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

## TL;DR

This study develops a new compound that blocks the Aryl Hydrocarbon Receptor, showing potential to enhance immunotherapy for colorectal cancer.

## Contribution

A novel pyrazolopyrimidine derivative with strong AhR antagonistic activity and antitumor effects is introduced.

## Key findings

- Compound 7k showed significant AhR antagonistic activity with an IC50 of 13.72 nM.
- 7k selectively inhibited colorectal cancer cells and suppressed the PD-1/PD-L1 pathway.
- Combining 7k with anti-PD1 therapy enhanced antitumor immunity in mouse models.

## Abstract

Background: 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. Methods: A new series of pyrazolopyrimidine derivatives was synthesized and evaluated for AhR antagonistic activity. Results: Compound 7k exhibited significant antagonistic activity against AhR in a transgenic zebrafish model. In addition, 7k exhibited good AhR antagonist activity, with a half-maximal inhibitory concentration (IC50) of 13.72 nM. Compound 7k showed a good pharmacokinetic profile with an oral bioavailability of 71.0% and a reasonable half-life of 3.77 h. Compound 7k 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 7k exhibited potent antitumor activity in syngeneic colorectal cancer models. Importantly, the combination of anti-PD1 and compound 7k enhanced antitumor immunity by augmenting cytotoxic T lymphocyte (CTL)-mediated activity. Conclusions: Collectively, a new pyrazolopyrimidine derivative, 7k, shows promise as a potential therapeutic agent for treating colorectal cancer.