In a screening program using-the agar diffusion-plaque inhibition test, 22 out of 27 substituted aminopyrazoles tested showed marked antiviral activity against mengovirus in FL cell cultures. The efficacy of the different derivatives was compared by determining their effect on the yield of infectious virus after a one-step growth cycle. The antiviral activity against mengovirus correlated with the chain length of the substituents and increased or decreased according to the position of the side chain.Our studies are concerned with pyrazolo- [3,4-d]pyrimidines, which were synthesized to be possible purine antagonists and, in this context, to exhibit antiviral activity. In the course of these studies we found that even the 5-amino-4-cyanopyrazoles (Fig. 1I) 49/20, 1974). So far little is known about the antiviral activity of pyrazolo-[3,4-dipyrimidines. Roby et al. (3) tested 1-H pyrazolo-[3,4-d]pyrimidine on myxoma and fibroma viruses in vitro but did not find antiviral activity. Antiviral effects of substituted 5-amino-4-cyanopyrazoles of structure I (Fig. 1) have not been described yet. However, this class of compounds bears a slight resemblance to Ribavirin (Virazole) (Fig. 1II), an agent recently reported to exhibit broad-spectrum antiviral activity (8), as well as to the antiviral antibiotic Pyrazomycin ( Fig. 1III) , 158th, 1969, abstr. 38). Furthermore, there are structural relationships among compounds with structure I and the cytostatic agents 5-amino-13-D-ribofuranosyl-pyrazole-4-carboxamidine (Fig. lIV) (1) and 5-amino-l,3-D-ribofuranosyl-1,2,3-triazole-4-carboxamidine ( Fig. 1V) (1).The aim of the present study was to elucidate structure-activity relationships by variation of structure I substituents R,, R2, and R%, using the model mengovirus-FL cells in vitro.
MATERIALS AND METHODSCell cultures. FL cells, media, and the cultivation of the cells were previously described (5).Virus. A plaque-purified strain of mengovirus selected to grow on FL cells was used (5). Screening method. The agar diffusion-plaque inhibition test described by Tonew and Tonew (4, 5) was used. A cell monolayer (4 x 106 to 5 x 106f cells) was infected with a concentration of approximately 200 plaque-forming units per bottle of virus, unadsorbed virus was removed, and the cell sheet was overlaid with agar. Standard sized paper disks (Schleicher and Schull [Dassel/Einbeck, FRG]; diameter, 6 mm) containing test compounds were placed on the agar. After 2 days of incubation at 37 C and staining with neutral red, plaque-free areas were recorded.The therapeutic index was estimated by using different concentrations (50 mM and lower) of compounds and determining the quotients of the maximally tolerated and the minimally active concentrations.Determination of effect of test substances on infectious virus yield. Cultures of FL cells (tubes with 4 x 105 cells) were infected with 20 mean tissue culture infective doses of mengovirus per cell. After 40 min of adsorption at 37 C, the residual unadsorbed virus was removed, cells were washed th...
