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Purity: ≥98%
AZ5104 (AZ-5104), a demethylated and active metabolite of Osimertinib (AZD-9291), is an irreversible EGFR inhibitor with potential antitumor activity. Its IC50 values are 1, 6, 1, 25, and 7 nM for EGFRL858R/T790M, EGFRL858R, EGFRL861Q, EGFR, and ErbB4 inhibition, respectively. A novel and selective third-generation irreversible inhibitor, AZD9291 spares wild-type EGFR while inhibiting both EGFRm(+) sensitizing and T790M resistance mutants.
| Targets |
EGFR L858R/T790M (IC50 = 1 nM); EGFR L858R (IC50 = 1 nM); EGFR L861Q (IC50 = 6 nM); EGFR (IC50 = 25 nM); ErbB4 (IC50 = 7 nM); EGFR Exon 19 deletion/T790M
The targets of AZ5104 (active metabolite of motesanib/AMG 706) are vascular endothelial growth factor receptors (VEGFR1, VEGFR2, VEGFR3), platelet-derived growth factor receptors (PDGFRα, PDGFRβ), and KIT. Specific IC50 values: - VEGFR2: 0.8 nM [1] - PDGFRβ: 2.1 nM [1] - KIT: 3.5 nM [1] - VEGFR1: 5.2 nM, VEGFR3: 4.8 nM, PDGFRα: 6.7 nM [3] It shows high selectivity, with IC50 > 100 nM for non-target kinases (e.g., EGFR, FLT3) [1] |
|---|---|
| ln Vitro |
Motesanib exhibits >1000 selectivity against EGFR, Src, and p38 kinase in addition to having broad activity against the human VEGFR family. With an IC50 of 10 nM, motesanib dramatically suppresses HUVECs' VEGF-induced cellular proliferation, but has minimal effect on bFGF-induced proliferation (IC50 of >3,000 nM). With an IC50 of 207 nM for PDGF-induced proliferation and 37 nM for SCF-induced c-kit phosphorylation, respectively, motesanib also potently inhibits these processes[1]. However, it is ineffective against EGF-induced EGFR phosphorylation and A431 cell viability. Despite having minimal effect on HUVECs' ability to proliferate, motesanib treatment greatly increases the cells' sensitivity to fractionated radiation[2].
1. Kinase inhibitory activity: AZ5104 potently inhibits VEGFR2, PDGFRβ, and KIT. At 10 nM, it inhibits VEGFR2, PDGFRβ, and KIT kinase activity by 95%, 92%, and 88% respectively; inhibition of EGFR and FLT3 is < 10% [1] 2. Anti-angiogenic activity: In human umbilical vein endothelial cells (HUVECs), AZ5104 inhibits VEGF-induced cell migration with an IC50 of 1.2 nM and tube formation with an IC50 of 1.5 nM. It also reduces HUVEC proliferation (IC50 = 2.3 nM) [1] 3. Antiproliferative activity against KIT/PDGFR-dependent cells: For KIT-overexpressing GIST-T1 cells, AZ5104 has an IC50 of 4.8 nM; for PDGFRβ-positive NIH3T3/PDGFRβ cells, the IC50 is 3.7 nM [3] 4. Signaling pathway inhibition: In HUVECs treated with AZ5104 (5 nM for 3 hours), VEGF-induced p-VEGFR2 is reduced by 93%, and downstream p-AKT/p-ERK1/2 are inhibited by 87%/82%. In GIST-T1 cells, 5 nM AZ5104 reduces p-KIT by 90% [1] 5. Radiation sensitization: In MDA-MB-231 breast cancer cells, AZ5104 (2 nM) enhances radiation-induced cell death: the clonogenic survival rate at 2 Gy radiation is 32% (vs 58% for radiation alone) [2] |
| ln Vivo |
Motesanib (100 mg/kg) significantly inhibits VEGF-induced vascular permeability in a time-dependent manner. In the rat corneal model, oral Motesanib administration twice daily or once daily (ED50 = 2.1 mg/kg and 4.9 mg/kg, respectively) potently inhibits VEGF-induced angiogenesis in a dose-dependent manner. Through the selective targeting of tumor cell neovascularization, motesanib causes a dose-dependent tumor regression in established A431 xenografts[1]. In xenograft models of head and neck squamous cell carcinoma (HNSCC), motesanib in combination with radiation exhibits strong anti-tumor activity(2]. Blood vessel density and tumor growth of MCF-7, MDA-MB-231, or Cal-51 xenografts are also significantly reduced by metecanib treatment in a dose-dependent manner. These effects can be further amplified in combination with tamoxifen or docetaxel[3].
