Evaluation of relative MS response factors of drug metabolites for semi-quantitative assessment of chemical liabilities in drug discovery.

Drug metabolism studies are performed in drug discovery to identify metabolic soft spots, detect potentially toxic or reactive metabolites and provide an early insight into potential species differences. The relative peak area approach is often used to semi-quantitatively estimate the abundance of metabolites. Differences in the liquid chromatography-mass spectrometry responses result in an underestimation or overestimation of the metabolite and misinterpretation of results. The relative MS response factors (RF) of 132 structurally diverse drug candidates and their 233 corresponding metabolites were evaluated using a capillary-liquid chromatography/high-resolution mass spectrometry system. All of the synthesized metabolites discussed here were previously identified as key biotransformation products in discovery investigations or predicted to be formed. The most commonly occurring biotransformation mechanisms such as oxygenation, dealkylation and amide cleavage are represented within this dataset. However, relatively few phase II metabolites were evaluated because of the limited availability of authentic standards. Approximately 85% of these metabolites had a relative RF in the range between 0.2 (fivefold under-prediction) and 2.0 (twofold over-prediction), and the median MS RF was 0.6. Exceptions to this included very small metabolites that were hardly detectable. Additional experiments performed to understand the impact of the MS platform, flow rate and concentration suggested that these parameters do not have a significant impact on the RF of the compounds tested. This indicates that the use of relative peak areas to semi-quantitatively estimate the abundance of metabolites is justified in the drug discovery setting in order to guide medicinal chemistry efforts. Copyright © 2017 John Wiley & Sons, Ltd.

Micropreparative isolation and NMR structure elucidation of metabolites of the drug candidate 1-isopropyl-4-(4-isopropylphenyl)-6-(prop-2-yn-1-yloxy) quinazolin-2(1H)-one from rat bile and urine.

LC-MS based drug metabolism studies are effective in the optimization stage of drug discovery for rapid partial structure identification of metabolites. However, these studies usually do not provide unambiguous structural characterization of all metabolites, due to the limitations of MS-based structure identification. LC-MS-SPE-NMR is a technique that allows complete structure identification, but is difficult to apply to complex in vivo samples (such as bile collected during in vivo drug metabolism studies) due to the presence, at high concentrations, of interfering endogenous components, and potentially also dosage excipient components (e.g. polyethylene glycols). Here, we describe the isolation and structure characterization of seven metabolites of the drug development candidate 1-isopropyl-4-(4-isopropylphenyl)-6-(prop-2-yn-1-yloxy) quinazolin-2(1H)-one from a routine metabolism study in a bile-duct cannulated rat by LC-MS-SPE. The metabolites were isolated from bile and urine by repeated automatic trapping of the chromatographic peak of each metabolite on separate Oasis HLB SPE columns. The micropreparative HPLC/MS was performed on an XBridge BEH130 C18 HPLC column using aqueous formic acid/acetonitrile/methanol as mobile phase for the gradient elution. Mass spectrometric detection was performed on a LTQ XL linear ion trap mass spectrometer using electrospray ionization. Desorption of each metabolite was performed after the separation sequence. NMR spectra ((1)H, (13)C, 2D ROESY, HSQC and HMBC were measured on a Bruker AVANCE III spectrometer (600 MHz proton frequency) equipped with a 1.7 mm (1)H{(13)C,(15)N} Bruker Biospin's TCI MicroCryoProbe™.

Complementary use of ion trap/time-of-flight mass spectrometry in combination with capillary high-pressure liquid chromatography: early characterization of in vivo metabolites of the cathepsin K inhibitor NVP-AAV490 in rat.

Cathepsin K is a cysteine proteinase, primarily expressed in osteoclasts, which has a strong collagenolytic activity and plays an essential role involved in bone matrix degradation. Its inhibition could provide a novel approach to the treatment and prevention of osteoporosis. One structural class of lead compounds in our cathepsin K inhibitors program is based on an arylaminoethyl amide scaffold, which has potential metabolic weak points that might be stabilized by appropriate chemical modification(s). For the identification of potential metabolic "soft spots" and the rational design of improved derivatives, early biotransformation of a potent arylaminoethyl amide cathepsin K inhibitor (NVP-AAV490-NX) was investigated in plasma, urine and liver homogenates of rats after intravenous bolus administration of 10 mg/kg. The detection and identification of metabolites was achieved by high-resolution mass spectrometry (time-of-flight MS) and multi-dimensional mass spectrometry (ion trap MS). Both mass spectrometers were combined with reversed-phase capillary high-performance liquid chromatography columns. It was demonstrated that both mass analyzers complement each other and that, even in the sub-nanogram range, the resulting set of MS data can be successfully used to elucidate most of the metabolic changes unambiguously, solely by mass spectrometric techniques. The proposed metabolite structures were additionally corroborated by exact mass measurement of the protonated molecular ions to confirm the predicted elemental composition, by determination of the number of the exchangeable hydrogen atoms replacing water against deuterium oxide as mobile phase and, in one case, by an MS(3) product ion experiment in order to elucidate the site of conjugation.