Discovery of 2-(6-(5-Chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide (PF-06282999): A Highly Selective Mechanism-Based Myeloperoxidase Inhibitor for the Treatment of Cardiovascular Diseases

Abstract

Myeloperoxidase (MPO) is a heme peroxidase that catalyzes the production of hypochlorous acid. Clinical evidence suggests a causal role for MPO in various autoimmune and inflammatory disorders including vasculitis and cardiovascular and Parkinson's diseases, implying that MPO inhibitors may represent a therapeutic treatment option. Herein, we present the design, synthesis, and preclinical evaluation of N1-substituted-6-arylthiouracils as potent and selective inhibitors of MPO. Inhibition proceeded in a time-dependent manner by a covalent, irreversible mechanism, which was dependent upon MPO catalysis, consistent with mechanism-based inactivation. N1-Substituted-6-arylthiouracils exhibited low partition ratios and high selectivity for MPO over thyroid peroxidase and cytochrome P450 isoforms. N1-Substituted-6-arylthiouracils also demonstrated inhibition of MPO activity in lipopolysaccharide-stimulated human whole blood. Robust inhibition of plasma MPO activity was demonstrated with the lead compound 2-(6-(5-chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide (PF-06282999, 8) upon oral administration to lipopolysaccharide-treated cynomolgus monkeys. On the basis of its pharmacological and pharmacokinetic profile, PF-06282999 has been advanced to first-in-human pharmacokinetic and safety studies.

Species Differences in the Oxidative Desulfurization of a Thiouracil-Based Irreversible Myeloperoxidase Inactivator by Flavin-Containing Monooxygenase Enzymes

“…The synthesis of compound 1 (chemical purity . 99% by highperformance liquid chromatography [HPLC] and NMR) has been previously reported elsewhere (Ruggeri et al, 2015). The preparation of the M1 metabolite is described in our Supplemental Data.…”

“…We recently reported structure-activity relationship studies on N1-substituted-6-aryl-2-thiouracil derivatives as irreversible, mechanism-based inactivators of the hemoprotein myeloperoxidase (MPO, EC 1.11.2.2) with a high degree of selectivity for MPO relative to peroxidases such as thyroid peroxidase (TPO) and cytochrome P450 (P450) enzymes (Ruggeri et al, 2015). The thiouracil analogs behave as suicide substrates of MPO and covalently adduct to the heme prosthetic group through an oxidized sulfur species (presumably a thiol radical) generated during catalysis (Tidén et al, 2011;Ruggeri et al, 2015).…”

“…The thiouracil analogs behave as suicide substrates of MPO and covalently adduct to the heme prosthetic group through an oxidized sulfur species (presumably a thiol radical) generated during catalysis (Tidén et al, 2011;Ruggeri et al, 2015). The antithyroid drug propylthiouracil (PTU, Fig.…”

“…The antithyroid drug propylthiouracil (PTU, Fig. 1), which irreversibly inhibits MPO and TPO in a nonselective fashion (Lee et al, 1990;Ruggeri et al, 2015), was used as a starting point in our structure-activity relationship work to identify selective MPO inhibitors. Introduction of polar N1 substituents and replacement of the C6 propyl group in PTU with electronrich aromatic functionalities resulted in significant improvements in MPO dx.doi.org/10.1124/dmd.116.070185. s This article has supplemental material available at dmd.aspetjournals.org.…”

“…Intramolecular trapping of reactive species was demonstrated by reacting compound 1 with excess hydrogen peroxide (H 2 O 2 ) (Kitamura, 1934;Kalm, 1961), which quantitatively converted compound 1 to the pharmacologically inactive cyclic ether 5-(2,4-dimethoxyphenyl)-2,3-dihydro-7H-oxazolo[3,2-a]pyrimidin-7-one (M1, Fig. 1), presumably via an unstable oxidized sulfur intermediate (Ruggeri et al, 2015).…”