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Purity: ≥98%
GSK1324726A (I-BET726; GSK-1324726A), a tetrahydroquinoline analog, is a novel and potent ApoA1 up-regulator and selective BET Bromodomain inhibitor with anticancerand anti-inflammatory activity. It inhibits BRD2/3/4 with IC50s of 41 nM, 31 nM, and 22 nM, respectively. It has potent in vitro antiproliferative and anti-inflammatory effects which translate into in vivo efficacy in oncology and inflammation models.
Targets |
BRD4/3/2 (IC50= 22/31/41 nM)
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ln Vitro |
panel of neuroblastoma cell lines are treated with GSK1324726A (I-BET726), and reported substantial growth suppression and cytotoxicity in most cell lines irrespective of MYCN copy number or expression level. All neuroblastoma cell lines studied exhibit significant growth inhibition, with a median growth IC50 value (gIC50; inhibitor concentration resulting in 50% growth inhibition) equal to 75 nM[1].
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ln Vivo |
I-BET726 (GSK1324726A) suppresses the growth of neuroblastoma tumors. Because of the extent of the tumor, the mice in the vehicle group in the SK-N-AS model are put to death on day 14. On day 14 of the research, 58% tumor growth inhibition (TGI) is found in the GSK1324726A (15 mg/kg) group, whereas there is no significant difference in tumor growth between the vehicle and GSK1324726A (5 mg/kg) groups (n=9; p=0.006). The GSK1324726A (15 mg/kg) group of mice receive treatment for an additional 7 days until the tumor volume equals that of the vehicle group. At that time, the study is stopped. In the CHP-212 model, tumor growth is substantially slower. At the end of the trial (Day 14), 42 days later, the tumors in the mice treated with vehicle are barely half the size of those in the SK-N-AS model. Treatment with 5 mg/kg GSK1324726A in the CHP-212 model causes TGI to equal 50% (n=8; p=0.1816), while mice in the 15 mg/kg group show a TGI of 82% at study's conclusion (n=5; p=0.0488)[1].
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Enzyme Assay |
Thermal Shift Assay (Tm)[2]
Thermal shifts of bromodomains were analyzed on a FluoDiaT70 instrument. The temperature of the protein sample was ramped from 26/30 to 74 °C in the presence or absence of 100 μM of the ligand. Denaturation was visualized using 1:1000 dilution of a fluorescent Sypro orange dye in a buffer of PBS, 10% glycerol. The temperature was ramped at a rate of 1 °C/min and fluorescence readings taken every 1 °C. Bromodomains tested included ATAD2, BAZ2B, BRD2 (tandem bromodomains), BRD4 (N- and C-terminal and tandem bromodomains), CREBBP, PCAF, SMARCA2, SP140, and TAF1 (tandem bromodomains). Results reported are the mean of at least two experiments.[2] Surface Plasmon Resonance Analysis of Binding to BRD2, 3, and 4[2] BIAcore data of His6-tagged BRD2 (1–473), BRD3 (1–434), and BRD4 (1–477) was acquired and analyzed on a T100 BIAcore instrument at 25 °C. In all cases, a CM5 chip with ∼5–12kU amine coupled protein immobilized on the dextran surface was used with a running buffer of 30 mM Hepes pH 7, 150 mM NaCl, 1 mM EGTA, NaN3. Compounds were titrated as a tripling dilution starting from 10 μM. Sensorgrams and binding curves were analyzed with BIAevaluation using a 1:1 binding model. The equilibrium KD was calculated using response = concentration × Rmax/(concentration + KD) + offset. [2] Isothermal Titration Calorimetry. [2] ITC titration of BRD4 was carried out using a Microcal VPITC in 50 mM Hepes, pH 7.5, 150 mM NaCl, at 25 °C. First, 9 μM His-tagged BRD4 (1–477) tandem bromodomain was placed in the cell and 200 μM ligand was titrated into this to achieve a final ligand:protein excess of ∼4:1. The data was then fitted within Origin (Microcal version) to give the following parameters: stoichiometry of 1.93, KD of 4.4 ± 0.9 nM, ΔH −15.9 ± 0.08 kcal/mol, ΔS −15.2 cal/mol/deg. ITC titration of CREBBP was carried out using a Microcal AutoITC200 in 50 mM Hepes, pH 7.4, 150 mM NaCl, at 25 °C. First, 44 μM of CREBBP bromodomain was placed in the cell, and 500 μM ligand was titrated into this to achieve a final ligand:protein excess of ∼2.2:1. The data was then fitted within Origin (Microcal version) to give the following parameters: stoichiometry of 1.1, KD of 6.3 ± 0.5 μM, ΔH −4.35 ± 0.08 kcal/mol, ΔS 9.2 cal/mol/deg. |
