Miniaturized Synthesis of Structurally Diverse Heterocyclic Libraries for Direct-to-Biology Antiparasitic Screening

Miniaturized reactions in 96-well plates can accelerate hit optimization¹ only if crude-screening data reliably reflect the activity of purified compounds. Here, we evaluated the performance of Direct-to-Biology (D2B) approaches by generating structurally diverse heterocyclic libraries and systematically comparing biological profiles obtained from crude reaction mixtures with those of resynthesized analogues. Libraries containing nitrogen-rich scaffolds were prepared and characterized by LC–MS and semi-quantitative ¹H NMR before phenotypic evaluation against Trypanosoma and Leishmania parasites. Active and inactive mixtures were subsequently resynthesized, purified, and biologically validated. Most prioritized compounds retained their antiparasitic activity after purification, demonstrating that D2B screening accurately identifies genuine hits and supports early SAR analysis. False-positive responses were restricted to a small subset of samples associated with residual reactive species. These findings establish miniaturized synthesis as a robust strategy for expanding antiparasitic chemical space while improving the efficiency and reliability of early-stage medicinal chemistry campaigns.