Compound A723U, a 2-acetylpyridine thiosemicarbazone, produced apparent inactivation of herpes simplex virus type 1 (HSV-1) ribonucleotide reductase. Inactivation occurred after A7M3U formed a reversible complex with the enzyme and only while the enzyme was catalyzing the formation of deoxynucleotides. A723U inhibited HSV-1 replication at concentrations that were not toxic to the confluent host cells. Most importantly, A723U and acyclovir (ACV) were found to exhibit mutual potentiation of their antiviral activities. Subinhibitory concentrations of either compound greatly reduced the ED50 (median effective dose) of the other. Studies of the deoxynucleotide pool sizes and the levels of ACV triphosphate, (ACV-P3) revealed that A723U not only significantly reduced the pool of dGTP but also increased the level of ACV-P3 in infected cells. The net result was an 80-fold increase in the ratio of ACV-P3 to dGTP. This should greatly facilitate the initial binding of ACV-P3 to HSV-1 DNA polymerase and probably accounts for the mechanism of potentiation.Acyclovir (ACV), a clinically useful antiherpetic agent, is selectively phosphorylated by herpes simplex virus (HSV)-encoded thymidine kinase (1, 2). Cellular enzymes (3, 4) then catalyze the conversion of ACV monophosphate to the triphosphate form (ACV-P3), which is the fully activated metabolite that is toxic to the virus. Its mechanism of action appears to be the inhibition ofviral DNA synthesis (5), which is probably the result of the selective inactivation by ACV-P3 of the HSV type 1 (HSV-1)-encoded DNA polymerase (6). Prior to inactivation, an obligate reversible complex (Michaelis type) is formed between ACV-P3 and the susceptible enzyme (6). Since dGTP can competitively prevent this initial binding of ACV-P3 (6-8), and since the level of dGTP characteristically increases after treatment of HSV-1-infected cells with ACV (9), dGTP has the potential to protect the polymerase from inactivation. Furthermore, an agent that blocks the synthesis of dGDP would decrease the intracellular dGTP pool. It follows that such an agent should promote the ACV-P3 inactivation ofthe HSV DNA polymerase. In the present study, we have investigated an inhibitor of HSV-1 ribonucleotide reductase, the enzyme that catalyzes the synthesis of dGDP in HSV-1 § and appears to be essential to HSV replication (11,12). This inhibitor was found to be tolerated by the host cells and to significantly potentiate the antiviral activity of ACV. In addition to its predicted suppression of the dGTP pool, this ribonucleotide reductase inhibitor also unexpectedly produced a marked increase in the level of ACV-P3. MATERIALS AND METHODSChemical Synthesis. Burroughs Wellcome compound A723U [2-acetylpyridine 4-(2-morpholinoethyl)thiosemicarbazone] was prepared by refluxing 2-acetylpyridine and 4-(2-morpholino)thiosemicarbazide (molar ratio, 1.1-1.0) for 75 min in 95% ethanol containing 2% (vol/vol) glacial acetic acid. The product was obtained as yellow-tinted crystals by recrystallization from 95% ethanol (...

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(9) and to potentiate the antiherpetic activities of ACV. In addition to producing the expected decrease in the dGTP pool, A723U also caused the pool of ACVTP to increase by 10-fold (17).In the present study, the properties of 2-acetylpyridine 5-[(dimethylamino)thiocarbonyl]thiocarbonohydrazone (A111OU) were investigated. This compound was found to be considerably more potent than A723U. At submicromolar concentrations, A111OU inactivated the ribonucleotide reductases of these three herpes viruses. It also markedly potentiated the activity of ACV against the viruses in vitro. Furthermore, as reported elsewhere ( §, ¶), A111OU and ACV produced significantly synergistic therapy as topical treatment of HSV-infected animals. MATERIALS AND METHODSReagents. All reagents not described below were obtained as previously described (11).Synthesis of A111OU. A mixture of 24.2 g (0.203 mol) of 4,4-dimethylthiosemicarbazide, 26.6 g (0.220 mol) of 2-acetylpyridine, 2.5 ml of glacial acetic acid, and 100 ml of 95% (vol/vol) ethanol was refluxed for 1.5 hr and then incubated overnight at room temperature. Thick orange needles contaminated with a small amount of a light-colored solid were collected, washed with 20 ml of ethanol, and, while still damp, added to 450 ml of boiling methanol. After 10 min, the mixture was filtered. The undissolved solid was re-incubated with 50 ml of boiling methanol for 3 min and then filtered. After 15 min at room temperature, the yellow product recrystallized from the combined filtrates and was collected by filtration.

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