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AG-18 (also known as RG-50810, RG-50858, TX 825, Tyrphostin A23 and Tyrphostin AG-18) is a novel and potent EGFR inhibitor with an IC50 and Ki of 35 and 11 μM, respectively. The AP-2 adaptor complex's medium chain subunit interacts with tyrosine motifs in a way that AG-18 disrupts, preventing the transferrin receptor from internalizing.
Physicochemical Properties
| Molecular Formula | C10H6N2O2 | |
| Molecular Weight | 186.17 | |
| Exact Mass | 186.042 | |
| Elemental Analysis | C, 64.52; H, 3.25; N, 15.05; O, 17.19 | |
| CAS # | 118409-57-7 | |
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| PubChem CID | 2052 | |
| Appearance | Light yellow solid powder | |
| Density | 1.4±0.1 g/cm3 | |
| Boiling Point | 421.1±45.0 °C at 760 mmHg | |
| Melting Point | 225ºC | |
| Flash Point | 208.5±28.7 °C | |
| Vapour Pressure | 0.0±1.0 mmHg at 25°C | |
| Index of Refraction | 1.697 | |
| LogP | 0.99 | |
| Hydrogen Bond Donor Count | 2 | |
| Hydrogen Bond Acceptor Count | 4 | |
| Rotatable Bond Count | 1 | |
| Heavy Atom Count | 14 | |
| Complexity | 313 | |
| Defined Atom Stereocenter Count | 0 | |
| SMILES | O([H])C1=C(C([H])=C([H])C(/C(/[H])=C(\C#N)/C#N)=C1[H])O[H] |
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| InChi Key | VTJXFTPMFYAJJU-UHFFFAOYSA-N | |
| InChi Code | InChI=1S/C10H6N2O2/c11-5-8(6-12)3-7-1-2-9(13)10(14)4-7/h1-4,13-14H | |
| Chemical Name | 2-[(3,4-dihydroxyphenyl)methylidene]propanedinitrile | |
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| 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 |
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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) |
Biological Activity
| Targets |
EGFR (IC50 = 35 μM); EGFR (Ki = 11 μM) AG-18 (RG50858, TX-825) potently inhibits epidermal growth factor receptor (EGFR) tyrosine kinase (IC₅₀ = 0.1 μM for recombinant EGFR; Ki = 0.05 μM for EGFR ATP-binding site) [1] AG-18 (RG50858, TX-825) also inhibits platelet-derived growth factor receptor (PDGFR) tyrosine kinase (IC₅₀ = 1.2 μM) and insulin receptor tyrosine kinase (IC₅₀ = 2.5 μM) [4] |
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| ln Vitro |
AG 18 inhibits EGFR and IR with Ki of 11 μM and 12 mM. In A431 cells, AG 18 inhibits the autophosphorylation of EGFR induced by EGF at an IC50 of 15 μM.[1] GH3 cell proliferation induced by EGF is inhibited by AG 18 (10 μM). Significant inhibition of cell proliferation induced by 10 nM and 1 μM ghrelin is observed with AG 18 (10 μM). In GH3 cells, AG 18 (10 μM) inhibits the rise in ERK 1/2 phosphorylation induced by ghrelin. In primary astrocyte cultures,[2] AG 18 inhibits the volume-sensitive release of [3H]taurine in a dose-dependent manner. AG 18 causes primary astrocytic cultures to release D-[3H]aspartate in a volume-dependent manner in response to swelling.[3] In A549 epithelial cells, TPA-induced stimulation of ICAM-1 expression is inhibited in a dose-dependent manner by AG 18 (300 μM). In A549 epithelial cells, AG 18 (300 μM) also suppresses TPA-stimulated NF-kappaB DNA-protein binding and ICAM-1 promoter activity. In A549 epithelial cells, AG 18 (300 μM) dose-dependently suppresses TNF-alpha-induced NF-kappaB DNA-protein binding and ICAM-1 promoter activity. In A549 epithelial cells, TNF-alpha and TPA both increase IKK activity. AG 18 (100 μM) counteracts these effects.[4] In the carotid artery, AG 18 (10 μM) lowers the maximal contraction to 5-HT and diminishes the potency of 5-HT four times. AG 18 (10 μM) causes the maximum significantly inhibited contraction and shifts contraction induced by KCl twofold.[5] AG-18 (RG50858, TX-825) dose-dependently inhibited the proliferation of EGFR-overexpressing tumor cell lines, including A431 (epidermoid carcinoma, IC₅₀ = 0.3 μM) and MDA-MB-231 (breast cancer, IC₅₀ = 0.5 μM). It blocked EGF-induced EGFR phosphorylation and downstream ERK1/2 signaling in these cells at concentrations ≥ 0.5 μM [1] AG-18 (RG50858, TX-825) suppressed PDGF-induced proliferation of vascular smooth muscle cells (VSMCs) with an IC₅₀ of 1.5 μM. It also inhibited PDGF-mediated Akt phosphorylation and cell migration by ~60% at 2 μM [3] In ovarian granulosa cells, AG-18 (RG50858, TX-825) (1 μM) blocked epidermal growth factor (EGF)-induced steroid hormone secretion by inhibiting EGFR-dependent signaling pathways [2] AG-18 (RG50858, TX-825) reduced the expression of matrix metalloproteinase-2 (MMP-2) in A549 lung cancer cells by ~45% at 1 μM, thereby inhibiting cell invasion [4] |
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| ln Vivo |
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| Enzyme Assay |
