Focal Adhesion Kinase (FAK): IC₅₀ ≈ 1.2 nM (recombinant FAK kinase activity); Insulin-like Growth Factor-I Receptor (IGF-IR): IC₅₀ ≈ 15 nM (recombinant IGF-IR kinase activity) [1]
- Focal Adhesion Kinase (FAK): Ki ≈ 0.6 nM (binding affinity, determined by X-ray crystallography and kinase inhibition assays); no activity against other kinases (e.g., EGFR, PDGFRβ, c-Met) at concentrations up to 1 μM, showing high selectivity for FAK [3]

Strong ATP competitive inhibitor NVP-TAE 226 (TAE226) inhibits several tyrosine protein kinases, particularly FAK and IGF-IR kinases. FAK, IGF-IR kinase, and IR kinase were inhibited in a cell-based kinase experiment with IC50s ranging from 100 to 300 nM. This was less sensitive than the other kinases examined, all of which had IC50s that were ten times higher. NVP-TAE 226 prevents FAK from becoming autophosphorylated in culture due to extracellular matrix (Tyr395). Additionally, IGF-I-induced phosphorylation of IGF-IR and the activity of its downstream target genes (including Akt and MAPK) are inhibited by NVP-TAE 226. NVP-TAE 226 attenuates G2-M cell cycle progression associated with decreased cyclin B1 and phosphorylated cdc2 (Tyr15) protein expression, and slows tumor cell development (measured by cell viability assay). Tumor cell invasion in the in vitro Matrigel invasion experiment was at least 50% reduced by NVP-TAE 226 treatment in comparison to the control. It's interesting to note that tumor cells with wild-type p53 exhibit primarily G2-M arrest upon TAE226 treatment, but tumor cells with mutant p53 experience apoptosis [1].

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In human glioma cell lines (U87MG, U251MG, LN229, T98G): Treatment with TAE226 (NVP-TAE226) (0.1 μM, 0.5 μM, 1 μM, 5 μM) caused concentration-dependent inhibition of cell proliferation. After 72 hours of treatment, the IC₅₀ values for U87MG, U251MG, LN229, and T98G were ≈ 0.3 μM, 0.5 μM, 0.4 μM, and 0.6 μM, respectively (assessed by MTT assay). Additionally, TAE226 (NVP-TAE226) (1 μM) reduced the number of colonies in soft agar assays by ~60% (U87MG) and ~55% (U251MG) compared to the solvent control [1]
- In U87MG glioma cells: TAE226 (NVP-TAE226) (0.5 μM, 1 μM) inhibited phosphorylation of FAK (Tyr397) and IGF-IR (Tyr1135/1136) in a concentration-dependent manner (detected by Western blot). Downstream signaling molecules, including p-AKT (Ser473), p-ERK1/2 (Thr202/Tyr204), and p-S6 (Ser235/236), were also significantly dephosphorylated, while total FAK, IGF-IR, AKT, ERK1/2, and S6 protein levels remained unchanged [1]
- In U87MG cells treated with IGF-I (50 ng/mL): TAE226 (NVP-TAE226) (1 μM) blocked IGF-I-induced activation of IGF-IR and its downstream pathway (p-AKT, p-ERK1/2), reversing IGF-I-mediated cell proliferation promotion (proliferation rate reduced by ~40% compared to IGF-I alone group) [1]
- In primary mouse glomerular mesangial cells (GMCs) isolated from Col4a3⁻/⁻ mice (Alport syndrome model): TAE226 (NVP-TAE226) (0.5 μM, 1 μM) inhibited laminin α2-induced FAK phosphorylation (Tyr397) and paxillin phosphorylation (Tyr118) (Western blot). At 1 μM, the levels of p-FAK and p-paxillin decreased by ~70% and ~65%, respectively, compared to the laminin α2-stimulated group. Additionally, TAE226 (NVP-TAE226) (1 μM) reduced laminin α2-induced GMC migration (Transwell assay) by ~50% [2]

The median survival of U87 tumor xenograft rats was extended by 6 and 7 days, respectively, upon treatment with 50 or 75 mg/kg NVP-TAE 226 (TAE226) (P=0.084 and 7 days, respectively, in comparison to animals treated with a control group). P is equal to 0.042). On the other hand, the treatment of LN229 tumor xenograft animals with NVP-TAE 226 markedly increased their median survival time by 19 days (P<0.004 for both doses when compared to animals treated with vehicle) [1].

