Several secondary tropomyosin receptor kinase (TRK) mutations located in the solvent front, xDFG, and gatekeeper regions, are a common cause of clinical resistance. Mutations in the xDFG motif in particular limit sensitivity to second-generation TRK inhibitors, which represent an unmet clinical need. We designed a series of 3-pyrazolyl-substituted pyrazolo[1,5-a]pyrimidine derivatives toward these secondary mutations using ring-opening and scaffold-hopping strategies. Compound 5n was the most potent, with IC₅₀ values of 2.3 nM, 0.4 nM, and 0.5 nM against TRKA^G667C, TRKA^F589L, and TRKA^G595R, compared to selitrectinib with IC₅₀ values of 12.6 nM, 5.8 nM, and 7.6 nM, respectively (approximately 5.4, 14.5, and 15.2-fold increases). Furthermore, 5n displayed favorable pharmacokinetic properties and satisfactory antitumor efficacy (tumor growth inhibition of 97% at 30 mg/kg and 73% at 100 mg/kg) in TRKA^WT and TRKA^G667C xenograft mouse models. Collectively, 5n is a promising TRK inhibitor lead compound for overcoming clinically acquired resistance to second-generation inhibitors, particularly for resistant tumors harboring the TRKA^G667C mutation in the xDFG motif.

位于溶剂前沿、xDFG 和看门人区域的几个继发性原肌球蛋白受体激酶 (TRK) 突变是临床耐药的常见原因。xDFG 基序中的突变尤其限制了对第二代 TRK 抑制剂的敏感性，这代表了未满足的临床需求。我们使用开环和支架跳跃策略设计了一系列 3-吡唑基取代的吡唑并[1,5- a ]嘧啶衍生物，以应对这些二级突变。化合物5n是最有效的，与具有 IC 50 的 selitrectinib 相比，针对 TRKA G667C、TRKA F589L 和 TRKA G595R 的 IC ₅₀值为^2.3 nM ^、 0.4 ^nM_和0.5 nM值分别为 12.6 nM、5.8 nM 和 7.6 nM（大约增加了 5.4、14.5 和 15.2 倍）。此外，5n在 TRKA ^WT和 TRKA ^G667C异种移植小鼠模型中表现出良好的药代动力学特性和令人满意的抗肿瘤功效（30 mg/kg 时肿瘤生长抑制率为 97%，100 mg/kg 时为 73%） 。总的来说，5n是一种很有前途的 TRK 抑制剂先导化合物，用于克服临床获得性对第二代抑制剂的耐药性，特别是对于在 xDFG 基序中具有 TRKA ^G667C突变的耐药性肿瘤。