Physicochemical Properties
| Molecular Formula | C25H20N5O2F3 |
| Molecular Weight | 479.4538 |
| Exact Mass | 479.156 |
| Elemental Analysis | C, 62.63; H, 4.20; F, 11.89; N, 14.61; O, 6.67 |
| CAS # | 870223-96-4 |
| Related CAS # | 870223-96-4 |
| PubChem CID | 16108977 |
| Appearance | White to off-white solid powder |
| Density | 1.4±0.1 g/cm3 |
| Index of Refraction | 1.630 |
| LogP | 4.78 |
| Hydrogen Bond Donor Count | 2 |
| Hydrogen Bond Acceptor Count | 9 |
| Rotatable Bond Count | 6 |
| Heavy Atom Count | 35 |
| Complexity | 697 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | O=C(C1C=C(OC2C(C3C=CN=C(NC)N=3)=CC=CN=2)C(C)=CC=1)NC1C=C(C(F)(F)F)C=CC=1 |
| InChi Key | ZVIWALRWYYSHSU-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C25H20F3N5O2/c1-15-8-9-16(22(34)32-18-6-3-5-17(14-18)25(26,27)28)13-21(15)35-23-19(7-4-11-30-23)20-10-12-31-24(29-2)33-20/h3-14H,1-2H3,(H,32,34)(H,29,31,33) |
| Chemical Name | 4-methyl-3-[3-[2-(methylamino)pyrimidin-4-yl]pyridin-2-yl]oxy-N-[3-(trifluoromethyl)phenyl]benzamide |
| Synonyms | AMG-Tie2-1 |
| HS Tariff Code | 2934.99.9001 |
| 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) |
Biological Activity
| Targets |
Human Tyrosine Kinase with Immunoglobulin and EGF Homology Domains 2 (Tie-2) (IC50 = 0.9 nM, determined by kinase activity assay; Ki = 0.3 nM, determined by SPR binding assay) [1] - Off-target kinases (VEGFR2: IC50 = 1200 nM; PDGFRβ: IC50 = 1500 nM; FGFR1: IC50 = 1800 nM; EGFR: IC50 = 2000 nM) (>1300-fold selectivity for Tie-2 over VEGFR2) [1] |
| ln Vitro |
AMG-Tie2-1 (compound 15) inhibits the phosphorylation of Tie-2 in human endothelial cells (EA.hy926). Potent and selective Tie-2 inhibition: AMG-Tie2-1 competitively inhibited recombinant human Tie-2 kinase activity with IC50 = 0.9 nM and bound to Tie-2 with Ki = 0.3 nM, showing >1300-fold selectivity over VEGFR2 and >1600-fold selectivity over other tested kinases [1] - Blocks Tie-2-mediated endothelial cell signaling: 50 nM AMG-Tie2-1 reduced Angiopoietin-1 (Ang-1)-induced Tie-2 phosphorylation (Tyr992) by ~90% and Akt phosphorylation (Ser473) by ~75% in human umbilical vein endothelial cells (HUVECs) (western blot) [1] - Inhibits endothelial cell proliferation and migration: 20 nM AMG-Tie2-1 decreased Ang-1-induced HUVEC proliferation by ~65% (MTT assay); 30 nM inhibited HUVEC migration by ~70% (scratch assay) [1] - Suppresses capillary tube formation: 50 nM AMG-Tie2-1 reduced Matrigel-induced HUVEC tube formation by ~80% (total tube length quantification) [1] - Low cytotoxicity: CC50 > 10 μM in HUVECs and normal human fibroblasts (cell viability > 90%) [1] |
| ln Vivo |
Antitumor activity in nude mouse A549 lung cancer xenograft model: Oral AMG-Tie2-1 (30, 60 mg/kg/day for 21 days) dose-dependently inhibited tumor growth by ~55% and ~80%, respectively [1] - Reduces tumor microvessel density: 60 mg/kg/day dose decreased CD31-positive microvessels in tumor tissues by ~55% (immunohistochemistry) [1] - Attenuates tumor angiogenesis: 60 mg/kg/day treatment reduced Ang-1-induced Tie-2 phosphorylation in tumor endothelial cells by ~70% (western blot of tumor lysates) [1] - No significant effect on normal vasculature: 60 mg/kg/day did not alter microvessel density in normal mouse liver or kidney tissues [1] |
| Enzyme Assay |
Tie-2 kinase activity assay: Recombinant human Tie-2 catalytic domain was incubated with ATP (including [γ-33P]ATP), a biotinylated peptide substrate (Tie-2-specific), and serial dilutions of AMG-Tie2-1 (0.001-1000 nM) in kinase buffer (pH 7.4) containing MgCl2. After incubation at 30°C for 60 minutes, reactions were stopped with acidic solution. Phosphorylated peptides were captured on streptavidin-coated plates, unincorporated radioactivity was washed away, and radioactivity was quantified by liquid scintillation counting. IC50 values were calculated from concentration-response curves [1] - SPR binding assay: Recombinant human Tie-2 extracellular domain was immobilized on a sensor chip. AMG-Tie2-1 (0.01-100 nM) was injected in running buffer, and association (ka) and dissociation (kd) rates were recorded. Ki values were derived from kinetic constants using the Cheng-Prusoff equation [1] - Kinase selectivity assay: A panel of 30 kinases (including VEGFR2, PDGFRβ, FGFR1, EGFR) was screened using the same kinase activity assay protocol as Tie-2. AMG-Tie2-1 (1 μM) was tested to assess off-target inhibition and selectivity ratios [1] |
| Cell Assay |
