Part I. Design, Synthesis and Biological Evaluation of Pyrazolopyrimidine Derivatives as Aryl Hydrocarbon Receptor Antagonists for Colorectal Cancer Immunotherapy Part II. Identification of New Diacylglycerol Kinase ζ Degraders for Colorectal Cancer

Abstract

Part I. 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 have identified effective small-molecule AhR antagonists for cancer immunotherapy. A new series of pyrazolopyrimidine derivatives was synthesized and evaluated for AhR antagonistic activity. 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 synergistically enhanced antitumor immunity by augmenting cytotoxic T lymphocyte (CTL)-mediated activity. Collectively, a new pyrazolopyrimidine derivative, 7k shows promise as a potential therapeutic agent for treating colorectal cancer. Part II. Diacylglycerol kinase zeta (DGKζ) is an intracellular checkpoint that suppresses T-cell activation by attenuating diacylglycerol signaling. To target this pathway, we developed PROTAC degraders designed to eliminate DGKζ. Compound 16 exhibited potent degradation (DC₅₀= 27.2 nM) with moderate metabolic stability and favorable pharmacokinetics. In an MC38 colorectal cancer model, 16 significantly suppressed tumor growth (TGI= 74%). These findings identify compound 16 as a DGKζ PROTAC with strong antitumor efficacy and favorable drug like properties for colorectal cancer immunotherapy.