Afatinib (BIBW2992) Dimaleate

Alias: Afatinib dimaleate; BIBW 2992; BIBW-2992; BIBW2992. Afatinib free base; trade name: Gilotrif, Tomtovok and Tovok
Cat No.:V0542 Purity: ≥98%
Afatinib dimaleate (the maleate salt form of Afatinib, also know as BIBW-2992; BIBW2992) is a potent, covalent/irreversible,and orally bioavailable dual EGFR/HER2 receptor tyrosine kinase (RTK) inhibitor with potential antineoplastic activity.
Afatinib (BIBW2992) Dimaleate Chemical Structure CAS No.: 850140-73-7
Product category: EGFR
This product is for research use only, not for human use. We do not sell to patients.
Size Price Stock Qty
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50mg
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Other Forms of Afatinib (BIBW2992) Dimaleate:

  • Afatinib (BIBW2992)
  • Afatinib oxalate
Official Supplier of:
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Top Publications Citing lnvivochem Products
InvivoChem's Afatinib (BIBW2992) Dimaleate has been cited by 1 publication
Purity & Quality Control Documentation

Purity: ≥98%

Product Description

Afatinib dimaleate (the maleate salt form of Afatinib, also know as BIBW-2992; BIBW2992) is a potent, covalent/irreversible, and orally bioavailable dual EGFR/HER2 receptor tyrosine kinase (RTK) inhibitor with potential antineoplastic activity. Afatinib is an FDA-approved anticancer medication used to treat lung cancer that is not small cell (NSCLC). In the USA, Gilotrif is the brand name under which it is sold. It is 100 times more active against the Gefitinib-resistant L858R-T790M EGFR mutant. It irreversibly binds to and inhibits EGFR/HER2, including EGFR(wt), EGFR(L858R), EGFR(L858R/T790M), and HER2. In cell-free assays, its IC50 values are 0.5 nM, 0.4 nM, 10 nM, and 14 nM, respectively.

Biological Activity I Assay Protocols (From Reference)
Targets
EGFRL858R (IC50 = 0.4 nM); EGFR (IC50 = 0.5 nM); EGFRL858R/T790M (IC50 = 10 nM); HER2 (IC50 = 14 nM); HER3
ln Vitro

BIBW2992 is more efficient at preventing lung cancer cell lines carrying wild-type (H1666) or L858R/T790M (NCI-H1975) EGFR from surviving than erlotinib, gefitinib, or lapatinib. Comparably effective against NSCLC lines expressing EGFR E746_A750del (HCC827) or HER2 776insV (NCI-H1781), BIBW2992 exhibits no activity against A549 cells, which express EGFR and HER2 in their wild-type forms.[1] Afatinib increases the apoptosis that topotecan and mitoxantrone induce in SP cells as well as the cytotoxicity of these substances to SP cells.[2]

