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

Abstract

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'-<i>O</i>-[(phosphonomethyl)phosphonic acid] (<b>15</b>, <b>16</b>) being the most potent inhibitors with <i>K</i>_i 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 (<b>21</b>) 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 <i>N</i>⁶-substituents. Moreover, it provides valuable insights into the binding modes of competitive CD73 inhibitors, representing an excellent basis for drug development.