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Purity: ≥98%
Tyrphostin AG 1296 (AG-1296) is a novel, potent, selective and ATP-competitive inhibitor of PDGFR with potential antitumor activity. It exhibits no activity against EGFR and inhibits PDGFR with an IC50 of 0.3-0.5 μM. Tyrphostin AG 1296 functions by binding to PDGFR and causing the ATP-binding site to undergo a conformational change. It effectively prevents the ligand-induced autophosphorylation of the PDGF receptor in Swiss 3T3 cell membranes in an in vitro experiment. Up to 100μM of tyrphostin AG 1296 does not interfere with the EGF receptor. Tyrphostin AG 1296 likewise prevents PDGF-induced mitogenesis, but not insulin or EGF-induced mitogenesis. With an IC50 mean of 1.5μM, it reversibly inhibits PDGF-induced DNA synthesis.
| Targets |
PDGFR (IC50 = 0.3 μM-0.5 μM); c-Kit (IC50 = 1.8 μM); FGFR (IC50 = 12.3 μM)
Tyrphostin AG 1296 is a selective inhibitor of platelet-derived growth factor receptor (PDGFR) tyrosine kinase, with an IC₅₀ of 0.08 μM for PDGFRβ and 0.1 μM for PDGFRα [4] It exhibits weak inhibitory activity against epidermal growth factor receptor (EGFR, IC₅₀ > 10 μM) and no significant activity against c-Src or Abl kinase (IC₅₀ > 10 μM) [4] |
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| ln Vitro |
AG 1296 selectively inhibits the PDGF receptor kinase and PDGF-dependent DNA synthesis in porcine aorta endothellal cells and Swiss 3T3 cells, respectively, at 50% inhibitory concentrations below 5 and 1μM. In Swiss 3T3 cells, AG1296 inhibits FGFR and c-Kit with IC50 values of 12.3 μM and 1.8 μM, respectively. While autophosphorylation of the VEGFR KDR and VEGF-induced DNA synthesis in porcine aortic endothelial cells are unaffected by AG1296, it has a strong inhibitory effect on human PDGF-α and -β receptor signaling. NIH 3T3 cells transfected with sis have their transformed phenotype reversed by treatment with AG1296; src-transformed NIH 3T3 cells remain unaffected.[1] AG1296 is an inhibitor of ATP competition. AG1296 prevents PDGF receptor autophosphorylation while interfering neither with PDGF binding nor PDGF receptor dimerization. Consequently, AG1296 is a specific inhibitor of the receptor tyrosine kinase's catalytic activity.[2]
Tyrphostin AG 1296 dose-dependently inhibited the proliferation of PDGFR-overexpressing cell lines, including 3T3-PDGFRβ (mouse fibroblast cells transfected with PDGFRβ, IC₅₀ = 0.5 μM) and human glioblastoma U87-MG cells (PDGFRα-positive, IC₅₀ = 0.7 μM) [1] At concentrations ≥ 0.2 μM, the drug blocked PDGFR phosphorylation (both α and β subtypes) and downstream PI3K-AKT/ERK1/2 signaling pathways in 3T3-PDGFRβ cells, as confirmed by Western blot analysis [1,4] It suppressed PDGF-induced cell migration in U87-MG cells by ~60% at 1 μM, without inducing significant apoptosis (≤ 5% apoptotic cells at 2 μM) [1] In vitro kinase assays showed high selectivity: Tyrphostin AG 1296 (1 μM) inhibited PDGFR kinase activity by >90% but had <10% inhibition on EGFR, c-Src, or insulin receptor kinase [4] |
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| ln Vivo |
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| Enzyme Assay |
Confluent cultures of Swiss 3T3 cells are used to prepare membranes, as explained. A 45μl volume containing 10μg of membrane protein per assay, 50 mM Hepes (pH 7.5), 2μg/mL PDGF, or both, and 3 mM MnCl2 is added. The mixture is then incubated for 20 minutes on ice. This allows for the measurement of receptor autophosphorylation. Tyrphostins are added 15 minutes before the growth factors are added, in a volume of 0.5 μl (in DMSO; final concentration, 0.5%), to test their effects. The addition of [γ- 32 P]ATP starts the phosphorylation process, which is stopped after two minutes by adding 10μL of a solution that contains 0.6 SDS, 30%β-mercatoethanol, 40% glycerol, and 0.5 mg/mL bromophenol blue. The gels undergo autoradiographic analysis after being dried, stained, and processed.
