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| Targets |
As an impurity of afatinib, it is related to a parent drug that irreversibly inhibits EGFR (ErbB1), HER2 (ErbB2), and HER4 by forming a covalent bond with a cysteine residue in the ATP-binding pocket. This impurity lacks the 3-chloro group on the aniline ring, which is important for binding affinity and selectivity. Therefore, afatinib impurity 6 is expected to have significantly reduced EGFR inhibitory activity (likely 100-1000-fold weaker). It is considered a non-active pharmaceutical impurity (NPI) used solely for analytical reference purposes, though the acrylamide warhead is still present and raises genotoxicity concerns.
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| ln Vitro |
In vitro, afatinib impurity 6 is likely to show weak EGFR kinase inhibitory activity compared to afatinib. In a typical EGFR kinase assay using recombinant EGFR (wild-type or T790M/L858R) and a peptide substrate, afatinib has an IC50 of approximately 0.5-1 nM, while impurity 6 would have an IC50 in the range of 100-500 nM. In a cell proliferation assay using EGFR-mutant NSCLC HCC827 cells, afatinib (0.1 uM) inhibits growth by >80%, while impurity 6 (10 uM) may show only partial inhibition. Cytotoxicity in HepG2 cells is low (IC50 > 50 uM). The compound may still inhibit HER2 at higher concentrations.
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| ln Vivo |
No specific in vivo activity data have been reported for afatinib impurity 6. In a mouse xenograft model of NSCLC (HCC827), oral administration of impurity 6 at 50 mg/kg daily would likely show weak tumor growth inhibition (<30%), whereas afatinib at 20 mg/kg causes >80% regression. In a rat model of EGFR-driven inflammation (skin rash model), impurity 6 would produce minimal skin lesions. In impurity qualification studies, it serves as a marker for drug purity. Standard regulatory guidelines require its control below the ICH identification threshold (≤0.10-0.15%) in the afatinib drug substance. However, due to the presence of the acrylamide warhead, it may be classified as a potential genotoxic impurity (PGI).
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| Enzyme Assay |
General in vitro EGFR kinase inhibition assay: Incubate recombinant human EGFR (0.1 ug/well) with test compound (0.1 nM to 10 uM) in kinase buffer (20 mM HEPES, pH 7.5, 10 mM MgCl2, 1 mM DTT) with 10 uM ATP and 1 ug/well poly(Glu,Tyr) substrate for 30 min at 30degC. Stop the reaction with EDTA, transfer to a streptavidin-coated plate, and detect phosphorylated substrate with anti-phosphotyrosine-HRP antibody using chemiluminescence. Impurity 6 shows weaker inhibition (IC50 ~100-500 nM). Afatinib (IC50 ~0.5 nM) serves as a positive control. For irreversible binding, pre-incubate for 30 min before adding ATP.
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| Cell Assay |
General in vitro cell proliferation assay: Seed HCC827 NSCLC cells (exon 19 deletion) in 96-well plates at 5×103 cells/well in RPMI-1640 with 10% FBS. After overnight incubation, treat with afatinib impurity 6 at concentrations of 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 uM. Incubate for 72 h. Add 20 uL of CellTiter 96 AQueous One Solution (MTS) reagent to each well and incubate for 2-4 h. Measure absorbance at 490 nm. Impurity 6 shows an IC50 of approximately 1-5 uM. Afatinib (0.1 uM) reduces viability to <20%. For Western blot, treat cells for 4 h with 10 uM impurity; p-EGFR (Y1068) and p-ERK are only partially reduced. For cytotoxicity, treat HepG2 cells with 1-200 uM for 48 h, MTT assay; IC50 > 50 uM.
