| Field | Specification |
|---|---|
| Target | |
| Alternative names | BIBW 2992MA2 |
| CAS no. | |
| Applications | |
| Molecular weight | |
| Molecular formula | C32H33ClFN5O11 |
| Purity | |
| SMILES | |
| Form | Solid |
| Storage | |
| Shipping | |
| Catalog no. (Mfr.) | |
| Main SKU |
Compound Overview
Afatinib dimaleate, also known as BIBW 2992MA2, is an orally active, potent, irreversible dual-specificity inhibitor of the ErbB family (EGFR and HER2), with IC50 values of 0.5 nM for EGFR wt, 0.4 nM for EGFR L858R, 10 nM for EGFR L858R/T790M, and 14 nM for HER2. It can be used to study esophageal squamous cell carcinoma (ESCC), non-small cell lung cancer (NSCLC), and gastric cancer[1][2][3][4]. It is supplied as a white to yellow solid (C32H33ClFN5O11, MW 718.08) at 99.74% purity.
Physical & Chemical Properties
| CAS Number | 850140-73-7 |
|---|---|
| Molecular Formula | C32H33ClFN5O11 |
| Molecular Weight | 718.08 g/mol |
| Purity | 99.74% |
| Appearance | Solid |
| Color | White to yellow |
| SMILES | O=C(NC1=C(C=C2C(C(NC3=CC(Cl)=C(C=C3)F)=NC=N2)=C1)O[C@H]4CCOC4)/C=C/CN(C)C.O=C(O)/C=C\C(O)=O.O=C(O)/C=C\C(O)=O |
| Target | EGFR L858R, EGFR, EGFR L858R/T790M, HER2, HER3 |
| Signaling Pathway | JAK/STAT Signaling; Protein Tyrosine Kinase/RTK; Autophagy; Apoptosis; PI3K/Akt/mTOR; MAPK/ERK Pathway |
| Solubility | In Vitro: DMSO: 100 mg/mL (139.26 mM; Requires sonication; Hygroscopic DMSO has a significant impact on the solubility of product, please use newly opened DMSO) H2O: 50 mg/mL (69.63 mM; Requires sonication) |
| Storage | 4°C, sealed storage, away from moisture. In solvent: -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture). |
| Shipping | Room temperature in continental US; may vary elsewhere. |
Biological Activity
IC50 & Target[1]
|
EGFRL858R 0.4 nM (IC50) |
EGFR 0.5 nM (IC50) |
EGFRL858R/T790M 10 nM (IC50) |
HER2 14 nM (IC50) |
Literature Cited
Sources cited in this description and in the In Vitro & In Vivo Data tab. Peer-reviewed publications that used this product are listed under References.
Safety
For Research Use Only. Not for use in diagnostic or therapeutic procedures, and not for human or veterinary use. Handle in accordance with the Safety Data Sheet and your institution's chemical hygiene plan.
In Vitro
| Solvent | Solubility | Notes |
|---|---|---|
| DMSO | 100 mg/mL (139.26 mM) | requires sonication; use freshly opened DMSO (absorbed moisture lowers solubility) |
| H2O | 50 mg/mL (69.63 mM) | requires sonication |
Aliquot the stock solution and store it at -80°C (up to 6 months) or -20°C (up to 1 month); sealed storage, away from moisture; avoid repeated freeze-thaw cycles.
If water is used as the stock solvent, dilute to the working solution and sterilize it through a 0.22 μm filter before use.
In Vivo
Choose the formulation that suits the animal model and route of administration. Percentages are volume ratios of the final working solution. Prepare the working solution fresh on the day of dosing; if precipitation or phase separation occurs, gentle warming or sonication can help.
Direct preparation of the working solution
These formulations are prepared directly, without a DMSO stock; use them promptly after preparation.
Protocol 1
| Composition | PBS |
|---|---|
| Result | 100 mg/mL (139.26 mM); clear solution; requires sonication |
Data provided by the manufacturer.
In Vitro
Afatinib dimaleate at 100 nM is sufficient to prevent heregulin-stimulated HER3 phosphorylation[1]. At 0-10000 nM, Afatinib dimaleate effectively blocks anchorage-independent proliferation of NIH-3T3 cells that ectopically express EGFR mutants, and also blocks proliferation of H1666, H3255, and NCI 1975 cells[1]. Growth inhibition by Afatinib dimaleate (48-72 h) is seen in HKESC-1, HKESC-2, SLMT-1, and EC-1 cells[2]. Afatinib dimaleate (0-1 μM, 24-48 h) inhibits the AKT and MAPK pathways as well as EGFR and AKT phosphorylation in ESCC cell lines[2]. In HKESC-2 and EC-1, Afatinib dimaleate (0-1 μM, 16-48 h) induces G0/G1 cell cycle arrest[2]. Apoptotic cell death is effectively induced by Afatinib dimaleate (0-1 μM, 24-48 h) in HKESC-2 and EC-1[2].
