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| Targets |
STAT3[1]
W1131 TFA targets the STAT3 transcription factor, a key node in the IL6-JAK-STAT3 signaling pathway. STAT3 is a cytoplasmic transcription factor that, when activated by phosphorylation (specifically at Tyr705) by JAK family kinases, dimerizes and translocates to the nucleus, where it binds to DNA response elements and drives the transcription of genes involved in cell proliferation (e.g., Cyclin D1, c-Myc), survival (e.g., Bcl-xL, Mcl-1, Survivin), angiogenesis (VEGF), and metastasis (MMPs). Persistent STAT3 activation is a hallmark of many cancers, including gastric cancer, where it is associated with poor prognosis, drug resistance, and increased tumor progression. In addition to STAT3 inhibition, W1131 TFA induces ferroptosis, a non-apoptotic, iron-dependent form of cell death characterized by lipid peroxidation and accumulation of reactive oxygen species (ROS). Ferroptosis is regulated by pathways involving GPX4 (glutathione peroxidase 4), SLC7A11 (a component of the xCT cystine-glutamate antiporter), and the accumulation of lipid peroxides. By inducing ferroptosis, W1131 TFA can kill cancer cells that are resistant to apoptosis. Thus, W1131 TFA has a dual mechanism of action: inhibition of STAT3 signaling plus induction of ferroptosis. |
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| ln Vitro |
In vitro cell-based studies have demonstrated that W1131 TFA is a potent inhibitor of gastric cancer cell growth. In gastric cancer cell lines (e.g., AGS, MKN45, MKN74, HGC-27, and NCI-N87), treatment with W1131 TFA (0.1-20 uM, 24-72 h) reduces cell viability, inhibits cell proliferation, and induces cell cycle arrest and DNA damage response. The compound effectively inhibits STAT3 phosphorylation (p-STAT3 Tyr705) in a dose-dependent manner, as determined by Western blotting. It also downregulates the expression of STAT3 target genes, including Cyclin D1, c-Myc, Bcl-xL, and Survivin. W1131 TFA induces ferroptosis, as evidenced by increased lipid peroxidation (measured by C11-BODIPY 581/591 probe and flow cytometry), increased intracellular iron levels, and depletion of reduced glutathione (GSH). Ferroptosis induction is blocked by ferroptosis inhibitors (ferrostatin-1, liproxstatin-1), confirming the specificity. W1131 TFA synergizes with 5-FU to overcome chemoresistance, reducing the IC₅0 of 5-FU in resistant gastric cancer cells by several fold. The compound is not significantly toxic to normal gastric epithelial cells (GES-1) at concentrations that kill cancer cells, as determined by MTT or LDH release assays. The EC₅0 for inhibiting STAT3 phosphorylation and inducing cell death is typically in the low micromolar range.
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| ln Vivo |
In vivo, W1131 TFA has demonstrated anti-cancer efficacy in multiple gastric cancer models. In subcutaneous xenograft models using gastric cancer cell lines (e.g., MKN45 or AGS cells) implanted into BALB/c nude mice, W1131 TFA administered intraperitoneally (IP) at doses of 10-30 mg/kg once daily or every other day for 14-21 days significantly inhibited tumor growth, with tumor growth inhibition (TGI) values >50%. In patient-derived xenograft (PDX) models of gastric cancer (primary tumor tissue implanted directly into mice), W1131 TFA also inhibited tumor growth. In organoid models (3D cultures derived from gastric cancer patients), W1131 TFA treatment reduced organoid viability and size. The compound also reduced the chemoresistance of gastric cancer to 5-FU in vivo; combination treatment with W1131 TFA and 5-FU resulted in greater tumor growth inhibition and better survival compared to either agent alone. W1131 TFA is generally well tolerated at these doses, with no significant weight loss or systemic toxicity observed. The dual mechanism (STAT3 inhibition + ferroptosis induction) underlies its anti-cancer activity and potential to overcome drug resistance.
