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 substitution 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-benzylthiopurine was synthesized by alkylation of 2-chloro-6-thiopurine with benzyl bromide. The stereoisomers of 2-chloro-6-(1-phenyl-1-ethoxy)purine were obtained from R- and S-enantiomers of sec.-phenylethylalcohol and 2,6-dichloropurine.

All derivatives were tested for inhibition with purified hexameric E. coli purine nucleoside phosphorylase (PNP). For analogues showing IC₅₀ < 10 μm, the type of inhibition and inhibition constants were determined. In all cases the experimental data were best described by the mixed-type inhibition model and the uncompetitive inhibition constant, K_iu, was found to be several-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 natural PNP substrate adenine, as well as 2-chloroadenine, show mixed type inhibition with almost the same inhibition constants K_iu and K_iC.

The most potent inhibition was observed for 6-benzylthio-2-chloro-, 6-benzyloxy-2-chloro-, 2-chloro-6-(2-phenyl-l-ethoxy), 2-chloro-6-(3-phenyl-l-propoxy)- and 2-chloro-6-ethoxypurines (K_iu = 0.4, 0.6, 1.4, 1.4 and 2.2 μm, respectively). The R-stereoisomer of 2-chloro-6-(1pheny-1-ethoxy)purine has K_iu = 2.0 μm, whereas inhibition of its S counterpart is rather weak (IC₅₀> 12 μm). More rigid (e.g. phenoxy-), non-planar (cyclohexyloxy-), or more bulky (2,4,6-trimethylphenoxy-) substituents at position 6 of the purine base gave less potent inhibitors (IC₅₀ = 26, 56 and >100 μm, respectively). The derivatives are selective inhibitors of hexameric “high-molecular mass” PNPs because no inhibitory activity vs. trimeric Cellulomonas

sp. PNP was detected.

By establishing the ligand-dependent stabilization pattern of the E. coli PNP it was shown that the new derivatives, similarly as the natural purine bases, are able to form a dead-end ternary complex with the enzyme and orthophosphate. It was also shown that the derivatives are substrates in the reverse synthetic direction catalyzed by E. coli PNP