Elucidation of the in vitro metabolic profile of stable isotope labeled BAL19403 by accurate mass capillary liquid chromatography/quadrupole time‐of‐flight mass spectrometry and isotope exchange

Abstract

The in vitro metabolic pattern of BAL19403, a novel macrolide antibiotic, was investigated by capillary liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/QTOF-MS) in incubations with human microsomes. For the elucidation of the metabolic pathway, BAL19403 labeled with four deuterium atoms (D4) was used, and detection of metabolites performed using mixtures of the unlabeled (H4) BAL19403 and its D4 analogue (1:1) as substrate. All metabolites appeared with similar chromatographic behavior. MS/MS spectra of BAL19403 and its metabolites are dominated by non-informative fragment ions. Therefore, the structure of the metabolites was elucidated mainly by accurate mass measurements with subsequent proposals of elemental compositions. Main biotransformations were N-demethylation, lactone ring hydrolysis, and oxidation. Additionally, N-dealkylation of the aromatic moiety was identified. This dealkylation results not only in formation of an aldehyde, according to the classical pathway, but also in formation of the corresponding alcohol and carboxylic acid. Final elucidation of their structures was possible, since this dealkylation takes place vicinal to the deuterium-labeled part of BAL19403 and interferes with D/H exchange. The degree of D/H exchange, determined by analysis of the metabolite isotopic pattern, was used to elucidate the adjacent functional group.

Natural products to drugs: natural product-derived compounds in clinical trials

“…181 BAL-19403 52 was obtained through the optimisation of binding to Propionibacterium acnes ribosomes to ensure that 52 had a relatively narrow spectrum of activity and good anti-inflammatory activity. [181][182][183] Tiacumicin B (difimicin, OPT-80, PAR-101) 54, which is identical to lipiarmycin A3 184 and clostomicin B1, 185 is an actinomycete-derived macrolactone isolated by Abbott as the major component of the tiacumicin antibiotic complex. 186 Its antibacterial activity is due to inhibition of bacterial RNA synthesis 187,188 and Optimer Pharmaceuticals are evaluating tiacumicin B 54 in several Phase III trials for the treatment of CDAD.…”

Advances in structure elucidation of small molecules using mass spectrometry

“…Stable isotopic labeling studies [193, 299] and hydrogen/deuterium exchange reactions [300–302] are commonly applied in drug metabolism studies. Proteomics approaches use labeling studies for the quantification of peptides and proteins [303–305] as well as mass defect isotopomer studies [306, 307].…”

Stable Isotope- and Mass Spectrometry-based Metabolomics as Tools in Drug Metabolism: A Study Expanding Tempol Pharmacology

“…Mass spectrometry and stable isotope-labeled drugs has been applied to metabolite identification in in vivo and in vitro metabolism studies. − However, not to be overlooked, the use of conventional approaches including radiolabeled tracers as well as making use of naturally occurring isotopes, such as 37 Cl, have been instrumental in helping to detect and identify drug metabolites. , In this report, we hypothesized that stable isotope- and mass spectrometry-based metabolomics may simultaneously facilitate a better understanding of both the drug metabolism route as well as to identify endogenous biomarkers of drug action (Figure ). To accomplish this, ultraperformance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC–ESI-QTOFMS)-based metabolomics combined with multivariate data analysis (MDA) was employed to analyze the metabolites generated from tempol and deuterated tempol and the impact of tempol on endogenous metabolism.…”