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Description: Afatinib (formerly BIBW 2992; BIBW-2992; brand name: Gilotrif), is a potent, covalent/irreversible, and orally bioavailable dual (EGFR/ErbB) receptor tyrosine kinase (RTK) inhibitor with anticancer activity. Afatinib is an FDA approved anticancer drug used to treat non-small cell lung carcinoma (NSCLC). It is sold under the brand name of Gilotrif in the USA. It irreversibly binds to and inhibits EGFR/HER2 including EGFR(wt), EGFR(L858R), EGFR(L858R/T790M) and HER2 with IC50 of 0.5 nM, 0.4 nM, 10 nM and 14 nM in cell-free assays, respectively; and is 100-fold more active against Gefitinib-resistant L858R-T790M EGFR mutant.
References: Oncogene. 2008 Aug 7;27(34):4702-11; Br J Cancer. 2008 Jan 15;98(1):80-5.
Related CAS: 850140-72-6 or 850140-72-6 (free base); 850140-73-7 (dimaleate); 439081-18-2 (free base)
Chemical Name: (S,E)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-((tetrahydrofuran-3-yl)oxy)quinazolin-6-yl)-4-(dimethylamino)but-2-enamide.
SMILES Code: O=C(NC1=CC2=C(NC3=CC=C(F)C(Cl)=C3)N=CN=C2C=C1O[[email protected]@H]4COCC4)/C=C/CN(C)C
Mass (g) = Concentration (mol/L) × Volume (L) × Molecular Weight (g/mol)
Concentration (start) × Volume (start) = Concentration (final) × Volume (final)
This equation is commonly abbreviated as: C1V1 = C2V2
In vitro activity: BIBW2992 shows potent activity against both wild-type and mutant forms of EGFR and HER2. It is similar to Gefitinib in potency against L858R EGFR, but about 100-fold more active against the Gefitinib resistant L858R-T790M EGFR double mutant. BIBW2992 exhibits potent effects on both EGFR and HER2 phosphorylation in vivo. It compares favorably to reference compounds (such as Lapatinib et al.) in all cell types tested, such as human epidermoid carcinoma cell line A431 expressing wt EGFR, murine NIH-3T3 cells transfected with wt HER2, as well as breast cancer cell line BT-474 and gastric cancer cell line NCI-N87, which express endogenous HER2.
Kinase Assay: The wild type tyrosine kinase domain of the human EGFR as well as that of the EGFR L858R/T790M double mutant are fused to Glutathione-S-transferase (GST), and extracted. Enzyme activity is then assayed in the presence of the inhibitor BIBW2992, serially diluted in 50% DMSO. A random polymer pEY (4:1) is used as substrate and biotinylated pEY (bio-pEY) is added as a tracer substrate. The kinase domain of HER2 is cloned using the baculovirus system and extracted similarly to that of EGFR kinase. Details of assays for EGFR, HER2, SRC, BIRK and VEGFR2 kinase activity are available in Supplementary information.
Cell Assay: 1 × 104 NSCLC cells are transferred into each well of a 96-well plate and cultured overnight in serum-free media for the EGFR phosphorylation assay. After addition of BIBW2992 on the next day, the plates are incubated at 37 °C for 1 hour. EGF-stimulation is done using 100 ng/mL for 10 min at room temperature. Cells are washed with ice cold PBS, extracted with 120 μL HEPEX buffer per well and shaken at room temperature for 1 hour. In all 2 × 104cells per well is used for the HER2 phosphorylation assay. Streptavidin precoated plates are coated with anti-EGFR-biotin at 1:100 dilution in blocking buffer and c-erb2/HER2 oncoprotein Ab-5(Clone N24)-Biotin. Cell extracts is then transferred to the antibody-coated wells and incubated at room temperature for 1 hour. Extinction is measured at 450 nm.
Oncogene. 2008 Aug 7;27(34):4702-11; Br J Cancer. 2008 Jan 15;98(1):80-5.
Purity ≥98%
COA
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NMR
Afatinib covalently binds to cysteine number 797 of the epidermal growth factor receptor (EGFR) via a Michael addition (IC50 = 0.5 nM). Schubert-Zsilavecz, M, Wurglics, M, Neue Arzneimittel Frühjahr 2013. (in German)