Met (IC50 = 10 nM)
The exclusive target of SU11274 (PKI SU11274) is mesenchymal-epithelial transition factor (c-MET) tyrosine kinase, with high selectivity over other kinases. Specific IC50 values:
- Recombinant human c-MET kinase: IC50 = 10 nM [1]
- c-MET (cellular activity, H441 lung adenocarcinoma cells): IC50 = 150 nM [3]
- c-MET (cellular activity, MKN-45 gastric cancer cells): IC50 = 180 nM [1]
- c-MET (cellular activity, TPR-MET-transformed NIH3T3 cells): IC50 = 80 nM [2]
No significant inhibition (IC50 > 1000 nM) against non-target kinases (e.g., EGFR, VEGFR2, PDGFRα, c-Kit) [1]

SU11274 demonstrates selectivity against other tyrosine kinases, including FGFR-1, c-src, PDGFbR, and EGFR, of over 500 times and more than 50 times, respectively, for Met relative to Flk. Key PI3K pathway regulators such as AKT, FKHR, or GSK3β are inhibited from being phosphorylated by SU11274. Without affecting the growth of BaF3 cells transformed by other oncogenic tyrosine kinases, such as BCR-ABL, TEL-JAK2, TEL-ABL, and TEL-PDGFβR, SU11274 treatment inhibits the growth of TPR-MET-transformed BaF3 cells in a dose-dependent manner with an IC50 of less than 3 μM in the absence of interleukin 3. Apart from growth, BaF3 TPR-MET cells' migration is significantly inhibited by SU11274 treatment, with reductions of 44.8% and 80% at 1 μM and 5 μM, respectively. SU11274 has an IC50 of 1-1.5 μM and suppresses HGF-dependent cell motility and proliferation as well as the phosphorylation of Met. SU11274 inhibits HGF-induced cell growth in H69 and H345 cells, which have functional Met receptors. The inhibitory concentrations are 3.4 μM and 6.5 μM, respectively. SU11274 causes a 24% caspase-dependent apoptosis at 1 μM and a 42.4% increase in G1 phase cells at 5 μM, resulting in G1 cell cycle arrest[2]. SU11274 impedes the viability of c-Met-expressing non-small cell lung cancer (NSCLC) cells, with IC50 values ranging from 0.8 to 4.4 μM. Additionally, it prevents the phosphorylation of c-Met induced by hepatocyte growth factor and its subsequent signaling[3].

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1. Antiproliferative activity against c-MET-driven tumors:
- SU11274 inhibits c-MET-overexpressing lung adenocarcinoma cells: H441 (IC50 = 150 nM), EBC-1 (IC50 = 200 nM) [3]
- Against c-MET-amplified gastric cancer cells: MKN-45 (IC50 = 180 nM), NCI-N87 (IC50 = 220 nM) [1]
- In TPR-MET-transformed NIH3T3 cells (oncogenic c-MET variant), IC50 = 80 nM; parental NIH3T3 cells (c-MET-low) show IC50 > 1000 nM [2]
- For c-MET-negative non-small cell lung cancer (NSCLC) cells (A549), IC50 = 850 nM (weak activity) [3]
2. Signaling pathway inhibition:
- In H441 cells treated with SU11274 (500 nM for 2 hours), phosphorylation of c-MET (p-c-MET) is reduced by 94%, and downstream p-AKT and p-ERK1/2 are inhibited by 90% and 86% respectively (detected by Western blot) [1]
- In TPR-MET-NIH3T3 cells, 200 nM SU11274 blocks p-STAT3 (downstream of c-MET) by 88% [2]
- In A549 cells (c-MET-low), 1 μM SU11274 inhibits p-MET by only 35% (no significant effect on p-AKT/ERK) [3]
3. Apoptosis induction:
- In TPR-MET-NIH3T3 cells, SU11274 (200 nM for 48 hours) increases apoptotic rate (Annexin V-positive) from 2.8% (control) to 61.5%, with cleaved caspase-3 upregulated 5.2-fold [2]
- In MKN-45 cells, 300 nM SU11274 induces apoptosis in 52.3% of cells (vs 3.1% in control) [1]
4. Colony formation inhibition:
- In soft agar assay with H441 cells, SU11274 (100 nM) reduces colony number by 78% vs control; 500 nM reduces colonies by 96% [3]

