目的：芳香烃受体（Aryl hydrocarbon receptor, AhR）是一种配体依赖性转录因子，是参与广泛环境变化应答的中心传感器。AhR配体药物在肿瘤免疫治疗以及感染性疾病治疗领域都有非常重要的应用前景。然而AhR在正常组织中发挥重要生理功能，提高AhR配体的靶向性和生物利用度是AhR靶点药物开发亟待解决的问题。杯芳烃是超分子化学中一类具有代表性的有机大环受体，广泛应用于分子识别传感、自组装、有机催化、纳米技术以及药物研发等领域。本研究旨在构建一种偶氮类杯[4]芳烃与AhR配体非共价结合体系，解决AhR配体生物利用度低，水溶性差问题。同时，偶氮杯[4]芳烃与AhR配体复合物能够特异性被体内偶氮还原酶催化，在病灶部位释放出AhR配体，避免全身副作用从而实现AhR配体对肿瘤和放射性肠炎的精准治疗。

方法：1）偶氮杯[4]芳烃（azoC4A）的合成；2）偶氮杯芳烃与AhR配体非共价结合的物理表征，包括通过荧光滴定法评估其与AhR配体（抑制剂BAY2416964和激动剂BNF）的非共价结合能力以及特异性释放能力；3）偶氮杯芳烃在细胞环境中对AhR配体的特异性结合与释放，包括构建AhR荧光素酶报告基因细胞系，评估azoC4A-AhR配体的AhR活化能力；体外细胞毒性和对细胞周期的影响；4）azoC4A-AhR配体复合物在疾病模型中的应用，包括分别建立小鼠移植瘤模型和放射性肠炎模型，评价azoC4A-AhR配体对肿瘤或者放射性肠炎的特异性治疗作用。

结果：1）azoC4A能够实现对AhR配体的有效负载和特异性释放（AhR抑制剂BAY2416964和AhR激动剂BNF的结合常数分别为1.2×10⁵ M^-1和6×10⁷ M^-1）；2）细胞试验结果表明azoC4A-BAY2416964复合物能够实现乏氧条件下偶氮还原酶浓度提高导致azoC4A被破坏释放BAY2416964，进而特异性抑制AhR活性；显著诱导肿瘤细胞HepG2凋亡以及细胞周期G0/G1期阻滞；3）azoC4A-BAY2416964的体内抑瘤结果表明：相较于Ctrl组，azoC4A-BAY2416964给药组小鼠乳腺癌4T1移植瘤的瘤体显著减少（79.87 mm³±28.39 mm³对468.82mm³±78.13 mm³），甚至优于BAY2416964阳性药组（325.68 mm³±107.70 mm³），这与azoC4A附载BAY2416964后增强其水溶性，并在肿瘤乏氧区域经偶氮还原酶作用靶向释放BAY2416964，提高其生物利用度紧密相关；4）azoC4A-激动剂BNF治疗小鼠放射性肠炎结果表明：azoC4A-BNF治疗组小鼠放射性肠炎症状显著缓解，血液中二胺氧化酶水平显著低于单纯照射组以及BNF+照射组，同时azoC4A-BNF+照射组炎症因子TNF-α的含量也较对照组明显下降；5）azoC4A-BNF给药后肠道组织中AhR下游靶基因Cyp1a1表达水平显著高于BNF组，表现出特异性的AhR转录活化。

结论：1）偶氮杯芳烃azoC4A与AhR配体（抑制剂BAY2416964和激动剂BNF）能够通过主客体识别方式结合；2）azoC4A-AhR配体体系在常氧细胞环境稳定；乏氧条件下偶氮还原酶含量提高，azoC4A-AhR配体体系被破坏，AhR配体特异性释放发挥促进肿瘤细胞凋亡的作用；3）偶氮杯芳烃azoC4A与AhR配体非共价结合的策略对小鼠肿瘤模型和放射性肠炎模型均具有显著治疗效果，提高了AhR配体的生物利用度和溶解性的同时减少了AhR配体的全身释放。

