Synthesis of N3-substituted carboranyl thymidine bioconjugates and their evaluation as substrates of recombinant human thymidine kinase 1

Four different libraries of overall twenty three N3-substituted thymidine (dThd) analogues, including eleven 3-carboranyl thymidine analogues (3CTAs), were synthesized. The latter are potential agents for Boron Neutron Capture Therapy (BNCT) of cancer. Linker between the dThd scaffold and the m-carborane cluster at the N3-position of the 3CTAs contained amidinyl-(3e and 3f), guanidyl-(7e–7g), tetrazolylmethyl-(9b1/2–9d1/2), or tetrazolyl groups (11b1/2–11d1/2) to improve human thymidine kinase 1 (hTK1) substrate characteristics and water solubilities compared with 1st generation 3CTAs, such as N5 and N5–2OH. The amidinyl- and guanidyl-type N3-substitued dThd analogues (3a–3f and 7a–7g) had hTK1 phosphorylation rates of <30% relative to that of dThd, the endogenous hTK1 substrate, whereas the tetrazolyl-type N3-substitued dThd analogues (9a, 9b1/2–9d1/2 and 11a, 11b1/2–11d1/2) had relative phosphorylation rates (rPRs) of >40%. Compounds 9a, 9b1/2–9d1/2 and 11a, 11b1/2–11d1/2 were subjected to in-depth enzyme kinetics studies and the obtained rkcat/Km (kcat/Km relative to that of dThd) ranged from 2.5 to 26%. The tetrazolyl-type N3-substitued dThd analogues 9b1/2 and 11d1/2 were the best substrates of hTK1 with rPRs of 52.4% and 42.5% and rkcat/Km values of 14.9% and 19.7% respectively. In comparison, the rPR and rkcat/Km values of N5–2OH in this specific study were 41.5% and 10.8%, respectively. Compounds 3e and 3f were >1900 and >1500 times, respectively, better soluble in PBS (pH 7.4) than N5–2OH whereas solubilities for 9b1/2–9d1/2 and 11b1/2–11d1/2 were only 1.3–13 times better.

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► Four libraries of N3-carboranyl thymidine analogues (3CTAs) were synthesized.
► 3CTAs were evaluated for improved binding to human thymidine kinase 1.
► Selected 3CTAs were subjected to in-depth enzyme kinetics studies (rkcat/Km).
► Solubility studies of 3CTAs were conducted in PBS at pH 7.4.