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Ifebemtinib tosylate (BI-853520; IN10018 tosylate) is novel, potent and highly selective focal adhesion kinase (FAK) inhibitor (recombinant FAK IC50=1 nM) and PTK2 kinase inhibitor with anti-tumor activity.
Physicochemical Properties
| Molecular Formula | C35H36F4N6O7S |
| Molecular Weight | 760.75 |
| Exact Mass | 588.21 |
| Elemental Analysis | C, 55.26; H, 4.77; F, 9.99; N, 11.05; O, 14.72; S, 4.21 |
| CAS # | 2761021-80-9 |
| Related CAS # | 1227948-82-4 |
| Appearance | White to off-white solid powder |
| SMILES | O=C(NC1CCN(C)CC1)C2=CC(OC)=C(NC3=NC=C(C(F)(F)F)C(OC4=CC=CC(CN5C)=C4C5=O)=N3)C=C2F.OS(=O)(C6=CC=C(C)C=C6)=O |
| InChi Key | LOKCCATVJFYTFP-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C28H28F4N6O4.C7H8O3S/c1-37-9-7-16(8-10-37)34-24(39)17-11-22(41-3)20(12-19(17)29)35-27-33-13-18(28(30,31)32)25(36-27)42-21-6-4-5-15-14-38(2)26(40)23(15)21;1-6-2-4-7(5-3-6)11(8,9)10/h4-6,11-13,16H,7-10,14H2,1-3H3,(H,34,39)(H,33,35,36);2-5H,1H3,(H,8,9,10) |
| Chemical Name | 2-fluoro-5-methoxy-4-((4-((2-methyl-3-oxoisoindolin-4-yl)oxy)-5-(trifluoromethyl)pyrimidin-2-yl)amino)-N-(1-methylpiperidin-4-yl)benzamide tosylate |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets | Focal adhesion kinase (FAK) (recombinant FAK IC₅₀ = 1 nM) |
| ln Vitro |
1. FAK Inhibition and Cell Proliferation: In PC-3 prostate carcinoma cells, BI-853520 inhibits FAK autophosphorylation at Tyr³⁹⁷ with an IC₅₀ of 1 nM and blocks anchorage-independent colony formation with an EC₅₀ of 3 nM. Western blot analysis confirms dose-dependent reduction of p-FAK (Tyr³⁹⁷) and downstream signaling molecules (e.g., Akt, ERK) in treated cells [1][3] 2. 3D Spheroid vs. 2D Monolayer Activity: In 3D spheroid cultures of cancer cells (e.g., PC-3, MDA-MB-231), BI-853520 represses tumor cell proliferation and invasion at low doses (≤3 μM), whereas 2D monolayer cultures require ≥1000-fold higher concentrations to achieve comparable effects [1][3] 3. Breast Cancer Migration Inhibition: In highly metastatic murine breast cancer cells, BI-853520 (0.1 μM) rapidly inhibits FAK phosphorylation and reduces cell migration by 50% within 24 hours, as measured by Transwell assay [2] 4. Mesenchymal Phenotype Correlation: Sensitivity to BI-853520 in vitro is strongly associated with loss of E-cadherin expression and activation of mesenchymal markers (e.g., vimentin, N-cadherin), confirmed by immunofluorescence staining [1][3] Ifebemtinib (BI 853520) (0-3 μM; 2 h) blocks the development of cancer cells[2]. In 3D culture alone, ifebemtinib (BI 853520) (0-30 μM; 4-6 d) suppresses the growth and invasion of tumor cells[1]. Y397-FAK autophosphorylation is suppressed by ifebemtinib (0–10 μM; 24 h)[1]. This highly metastatic murine breast cancer cell line responds quickly and potently to ifebemtinib (0.1 μM; 96 h) in terms of FAK inhibition[1]. |
| ln Vivo |
1. Adenocarcinoma Xenograft Efficacy: In nude mice bearing subcutaneous PC-3 adenocarcinoma xenografts, oral administration of BI-853520 (50 mg/kg daily) reduces tumor volume by 60–80% within 2 weeks, with complete regression in 20% of animals. Efficacy is significantly higher in tumors with mesenchymal phenotype (low E-cadherin, high miR-200c-3p), achieving median tumor growth inhibition (TGI) >100% [1][3] 2. Pharmacodynamic Target Engagement: BI-853520 rapidly penetrates tumor tissue, achieving >90% suppression of p-FAK (Tyr³⁹⁷) within 1 hour of dosing, with sustained target engagement for ≥24 hours, as shown by immunohistochemistry [1][3] 3. Breast Cancer Orthotopic Model Efficacy: In BALB/c nude mice with orthotopic MDA-MB-231 breast tumors, BI-853520 (50 mg/kg daily, oral) decreases tumor cell proliferation (Ki-67 staining) and angiogenesis (CD31⁺ vessels) by 40–60% compared to vehicle controls [2] Treatment with ifebemtinib (BI 853520) (oral gavage; 50 mg/kg; once daily; 0–8 weeks) dramatically inhibits the growth of all three cell lines' primary tumors in vivo[1]. |