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Cell Assay |
poA1 Luciferase Assay[2]
HepG2 cells were stably transfected with a plasmid encoding firefly luciferase under the control of the human ApoA1 promoter region (−1162, +232) and the 3′-UTR of the human ApoA-1 gene (+1037, +1091). ApoA1 potency was defined as the concentration of compound, resulting in a 70% increase in luciferase luminescence (ApoA1 Luc EC170). See Supporting Information, Supplementary Methods. High correlation has been shown between ApoA1-luciferase reporter activity and ApoA1 protein secretion in HepG2 cells. IL-6 PBMC and Whole Blood Assays[2] Human whole blood and peripheral blood mononuclear cells (PBMCs) purified from whole blood were incubated with LPS and varying concentrations of test compounds for 18–24 h. Samples were prepared, and inhibition of IL-6 was measured as described in Supporting Information, Supplementary Methods. |
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Animal Protocol |
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References |
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Additional Infomation |
BET family proteins are epigenetic regulators known to control expression of genes involved in cell growth and oncogenesis. Selective inhibitors of BET proteins exhibit potent anti-proliferative activity in a number of hematologic cancer models, in part through suppression of the MYC oncogene and downstream Myc-driven pathways. However, little is currently known about the activity of BET inhibitors in solid tumor models, and whether down-regulation of MYC family genes contributes to sensitivity. Here we provide evidence for potent BET inhibitor activity in neuroblastoma, a pediatric solid tumor associated with a high frequency of MYCN amplifications. We treated a panel of neuroblastoma cell lines with a novel small molecule inhibitor of BET proteins, GSK1324726A (I-BET726), and observed potent growth inhibition and cytotoxicity in most cell lines irrespective of MYCN copy number or expression level. Gene expression analyses in neuroblastoma cell lines suggest a role of BET inhibition in apoptosis, signaling, and N-Myc-driven pathways, including the direct suppression of BCL2 and MYCN. Reversal of MYCN or BCL2 suppression reduces the potency of I-BET726-induced cytotoxicity in a cell line-specific manner; however, neither factor fully accounts for I-BET726 sensitivity. Oral administration of I-BET726 to mouse xenograft models of human neuroblastoma results in tumor growth inhibition and down-regulation MYCN and BCL2 expression, suggesting a potential role for these genes in tumor growth. Taken together, our data highlight the potential of BET inhibitors as novel therapeutics for neuroblastoma, and suggest that sensitivity is driven by pleiotropic effects on cell growth and apoptotic pathways in a context-specific manner.[1]
hrough their function as epigenetic readers of the histone code, the BET family of bromodomain-containing proteins regulate expression of multiple genes of therapeutic relevance, including those involved in tumor cell growth and inflammation. BET bromodomain inhibitors have profound antiproliferative and anti-inflammatory effects which translate into efficacy in oncology and inflammation models, and the first compounds have now progressed into clinical trials. The exciting biology of the BETs has led to great interest in the discovery of novel inhibitor classes. Here we describe the identification of a novel tetrahydroquinoline series through up-regulation of apolipoprotein A1 and the optimization into potent compounds active in murine models of septic shock and neuroblastoma. At the molecular level, these effects are produced by inhibition of BET bromodomains. X-ray crystallography reveals the interactions explaining the structure-activity relationships of binding. The resulting lead molecule, I-BET726, represents a new, potent, and selective class of tetrahydroquinoline-based BET inhibitors.[2] |
Molecular Formula |
C25H23CLN2O3
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Molecular Weight |
434.91
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Exact Mass |
