Design, Synthesis and Biological Evaluation of Pyrazolopyrimidine Derivatives as Aryl Hydrocarbon Receptor Antagonists for Colorectal Cancer Immunotherapy

<b>Background:</b> Aryl hydrocarbon receptor (AhR) is a transcription factor that is involved in the regulation of immunity. AhR inhibits T cell activation in tumors, which induces immune suppression in the blood and solid tumors. We identified effective small-molecule AhR antagonists for cancer immunotherapy. <b>Methods:</b> A new series of pyrazolopyrimidine derivatives was synthesized and evaluated for AhR antagonistic activity. <b>Results:</b> Compound <b>7k</b> exhibited significant antagonistic activity against AhR in a transgenic zebrafish model. In addition, <b>7k</b> exhibited good AhR antagonist activity, with a half-maximal inhibitory concentration (IC₅₀) of 13.72 nM. Compound <b>7k</b> showed a good pharmacokinetic profile with an oral bioavailability of 71.0% and a reasonable half-life of 3.77 h. Compound <b>7k</b> selectively exerted anti-proliferative effects on colorectal cancer cells without affecting normal cells, concurrently suppressing the expression of AhR-related genes and the PD-1/PD-L1 signaling pathway. Compound <b>7k</b> exhibited potent antitumor activity in syngeneic colorectal cancer models. Importantly, the combination of anti-PD1 and compound <b>7k</b> enhanced antitumor immunity by augmenting cytotoxic T lymphocyte (CTL)-mediated activity. <b>Conclusions:</b> Collectively, a new pyrazolopyrimidine derivative, <b>7k</b>, shows promise as a potential therapeutic agent for treating colorectal cancer.