Improvement of Metabolic Weakness of New Dihydroorotate Dehydrogenase Inhibitors Based on 2-Hydroxypyrazolo[1,5-a]pyridine Scaffold to Target Myeloid Leukemias

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 differentiation1. In recent years, we designed potent hDHODH inhibitors applying a scaffold-hopping approach to brequinar’s structure2. By investigating the lead compound’s SAR, we recently discovered compound 12, 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 enzymes3. In this occasion, we investigated on the metabolic hydroxylation site through the synthesis of the possible products of cytochrome metabolism according to literature4 and their comparison to microsome-incubated compound 1 in high-resolution mass spectrometry; then we developed new hDHODH inhibitors protected from metabolic oxidation on the alkoxy side chain. Design, synthesis, in vitro metabolism and biological characterization of the new compounds are here described and discussed. 1. Sykes, D. B. et al. Inhibition of Dihydroorotate Dehydrogenase Overcomes Differentiation Blockade in Acute Myeloid Leukemia. Cell 2016, 167(1), 171-186 2. Sainas, S. et al. Targeting Myeloid Differentiation Using Potent 2-Hydroxypyrazolo[1,5- a]pyridine Scaffold-Based Human Dihydroorotate Dehydrogenase Inhibitors. J. Med. Chem. 2018, 61, 6034-6055. 3. Sainas, S. et al. Targeting Acute Myelogenous Leukemia using potent human dihydroorotate dehydrogenase inhibitors based on the 2-hydroxypyrazolo[1,5- a]pyridine scaffold: SAR of the biphenyl moiety. J. Med. Chem. 2021, 64, 5404-5428 4. Testa, B. and Clement, B. Biotransformation Reactions and Their Enzymes. The Practice of Medicinal Chemistry: Fourth Ed. Academic Press. 2015, 561-584.