Base-modified thymidines capable of terminating DNA synthesis are novel bioactive compounds with activity in cancer cells

Abstract

Current FDA-approved chemotherapeutic antimetabolites elicit severe side effects that warrant their improvement; therefore, we designed compounds with mechanisms of action focusing on inhibiting DNA replication rather than targeting multiple pathways. We previously discovered that 5-(α-substituted-2-nitrobenzyloxy)methyluridine-5′-triphosphates were exquisite DNA synthesis terminators; therefore, we synthesized a library of 35 thymidine analogs and evaluated their activity using an MTT cell viability assay of MCF7 breast cancer cells chosen for their vulnerability to these nucleoside derivatives. Compound 3a, having an α-tert-butyl-2-nitro-4-(phenyl)alkynylbenzyloxy group, showed an IC50 of 9 ± 1 μM. The compound is more selective for cancer cells than for fibroblast cells compared with 5-fluorouracil. Treatment of MCF7 cells with 3a elicits the DNA damage response as indicated by phosphorylation of γ-H2A. A primer extension assay of the 5′-triphosphate of 3a revealed that 3aTP is more likely to inhibit DNA polymerase than to lead to termination events upon incorporation into the DNA replication fork.

Base-modified thymidine and thymine analogs with low cytotoxicity effectively obstruct DNA replication in papovaviridae

“…The data reflecting quantitative inhibition of human papilloma virus replication is summarized in Table 1. The trends revealed by structure-activity relationship are quite different from those observed for cytotoxicity toward cancer cells [27]. First, the α-tert-butyl and 2-nitrobenzyl groups were not the best substituents, as evidenced by the higher EC 50 of 3a compared to 10a, as well as 4a versus 11a, and 4a versus 5a.…”

“…The bioactive compounds were obtained by heating 5-bromomethyl-3-N-(tert-butyloxy)carbobyl-3' ,5'-bis-(tert-butyl) dimethylsilyl-O-2'-deoxyuridine (1) [28] or 5-chloromethyluracil (2) with an appropriate alcohol under neat, anhydrous conditions, as reported previously [27]. This transformation yielded nucleobases 3b-18b; removal of the residual TBS groups using tetra-n-butylammonium fluoride yielded nucleoside derivatives 3a-18a (Figure 1).…”

“…The solvent was removed under reduced pressure and the residue was purified by silica gel (chloroform/methanol=1:0 to 10:1) and then by C8 reverse-phase column chromatography (water/methanol=19:1 to 1:4) to yield the product as a waxy solid. Compounds 3a-11a and 14a-18a were obtained as reported previously [27].…”

“…Recently, we have discovered base-modified thymidine analogs that show higher cytotoxicity toward cancerous cells compared to benign [27]. Although the modifying moiety attached to the nucleobase obstructs further DNA synthesis upon incorporation of the 5'-triphosphate of such nucleoside into a partial double helix DNA primer [28], the preliminary insight into the mechanism of action strongly suggested inhibition of a DNA polymerase instead [27].…”

“…Although the modifying moiety attached to the nucleobase obstructs further DNA synthesis upon incorporation of the 5'-triphosphate of such nucleoside into a partial double helix DNA primer [28], the preliminary insight into the mechanism of action strongly suggested inhibition of a DNA polymerase instead [27]. However, it was still evident that termination of DNA synthesis occurs only in the presence of a certain bulky group attached at the 5-methyl group of the thymine nucleobase.…”