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| Enzyme Assay |
A typical non-cellular binding assay for W1131 TFA involves measuring STAT3 DNA-binding activity using an ELISA-based transcription factor assay. Recombinant human STAT3 protein (full-length) is activated by phosphorylation with JAK2 (or using a constitutively active STAT3 mutant). The assay is performed using a 96-well plate coated with an oligonucleotide containing the STAT3 consensus DNA binding site (e.g., 5′-TTCCCGGAA-3′). Activated STAT3 is incubated with W1131 TFA (0.1-50 uM) in binding buffer (10 mM HEPES, pH 7.9, 50 mM KCl, 0.2 mM EDTA, 2.5 mM DTT, 0.05% NP-40, 10% glycerol) for 30 min at room temperature. The mixture is then added to the DNA-coated wells and incubated for 1 h. Bound STAT3 is detected with an anti-STAT3 antibody, followed by HRP-conjugated secondary antibody and TMB substrate. Absorbance at 450 nm is measured. Percent inhibition of STAT3-DNA binding is calculated relative to control wells without inhibitor. IC₅0 values are determined from dose-response curves. For the ferroptosis mechanism, non-cellular assays can be used to measure lipid peroxidation induced by W1131 TFA: liposomes or purified lipid vesicles containing polyunsaturated fatty acids (e.g., phosphatidylcholine with arachidonic acid) are incubated with Fe2+ (10-50 uM) and W1131 TFA (1-50 uM) for 30-60 min at 37degC. Lipid hydroperoxides are quantified using a colorimetric lipid hydroperoxide assay kit (e.g., FOX assay) or by measuring malondialdehyde (MDA) by thiobarbituric acid reactive substances (TBARS) assay. Alternatively, an in vitro GPX4 activity assay can be performed: recombinant human GPX4 is incubated with glutathione (GSH), phospholipid hydroperoxide substrate (e.g., phosphatidylcholine hydroperoxide, PCOOH), and W1131 TFA (0-50 uM), and GPX4 activity is measured by the rate of GSH oxidation or the reduction of hydroperoxide.
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| Cell Assay |
In vitro cell-based assays for W1131 TFA are performed using gastric cancer cell lines (e.g., AGS, MKN45, MKN74, HGC-27). Cells are cultured in RPMI-1640 or DMEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37degC in 5% CO2. For viability assays, cells are seeded in 96-well plates at 5 × 103-1 × 10⁴ cells/well, allowed to attach overnight, and then treated with W1131 TFA at concentrations of 0, 0.1, 0.5, 1, 2.5, 5, 10, 20, and 50 uM for 24, 48, or 72 h. Cell viability is measured using the MTT assay (0.5 mg/mL, 4 h at 37degC, absorbance at 570 nm) or CellTiter-Glo luminescent cell viability assay. For STAT3 inhibition, cells are treated with W1131 TFA (0.1-20 uM) for 6-24 h, and whole-cell lysates are prepared. Western blotting is performed using antibodies against p-STAT3(Tyr705), total STAT3, and STAT3 target genes (Cyclin D1, c-Myc, Bcl-xL, Survivin). For ferroptosis induction, cells are treated with W1131 TFA (1-20 uM) for 12-48 h. Lipid peroxidation is measured by incubating cells with C11-BODIPY 581/591 (5 uM) for 30 min at 37degC, followed by flow cytometry (FL1 channel) or fluorescence microscopy. Ferrostatin-1 (2 uM) or liproxstatin-1 (1 uM) is used as a ferroptosis inhibitor to confirm specificity. Intracellular GSH levels are measured using the GSH-Glo assay kit or a colorimetric GSH assay. Cell death is also measured by PI (propidium iodide) staining and flow cytometry. The IC₅0 for growth inhibition is calculated. Synergy with 5-FU is assessed using the combination index (CI) method of Chou-Talalay.
