Licochalcone A Derivatives as Selective Dipeptidyl Peptidase 4 Inhibitors with Anti-Inflammatory Effects

A set of 22 analogs of licochalcone A was designed and synthesized to explore their potentials as Dipeptidyl Peptidase 4 (DPP4) inhibitors with anti-inflammatory effects. The anti-DPP4 effects of these analogs were evaluated using the fluorescent substrate Gly-Pro-N-butyl-4-amino-1,8-naphthalimide (GP-BAN). The nitro-substituted analogue 27 exhibited the most potent activity (K_i = 0.96 μM). A structure-activity relationship investigation revealed that 4-hydroxyl and 5-chloro substituents are essential for DPP4 inhibition, while the 3'-nitro substituent improved both DPP4 inhibition and microsomal stability. Furthermore, compound 27 demonstrated good selectivity for DPP4 over Other proteases, including Dipeptidyl Peptidase 9 (DPP9), Thrombin, prolyl endopeptidase (PREP), and fibroblast activation protein (FAP). The cytotoxic effect of 27 was evaluated in Cancer cell lines HepG-2 and Caco-2 and in somatic RAW264.7 cells and RPTECs. Compound 27 showed no toxicity to normal cells and weak toxicity to Cancer cells. In a living cell imaging assay, 27 blocked the dipeptidase activity of DPP4 in both Caco-2 and HepG-2 cells. This compound also dose-dependently suppressed the expression levels of the chemokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β).