EGFR-IN-1 HCl is an orally bioactive, irreversible, mutant-selective L858R/T790M EGFR inhibitor. EGFR-IN-1 HCl has a 100-fold inhibitory activity against Gefitinib-resistant EGFR L858R/T790M compared to wild-type EGFR. EGFR-IN-1 HCl has strong anti-proliferation activity on H1975 cells and HCC827 cells. Has anti-tumor activity.

Physicochemical Properties

Molecular Formula	C28H31CLN6O4
Molecular Weight	551.04
Exact Mass	550.209
CAS #	2227455-78-7
Related CAS #	EGFR-IN-1 TFA;2753348-63-7
PubChem CID	146026038
Appearance	Typically exists as solid at room temperature
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	8
Rotatable Bond Count	10
Heavy Atom Count	39
Complexity	866
Defined Atom Stereocenter Count	0
InChi Key	SDTFYMHVYLJYFV-UHFFFAOYSA-N
InChi Code	InChI=1S/C28H30N6O4.ClH/c1-6-25(35)30-19-8-7-9-20(15-19)34-26(36)14-18(2)22-17-29-28(32-27(22)34)31-23-11-10-21(16-24(23)37-5)38-13-12-33(3)4;/h6-11,14-17H,1,12-13H2,2-5H3,(H,30,35)(H,29,31,32);1H
Chemical Name	N-[3-[2-[4-[2-(dimethylamino)ethoxy]-2-methoxyanilino]-5-methyl-7-oxopyrido[2,3-d]pyrimidin-8-yl]phenyl]prop-2-enamide;hydrochloride
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

ln Vitro	EGFR-IN-1 hydrochloride (compound 24) had a substantial antiproliferative effect on H1975 and HCC827 cells, with IC50 values of 4 and 28 nM, respectively, after being applied for 72 hours (10 μM) [1]. In H1975 and HCC827 cells, EGFR-IN-1 hydrochloride suppresses p-EGFR with IC50 values of 4 and 9 nM, respectively. A panel of 100 kinases is highly selective for EGFR-IN-1 [1].
ln Vivo	EGFR-IN-1 hydrochloride (30 mg/kg; oral; once daily for 2 weeks) revealed considerable tumor growth inhibition without noticeable weight loss [1]. EGFR-IN-1 hydrochloride was assessed in a time-course PD experiment at an oral dose of 30 mg/kg. EGFR-IN-1 reduces EGFR phosphorylation by >50%, lasting >12 hours. EGFR-IN-1 achieved a maximum concentration of 0.10 μM at 2 hours, with a systemic exposure (AUC0-inf.) of 0.33 μM. h[1].
Cell Assay	Cell proliferation experiment [1] Cell Types: NSCLC cell line H1975 (T790M/L858R), HCC827 (Δ746-750) Tested Concentrations: 10 μM Incubation Duration: 72 hrs (hours) Experimental Results: Inhibition of H1975 non-small cell lung cancer cell line and the first mutant The IC50 for HCC827 cell line was 4 and 28 nM, respectively.
Animal Protocol	Animal/Disease Models: Female athymic nude mice (H1975 tumor xenograft) [1] Doses: 30 mg/kg Route of Administration: po; one time/day for 2 weeks Experimental Results: resulted in significant tumor growth inhibition, and no body weight was observed alleviate.
References	[1]. Wurz RP, et al. Oxopyrido[2,3-d]pyrimidines as Covalent L858R/T790M Mutant Selective Epidermal Growth Factor Receptor (EGFR) Inhibitors. ACS Med Chem Lett. 2015 Jul 27;6(9):987-92.

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	1.8148 mL	9.0738 mL	18.1475 mL
	5 mM	0.3630 mL	1.8148 mL	3.6295 mL
	10 mM	0.1815 mL	0.9074 mL	1.8148 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.