Structure–activity relationships in a series of dihydrouracil–JQ1 conjugates: discovery of highly potent BRD4 degraders

Abstract

A detailed study of the structure–activity relationship (SAR) in a series of phenyl dihydrouracil (PDHU) derivatives as promising chemotypes for protein degrader discovery is reported. Evaluation of cereblon binding for several dozen PDHU-derived building blocks showed that over 20 compounds with various substituents at the phenyl ring showed over 50% displacement of labeled thalidomide at 10 μM. Most potent representatives bearing methyl substituents were used to synthesize 72 PROTACs by conjugating with JQ1 through a series of linear linkers with or without additional heteroatoms. A series of assays (including binary and ternary complex formation, as well as cell-based BRD4 degradation) allowed the establishment of SAR trends related to the linker nature and length. Compounds with (CH₂)₅, (CH₂)₆, and especially (CH₂)₁₁ linkers demonstrated the best results in both BRD4 degradation (DC₅₀ = 16–52 nM) and cell viability assays, outperforming dBET1, a well-known thalidomide-based protein degrader, in the HepG2 cell line. Mechanistic experiments performed in the presence of a proteasome inhibitor, a NEDD8-activating enzyme inhibitor, and excess of cereblon or BRD4 ligands, as well as Western blot assays, confirmed that BRD4 degradation is indeed involved in the compound's cellular effects. The most potent representatives (2/5-Me-PDHU11) can be promising tools for biomedical research.