Cell Proliferation Assay[1]
- Cell Line: NIH-3T3 cells, H1666, H3255, and NCI 1975 cells
- Concentration: 0, 1, 10, 100, 1000, 10000 nM
- Incubation Time:
- Result: Effectively inhibited anchorage-independent proliferation of NIH-3T3 cells ectopically expressing EGFR mutants. Showed inhibition of anchorage independent cell proliferation of various lung cancer cell lines (H1666, H3255, and NCI 1975 cells), with IC50 values of 60 nM, 0.7 nM and 99 nM, respectively.
Cell Viability Assay[2]
- Cell Line: HKESC-1, HKESC-2, SLMT-1 and EC-1 cell lines
- Concentration:
- Incubation Time: 48 and 72 hours
- Result: Observed over 95% of growth inhibition. The respective IC50 concentrations at 48 hours (HKESC-1=0.078 μM, HKESC-2=0.115 μM, KYSE510=3.182 μM, SLMT-1=4.625 μM and EC-1=1.489 μM) and 72 hours (HKESC-1=0.002 μM, HKESC-2=0.002 μM, KYSE510=1.090 μM, SLMT-1=1.161 μM and EC-1=0.109 μM) were all in lower micro-molar range.
Western Blot Analysis[2]
| Cell Line | HKESC-2 cells and EC-1 cells |
|---|---|
| Concentration | 0, 0.01, and 0.1 μM (HKESC-2 cells), 0, 0.1 and 1 μM (EC-1 cells) |
| Incubation Time | 24 and 48 hours |
| Result | Reduced the phosphorylation of EGFR and the endogenous expression level of HER2 receptors in ESCC cells. Suppressed AKT phosphorylation in a dose and time dependent manner. Significantly reduced the phosphorylation level of the downstream effectors of the AKT-mTOR axis especially in HKESC-2 cells. Inhibited the two major downstream pathways of the ErbB/HER axis, namely, AKT and MAPK pathways in ESCC cell lines. |
Cell Cycle Analysis[2]
| Cell Line | HKESC-2 cells and EC-1 cells |
|---|---|
| Concentration | 0, 0.01, and 0.1 μM (HKESC-2 cells), 0, 0.1 and 1 μM (EC-1 cells) |
| Incubation Time | 16, 24, and 48 hours |
| Result | Induced G0/G1 cell cycle arrest in both tested ESCC cell lines in a time and dose dependent manner. In HKESC-2 cells, the percentage of cells in G0/G1 phase was increased from 38.2% to 68.1% at 0.01 μM of afatinib and to 74.7% at 0.1 μM of afatinib, from 24 hours (82.4% G0/G1 arrest at 0.01 μM and 86.2% at 0.1 μM) to 48 hours (from 74.7% to 88.2% for 0.01 μM and 91.0% for 0.1 μM). In EC-1 cells, the percentage of cells arrested in the G0/G1 phase was increased from 59.1% to 66.6% and 72.2% at 24 and 48 hours respectively. |
Apoptosis Analysis[2]
| Cell Line | HKESC-2 cells and EC-1 cells |
|---|---|
| Concentration | 0, 0.01, and 0.1 μM (HKESC-2 cells), 0, 0.1 and 1 μM (EC-1 cells) |
| Incubation Time | 24 and 48 hours |
| Result | Effectively induced cell death by triggering apoptotic mechanisms in ESCC cell lines. Showed a stronger expression level of cleaved Poly (ADP-ribose) polymerase (PARP) in these cell lines. |
In Vivo
Oral dosing of Afatinib dimaleate (0-20 mg/kg, daily for 25 days) produces dramatic tumor regression and downregulates phosphorylation of EGFR, HER2, HER3, and AKT[1]. Afatinib dimaleate given orally (15 mg/kg; two weeks on a schedule of 5 days on plus 2 days off) strongly inhibits growth of HKESC-2 tumor[2].
| Animal Model | Athymic NMRI-nu/nu female mice (21–31 g, five to six-week-old, transgenic murine lung cancer model and xenograft models)[1] |
|---|---|
| Dosage | 15 mg/kg, 20 mg/kg |
| Administration | Orally, daily for 25 days |
| Result | Resulted in dramatic tumor regression with a cumulative treated/control tumor volume ratio (T/C ratio) of 2% in a standard xenograft model of the epidermoid carcinoma cell line A431, and downregulation of EGFR and AKT phosphorylation. Induced regression of large tumors in this HER2-driven model, effectively controlled xenograft tumor formation by the NCIH1975 cell line, expressing EGFR L858R/T790M, with a T/C value of 12% for doses of 20 mg/kg. Induced more than 50% percent tumor reduction after a 4-week treatment period. Downregulated EGFR, HER2 and HER3 phosphorylation. |
| Animal Model | Six weeks old female athymic nude mice (nu/nu) (16-20 g)[2] |
|---|---|
| Dosage | 15 mg/kg |
| Administration | Oral gavage in a schedule of 5 days on plus 2 days off, for two weeks |
| Result | Strongly inhibited the growth of HKESC-2 tumor. Average tumor sizes of vehicle and treatment at end point are 348 ± 24 mm3 and 108 ± 36 mm3 respectively. |
Data provided by the manufacturer. Numbered citations refer to the Literature Cited list in the product description.
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