Separation, characterization and cytotoxicity of unknown forced degradation impurity of selpercatinib using Prep-LC, HRMS and NMR

Abstract

Selpercatinib (LOXO-292) is a newly marketed oral selective receptor tyrosine kinase inhibitor targeting rearranged during transfection (RET), demonstrating precise therapeutic effects against RET-positive non-small cell lung cancer and thyroid cancer. In this study, an unknown acid forced degradation impurity of selpercatinib, designated sel-1, was isolated and purified using semi-preparative liquid chromatography (semi-Prep-LC). The purified sel-1 showed a chromatographic purity of 99.1 % as determined by high-performance liquid chromatography (HPLC). It appeared as a white amorphous powder, with a maximum absorption peak at 235 nm and a chemical formula of C₂₈H₂₉N₇O₃. Its molecular structure was elucidated using high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR). sel-1 was identified as 6-(2-hydroxy-2-methylpropoxy)-4-(6-(6-((6-oxo-1,6-dihydropyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile. In vitro MTT assays revealed that sel-1 exhibited significant antitumor activity, particularly against HepaRG and MKN-1 cell lines, with stronger inhibition than selpercatinib. The study contributes to enhancing the quality control standards for selpercatinib and suggests that sel-1 holds potential for further drug development.