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| 25mg |
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Purity: ≥98%
AT7867 is a novel, highly potent, orally bioavailable and ATP-competitive inhibitor of Akt1/Akt2/Akt3 and p70S6K/PKA (protein kinase A) with potential anticancer activity. it inhibits Akt1/Akt2/Akt3 and p70S6K/PKA with IC50 values of 32 nM/17 nM/47 nM and 85 nM/20 nM, respectively.
| Targets |
Akt2 (IC50 = 17 nM); p70S6K (IC50 = 85 nM); Akt1 (IC50 = 32 nM); Akt3 (IC50 = 47 nM); PKA (IC50 = 20 nM)
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| ln Vitro |
The inhibition of AKT2 by AT7867 is shown to be ATP-competitive with a Ki of 18nM. Although AT7867 exhibits a pronounced window of selectivity against kinases from other kinase sub-families, it also exhibits potent activity against the structurally related AGC kinases p70S6K and PKA. Studies on the inhibition of in vitro growth have revealed that AT7867 inhibits proliferation in a number of human cancer cell lines. AT7867 appears to be least effective in the two prostate lines tested (IC50 values range from 10–12 M) and most potent at inhibiting proliferation in the MES-SA uterine, MDA-MB-468 and MCF-7 breast, HCT116 and HT29 colon lines (IC50 values range from 0.9–3 M)[1].
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| ln Vivo |
Following oral administration at 20 mg/kg, the elimination of AT7867 from plasma appears to be similar to that observed after i.v. administration. Following an oral dose of 20 mg/kg of AT7867, plasma levels stay above 0.5 M for at least 6 hours. The bioavailability through the oral route is calculated to be 44% under the assumption that linear pharmacokinetics occur after intravenous administration. Therefore, this model is used for in vivo pharmacodynamic (PD) biomarker studies. Athymic mice with MES-SA tumors are given doses of AT7867 (90 mg/kg p.o. or 20 mg/kg i.p.) after pharmacokinetic and tolerability studies, and the phosphorylation status of GSK3 and S6RP in tumors is tracked over time. At 2 and 6 hours after AT7867 treatment, a definite inhibition of the phosphorylation of the two markers of pathway activity is observed. Total levels of GSK3 and S6RP are significantly decreased by 24 hours[1].
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| Enzyme Assay |
AKT2, PKA, p70S6K, and CDK2/cyclinA kinase assays are all conducted using a radiometric filter binding format. Reactions for the assay are set up with the compound present. For AKT2, the AKT2 enzyme is incubated for 4 hours with 25 mM AKTide-2T peptide (HARKRERTYSFGHHA), 20 mM MOPS, pH 7.2, 5 mM EDTA, 15 mM MgCl2, 1 mM sodium orthovanadate, 1 mM DTT, 10 g/mL BSA, and 30 mM ATP (1.16 Ci/mmol). For PKA, 50 mM of the peptide (GRTGRRNSI) and the PKA enzyme are incubated for 20 minutes in a solution containing 2 mM MOPS, pH 7.2, 25 mM -glycerophosphate, 5 mM EDTA, 15 mM MgCl2, 1 mM orthovanadate, 1 mM DTT, and 40 mM ATP (0.88 Ci/mmol). For p70S6K, the enzyme and 25 M peptide substrate (AKRRRLSSLRA) are incubated for 60 minutes in 10 mM MOPS, pH 7, 0.2 mM EDTA, 1 mM MgCl2, 0.01% β-mercaptoethanol, 0.1 mg/mL BSA, 0.001% Brij-35, 0.5% glycerol, and 15μM ATP (2.3 Ci/mmol).
For CDK2, the CDK2/cyclinA enzyme and 0.12 g/ml histone H1 are incubated for 4 hours in 20 mM MOPS, pH 7.2, 25 mM β-glycerophosphate, 5 mM EDTA, 15 mM MgCl2, 1 mM sodium orthovanadate, 1 mM DTT, 0.1 mg/ml BSA and 45 μM ATP (0.78 Ci/mmol) for 4 hours. Orthophosphoric acid is used to stop assay reactions, and the stopped reaction mixture is then transferred to Millipore MAPH filter plates where it is filtered. The plates are then washed, scintillant added and radioactivity measured by scintillation counting on a Packard TopCount. IC50 values are calculated from replicate curves using GraphPad Prism software. AKT1 and 3 enzyme assays are carried out, while all other enzyme assays are performed[1].