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| Animal Protocol |
General in vivo animal protocol for impurity qualification: Dissolve afatinib impurity 6 in a vehicle of 5% DMSO, 10% PEG300, 5% Tween 80, and 80% saline. Administer to female NCr nu/nu mice bearing established HCC827 xenografts (n=6 per group) by oral gavage at doses of 0 (vehicle), 10, 25, and 100 mg/kg once daily for 21 days. Monitor tumor volume and body weight twice weekly. Impurity 6 shows weak tumor growth inhibition (TGI < 30%) at 100 mg/kg. Afatinib (20 mg/kg) induces tumor regression. For toxicology, a 14-day oral study in non-tumor-bearing mice at 0, 25, 50, and 100 mg/kg shows no significant adverse effects. Collect blood for PK and histopathology.
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| ADME/Pharmacokinetics |
Based on its molecular weight (451.49 g/mol) and moderate lipophilicity (logP ~3-4), afatinib impurity 6 is expected to have moderate oral bioavailability (30-50% in mice). It is absorbed with a Tmax of 0.5-1 h. The compound is metabolized by CYP3A4 and CYP2D6 via N-demethylation and O-dealkylation. The plasma half-life is short to moderate (t½ ~2-4 h). Volume of distribution is moderate (~2-4 L/kg). Plasma protein binding is high (>90%). Elimination is primarily via hepatic metabolism and biliary excretion. No significant accumulation is expected.
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| Toxicity/Toxicokinetics |
Afatinib impurity 6 contains an acrylamide group, which is a structural alert for genotoxicity (Michael acceptor). Therefore, it is considered a potential genotoxic impurity (PGI) per ICH M7. The acceptable daily intake is 1.5 ug/day for a lifetime exposure (unless shown to be non-mutagenic in an Ames test). For a 40 mg daily dose of afatinib, the limit would be 1.5 ug/day / 40 mg = 37.5 ppm (0.00375%), which is much stricter than the standard identification threshold. An Ames test (TA98, TA100, TA1535, TA1537, WP2 uvrA with and without S9) must be performed. If negative, it can be controlled at 0.15%; if positive, strict ppm control is required. In a 28-day oral toxicity study, the predicted NOAEL is 100 mg/kg/day if genotoxicity is ruled out.
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| Additional Infomation |
Appearance: white to off-white solid powder. Molecular formula: C24H2₆FN₅O3. Storage: powder at -20degC (3 years) or 4degC (2 years); in solvent at -80degC (6 months) or -20degC (1 month), protect from light and air. Solubility: soluble in DMSO and DMF; sparingly soluble in ethanol; practically insoluble in water. The compound is typically analyzed by reversed-phase HPLC with UV detection at 254 nm or by LC-MS/MS in positive ion mode. Other names: (S,E)-4-(Dimethylamino)-N-(4-((3-fluorophenyl)amino)-7-((tetrahydrofuran-3-yl)oxy)quinazolin-6-yl)but-2-enamide; Afatinib des-chloro impurity; Afatinib impurity 6. Safety: potential genotoxic impurity; handle with care in a fume hood.
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| Molecular Formula |
C16H14CLFN4O
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| Molecular Weight |
332.77
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| Exact Mass |
332.084
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| CAS # |
1269662-90-9
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| PubChem CID |
66584354
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| Appearance |
Off-white to yellow solid powder
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| Hydrogen Bond Donor Count |
2
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
23
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| Complexity |
391
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CCOC1=C(C=C2C(=C1)N=CN=C2NC3=CC(=C(C=C3)F)Cl)N
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| InChi Key |
PDLCDTRXIQXHIL-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C16H14ClFN4O/c1-2-23-15-7-14-10(6-13(15)19)16(21-8-20-14)22-9-3-4-12(18)11(17)5-9/h3-8H,2,19H2,1H3,(H,20,21,22)
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| Chemical Name |
4-N-(3-chloro-4-fluorophenyl)-7-ethoxyquinazoline-4,6-diamine
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~25 mg/mL (~75.13 mM; with sonication)
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| 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
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] 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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.0051 mL | 15.0254 mL | 30.0508 mL | |
| 5 mM | 0.6010 mL | 3.0051 mL | 6.0102 mL | |
| 10 mM | 0.3005 mL | 1.5025 mL | 3.0051 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.
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.
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