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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.
| Targets |
Focal adhesion kinase (FAK) (recombinant FAK IC₅₀ = 1 nM)
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| 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, the bioavailability of oral BI-853520 (50 mg/kg) was >80%. Peak plasma concentration (Cmax) of 2-3 μM was reached 1-2 hours after administration, with a terminal half-life (t₁/₂) of 8-10 hours, supporting once-daily administration [1][3]
2. Tissue distribution and excretion: BI-853520 is widely distributed in various tissues, with a tumor/plasma concentration ratio >2:1 at steady state (day 7 of daily administration). It is mainly metabolized by hepatic CYP3A4/5, with only <5% of the dose excreted unchanged in the urine within 24 hours [1][3] |
| Toxicity/Toxicokinetics |
1. Preclinical acute and chronic toxicity: In Sprague-Dawley rats (n=6 per group), no significant changes in body weight, organ weight (liver, kidney, heart), or hematological parameters (erythrocytes, leukocytes, platelets) were observed after 28 consecutive days of oral administration of BI-853520 (50, 100, or 200 mg/kg daily). In the 200 mg/kg dose group, 10–15% of rats experienced mild transient proteinuria (<30 mg/dL), which resolved within 48 hours after drug withdrawal [1][3]. 2. Tumor tissue toxicity: In xenograft models, HE staining showed that BI-853520 (50 mg/kg daily) did not cause significant necrosis or inflammation in normal tissues (e.g., liver, kidneys) [1][3].
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| References |
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| Additional Infomation |
1. Mechanism of action: BI-853520 is an ATP-competitive FAK inhibitor that disrupts integrin-mediated signaling pathways, thereby reducing tumor cell adhesion, migration, and survival. It can also inhibit FAK-dependent stromal cell recruitment, thereby inhibiting the supportive role of the tumor microenvironment [1][3]
2. Sensitivity biomarkers: Preclinical studies have confirmed that the absence of E-cadherin and low expression of miR-200c-3p are predictive biomarkers for the efficacy of BI-853520, as the response rate of stromal phenotype tumors is significantly higher than that of epithelial phenotype tumors [1][3] 3. Clinical relevance to breast cancer: In breast cancer models, BI-853520 targets metastatic potential by inhibiting FAK-mediated cell migration, supporting its development in the treatment of metastatic breast cancer [2] 4. Formulation advantages: The oral formulation of BI-853520 (0.5% methylcellulose) has good stability and bioavailability, which is conducive to the translation from preclinical studies to clinical practice [1][3] |
| Molecular Formula |
C35H36F4N6O7S
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|---|---|
| Molecular Weight |
760.75
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| Exact Mass |
588.21
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| Elemental Analysis |
C, 55.26; H, 4.77; F, 9.99; N, 11.05; O, 14.72; S, 4.21
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| CAS # |
2761021-80-9
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| Related CAS # |
1227948-82-4
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| Appearance |
White to off-white solid powder
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| 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
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| InChi Key |
LOKCCATVJFYTFP-UHFFFAOYSA-N
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| 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)
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| 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
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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: 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)
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| Solubility (In Vitro) |
DMSO : ~66.67 mg/mL (~113.28 mM)
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| 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 |
*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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