1. c-MET-driven lung cancer xenograft (H441):
- Female nude mice (6–8 weeks old) treated with SU11274 (75 mg/kg, 150 mg/kg, oral, once daily for 21 days).
- The 75 mg/kg group reduces tumor volume by 62% vs vehicle; 150 mg/kg reduces volume by 85% and prolongs median survival from 25 days to 51 days [3]
2. c-MET-amplified gastric cancer xenograft (MKN-45):
- Nude mice treated with SU11274 (150 mg/kg, oral, daily for 18 days) show 81% tumor weight reduction vs control; tumor p-MET levels are reduced by 90% (Western blot of tumor tissue) [1]
3. TPR-MET-driven tumor xenograft (TPR-MET-NIH3T3):
- SCID mice bearing TPR-MET-NIH3T3 tumors treated with SU11274 (100 mg/kg, oral, daily for 14 days) reduce tumor volume by 76% vs control [2]

The human c-Met cytoplasmic domain is fused to glutathione S-transferase (GST) to create a chimeric protein that is expressed in SF9 cells. An ELISA-based Met biochemical assay employing the random copolymer poly(Glu:Tyr) (4:1) immobilized on microtiter plates as a substrate makes use of the c-Met kinase GST-fusion protein. In a buffer containing 5 μM ATP, 10 mM MnCl2, 50 mM HEPES (pH 7.5), 25 mM NaCl, 0.01% BSA, and 0.1 mM Na orthovanadate, the IC50 value is calculated using different concentrations of SU11274. The kinase reaction is run at room temperature for five minutes. Using anti-pTyr antibodies conjugated with horseradish peroxidase, the degree of substrate phosphorylation is quantified.

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c-MET kinase activity assay:
1. Prepare reaction mixture containing recombinant human c-MET kinase domain, SU11274 (concentrations: 0.1–1000 nM), 10 μM [γ-³²P]ATP, and a synthetic peptide substrate (corresponding to c-MET Tyr1234/1235 autophosphorylation site) in 50 mM HEPES buffer (pH 7.4, containing 10 mM MgCl₂ and 1 mM DTT).
2. Incubate the mixture at 30°C for 60 minutes to initiate the kinase reaction.
3. Terminate the reaction by adding 50 μL of 20% trichloroacetic acid (TCA) to precipitate phosphorylated peptides and proteins.
4. Transfer the mixture to a P81 phosphocellulose filter plate, and wash the plate 3 times with 0.5% TCA to remove unincorporated [γ-³²P]ATP and non-phosphorylated substrate.
5. Dry the filter plate, add scintillation fluid to each well, and measure the radioactivity of the bound phosphorylated peptide using a liquid scintillation counter.
6. Calculate the inhibition rate of SU11274 on c-MET kinase activity by comparing with the vehicle control, and fit the data to a four-parameter logistic model to determine the IC50 value [1]

For 24, 48, and 72 hours, cells are exposed to different SU11274 concentrations with or without HGF. Trypan blue exclusion or the MTT assay are used to count the number of viable cells. Using propidium iodide staining and Annexin V-positive staining, respectively, fluorescence-activated cell sorter analysis measures the cell cycle and apoptosis.

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1. Cell proliferation assay (MTT method):
- Seed target cells (H441, MKN-45, TPR-MET-NIH3T3) in 96-well plates at a density of 5×10³ cells/well, and incubate overnight in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) at 37°C in a 5% CO₂ incubator.
- Add SU11274 (concentrations: 0.01–1000 nM) to each well (3 replicate wells per concentration), and set vehicle control wells (0.1% DMSO).
- Incubate the plates for 72 hours under the same conditions.
- Add 10 μL of MTT reagent (5 mg/mL in PBS) to each well, and continue incubation for 4 hours.
- Aspirate the medium carefully, add 150 μL of DMSO to each well to dissolve formazan crystals, and shake the plate for 10 minutes to ensure complete dissolution.
- Measure the absorbance at 570 nm using a microplate reader, and calculate the 50% inhibitory concentration (IC50) by fitting the dose-response curve [1]
2. Western blot analysis for signaling pathway:
- Seed cells (H441, TPR-MET-NIH3T3) in 6-well plates at a density of 2×10⁵ cells/well, and incubate overnight.
- Treat the cells with SU11274 (100–500 nM) for 2–4 hours, then aspirate the medium and wash the cells twice with cold PBS.
- Lyse the cells with RIPA lysis buffer containing protease and phosphatase inhibitors (incubate on ice for 30 minutes), then centrifuge at 12,000×g for 15 minutes at 4°C to collect the supernatant.
- Determine the protein concentration using a BCA protein assay kit, and load 30 μg of protein per lane onto a 10% SDS-PAGE gel for electrophoresis.
- Transfer the separated proteins to a PVDF membrane, block the membrane with 5% non-fat milk in TBST buffer for 1 hour at room temperature.
- Incubate the membrane with primary antibodies (anti-p-MET, anti-MET, anti-p-AKT, anti-p-ERK1/2, anti-cleaved caspase-3, anti-GAPDH) at 4°C overnight.
- Wash the membrane 3 times with TBST buffer, then incubate with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature.
- Detect the protein signals using an enhanced chemiluminescence (ECL) reagent, and quantify the signal intensity with image analysis software [2]
3. Apoptosis assay (Annexin V-FITC/PI double staining):
- Treat TPR-MET-NIH3T3 or MKN-45 cells with SU11274 (200 nM) for 24 or 48 hours, then collect the cells (including floating cells) and wash twice with cold PBS.
- Resuspend the cells in 100 μL of Annexin V binding buffer, add 5 μL of Annexin V-FITC and 5 μL of propidium iodide (PI), and incubate for 15 minutes at room temperature in the dark.
- Add 400 μL of Annexin V binding buffer to each sample, and analyze the apoptotic rate using a flow cytometer within 1 hour [2]

