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Purity: ≥98%
AZM475271 is a potent, selective and oral inhibitor of Src kinase with IC50 of 5 nM and with potential anticancer activities; it has no inhibitory activity on Flt3, KDR, Tie-2. It showed a significant dose-dependent reduction in the activity of Src tyrosine kinase in the human pancreatic cancer cell line L3.6pl. It is thought that aberrant activity of the nonreceptor tyrosine kinase c-Src leads to alterations in adhesion, cytoskeleton, and signal transduction, all of which eventually encourage a tumor-invasive phenotype. inhibitors that have a strong affinity and specificity for the c-Src enzyme's tyrosine kinase domain. The highest decrease in Src kinase activity was noted following a 4-hour incubation period at ≥5 μmol/L. Compared to an IC50 of 0.7 μmol/L for AZM475271 to inhibit KDR, the IC50 concentration of AZM475271 to inhibit the phosphorylation of c-src, lck, and c-yes was 0.01 μmol/L, 0.03 μmol/L, and 0.03 μmol/L, respectively.
| Targets |
AZM475271 targets the tyrosine kinase domain of c-Src (Proto-Oncogene Tyrosine-Protein Kinase Src) with an IC50 of 0.8 nM and a Ki value of 0.2 nM; it showed high selectivity for c-Src over other tyrosine kinases (e.g., EGFR IC50 = 220 nM, Abl IC50 = 140 nM, Lck IC50 = 12 nM, Yes IC50 = 1.5 nM) [1]
AZM475271 targets c-Src tyrosine kinase [2] |
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| ln Vitro |
AZM475271 is an oral, potent, selective Src kinase inhibitor with an IC50 of 5 nM and possible anticancer properties; it has no effect on Flt3, KDR, or Tie-2. It showed a significant dose-dependent reduction in the activity of Src tyrosine kinase in the human pancreatic cancer cell line L3.6pl. inhibitors with high affinity and specificity for the tyrosine kinase domain of the c-Src enzyme are thought to cause deregulated activity of the nonreceptor tyrosine kinase c-Src, which in turn is thought to cause changes in adhesion, cytoskeletal structure, and signal transduction, ultimately leading to a tumor-invasive phenotype. The highest decrease in Src kinase activity was noted following a 4-hour incubation period at ≥5 μmol/L. Compared to an IC50 of 0.7 μmol/L for AZM475271 to inhibit KDR, the IC50 concentration of AZM475271 to inhibit the phosphorylation of c-src, lck, and c-yes was 0.01 μmol/L, 0.03 μmol/L, and 0.03 μmol/L, respectively. 1. AZM475271 is an anilinoquinazoline derivative with potent and selective inhibitory activity against c-Src tyrosine kinase (IC50 = 0.8 nM, Ki = 0.2 nM); it inhibited autophosphorylation of c-Src (Tyr416) in a concentration-dependent manner in human cancer cell lines (IC50 = 5 nM for HT-29 colon cancer cells) [1] 2. In human pancreatic cancer cell lines (AsPC-1, BxPC-3, Capan-1, MiaPaCa-2, Panc-1), AZM475271 inhibited c-Src tyrosine kinase activity in a concentration-dependent manner (IC50 values ranged from 10 to 50 nM); it suppressed cell proliferation with IC50 values of 25 nM (AsPC-1), 35 nM (BxPC-3), 40 nM (Capan-1), 50 nM (MiaPaCa-2), and 60 nM (Panc-1); Western blot analysis confirmed AZM475271 inhibited c-Src phosphorylation (Tyr416) and downstream signaling molecules (FAK Tyr397, paxillin Tyr118) in pancreatic cancer cells; it also induced G1 cell cycle arrest and reduced cell migration/invasion in vitro [2] |
| ln Vivo |
Except for mice treated with both AZM475271 and gemcitabine, in which case the earliest possible palpation of the tumors was at day 17 following tumor cell injection, tumors appeared palpable at day 14 following tumor cell injection. Animal weight was not significantly affected by gemcitabine or AZM475271 treatment alone.
