Investigations into specificity of azepinomycin for inhibition of guanase: Discrimination between the natural heterocyclic inhibitor and its synthetic nucleoside analogues

In our long and broad program to explore structure–activity relationships of the natural product azepinomycin and its analogues for inhibition of guanase, an important enzyme of purine salvage pathway of nucleic acid metabolism, it became necessary to investigate if the nucleoside analogues of the heterocycle azepinomycin, which are likely to be formed in vivo, would be more or less potent than the parent heterocycle. To this end, we have resynthesized both azepinomycin (1) and its two diastereomeric nucleoside analogues (2 and 3), employing a modified, more efficient procedure, and have biochemically screened all three compounds against a mammalian guanase. Our results indicate that the natural product is at least 200 times more potent toward inhibition of guanase as compared with its nucleoside analogues, with the observed Ki of azepinomycin (1) against the rabbit liver guanase=2.5 (±0.6)×10−6M, while Ki of Compound 2=1.19 (±0.02)×10−4M and that of Compound 3=1.29 (±0.03)×10−4M. It is also to be noted that while IC50 value of azepinomycin against guanase in cell culture has long been reported, no inhibition studies nor Ki against a pure mammalian enzyme have ever been documented. In addition, we have, for the first time, determined the absolute stereochemistry of the 6-OH group of 2 and 3 using conformational analysis coupled with 2-D 1H NMR NOESY