Synthesis,Evaluation And Application Of Novel TLR7 Agonists As Antiviral Agents And Vaccine Adjuvants

Toll-like receptors（TLRs）are a class of pattern recognition receptors widely expressed in various immune cells,highly conserved in evolution,and can be activated by pathogen-associated molecular patterns or agonists to initiate innate immune responses.T LR7,a member of the Toll-like receptor family,is mainly expressed in antigen-presenting cells,recognizing single-stranded RNA and small molecule ligands,initiating signal cascade reactions after dimerization,promoting transcription factor phosphorylation,regulating downstream cytokine expression,and exerting antiviral effects.TLR7agonists play important roles in antiviral,antitumor,and immune activation by stimulating the innate immune system.Currently,there are few TLR7 agonists approved for clinical use.This study aims to expand the structures of existing compounds,design and synthesize quinazoline derivatives and pyrrolo[3,2-d]pyrimidine derivatives as novel TLR7 agonists,evaluate their safety and TLR7 pathway activation ability on cells,assess their antiviral activity against pseudorabies virus（PRV）and vesicular stomatitis virus（VSV）,and screen for an optimized compound.Then,evaluate the antiviral effects of the selected compound on cells and mice systematically;furthermore,evaluate the effects of the selected compound as vaccine adjuvant.The results are as follows:1.Synthesis of quinazoline derivatives and pyrrolo[3,2-d]pyrimidine derivatives.The chemical structures of quinazoline and pyrrolo[3,2-d]pyrimidine were expanded,and various quinazoline derivatives and pyrrolo[3,2-d]pyrimidine derivatives were designed.These compounds were chemically synthesized and structurally characterized.The results showed that a total of 8 compounds were successfully synthesized through analysis using ¹H NMR,¹³C NMR,and HRMS.They were 3quinazoline derivatives（compound 3,5,and 6）and 5 pyrrolo[3,2-d]pyrimidine derivatives（compound10,11,12,13,and 14）.2.Evaluation of the safety and biological activity of novel TLR7 agonists.PK-15 cells were treated with compounds for 24 hours or 48 hours,and the effects of compounds on cell proliferation,cell cycle,and apoptosis were analyzed.The results showed that compounds 3,6,11,12,and 14 at a concentration of 3μM exhibited no significant cytotoxicity.The activation of NF-κB promoter and interferon-stimulated response element（ISRE）promoter by compounds was evaluated using a dual-luciferase reporter assay.Results indicated that compounds 6,3,10,and 11 were the most potent in activating the NF-κB promoter,and compounds 11,3,13,and 14 were the most potent in activating the ISRE promoter.The antiviral activity of compounds against PRV and VSV was evaluated using PK-15cells infected with these viruses,demonstrating that compounds 5,10,11,and 14 exhibited good antiviral effects compared to reported antiviral agents（TLR7 agonist 2,Apilimod,and Tamoxifen）.Compound 11 was selected for further investigation based on its safety in cells,activation ability of the TLR7 signaling pathway,and antiviral activity.3.Evaluation of the antiviral effects of compound 11 in vitro and in vivo.Firstly,the effects of compound 11 on cell proliferation,cell cycle,and apoptosis in PK-15 cells were evaluated.The results showed that there was no significant cytotoxicity within the concentration of 0-3μM,and the half-maximal toxic concentration was 19.886μM.Moreover,the effect of compound 11 on activation of TLR7 signaling pathways was confirmed by using TLR7^WTand TLR7^-/-PK-15 cells.Subsequently,the inhibitory effects of compound 11 on PRV and VSV proliferation in PK-15 cells were revealed.These results showed that compound 11 exert antiviral effects by activating the TLR7 signaling pathway.Then,the safety of compound 11 in mice was also assessed,showing no adverse effects on weight,liver function,or tissue morphology at the concentration of 25 mg/kg.Finally,the the antiviral effect of compound 11 in vivo was assessed,and compound 11 could alleviate tissue damage caused by PRV infection and improve mouse survival rates.These results indicate that compound 11 is a TLR7 agonist,exerting antiviral effects during the replication stage of the PRV life cycle,and demonstrating good antiviral effects both in vitro and in vivo.4.Evaluation of the effect of compound 11 as vaccine adjuvant.To assess the potential of compound 11 as a vaccine adjuvant,its effects on immune cell were studied in vitro using DC2.4 cells,mouse-derived bone marrow dendritic cells,and splenic lymphocytes.Compound 11 enhanced the ability of antigen phagocytosis of dendritic cells,promoted dendritic cell maturation,and increased splenic T lymphocyte proliferation.In a live imaging study,compound 11 facilitated the transfer of antigen from foot pad to lymph nodes and increased dendritic cell proportions in lymph nodes.Immune responses were evaluated after immunizing mice with aluminum-adjuvanted vaccines containing OVA and inactivated PRV with or without compound 11.The results indicated that compound 11 promoted antigen-specific Ig G expression and enhanced cellular immune responses.Notably,postimmunization with PRV-inactivated vaccine,the survival rate of mice infected with PRV-QXX was increased from62.5%to 75%when compound 11 was used.In conclusion,3 quinazoline derivatives and 5 pyrrolo[3,2-d]pyrimidine derivatives were successfully synthesized.Based on safety,TLR7 activation,antiviral activity against PRV and VSV,compound 11 was identified as a promising TLR7 agonist with demonstrated in vitro and in vivo antiviral efficacy,and potential usage as a vaccine adjuvant,providing new insights for the prevention and control of infectious diseases.