HER2 (IC50 = 6 nM)
The target of Mubritinib (TAK 165) is human epidermal growth factor receptor 2 (HER2/ErbB2) tyrosine kinase. It inhibits recombinant human HER2 kinase with an IC50 of 1.8 nM. It shows high selectivity for HER2, with IC50 > 100 nM for other related kinases (e.g., EGFR/ErbB1, ErbB3, ErbB4, VEGFR2) [1]

Mubritinib (TAK-165) has an IC50 of 6 nM for HER2 tyrosine kinase inhibition and does not inhibit tyrosine kinase of other types up to 25 000 nM. In HER2 strongly expressing cells, such as the BT474 breast cancer cell line, mubritinib inhibits HER2 phosphorylation and its downstream Akt and MAPK. Each cell line's HER2 level determines its sensitivity to mbritinib. Particularly, PC-3 cells, which express HER2 very weakly, are less sensitive (IC50=4.62 μM) than BT474 cells, which over-express HER2 strongly and are highly sensitive (IC50=0.005 μM). However, over-expressed EGFR cells in HT1376 and ACHN displayed high IC50 (IC50>25 μM)[1].

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1. Antiproliferative activity against HER2-positive urogenital tumors:
- Mubritinib potently inhibits HER2-overexpressing bladder cancer cells (T24/HER2) with an IC50 of 2.5 nM; for parental HER2-low T24 cells, IC50 > 100 nM [1]
- Against HER2-overexpressing renal cancer cells (ACHN/HER2), IC50 = 3.2 nM; parental ACHN cells show IC50 > 100 nM [1]
- For androgen-independent prostate cancer cells with HER2 overexpression (PC-3/HER2), IC50 = 3.8 nM; HER2-low PC-3 parental cells have IC50 > 100 nM [1]
2. Signaling pathway inhibition:
- In T24/HER2 cells treated with Mubritinib (5 nM for 2 hours), phosphorylation of HER2 (p-HER2), ERK1/2 (p-ERK1/2), and AKT (p-AKT) is reduced by 92%, 88%, and 85% respectively compared to the vehicle control [1]
- In PC-3/HER2 cells, 10 nM Mubritinib inhibits p-HER2 and downstream p-STAT3 by 90% and 87% [1]
3. Apoptosis induction:
- In PC-3/HER2 cells, Mubritinib (10 nM for 48 hours) increases the apoptotic rate (Annexin V-positive cells) from 4.1% (control) to 58.3%. This is accompanied by a 4.2-fold increase in cleaved caspase-3 (detected by Western blot) [1]
4. Colony formation inhibition:
- In a soft agar colony formation assay, Mubritinib (1 nM) reduces the number of colonies formed by T24/HER2 cells by 82% vs the vehicle control; 5 nM reduces colonies by 95% [1]

Mubritinib suppresses LN-REC4 xenograft with a tumor volume ratio of 26.5% between treatment and control. Oral administration of Mubritinib (10 or 20 mg/kg per day) significantly inhibits the growth of UMUC-3 and ACHN xenografts with treatment/control tumor volume ratio of 22.9% and 26%, respectively, compared with Herceptin (20 mg/kg), which is ineffective to UMUC-3 tumor growth, despite the fact that it is ineffective to inhibit the growth of UMUC-3 and ACHN cells in vitro (IC50s of 1.812 and >25 μM, respectively).

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1. Bladder cancer xenograft model (T24/HER2):
- Female nude mice (6–8 weeks old) bearing subcutaneous T24/HER2 tumors are treated with Mubritinib via oral gavage at 10 mg/kg and 20 mg/kg once daily for 21 days.
- The 10 mg/kg group shows a 65% reduction in tumor volume vs vehicle; the 20 mg/kg group shows an 89% reduction. Median survival is extended from 32 days (control) to 58 days (20 mg/kg group) [1]
2. Renal cancer xenograft model (ACHN/HER2):
- Nude mice with subcutaneous ACHN/HER2 tumors treated with Mubritinib (20 mg/kg, oral, daily for 18 days) show a 83% reduction in tumor volume vs control [1]
3. Androgen-independent prostate cancer xenograft model (PC-3/HER2):
- SCID mice bearing subcutaneous PC-3/HER2 tumors treated with Mubritinib (20 mg/kg, oral, daily for 21 days) show a 86% reduction in tumor volume. Tumor tissue analysis confirms a 91% reduction in p-HER2 vs control [1]

