Identification of alkynyl nicotinamide HSN748 as a RET solvent-front mutant inhibitor with intracranial efficacy

RET solvent-front G810C/R/S mutations confer resistance to the currently approved RET protein tyrosine kinase inhibitors (TKIs) selpercatinib and pralsetinib. Moreover, RET fusion-positive lung adenocarcinoma frequently metastasizes to the brain. To address these challenges, it is imperative to develop a RET TKI that is effective against solvent-front mutations and exhibits intracranial activity. We synthesized alkynyl nicotinamide-based RET TKIs and tested their efficacy in cell cultures in inhibiting selpercatinib/pralsetinib-resistant RET solvent-front mutants G810C/R/S found in cancer patients, and in BaF3/KIF5B-RET(G810C) cell-derived subcutaneous and intracranial tumors <i>in vivo</i>. We also evaluated alkynyl nicotinamide RET TKIs in KIF5B-RET-induced lung tumors in immune competent transgenic mice, and in CCDC6-RET fusion-positive thyroid patient-derived xenograft PDX.003.047 tumors. <i>In vivo</i> pharmacokinetics (PK) studies were conducted to determine drug concentrations in plasma and brain. HSN748, HSND19, and HSND14 demonstrated potent inhibition of RET G810C/R/S mutants, with low nanomolar IC₅₀ values. HSN748 induced regression of subcutaneous B/KR(G810C) tumors without causing body weight loss. Both HSN748 and HSND19 significantly reduced KIF5B-RET-driven lung tumors in transgenic mice, and inhibited growth of CCDC6-RET-positive PDX tumors. Among three compounds (HSN748, HSND19, and HSN608) evaluated for B/KR(G810C) brain tumors, HSN748 exhibited significant intracranial tumor inhibition. PK analysis indicated that HSN748 has a brain/plasma partition coefficient (<i>K</i> _p) of 0.4, demonstrating its capability to penetrate the central nervous system (CNS).