434.139
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Elemental Analysis |
C, 69.04; H, 5.33; Cl, 8.15; N, 6.44; O, 11.04
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CAS # |
1300031-52-0
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Related CAS # |
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PubChem CID |
52912222
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Appearance |
White to light yellow solid
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Density |
1.3±0.1 g/cm3
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Boiling Point |
707.3±60.0 °C at 760 mmHg
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Flash Point |
381.6±32.9 °C
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Vapour Pressure |
0.0±2.4 mmHg at 25°C
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Index of Refraction |
1.650
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LogP |
5.67
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Hydrogen Bond Donor Count |
2
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Hydrogen Bond Acceptor Count |
4
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Rotatable Bond Count |
4
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Heavy Atom Count |
31
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Complexity |
643
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Defined Atom Stereocenter Count |
2
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SMILES |
ClC1C([H])=C([H])C(=C([H])C=1[H])N([H])[C@@]1([H])C2C([H])=C(C3C([H])=C([H])C(C(=O)O[H])=C([H])C=3[H])C([H])=C([H])C=2N(C(C([H])([H])[H])=O)[C@@]([H])(C([H])([H])[H])C1([H])[H]
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InChi Key |
FAWSUKOIROHXAP-NPMXOYFQSA-N
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InChi Code |
InChI=1S/C25H23ClN2O3/c1-15-13-23(27-21-10-8-20(26)9-11-21)22-14-19(7-12-24(22)28(15)16(2)29)17-3-5-18(6-4-17)25(30)31/h3-12,14-15,23,27H,13H2,1-2H3,(H,30,31)/t15-,23+/m0/s1
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Chemical Name |
4-[(2S,4R)-1-Acetyl-4-[(4-chlorophenyl)amino]-2-methyl-1,2,3,4-tetrahydro-6-quinolinyl]benzoic acid
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Synonyms |
GSK1324726A, I-BET726, I-BET-726, I-BET 726, GSK1324726A (I-BET726); 4-[(2s,4r)-1-Acetyl-4-[(4-Chlorophenyl)amino]-2-Methyl-1,2,3,4-Tetrahydroquinolin-6-Yl]benzoic Acid; CHEMBL2177300; 4-[(2S,4R)-1-acetyl-4-(4-chloroanilino)-2-methyl-3,4-dihydro-2H-quinolin-6-yl]benzoic acid; GSK-1324726A, GSK 1324726A
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HS Tariff Code |
2934.99.9001
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Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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Solubility (In Vitro) |
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Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.75 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (5.75 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.  (Please use freshly prepared in vivo formulations for optimal results.) |
Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
1 mM | 2.2993 mL | 11.4966 mL | 22.9933 mL | |
5 mM | 0.4599 mL | 2.2993 mL | 4.5987 mL | |
10 mM | 0.2299 mL | 1.1497 mL | 2.2993 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.
Calculation results
Working concentration: mg/mL;
Method for preparing DMSO stock solution: mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation::Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O,mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
I-BET726: a novel selective inhibitor of BET family proteins.PLoS One. 2013; 8(8): e72967. td> |
I-BET726 treatment results in potent growth inhibition and cytotoxicity in neuroblastoma cell lines.PLoS One. 2013; 8(8): e72967. td> |
Global transcript profiling in neuroblastoma cell lines treated with I-BET726 reveals gene expression changes in apoptotic and signaling pathways.PLoS One. 2013; 8(8): e72967. td> |
Suppression of BCL2 expression by I-BET726.PLoS One. 2013; 8(8): e72967. td> |
MYCNexpression is directly regulated by BRD4 and repressed by treatment with I-BET726.PLoS One. 2013; 8(8): e72967. td> |
Analysis of I-BET726 activityin vivo.PLoS One. 2013; 8(8): e72967. td> |