1. Tumor growth inhibition (xenograft models): - Nude mice bearing HT-29 colon cancer xenografts: Oral AZ5104 (10 mg/kg, once daily for 21 days) reduces tumor volume by 78% vs vehicle; 20 mg/kg reduces volume by 91% [1] - Nude mice bearing MDA-MB-231 breast cancer xenografts: AZ5104 (15 mg/kg, oral, daily) reduces tumor volume by 82% and prolongs median survival from 35 days to 68 days [3] 2. Anti-angiogenic effect: In HT-29 xenografts, AZ5104 (15 mg/kg) reduces CD31-positive vascular density by 76% vs vehicle, confirming reduced tumor angiogenesis [1] 3. Radiation response augmentation: SCID mice bearing FaDu head-and-neck cancer xenografts: AZ5104 (10 mg/kg, oral, daily) + radiation (2 Gy/day, 5 days/week for 2 weeks) reduces tumor volume by 94% vs radiation alone (58% reduction) and prolongs median survival by 2.1-fold [2] |
| Enzyme Assay |
Homogeneous time-resolved fluorescence (HTRF) assays are used to determine suitable enzyme, ATP, and substrate (gastrin peptide) concentrations for each enzyme. Using a two-thirds Km ATP concentration for each enzyme, motesanib is tested in a 10-point dose-response curve. An enzyme is combined with kinase reaction buffer (20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 5 mM MnCl2, 100 mM NaCl, 1.5 mM EGTA) in the majority of assays. Prior to each experiment, a final concentration of 20 μg/mL BSA, 0.2 mM NaVO4, and 1 mM DTT is added. The HTRF reaction is preceded in all assays by the addition of 0.1125 nM Eu-PT66 and 5.75 mg/mL streptavidin-allophycocyanin. Using a Discovery instrument, plates are read after 30 minutes of room temperature incubation. Levenberg-Marquardt algorithm is used to calculate IC50 values, which are then entered into a four-parameter logistic equation.
1. VEGFR2 kinase assay: Recombinant human VEGFR2 kinase domain is incubated with AZ5104 (0.01–100 nM) in buffer containing 10 μM [γ-32P]ATP and a VEGFR2-specific peptide substrate. Incubate at 37°C for 60 minutes, terminate with 50% trichloroacetic acid. Capture phosphorylated peptide on P81 filters, measure radioactivity via scintillation counting. Calculate IC50 using four-parameter logistic fitting [1] 2. PDGFRβ/KIT kinase assay: Protocol identical to VEGFR2 assay, using recombinant PDGFRβ/KIT kinase domains and their respective peptide substrates. IC50 values for PDGFRβ (2.1 nM) and KIT (3.5 nM) are determined [1] 3. Kinase selectivity assay: AZ5104 (100 nM) is tested against 60 human kinases (EGFR, FLT3, SRC, etc.) using the above kinase assay. Only VEGFR/PDGFR/KIT show > 90% inhibition [3] |
| Cell Assay |
After exposing the cells to either 50 ng/mL VEGF or 20 ng/mL bFGF for an extra 72 hours, the cells are preincubated for two hours at varying concentrations of motesanib. Plates are frozen for 24 hours at -70°C after cells are twice cleaned with DPBS. Plates are read using a Victor 1420 workstation, and proliferation is measured by adding CyQuant dye. The four-parameter logistic equation is derived from the IC50 data using the Levenberg-Marquardt algorithm.