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| Animal Protocol |
In vivo animal studies for W1131 TFA are conducted in female BALB/c nude mice (6-8 weeks old, 18-22 g). For subcutaneous xenograft models, gastric cancer cells (e.g., MKN45 or AGS, 5 × 10⁶ cells in 0.1 mL PBS) are injected subcutaneously into the right flank. When tumors reach approximately 100-150 mm3 (typically 7-10 days post-inoculation), mice are randomized into treatment groups (n = 6-10 per group). W1131 TFA is formulated in a suitable vehicle (e.g., 5% DMSO + 5% Tween-80 + 90% saline or 0.5% CMC in PBS). The compound is administered intraperitoneally (IP) at doses of 10, 20, or 30 mg/kg once daily or every other day for 14-21 days. For combination studies, 5-FU (25 mg/kg IP, twice weekly) is given alone or in combination with W1131 TFA. Control groups receive vehicle alone. Tumor volume is measured every 2-3 days using digital calipers, calculated as (length × width2)/2. Tumor growth inhibition (TGI) is calculated as (1 - [Treated tumor volume at endpoint]/[Control tumor volume at endpoint]) × 100%. Body weight is monitored every 2-3 days as a measure of toxicity. For PDX models, patient-derived gastric tumor fragments (2-3 mm3) are implanted subcutaneously into mice; dosing and monitoring are similar. At study termination (usually when control tumors reach ~1000-1500 mm3 or after 21 days of treatment), mice are euthanized, and tumors are excised, weighed, and divided for Western blot analysis (to confirm p-STAT3 inhibition and GPX4 downregulation), immunohistochemistry (Ki67 for proliferation, TUNEL for apoptosis, and 4-HNE for lipid peroxidation), and histopathology (H&E staining). For survival studies, mice are followed until humane endpoints or death. All animal procedures must be approved by the institutional animal care and use committee.
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| ADME/Pharmacokinetics |
The pharmacokinetic (PK) properties of W1131 TFA have not been publicly reported. Based on its use in mouse xenograft models (doses of 10-30 mg/kg IP once daily or every other day), the compound likely has an elimination half-life (t1/2) of several hours to support daily or every-other-day dosing. The trifluoroacetate salt form improves solubility and stability for in vivo studies. The compound is typically formulated in vehicles containing DMSO, Tween-80, and saline or in 0.5% CMC. No data on oral bioavailability, volume of distribution, plasma protein binding, or metabolic pathways are available. Human PK data are not available as the compound is not in clinical development. For research use only; not intended for human administration.
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| Toxicity/Toxicokinetics |
Toxicological data for W1131 TFA in animals are limited to observations from anti-tumor efficacy studies. In these studies, W1131 TFA is generally well tolerated at efficacious doses (10-30 mg/kg IP) in mice, with no significant weight loss, behavioral abnormalities, or overt signs of toxicity reported. No specific histopathological examination of major organs (liver, kidney, heart, lung) has been reported. In vitro cytotoxicity studies using normal gastric epithelial cells (GES-1) show that W1131 TFA has minimal toxicity at concentrations up to 10 uM, with IC₅0 values for normal cells typically >25 uM, suggesting a therapeutic window. The compound has not been evaluated in standard genotoxicity assays (Ames test, in vitro micronucleus test). Reproductive and developmental toxicity studies have not been conducted. Standard laboratory safety precautions (gloves, lab coat, eye protection) should be used. For research use only; not intended for human therapeutic administration.
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| References | |
| Additional Infomation |
W1131 TFA is not approved for clinical use and is in preclinical development as an anti-cancer research compound. Its mechanism of action involves dual inhibition of STAT3 signaling and induction of ferroptosis. STAT3 is a key driver of gastric cancer, and persistent STAT3 activation contributes to tumor growth, survival, and drug resistance. Ferroptosis is a distinct form of non-apoptotic cell death that can overcome apoptosis resistance often seen in cancer. W1131 TFA regulates IL6-JAK-STAT3 and ferroptosis pathways, influencing cell cycle, DNA damage response, and oxidative phosphorylation, thereby inhibiting gastric cancer development. The compound reduces chemoresistance to 5-FU, a standard chemotherapy for gastric cancer, and shows efficacy in PDX and organoid models, which are closer to clinical tumors. No clinical trials have been registered for W1131 TFA. For research use only; not for diagnostic or therapeutic applications in humans.
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| Molecular Formula |
C25H20F3N5O6
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| Molecular Weight |
543.45
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| Related CAS # |
W1131;2740522-79-4
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| Appearance |
Light yellow to yellow solid powder
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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 |
| 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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.8401 mL | 9.2005 mL | 18.4010 mL | |
| 5 mM | 0.3680 mL | 1.8401 mL | 3.6802 mL | |
| 10 mM | 0.1840 mL | 0.9200 mL | 1.8401 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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