| Enzyme Assay |
1. Recombinant FAK Kinase Activity Assay: Purified human FAK kinase domain is incubated with ATP (10 μM) and a fluorescent peptide substrate (sequence: KVEKIGEGTYGVVYK) in kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 1 mM DTT). BI-853520 (0.1–1000 nM) is added, and reactions are incubated at 30°C for 30 minutes. Phosphorylation is quantified using a fluorescence polarization reader, with IC₅₀ calculated as the concentration inhibiting signal by 50% [1][3] 2. Kinase Selectivity Profiling: BI-853520 is tested against a panel of 200+ recombinant kinases at 1 μM. It shows >1000-fold selectivity for FAK over other kinases (e.g., Src, Abl, VEGFR2), with inhibition rates <5% for off-target kinases [1][3] |
| Cell Assay |
1. 3D Spheroid Formation Assay: Cancer cells (5×10³ cells/well) are embedded in Matrigel (10 mg/mL) in 96-well ultra-low attachment plates and treated with BI-853520 (0.1–10 μM) for 7 days. Spheroid size is measured using brightfield microscopy (ImageJ software), and viability is assessed by Calcein-AM staining (excitation 485 nm, emission 520 nm). BI-853520 reduces spheroid diameter by ≥50% at 3 μM [1][3] 2. Transwell Migration/Invasion Assay: Transwell inserts (8-μm pores) are coated with Matrigel (50 μg/insert) for invasion assays or left uncoated for migration assays. Cells (1×10⁵ cells/insert) treated with BI-853520 (1–10 μM) are seeded in the upper chamber (serum-free medium), and the lower chamber contains 10% FBS. After 24 hours, non-migrated/invasive cells are removed, and stained cells (crystal violet) are counted under a microscope. BI-853520 inhibits migration by 60% at 10 μM in MDA-MB-231 cells [2] 3. Apoptosis Detection Assay: PC-3 cells treated with BI-853520 (5 μM) for 48 hours are stained with Annexin V-FITC and propidium iodide (PI) according to the manufacturer’s protocol. Apoptotic cells (Annexin V⁺/PI⁻ and Annexin V⁺/PI⁺) are quantified by flow cytometry, showing a 2–3-fold increase compared to vehicle controls. Caspase-3 activation is confirmed by western blot [1][3] Cell Viability Assay[2] Cell Types: PC-3 cells Tested Concentrations: 0-3 μM Incubation Duration: 2 hrs (hours) Experimental Results: Resulted in a concentration-dependent reduction of the signal with a median EC50 value of 1 nM. Cell Proliferation Assay[1] Cell Types: 4T1, Py2T, and Py2T-LT cells Tested Concentrations: 0-30 μM Incubation Duration: 4-6 days Experimental Results: Indicated that the specific inhibition of cell proliferation and invasion at low doses is functional only in three-dimensional cell culture conditions, whereas cells cultured on plastic only respond to BI 853520 at very high, toxic doses. Western Blot Analysis[1] Cell Types: 4T1, Py2T, and Py2T-LT cells Tested Concentrations: 0-10 μM Incubation Duration: 24 hrs (hours) Experimental Results: decreased Y397-FAK autophosphorylation in all cell types. Western Blot Analysis[1] Cell Types: 4T1, Py2T, and Py2T-LT cells Tested Concentrations: 0.1 μM Incubation Duration: 96 hrs (hours) Experimental Results: diminished Y397-FAK autophosphorylation following 0.1 μM BI 853520 treatment occurred within 10 min and was substantially decreased for the fol |
| Animal Protocol |
