Purine analog inhibitors of xanthine oxidase ‐ structure activity relationships and proposed binding of the molybdenum cofactor

Abstract A number of new hypoxanthine analogs have been prepared as substrate inhibitors of xanthine oxidase. Most noteworthy inhibitory new hypoxanthine analogs are 3‐(m‐tolyl)pyrazolo[1,5‐a]pyrimidin‐7‐one (47), ID50 0.06 μM and 3‐phenylpyrazolo[1,5‐a]pyrimidin‐7‐one (46), ID50 0.40 μM. 5‐(p‐Chlorophenyl)pyrazolo[1,5‐a]pyrimidin‐7‐one (63) and the corresponding 5‐nitrophenyl derivative 64 exhibited an ID50 of 0.21 and 0.23 μM, respectively. 7‐Phenylpyrazolo[1,5‐a]‐s‐triazin‐4‐one (40) is shown to exhibit an ID50 of 0.047 μM. The structure‐activity relationships of these new phenyl substituted hypoxanthine analogs are discussed and compared with the xanthine analogs 3‐m‐tolyl‐ and 3‐phenyl‐7‐hydroxypyrazolo[1,5‐a]pyrimidin‐5‐ones (90) and (91), previously reported from our laboratory to have ID50 of 0.025 and 0.038 μM, respectively. The presence of the phenyl and substitutedphenyl groups contribute directly to the substrate binding of these potent inhibitors. This work presents an updated study of structure‐activity relationships and binding to xanthine oxidase. In view of the recent elucidation of the pterin cofactor and the proposed binding of this factor to the molybdenum ion in xanthine oxidase, a detailed mechanism of xanthine oxidase oxidation of hypoxanthine and xanthine is proposed. Three types of substrate binding are viewed for xanthine oxidase. The binding of xanthine to xanthine oxidase is termed Type I binding. The binding of hypoxanthine is termed Type II binding and the specific binding of alloxanthine is assigned as Type III binding. These three types of substrate binding are analyzed relative to the most potent compounds known to inhibit xanthine oxidase and these inhibitors have been classified as to the type of inhibitor binding most likely to be associated with specific enzyme inhibition. The structural requirements for each type of binding can be clearly seen to correlate with the inhibitory activity observed. The chemical syntheses of the new 3‐phenyl‐ and 3‐substituted phenylpyrazolo[1,5‐a]pyrimidines with various substituents are reported. The syntheses of various 8‐phenyl‐2‐substituted pyrazolo‐[1,5‐a]‐s‐triazines, certain s‐triazolo[1,5‐a]‐s‐triazines and s‐triazolo[1,5‐a]pyrimidine derivatives prepared in connection with the present study are also described.