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 22 significantly inhibited the proliferation of colorectal cancer cells HCT116 (IC₅₀ = 162 nM), with a 20-fold increase in antiproliferative activity compared to inhibitor 14. The results of WesternBlot showed that compound 22 could down-regulate c-MYC protein levels and up-regulate HEXIM1 expression and modulate apoptosis through intrinsic pathways. In addition, compound 22 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 22 has remarkable protein inhibitory effect and antitumor activity in vitro and in vivo. A protein binding model of compound 22 is being further analyzed, which will facilitate the development of bivalent BRD4 inhibitors and probe the biological function of BRD4.

中文翻译：

---

鉴定 3,5-二甲基异恶唑衍生物作为治疗结直肠癌的 BRD4 抑制剂

含溴结构域的蛋白质 4 (Brd4) 在基因转录中起着关键的调节作用，最近被认为是一种有前途的癌症治疗策略。基于包含两个串联溴结构域结构的 BRD4 蛋白，BD1 和 BD2，我们设计并合成了一系列同时靶向两个溴结构域的 3,5-二甲基异恶唑衍生物二聚体，以增强蛋白质结合效力。其中，化合物22显着抑制结直肠癌细胞HCT116的增殖（IC ₅₀ = 162 nM），与抑制剂14相比，抗增殖活性提高了20倍。WesternBlot 结果表明，化合物22可以下调 c-MYC 蛋白水平并上调 HEXIM1 表达并通过内在途径调节细胞凋亡。此外，化合物22在CT-26肿瘤小鼠模型中表现出优异的抗肿瘤功效，肿瘤抑制率为56.1%。综上所述，3,5-二甲基异恶唑衍生物二聚体22在体内外均具有显着的蛋白质抑制作用和抗肿瘤活性。正在进一步分析化合物22的蛋白质结合模型，这将促进二价BRD4抑制剂的开发并探索BRD4的生物学功能。