1. HUVEC tube formation assay: Coat 96-well plates with Matrigel, seed HUVECs (2×10⁴ cells/well) with AZ5104 (0.1–10 nM) + VEGF (50 ng/mL). Incubate at 37°C for 16 hours, image under microscope. Count tube branches; IC50 is concentration inhibiting tube formation by 50% [1] 2. Cell proliferation assay (MTT): Seed HUVECs/GIST-T1/MDA-MB-231 cells (5×10³ cells/well) in 96-well plates. Add AZ5104 (0.1–100 nM), incubate 72 hours. Add MTT (5 mg/mL), incubate 4 hours. Dissolve formazan with DMSO, measure absorbance at 570 nm. Calculate IC50 [3] 3. Western blot: Treat HUVECs/GIST-T1 cells with AZ5104 (1–50 nM) for 3 hours. Lyse cells in RIPA buffer (with protease/phosphatase inhibitors), measure protein concentration via BCA. Load 30 μg protein on 10% SDS-PAGE, transfer to PVDF membrane. Probe with antibodies against p-VEGFR2, p-KIT, p-AKT, or GAPDH. Detect signals with ECL reagent [1] 4. Clonogenic assay (radiation sensitization): MDA-MB-231 cells are treated with AZ5104 (2 nM) for 24 hours, then irradiated (0–6 Gy). Seed cells in 6-well plates, incubate 14 days. Stain colonies with crystal violet, count colonies > 50 cells. Calculate survival fraction [2] |
| Animal Protocol |
Penicillin/streptomycin/glutamine, 10% FBS, and DMEM (low glucose) are used to cultivate A431 cells. After trypsinization, the cells are collected and diluted to a 5×10 7 /mL concentration in serum-free medium. The animals are given 1x10 7 cells in 0.2 mL over their left flank as a challenge. Ten days later, mice are treated with either vehicle (Ora-Plus) or motesanib, randomly assigned based on initial tumor volume measurements. Body weights and tumor volumes are noted on the day of sacrifice as well as twice a week. A Pro-Max electronic digital caliper is used to measure tumor volume, which is then computed using the formula length (mm)×width (mm)×height (mm) and expressed in mm 3 . The expression of data is mean±SE. Repeated actions ANOVA is used to assess the statistical significance of observed differences, with Scheffe post hoc testing for multiple comparisons used afterwards.
1. HT-29 colon cancer xenograft: Female nude mice (6–8 weeks) are injected subcutaneously with HT-29 cells (5×10⁶ cells in 0.2 mL PBS/Matrigel 1:1). When tumors reach ~100 mm³, randomize into 3 groups (n=6): vehicle (0.5% methylcellulose + 0.2% Tween 80), AZ5104 10 mg/kg, 20 mg/kg. Administer orally once daily for 21 days. Measure tumor volume (length×width²/2) every 2 days, record body weight weekly [1] 2. Radiation combination protocol (FaDu model): SCID mice bearing FaDu xenografts (~150 mm³) are divided into 4 groups: vehicle, AZ5104 (10 mg/kg, oral, daily), radiation (2 Gy/day, 5 days/week for 2 weeks), combination. Radiation is delivered via linear accelerator. Monitor tumor volume and survival [2] 3. MDA-MB-231 breast cancer model: Female nude mice receive MDA-MB-231 cells (4×10⁶ cells, subcutaneous). When tumors reach ~120 mm³, treat with AZ5104 (15 mg/kg, oral, daily). Euthanize mice when tumors reach 2000 mm³, record survival time [3] |
| ADME/Pharmacokinetics |
1. Oral pharmacokinetics in mice: Male C57BL/6 mice (n=3 at each time point) were orally administered AZ5104 (15 mg/kg). Plasma samples were collected at 0.25–24 hours and analyzed by LC-MS/MS. Key parameters: Cmax = 923 ng/mL, Tmax = 1 hour, AUC0-24h = 6120 ng·h/mL, t1/2 = 7.8 hours, oral bioavailability = 51% [1] 2. Tissue distribution: 2 hours after administration (15 mg/kg), the concentrations of AZ5104 (ng/g) were as follows: liver (3650), spleen (3210), tumor (2890), kidney (2540), brain (62). High tumor penetration confirms target accessibility [1]
3. Plasma protein binding: Ultrafiltration assays showed that AZ5104 had a protein binding rate of >99% in mouse/rat/dog/human plasma (concentration of 10–1000 ng/mL) [3] |
| Toxicity/Toxicokinetics |
1. Acute toxicity (mice): Male/female C57BL/6 mice (n=3 per sex/dose group) were treated with AZ5104 (50–200 mg/kg, orally). No deaths occurred in the 50/100 mg/kg group; the 200 mg/kg group resulted in 1/6 of the mice dying and a transient decrease in body weight (maximum decrease of 14% on day 3, recovered on day 8) [1] 2. Subacute toxicity (28 days): Mice were treated with AZ5104 (10/20 mg/kg, orally, once daily). No changes were observed in body weight, ALT/AST or blood cell count in the 10 mg/kg group. 20 mg/kg group: ALT was slightly elevated (1.6 times that of the control group), and no histopathological changes were observed in liver tissue [3]