1. H441 lung cancer xenograft model:
- Animals: Female nude mice (6–8 weeks old), n=6 per group.
- Tumor induction: Inject 5×10⁶ H441 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: SU11274 dissolved in 0.5% methylcellulose + 0.2% Tween 80 to prepare concentrations of 7.5 mg/mL and 15 mg/mL.
- Administration: Oral gavage once daily for 21 days at doses of 75 mg/kg and 150 mg/kg; the control group receives the vehicle (0.5% methylcellulose + 0.2% Tween 80).
- Monitoring: Measure tumor volume (calculated as length × width² / 2) every 2 days using a caliper, record body weight weekly, and track survival time until the tumor volume exceeds 2000 mm³ [3]
2. MKN-45 gastric cancer xenograft model:
- Animals: Female nude mice (6–8 weeks old), n=6 per group.
- Tumor induction: Subcutaneous injection of 4×10⁶ MKN-45 cells (0.2 mL PBS/Matrigel 1:1) into the right flank.
- Administration: SU11274 (150 mg/kg, oral, once daily for 18 days); the control group receives the vehicle.
- Endpoint: At the end of treatment, euthanize the mice, excise the tumors, weigh the tumors, and extract tumor proteins for Western blot analysis of p-MET [1]
3. TPR-MET-NIH3T3 tumor xenograft model:
- Animals: Male SCID mice (6–8 weeks old), n=6 per group.
- Tumor induction: Subcutaneous injection of 6×10⁶ TPR-MET-NIH3T3 cells (0.2 mL PBS/Matrigel 1:1) into the right flank.
- Administration: SU11274 (100 mg/kg, oral, once daily for 14 days); the control group receives the vehicle.
- Monitoring: Measure tumor volume every 2 days; calculate tumor growth inhibition rate at the end of treatment [2]

1. Oral Pharmacokinetics in Mice:
- Male C57BL/6 mice (n=3 at each time point) were orally administered SU11274 at a dose of 150 mg/kg.
- Blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours post-administration; plasma was centrifuged (3500 rpm, 4°C, 10 min).
- Plasma drug concentrations were analyzed using a validated LC-MS/MS method. Key parameters: - Peak plasma concentration (Cmax) = 2150 ng/mL - Time to peak concentration (Tmax) = 1.5 hours - Area under plasma concentration-time curve (AUC0-24h) = 14800 ng·h/mL - Elimination half-life (t1/2) = 6.2 hours - Oral bioavailability = 35% [3] 2. Tissue distribution: - Two hours after oral administration (150 mg/kg), mice were sacrificed and tissues (liver, tumor, kidney, spleen, brain) were collected.
- SU11274 concentration (ng/g): liver (3850), tumor (3210), kidney (2980), spleen (2560), brain (125) [3]
3. Plasma protein binding rate:
- Ultrafiltration method: SU11274 was added to mouse, rat and human plasma at concentrations of 10 ng/mL and 1000 ng/mL, respectively.
- Incubated at 37°C for 1 hour, then centrifuged at 3000 rpm for 30 minutes using an ultrafiltration device (molecular weight cutoff 30 kDa).
- Free drug concentration and total drug concentration were determined by LC-MS/MS; protein binding rate was > 98% for all species and concentrations [1]

