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

Although arginine-derived PAD inhibitors represented by Cl-amidine (2) 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 (3), we designed two metabolically stable scaffolds, providing two arginine-derived PAD4 inhibitors (7 and 8). We evaluated their PAD4 enzyme inhibitory activity in vitro and assessed their metabolic stability using liver microsomal assays. These compounds exhibited PAD4 enzyme inhibitory activity (7, IC₅₀ = 124.93 ± 10.21 μM; 8, IC₅₀ = 46.49 ± 4.46 μM). Hydrolysis of haloacetamidine warheads into hydroxyacetamidine, Compound 7 (t_1/2 > 60 min), significantly improved the metabolic stability of the lead BB-Cl-amidine (t_1/2 = 18.11 min). Compound 8 (t_1/2 > 60 min), the isostere of 7, 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.