1 that leads to polyubiquitination of cyclin K and its subsequent degradation.

中文翻译：

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细胞周期蛋白依赖性激酶和细胞周期蛋白 K 降解剂的 3,5,7-取代吡唑并[4,3-d]嘧啶抑制剂

3,5,7-三取代的吡唑并[4,3- d ]嘧啶已被确定为细胞周期蛋白依赖性激酶 (CDK) 的有效抑制剂，CDK 是既定的药物靶点。在此，我们描述了它们进一步的结构修饰，从而产生具有强抗增殖活性的新型纳摩尔抑制剂。我们用 5-(2-amino-1-ethyl)thio-3-cyclobutyl-7-[4-(pyrazol-1-yl)benzyl]amino-1(2) H确定了完全活性 CDK2/A2 的晶体结构-吡唑并[4,3- d ]嘧啶（24) 在 1.7 Å 分辨率下，确认抑制的竞争模式。淋巴瘤细胞系中的生化和细胞分析证实了预期的作用机制，即通过视网膜母细胞瘤蛋白和 RNA 聚合酶 II 的去磷酸化，从而诱导细胞凋亡。重要的是，我们还揭示了化合物24在体外和体内源自患者的异种移植物中诱导蛋白酶体依赖性细胞周期蛋白 K 降解的有趣能力。我们提出24具有双重作用机制，既作为激酶抑制剂，又作为分子胶诱导 CDK12 和 DDB1 之间的相互作用，从而导致细胞周期蛋白 K 的多泛素化及其随后的降解。