EGFR-IN-44 (Compound 6a) is a potent, orally bioactive EGFR tyrosine kinase inhibitor (TKI) (antagonist) with IC50 of 4.11 nM. EGFR-IN-44 causes apoptosis, and its oral bioavailability (F) is 33.57%. EGFR-IN-44 may be utilized in study/research of non-small cell lung cancer.

Physicochemical Properties

Molecular Formula	C27H29CLN6O2S
Molecular Weight	537.08
Appearance	Typically exists as solid at room temperature
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	Tumor cell lines are resistant to the anti-proliferative effects of EGFR-IN-44 (Compound 6a) (0–10 µM, 72 h)[1]. The EGFR ATP binding site is bound by EGFR-IN-44[1]. EGFR-IN-44 (0-10 nM, 48 h) inhibits cell migration, stops the cell cycle in the G0/G1 phase, and causes H1975 cell death via the mitochondrial pathway[1]. In 48 and 72 hours, EGFR-IN-44 (0–10 nM) exhibits hypotoxicity toward normal cells[1].
ln Vivo	With no discernible toxicity, EGFR-IN-44 (Compound 6a) (25 mg/kg; ir; daily, 7 days) exhibits potent anticancer activity[1].
Cell Assay	Cell Proliferation Assay[1] Cell Types: H1975 (EGFRT790M/L858R), PC9 (EGFRdel19), and H292 (EGFRWT) Tested Concentrations: 0-10 µM Incubation Duration: 72 h Experimental Results: demonstrated anti-proliferative activities with IC50 values of 0.0022 ± 0.001, 0.0048 ± 0.001, and 4.499 ± 0.057 µM against H1975, PC9, and H292 cells, respectively. Apoptosis Analysis[1] Cell Types: H1975 Tested Concentrations: 1, 5, and 10 nM Incubation Duration: 48 h Experimental Results: Effectively induced cell apoptosis in a dose-dependent manner. Resulted in 33.7%, 52.4%, and 56.2% apoptosis at 1, 5, and 10 µM, respectively, compared to 5.81% apoptosis in the control group. Western Blot Analysis[1] Cell Types: H1975 Tested Concentrations: 5, 10, and 25 nM Incubation Duration: 48 h and 72 h Experimental Results: Dose-dependently upregulated the expression levels of the proapoptotic proteins Bad and Bax and downregulated the expression level of the antiapoptotic protein Bcl-2. Sufficiently decreased the phosphorylation of EGFR and AKT. Cell Cycle Analysis[1] Cell Types: H1975 Tested Concentrations: 5
Animal Protocol	Animal/Disease Models: Male BALB/c nude mice (5 weeks old, 18 - 20 g ), H1975 xenograft model[1] Doses: 25 mg/kg Route of Administration: intragastric (po) administration, daily, 7 days Experimental Results: demonstrated strong tumor inhibition (TGI = 90.24%) without obvious toxicity. Animal/Disease Models: Male Sprague–Dawley (SD) rats [1] Doses: 1 mg/kg and 5 mg/kg Route of Administration: intravenous (iv) injection and oral administration (pharmacokinetic/PK Analysis) Experimental Results: In Vivo PK parameters of EGFR-IN-44 [1] Parameters Dose(mg/kg) EGFR-IN -44 5 (po) 1 (iv) t1/2 (h) 8.60 ± 1.8 1.42 ± 0.1 Tmax (h) 4.00 ± 0.002 / Cmax (ng/mL) 80.40 ± 2.7 / Vz F_pred (L/kg) 220.80 ± 41.2 6.83 ± 08 AUC0-t (H.ng/mL) 490.41 ± 29.9 291.91 ± 38.2 AUC0-∞ (H.ng/mL) 491.02 ± 44.2 295.76 ± 38.8 MRT0-last (h) 7.93 ± 0.8 1.35 ± 01 CL (mL /h/kg) 17.79 ± 3.9 3.12 ± 0.4 F(%) 33.57 ± 5.9 / F = (AUC0-inf-PO × DOSEIV)/(AUC0-inf-IV × DOSE PO)*100%.
References	[1]. Novel third-generation pyrimidines-based EGFR tyrosine kinase inhibitors targeting EGFR T790M mutation in advanced non-small cell lung cancer. Bioorg Chem. 2022 May;122:105743.

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	1.8619 mL	9.3096 mL	18.6192 mL
	5 mM	0.3724 mL	1.8619 mL	3.7238 mL
	10 mM	0.1862 mL	0.9310 mL	1.8619 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.