Multicomponent reaction synthesis and evaluation of novel 3,4-dihydropyrazine[1,2-b]Indazole-1(2H)-one derivatives as inhibitors of pyroptosis and inflammation.

Inflammation is a protective response by the body aimed at maintaining tissue homeostasis by eliminating pathogenic microbial infection, irritants, or tissue damage. However, dysregulated inflammation is pathological and involved in various diseases such as metabolic disorders, cancer, and neurodegenerative diseases. In this study, multicomponent reaction, an efficient tool for the synthesis of complex compounds with potential biological activities, was employed to synthesize twenty 3,4-dihydro-pyrazine[1,2-b]indazole-1(2H)-one derivatives and two 6,7-dihydropyrazolo[1,5-a]pyrazin-4(5H)-one analogues. We next identified compounds 6e and 6r as potential inhibitors for NLRP3 inflammasome-driven pyroptosis through activity-based screening and investigated their potential binding modes with the NLRP3 protein via molecular docking. Further studies on anti-inflammatory activity showed that compounds 6e and 6r also significantly inhibited LPS-induced NO release, among which compound 6e had better anti-inflammatory activity, with an IC₅₀ of 8.55 ± 0.32 μM in inhibiting NO release. Additionally, qPCR analysis indicated that compound 6e notably suppressed the gene transcription of the pro-inflammatory cytokine IL-6. In conclusion, this study identifies compound 6e, featuring a novel 3,4-dihydro-pyrazine[1,2-b]indazole-1(2H)-one scaffold, as a promising hit compound with inhibitory activity against pyroptosis and key inflammatory mediators. These findings highlight this chemotype as a valuable starting point for the development of a new class of anti-inflammatory agents.