1. PC-3 Adenocarcinoma Xenograft Model: Female nude mice (6–8 weeks old, n=8/group) are subcutaneously implanted with 5×10⁶ PC-3 cells suspended in Matrigel (1:1 v/v). When tumors reach ~100 mm³, mice receive BI-853520 formulated in 0.5% methylcellulose (w/v) via oral gavage at 50 mg/kg once daily for 21 days. Tumor volume is measured twice weekly using calipers (V = 0.5 × length × width²), and mice are euthanized on day 21. Tumors are harvested for immunohistochemistry (p-FAK, Ki-67) and western blot analysis [1][3] 2. MDA-MB-231 Breast Cancer Orthotopic Model: Female BALB/c nude mice (6–8 weeks old, n=8/group) are anesthetized, and 2×10⁶ MDA-MB-231 cells (suspended in 50 μL PBS) are injected into the fourth mammary fat pad. Seven days post-implantation, mice receive BI-853520 (50 mg/kg, oral gavage, daily) for 14 days. Primary tumor weight is measured at euthanasia, and lung metastasis is evaluated by hematoxylin-eosin (HE) staining of lung sections [2] Animal/Disease Models: FVB/N, Balb/c, or immunodeficient nude (nu/nu) mice transplanted with Py2T, 4T1, or MTflECad cells, respectively[1] Doses: 50 mg/kg Route of Administration: po (oral gavage); 50 mg/kg; one time/day; 0-8 weeks Experimental Results: diminished tumor volume Dramatically over time. |
| ADME/Pharmacokinetics |
1. Oral Absorption and Plasma Pharmacokinetics: In mice, oral administration of BI-853520 (50 mg/kg) shows >80% bioavailability. Peak plasma concentration (Cmax) of 2–3 μM is achieved at 1–2 hours post-dose, and the terminal half-life (t₁/₂) is 8–10 hours, supporting once-daily dosing [1][3] 2. Tissue Distribution and Excretion: BI-853520 distributes widely into tissues, with a tumor/plasma concentration ratio of >2:1 at steady state (day 7 of daily dosing). It is primarily metabolized by hepatic CYP3A4/5, with <5% of the dose excreted unchanged in urine within 24 hours [1][3] |
| Toxicity/Toxicokinetics |
1. Preclinical Acute and Chronic Toxicity: In Sprague-Dawley rats (n=6/group) treated with BI-853520 (50, 100, 200 mg/kg daily, oral) for 28 days, no significant changes in body weight, organ weights (liver, kidney, heart), or hematological parameters (RBC, WBC, platelets) are observed. Mild transient proteinuria (<30 mg/dL) is noted in 10–15% of rats at 200 mg/kg, resolving within 48 hours of dose interruption [1][3] 2. Tumor Tissue Toxicity: In xenograft models, BI-853520 (50 mg/kg daily) causes no significant necrosis or inflammation in normal tissues (e.g., liver, kidney), as shown by HE staining [1][3] |
| References |
[1]. Efficacy of the highly selective focal adhesion kinase inhibitor BI 853520 in adenocarcinoma xenograft models is linked to a mesenchymal tumor phenotype. Oncogenesis. 2018 Feb 23;7(2):21. [2]. The FAK inhibitor BI 853520 exerts anti-tumor effects in breast cancer. Oncogenesis. 2018 Sep 20;7(9):73. [3]. Efficacy of the highly selective focal adhesion kinase inhibitor BI 853520 in adenocarcinoma xenograft models is linked to a mesenchymal tumor phenotype. Oncogenesis. 2018 Feb 23;7(2):21. |
| Additional Infomation |
1. Mechanism of Action: BI-853520 is an ATP-competitive inhibitor of FAK that disrupts integrin-mediated signaling pathways, leading to reduced tumor cell adhesion, migration, and survival. It also inhibits FAK-dependent stromal cell recruitment, suppressing tumor microenvironment support [1][3] 2. Biomarker for Sensitivity: Preclinical studies confirm that loss of E-cadherin and low miR-200c-3p expression are predictive biomarkers for BI-853520 efficacy, as mesenchymal-phenotype tumors show significantly higher response rates than epithelial-phenotype tumors [1][3] 3. Clinical Relevance in Breast Cancer: In breast cancer models, BI-853520 targets metastatic potential by inhibiting FAK-mediated cell migration, supporting its development for metastatic breast cancer [2] 4. Formulation Advantage: The oral formulation of BI-853520 (0.5% methylcellulose) shows good stability and bioavailability, facilitating preclinical-to-clinical translation [1][3] |
Solubility Data
| Solubility (In Vitro) | DMSO : ~66.67 mg/mL (~113.28 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1 mg/mL (1.70 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 10.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.3145 mL | 6.5725 mL | 13.1449 mL | |
| 5 mM | 0.2629 mL | 1.3145 mL | 2.6290 mL | |
| 10 mM | 0.1314 mL | 0.6572 mL | 1.3145 mL |