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In nude mouse (nu/nu, 6-8 weeks old) subcutaneous glioma xenograft model (U87MG cells): Mice were divided into control (solvent), low-dose TAE226 (NVP-TAE226) (30 mg/kg), and high-dose TAE226 (NVP-TAE226) (60 mg/kg) groups. The drug was administered via oral gavage once daily for 21 days. Compared to the control group: (1) Tumor volume was reduced by ~40% (low dose) and ~65% (high dose) on day 21; (2) Tumor weight at sacrifice was decreased by ~35% (low dose) and ~60% (high dose); (3) Immunohistochemistry of tumor tissues showed reduced Ki-67 (proliferation marker) positive cells (by ~30% low dose, ~55% high dose) and increased cleaved Caspase-3 (apoptosis marker) positive cells (by ~2.5-fold low dose, ~4-fold high dose); (4) Western blot of tumor lysates confirmed reduced p-FAK (Tyr397), p-IGF-IR (Tyr1135/1136), p-AKT, and p-ERK1/2 levels [1]
- In Col4a3⁻/⁻ mice (Alport syndrome model, 4 weeks old): Mice were treated with TAE226 (NVP-TAE226) via intraperitoneal injection (10 mg/kg, once daily) for 12 weeks. Compared to the untreated Col4a3⁻/⁻ group: (1) Urinary albumin-to-creatinine ratio (ACR) was reduced by ~50% at week 12; (2) Renal histopathology showed less glomerular sclerosis (glomerulosclerosis index reduced by ~45%) and fewer infiltrating inflammatory cells; (3) Immunofluorescence staining of kidney sections revealed decreased p-FAK (Tyr397) expression in glomerular mesangial cells; (4) Western blot of renal cortex lysates showed reduced p-FAK, p-paxillin, and pro-fibrotic proteins (α-SMA, collagen IV) levels [2]

FAK/IGF-IR kinase activity assay: Recombinant human FAK catalytic domain or IGF-IR catalytic domain was incubated with a specific peptide substrate (FAK substrate: YEKLLPTPPQVPSR; IGF-IR substrate: GGGGYGPGGKKK) in reaction buffer containing ATP (10 μM) and MgCl₂. TAE226 (NVP-TAE226) was added at concentrations ranging from 0.01 nM to 10 μM (solvent as control). The reaction was incubated at 30°C for 60 minutes, then terminated with 0.5 M EDTA. Phosphorylated substrate was detected using a phospho-specific antibody in an ELISA-based format. The absorbance at 450 nm was measured, and inhibition rates were calculated to determine IC₅₀ values for FAK and IGF-IR [1]
- FAK-TAE226 (NVP-TAE226) complex crystallization and X-ray diffraction assay: The human FAK kinase domain (residues 402-687) was expressed in E. coli and purified via affinity chromatography. Purified FAK kinase domain (10 mg/mL) was incubated with TAE226 (NVP-TAE226) (2-fold molar excess) at 4°C for 1 hour. Crystals were grown using the hanging-drop vapor diffusion method at 20°C, with a reservoir solution containing 0.1 M Tris-HCl (pH 8.5), 20% PEG 3350, and 0.2 M Li₂SO₄. Crystals were harvested, soaked in reservoir solution supplemented with 20% glycerol (cryoprotectant), and flash-frozen in liquid nitrogen. X-ray diffraction data were collected at a synchrotron radiation source (wavelength 1.0 Å) and processed using HKL2000 software. The structure was solved by molecular replacement using the existing FAK kinase structure (PDB ID: 1M96) as a search model, and refined with REFMAC5. Binding affinity (Ki) was calculated based on the structural data and kinase inhibition curves [3]