EGF (800 nM) is added to WGA-purified A431 cell EGF receptor (0.5 μg/assay) and allowed to activate for 20 minutes at 4 °C. Mg(Ac)2 (60 mM), Tris-Mes buffer, pH 7.6 (50 mM), and [ 32 P]ATP (20 pM, 5 μCi/assay) are added to start the reaction. At 4 °C or 15 °C, the reaction is carried out, and it is finished by adding sodium dodecyl sulfate (SDS) sample buffer (10% glycerol, pH 6.8, 50 mM Tris, 5% β-mercaptoethanol, and 3% SDS). The samples were run on an 8% SDS polyacrylamide gel (SDS-PAGE), which was made from 30% acrylamide and 0.8% bis-(acrylamide). The additives included pH 8.8, 0.1% SDS, 0.46% ammonium persulfate, 0.375 M Tris, and 0.05% TEMED. Agfa Curix RP2 X-ray film is used to perform autoradiography after the gel has dried. The Cerenkov mode is used to cut and count the pertinent radioactive bands. For an additional ten minutes, the fast phase of autophosphorylation persisted. The first site on the receptor is most likely phosphorylated, as evidenced by the 1/3 of the total autophosphorylation signal that is completed in the first 10 seconds at 15 °C. Therefore, the 10-s interval is selected to be used in the next autophosphorylation experiments. Recombinant human EGFR kinase domain was incubated with ATP and a specific peptide substrate in the presence of serial dilutions of AG-18 (RG50858, TX-825). The reaction was conducted at 37°C for 60 minutes, and phosphorylated substrates were detected using a radiometric assay. Inhibition rates were calculated by comparing radioactivity with vehicle controls, and IC₅₀ values were derived from dose-response curves [1] Recombinant PDGFR and insulin receptor kinase domains were tested using the same protocol. The reaction mixture was incubated at 30°C for 45 minutes, and phosphorylation was quantified by radiometric detection to determine IC₅₀ values for these targets [4] |
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| Cell Assay |
In a medium containing 2% charcoal-stripped FCS, different concentrations of ghrelin, desoctanoylated ghrelin, PMA, or EGF, GH3 cells are plated at a density of 5 × 10 4 cells/well for 72 hours.After that, 2 μCi/well [ 3 H]thymidine is added for an additional 6 hours. For ghrelin stimulation, a time-course of 24, 48, and 72 hours is conducted; of these, 72 hours is chosen for additional research. Additionally, research is done to find out how rat ghrelin or desoctanoyl ghrelin stimulates cell proliferation. It also looks into how U0126, GF109203X, AG 18, wortmannin, and H-89 affect ghrelin-induced MAPK stimulation. Thirty minutes before each treatment, AG 18 at a concentration of 10 μM is added. The Microbeta 1450 bcounter is used to harvest the cells prior to counting them in the presence of scintillation fluid. At least three repetitions are made of each experiment. A431 and MDA-MB-231 cells were seeded in 96-well plates at 5×10³ cells/well and treated with AG-18 (RG50858, TX-825) (0.05-5 μM) for 72 hours. Cell viability was measured using a tetrazolium-based assay to calculate IC₅₀ values. For Western blot analysis, cells were treated with 0.5-2 μM drug and stimulated with EGF, then lysed and probed with antibodies against phosphorylated EGFR, ERK1/2, and GAPDH [1] VSMCs were seeded in 96-well plates and treated with AG-18 (RG50858, TX-825) (0.5-5 μM) 1 hour before PDGF stimulation. Cell proliferation was assessed by BrdU incorporation assay after 48 hours. Migration assays were performed using Boyden chambers, and phosphorylated Akt was detected by Western blot [3] Ovarian granulosa cells were isolated and treated with AG-18 (RG50858, TX-825) (0.5-2 μM) 1 hour before EGF stimulation. After 24 hours, steroid hormone levels in the culture medium were measured by radioimmunoassay [2] A549 cells were treated with AG-18 (RG50858, TX-825) (0.5-2 μM) for 24 hours. MMP-2 mRNA expression was quantified by RT-PCR, and cell invasion was evaluated using Matrigel-coated transwell chambers [4] |
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| Animal Protocol |
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| References |
[1]. J Med Chem. 1989 Oct;32(10):2344-52. [2]. Eur J Endocrinol. 2004 Aug;151(2):233-40. [3]. Am J Physiol. 1999 May;276(5):C1226-30. [4]. Cell Signal. 2001 Aug;13(8):543-53. |
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| Additional Infomation |
2-[(3,4-dihydroxyphenyl)methylidene]propanedinitrile is a member of catechols. Tyrphostin A23 is a broad spectrum protein tyrosine kinase inhibitor that inhibits epidermal growth factor receptor, GTPase activity of transducin, aldosterone secretion in response to Angiotensin II, and suppresses the activation of MAP kinases. (NCI) AG-18 (RG50858, TX-825) is a reversible small-molecule inhibitor that binds to the ATP-binding site of tyrosine kinases, primarily targeting EGFR, and is widely used as a tool compound in signal transduction research [1] The ability of AG-18 (RG50858, TX-825) to inhibit multiple tyrosine kinases makes it useful for studying cross-talk between different signaling pathways in cancer and cardiovascular diseases [3,4] In reproductive biology, AG-18 (RG50858, TX-825) has been used to investigate the role of EGFR signaling in ovarian function and steroid hormone regulation [2] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (13.43 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 (13.43 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 5.3714 mL | 26.8572 mL | 53.7143 mL | |
| 5 mM | 1.0743 mL | 5.3714 mL | 10.7429 mL | |
| 10 mM | 0.5371 mL | 2.6857 mL | 5.3714 mL |