A One‐Min, FDA‐Validated UPLC–MS/MS Method for the AML Smoothened Inhibitor Glasdegib Quantification and Metabolic Stability Assessment in Human Liver Microsomes: In Silico Metabolic Lability and Toxicity Screening

ABSTRACT Glasdegib (GSB) is an orally bioavailable Smoothened (Hedgehog pathway) inhibitor approved, in combination with low‐dose cytarabine, for the treatment of newly diagnosed acute myeloid leukemia (AML) in patients who are unfit for intensive induction chemotherapy. Although GSB is predominantly cleared by hepatic CYP3A4‐mediated oxidation, no human liver microsomes (HLM) metabolic stability method has been reported; existing assays are limited to plasma matrices. Here, we describe an ultra‐fast validated UPLC–MS/MS method for GSB and apply it to the first assessment of metabolic stability in HLM. StarDrop WhichP450 (CSL = 0.8928) and DEREK alerts were performed to localize metabolic soft spots and safety liabilities. Chromatography was performed on an SB‐C18 column in 1 min. The method was linear over 1–4000 ng/mL ( y = 1.2753 x + 0.4567; r 2 = 0.9992), with accuracy (−3.10 to +10.25 %E) and precision (≤ 3.56 %RSD). GSB exhibited an in vitro t 1/2 of 44.15 min and an intrinsic clearance of 18.37 mL/min/kg (moderate‐clearance compound). The predicted soft spots (the N‐methylpiperidine ring [65%] and the benzimidazole moiety [29%]) are consistent with the known human N‐desmethyl metabolite and CYP3A4 dominance. The combined dataset provides a soft‐spot‐guided rationale for the metabolic optimization of future Hedgehog‐pathway inhibitors.