New structural scaffolds to enhance the metabolic stability of arginine-derived PAD4 inhibitors

Although arginine-derived PAD inhibitors represented by Cl-amidine (<b>2</b>) showed strong inhibition of PAD4 enzymes and exhibited efficacies in a variety of cellular assays and animal studies, their metabolic instability is a significant challenge for pre-clinical and clinical research. On the basis of the structure of a well-known PAD4 inhibitor BB-Cl-amidine (<b>3</b>), we designed two metabolically stable scaffolds, providing two arginine-derived PAD4 inhibitors (<b>7</b> and <b>8</b>). We evaluated their PAD4 enzyme inhibitory activity <i>in vitro</i> and assessed their metabolic stability using liver microsomal assays. These compounds exhibited PAD4 enzyme inhibitory activity (<b>7</b>, IC₅₀ = 124.93 ± 10.21 μM; <b>8</b>, IC₅₀ = 46.49 ± 4.46 μM). Hydrolysis of haloacetamidine warheads into hydroxyacetamidine, Compound <b>7</b> (t_1/2 > 60 min), significantly improved the metabolic stability of the lead BB-Cl-amidine (t_1/2 = 18.11 min). Compound <b>8</b> (t_1/2 > 60 min), the isostere of <b>7</b>, also displayed enhanced metabolic stability. Therefore, these two structural scaffolds represent promising new leads for stable PAD4 inhibitors and valuable tools for exploring the reactive cavity of PAD enzymes.