Design, Synthesis, and Antitumor Activity Evaluation of 2‑Arylmethoxy-4-(2,2′-dihalogen-substituted biphenyl-3-ylmethoxy) Benzylamine Derivatives as Potent PD-1/PD-L1 Inhibitors

Novel 2-arylmethoxy-4-(2,2′-dihalogen-substituted
biphenyl-3-ylmethoxy)
benzylamine derivatives were designed, synthesized, and evaluated in vitro and in vivo against cancers as
PD-1/PD-L1 inhibitors. Through the computer-aided structural optimization
and the homogeneous time-resolved fluorescence (HTRF) assay, compound A56 was found to most strongly block the PD-1/PD-L1 interaction
with an IC50 value of 2.4 ± 0.8 nM and showed the
most potent activity. 1H NMR titration results indicated
that A56 can tightly bind to the PD-L1 protein with KD < 1 μM. The X-ray diffraction data
for the cocrystal structure of the A56/PD-L1 complex
(3.5 Å) deciphered a novel binding mode in detail, which can
account for its most potent inhibitory activity. Cell-based assays
further demonstrated the strong ability of A56 as an
hPD-1/hPD-L1 blocker. Especially in an hPD-L1 MC38 humanized mouse
model, A56 significantly inhibited tumor growth without
obvious toxicity, with a TGI rate of 55.20% (50 mg/kg, i.g.). In conclusion, A56 is a promising clinical candidate worthy of further development.