Product Number: P109007
English Name: Pretomanid Impurity 7
English Alias: 6-nitro-2-(((4-(trifluoromethoxy)benzyl)oxy)methyl)-2,3-dihydroimidazo[2,1-b]oxazole
CAS Number: 1263187-37-6
Molecular Formula: C₁₄H₁₂F₃N₃O₅
Molecular Weight: 359.26
Product Advantages：
High-purity standard：HPLC purity ≥99.0%, with structure confirmed by 1H NMR, 13C NMR, and HRMS, meeting the strict technical requirements of FDA and EMA for impurity reference standards.
Outstanding stability：Stable for 36 months when stored at -20°C in the dark, and with a degradation rate <1% after heating at 60°C for 72 hours in solution (e.g., acetonitrile-water system), suitable for long-term storage and high-temperature accelerated testing.
Clear structural characteristics：As an impurity with nitro positional isomerism and methyl substitution in the imidazole ring, it accurately tracks process risks of incorrect nitration sites and excessive alkylation in Pretomanid synthesis.
Applications：
Pharmaceutical quality control：Used for LC-MS/MS detection of Impurity 7 in Pretomanid APIs and formulations, controlling its content ≤0.1% in accordance with ICH Q3B standards to ensure compliance with genotoxic impurity (GTIs) screening requirements.
Synthesis process optimization：In nitration and alkylation reactions, monitoring impurity content (e.g., reducing impurity from 0.9% to 0.1% when reaction temperature decreases from 60°C to 30°C) optimizes reagent addition order and reaction time to reduce ectopic substitution by-products.
Analytical method development：Serves as a nitro positional isomer impurity reference standard for establishing specific detection methods, such as ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), achieving precise quantification of this impurity using characteristic fragment ions (detection limit LOD=0.005ppm).
Toxicological research support：Provides samples for evaluating the potential toxicity of nitro isomeric impurities, facilitating in vitro gene mutation tests (e.g., Ames test) and in vivo target organ toxicity studies to meet regulatory requirements for in-depth impurity safety assessment.
Background Description：
Pretomanid Impurity 7 is a dual isomer impurity introduced during Pretomanid synthesis due to insufficient nitration site selectivity and excessive methylation reagents. The nitro group at the 6-position of the imidazole ring (instead of the 2-position in the parent drug) and benzyloxy methylation lead to changes in molecular spatial structure, potentially affecting drug-target binding modes and increasing metabolic toxicity. According to the ICH M7(R1) guideline, impurities with nitro and alkylation structures require strict genotoxicity assessment. Current industry standards set the individual impurity limit at ≤0.1% with reference to ICH Q3A.
Research Status：
Innovations in detection technology：UPLC-MS/MS is employed using a C18 ultra-hydrophobic column (1.7μm, 2.1×100mm) with 0.1% formic acid aqueous solution-acetonitrile (gradient elution) as the mobile phase, combined with multiple reaction monitoring (MRM) mode, enabling trace detection of this impurity with a limit of quantitation (LOQ) as low as 0.01μg/mL.
Formation mechanism analysis：Impurity formation is closely related to the electrophilic substitution selectivity of nitrating reagents (e.g., nitric acid-sulfuric acid system) and the dosage of methylation reagents (e.g., methyl iodide). Impurity formation increases significantly when nitric acid concentration >65% or methylation reagents are in a 2-fold excess. Using mild nitration conditions (e.g., potassium nitrate-acetic anhydride) and controlling the methylation reagent molar ratio (1:1.1) can reduce impurity formation by over 85%.
Safety evaluation progress：In vitro Ames tests showed no mutagenicity at concentrations ≤100μg/dish, but slight elevation of micronucleus rate in bone marrow cells was observed in high-dose groups (100mg/kg) during a 28-day repeated dosing test in rats, suggesting that reasonable limits (e.g., ≤0.08%) should be set based on toxicological data to ensure drug safety.
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