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Purity: ≥98%
BX912 (BX-912) is a novel, potent and specific inhibitor of PDK-1 (phosphoinositide-dependent kinase 1) inhibitor with potential antitumor activity. In cell-free assays, it exhibits selectivity for PDK1 over PKA and PKC of 9 and 105 folds, respectively, and inhibits PDK-1 with an IC50 of 12 nM. By specifically targeting the PDK1/Akt signaling pathway, it demonstrated strong antiproliferative activity in vitro. This pathway is a promising target for anticancer medications because it is essential for the growth, survival, and angiogenesis of cancer cells.
| Targets |
PDK-1 (IC50 = 2 nM); PKA (IC50 = 110 nM); KDR (IC50 = 410 nM); CDK2/CyclinE (IC50 = 650 nM); Chk1 (IC50 = 830 nM)
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| ln Vitro |
BX912 prevents ChcK1, PKA, c-kit, and KDR with IC50 of 0.83, 0.11, 0.85, and 0.41 μM, resepectively. BX912 inhibits PDK1/Akt signaling in tumor cells and either induces apoptosis or suppresses the anchorage-dependent growth of several tumor cell lines, including PC-3 cells, in culture. The PDK1/Akt signaling pathway's role in cell survival is supported by the fact that a number of cancer cell lines (including the MDA-468 breast cancer cell line) with elevated Akt activity are >30-fold more sensitive to growth inhibition by PDK1 inhibitor BX912 in soft agar than on tissue culture plastic, which is crucial for unattached cells in particular. In a direct kinase assay format, BX912 effectively inhibits PDK1 enzyme activity, but it is unable to inhibit preactivated AKT2 activity (IC50 > 10 μM). BX-912 is therefore a PDK1 direct inhibitor. BX912 may bind to the ATP-binding pocket of PDK1 because it inhibits the enzyme's ability to use its substrate, ATP, in a competitive manner. The aminopyrimidine backbone of BX912 adopts a similar orientation in the active site of PDK1. BX912 promotes a pronounced increase in the population of MDA-468 cells with 4 N DNA content, indicative of a block at the G2/M phase of the cell cycle. BX912 also potently inhibits the growth of HCT-116 cells in soft agar, showing a 96% inhibitory effect at a dose of 1 μM. BX912 potently inhibits the growth of PC-3 cells in soft agar, displaying IC50 of 0.32 μM. [1]
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| ln Vivo |
NA
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| Enzyme Assay |
AKT2 is activated by PDK1 and PtdIns-3,4-P2 in a PDK1 and coupled assay format. For the coupled assay, the final assay mixture (60 μL) contained: 15 mM MOPS, pH 7.2, 1 mg/mL bovine serum albumin, 18 mM β-glycerol phosphate, 0.7 mM dithiothreitol, 3 mM EGTA, 10 mM MgOAc, 7.5 μM ATP, 0.2 μCi of [γ- 33P]ATP, 7.5 μM biotinylated peptide substrate (biotin-ARRRDGGGAQPFRPRAATF), 0.5 μL of PtdIns-3,4-P2-containing phospholipid vesicles, 60 pg of purified recombinant human PDK1, and 172 ng of purified recombinant human AKT2. The biotin-labeled peptide is captured from 10 L of the assay mixture on streptavidin-coated SPA beads after incubating for 2 hours at room temperature. Product formation is then measured by scintillation proximity in a Wallac MicroBeta counter. The amount of PDK1 and inactive AKT2 added, along with the time of incubation, determine the amount of product that forms. PDK1 is added at suboptimal levels so that the assay could sensitively detect inhibitors of AKT2 activation as well as direct inhibitor BX912 of PDK1 or AKT2. To measure PDK1 activity directly, the final assay mixture (60 μL) contained 50 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, 0.1 mM EDTA, 0.1% β-mercaptoethanol, 1 mg/mL bovine serum albumin, 10 mM MgOAc, 10 μM ATP, 0.2 μCi of [γ-33P]ATP, 7.5 μM substrate peptide (H2N-ARRRGVTTKTFCGT), and 60 ng of purified recombinant human PDK1. After 4 hours at room temperature, 25 mM EDTA is added, and some of the reaction mixture is spotted on P81 phosphocellulose paper. Three times in 0.75% phosphoric acid and once in acetone are used to wash the filter paper. A phosphorimager is used to measure the labeled peptide that is bound to the filter after drying.
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| Cell Assay |
Cells such as MDA-468, MDA-453 are seeded at a low density (1.5-3 × 103 cells/well, 0.1 mL/well, 96-well plates) and are incubated overnight. BX912 treatments are made by adding 10 μL/well of the compound in 1% dimethyl sulfoxide and growth medium (final concentration of dimethyl sulfoxide, 0.1%), followed by brief shaking. The metabolic dye WST-1 is added to treated cells after 72 hours of incubation to determine viability. The net signal is calculated by subtracting a no cell, or zero time cell, background from the WST-1 signal, which is read at 450 nm on a plate reader.
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| Animal Protocol |
NA;
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| References | |
| Additional Infomation |
N-[3-[[5-bromo-4-[2-(1H-imidazol-5-yl)ethylamino]-2-pyrimidinyl]amino]phenyl]-1-pyrrolidinecarboxamide is a member of ureas.
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| Molecular Formula |
C20H23BRN8O
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|---|---|
| Molecular Weight |
471.35
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| Exact Mass |
470.117
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| Elemental Analysis |
C, 50.96; H, 4.92; Br, 16.95; N, 23.77; O, 3.39
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| CAS # |
702674-56-4
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| Related CAS # |
702674-56-4
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| PubChem CID |
11754511
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| Appearance |
White solid powder
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| Density |
1.6±0.1 g/cm3
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| Index of Refraction |
1.750
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| LogP |
1.38
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
30
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| Complexity |
549
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| Defined Atom Stereocenter Count |
0
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| SMILES |
BrC1=C(NCCC2=CNC=N2)N=C(NC3=CC(NC(N4CCCC4)=O)=CC=C3)N=C1
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| InChi Key |
DMMILYKXNCVKOJ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C20H23BrN8O/c21-17-12-24-19(28-18(17)23-7-6-16-11-22-13-25-16)26-14-4-3-5-15(10-14)27-20(30)29-8-1-2-9-29/h3-5,10-13H,1-2,6-9H2,(H,22,25)(H,27,30)(H2,23,24,26,28)
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| Chemical Name |
N-(3-((4-((2-(1H-imidazol-4-yl)ethyl)amino)-5-bromopyrimidin-2-yl)amino)phenyl)pyrrolidine-1-carboxamide
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| Synonyms |
BX 912; BX912; BX912
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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 |
| 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) |
DMSO: ~94 mg/mL (~199.4 mM)
Water: <1 mg/mL Ethanol: N/A |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.75 mg/mL (5.83 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 27.5 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: ≥ 2.75 mg/mL (5.83 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 27.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. 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. View More
Solubility in Formulation 3: ≥ 2.75 mg/mL (5.83 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 30% PEG400+0.5% Tween80+5% propylene glycol: 30mg/mL |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1216 mL | 10.6078 mL | 21.2157 mL | |
| 5 mM | 0.4243 mL | 2.1216 mL | 4.2431 mL | |
| 10 mM | 0.2122 mL | 1.0608 mL | 2.1216 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.
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