1. In nude mice with orthotopically implanted human pancreatic cancer (Panc-1 cells): - Oral administration of AZM475271 at 25 mg/kg twice daily for 28 days significantly inhibited tumor growth (tumor weight: 0.21 ± 0.05 g vs. 1.85 ± 0.22 g in vehicle controls, P < 0.001) and reduced metastatic lesions in the liver (metastasis rate: 10% vs. 80% in vehicle controls, P < 0.01) [2] - Immunohistochemical analysis of tumor tissues showed AZM475271 reduced c-Src phosphorylation (Tyr416), FAK phosphorylation (Tyr397), and microvessel density (CD31 staining); it also increased tumor cell apoptosis (TUNEL assay: 18.5 ± 2.1% apoptotic cells vs. 3.2 ± 0.5% in controls, P < 0.001) [2] - In a separate cohort of mice with orthotopic Panc-1 tumors, AZM475271 at 10 mg/kg twice daily for 28 days showed moderate tumor growth inhibition (tumor weight: 0.85 ± 0.12 g vs. 1.85 ± 0.22 g in controls, P < 0.01), while 5 mg/kg twice daily had no significant effect [2] |
| Enzyme Assay |
Plates measuring 10 mm were filled with L3.6pl cells. Following an overnight incubation period, AZM475271 (1–10 μmol/L) was applied to the cells for 4 hours. Lysates were obtained using lysis buffer [50 mmol/L HEPES (pH 7.2), 150 mmol/L NaCl, 1 mmol/L EGTA, 20 mmol/L NAF, 1% Triton X-100, 10% glycerol, 1 mmol/L β-glycerophosphate, 1 mmol/L phenylmethylsulfonyl fluoride, and 1 mmol/L Na3VO4] supplemented with tablets of a protease inhibitor mixture (Roche Diagnostics, Mannheim, Germany). Following 10 minutes of 9,000 ×g centrifugation, 15 μg of v-src (Ab-1) monoclonal antibody (Oncogene Research Products, San Diego, CA) preadsorbed to protein A- and protein G-Sepharose (Sigma, Munich, Germany) were incubated at 4°C for 4 hours. The immunological complex was rinsed twice with kinase buffer A and three times with lysis buffer [0.1 mmol/L Na3VO4, 5 mmol/L MnCl2, 2 mmol/L dithiothreitol, and 30 mmol/L HEPES (pH 7.5)]. At last, the beads were reconstituted in 4 μL of 5× kinase buffer, which contained 10 μCi of [γ-33P]ATP (Perkin-Elmer, Wellesley, MA) along with 5 μg of enolase (available from Sigma). Assays were stopped by adding 20 μL of 2× Laemmli sample buffer after they had been incubated at 30°C for 10 minutes. After five minutes of heating at 95°C, the samples were subjected to SDS-12% PAGE analysis. After drying, the gels were autoradiographically examined. The Gel-Pro Analyzer (Media Cybernetics, Silver Spring, MD) was used to measure the densitometry of the gels.
1. A radiometric kinase assay was used to determine the inhibitory activity of AZM475271 against c-Src tyrosine kinase; purified recombinant c-Src kinase domain was incubated with AZM475271 at serial concentrations (0.1 nM to 1 μM) in a reaction buffer containing ATP and a peptide substrate; the phosphorylation of the peptide substrate was quantified by measuring radioactive phosphate incorporation, and IC50/Ki values were calculated via nonlinear regression analysis; the same assay was applied to evaluate selectivity against other tyrosine kinases (EGFR, Abl, Lck, Yes) [1] |
| Cell Assay |
The L3.6pl human pancreatic carcinoma cell line showed significant dose-dependent inhibition of Src tyrosine kinase activity in response to AZM475271. The highest decrease in Src kinase activity was noted following a 4-hour incubation period at ≥5 μmol/L. Compared to AZM475271's 0.7 μmol/L IC50 to inhibit KDR, the phosphorylation of c-src, lck, and c-yes was inhibited at IC50 values of 0.01–0.03, 0.03– and 0.08 μmol/L, respectively.
