Improvement of metabolic weakness of new human Dihydroorotate Dehydrogenase inhibitors based on 2-hydroxypyrazolo[1,5-a]pyridine scaffold

Human Dihydroorotate Dehydrogenase (hDHODH), a mitochondrial enzyme that plays a pivotal role in the de novo pyrimidine biosynthesis, has been associated to Acute Myelogenous Leukemia (AML), as hDHODH inhibitors are able to restore myeloid differentiation (1). In recent years, we designed potent hDHODH inhibitors applying a scaffold-hopping approach to brequinar’s structure (2). By investigating the lead compound’s SAR, we recently discovered compound 1 (Figure), a candidate superior to brequinar in terms of in vitro potency. Unfortunately, compound 1 showed in vitro metabolic instability, as it soon undergoes to hydroxylation on alkoxy side chain by microsomal enzymes (3). In this occasion, we investigated on the metabolic hydroxylation site through the synthesis of the possible products of microsomal metabolism according to literature (4) and their comparison to compound 1’ s own metabolite in high resolution mass spectrometry (HR-MS); then we designed a new generation of hDHODH inhibitors protected from metabolic oxidation on the alkoxy side chain. Design, synthesis, in vitro metabolism and biological characterization of the new developed compounds are here described and discussed.