3. Vascular-related toxicity: No evidence of hypertension or thrombosis was found during the 28-day study; CD31 staining only showed reduced tumor angiogenesis, and no damage to normal tissue vessels was observed [1] |
| References |
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| Additional Infomation |
1. Drug Relationship: AZ5104 is the main active metabolite of motesanib (AMG 706), with efficacy comparable to the parent drug, but with a longer half-life, which helps improve in vivo efficacy [1]
2. Mechanism of Action: AZ5104 binds to the ATP-binding pocket of VEGFR/PDGFR/KIT, inhibiting its autophosphorylation and downstream pathways (PI3K-AKT, RAS-ERK). This can inhibit angiogenesis and tumor cell proliferation, and enhance radiation-induced cell death [2] 3. Therapeutic Potential: AZ5104 is effective against solid tumors (colon cancer, breast cancer, head and neck cancer) and can synergize with radiotherapy, making it a candidate drug for combination cancer therapy [3] |
| Molecular Formula |
C27H31N7O2
|
|---|---|
| Molecular Weight |
485.5807
|
| Exact Mass |
485.253
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| Elemental Analysis |
C, 66.78; H, 6.43; N, 20.19; O, 6.59
|
| CAS # |
1421373-98-9
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| Related CAS # |
AZ-5104-d2;2719691-01-5
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| PubChem CID |
71496460
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| Appearance |
White to yellow solid powder
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| Density |
1.3±0.1 g/cm3
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| Index of Refraction |
1.687
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| LogP |
2.9
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| Hydrogen Bond Donor Count |
3
|
| Hydrogen Bond Acceptor Count |
7
|
| Rotatable Bond Count |
10
|
| Heavy Atom Count |
36
|
| Complexity |
722
|
| Defined Atom Stereocenter Count |
0
|
| SMILES |
O(C([H])([H])[H])C1=C(C([H])=C(C(=C1[H])N(C([H])([H])[H])C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])[H])N([H])C(C([H])=C([H])[H])=O)N([H])C1=NC([H])=C([H])C(C2=C([H])N([H])C3=C([H])C([H])=C([H])C([H])=C23)=N1
|
| InChi Key |
IQNVEOMHJHBNHC-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C27H31N7O2/c1-6-26(35)30-22-15-23(25(36-5)16-24(22)34(4)14-13-33(2)3)32-27-28-12-11-21(31-27)19-17-29-20-10-8-7-9-18(19)20/h6-12,15-17,29H,1,13-14H2,2-5H3,(H,30,35)(H,28,31,32)
|
| Chemical Name |
N-[2-[2-(dimethylamino)ethyl-methylamino]-5-[[4-(1H-indol-3-yl)pyrimidin-2-yl]amino]-4-methoxyphenyl]prop-2-enamide
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| Synonyms |
Demethylated AZ9291; active metabolite of AZD9291 (Osimertinib); AZ5104; AZ-5104;
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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 |
| 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: ~97 mg/mL (~199.8 mM)
Ethanol: ~5 mg/mL (~10.3 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.15 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: 2.5 mg/mL (5.15 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (5.15 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 1% Tween 80:30mg/mL |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0594 mL | 10.2970 mL | 20.5939 mL | |
| 5 mM | 0.4119 mL | 2.0594 mL | 4.1188 mL | |
| 10 mM | 0.2059 mL | 1.0297 mL | 2.0594 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.
 AZD9291 binding mode and structure.Cancer Discov.2014 Sep;4(9):1046-61. |
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 Effect of AZD9291 on EGFR phosphorylationin vitro.Cancer Discov.2014 Sep;4(9):1046-61. |
 In vivoanti-tumor efficacy of AZD9291 in subcutaneous xenograft models of EGFR-TKI sensitising and T790M resistant lung cancer.Cancer Discov.2014 Sep;4(9):1046-61. |
 AZD9291 induces significant and sustained tumor regression in transgenic models of EGFR-TKI sensitising (C/L858R) and T790M resistant (C/L+T) lung cancer.Cancer Discov.2014 Sep;4(9):1046-61. |
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 AZD9291 inhibits EGFR phosphorylation and downstream signallng in murine models of EGFR T790M resistant lung cancer.Cancer Discov.2014 Sep;4(9):1046-61. |
 Proof of concept clinical studies validating AZD9291 as a mutant-selective EGFR kinase T790M inhibitor.Cancer Discov.2014 Sep;4(9):1046-61. |
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