Structure-Activity Relationships in a Series of Dihydrouracil-JQ1 Conjugates: Discovery of Highly Potent BRD4 Degraders

Abstract

A detailed study of 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 substituent were used to synthesize 72 PROTACs by conjugating with JQ1 though 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 establishing SAR trends regarding 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, excess of cereblon or BRD4 ligands, as well as Western blot assay confirmed that BRD4 degradation is indeed involved into the compound’s cellular effects. The most potent representatives (2/5-Me-PDHU11) can be promising tools for biomedical research.