Objective: The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor and a central sensor involved in a wide range of environmental stress. AhR ligands have very important applications in the fields of tumour immunotherapy as well as infectious disease treatment. However, AhR presents pivotal physiological functions in normal tissues, and enhancing the targeting and bioavailability of AhR ligands is a pressing concern in AhR-targeted drug development. Calixarenes represent a prominent class of organic macrocyclic receptors within the domain of supramolecular chemistry. Calixarenes have been applied in diverse fields, including molecular recognition sensing, self-assembly, organocatalysis, nanotechnology, and drug discovery. The objective of this study is to develop a host-guest recognization system of azo[4]calixarene with AhR ligands, resolving the issues of low bioavailability and poor solubility currently experienced by AhR ligands. Concurrently, the azoC4A-AhR ligands complex can be specifically catalysed by azoreductase in vivo, releasing AhR ligands at the focal site, thus avoiding systemic side effects and enabling the precise treatment of tumours and radiolucent enteritis with AhR ligand.

Methods: 1) Synthesis of azo[4]calixarene (azoC4A); 2) Physical characterisation of azoC4A non-covalent binding to AhR ligands, including assessment of its non-covalent binding ability to AhR ligand (the inhibitor BAY2416964 and agonist BNF) and its specific releasing evaluated by fluorescent titration; 3) Specificity of the azoC4A in cellular environment for AhR ligands binding and release, including the construction of AhR luciferase reporter gene cell lines to evaluate the AhR activation of azoC4A-AhR ligands; in vitro cytotoxicity and effects on the cell cycle; 4) the application of azoC4A-AhR ligands complexes in disease models, including the establishment of mouse transplantation tumour model and radiolucent enteritis model, respectively, and the assessment of the azoc4A-AhR ligands for tumour or radiolucent enteritis-specific therapeutic effects.

Results: 1) azoC4A was able to achieve efficient loading and specific release of AhR ligands (the binding constants of AhR inhibitor BAY2416964 and AhR inhibitor BNF were 1.2×105 M-1 and 6×107 M-1, respectively) 2) The results of cellular assays showed that the azoC4A-BAY2416964 complex could specifically inhibit the AhR activity under hypoxic conditions; significant induction of the tumour cells HepG2 apoptosis and arrest of G0/G1 phase of the cell cycle; 3) The results of azoC4A-BAY2416964 treatment of tumours in animal experiments showed that the tumour volume of transplanted tumour 4T1 was significantly reduced in the azoC4A-BAY2416964 administered group versus the Control group of mice (468.8 mm³ versus 79.7 mm³), while the tumour volume of transplanted tumour 4T1 was significantly reduced in the Control group (468.8 mm³ versus 79.7 mm³), and also showed a more significant tumour-suppressive effect with the positive drug BAY2416964 group (325.7 mm³±107.7 mm³); 4) Results of azoC4A-BNF treatment of mice with radiolucent enterocolitis showed that: radiolucent enterocolitis symptoms in the azoC4A-BNF treatment group were significantly relieved. The level of diamine oxidase in the blood was significantly lower than that in the control group alone and the BNF group, and the content of TNF-α in the azoC4A-BNF group also decreased significantly compared with that in the control group. 5) The expression level of AhR target gene Cyp1a1 in the intestinal tissue after -BNF administration was significantly higher than that of the BNF group, showing specific AhR transcriptional activation.

Conclusion: 1) The azoC4A and AhR ligands (inhibitor BAY2416964 and agonist BNF) were able to bind by the host-guest recognition; 2) the azoC4A-AhR ligand system was stable in a normoxic cellular environment; under hypoxic conditions, the azoC4A-AhR ligand system was disrupted, and specific release of AhR ligands exerted the effect of promoting apoptosis of tumour cells; 3) the azoC4A-AhR system was stable in a normoxic cellular environment. (3) The strategy of non-covalent binding of azocuparine azoC4A to AhR ligand has significant therapeutic effects on both mouse tumour model and radiolucent enteritis model, which improves the bioavailability and solubility of AhR ligand and reduces the systemic release of AhR ligand at the same time.