BRD4 Inhibitor-20 is a potent, orally bioactive, bromodomain protein 4 (BRD4) inhibitor. BRD4 Inhibitor-20 has inhibitory activity against BRD4 (BD1) and BRD4 (BD2) with IC50s of 19 nM and 28 nM, respectively. BRD4 Inhibitor-20 also has antiproliferation activity in cancer/tumor cell lines. BRD4 Inhibitor-20 may be utilized in research on a variety of cancers like colon cancer.

Physicochemical Properties

Molecular Formula	C18H18N2O4S
Molecular Weight	358.411523342133
Exact Mass	358.098
CAS #	2490311-14-1
PubChem CID	162653363
Appearance	Brown to reddish brown solid powder
LogP	2.8
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	4
Heavy Atom Count	25
Complexity	649
Defined Atom Stereocenter Count	0
SMILES	C1(S(NC2C=CC3=C(C=2)/C(=C(/C)\C)/C(=O)N3)(=O)=O)=CC=CC=C1OC
InChi Key	CDERLIUJSXSXFH-UHFFFAOYSA-N
InChi Code	InChI=1S/C18H18N2O4S/c1-11(2)17-13-10-12(8-9-14(13)19-18(17)21)20-25(22,23)16-7-5-4-6-15(16)24-3/h4-10,20H,1-3H3,(H,19,21)
Chemical Name	2-methoxy-N-(2-oxo-3-propan-2-ylidene-1H-indol-5-yl)benzenesulfonamide
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

ln Vitro	Excellent BRD4 and BRD2 inhibitory action (BD1, IC50=19 nM; BD2, IC50=28 nM; BD1, IC50=24 nM; BD2, IC50=18 nM) is demonstrated by BRD4 Inhibitor-20 (compound 12j) [1]. C-Myc expression is decreased by BRD4 Inhibitor-20 (0.5, 2.5, and 5.0 μM; 24 hours) [1]. In HT-29, HL-60, and WI-38 cells, BRD4 Inhibitor-20 (72 h) has anti-proliferative effectiveness with IC50 values of 4.75 μM, 1.35 μM, and 44.07 μM, respectively[1]. HT-29 cells can be stopped from progressing through the cell cycle and going into the G1 phase by BRD4 Inhibitor-20 (2.5, 5.0, 10.0 μM; 24 h) [1].
ln Vivo	Compound 12j, also known as BRD4 Inhibitor-20, has good oral pharmacokinetic properties (iv, 5 mg/kg; po, 15 mg/kg) [1].
Cell Assay	Western Blot analysis [1] Cell Types: HT-29 Cell Tested Concentrations: 0.5, 2.5, 5.0 μM Incubation Duration: 24 h Experimental Results: Significant inhibitory effect on c-Myc protein expression. Cell proliferation experiment [1] Cell Types: HT-29, HL-60, WI-38 Cell Tested Concentrations: Incubation Duration: 72 h Experimental Results: Strong anti-proliferative activity and weak toxicity. Cell cycle analysis[1] Cell Types: HT-29 Cell Line Tested Concentrations: 2.5, 5.0, 10.0 μM Incubation Duration: 24 hrs (hours) Experimental Results: Blocked cell cycle progression of cell line into G1 phase and percentage of cells in G1 phase 2.5, 5.0 and 10.0 The phases after treatment with μM concentration were 85.98%, 86.49% and 86.05% respectively.
Animal Protocol	Animal/Disease Models: Rat[1] Doses: 5 mg/kg, 15 mg/kg Route of Administration: intravenous (iv) (iv)administration (iv) or oral administration (po) Experimental Results: PK parameters iv (5 mg/kg) po (15 mg/kg) Cmax (μg/L) - 2175 Tmax(h) - 1.00 t1/2(h) 1.56 3.59 CLz/F (L/h/kg) 0.68 1.03 AUC0−t(μg/L*h) 7296 14384 AUC0−∞(μg) /L*h) 7340 14600 F (%) - 66%
References	[1]. Design, synthesis and biological evaluation of indole-2-one derivatives as potent BRD4 inhibitors. Eur J Med Chem. 2020 Dec 15;208:112780.

Solubility Data

Solubility (In Vitro)

DMSO : ~125 mg/mL (~348.76 mM)

Solubility (In Vivo)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.7901 mL	13.9505 mL	27.9010 mL
	5 mM	0.5580 mL	2.7901 mL	5.5802 mL
	10 mM	0.2790 mL	1.3951 mL	2.7901 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.