2-Substituted α,β-Methylene-ADP Derivatives: Potent Competitive Ecto-5'-nucleotidase (CD73) Inhibitors with Variable Binding Modes

J Med Chem. 2020 Mar 26;63(6):2941-2957. doi: 10.1021/acs.jmedchem.9b01611. Epub 2020 Feb 28.


CD73 inhibitors are promising drugs for the (immuno)therapy of cancer. Here, we present the synthesis, structure-activity relationships, and cocrystal structures of novel derivatives of the competitive CD73 inhibitor α,β-methylene-ADP (AOPCP) substituted in the 2-position. Small polar or lipophilic residues increased potency, 2-iodo- and 2-chloro-adenosine-5'-O-[(phosphonomethyl)phosphonic acid] (15, 16) being the most potent inhibitors with Ki values toward human CD73 of 3-6 nM. Subject to the size and nature of the 2-substituent, variable binding modes were observed by X-ray crystallography. Depending on the binding mode, large species differences were found, e.g., 2-piperazinyl-AOPCP (21) was >12-fold less potent against rat CD73 compared to human CD73. This study shows that high CD73 inhibitory potency can be achieved by simply introducing a small substituent into the 2-position of AOPCP without the necessity of additional bulky N6-substituents. Moreover, it provides valuable insights into the binding modes of competitive CD73 inhibitors, representing an excellent basis for drug development.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 5'-Nucleotidase / antagonists & inhibitors*
  • 5'-Nucleotidase / chemistry
  • 5'-Nucleotidase / metabolism
  • Adenosine Diphosphate / analogs & derivatives*
  • Adenosine Diphosphate / chemistry
  • Adenosine Diphosphate / pharmacology
  • Animals
  • Crystallography, X-Ray
  • Enzyme Inhibitors / chemistry*
  • Enzyme Inhibitors / pharmacology*
  • GPI-Linked Proteins / antagonists & inhibitors
  • GPI-Linked Proteins / chemistry
  • GPI-Linked Proteins / metabolism
  • Humans
  • Molecular Docking Simulation
  • Protein Binding
  • Rats
  • Structure-Activity Relationship


  • Enzyme Inhibitors
  • GPI-Linked Proteins
  • alpha,beta-methyleneadenosine 5'-diphosphate
  • Adenosine Diphosphate
  • 5'-Nucleotidase
  • NT5E protein, human