Process Development of the Novel LpxC Inhibitor T-1228. Part 4: Control of Mutagenic Impurities during API Synthesis

In this study, we performed a hazard assessment of actual and potential impurities involved in the synthesis of a newly developed LpxC inhibitor T-1228 in accordance with the ICH M7(R2) guideline. We identified eight mutagenic/carcinogenic impurities in need of control: benzyl chloride, 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride, O-(tetrahydro-2H-pyran-2-yl)hydroxylamine, 2-(4-iodophenyl)-2-oxoethyl acetate, (S)-1-(4-ethynylphenyl)ethane-1,2-diol, isopropyl methanesulfonate, hydroxylamine, and (S)-4-((4-(1,2-dihydroxyethyl)phenyl)ethynyl)-N-hydroxybenzamide. We then conducted risk assessments of the first six impurities using purge factors, and determined that Option 4 control of ICH M7(R2) guideline was the optimal approach for controlling these impurities. Furthermore, as (S)-1-(4-ethynylphenyl)ethane-1,2-diol and isopropyl methanesulfonate are impurities introduced during the later stages of active pharmaceutical ingredient (API) synthesis, we obtained additional experimental data (verification of reactivity, solubility, spike experiments, and analytical experiments) to verify the validity of the purge-based risk assessment. Regarding the final two impurities, hydroxylamine may be produced as a byproduct in the final intermediate and final processes; therefore, we determined that Option 1 control of ICH M7(R2) guideline was the optimal control strategy and set appropriate API criteria. Although (S)-4-((4-(1,2-dihydroxyethyl)phenyl)ethynyl)-N-hydroxybenzamide is a degradation product of API, in vivo mutagenicity assay conducted according to the ICH M7(R2) guideline showed negative results. Therefore, we determined that (S)-4-((4-(1,2-dihydroxyethyl)phenyl)ethynyl)-N-hydroxybenzamide can be managed as a nonmutagenic impurity according to the ICH Q3A(R2) guideline. The risk assessment of mutagenic impurities related to the T-1228 API manufacturing method, performed in accordance with ICH M7(R2) guideline, can guarantee the quality of this manufacturing method for initial clinical trials.