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Among 46 novel pyrimido [5.4-d] pyrimidine derivatives, 26 compounds were found to exhibit antiviral activity as revealed in a test programme against Mengo, Coxsackie B1, fowl plague, vaccinia and pseudorabies viruses, as concerns inhibition of plaque formation and of infectious virus yield. Attempts to disclose structure-activity relationships by discriminant analysis pointed to a possible importance of hydrophobic substitution for the antiviral effectiveness against Mengo virus of the derivatives investigated.