AZ14289671 (1 μM) exhibited high kinase selectivity, with only a few non-EGFR family kinases showing inhibition rates exceeding 50%[1]. AZ14289671 (2 h) effectively inhibited EGFR phosphorylation in the EGFRExon20Ins cell line (mean IC50 = 17-41 nM), but its activity was significantly reduced in the EGFRWT cell line (mean IC50 = 480-832 nM), exhibiting high mutant selectivity[1]. AZ14289671 (2 h) effectively inhibited ERK phosphorylation in LXF2478ASV EGFR Exon20Ins cells, with a mean IC50 of 32 nM[1]. AZ14289671 (10-1000 nmol/L; 2 h, 6 h) effectively inhibited MAPK pathway signaling (including phosphorylation of ERK, AKT and S6) in LXF2478ASV EGFRExon20Ins cells after treatment for 2 h and 6 h [1]. AZ14289671 (30-1000 nM; 6 h) dose-dependently inhibited gene expression of the MAPK pathway in LXF2478ASV EGFR exon 20 insertion mutant cells [1]. AZ14289671 (2 hours) effectively inhibited EGFR phosphorylation in EGFRExon20Ins/T790M, EGFRL858R, EGFRL858R/T790M and EGFREx19del cell lines (mean IC50 of 7-45 nM), and also inhibited HER2 phosphorylation in BT-474HER2WT and H2170HER2YVMA cells, with mean IC50 values of 75 nM and 125 nM, respectively [1]. AZ14289671 (1 μM; 2 hours) showed a low tendency to act as a substrate for efflux transporters, with an efflux ratio of 1.5 in MDCKII-MDR1-BCRP cells and 4.7 in MCKII-MDR1 cells [1].

AZ14289671 (6.25-50 mg/kg; orally; twice daily for 14 days) induced dose-dependent tumor growth inhibition in the LXF2478ASV xenograft mouse model [1]. AZ14289671 (6.25-50 mg/kg; orally; twice daily for 14 days) showed significantly lower tumor growth inhibition activity (maximum tumor growth inhibition rate of 62%) and EGFR phosphorylation inhibition in the H2073WT xenograft mouse model [1]. AZ14289671 (50 mg/kg; orally; twice daily for 16 days) induced 86% tumor growth inhibition in the A431WT xenograft mouse model [1]. AZ14289671 (6.25-50 mg/kg; orally; twice daily for 28 days) induced dose-dependent tumor growth inhibition in the LU3075DNP xenograft mouse model [1]. AZ14289671 (25-50 mg/kg; orally; twice daily for 28 days) induced dose-dependent tumor growth inhibition in the LU0387NPH xenograft mouse model [1]. AZ14289671 (2 μmol/kg/h; intravenously; continuous infusion; 4 hours) crossed the blood-brain barrier in healthy rats with a Kpuu value of 0.17 [1].

Western Blot Analysis [1]
Cell Types: LXF2478ASV EGFRExon20Ins Cell Line
Tested Concentrations: 10, 30, 100, 300 and 1000 nM
Incubation Duration: 2 hours; 6 hours
Experimental Results: After treatment for 2 hours and 6 hours, downstream EGFR signaling pathways were inhibited, with the inhibition of ERK phosphorylation being the most significant. AKT and S6 phosphorylation were also significantly inhibited in a dose-dependent manner.

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Real-time quantitative PCR[1]
Cell Types: LXF2478ASV EGFRExon20Ins cell line
Tested Concentrations: 30, 100, 300 and 1000 nM
Incubation Duration: 6 hours
Experimental Results: At concentrations of 30 nM and above, MAPK pathway gene expression was significantly inhibited in a dose-dependent manner. Significant downregulation of the expression of CCND1, DUSP6, EPHA2, EPHA4, ETV5 and SPRY4 was observed, consistent with the reduced MAPK signaling pathway activity shown in the immunoblotting results.