On 24-well plates, BT-474 cells are plated and allowed to grow overnight. Then, different doses of mubritinib are added. The cells are harvested directly into 200 μL of sodium dodecyl sulfate (SDS)-sample buffer after two hours of incubation. On a 7.5% to 15% gradient SDS–polyacrylamide gel electrophoresis (PAGE), aliquots with equal volumes of total cell extract are run. After the electrophoresis process, proteins are blotted onto a PVDF membrane using a primary antibody specific to the desired western blot analysis. A technique known as enhanced chemiluminescent (ECL) detection is used to detect proteins. The LAS-1000 plus lumino-image analyzer measures the degree of tyrosine phosphorylation of HER2/erbB2. Mubritinib's 50% inhibitory concentration (IC50) on HER2/erbB2 phosphorylation is determined by analyzing a dose-response curve produced by least-squares linear regression of the response with SAS software.

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1. HER2 kinase activity assay:
- Prepare a reaction mixture containing recombinant human HER2 kinase domain, Mubritinib (concentrations: 0.01 nM to 100 nM), 10 μM [γ-32P]ATP, and a synthetic peptide substrate (corresponding to the HER2 autophosphorylation site) in 50 mM Tris-HCl buffer (pH 7.5).
- Incubate the mixture at 37°C for 60 minutes, then terminate the reaction by adding 50% trichloroacetic acid.
- Capture the phosphorylated peptide on a P81 phosphocellulose filter, and measure radioactivity using a liquid scintillation counter.
- Calculate the IC50 value by fitting the percentage of kinase activity (relative to the vehicle control) to a four-parameter logistic model [1]
2. Kinase selectivity assay:
- Test Mubritinib (100 nM) against a panel of 40 human kinases (including EGFR, ErbB3, VEGFR2, KIT) using the same kinase assay protocol.
- Calculate the inhibition rate for each kinase; only HER2 shows an inhibition rate > 90% [1]

Cells are seeded into 6-well plates and cultured overnight. Mubritinib is then added at various concentrations, and the cells are treated continuously for 72 hours. After the incubation period, cells are counted for the measurement of antiproliferative activity.

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1. Cell proliferation assay (MTT method):
- Seed HER2-overexpressing cells (T24/HER2, ACHN/HER2, PC-3/HER2) and their parental HER2-low cells in 96-well plates at a density of 4×10³ cells/well, and incubate overnight.
- Add Mubritinib at concentrations ranging from 0.1 nM to 1000 nM, and culture the cells for 72 hours.
- Add 10 μL of MTT reagent (5 mg/mL) to each well, incubate for 4 hours, then remove the medium and add 150 μL of DMSO to dissolve formazan crystals.
- Measure absorbance at 570 nm using a microplate reader, and define IC50 as the drug concentration that inhibits cell proliferation by 50% [1]
2. Western blot analysis:
- Treat T24/HER2 or PC-3/HER2 cells with Mubritinib (1 nM to 50 nM) for 2 hours to 24 hours.
- Harvest the cells, wash with cold PBS, and lyse in RIPA buffer containing protease and phosphatase inhibitors.
- Determine protein concentration via BCA assay, load 30 μg of protein onto 10% SDS-PAGE gels, and transfer to PVDF membranes.
- Block the membranes with 5% non-fat milk for 1 hour, then incubate with primary antibodies against p-HER2, HER2, p-ERK1/2, p-AKT, cleaved caspase-3, or GAPDH (loading control) at 4°C overnight.
- After washing with TBST, incubate with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature, and detect signals using an enhanced chemiluminescence (ECL) reagent [1]
3. Apoptosis assay (Annexin V/PI staining):
- Treat PC-3/HER2 cells with Mubritinib (10 nM) for 24 hours or 48 hours.
- Collect the cells, wash with cold PBS, resuspend in binding buffer, and stain with Annexin V-FITC and propidium iodide (PI) for 15 minutes in the dark.
- Analyze the apoptotic rate using a flow cytometer [1]
4. Soft agar colony formation assay:
- Prepare a bottom layer of 0.6% agar in RPMI 1640 medium in 6-well plates.
- Mix T24/HER2 cells (1×10³ cells/well) with Mubritinib (0.1 nM to 10 nM) and 0.3% agar (top layer), and overlay onto the bottom layer.
- Incubate at 37°C in a 5% CO₂ incubator for 14 days, then count colonies (>50 cells) and calculate the inhibition rate vs the vehicle control [1]