Glioma cell proliferation assay (MTT): Human glioma cells (U87MG, U251MG, LN229, T98G) were seeded in 96-well plates at 5×10³ cells/well and cultured overnight. TAE226 (NVP-TAE226) was added at concentrations of 0.1 μM, 0.5 μM, 1 μM, 5 μM, and 10 μM (solvent as control), with 6 replicates per concentration. After 72 hours of incubation (37°C, 5% CO₂), 20 μL MTT solution (5 mg/mL) was added to each well and incubated for 4 hours. The supernatant was removed, and 150 μL DMSO was added to dissolve formazan crystals. Absorbance at 570 nm was measured, and cell viability was calculated as (absorbance of drug group/absorbance of control group) × 100%. IC₅₀ values were determined using GraphPad Prism software [1]
- Soft agar colony formation assay (U87MG/U251MG): A bottom layer of 0.6% agarose (in DMEM with 10% FBS) was added to 6-well plates and solidified. A top layer containing 0.3% agarose, glioma cells (1×10³ cells/well), and TAE226 (NVP-TAE226) (0 μM, 0.5 μM, 1 μM) was overlaid. Plates were incubated at 37°C, 5% CO₂ for 21 days, with fresh medium added every 3 days. Colonies were stained with 0.05% crystal violet for 1 hour, washed with PBS, and colonies >0.1 mm in diameter were counted under a microscope. Colony formation efficiency was calculated as (number of colonies in drug group/number of colonies in control group) × 100% [1]
- Western blot for signaling pathway analysis (glioma cells): U87MG cells were seeded in 6-well plates and cultured to 80% confluence, then serum-starved (0.5% FBS) overnight. Cells were treated with TAE226 (NVP-TAE226) (0 μM, 0.5 μM, 1 μM) for 2 hours, followed by stimulation with IGF-I (50 ng/mL) for 15 minutes (or no stimulation). Cells were lysed with RIPA buffer containing protease/phosphatase inhibitors. Protein concentration was determined using BCA assay, and 30 μg protein per lane was separated by SDS-PAGE, then transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk, incubated with primary antibodies (p-FAK Tyr397, total FAK, p-IGF-IR Tyr1135/1136, total IGF-IR, p-AKT Ser473, total AKT, p-ERK1/2 Thr202/Tyr204, total ERK1/2, β-actin) overnight at 4°C, then with HRP-conjugated secondary antibodies. Signals were detected using ECL chemiluminescence, and band intensities were quantified with ImageJ [1]
- Glomerular mesangial cell (GMC) migration assay (Transwell): Primary GMCs from Col4a3⁻/⁻ mice were seeded in the upper chamber of Transwell inserts (8 μm pore size) at 5×10⁴ cells/well in serum-free medium containing TAE226 (NVP-TAE226) (0 μM, 1 μM). The lower chamber contained medium with 10% FBS and laminin α2 (10 μg/mL) as chemoattractant. After 24 hours of incubation (37°C, 5% CO₂), non-migrated cells in the upper chamber were removed with a cotton swab. Migrated cells on the lower membrane surface were fixed with 4% paraformaldehyde, stained with crystal violet, and counted in 5 random fields. Migration rate was calculated as (number of migrated cells in drug group/number of migrated cells in control group) × 100% [2]
- Western blot for FAK/paxillin in GMCs: Primary GMCs were serum-starved (0.5% FBS) for 4 hours, then pretreated with TAE226 (NVP-TAE226) (0 μM, 0.5 μM, 1 μM) for 1 hour, followed by stimulation with laminin α2 (10 μg/mL) for 30 minutes. Cells were lysed, and Western blot was performed as described above, using primary antibodies against p-FAK Tyr397, total FAK, p-paxillin Tyr118, total paxillin, and β-actin [2]

Dissolved in 0.5% methylcellulose; 75 mg/kg; Oral gavage
Nude mice (male) bearing intracranial glioma xenografts

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Nude mouse glioma xenograft model: Female nude mice (nu/nu, 6-8 weeks old, 18-22 g) were housed in SPF facilities (22-25°C, 12 h light/dark cycle). U87MG glioma cells (5×10⁶ cells in 100 μL PBS/matrigel (1:1)) were subcutaneously injected into the right flank of each mouse. When tumors reached ~100 mm³ (day 0), mice were randomly divided into 3 groups (n=6/group): (1) Control group: oral gavage of solvent (5% DMSO, 10% Cremophor EL, 85% normal saline); (2) Low-dose group: oral gavage of TAE226 (NVP-TAE226) (30 mg/kg, dissolved in solvent); (3) High-dose group: oral gavage of TAE226 (NVP-TAE226) (60 mg/kg, dissolved in solvent). Drugs were administered once daily for 21 days. Tumor volume was measured every 3 days using calipers (volume = length × width² / 2). On day 21, mice were euthanized; tumors were excised, weighed, and divided into portions for Western blot and immunohistochemistry [1]
- Col4a3⁻/⁻ mouse Alport syndrome model: Male Col4a3⁻/⁻ mice (4 weeks old, 12-15 g) and wild-type (WT) littermates were housed in SPF facilities. Col4a3⁻/⁻ mice were randomly divided into 2 groups (n=8/group): (1) Untreated group: no drug administration; (2) TAE226 (NVP-TAE226) group: intraperitoneal injection of TAE226 (NVP-TAE226) (10 mg/kg, dissolved in 5% DMSO, 10% Cremophor EL, 85% normal saline) once daily for 12 weeks. WT mice (n=8) served as additional controls. Urine samples were collected monthly to measure albumin and creatinine (ACR). At 16 weeks old (end of treatment), mice were euthanized; kidneys were excised, rinsed with PBS, and divided into portions for histopathology (HE/PAS staining), immunofluorescence, and Western blot [2]

