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| Targets |
EGFR-IN-69 targets mutant forms of the epidermal growth factor receptor (EGFR), a receptor tyrosine kinase that is frequently mutated in non-small cell lung cancer (NSCLC). It is a potent inhibitor of the triple-mutant EGFR L858R/T790M/C797S (IC50 = 4.3 nM), the double-mutant EGFR L858R/T790M (IC50 = 6.6 nM), and the triple-mutant EGFR 19del/T790M/C797S (IC50 = 25.6 nM). By binding to the ATP-binding pocket of these mutant EGFR kinases, it blocks downstream signaling pathways such as the MAPK/ERK and PI3K/AKT pathways, leading to inhibition of cancer cell proliferation.
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| ln Vitro |
In vitro, EGFR-IN-69 potently inhibits the kinase activity of various EGFR mutants. In enzyme assays, it demonstrates IC50 values of 4.3 nM against EGFR L858R/T790M/C797S, 6.6 nM against EGFR L858R/T790M, and 25.6 nM against EGFR 19del/T790M/C797S. In cell-based assays using NSCLC cell lines harboring these EGFR mutations, EGFR-IN-69 suppresses cell proliferation and induces apoptosis. It is a valuable research tool for studying resistance mechanisms to EGFR inhibitors, particularly the C797S mutation, which confers resistance to third-generation EGFR inhibitors like osimertinib.
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| ln Vivo |
In vivo, EGFR-IN-69 has shown efficacy in mouse xenograft models of NSCLC. In mice bearing tumors with EGFR L858R/T790M/C797S mutations, treatment with EGFR-IN-69 results in significant tumor growth inhibition or regression. The compound is suitable for use in in vivo xenograft models to investigate drug resistance mechanisms and to evaluate its potential as a fourth-generation EGFR inhibitor for patients who have progressed on osimertinib. Detailed efficacy data is available in the primary literature describing compound 17g.
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| Enzyme Assay |
For non-cell-based kinase inhibition assays, a standard protocol uses an HTRF (Homogeneous Time-Resolved Fluorescence) kinase assay. Recombinant EGFR kinase domains (e.g., EGFR L858R/T790M/C797S) are incubated with varying concentrations of EGFR-IN-69 (0.001-10,000 nM), a biotinylated substrate peptide, and ATP (at the Km for the enzyme) in a reaction buffer. After 60-120 minutes at room temperature, the reaction is stopped, and the phosphorylated product is detected using a streptavidin-conjugated donor fluorophore and a phospho-specific antibody conjugated to an acceptor fluorophore. The HTRF signal is measured, and IC50 values are calculated.
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| Cell Assay |
For in vitro cell-based assays, NSCLC cell lines expressing specific EGFR mutations (e.g., NCI-H1975 harboring EGFR L858R/T790M; or isogenic cell lines engineered to express EGFR L858R/T790M/C797S) are seeded in 96-well plates. After 24 hours, cells are treated with a dilution series of EGFR-IN-69 (0.1 nM - 10 uM) for 72 hours. Cell viability is assessed using the CellTiter-Glo luminescent assay. For mechanism-of-action studies, cells are treated with 1-100 nM of EGFR-IN-69 for 4 hours, lysed, and analyzed by Western blotting with antibodies against p-EGFR, total EGFR, p-AKT, total AKT, p-ERK, and total ERK.
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| Animal Protocol |
For in vivo animal studies, a mouse xenograft model is used. Immunodeficient nude mice are subcutaneously injected with NSCLC cells harboring EGFR mutations (e.g., NCI-H1975 cells, or patient-derived xenograft cells with the C797S mutation). When tumors reach approximately 150-200 mm3, mice are randomized into treatment groups. EGFR-IN-69 is administered orally by gavage once daily at doses ranging from 3-50 mg/kg. Control groups receive vehicle or a comparator EGFR inhibitor (e.g., osimertinib). Tumor volumes are measured by calipers twice per week. Body weight is monitored for signs of toxicity. At study termination (e.g., 21-28 days), tumors are excised, weighed, and analyzed for p-EGFR, p-ERK, and p-AKT by Western blot or immunohistochemistry.
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| ADME/Pharmacokinetics |
EGFR-IN-69 is a small molecule designed for oral administration. Based on its structure, it is expected to have moderate oral bioavailability and a reasonable half-life suitable for once-daily dosing in preclinical species. The compound has a molecular weight of approximately 658.6 g/mol and a ClogP suggesting moderate lipophilicity, which may facilitate membrane permeability. Detailed PK parameters (Cmax, T1/2, AUC, clearance, volume of distribution) would be determined during preclinical development and are not publicly available. The compound is soluble in DMSO for in vitro studies.
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| Toxicity/Toxicokinetics |
Toxicology data for EGFR-IN-69 is not publicly available, as it is a preclinical research compound. As a potent EGFR inhibitor, potential mechanism-based toxicities include skin rash, diarrhea, and stomatitis, which are common class effects of EGFR inhibitors due to the role of EGFR in maintaining skin and gastrointestinal epithelial integrity. The compound's selectivity for mutant EGFR over wild-type EGFR would determine its therapeutic window. Standard safety pharmacology studies would include hERG assessment for cardiotoxicity, CYP450 inhibition screening for drug-drug interaction potential, and a 14-day repeat-dose toxicity study in rats.
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| References | |
| Additional Infomation |
EGFR-IN-69 is a research compound and is not approved for clinical use. It is a fourth-generation EGFR inhibitor designed to overcome resistance mutations, particularly the C797S mutation that arises in patients treated with osimertinib. The C797S mutation prevents covalent binding of osimertinib to the C797 residue, rendering the drug ineffective. EGFR-IN-69, as a non-covalent inhibitor, retains activity against the C797S mutation. The compound is also known as compound 17g and is a valuable chemical probe for studying mechanisms of resistance to EGFR-targeted therapies in non-small cell lung cancer. It is for research use only.
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| Molecular Formula |
C31H37CL2N7O3S
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| Molecular Weight |
658.641583204269
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| Exact Mass |
657.205
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| CAS # |
2433837-65-9
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| PubChem CID |
151183629
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
5.6
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
44
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| Complexity |
1040
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1(NC2=CC(Cl)=C(N3CCC(N4CCN(C)CC4)CC3)C=C2OC)=NC=C(Cl)C(C2C3=C(N(S(CC)(=O)=O)C=2)C=CC=C3)=N1
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| InChi Key |
NEVDBLDTOAJSBN-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C31H37Cl2N7O3S/c1-4-44(41,42)40-20-23(22-7-5-6-8-27(22)40)30-25(33)19-34-31(36-30)35-26-17-24(32)28(18-29(26)43-3)39-11-9-21(10-12-39)38-15-13-37(2)14-16-38/h5-8,17-21H,4,9-16H2,1-3H3,(H,34,35,36)
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| Chemical Name |
5-chloro-N-[5-chloro-2-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl]-4-(1-ethylsulfonylindol-3-yl)pyrimidin-2-amine
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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 : ~12.5 mg/mL (~18.98 mM)
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| 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
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] 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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.5183 mL | 7.5914 mL | 15.1828 mL | |
| 5 mM | 0.3037 mL | 1.5183 mL | 3.0366 mL | |
| 10 mM | 0.1518 mL | 0.7591 mL | 1.5183 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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