The identification of metabolites is almost exclusively done with liquid chromatography/tandem mass spectrometry (LC/MSMS) and despite the enormous progress in the development of these techniques and software for handling of data this is a time-consuming task. In this study the use of quadrupole time-of-flight (QTOF)-generated MS(E) and MS/MS data were compared with respect to rationalization of metabolites. In addition Mass-MetaSite, a semi-automated software for metabolite identification, was evaluated. The program combines the information from MS raw data, in the form of collision-induced dissociation spectra, with a prediction of the site of metabolism in order to assign the structure of a metabolite. The aim of the software is to mimic the rationalization of fragment ions performed by a biotransformation scientist in the process of structural elucidation. For this evaluation, metabolite identification in human liver microsomes was accomplished for 19 commercially available compounds and 15 in-house compounds. The results were very encouraging and for 96% of the metabolites the same structures were assigned using MS(E) compared with MSMS acquired data. The possibility of using MS(E) could considerably reduce the analysis time. Moreover, Mass-MetaSite performed well and the correct assigned structure, compared to manual inspection of the data, was picked in the first rank in ∼80% of the cases. In conclusion MS(E) could be successfully used for metabolite identification in order to reduce time of analysis and Mass-MetaSite could alleviate the work of a biotransformation scientist and decrease the workload by assigning the structure for a majority of the metabolites.
PI3Kδ is a lipid kinase that is believed to be important in the migration and activation of cells of the immune system. Inhibition is hypothesized to provide a powerful yet selective immunomodulatory effect that may be beneficial for the treatment of conditions such as asthma or rheumatoid arthritis. In this work, we describe the identification of inhibitors based on a thiazolopyridone core structure and their subsequent optimization for inhalation. The initially identified compound (13) had good potency and isoform selectivity but was not suitable for inhalation. Addition of basic substituents to a region of the molecule pointing to solvent was tolerated (enzyme inhibition pIC > 9), and by careful manipulation of the pK and lipophilicity, we were able to discover compounds (20b, 20f) with good lung retention and cell potency that could be taken forward to in vivo studies where significant target engagement could be demonstrated.
While
bronchodilators and inhaled corticosteroids are the mainstay
of asthma treatment, up to 50% of asthmatics remain uncontrolled.
Many studies show that the cysteinyl leukotriene cascade remains highly
activated in some asthmatics, even those on high-dose inhaled or oral
corticosteroids. Hence, inhibition of the leukotriene C4 synthase
(LTC4S) enzyme could provide a new and differentiated core treatment
for patients with a highly activated cysteinyl leukotriene cascade.
Starting from a screening hit (3), a program to discover
oral inhibitors of LTC4S led to (1S,2S)-2-({5-[(5-chloro-2,4-difluorophenyl)(2-fluoro-2-methylpropyl)amino]-3-methoxypyrazin-2-yl}carbonyl)cyclopropanecarboxylic
acid (AZD9898) (36), a picomolar LTC4S inhibitor (IC50 = 0.28 nM) with high lipophilic ligand efficiency (LLE =
8.5), which displays nanomolar potency in cells (peripheral blood
mononuclear cell, IC50,free = 6.2 nM) and good in vivo
pharmacodynamics in a calcium ionophore-stimulated rat model after
oral dosing (in vivo, IC50,free = 34 nM). Compound 36 mitigates the GABA binding, hepatic toxicity signal, and
in vivo toxicology findings of an early lead compound 7 with a human dose predicted to be 30 mg once daily.
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