Synthesis and structure-activity relationships of α,β-methylene-ADP derivatives: potent and selective ecto-5-nucleotidase inhibitors

Abstract

<em>Ecto-5'</em>-nucleotidase (<em>ecto-5'</em>-NT, <em>e</em>N, CD73, EC 3.1.3.5) is a member of the group of <em>ecto</em>-nucleotidases which dephosphorylate extracellular nucleotides. <em>e</em>N catalyzes the dephosphorylation of nucleoside monophosphates and its main substrate is AMP. Further members of the membrane-bound group of ecto-nucleotidases include nucleoside triphosphate diphosphohydrolases (NTPDases; subtypes 1, 2, 3 and 8), nucleotide pyrophosphatase/phosphodiesterases (NPPs 1-4) and alkaline phosphatases (APs; tissue non-specific, intestinal, placental and germ cell APs). NTPDase and NPPs are ATP- and ADP-hydrolyzing <em>ecto</em>-nucleotidases, which prevent ATP, ADP and other nucleotides from acting on purinergic P2X and P2Y receptors. They produce AMP which is further hydrolyzed by <em>e</em>N thereby elevating extracellular concentrations of adenosine which activates adenosine receptors. <br /> Recently it was shown that inhibition of <em>e</em>N with monoclonal antibodies, siRNA, or drug-like inhibitors delays tumor growth and metastasis. Thus, <em>e</em>N inhibitors have potential as novel therapeutics, e.g. for melanomas, lung, prostate and breast cancers. Only very few, moderately potent <em>e</em>N inhibitors are currently known. In the present study we used the ADP analog <em>α,β</em>-methylene-ADP (AOPCP, adenosine-<em>5-O'</em>-[(phosphonomethyl)phosphonic acid]) as a lead structure for the development of potent, selective and metabolically stable <em>e</em>N inhibitors. Derivatives substituted at the <em>N⁶</em>-, C-8- or C-2-positions and/or at the methylene diphosphonate-side chain were synthesized to improve potency and metabolic stability. All new compounds were tested for inhibition of rat recombinant <em>e</em>N.<br /> For the preparation of the target compounds with 2-, 6- or 8-substitution and for 2,6-disubstituted derivatives, a convergent synthetic strategy was applied which involves the initial preparation of the intermediate nucleosides followed by phosphorylation with methylenebis(phosphonic dichloride) to provide the desired AOPCP derivatives. For side-chain-modified analogs of AOPCP commercially available substituted bis(phosphonic acid) derivatives were employed for 5 phosphorylation. Altogether 60 AOPCP derivatives and analogs were obtained in good yields and high purity by an optimized method for their preparation. 6 (Ar)alkylamino-substitution, 2-amino-, 2-halo-, and 2-thioalkyl-substitution significantly improved potency. The most potent nucleotides were 2-chloro-<em>N⁶</em>-(2 chlorobenzyl)¬purine riboside-<em>5-O</em>-[(phosphonomethyl)¬phosphonic acid] (<strong>144</strong>, K_i= 0.34 nM), and 2-chloro-<em>N⁶</em>-benzyl--<em>N⁶</em>-methylpurine riboside-<em>5-O'</em>-[(phosphonomethyl)phosphonic acid] (<strong>149</strong>, Ki= 0.88 nM). The compounds displayed high selectivity versus other ecto-nucleotidases and ADP-activated P2Y receptors. They also showed high metabolic stability upon incubation with liver microsomes and blood plasma. These compounds are the most potent <em>e</em>N inhibitors known to date and may serve as valuable pharmacological tools to further elucidate the enzyme’s (patho)physiological roles.