To expand the variety of density functionalized compounds evaluated against severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2), we decided to prepare new acetylated and disubstituted 3-hydroxy bis-oxindoles and isoindigos coupling compounds using known protocols. The corresponding isatin derivatives were synthesized by ZrCl4/EtOH/reflux or HCl/AcOH/reflux coupling conditions using oxindole and functionalized isatins, furnishing new 3-hydroxy bis-oxindoles, which were dehydrated into new isoindigos. A total of 27 compounds bearing halogen, nitro and/or hydroxy groups on the isatin moiety at the 3-, 5- and 7-positions, were prepared, including 5 new 3-hydroxy bis-oxindoles and 3 new halogenated isoindigos prepared according to adapted procedures described in the literature. This library of nitrogen-isatin derivatives was evaluated against SARS-CoV-2 using a phenotypic screening assay. In this investigation, isatin derivatives 3d, 3e, 3h and 3i showed antiviral activity when tested at a single concentration. Compound 3e showed antiviral activity against SARS-CoV-2 in the concentration-response assay; however, it showed cellular toxicity in Vero cells. The present study identified substituted isatins as a promising new starting point for the development of anti-SARS-CoV-2 agents. SARSCoV2, SARSCoV2 SARSCoV SARS CoV , (SARS-CoV-2) 3hydroxy bisoxindoles bis oxindoles protocols ZrCl4EtOHreflux ZrClEtOHreflux ZrCl4 EtOH reflux ZrCl HClAcOHreflux HCl AcOH bisoxindoles, oxindoles, halogen andor 3, 3- 7positions, 7positions positions 7 positions, 7-positions literature nitrogenisatin nitrogen SARS-CoV- assay investigation 3d d e h i concentration concentrationresponse response however cells antiSARSCoV2 antiSARSCoV anti anti-SARS-CoV- agents (SARS-CoV-2 EtOHreflux SARS-CoV anti-SARS-CoV (SARS-CoV- (SARS-CoV

This opinion piece presents an approach to standardisation of an important aspect of Chagas disease drug discovery and development: selecting Trypanosoma cruzi strains for in vitro screening. We discuss the rationale for strain selection representing T. cruzi diversity and provide recommendations on the preferred parasite stage for drug discovery, T. cruzi discrete typing units to include in the panel of strains and the number of strains/clones for primary screens and lead compounds. We also consider experimental approaches for in vitro drug assays. The Figure illustrates the current Chagas disease drug-discovery and development landscape.