Synergistic Effects of Stereochemistry and Appendages on the Performance Diversity of a Collection of Synthetic Compounds

Target-
and phenotype-agnostic assessments of biological activity
have emerged as viable strategies for prioritizing scaffolds, structural
features, and synthetic pathways in screening sets, with the goal
of increasing performance diversity. Here, we describe the synthesis
of a small library of functionalized stereoisomeric azetidines and
its biological annotation by “cell painting,” a multiplexed,
high-content imaging assay capable of measuring many hundreds of compound-induced
changes in cell morphology in a quantitative and unbiased fashion.
Using this approach, we systematically compare the degrees to which
a core scaffold’s biological activity, inferred from its effects
on cell morphology, is affected by variations in stereochemistry and
appendages. We show that stereoisomerism and appendage diversification
can produce effects of similar magnitude, and that the concurrent
use of these strategies results in a broader sampling of biological
activity.