Synthesis of 6-aryloxy- and 6-arylalkoxy-2-chloropurines and their interactions with purine nucleoside phosphorylase from Escherichia coli

The phase transfer method was applied to perform the nucleophilic substitut ion of 2,6-dichloropurines by modified arylalkyl alcohol or phenols, Since under these conditions only the 6-halogen is exchanged, this method gives 2 -chloro-6-aryloxy- and 2-chloro-6-arylalkoxypurines. 2-Chloro-6-benzylthiop urine was synthesized by alkylation of 2-chloro-6-thiopurine with benzyl br omide. The stereoisomers of 2-chloro-6-(1-phenyl-1-ethoxy)purine were obtai ned from R- and S-enantiomers of sec.-phenylethylalcohol and 2,6-dichloropu rine. All derivatives were tested for inhibition with purified hexameric E. coli purine nucleoside phosphorylase (PNP). For analogues showing IC50 < 10 mu M , the type of inhibition and inhibition constants were determined. In all c ases the experimental data were best described by the mixed-type inhibition model and the uncompetitive inhibition constant, K-iu, was found to be sev eral-fold lower than the competitive inhibition constant, K-ic. This effect seems to be due to the 6-aryloxy- or 6-arylalkoxy substituent, because a n atural PNP substrate adenine, as well as 2-chloroadenine, show mixed type i nhibition with almost the same inhibition constants K-iu and K-ic. The most potent inhibition was observed for 6-benzylthio-2-chloro-, 6-benzy loxy-2-chloro-, 2-chloro-6-(2-phenyl-1-ethoxy), 2-chloro-6-(3-phenyl-1-prop oxy)- and 2-chloro-6-ethoxypurines (K-iu = 0.4, 0.6, 1.4, 1.4 and 2.2 mu M, respectively). The R-stsreoisomer of 2-chloro-6-(1-pheny-1-ethoxy)purine h as K-iu = 2.0 mu M, whereas inhibition of its S counterpart is rather weak (IC50 > 12 mu M). More rigid (e.g, phenoxy-), non-planar (cyclohexyloxy-), or more bulky (2,4,6-trimethylphenoxy-) substituents at position 6 of the p urine base gave less potent inhibitors (IC50 = 26, 56 and > 100 mu M respec tively). The derivatives are selective inhibitors of hexameric "high-molecu lar mass" PNPs because no inhibitory activity vs. trimeric Cellulomonas sp. PNP was detected. By establishing the ligand-dependent stabilization pattern of the E. coil P NP it was shown that the new derivatives, similarly as the natural purine b ases, are able to form a dead-end ternary complex with the enzyme and ortho phosphate. It was also shown that the derivatives are substrates in the rev erse synthetic direction catalyzed by E. coli PNP.