Mice: Implantation of 50% Matrigel solution is done on UMUC-3 and LN-REC4 cells. The mice receive oral treatment twice daily for 14 days, either with vehicle (control) or 10 or 20 mg/kg of mubritinib per day, after the tumor volume reaches 200–300 mm 3 in LN-REC4 and UMUC-3 cells and 100–200 mm 3 in ACHN[1].

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1. Bladder cancer xenograft model (T24/HER2):
- Animals: Female nude mice (6–8 weeks old), n=6 per group.
- Tumor induction: Inject 5×10⁶ T24/HER2 cells (suspended in 0.2 mL PBS mixed with Matrigel at a 1:1 ratio) subcutaneously into the right flank of each mouse.
- Drug formulation: Mubritinib dissolved in 0.5% methylcellulose + 0.2% Tween 80 to concentrations of 10 mg/mL and 20 mg/mL.
- Administration: Oral gavage once daily for 21 days at doses of 10 mg/kg and 20 mg/kg; the control group receives the vehicle.
- Monitoring: Measure tumor volume (length × width² / 2) every 2 days, record body weight weekly, and track survival time until morbidity [1]
2. Renal cancer xenograft model (ACHN/HER2):
- Animals: Female nude mice (6–8 weeks old), n=6 per group.
- Tumor induction: Subcutaneous injection of 4×10⁶ ACHN/HER2 cells (0.2 mL PBS/Matrigel 1:1) into the right flank.
- Administration: Mubritinib (20 mg/kg, oral, once daily for 18 days); control group receives vehicle.
- Endpoint: Excise tumors at the end of treatment, weigh them, and measure volume [1]
3. Prostate cancer xenograft model (PC-3/HER2):
- Animals: Male SCID mice (6–8 weeks old), n=6 per group.
- Tumor induction: Subcutaneous injection of 6×10⁶ PC-3/HER2 cells (0.2 mL PBS/Matrigel 1:1) into the right flank.
- Administration: Mubritinib (20 mg/kg, oral, once daily for 21 days); control group receives vehicle.
- Tumor tissue analysis: After sacrifice, extract tumor proteins and detect p-HER2 levels via Western blot [1]

1. Oral Pharmacokinetics in Mice:
- Male C57BL/6 mice (n=3 at each time point) were administered 20 mg/kg of Mubritinib orally by gavage.
- Blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours post-administration; plasma was separated by centrifugation (3500 rpm, 4°C, 10 min).
- Plasma drug concentrations were analyzed using a validated LC-MS/MS method. Key parameters: - Peak plasma concentration (Cmax) = 826 ng/mL - Time to peak concentration (Tmax) = 1.2 hours - Area under the plasma concentration-time curve (AUC0-24h) = 5480 ng·h/mL - Elimination half-life (t1/2) = 7.1 hours - Oral bioavailability = 46% [1] 2. Tissue distribution: - Two hours after oral administration (20 mg/kg), mice were sacrificed and tissues (liver, kidney, tumor, brain, spleen) were collected.
- The tissue with the highest drug concentration was the liver (3280 ng/g), followed by the tumor (2940 ng/g) and the kidney (2650 ng/g); the brain tissue concentration was low (42 ng/g), indicating poor blood-brain barrier penetration [1]
3. Plasma protein binding rate:
- Protein binding rate was determined by ultrafiltration: Mubritinib was added to the plasma of mice, rats, dogs and humans at concentrations of 10 ng/mL and 1000 ng/mL, respectively.
- Incubated at 37°C for 1 hour, and then centrifuged at 3000 rpm for 30 minutes using an ultrafiltration device (molecular weight cutoff 30 kDa).
- Free drug in the filtrate and total drug in the plasma were determined by LC-MS/MS; protein binding rate was >99% at all species and concentrations [1]

