Discovery, synthesis, and biological evaluation of N,N'-diacyl-m-phenylenediamine derivatives as novel PCSK9 small-molecule inhibitors.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a validated therapeutic target for regulating low-density lipoprotein cholesterol (LDL-C) levels and treating dyslipidemia-related cardiovascular diseases. In this study, pharmacophore-based virtual screening identified 4 (ZINC4681905) as a lead compound, which was subsequently optimized into a series of N,N'-diacyl-m-phenylenediamine derivatives. A total of fourteen compounds were synthesized and evaluated using a fluorescence polarization-based assay. Among them, compound 4a exhibits the highest potency with an IC50 value of 1.07 μM, surpassing that of the positive control berberine (IC50 = 2.59 μM), while compound 4f shows comparable activity. Structure-activity relationship (SAR) analysis revealed that the inhibitory activity of this N,N'-diacyl-m-phenylenediamine scaffold is influenced by the cooperative interplay of the two terminal substituents, with a combination of a multisubstituted hydrophobic aryl group and a flexible, bulky alicyclic moiety affording favorable potency. In cell-based assays, 4a reduced PCSK9 expression and concurrently increased low-density lipoprotein receptor (LDLR) levels in HK-2 cells, and this effect was partially rescued by PCSK9 overexpression, providing supportive cell-based evidence for the involvement of the PCSK9 pathway. Collectively, these findings support further optimization of this scaffold as a potential class of small molecule PCSK9 inhibitors. However, direct LDL uptake assays and assessment of PCSK9-LDLR interaction disruption have not been performed; therefore, the functional evidence remains preliminary.