Practical and Economical Implementation of Online H/D Exchange in LC-MS

Structural elucidation is an integral part of drug discovery and development. In recent years, due to acceleration of the drug discovery and development process, there is a significant need for highly efficient methodologies for structural elucidation. In this work, we devised and standardized a simple and economical online hydrogen–deuterium exchange methodology, which can be used for structure elucidation purposes.Deuterium oxide (D₂O) was infused as a postcolumn addition using the syringe pump at the time of elution of the analyte. The obtained hydrogen/deuterium (H/D) exchange spectrum of the unknown analyte was compared with the nonexchanged spectrum, and the extent of deuterium incorporation was delineated by using an algorithm to deconvolute partial H/D exchange, which confirmed the number of labile hydrogen(s) in the analyte. The procedure was standardized by optimizing flow rates of LC output, D₂O infusion, sheath gas, and auxiliary gas using the model compound sulfasalazine. The robustness of the methodology was demonstrated by performing sensitivity analysis of various parameters such as concentrations of analyte, effect of matrices, concentrations of aqueous mobile phase, and types of LC modifiers. The optimized technique was also applied to chemically diverse analytes and tested on various mass spectrometers. Moreover, utility of the technique was demonstrated in the areas of impurity profiling and metabolite identification, taking pravastatin-lactone and N-oxide desloratidine, as examples

Electrophilicity of Pyridazine-3-carbonitrile, Pyrimidine-2-carbonitrile, and Pyridine-carbonitrile Derivatives: A Chemical Model To Describe the Formation of Thiazoline Derivatives in Human Liver Microsomes

Certain aromatic nitriles are well-known inhibitors of cysteine proteases. The mode of action of these compounds involves the formation of a reversible or irreversible covalent bond between the nitrile and a thiol group in the active site of the enzyme. However, the reactivity of these aromatic nitrile-substituted heterocycles may lead inadvertently to nonspecific interactions with DNA, protein, glutathione, and other endogenous components, resulting in toxicity and complicating the use of these compounds as therapeutic agents. In the present study, the intrinsic reactivity and associated structure–property relationships of cathepsin K inhibitors featuring substituted pyridazines [6-phenylpyridazine-3-carbonitrile, 6-(4-fluorophenyl)­pyridazine-3-carbonitrile, 6-(4-methoxyphenyl)­pyridazine-3-carbonitrile, 6-<i>p</i>-tolylpyridazine-3-carbonitrile], pyrimidines [5-<i>p</i>-tolylpyrimidine-2-carbonitrile, 5-(4-fluorophenyl)­pyrimidine-2-carbonitrile], and pyridines [5-<i>p</i>-tolylpicolinonitrile and 5-(4-fluorophenyl)­picolinonitrile] were evaluated using a combination of computational and analytical approaches to establish correlations between electrophilicity and levels of metabolites that were formed in glutathione- and <i>N</i>-acetylcysteine-supplemented human liver microsomes. Metabolites that were characterized in this study featured substituted thiazolines that were formed following rearrangements of transient glutathione and <i>N</i>-acetylcysteine conjugates. Peptidases including γ-glutamyltranspeptidase were shown to catalyze the formation of these products, which were formed to lesser extents in the presence of the selective γ-glutamyltranspeptidase inhibitor acivicin and the nonspecific peptidase inhibitors phenylmethylsulfonyl fluoride and aprotinin. Of the chemical series mentioned above, the pyrimidine series was the most susceptible to metabolism to thiazoline-containing products, followed, in order, by the pyridazine and pyridine series. This trend was in keeping with the diminishing electrophilicity across these series, as demonstrated by <i>in silico</i> modeling. Hence, mechanistic insights gained from this study could be used to assist a medicinal chemistry campaign to design cysteine protease inhibitors that were less prone to the formation of covalent adducts

Selective <i>I</i>_Kur Inhibitors for the Potential Treatment of Atrial Fibrillation: Optimization of the Phenyl Quinazoline Series Leading to Clinical Candidate 5‑[5-Phenyl-4-(pyridin-2-ylmethylamino)quinazolin-2-yl]pyridine-3-sulfonamide

We have recently disclosed 5-phenyl-<i>N</i>-(pyridin-2-ylmethyl)-2-(pyrimidin-5-yl)­quinazolin-4-amine <b>1</b> as a potent <i>I</i>_Kur current blocker with selectivity versus <i>h</i>ERG, Na and Ca channels, and an acceptable preclinical PK profile. Upon further characterization <i>in vivo</i>, compound <b>1</b> demonstrated an unacceptable level of brain penetration. In an effort to reduce the level of brain penetration while maintaining the overall profile, SAR was developed at the C2′ position for a series of close analogues by employing hydrogen bond donors. As a result, 5-[5-phenyl-4-(pyridin-2-ylmethylamino)­quinazolin-2-yl]­pyridine-3-sulfonamide (<b>25</b>) was identified as the lead compound in this series. Compound <b>25</b> showed robust effects in rabbit and canine pharmacodynamic models and an acceptable cross-species pharmacokinetic profile and was advanced as the clinical candidate. Further optimization of <b>25</b> to mitigate pH-dependent absorption resulted in identification of the corresponding phosphoramide prodrug (<b>29</b>) with an improved solubility and pharmacokinetic profile