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The relative in vitro antiviral activities of three related nucleoside carboxamides, ribavirin (1-3-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), tiazofurin (2-3-D-ribofuranosylthiazole-4-carboxamide), and selenazole (2-p-D-ribofuranosylselenazole-4-carboxamide), were studied against selected DNA and RNA viruses. Although the activity of selenazole against different viruses varied, it was significantly more potent than ribavirin and tiazofurin against all tested representatives of the families Paramyxoviridae (parainfluenza virus type 3, mumps virus, measles virus), Reoviridae (reovirus type 3), Poxviridae (vaccinia virus), Herpesviridae (herpes simplex virus types 1 and 2), Togaviridae (Venezuelan equine encephalomyelitis virus, yellow fever virus, Japanese encephalitis virus), Bunyaviridae (Rift Valley fever virus, sandfly fever virus [strain Sicilian], Korean hemorrhagic fever virus), Arenaviridae (Pichinde virus), Picornaviridae (coxsackieviruses B1 and B4, echovirus type 6, encephalomyocarditis virus), Adenoviridae (adenovirus type 2), and Rhabdoviridae (vesicular stomatitis virus). The antiviral activity of selenazole was also cell line dependent, being greatest in HeLa, Vero-76, and Vero E6 cells. Selenazole was relatively nontoxic for Vero, Vero-76, Vero E6, and HeLa cells at concentrations of up to 1,000 ,g/ml. The relative plating efficiency at that concentration was over 90%. The effects of selenazole on viral replication were greatest when this agent was present at the time of viral infection. The removal of selenazole from the medium of infected cells did not reverse the antiviral effect against vaccinia virus, but there was a gradual resumption of viral replication in cells infected with parainfluenza type 3 or herpes simplex virus type 1 (strain KOS). However, the antiviral activity of ribavirin against the same viruses was reversible when the drig was removed.

A B S T R A C T 2-Methylthio-ADP and its radioactive analogue [,_-32P]2-methylthio-ADP were synthesized and used to investigate platelet receptors for ADP. 2-Methylthio-ADP induced platelet aggregation and shape change, and inhibited cyclic AMP accumulation in platelets exposed to prostaglandin El. Compared with ADP, 2-methylthio-ADP was 3-5 times as active as an aggregating agent and 150-200 times as active as an inhibitor of cyclic AMP accumulation. Binding of [#-32P]2-methylthio-ADP to platelets was measured after centrifuging them through silicone oil to separate platelets from their suspension medium. Binding was reversible, saturable, and specific, with between 400 and 1,200 sites/cell in different platelet preparations. There was no evidence for a second class of binding sites with different affinity. The second order association rate constant was -3.5 X 10' M`so', and the first order dissociation rate was 0.024 s-', both measured at 230C. The dissociation equilibrium constant (-15 nM) was about three times higher than the concentration giving half-maximal inhibition of prostaglandin El-stimulated cyclic AMP accumulation in platelet-rich plasma. Binding was inhibited by ADP (Kj = 3.5 MM), ATP (7 AM), 2-azido-ADP (0.12 AM), inosine diphosphate (IDP, 150 AM), guanosine diphosphate (GDP, 350 MM), and AMP (800 MM). Binding of 2-methylthio-ADP was also blocked by the noncell-penetrating thiol reagent, p-mercuribenzene sulphonate, a reagent that blocks the inhibition of adenylate cyclase by ADP, but which does not block the ability of ADP to induce aggregation or platelet shape change. The amount of 2-methylthio-ADP bound at saturation was independent of pH in the range 6-8, but the affinity was reduced at pH 6 compared with pH 6.5-8.0. The dissociation constant was not temperature dependent in the range 320-40'C, whereas the rate of dissociation of 2-methylthio-ADP from platelets after the addition of an excess of ADP approximately doubled over this range. The activation energy for dissociation was -15 kcal/mol. Our results support the conclusion that platelets have a receptor for ADP, which inhibits cyclic AMP accumulation, and which has a sulphydryl group in the binding pocket. INTRODUCTION ADP has two distinguishable effects on blood platelets (1): firstly, it induces aggregation of the cell by stimulating a change in their shape and exposure of fibrinogen-binding sites, and, secondly, it inhibits the adenylate cyclase of membrane preparations of platelets and prevents the accumulation of cyclic AMP by intact platelets (2). Inhibition of aggregation by pros-

Treatment of 2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl-1-carbonitrile with hydrogen selenide provided 2,5-anhydro-3,4,6-tri-O-benzoyl-D-allonselenoamide (3). Compound 3 was treated with ethyl bromopyruvate to provide ethyl 2-(2,3,5-tri-O-benzoyl-D-ribofuranosyl)selenazole-4-carboxylates, which after ammonolysis were converted to 2-beta-D-ribofuranosylselenazole-4-carboxamide (6) and its alpha-analogue 7, respectively. Acetylation of nucleoside 6 provided 2-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)selenazole-4-carboxamide, and phosphorylation of 6 provided the corresponding 5'-phosphate 9. Compounds 6 and 9 were found to be cytotoxic toward P388 and L1210 cells in culture and effective against Lewis lung carcinoma in mice.

A general reaction of glycosyl cyanides with liquid hydrogen sulfide in the presence of 4-dimethylaminopyridine to provide the corresponding glycosylthiocarboxamides is described. These glycosylthiocarboxamides were utilized as the precursors for the synthesis of 2-D-ribofuranosylthiazole-4-carboxamide and 2-beta-D-ribofuranosylthiazole-5-carboxamide (23). The structural modification of 2-beta-D-ribofuranosylthiazole-4-carboxamide (12) into 2-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)thiazole-4-carboxamide (15), 2-beta-D-ribofuranosylthiazole-4-thiocarboxamide (17), and 2-(5-deoxy-beta-D-ribofuranosyl)thiazole-4-carboxamide (19) is also described. These thiazole nucleosides were tested for in vitro activity against type 1 herpes virus, type 3 parainfluenza virus, and type 13 rhinovirus and an in vivo experiment was run against parainfluenza virus. They were also evaluated as potential inhibitors of purine nucleotide biosynthesis. It was shown that the compounds (12 and 15) which possessed the most significant antiviral activity were also active inhibitors (40-70%) of guanine nucleotide biosynthesis.