α,β-Methylene-ADP (AOPCP) Derivatives and Analogues: Development of Potent and Selective ecto-5′-Nucleotidase (CD73) Inhibitors

ecto-5'-Nucleotidase (eN, CD73) catalyzes the hydrolysis of extracellular AMP to adenosine. eN inhibitors have potential for use as cancer therapeutics. The eN inhibitor α,β-methylene-ADP (AOPCP, adenosine-5'-O-[(phosphonomethyl)phosphonic acid]) was used as a lead structure, and derivatives modified in various positions were prepared. Products were tested at rat recombinant eN. 6-(Ar)alkylamino substitution led to the largest improvement in potency. N(6)-Monosubstitution was superior to symmetrical N(6),N(6)-disubstitution. The most potent inhibitors were N(6)-(4-chlorobenzyl)- (10l, PSB-12441, Ki 7.23 nM), N(6)-phenylethyl- (10h, PSB-12425, Ki 8.04 nM), and N(6)-benzyl-adenosine-5'-O-[(phosphonomethyl)phosphonic acid] (10g, PSB-12379, Ki 9.03 nM). Replacement of the 6-NH group in 10g by O (10q, PSB-12431) or S (10r, PSB-12553) yielded equally potent inhibitors (10q, 9.20 nM; 10r, 9.50 nM). Selected compounds investigated at the human enzyme did not show species differences; they displayed high selectivity versus other ecto-nucleotidases and ADP-activated P2Y receptors. Moreover, high metabolic stability was observed. These compounds represent the most potent eN inhibitors described to date.