Primer sequences used for qPCR.

Toll-like receptor 7 (TLR7) plays a key role in the signaling pathways involved in immunity by recognizing pathogen-associated molecular patterns. Due to its broad effectiveness in various diseases and the limited number of drugs on the market that act on this receptor, it remains an ideal target for pharmaceutical scientists. To find novel TLR7 ligands, chemical feature-based pharmacophore models were prepared for TLR7 using Pharmit. This model was used for virtual screening in related databases, and the identified compounds were ranked using the Autodock docking method and minimum binding affinity. Among the identified compounds, the triazoloquinoxaline amine scaffold was selected as the lead structure, and its different derivatives were designed and docked again. Finally, two new series of [1,2,4]triazolo[4,3-a]quinoxalin-4-amine and 1-methyl-[1,2,4]triazolo[4,3-a]quinoxalin-4-amine derivatives were selected and synthesized using green chemistry routes. The cytotoxicity of the synthetic compounds was assessed using a macrophage cell line, and non-toxic compounds were then selected for evaluating cytokine stimulation. Ultimately, compound 7f emerged as the most promising candidate for boosting the immune system based on fold change assessment compared to the control group. It exhibited the highest production of interleukin-1 (IL-1), tumor necrosis factor (TNF-α), and interferon beta (IFN-β) cytokines. Interestingly, the results of molecular docking aligned with the biological findings.