Fabrication of copper-organic complex and copper-organic salt for non-substrate-like urease inhibitors

Urease, a nickel-dependent enzyme that significantly accelerates urea hydrolysis, leads to substantial economic losses, underscoring the importance of developing efficient urease inhibitors (UIs). Traditional chemical inhibitors are limited in application due to their high activity yet low efficacy. Recently, metal-organic compounds have garnered significant attention in the field of urease inhibition owing to their unique architectures. In this study, two copper-based compounds with distinct coordination modes, [Cu( L )Cl 2 ] ( UI-MOC , a metal-organic complex) and [(CuCl 4 )(H 2 L )] ( UI-MOS , a metal-organic salt), were synthesized from a flexible N-bidentate ligand (N,N,N',N'-tetrabenzylethylenediamine, L ) and copper ions via different synthetic routes. Structural characterization confirmed their spatial configuration differences: in UI-MOC , the metal center is directly coordinated to the ligand via covalent bonds, resulting in a more stable structure, whereas in UI-MOS , the components are connected through non-covalent interactions such as hydrogen bonds. In vitro urease inhibition assays demonstrated that both UI-MOC and UI-MOS exhibit excellent inhibitory performance, with IC₅₀ values of 0.52 ± 0.02 μM and 1.96 ± 0.02 μM, respectively, surpassing most reported inhibitors. Kinetic studies revealed that both are non-competitive inhibitors, with UI-MOC possessing a lower inhibition constant (Kᵢ = 3.6 μM), indicating tighter binding to urease. Molecular docking results showed that UI-MOC has a higher binding energy (–8.87 kcal/mol), and its metal center is closer to the active site of urease. Furthermore, UI-MOC forms extensive hydrophobic interactions with multiple key amino acid residues, enhancing inhibitory stability. Density functional theory (DFT) calculations further revealed that UI-MOC has a higher energy gap (ΔE = 3.8896 eV) and greater overall electronegativity, contributing to its superior structural stability, reactivity, and enhanced interaction with urease. This work not only successfully develops two highly efficient urease inhibitors but also systematically compares the influence of different coordination modes on inhibitory performance, providing theoretical insights and experimental guidance for the future design of novel UIs based on coordination chemistry. Copper-organic complex and copper-organic salt with different structures and urease inhibitory activities were prepared and systematically characterized. The influence of the preparation method on the urease performance was investigated in detail. • Two types of UIs, UI-MOC and UI-MOS , were synthesized and investigated in detail. • Both UI-MOC and UI-MOS exhibited good urease inhibitory activities. • The inhibition mechanisms of UI-MOC and UI-MOS on urease were investigated in detail. • The stability of materials played a crucial role in urease inhibition.