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| Cell Assay |
Prior to AT7867 treatment, cells are grown for 24 hours in 10% FBS-supplemented medium in 96-well microplates at a density of 16,000 cells per well. The cells are supplemented for an hour with AT7867 or vehicle control. A phospho-GSK3 (serine 9) antibody is then incubated with cells overnight before being fixed with 3% paraformaldehyde, 0.25% glutaraldehyde, and 0.25% Triton-X100, washed, and blocked with 5% milk in tris-buffered saline with 0.1% Tween-20 (TBST). Following a secondary antibody addition and plate washing, DELFIA reagents are used to boost the signal. Using non-linear regression analysis and a sigmoidal dose-response (variable slope) equation, the europium counts are normalized to the protein concentration, and the IC50 value for each inhibitor is determined[1].
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| Animal Protocol |
Mice: Male athymic BALB/c mice (nu/nu) are used. BALB/c mice receive a single dose of AT7867 at 5 mg/kg intravenously (i.v.) and 20 mg/kg orally (p.o.). At each of the following time points—0.083, 0.167, 0.33, 0.67, 1, 2, 4, 6, 16 and 24 hours after an intravenous dose—as well as at 0.25, 0.5, 1, 2, 4, 6 and 24 hours after an oral dose—plasma samples are taken from duplicate animals. Mice are bled via cardiac puncture, and after all blood samples are centrifuged to obtain plasma, the plasma is frozen at -20°C until analysis. All plasma samples are prepared for bioanalysis using acetonitrile-containing internal standard protein precipitation. A standard calibration line made with AT7867 is used to quantify sample extracts, and an inhibitor-specific liquid chromatography tandem mass spectrometry (LC-MS/MS) technique is used for this. The parameters of pharmacokinetics are established.
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| References | |
| Additional Infomation |
AT7867 is a member of the class of piperidines carrying two aryl substituents (4-chlorophenyl and 4-(pyrazol-4-yl)phenyl) at position 4. It has a role as an EC 2.7.11.1 (non-specific serine/threonine protein kinase) inhibitor and an antineoplastic agent. It is a member of pyrazoles, a member of piperidines and a member of monochlorobenzenes.
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| Molecular Formula |
C20H20N3CL
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|---|---|
| Molecular Weight |
337.8459
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| Exact Mass |
337.134
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| Elemental Analysis |
C, 71.10; H, 5.97; Cl, 10.49; N, 12.44
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| CAS # |
857531-00-1
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| Related CAS # |
AT7867 dihydrochloride;1431697-86-7
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| PubChem CID |
11175137
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| Appearance |
White to off-white solid powder
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| Density |
1.2±0.1 g/cm3
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| Boiling Point |
530.2±50.0 °C at 760 mmHg
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| Flash Point |
274.5±30.1 °C
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| Vapour Pressure |
0.0±1.4 mmHg at 25°C
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| Index of Refraction |
1.616
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| LogP |
4.41
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
2
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
24
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| Complexity |
394
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC1C=CC(C2(CCNCC2)C2C=CC(C3=CNN=C3)=CC=2)=CC=1
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| InChi Key |
LZMOSYUFVYJEPY-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C20H20ClN3/c21-19-7-5-18(6-8-19)20(9-11-22-12-10-20)17-3-1-15(2-4-17)16-13-23-24-14-16/h1-8,13-14,22H,9-12H2,(H,23,24)
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| Chemical Name |
4-(4-(1H-pyrazol-4-yl)phenyl)-4-(4-chlorophenyl)piperidine
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| Synonyms |
AT7867 dihydrochloride; AT7867 hydrochloride; AT7867 HCl; AT7867; AT 7867; AT-7867
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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: ~68 mg/mL (201.3 mM)
Water: <1 mg/mL Ethanol: 5 mg/mL (14.8 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1 mg/mL (2.96 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 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: ≥ 1 mg/mL (2.96 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 10.0 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: ≥ 1 mg/mL (2.96 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: 15% Captisol, pH 9: 10mg/mL |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.9599 mL | 14.7995 mL | 29.5989 mL | |
| 5 mM | 0.5920 mL | 2.9599 mL | 5.9198 mL | |
| 10 mM | 0.2960 mL | 1.4799 mL | 2.9599 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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