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

Abstract

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 rk(cat)/K(m) (k(cat)/K(m) 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 rk(cat)/K(m) values of 14.9% and 19.7% respectively. In comparison, the rPR and rk(cat)/K(m) 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.

Figure 1

Structures of N5 and N5-2OH.

Figure 2

Phosphorylation rates of dThd, N5-2OH and N3-substituted dThd analogues IPwith hTK1. Assay products were separated by PEI–cellulose TLC and quantified by β-radiography. (A). Compounds 3e, 3f and 7e-7g; (a) dThd monophosphate (dTMP), (b) compound-monophosphates (N5-2OH, 3e, 3f and 7e-7g) and (c) monophosphorylated degradation products, presumably dTMP. (B). Compounds 8, 9a, 9b1/2-9d1/2, (a) dTMP and (b) compound-monophosphates (8, 9a, 9b1/2-9d1/2). (C). Compounds 10, 11a, 11b1/2-11d1/2, (a) dTMP and (b) compound-monophosphates (11a, 11b1/2-11d1/2)

Scheme 1

Synthesis of N3-amidine-type dThd derivatives (3a-3f).

Scheme 2

Synthesis of N3-guanidine-type dThd derivatives (7a-7g).

Scheme 3

Synthesis of N3-tetrazolylmethyl-type dThd derivatives (9a, 9b1/2-9d1/2).

Scheme 4

Synthesis of N3-tetrazolyl-type dThd derivatives (11a, 11b1/2-11d1/2).