ln Vivo
BIBW2992 (20 mg/kg, p.o.) causes a significant tumor regression with a cumulative treated/control tumor volume ratio (T/C ratio) of 2% in the MDA-MB-453 xenograft model. It also results in the downregulation of EGFR and AKT phosphorylation.[1] Applying BIBW 2992 (30 mg/kg, p.o.) to xenograft models A7, A431, FaDu, UT-SCC-14, and UT-SCC-15 significantly extends the time for tumor growth.[3] Afatinib (30 mg/kg, p.o.) causes a notable inhibition of tumor growth and a notable extension of overall survival in HER2-amplified xenograft models. *[4] Afatinib (25 mg/kg, p.o.) causes a dramatic tumor volume regression within 4 days and nearly complete tumor resolution after 21 days of treatment in HER2-positive gastric cancer NCI-N87 xenograft.[5]
Enzyme Assay
EGFR kinase: 10 μL of inhibitor in 50% Me2SO, 20 μL of substrate solution (200 mM HEPES pH 7.4, 50 mM Mg-acetate, 2.5 mg/mL poly (EY), 5 μg/mL bio-pEY), and 20 µL enzyme preparation were included in each 100 µL enzyme reaction. The addition of 50 µL of a 100 µM ATP solution prepared in 10 mM MgCl2 initiates the enzymatic reaction. After 30 minutes of assaying at room temperature, 50 µL of stop solution (250 mM EDTA in 20 mM HEPES pH 7.4) is added to end the assay. 100 µL are added to a microtiterplate coated with streptavidin, and after 60 minutes of room temperature incubation, the plate is cleaned with 200 µL of wash solution (50 mM Tris, 0.05% Tween20). The wells are filled with a 100 µL aliquot of PY20H Anti-Ptyr:HRP, a 250 ng/mL HRPO-labeled anti-PY antibody. Following a 60-minute incubation period, the plate is three times cleaned using a 200 µL wash solution. Following that, 100µL of TMB Peroxidase Solution (A:B=1:1) is used to develop the samples. After ten minutes, the reaction is stopped. After the plate is placed in an ELISA reader, the extinction at OD450nm is calculated. The enzyme HER2-IC: The assay of enzyme activity is conducted in 50% Me2SO with or without serial inhibitor dilutions. Similar components as described for the EGFR kinase assay are included in each 100 µL reaction, along with the addition of 1000 µM Na3VO4. The addition of 50µL of a 500 µM ATP solution prepared in 10 mM magnesium acetate initiates the enzymatic reaction. The enzyme is diluted to the point where the amount of enzyme and the amount of time it takes for phosphate to be incorporated into bio-pEY are linear. The mixture of 20 mM HEPES pH 7.4, 130 mM NaCl, 0.05% Triton X-100, 1 mM DTT, and 10% glycerol is used to dilute the enzyme preparation. After 30 minutes of assaying at room temperature, 50 µL of stop solution is added to end the procedure. Src kinase assays: 10 µL of inhibitor in 50% Me2SO, 20 µL of enzyme preparation, and 20 µL of substrate solution enhanced with 1000 µM Na3VO4 were included in each 100 µL reaction. The addition of 50 µL of a 1000 µM ATP solution prepared in 10 mM Mg-acetate initiates the enzymatic reaction. Assay for BIRK kinase: 50 µL of a 2 mM ATP solution prepared in 8 mM MnCl2 and 20 mM Mg-acetate is added to 250 mM Tris pH 7.4, 10 mM DTT, 2.5 mg/mL poly(EY), and 5 mg/mL bio-pEY as the substrate solution to initiate the enzymatic reaction. HGFR kinase and VEGF2 assays: The assay is completed by adding 10 µL of 5% H3PO4 after it has been running at room temperature for 20 minutes. The precipitate is then collected using a 96 well filter mate universal harvester and trapped onto GF/B filters. The filter plate is thoroughly cleaned, dried for one hour at 50°C, sealed, and the radioactivity is measured using scintillation counting with either a TopCountTM or a Microbeta b counterTM.
Cell Assay
The MTT assay is used to determine cytotoxicity. The drug concentration that causes 50% of cells to die is known as the IC 50 value. The Bliss method is used to calculate the IC50 as well as the fitted sigmoidal dose response curve.
Animal Protocol
SCID mice harbouring ARK2 xenografts
25 mg/kg
p.o.
References

[1]. Oncogene. 2008 Aug 7; 27(34): 4702–4711.

[2]. Cancer Res . 2014 Aug 15;74(16):4431-45.

[3]. Radiat Oncol . 2014 Dec 2:9:261.

[4]. Br J Cancer . 2014 Oct 28;111(9):1750-6.

[5]. J Nucl Med . 2013 Jun;54(6):936-43.

