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Purity: ≥98%
AST487 (also known as NVP- AST487) is a novel and potent inhibitor of RET kinase with anticancer activity. Medullary thyroid cancers (MTC) and a subset of papillary thyroid cancers have shown interest in RET kinase as a potential target for treatment. With an IC(50) of 0.88 mumol/L on RET kinase, NVP-AST487, an N,N'-diphenyl urea, suppressed RET autophosphorylation and downstream effector activation. It also effectively stopped the growth of human thyroid cancer cell lines harboring activating RET mutations, but not of lines lacking such mutations. For xenografts of NIH3T3 cells expressing oncogenic RET and the MTC cell line TT, NVP-AST487 caused a dose-dependent growth inhibition in nude mice.
| Targets |
MET H1094Y (IC50 = 0.22 nM); MET Y1235D (IC50 = 1.7 nM); WT MET (IC50 = 4.2 nM); MET M1250T (IC50 = 6.5 nM); TRKA/NTRK1 (IC50 = 39 nM)
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| ln Vitro |
AST 487 (NVP-AST487) also exhibits similar inhibitory effects on several other kinases in the in vitro kinase assays, such as c-Kit (IC50=500 nM), c-Abl (IC50=20 nM), Flt-4 (IC50=790 nM), Flt-3 (IC50=520 nM), and KDR (IC50=170 nM). When human thyroid cancer cell lines with activating RET mutations are exposed to AST 487, it significantly slows down their growth, but not when the lines do not have similar mutations. By coincubating MTC-M cells with 100 nM of AST 487, both GDNF/GFRα1 and persephin-induced calcitonin mRNA are significantly inhibited[1]. A novel mutant inhibitor of FLT3 is AST 487. Flt-3 kinase activity inhibition is tested by AST 487 using biochemical assays. It is found that the Ki is 0.12 μM. In addition to Flt-3, NVP-AST487 has IC50 values less than 1 μM and inhibits RET, KDR, c-Kit, and c-Abl kinase. AST 487 treatment significantly reduces cell division in FLT3-ITD-Ba/F3 and D835Y-Ba/F3 cells (IC50<5 nM). When FLT3-ITD-Ba/F3 cells are treated with 0.01 μM AST 487, all cells are killed, whereas AML patient samples treated at the same concentration only kill about 50% of the cells[2].
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| ln Vivo |
Following a single oral dosage of 15 mg/kg of AST 487, OF1 mice reach a mean peak plasma level (Cmax) in 0.5 hours at 0.505±0.078 μM SE. Mice's plasma contains similar amounts of AST 487 up to 6 hours after oral administration, with a Clast of 21±4 nM at 24 hours. With a t1/2 terminal elimination of 1.5 h, the oral bioavailability is estimated to be 9.7%[1].
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| Enzyme Assay |
The kinase domains fused with glutathione S-transferase (GST) are purified using glutathione-sepharose after being expressed in baculovirus. Phosphorylation of a synthetic substrate [poly(Glu, Tyr)] by purified GST-fusion kinase domains of the corresponding protein kinase in the presence of radiolabeled ATP is how kinase activity is measured; ATP concentrations are optimized within the Km range for each specific kinase. In summary, every kinase is cultured in 20 mM of Tris-HCl (pH 7.5), 1 to 3 mM of MnCl2, 3 to 10 mM of MgCl2, 10 μM of Na3VO4, 1 mM of DTT, 0.2 μCi [33P]ATP, 1 to 8 μM of ATP, 3 to 8 μg/mL of poly(Glu/Tyr, 4:1), and 1% DMSO in a total volume of 30 μL, either in the presence or absence of NVP-AST487 for 10 minutes at room temperature. The reaction mixture is placed onto an Immobilon polyvinylidene difluoride membrane after 10 μL of 250 mM EDTA is added to stop the reaction. In a liquid scintillation counter, filters are counted after being cleaned (0.5% H3PO4), soaked in ethanol, and dried. A linear regression analysis of the percentage inhibition is used to determine the IC50s for AST 487.
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| Cell Assay |
The trypan blue exclusion assay is used to assess how many cells proliferate when NVP-AST 487 is present or absent during culture. Cell viability is expressed as a percentage of control (untreated) cells. Unless otherwise noted, data are the mean of two independent experiments. Each data point's standard error of the mean is shown by an error bar. The Annexin-V-Fluos Staining Kit is used to quantify the amount of apoptosis in drug-treated cells. Analysis of the cell cycle is done.
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| Animal Protocol |
Mice: Female athymic nude mice are housed in Makrolon type III cages with unrestricted access to food and water, and ideal hygienic conditions (maximum of 10 mice per cage). NIH3T3-RETC634W and TT cells, at 1×106 and 5×106, respectively, are injected subcutaneously into 100 μL of HBSS per mouse to form tumors. Tumors that could be treated, defined as having a mean tumor volume of 100 mm3, appeared 10 days after the injection of NIH3T3-RETC634W cells and 20 days after the injection of TT cells. NVP-AST487 is administered orally once a day via gavage. The right amount of powder is dissolved in N-methylpyrrolidone/PEG300 (1:10 v/v) to create the compound. The four treatment groups, each with eight mice, are randomly assigned to the mice. For three weeks, NVP-AST487 was given orally to the first three groups at 50, 30, and 10 mg/kg, respectively. A car was used to treat the fourth group. Body weights and tumor growth are recorded twice a week. The formula for calculating tumor volumes is length×diameter2×π/6. At the conclusion of the efficacy study, six hours after the last administration, tumors are gathered and frozen in liquid nitrogen.
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| References |
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| Molecular Formula |
C26H30F3N7O2
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|---|---|
| Molecular Weight |
529.557315349579
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| Exact Mass |
529.241
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| Elemental Analysis |
C, 58.97; H, 5.71; F, 10.76; N, 18.51; O, 6.04
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| CAS # |
630124-46-8
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| Related CAS # |
630124-46-8
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| PubChem CID |
11409972
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| Appearance |
White to off-white solid powder
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| LogP |
5.205
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
10
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
38
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| Complexity |
730
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| Defined Atom Stereocenter Count |
0
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| SMILES |
FC(C1C=C(C=CC=1CN1CCN(CC)CC1)NC(NC1C=CC(=CC=1)OC1C=C(N=CN=1)NC)=O)(F)F
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| InChi Key |
ODPGGGTTYSGTGO-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C26H30F3N7O2/c1-3-35-10-12-36(13-11-35)16-18-4-5-20(14-22(18)26(27,28)29)34-25(37)33-19-6-8-21(9-7-19)38-24-15-23(30-2)31-17-32-24/h4-9,14-15,17H,3,10-13,16H2,1-2H3,(H,30,31,32)(H2,33,34,37)
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| Chemical Name |
1-[4-[(4-ethylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl]-3-[4-[6-(methylamino)pyrimidin-4-yl]oxyphenyl]urea
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| Synonyms |
AST 487; NVP AST 487; NVP-AST487; AST487; AST-487; NVP-AST-487; NVP-AST 487; NVP AST-487; NVP AST487
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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: ~11 mg/mL (~20 mM)
Ethanol: ˂1 mg/mL Water: ˂1 mg/mL |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.72 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 (4.72 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 25.0 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: ≥ 2.5 mg/mL (4.72 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.8884 mL | 9.4418 mL | 18.8836 mL | |
| 5 mM | 0.3777 mL | 1.8884 mL | 3.7767 mL | |
| 10 mM | 0.1888 mL | 0.9442 mL | 1.8884 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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