Identification, toxicity evaluation, and HPLC-based quality control method development of process-related impurities...

As an oral small-molecule drug that selectively inhibits ROCK2, Belm exhibits unique advantages in the treatment of hormone-dependent/refractory cGVHD. However, impurity control and stability during the production of its API remain key challenges. This study conducted a systematic investigation into Belm's synthetic process, impurity analysis, toxicity evaluation, and quality control. First, the synthetic route was optimized to avoid the environmental hazards and low-yield issues of traditional routes. Nine relate impurities (including 5 new impurities: Imp-B, Imp-D, Imp-E, Imp-F and Imp-I) were successfully synthesized. An HPLC-based method for the separation and detection of Belm and its impurities was established, and method validation, Confirmed its applicability for API quality control. Impurity structures were elucidated by MS and NMR, and forced degradation studies (under acidic, basic, oxidative, thermal and photolysis conditions) were performed to reveal Belm's degradation behavior. Its mesylate form shows good stability under acidic and thermal conditions but is prone to forming new impurities (Imp-D1, Imp-D3 and Imp-D4) under basic, oxidative, and photolysis conditions. Toxicity prediction of impurities using ProTox-II and ADMETlab 3.0 indicated that Imp-D possesses significant immunotoxicity. Further network pharmacology analysis and molecular docking clarified the potential immunotoxicity mechanism of Imp-D. This study provides critical data support for the optimization of Belm's production process, formulation of impurity control strategies, and guarantee of stability. It also lays a foundation for its drug registration applications and full-life-cycle quality management.