These protocols are for reference only. InvivoChem does not independently validate these methods.
Physicochemical Properties
Molecular Formula
C32H33CLFN5O11
Molecular Weight
717.18
Exact Mass
717.1849127
Elemental Analysis
C, 53.52; H, 4.63; Cl, 4.94; F, 2.65; N, 9.75; O, 24.51
CAS #
850140-73-7
Appearance
White to off-white solid powder
SMILES
CN(C/C=C/C(=O)NC1=C(C=C2N=CN=C(C2=C1)NC3=CC(=C(C=C3)F)Cl)O[C@@H]4COCC4)C.C(=C\C(=O)O)\C(=O)O.C(=C\C(=O)O)\C(=O)O
InChi Key
USNRYVNRPYXCSP-JUGPPOIOSA-N
InChi Code
InChI=1S/C24H25ClFN5O3.2C4H4O4/c1-31(2)8-3-4-23(32)30-21-11-17-20(12-22(21)34-16-7-9-33-13-16)27-14-28-24(17)29-15-5-6-19(26)18(25)10-15;2*5-3(6)1-2-4(7)8/h3-6,10-12,14,16H,7-9,13H2,1-2H3,(H,30,32)(H,27,28,29);2*1-2H,(H,5,6)(H,7,8)/b4-3+;2*2-1-/t16-;;/m0../s1
Chemical Name
(Z)-but-2-enedioic acid;(E)-N-[4-(3-chloro-4-fluoroanilino)-7-[(3S)-oxolan-3-yl]oxyquinazolin-6-yl]-4-(dimethylamino)but-2-enamide
Synonyms
Afatinib dimaleate; BIBW 2992; BIBW-2992; BIBW2992. Afatinib free base; trade name: Gilotrif, Tomtovok and Tovok
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)
Solubility Data
Solubility (In Vitro)
DMSO: ~100 mg/mL (~139.4 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL
Solubility (In Vivo)
5% DMSO+30% PEG 300+ddH2O: 28 mg/mL
 (Please use freshly prepared in vivo formulations for optimal results.)
Preparing Stock Solutions 1 mg 5 mg 10 mg
1 mM 1.3944 mL 6.9718 mL 13.9435 mL
5 mM 0.2789 mL 1.3944 mL 2.7887 mL
10 mM 0.1394 mL 0.6972 mL 1.3944 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.

Calculator

Molarity Calculator allows you to calculate the mass, volume, and/or concentration required for a solution, as detailed below:

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An example of molarity calculation using the molarity calculator is shown below:
What is the mass of compound required to make a 10 mM stock solution in 5 ml of DMSO given that the molecular weight of the compound is 350.26 g/mol?
  • Enter 350.26 in the Molecular Weight (MW) box
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  • The answer of 17.513 mg appears in the Mass box. In a similar way, you may calculate the volume and concentration.

Dilution Calculator allows you to calculate how to dilute a stock solution of known concentrations. For example, you may Enter C1, C2 & V2 to calculate V1, as detailed below:

What volume of a given 10 mM stock solution is required to make 25 ml of a 25 μM solution?
Using the equation C1V1 = C2V2, where C1=10 mM, C2=25 μM, V2=25 ml and V1 is the unknown:
  • Enter 10 into the Concentration (Start) box and choose the correct unit (mM)
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  • The answer of 62.5 μL (0.1 ml) appears in the Volume (Start) box
g/mol

Molecular Weight Calculator allows you to calculate the molar mass and elemental composition of a compound, as detailed below:

Note: Chemical formula is case sensitive: C12H18N3O4  c12h18n3o4
Instructions to calculate molar mass (molecular weight) of a chemical compound:
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Definitions of molecular mass, molecular weight, molar mass and molar weight:
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  • Molar mass (molar weight) is the mass of one mole of a substance and is expressed in g/mol.
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In vivo Formulation Calculator (Clear solution)
Step 1: Enter information below (Recommended: An additional animal to make allowance for loss during the experiment)
Step 2: Enter in vivo formulation (This is only a calculator, not the exact formulation for a specific product. Please contact us first if there is no in vivo formulation in the solubility section.)
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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.

Clinical Trial Information
NCT Number Recruitment interventions Conditions Sponsor/Collaborators Start Date Phases
NCT04439136 Active
Recruiting
Drug: Afatinib Dimaleate Advanced Lymphoma
Refractory Lymphoma
National Cancer Institute
(NCI)
August 12, 2015 Phase 2
NCT02438722 Active
Recruiting
Drug: Afatinib Dimaleate
Biological: Cetuximab
Recurrent Non-Small Cell Lung
Carcinoma
Stage IV Non-Small Cell Lung
Cancer
SWOG Cancer Research Network May 7, 2015 Phase 2
Phase 3
NCT02465060 Active
Recruiting
Drug: Afatinib
Drug: Afatinib Dimaleate
Bladder Carcinoma
Glioma
National Cancer Institute
(NCI)
August 17, 2015 Phase 2
NCT03083678 Active
Recruiting
Drug: Afatinib Chordoma Leiden University Medical Center June 21, 2018 Phase 2
NCT03827070 Active
Recruiting
Drug: Afatinib
Drug: Talcum powder
Non Small Cell Lung Cancer Center Trials & Treatment Europe March 5, 2019 Phase 1
Biological Data
  • Afatinib (BIBW2992) Dimaleate

  • Afatinib (BIBW2992) Dimaleate
  • Afatinib (BIBW2992) Dimaleate

    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)

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