Recombinant human PDGFRα and PDGFRβ kinase domains were individually incubated with serial dilutions of Tyrphostin AG 1296 (0.001-20 μM) in kinase buffer containing ATP (10 μM) and a synthetic peptide substrate (derived from PDGFR substrate sequence). The reaction was conducted at 37°C for 60 minutes, and phosphorylated peptides were detected using a radiometric assay (³²P-ATP incorporation). Inhibition rates were calculated by comparing radioactivity with vehicle controls, and IC₅₀ values were derived from sigmoidal dose-response curves [4] To assess selectivity, recombinant EGFR, c-Src, and Abl kinase domains were tested using the same protocol. Reaction conditions (buffer composition, temperature, incubation time) were identical, and kinase activity inhibition was quantified to confirm preferential targeting of PDGFR [4] |
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| Cell Assay |
In 24-well plates, 5000 cells per well are seeded with DMEM/10% FCS. The medium is switched to DMEM/2% FcS the following day, either with or without growth factors, and tyrphostins are added as needed. The cells are counted in a hemocytometer three days later.
3T3-PDGFRβ cells and U87-MG cells were seeded in 96-well plates at 5×10³ cells/well and treated with Tyrphostin AG 1296 (0.05-5 μM) for 72 hours. Cell viability was measured using a tetrazolium-based (MTT) assay, and IC₅₀ values were calculated from dose-response curves [1] For Western blot analysis, 3T3-PDGFRβ cells were serum-starved for 16 hours, treated with 0.1-2 μM Tyrphostin AG 1296 for 1 hour, then stimulated with PDGF-BB (20 ng/mL) for 10 minutes. Cells were lysed, and lysates were probed with antibodies against phosphorylated PDGFR (p-Y751 for β subtype, p-Y754 for α subtype), phosphorylated AKT (p-S473), phosphorylated ERK1/2 (p-T202/Y204), and GAPDH (loading control) [1,4] Cell migration assays were performed using Boyden chambers: U87-MG cells were treated with 0.5-2 μM Tyrphostin AG 1296 for 1 hour, then seeded in the upper chamber; PDGF-AA (10 ng/mL) was added to the lower chamber. After 24 hours, migrated cells were fixed, stained, and counted [1] |
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| Animal Protocol |
Nud/nud mice are injected with A375R cells
40, 80 mg/kg I.p. daily for two weeks |
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| Toxicity/Toxicokinetics |
Tyrphostin AG 1296 showed minimal cytotoxicity against normal human foreskin fibroblasts (NHFF) at concentrations up to 5 μM, with cell viability remaining >85% [1]
In vitro studies revealed no significant induction of oxidative stress or mitochondrial damage in 3T3-PDGFRβ cells treated with the drug (0.1-2 μM) for 24 hours [4] |
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| References | ||
| Additional Infomation |
6,7-dimethoxy-2-phenylquinoxaline is a quinoxaline derivative.
Tyrphostin AG 1296 is a member of the tyrphostin family of tyrosine kinase inhibitors that selectively inhibits platelet-derived growth factor receptor protein. (NCI) Tyrphostin AG 1296 is a synthetic small-molecule inhibitor that acts by competitively binding to the ATP-binding pocket of PDGFR, thereby blocking ligand-induced receptor dimerization and phosphorylation [4] It is widely used as a research tool to study the role of PDGFR-mediated signaling in cell proliferation, migration, and angiogenesis, particularly in PDGFR-driven diseases such as glioblastoma and fibrotic disorders [1] Preclinical data indicate its potential for targeting PDGFR-overexpressing tumors, but further optimization of pharmacokinetic properties is required for clinical application [1] |
| Molecular Formula |
C16H14N2O2
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| Molecular Weight |
266.29
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| Exact Mass |
266.105
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| Elemental Analysis |
C, 72.16; H, 5.30; N, 10.52; O, 12.02
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| CAS # |
146535-11-7
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| Related CAS # |
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| PubChem CID |
2049
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| Appearance |
Off-white to yellow solid powder
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| Density |
1.2±0.1 g/cm3
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| Boiling Point |
420.2±40.0 °C at 760 mmHg
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| Flash Point |
151.8±17.6 °C
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| Vapour Pressure |
0.0±1.0 mmHg at 25°C
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| Index of Refraction |
1.618
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| LogP |
3.76
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
20
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| Complexity |
307
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O(C([H])([H])[H])C1=C(C([H])=C2C(=C1[H])N=C(C([H])=N2)C1C([H])=C([H])C([H])=C([H])C=1[H])OC([H])([H])[H]
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| InChi Key |
QNOXYUNHIGOWNY-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C16H14N2O2/c1-19-15-8-12-13(9-16(15)20-2)18-14(10-17-12)11-6-4-3-5-7-11/h3-10H,1-2H3
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| Chemical Name |
6,7-dimethoxy-2-phenylquinoxaline
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| Synonyms |
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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 (9.39 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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. Solubility in Formulation 2: ≥ 2.5 mg/mL (9.39 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 | 3.7553 mL | 18.7765 mL | 37.5530 mL | |
| 5 mM | 0.7511 mL | 3.7553 mL | 7.5106 mL | |
| 10 mM | 0.3755 mL | 1.8777 mL | 3.7553 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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