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Pyrazolo[3,4-<i>d</i>]pyrimidines represent an important class of heterocyclic compounds well-known for their anticancer activity exerted by the inhibition of eukaryotic protein kinases. Recently, pyrazolo[3,4-<i>d</i>]pyrimidines have become increasingly attractive for their potential antimicrobial properties. Here, we explored the activity of a library of in-house pyrazolo[3,4-<i>d</i>]pyrimidines, targeting human protein kinases, against <i>Staphylococcus aureus</i> and <i>Escherichia coli</i> and their interaction with ampicillin and kanamycin, representing important classes of clinically used antibiotics. Our results represent a first step towards the potential application of dual active pyrazolo[3,4-<i>d</i>]pyrimidine kinase inhibitors in the prevention and treatment of bacterial infections in cancer patients.