Synthesis of N³-substituted thymidine analogues for measurement of cellular kinase activity

N³-Substitued thymidine analogues that carry a carboranylalkyl moiety at the N³-position with various spacer lengths have been reported to be good substrates for thymidine kinase (TK1). As part of our continuing effort towards the development of new TK1 substrates for imaging tumor proliferative activity, we have synthesized a series of new N³-substituted analogues of thymidine that carry an aromatic ring with different spacer lengths. The overall yields for 6 and 7 were 13% and 39% in four steps and three steps, respectively, and those for 14, 16 and 18 were in the range of 13%-15% in six steps. The overall yield for 24 was 33% in three steps, and those for 25 and 26 were 64% and 58%, respectively, in one step. Most of these compounds have been tested for TK1 activity by enzymatic assay to identify a good substrate that can be radiolabeled for imaging. The phosphorylation rates of these compounds were 2%-6% compared with that of thymidine. The results from the in vitro enzymatic assays suggest that these N³-substituted thymidine analogues have some potential for imaging TK1 activity if radiolabeled with a suitable isotope.