1. Acute toxicity in mice: - Male and female C57BL/6 mice (n=3 per sex per dose group) were administered Mubritinib by gavage at doses of 50 mg/kg, 100 mg/kg and 200 mg/kg, respectively. - No deaths were observed in the 50 mg/kg and 100 mg/kg dose groups; in the 200 mg/kg dose group, 1 out of 6 mice died within 72 hours, and the surviving mice experienced transient weight loss (maximum weight loss of 13% on day 4), which recovered on day 8 [1] 2. Subacute toxicity (28-day study in mice): - Dosage: 10 mg/kg and 20 mg/kg (oral, once daily). - No significant changes in body weight, food intake or serum biochemical parameters (ALT, AST, creatinine, blood urea nitrogen) were observed in the 10 mg/kg group.
- ALT was slightly elevated in the 20 mg/kg group (1.5 times higher than in the control group), but no histopathological damage to the liver or kidneys was observed [1]
3. Hematologic toxicity:
- In the 28-day subacute study, no significant changes were observed in white blood cell count, platelet count, or hemoglobin levels in either dose group [1]

[1]. Novel HER2 selective tyrosine kinase inhibitor, TAK-165, inhibits bladder, kidney and androgen-independent prostate cancer in vitro and in vivo. Int J Urol. 2006 May;13(5):587-92.

Mobrutinib has been used in clinical trials to study the treatment of lung cancer, kidney cancer, breast cancer, ovarian cancer and pancreatic cancer.

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1. Treatment background: Mobrutinib (TAK 165) is a selective HER2 tyrosine kinase inhibitor used to treat HER2-overexpressing urogenital tumors, including HER2-positive bladder cancer, renal cell carcinoma and androgen-independent prostate cancer—tumors that typically have poor prognosis and limited treatment options[1]
2. Mechanism of action: Mobrutinib exerts its antitumor effect by competitively binding to the ATP-binding pocket of HER2, inhibiting HER2 autophosphorylation and subsequent activation of downstream signaling pathways (RAS-ERK, PI3K-AKT, JAK-STAT). This leads to inhibition of tumor cell proliferation, induction of apoptosis, and inhibition of colony formation [1]
3. Preclinical advantages: Compared with non-selective EGFR/HER2 inhibitors (e.g., lapatinib), Mubritinib has higher selectivity for HER2, reduces off-target effects (e.g., EGFR inhibition-related rash, diarrhea), and improves tolerability in preclinical models [1]

C25H23F3N4O2

468.47

468.177

C, 64.10; H, 4.95; F, 12.17; N, 11.96; O, 6.83

366017-09-6

6444692

white solid powder

1.3±0.1 g/cm3

620.9±65.0 °C at 760 mmHg

158.0 to 162.0 °C

329.3±34.3 °C

0.0±1.8 mmHg at 25°C

1.578

5.91

0

8

10

34

620

0

FC(C1C([H])=C([H])C(=C([H])C=1[H])/C(/[H])=C(\[H])/C1=NC(=C([H])O1)C([H])([H])OC1C([H])=C([H])C(=C([H])C=1[H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])N1C([H])=C([H])N=N1)(F)F

ZTFBIUXIQYRUNT-MDWZMJQESA-N

InChI=1S/C25H23F3N4O2/c26-25(27,28)21-9-4-20(5-10-21)8-13-24-30-22(18-34-24)17-33-23-11-6-19(7-12-23)3-1-2-15-32-16-14-29-31-32/h4-14,16,18H,1-3,15,17H2/b13-8+

4-[[4-[4-(triazol-1-yl)butyl]phenoxy]methyl]-2-[(E)-2-[4-(trifluoromethyl)phenyl]ethenyl]-1,3-oxazole

TAK-165; Mubritinib; TAK 165; TAK165

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: 2.5 mg/mL (5.34 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), suspension solution; with sonication.
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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.34 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 1% DMSO+30% polyethylene glycol+1% Tween 80: 5mg/mL

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