Design, synthesis, and evaluation of pyrrolidine based CXCR4 antagonists with in vivo anti-tumor metastatic activity

The chemokine receptor CXCR4 has been proposed as a drug target based on its important functions in HIV infection, inflammation/autoimmune diseases and cancer metastasis. Herein we report the design, synthesis and evaluation of novel CXCR4 antagonists based on a pyrrolidine scaffold. The structural exploration/optimization identified numerous potent CXCR4 antagonists, represented by compound 46, which displayed potent binding affinity to CXCR4 receptor (IC₅₀ = 79 nM competitively displacing fluorescent 12G5 antibody) and inhibited CXCL12 induced cytosolic calcium flux (IC₅₀ = 0.25 nM). Moreover, in a transwell invasion assay, compound 46 significantly mitigated CXCL12/CXCR4 mediated cell migration. Compound 46 exhibited good physicochemical properties (MW 367, logD_7.4 1.12, pKa 8.2) and excellent in vitro safety profiles (e.g., hERG patch clamp IC₅₀ > 30 μM and minimal CYP isozyme inhibition). Importantly, 46 displayed much improved metabolic stability in human and rat liver microsomes. Lastly, 46 demonstrated marked efficacy in a cancer metastasis model in mice. These results strongly support 46 as a prototypical lead for the development of promising CXCR4 antagonists as clinical candidates.