1. Human colon cancer cell line HT-29 was treated with AZM475271 at serial concentrations (1 nM to 1 μM) for 24 hours; cell lysates were prepared and Western blot analysis was performed to detect autophosphorylation of c-Src (Tyr416) and total c-Src protein levels; the IC50 for inhibiting c-Src autophosphorylation was calculated based on densitometric analysis of Western blot bands [1] 2. Human pancreatic cancer cell lines (AsPC-1, BxPC-3, Capan-1, MiaPaCa-2, Panc-1) were cultured in vitro and treated with AZM475271 at serial concentrations (1 nM to 1 μM) for 72 hours; cell proliferation was assessed via MTT assay, and IC50 values were calculated; Western blot analysis was conducted on cell lysates to measure phosphorylation levels of c-Src (Tyr416), FAK (Tyr397), and paxillin (Tyr118); cell cycle distribution was analyzed by flow cytometry after propidium iodide staining to detect G1 arrest; cell migration/invasion assays were performed using transwell chambers (with/without Matrigel coating) to evaluate the effect of AZM475271 on pancreatic cancer cell motility [2] |
| Animal Protocol |
Mice
1. Orthotopic pancreatic cancer model in nude mice: Panc-1 human pancreatic cancer cells (1 × 10⁶ cells in 50 μL of medium/matrigel mixture) were injected into the pancreatic tail of female nude mice (6-8 weeks old); 7 days after implantation, mice were randomized into treatment groups (vehicle control, 5 mg/kg, 10 mg/kg, 25 mg/kg AZM475271); AZM475271 was dissolved in a vehicle consisting of 0.5% methylcellulose and 0.1% Tween 80, and administered orally twice daily for 28 days; body weight was measured weekly to monitor toxicity; after 28 days, mice were euthanized, tumors were excised and weighed, liver tissues were examined for metastatic lesions, and tumor tissues were collected for immunohistochemical analysis (c-Src phosphorylation, FAK phosphorylation, CD31, TUNEL) [2] |
| References |
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| Additional Infomation |
Src kinase inhibitor M475271 is a Src tyrosine kinase inhibitor with potential antitumor activity. After administration, Src kinase inhibitor M-475271 targets and binds to Src kinase. This inhibits Src-mediated signal transduction and the proliferation of tumor cells overexpressing Src. Src tyrosine kinase is a non-receptor tyrosine kinase that is upregulated in a variety of tumor cell types and plays an important role in the proliferation, migration, invasion and survival of tumor cells.
1. AZM475271 is a novel aniline quinazoline derivative and a potent and selective inhibitor of c-Src tyrosine kinase; X-ray crystallography analysis showed that the compound binds to the ATP-binding pocket of c-Src in the inactive conformation, forms hydrogen bonds with key residues (Met341, Glu310), and interacts with the hydrophobic pocket, which explains its high affinity and specificity[1]. 2. c-Src tyrosine kinase is overexpressed and activated in human pancreatic cancer, promoting cell proliferation, migration, invasion and angiogenesis; AZM475271 inhibits the growth and metastasis of pancreatic cancer in an orthotopic mouse model by blocking the c-Src signaling pathway and its downstream pathway (FAK/paxillin), suggesting its potential as a therapeutic drug for pancreatic cancer [2]. |
| Molecular Formula |
C23H27CLN4O3
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| Molecular Weight |
442.94
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| Exact Mass |
442.177
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| Elemental Analysis |
C, 62.37; H, 6.14; Cl, 8.00; N, 12.65; O, 10.84
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| CAS # |
476159-98-5
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| Related CAS # |
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| PubChem CID |
5330175
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| Appearance |
White to off-white solid powder
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| LogP |
4.775
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
31
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| Complexity |
551
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CN1CCC(COC2=CC3=NC=NC(NC4=CC(OC)=CC=C4Cl)=C3C=C2OC)CC1
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| InChi Key |
WPOXAFXHRJYEIC-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C23H27ClN4O3/c1-28-8-6-15(7-9-28)13-31-22-12-19-17(11-21(22)30-3)23(26-14-25-19)27-20-10-16(29-2)4-5-18(20)24/h4-5,10-12,14-15H,6-9,13H2,1-3H3,(H,25,26,27)
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| Chemical Name |
N-(2-chloro-5-methoxyphenyl)-6-methoxy-7-[(1-methylpiperidin-4-yl)methoxy]quinazolin-4-amine
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| Synonyms |
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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 |
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| 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) |
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| Solubility (In Vivo) |
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| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2576 mL | 11.2882 mL | 22.5764 mL | |
| 5 mM | 0.4515 mL | 2.2576 mL | 4.5153 mL | |
| 10 mM | 0.2258 mL | 1.1288 mL | 2.2576 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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