Isolation, structural elucidation and cytotoxicity evaluation of novel degradation impurities of futibatinib.

Futibatinib (TAS-120) is a next-generation covalent inhibitor of fibroblast growth factor receptor (FGFR) 1-4 that has been authorized for the treatment of patients with previously treated intrahepatic cholangiocarcinoma carrying FGFR2 fusions or rearrangements. However, information regarding its degradation behavior and impurity profile remains limited. The stability behavior of futibatinib under a series of forced degradation conditions was comprehensively evaluated using an HPLC-based analytical approach. Forced degradation studies demonstrated that futibatinib was relatively stable under oxidative, thermal, and photolytic conditions, whereas significant degradation occurred under alkaline conditions. Two previously unreported degradation-related products, designated as Fut-1 and Fut-2, were generated under intensified alkaline conditions and subsequently isolated by preparative liquid chromatography. Fut-1 and Fut-2 were characterized using high-resolution mass spectrometry, nuclear magnetic resonance and heteronuclear multiple bond correlation spectroscopy. Based on comprehensive spectral analyses, Fut-1 was identified as (S)-3-((3,5-dimethoxyphenyl)ethynyl)-1-(pyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine, while Fut-2 was identified as (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)-3-methoxypropan-1-one. Structural analysis suggested that degradation primarily involved transformation of the acrylamide side chain, whereas the pyrazolo[3,4-d]pyrimidine core and the 3,5-dimethoxyphenyl-ethynyl moiety remained largely intact. In vitro cytotoxicity studies against five human tumor cell lines revealed that Fut-1 retained considerable cytotoxic activity and exhibited stronger inhibitory effects than futibatinib in several cell lines, whereas Fut-2 showed reduced activity. The results provide valuable insights into the structural transformation behavior, impurity profiling, and quality control considerations of futibatinib and may contribute to future investigations of structure-activity relationships of futibatinib-related compounds.