Design, synthesis, and enzymatic evaluation of multisubstrate analogue inhibitors of Escherichia coli thymidine phosphorylase

A series of acyclic phosphonate derivatives of thymine has been synthesized and tested as multisubstrate analogue inhibitors of Escherichia colt thymi dine phosphorylase. The compounds synthesized include 1-(phosphonoalkyl)thy mines with six to nine methylenes (1-4, respectively); 1-[(Z)-4-phosphonome thoxy-2-butenyl]thymine (5) and its butyl and 2,3-cis-dihydroxybutyl deriva tives (6 and 7, respectively); 1-[(Z)-(4-(phosphonomethoxy)methoxy)-2-buten yl]thymine (8) and also its butyl and 2,3-cis-dihydroxybutyl analogues (9 a nd 10); and 1-[((Z)-4-(phosphonomethoxy)-2-butenoxy)methyl]thymine(11), Eva luation of these compounds against E. coli revealed significant enzymatic i nhibition by 2, 3, 4, 6, and 8 at a concentration of 1000 mu M, 3 and 4 bei ng the most potent. Replacement of the thymine base in 3 by 6-amino-5-bromo uracil and 7-deazaxanthine afforded compounds 12 and 13, which showed a pro nounced improvement of TPase inhibition, comparable to 7-deazaxanthine. Whe n inorganic phosphate was used as a variable substrate, compounds 12 and 13 displayed competitive kinetics with respect to phosphate, indicating a dir ect interaction of these compounds with the phosphate binding site. Also co mpounds 12 and 13 were found to be competitive inhibitors of TPase against thymidine as a variable substrate. These results are consistent with the co mpounds being multisubstrate analogue inhibitors of E. coli TPase, and they represent the first example of such TPase inhibitors.