Thrombocytopenia Assays (CFU-MK Assays). Compounds 1, 2, 3, I-BET151 and iBET-BD2 were screened against a colony forming assay for megakaryocyte progenitors (CFU-MK) that investigates colony formation of megakaryocyte cells over several days as an indicator of toxicity. This assay serves as a predictive model for thrombocytopenia and has been used previously on other pan-BET inhibitors such as Synthesis of Boc-1 was carried out as reported in Divakaran et al. where the characterization of this intermediate was reported. For in vivo studies, 1 was isolated as an HCl salt, rather than the previous TFA salt. This was accomplished by adding Boc-1 (100 mg, 0.16 mmol, 1 eq) to a 2-dram vial with diethyl ether (0.5 mL) and stirred at room temperature. To the reaction mixture was added 2.0 N HCl in diethyl ether (0.8 mL, 1.6 mmol, 10 eq) and stirred for 4 h at room temperature, resulting in the product as a white solid. The solid was then isolated by filtration and the HCl salt of was quantified using 1 H NMR. In a single neck 50 mL round-bottom flask fitted with a magnetic stir bar and nitrogen inlet was charged 1 (60 mg, 0.094 mmol) in DMF. To the reaction mixture was charged K2CO3 (52 mg, 0.376 mmol) followed by TBDMS protected Bromoethanol (45 mg, 0.188 mmol). The reaction was heated to 80 o C and allowed to stir overnight at that temperature under nitrogen atmosphere. Upon completion of the reaction as indicated by TLC, the reaction mixture was cooled to r.t. The reaction mixture was extracted with ethyl acetate and sequentially washed with 20% LiCl and water. The washed organic layer was dried over anhydrous Mg2SO4.The dried organic layer was evaporated in vacuo to afford the crude product which was purified by Combiflash chromatography (0-20% MeOH in DCM) to afford the pure product 1a as a colorless oil (36 mg, 38%) In a 20 mL glass vial fitted with a magnetic stir bar was charged 1a (36 mg, 0.0529 mmol) in 2M HCl in ether (5 mL). The reaction mixture was allowed to stir for 24 h at ambient temperature. Upon completion of the reaction as indicated by TLC, the reaction mixture was evaporated in vacuo to afford a white sticky solid, which was lyophilized to furnish the product 2 x2HCl as a fine white powder (26 mg, 77%) that was used without further purification. Synthesis of 3, formerly DW34, followed the same synthetic route as outlined by our previous work, where spectra were identical to the previous report. Compounds were isolated as HCl salts by resuspending in 2.0 N HCl in diethyl ether, where the product was isolated as a white solid and quantified through quantitative 1 H NMR. Additional purity of 10t synthesized here provided through QC-HPLC. H NMR (500 MHz, MeOD) δ 8.07 (s, 1H), 7.49 (s, 1H), 7.19 (d, 7.62 Hz, 1H) 7.00 (s, 1H), 6.86 (b, 2H), 5.07 (b, 1H), 3.75 (m, 5H), 3.05 (s, 6H), 2.94 (b, 1H), 2.55 (m, 4H), 2.37 (s, 6H), 2.22 (b, 2H). Due to the reported nature of diazo-containing compounds as PAINS and ZL0454's strong colorimetric profile, we decided to test ZL0454 in a target-negative assay for our AlphaScreen platform known as TruHits. This targetnegative assay screens primarily for nickel chelators, singlet oxygen quenchers, and colorimetric quenchers. ZL0454 shows an IC50 of 4.6 μM in this assay, suggesting assay interference at the concentrations needed for BRD4 activity, but at concentrations well above that of its BRD2-D2 activity displayed in Table . Further, the UV-Vis profile does not suggest a strong absorbance band, indicating ZL0454 as a singlet oxygen quencher.