Molecular Pharmacology, Vol 14, 422-430, Copyright © 1978 by the American Society for Pharmacology and Experimental Therapeutics

5-Nitro-2'-deoxyuridine and 5-Nitro-2'-deoxyuridine 5'-Monophosphate: Antiviral Activity and Inhibition of Thymidylate Synthetase in Vivo

¹ Rega Institute for Medical Research, University of Leuven, B-3000 Leuven, Belgium
² Laboratory of Chemistry, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014

The antiviral activity of a series of nitrated uracil derivatives, including 5-nitrouracil, 1-methyl-5-nitrouracil, 1,3-dimethyl-5-nitrouracil, 5-nitro-2'-deoxyuridine (5-nitro-dU), 3'-O,5-dinitro-2'-deoxyuridine, 5-nitro-2'-deoxyuridine 5'-monophosphate (5-nitro-dUMP), and 3'-O,5-dinitro-2'-deoxyuridine 5'-monophosphate, was evaluated in primary rabbit kidney or human skin fibroblast cultures challenged with vaccinia, herpes simplex, or vesicular stomatitis virus. The most remarkable antiviral activity was shown by 5-nitro-dU and 5-nitro-dUMP, which inhibited the replication of vaccinia virus at concentrations as low as 0.1-0.4 µg/ml. Somewhat higher concentrations were required to inhibit the replication of herpes simplex virus (1-4 µg/ml for 5-nitro-dU and 40-100 µg/ml for 5-nitro-dUMP). Neither 5-nitro-dU nor its 5'-monophosphate was inhibitory to vesicular stomatitis virus at concentrations up to 100 µg/ml. Combination of 5-nitro-dU with 5-iodo-2'-deoxyuridine (5-iodo-dU) resulted in an increased antiviral activity over the activity of the compounds used individually, suggesting that 5-nitro-dU and 5-iodo-dU act at different steps in DNA biosynthesis. In fact, 5-nitro-dU and its 5'-monophosphate effectively blocked the incorporation of [¹⁴C]2'-deoxyuridine into host cell DNA, but neither compound inhibited the incorporation of [³H]2'-deoxythymidine, pointing to thymidylate synthetase as a specific target for the action of 5-nitro-dU (or 5-nitro-dUMP). Inhibition of thymidylate synthetase would account for the antiviral activity of 5-nitro-dU, since the inhibitory effect of 5-nitro-dU on vacciia virus replication could be readily reversed by 2'-deoxythymidine, but not by 2'-deoxyuridine or 2'-deoxycytidine. Other deoxythymidine analogues that were found to inhibit deoxyuridine incorporation, but not deoxythymidine incorporation, and could therefore be assumed to block thymidylate synthetase selectively in vivo, include 5-fluoro-, 5-trifluoromethyl-, 5-cyamo-, and 5-thiocyamato-2'-deoxyuridine.