HUVEC proliferation assay: HUVECs were seeded in 96-well plates (5×10³ cells/well) and allowed to adhere overnight. Cells were pre-treated with serial dilutions of AMG-Tie2-1 (0.01-1000 nM) for 1 hour, then stimulated with Ang-1 (100 ng/mL) for 72 hours. MTT reagent was added, and absorbance was measured to calculate cell viability and IC50 values [1] - HUVEC migration assay: HUVECs were seeded in 6-well plates and grown to confluence. A scratch was created with a pipette tip, and cells were treated with AMG-Tie2-1 (0.01-100 nM) and Ang-1 (100 ng/mL). Migration into the scratch area was imaged at 0 and 24 hours, and the migrated area was quantified [1] - Tube formation assay: Matrigel was coated onto 96-well plates and polymerized. HUVECs were suspended in medium containing AMG-Tie2-1 (0.01-100 nM) and Ang-1 (100 ng/mL), seeded onto Matrigel, and incubated at 37°C for 12 hours. Capillary-like tube structures were imaged, and total tube length was measured [1] - Tie-2 signaling assay: HUVECs were seeded in 6-well plates and treated with AMG-Tie2-1 (0.01-100 nM) for 1 hour, then stimulated with Ang-1 (100 ng/mL) for 15 minutes. Cells were lysed, and protein extracts were analyzed by western blot using phospho-Tie2 (Tyr992), phospho-Akt (Ser473), and total protein antibodies [1] |
| Animal Protocol |
Nude mouse A549 xenograft model: 6-8 week-old BALB/c nude mice were subcutaneously injected with 2×10⁶ A549 lung cancer cells. When tumors reached ~100 mm³, mice were randomly divided into vehicle and AMG-Tie2-1 treatment groups (30, 60 mg/kg/day). The drug was dissolved in 0.5% methylcellulose and administered orally once daily for 21 days. Tumor volume was measured every 3 days (volume = length × width² / 2). At the end of treatment, tumors were excised, weighed, and analyzed for microvessel density (CD31 immunohistochemistry) and Tie-2 phosphorylation (western blot) [1] - Normal vasculature assessment: A subset of mice treated with 60 mg/kg/day was sacrificed, and liver/kidney tissues were collected for CD31 immunohistochemistry to evaluate microvessel density changes [1] |
| ADME/Pharmacokinetics |
Oral bioavailability: 58% (rat), 63% (dog) [1] - Plasma half-life (t1/2): 4.2 hours (rat, oral), 5.7 hours (dog, oral) [1] - Peak plasma concentration (Cmax): 1.8 μg/mL (rat, 30 mg/kg oral), 2.5 μg/mL (dog, 30 mg/kg oral) [1] - Volume of distribution (Vss): 2.9 L/kg (rat), 3.6 L/kg (dog) [1] - Clearance (CL): 0.35 L/h/kg (rat), 0.28 L/h/kg (dog) [1] - Metabolism: Primarily metabolized by cytochrome P450 3A4; major metabolites are inactive [1] - Excretion: ~65% excreted in feces (as metabolites), ~30% in urine (as metabolites); unchanged drug < 5% [1] |
| Toxicity/Toxicokinetics |
Acute toxicity: LD50 > 200 mg/kg (oral in rat and mouse); no mortality or overt adverse effects (ataxia, lethargy) at doses up to 200 mg/kg [1] - Subchronic toxicity: Daily oral 60 mg/kg for 28 days in rats caused no significant changes in liver/kidney function (ALT, AST, creatinine) or hematological parameters [1] - Plasma protein binding rate: ~92% (human), ~90% (rat) [1] - No hepatotoxicity: Liver histopathology showed no inflammation or steatosis in treated mice [1] |
| References |
[1]. Evolution of a highly selective and potent 2-(pyridin-2-yl)-1,3,5-triazine Tie-2 kinase inhibitor. J Med Chem. 2007 Feb 22;50(4):611-26. |
| Additional Infomation |
AMG-Tie2-1 is a potent, highly selective, orally active 2-(pyridin-2-yl)-1,3,5-triazine-derived Tie-2 kinase inhibitor [1] - Core mechanism of action: Competitively binds to Tie-2 kinase domain, inhibits Tie-2 phosphorylation and downstream Akt signaling, blocks Ang-1-induced endothelial cell proliferation, migration, and tube formation, thereby suppressing tumor angiogenesis and tumor growth [1] - Potential therapeutic application: Solid tumors (e.g., lung cancer) by targeting tumor angiogenesis [1] - Distinguished by high selectivity for Tie-2 (>1300-fold over VEGFR2), good oral bioavailability, favorable pharmacokinetics, and low toxicity to normal vasculature [1] - Serves as a lead compound for optimizing Tie-2-targeted anticancer drugs with improved efficacy and safety [1] |
Solubility Data
| Solubility (In Vitro) | May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples |
| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *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. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0857 mL | 10.4286 mL | 20.8572 mL | |
| 5 mM | 0.4171 mL | 2.0857 mL | 4.1714 mL | |
| 10 mM | 0.2086 mL | 1.0429 mL | 2.0857 mL |