Tandem mass spectrometry study of p38α kinase inhibitors and related substances

Abstract

The p38 mitogen-activated protein kinase α (p38α) is an important drug target widely investigated for therapy of chronic inflammatory diseases. Its inhibitors are rather lipophilic and as such not very favourable lead compounds in drug discovery. Therefore, we explored various approaches to access new chemical space, create diversity, and generate lead libraries with improved solubility and reduced lipophilicity, based on known p38α inhibitors, e.g., BIRB796 and TAK-715. Compound modification strategies include incubation with human liver microsomes and bacterial cytochrome P450 mutants from Bacillus megaterium and treatment by electrochemical oxidation, H2O2, and intense light irradiation. The MS/MS fragmentation pathways of p38α inhibitors and their conversion products have been studied in an ion-trap-time-of-flight MS(n) instrument. Interpretation of accurate mass MS(n) data for four sets of related compounds revealed unexpected and peculiar fragmentation pathways that are discussed in detail. Emphasis is put on the usefulness of HRMS(n)-based structure elucidation in a screening setting and on peculiarities of the fragmentation with regard to the analytes and the MS instrument. In one example, an intramolecular rearrangement reaction accompanied by the loss of a bulky group is observed. For BIRB796, the double-charge precursor ion is used in MS(2), providing a wider range of fragment ions in our instrument. For TAK-715, a number of related compounds could be produced in a large-scale incubation with a Bacillus megaterium mutant, thus enabling comparison of the structure elucidation by (1)H NMR and MS(n). A surprisingly large number of homolytic cleavages are observed. Competition between two fragmentation pathways involving either the loss of CH3(•) or OH(•) radicals was observed for SB203580 and its conversion products.

Neutralization of Streptolysin S-Dependent and Independent Inflammatory Cytokine IL-1β Activity Reduces Pathology During Early Group A Streptococcal Skin Infection

“…As we utilized a fairly modest treatment dose in the studies described here, experimentation with higher doses may be beneficial. An additional complication that likely contributes to SB203580's variable efficacy in our in vivo infection system is its poor water solubility (Falck et al, 2013 ). Although few studies have been conducted to enhance SB203580's solubility for therapeutic use in animal models of disease, such approaches may be worth investigating in the future (Falck et al, 2013 ).…”

“…An additional complication that likely contributes to SB203580's variable efficacy in our in vivo infection system is its poor water solubility (Falck et al, 2013 ). Although few studies have been conducted to enhance SB203580's solubility for therapeutic use in animal models of disease, such approaches may be worth investigating in the future (Falck et al, 2013 ). In addition to optimizing drug solubility and dosage, continued efforts to utilize localized treatment approaches to treat subcutaneous GAS infection may be worthwhile.…”