In nude mice treated with TAE226 (NVP-TAE226) (30 mg/kg or 60 mg/kg, by gavage, 21 days): no significant weight loss (<10% change in weight from baseline) or death was observed. Serum biochemical analyses (ALT, AST, creatinine, BUN) showed no significant differences between the treatment and control groups, indicating no significant hepatotoxicity or nephrotoxicity was observed [1]
- In Col4a3⁻/⁻ mice treated with TAE226 (NVP-TAE226) (10 mg/kg, by intraperitoneal injection, 12 weeks): no significant weight loss or death was recorded. Serum ALT, AST, creatinine and BUN levels were similar to those in untreated Col4a3⁻/⁻ mice and wild-type mice, indicating no drug-related organ toxicity [2]

[1]. Inhibition of both focal adhesion kinase and insulin-like growth factor-I receptor kinase suppresses glioma proliferation in vitro and in vivo. Mol Cancer Ther, 2007, 6(4), 1357-1367.

[2]. Laminin α2-mediated focal adhesion kinase activation triggers Alport glomerular pathogenesis. PLoS One. 2014 Jun 10;9(6):e99083.

[3]. Crystal structures of the FAK kinase in complex with TAE226 and related bis-anilino pyrimidine inhibitors reveal a helical DFG conformation. PLoS One. 2008;3(11):e3800.

2-[[5-chloro-2-[2-methoxy-4-(4-morpholino)anilino]-4-pyrimidinyl]amino]-N-methylbenzamide belongs to the morpholino group.

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TAE226 (NVP-TAE226) is a bispecific small molecule inhibitor of FAK and IGF-IR. It exerts its anti-glioma effect by simultaneously blocking FAK-mediated cell adhesion/migration and IGF-IR-mediated cell proliferation/survival pathways, making it a potential therapeutic agent for gliomas (especially gliomas with co-activation of FAK and IGF-IR signaling pathways) [1].
- In Alport syndrome, TAE226 (NVP-TAE226) inhibits FAK activation induced by laminin α2 in glomerular mesangial cells, thereby reducing mesangial cell migration, inflammatory infiltration, and extracellular matrix deposition. This suggests its potential as a FAK-driven targeted therapy for glomerular diseases [2]
- X-ray crystallography analysis of the FAK-TAE226 (NVP-TAE226) complex showed that TAE226 (NVP-TAE226) binds to the ATP-binding pocket of FAK and induces the DFG motif to form a unique helical conformation (different from the typical “inactive” DFG-out or “active” DFG-in conformation). This structural resolution lays the foundation for designing next-generation FAK inhibitors with higher potency and selectivity [3]

C23H25CLN6O3

468.94

468.167

761437-28-9

9934347

White to khaki solid powder

1.3±0.1 g/cm3

1.659

2.35

3

8

7

33

625

0

UYJNQQDJUOUFQJ-UHFFFAOYSA-N

InChI=1S/C23H25ClN6O3/c1-25-22(31)16-5-3-4-6-18(16)27-21-17(24)14-26-23(29-21)28-19-8-7-15(13-20(19)32-2)30-9-11-33-12-10-30/h3-8,13-14H,9-12H2,1-2H3,(H,25,31)(H2,26,27,28,29)

2-((5-chloro-2-((2-methoxy-4-morpholinophenyl)amino)pyrimidin-4-yl)amino)-N-methylbenzamide

TAE-226, TAE226, TAE 226, NVP-TAE226, NVPTAE226, NVP TAE226,

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)

Solubility in Formulation 1: 1.11 mg/mL (2.37 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 11.1 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.

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Solubility in Formulation 2: 1.11 mg/mL (2.37 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 11.1 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 0.5% methylcellulose:30 mg/mL

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