Identification of 3,5-Dimethylisoxazole Derivatives as BRD4 Inhibitors for the Treatment of Colorectal Cancer.

Bromodomain-containing protein 4 (Brd4) plays a critical regulatory role in gene transcription that has been recently recognized as a promising strategy for cancer therapy. Based on the BRD4 protein containing two tandem bromodomain structures, BD1 and BD2, we designed and synthesized a series of 3,5-dimethylisoxazole derivative dimers targeting both bromodomains simultaneously to enhance protein binding potency. Among them, compound significantly inhibited the proliferation of colorectal cancer cells HCT116 (IC = 162 nM), with a 20-fold increase in antiproliferative activity compared to inhibitor . The results of WesternBlot showed that compound could down-regulate c-MYC protein levels and up-regulate HEXIM1 expression and modulate apoptosis through intrinsic pathways. In addition, compound exhibited outstanding antitumor efficacy in the CT-26 tumor mouse model with a tumor suppression rate of 56.1%. Taken together, 3,5-dimethylisoxazole derivative dimer has remarkable protein inhibitory effect and antitumor activity in vitro and in vivo. A protein binding model of compound is being further analyzed, which will facilitate the development of bivalent BRD4 inhibitors and probe the biological function of BRD4.