Design, Synthesis, and Anti-Inflammatory Activity of Functionalized 1,2,4,5-Tetrasubstituted-Pyrimidinone.

A highly efficient aza-Michael addition reaction of 5-amino pyrimidinones with α, β-unsaturated olefins under optimized reaction conditions is reported. The resulting functionalized amino-pyrimidinone-based aza-Michael mono-adducts were obtained in good to excellent yields. These synthesized compounds were screened for their anti-inflammatory potential in RAW264.7 macrophages. Among the synthesized compounds, we identified two derivatives, 3d and 3f, as potent anti-inflammatory compounds. Our findings demonstrated that 3d and 3f maintained normal cell viability at the bioactive concentrations (1, 5, and 10 µM), effectively reduced the production of proinflammatory mediators such as nitric oxide and proinflammatory cytokines including interleukin-6 and tumor necrosis factor-α. In addition, immunoblotting results revealed that 3d and 3f downregulate the protein expression of i-NOS and COX-2 in LPS-activated RAW264.7 macrophages. Furthermore, molecular docking was performed to elucidate the binding mechanism of these compounds and to establish a correlation between the in vitro results and the in silico data. Collectively, our results suggest that 3d and 3f attenuated the LPS-induced inflammatory response in macrophages and can be a structural lead for the development of novel anti-inflammatory agents.