Acyclic purine and pyrimidine nucleotide analogs as ecto-5′-nucleotidase (CD73) inhibitors

Ecto-5'-nucleotidase (CD73) is a novel target in cancer (immuno)therapy. Its blockade prevents the formation of immunosuppressive and cancer-promoting adenosine from AMP. Here, we report on the development of a series of small molecules that mimic adenine nucleotides, in which the ribose moiety was replaced by an alkyl chain. Its length was found to be crucial for potency. A crystal structure of the N⁶-disubstituted acyclic ADP analog 26 (N⁶-benzyl,N⁶-methyladenine-9-yl)pentyloxydiphosphonate) in complex with human CD73 revealed that the flexible pentyl linker adopts to interdomain rotation angles differing by up to 18.5°. The most potent CD73 inhibitor of the present series was analog 27 (N⁶-benzyl,N⁶-methyladenine-9-yl)hexyloxydiphosphonate, PSB-24000) which exhibited submicromolar potency at human CD73 (K_i 563 nM at soluble CD73; K_i 481 nM at membrane-bound CD73 of triple-negative breast cancer cells). Acyclic nucleotide analogs may be advantageous compared to the previously reported nucleotidic CD73 inhibitors due to their high chemical stability, and because less off-target effects are to be expected. The structure-activity relationships discovered in this study provide valuable insights which will be useful for the development of CD73 inhibitors as immunotherapeutic drugs.