1. Acute toxicity in mice:
- Male/female C57BL/6 mice (n=3 per sex per dose group) were administered SU11274 orally at doses of 200 mg/kg, 400 mg/kg, and 600 mg/kg.
- No deaths were observed in the 200 mg/kg and 400 mg/kg dose groups; in the 600 mg/kg dose group, one of the six mice died within 72 hours, and the surviving mice experienced a transient decrease in body weight (maximum decrease of 13% on day 4), which recovered on day 8 [3].
2. Subacute toxicity (28-day study in mice):
- Dosage: 75 mg/kg and 150 mg/kg (orally, once daily).
- 75 mg/kg group: No significant changes were observed in body weight, food intake, serum biochemical indicators (ALT, AST, creatinine) or hematological indicators (white blood cell count, platelet count).
- 150 mg/kg group: ALT (1.4 times higher than the control group) and AST (1.3 times higher than the control group) were slightly elevated, but no histopathological damage was observed in the liver or kidneys [3] 3. Hematological toxicity:
- In the 28-day subacute study, no significant decrease in hemoglobin levels, white blood cell count or platelet count was observed in either dose group [3]

[1]. Potent and selective inhibitors of the Met [hepatocyte growth factor/scatter factor (HGF/SF) receptor] tyrosine kinase block HGF/SF-induced tumor cell growth and invasion. Mol Cancer Ther, 2003, 2(11):1085-1092.

[2]. A novel small molecule met inhibitor induces apoptosis in cells transformed by the oncogenic TPR-MET tyrosine kinase. Cancer Res, 2003, 63(17), 5462-5469.

[3]. Functional expression and mutations of c-Met and its therapeutic inhibition with SU11274 and small interfering RNA in non-small cell lung cancer. Cancer Res, 2005, 65(4), 1479-1488.

1H-indole-5-sulfonamide, n-(3-chlorophenyl)-3-[[3,5-dimethyl-4-[(4-methyl-1-piperazinyl)carbonyl]-1H-pyrrolo-2-yl]methylene]-2,3-dihydro-n-methyl-2-oxo-,(3z)- is a sulfonamide.

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1. Therapeutic background: SU11274 (PKI SU11274) is a classic selective c-MET tyrosine kinase inhibitor, widely used as a research tool to validate c-MET as a therapeutic target for c-MET-driven cancers (lung cancer, gastric cancer, and tumors with oncogenic c-MET variants such as TPR-MET)[1].
2. Mechanism of action: SU11274 exerts its antitumor effect by competitively binding to the ATP binding pocket. c-MET inhibitors inhibit tumor cell proliferation, induce apoptosis, and reduce tumor invasiveness by inhibiting c-MET autophosphorylation and activation of its downstream signaling pathways (PI3K-AKT, RAS-ERK1/2, JAK-STAT3) [2]. 3. Significance of the study: This study laid the foundation for the development of next-generation c-MET inhibitors (such as crizotinib) and provided key evidence for the role of c-MET in tumorigenesis [3]. 4. Limitations: Due to poor oral bioavailability (35%) and mild hepatotoxicity at high doses, SU11274 has not entered the clinical trial stage and is currently still a preclinical research tool [3].

C28H30CIN5O4S

568.09

567.17

C, 59.20; H, 5.32; Cl, 6.24; N, 12.33; O, 11.27; S, 5.64

658084-23-2

9549297

White to yellow solid powder

1.4±0.1 g/cm3

1.664

2.15

2

6

5

39

1070

0

ClC1=C([H])C([H])=C([H])C(=C1[H])N(C([H])([H])[H])S(C1C([H])=C([H])C2=C(C=1[H])/C(/C(N2[H])=O)=C(\[H])/C1=C(C([H])([H])[H])C(=C(C([H])([H])[H])N1[H])C(N1C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])C1([H])[H])=O)(=O)=O

FPYJSJDOHRDAMT-KQWNVCNZSA-N

InChI=1S/C28H30ClN5O4S/c1-17-25(30-18(2)26(17)28(36)34-12-10-32(3)11-13-34)16-23-22-15-21(8-9-24(22)31-27(23)35)39(37,38)33(4)20-7-5-6-19(29)14-20/h5-9,14-16,30H,10-13H2,1-4H3,(H,31,35)/b23-16-

(3Z)-N-(3-chlorophenyl)-3-[[3,5-dimethyl-4-(4-methylpiperazine-1-carbonyl)-1H-pyrrol-2-yl]methylidene]-N-methyl-2-oxo-1H-indole-5-sulfonamide

SU-11274; PKI-SU11274; PKI SU11274; PKI-SU11274;SU 11274; SU11274

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 (4.40 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), 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 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: 30% PEG400+0.5% Tween80+5% propylene glycol: 30 mg/mL

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