Animal/Disease Models:Hsd: Athymic nude mice - Foxn1nu (female; subcutaneous transplantation of EGFR exon 20 insertion mutant cells derived from LXF2478ASV patients) [1]
Doses: 6.25 mg/kg (twice daily); 12.5 mg/kg (twice daily; single dose); 25 mg/kg (twice daily; single dose); 50 mg/kg (twice daily; single dose)
Route of Administration: Oral; twice daily; 14 days; oral; single dose
Experimental Results: The 6.25 mg/kg dose group achieved 71% target drug concentration (TGI), the 12.5 mg/kg dose group achieved 81% TGI, the 25 mg/kg dose group achieved 112% TGI, and the twice daily dose group achieved 121% TGI after 14 days. All dosage groups showed a significant reduction in EGFR phosphorylation levels at 1 hour and 6 hours after a single dose, with the 50 mg/kg dose showing a reduction of over 90% at 6 hours. EGFR phosphorylation levels recovered at 16 hours and 24 hours after a single dose. Plasma free drug concentrations remained above IC90 for approximately 6 hours after a single dose.

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Animal/Disease Models:CB-17/lcrHanHsd-Prkdcscid (female; H2073WT EGFR wild-type cell subcutaneous xenograft) [1]
Doses: 6.25 mg/kg (twice daily); 12.5 mg/kg (twice daily; single dose); 25 mg/kg (twice daily; single dose); 50 mg/kg (twice daily; single dose)
Route of Administration: Oral; twice daily; 14 days; oral; single dose
Experimental Results: The TGI reached 47% at the 6.25 mg/kg dose, 53% at the 12.5 mg/kg dose, 55% at the 25 mg/kg dose, and 62% after 14 days of twice daily administration. Compared with the mutant model, the inhibition of EGFR phosphorylation was significantly reduced after a single dose, with only about 50% inhibition at a dose of 50 mg/kg 6 hours after administration. After a single dose, the free plasma concentrations in all dose groups did not exceed the IC50.

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Animal/Disease Models:CB-17/lcrHanHsd-Prkdcscid (female; subcutaneous xenograft of A431WT EGFR wild-type cells) [1]
Doses: 50 mg/kg
Route of Administration: Oral; twice daily; 16 days
Experimental Results: After 16 days of twice-daily administration, the TGI reached 86%. The drug was well tolerated, with a weight loss of <15%.

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Animal/Disease Models:BALB/cNj-Foxn1nu/Gpt (female; subcutaneous transplantation of EGFR D770_N771InsPG exon 20 insertion mutant cells derived from LU3075DNP patients) [1]
Doses: 6.25 mg/kg (twice daily); 12.5 mg/kg (twice daily; single dose); 25 mg/kg (twice daily; single dose); 50 mg/kg (twice daily; single dose)
Route of Administration: Oral; twice daily; 28 days; oral; single dose
Experimental Results: The TGI reached 27% at the 6.25 mg/kg dose, 81% at the 12.5 mg/kg dose, 159% at the 25 mg/kg dose, and 169% after 28 days of twice daily administration. Following a single dose, all dose levels significantly inhibited EGFR phosphorylation, with inhibition rates exceeding 75% at 1 hour and 6 hours after single administration of 25 mg/kg and 50 mg/kg.

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Animal/Disease Models:BALB/cNj-Foxn1nu/Gpt (female; EGFR N771_H773InsH exon 20 insertion mutant cells derived from LU0387NPH patients, subcutaneous xenograft) [1]
Doses: 25 mg/kg; 50 mg/kg
Route of Administration: Oral; twice daily; 28 days
Experimental Results: After 28 days of administration twice daily, the TGI of the 25 mg/kg dose group reached 45%, and the TGI of the 50 mg/kg dose group reached 133%.

[1]. AZ14289671 is a highly selective and blood-brain barrier penetrant irreversible TKI that targets EGFRExon20 insertions. Cell Rep Med. 2025;6(9):102305.

3101563-22-5

Typically exists as solids at room temperature

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

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

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.

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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).

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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)

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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).

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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

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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.

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 (Please use freshly prepared in vivo formulations for optimal results.)