Novel, Highly Potent Adenosine Deaminase Inhibitors Containing the Pyrazolo[3,4-<i>d</i>]pyrimidine Ring System. Synthesis, Structure−Activity Relationships, and Molecular Modeling Studies SettimoFederico Da PrimofioreGiampaolo MottaConcettina La TalianiSabrina SimoriniFrancesca MariniAnna Maria MugnainiLaura LavecchiaAntonio NovellinoEttore TuscanoDaniela MartiniClaudia 2005 This study reports the synthesis of a number of 1- and 2-alkyl derivatives of the 4-aminopyrazolo[3,4-<i>d</i>]pyrimidine (APP) nucleus and their evaluation as inhibitors of ADA from bovine spleen. The 2-substituted aminopyrazolopyrimidines proved to be potent inhibitors, most of them exhibiting <i>K</i><sub>i</sub> values in the nanomolar/subnanomolar range. In this series the inhibitory activity is enhanced with the increase in length of the alkyl chain, reaching a maximum with the <i>n</i>-decyl substituent. Insertion of a 2‘-hydroxy group in the <i>n</i>-decyl chain gave <b>3k</b>, whose (<i>R</i>)-isomer displayed the highest inhibitory potency of the series (<i>K</i><sub>i</sub> 0.053 nM), showing an activity 2 orders of magnitude higher than that of (+)-EHNA (<i>K</i><sub>i</sub> 1.14 nM), which was taken as the reference standard. Docking simulations of aminopyrazolopyrimidines into the ADA binding site were also performed, to rationalize the structure−activity relationships of this class of inhibitors.