Osimertinib-d6 is deuterium-labeled Osimtinib. Osimtinib is a covalent, orally bioactive, irreversible, and mutation-selective EGFR inhibitor that potently inhibits L858R (IC50=12 nM) and L858R/T790M (IC50=1 nM) EGFR. Osimtinib resolves EGFR inhibitor resistance mediated by T790M mutation in lung cancer.

Physicochemical Properties

Molecular Formula	C28H33N7O2
Molecular Weight	505.6443
Exact Mass	505.307
CAS #	1638281-44-3
Related CAS #	Osimertinib;1421373-65-0;Osimertinib mesylate;1421373-66-1
PubChem CID	87056174
Appearance	White to light yellow solid powder
LogP	3.7
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	10
Heavy Atom Count	37
Complexity	752
Defined Atom Stereocenter Count	0
SMILES	O(C([H])([H])[H])C1=C(C([H])=C(C(=C1[H])N(C([H])([H])[H])C([H])([H])C([H])([H])N(C([2H])([2H])[2H])C([2H])([2H])[2H])N([H])C(C([H])=C([H])[H])=O)N([H])C1=NC([H])=C([H])C(C2=C([H])N(C([H])([H])[H])C3=C([H])C([H])=C([H])C([H])=C32)=N1
InChi Key	DUYJMQONPNNFPI-XERRXZQWSA-N
InChi Code	InChI=1S/C28H33N7O2/c1-7-27(36)30-22-16-23(26(37-6)17-25(22)34(4)15-14-33(2)3)32-28-29-13-12-21(31-28)20-18-35(5)24-11-9-8-10-19(20)24/h7-13,16-18H,1,14-15H2,2-6H3,(H,30,36)(H,29,31,32)/i2D3,3D3
Chemical Name	N-[2-[2-[bis(trideuteriomethyl)amino]ethyl-methylamino]-4-methoxy-5-[[4-(1-methylindol-3-yl)pyrimidin-2-yl]amino]phenyl]prop-2-enamide
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 Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.(2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.
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	Osimertinib (AZD9291) (0-10 μM; 72 hours) has IC50s of 41, 26, 41, and 31 nM, respectively, which significantly inhibit cell proliferation[2]. With IC50s of 6, 7, and 74 nM, respectively, osimertinib (0-10 μM; 72 hours) inhibits the proliferation of Ba/F3 cells harboring a T790M mutation, exon 19del+T790M, or L858R+T790M. Ba/F3 cells with EGFR exon 20 insertion mutations are inhibited by osimertinib (0-10 μM; 72 hours); the IC50 ranges from 16-701 nM for A763_Y764insFQEA (FQEA), Y764_V765insHH (HH), A767_V769dupASV (ASV), and D770_N771insNPG (NPG) cells [2]. In sensitizing-mutant (mean IC50 of 8 nM in PC-9) and T790M (mean IC50 of 11 and 40 nM in H1975 and PC-9VanR, respectively) EGFR cell lines, osimertinib exhibits high levels of phenotypic potency. Compared to wild-type EGFR, osimertinib exhibits significantly less activity (mean IC50 of 650 and 461 nM in Calu3 and H2073, respectively)[1]. Ba/F3 cells are exposed to 0.1 μM of osimertinib for 48 hours, which causes apoptosis (rates of 40.9% and 90%, respectively, for EGFR exon 19del+T790M and EGFR L858R+T790M) [2].
ln Vivo	Both PC-9 (ex19del) and H1975 (L858R/T790M) tumor xenograft models exhibit significant dose-dependent regression when oximertinib (0.1–25 mg/kg; po; daily for 14 days) is administered[1].
Cell Assay	Cell Proliferation Assay[2] Cell Types: PC-9, H3255, PC-9ER, and H1975 cells Tested Concentrations: 0.0001, 0.001, 0.01, 0.1, 1, 10 μM Incubation Duration: 72 hrs (hours) Experimental Results: Dramatically inhibited cell proliferation (IC50=41,26, 41, 31 nM, respectively) Cell Proliferation Assay[2] Cell Types: Ba/F3 cells (harboring a T790M mutation, exon 19del+T790M, or L858R+T790M) Tested Concentrations: 0.0001, 0.001, 0.01, 0.1, 1, 10 μM Incubation Duration: 72 hrs (hours) Experimental Results: Inhibited cell proliferation (IC50 = 6, 7, 74 nM, respectively) Apoptosis Analysis[2] Cell Types: Ba/F3 cells (harboring EGFR exon 20 insertion mutations) Tested Concentrations: 0.0001, 0.001, 0.01, 0.1, 1, 10 μM Incubation Duration: 72 hrs (hours) Experimental Results: Inhibited cell proliferation (IC50 = 16, 701, 230, 38 nM, respectively) Apoptosis Analysis[2] Cell Types: Ba/F3 cells (harboring EGFR exon 19del+T790M or EGFR L858R+T790M) Tested Concentrations: 0.1 μM Incubation Duration: 48 hrs (hours) Experimental Results: Inducted apoptosis with the rate of 40.9% and 90% in EGFR T790M positive mutations cells respecti
Animal Protocol	Animal/Disease Models: PC-9 (ex19del) and H1975 (L858R/T790M) tumor xenograft models[1] Doses: 0.1-10 mg/kg (PC-9 xenograft models); 0.5- 25 mg/kg (H1975 xenograft models) Route of Administration: po; daily for 14 day Experimental Results: Induced significant dose-dependent regression in both PC-9 (ex19del) and H1975 (L858R/T790M) tumor xenograft models.
References	[1]. AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer. Cancer Discov. 2014 Sep;4(9):1046-61. [2]. Pharmacological and Structural Characterizations of Naquotinib, a Novel Third-Generation EGFR Tyrosine Kinase Inhibitor, in EGFR-Mutated Non-Small Cell Lung Cancer. Mol Cancer Ther. 2018 Apr;17(4):740-750.

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	1.9777 mL	9.8885 mL	19.7769 mL
	5 mM	0.3955 mL	1.9777 mL	3.9554 mL
	10 mM	0.1978 mL	0.9888 mL	1.9777 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.