Compounds 1 and 2 were both shown to be selective for BRD4-D1, however, 1 is significantly limited by observed toxicity in TSECs at higher concentrations. Compounds 2 and 3, in contrast, displayed improved cytotoxicity profiles, comparable to those of I-BET151 and iBETBD2. Compound 3 was also able to produce a considerable reduction in the expression of CXCL1 and CCL2 in cellular models of LPS-induced inflammatory signaling. Interestingly, treatment with iBETBD2 was not as effective at limiting the expression of inflammatory markers as I-BET151 compared to other inflammation models. Compounds 1 and 2 showed lesser reduction in chemokine expression at micromolar concentrations. The decreased antiinflammatory activity here can potentially be attributed to a cell permeability issue, as supported by CETSA NanoBRET, and Caco2 permeability assays. In this case, the modulation of cytokine expression and cellular target engagement occur at similar concentrations. The development of more permeable BRD4-D1 selective compounds will be necessary to more thoroughly evaluate the cellular effects of BRD4-D1 inhibition. In addition, while pharmacokinetic analysis showed sufficient liver exposure for all three experimental compounds 1-3, future designs will seek to improve the metabolic stability of 3.