Carbene reactive metabolite explains the hepatotoxicity of the hot flashes drug fezolinetant: A DFT investigation

Fezolinetant, the first-in-class hot flashes drug, was flagged in September 2024 by US-FDA for liver injury. It is alarming that an FDA-approved drug shows liver toxicity within a year. Fezolinetant's metabolic pathways and metabolites are not disclosed in the FDA label, clinical trial, or other literature reports. This creates a gap in understanding the mechanisms of metabolism and toxicity. In this manuscript, we have investigated novel metabolic pathways that generate reactive metabolites and rationalize hepatotoxicity. Quantum chemical calculations were performed to assess the reactivity of various sites using B3LYP/CC-PVTZ level of theory and H₂O₂ as the model oxidant. A combination of reaction free-energy (ΔG°) ≤ k_BT = 18.5 kcal/mol and docking-based site of metabolism (SOM) to Heme-Fe distance (D_AFe ≤ 6 ± 1.95 Å) criterion was used to assess metabolism feasibilities. Metabolic reactions and sites were considered potential only if both reactivity and accessibility criteria were met. A novel pathway involving Me oxidation at the thiadiazole ring to acid leading to reactive Fezo-carbene metabolite is most feasible (ΔG° and D_AFe: 11 kcal/mol and 6.84 Å). Other feasible pathways include radical formation at the triazolopiperazine ring forming the Fezo-pip-dehydro metabolite (12 kcal/mol and 7.96 Å). The epoxidation, N-oxidation, and S-oxidation of the fluorophenyl, triazolopiperazine, and thiadiazole ring cleavage have minor contributions. Global and local electrophilicity analysis also confirms the higher reactivity potential of Fezo-carbene (ω = 2.77 eV) metabolite. Differences in local electrophilicity (Δω_c⁺) further confirm the higher reactivity of the thiadiazole, fluorophenyl, and triazolopiperazine ring in Fezo-carbene, Fezo-epoxy, and Fezo-pip-dehydro metabolites respectively. Reactions with model nucleophiles (MeOH, H₂O, MeNH₂, and MeSH) show that Fezo-carbene is likely to form thiol adducts. In summary, these results give vital insights into the metabolic mechanisms of fezolinetant's hepatotoxicity and are likely to be useful for the design of less-toxic analogues.