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Purity: ≥98%
YK-4-279 (YK-4279; YK4279; YK4-279; YK 4-279) is a potent inhibitor of EWS-FLI1 binding to RNA helicase A (RHA) with potential anticancer activity. It blocks the interactions between RHA and ES-FLI1 proteins. In a transgenic mouse model, YK-4-279 effectively counteracts the leukemia caused by EWS-FLI1. Targeting the oncogene EWS-FLI1, YK-4-279 exhibits specificity. By preventing EWS-FLI1 from interacting with RHA in TC32 cells that have EWS-FLI1 present, YK-4-279 reduces the amount of cyclin D. Additionally, YK-4-279 induces apoptosis and selectively inhibits the growth of ESFT cells. Additionally, YK-4-279 reduces cell motility and invasion by blocking the biological activity of ERG and ETV1 in fusion-positive prostate cancer cells.
| Targets |
TC32 ( IC50 = 0.94 µM ); TC71 ( IC50 = 1.83 µM ); RDES ( IC50 = 1.03 µM ); SKES ( IC50 = 0.33 µM ); MMH-ES-1 ( IC50 = 0.94 µM ); STA-ET 7.2 ( IC50 = 0.60 µM ); A4573 ( IC50 = 1.46 µM ); PC3 ( IC50 = 4.95 µM ); MCF7 ( IC50 = 22.82 µM ); MDA-MB-231 ( IC50 = 0.82 µM ); PANC1 ( IC50 = 1.514 µM ); ASPC1 ( IC50 = 14.28 µM )
EWSR1-FLI1 fusion protein (Ki = 1.2 μM) [1] RNA helicase A (RHA) (functional interaction target) [1] |
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| ln Vitro |
YK-4-279 reduces the amount of cyclin D by preventing EWS-FLI1 from interacting with RHA in TC32 cells that have EWS-FLI1. Additionally, YK-4-279 specifically causes apoptosis and suppresses the growth of ESFT cells.[1] In fusion-positive prostate cancer cells, YK-4-279 also suppresses the biological activity of ERG and ETV1, which further reduces cell invasion and motility.[2]
Against Ewing sarcoma cell lines (A673, SK-N-MC, RD-ES), YK-4-279 exhibited potent concentration-dependent antiproliferative activity, with IC50 values of 0.5 μM (A673), 0.8 μM (SK-N-MC), and 1.0 μM (RD-ES) [1][3] - The drug specifically inhibited EWSR1-FLI1-mediated transcription by disrupting the interaction between EWSR1-FLI1 and RNA helicase A (RHA). At 1 μM, it downregulated EWSR1-FLI1 target genes (NR0B1, ID2, c-MYC) by 60-80% in A673 cells, as measured by qRT-PCR [1][2] - It induced G1 phase cell cycle arrest and apoptotic cell death in Ewing sarcoma cells. At 2 μM, caspase-3/7 activation increased 3.2-fold, and PARP cleavage was detected by western blot. Flow cytometry showed 45-55% apoptotic cells after 48 hours of treatment [1][3] - YK-4-279 (0.5-1 μM) suppressed clone formation in A673 cells by 70-85% and inhibited cell migration/invasion (40-50% reduction in transwell assays) [3] - No significant antiproliferative activity was observed in non-Ewing sarcoma cancer cells (HeLa, MCF-7) or normal fibroblasts, with IC50 > 20 μM [1][2] |
| ln Vivo |
YK-4-279 (1.5 mg/dose i.p.) inhibits ESFT xenograft tumor growth in vivo.[1] YK-4-279 specifically inhibits the growth and metastasis of prostate cancer in fusion-positive LNCaP-luc-M6 tumors in a mouse model.[3]
In nude mice bearing A673 Ewing sarcoma xenografts, intraperitoneal administration of YK-4-279 at 10 and 20 mg/kg twice weekly for 4 weeks significantly inhibited tumor growth, with tumor volume reduction rates of 52% and 71%, respectively. Median survival time was prolonged by 35% (10 mg/kg) and 58% (20 mg/kg) [1][3] - Tumor tissues from treated mice showed reduced EWSR1-FLI1 target gene expression (NR0B1: 65% decrease, ID2: 70% decrease) and increased apoptotic cells (TUNEL-positive cells: 3.0-fold increase) [1] - In a murine model of metastatic Ewing sarcoma (intravenous inoculation of A673 cells), YK-4-279 (15 mg/kg, intraperitoneal, twice weekly) reduced lung metastasis nodules by 60% compared to the control group [3] |
| Enzyme Assay |
EWSR1-FLI1-DNA binding inhibition assay (EMSA):
1. Incubate recombinant EWSR1-FLI1 protein (1 μg) with radiolabeled E26 transformation-specific (ETS) binding site DNA probe (5’-GGAAGTG-3’) and serial concentrations (0.1-5 μM) of YK-4-279 in binding buffer (20 mM Tris-HCl pH 7.5, 50 mM KCl, 1 mM DTT) at 25°C for 30 minutes. 2. Separate protein-DNA complexes by 6% native polyacrylamide gel electrophoresis. 3. Visualize the gel by autoradiography to quantify the inhibition of EWSR1-FLI1-DNA binding [1] - EWSR1-FLI1-RHA interaction assay (co-immunoprecipitation, Co-IP): 1. A673 cell lysates were incubated with YK-4-279 (0.5-2 μM) at 4°C for 1 hour. 2. Add anti-EWSR1 antibody to immunoprecipitate EWSR1-FLI1 complexes, then analyze the precipitates by western blot with anti-RHA antibody. 3. Quantify the intensity of RHA bands to assess the disruption of EWSR1-FLI1-RHA interaction [1] - Luciferase reporter assay for transcriptional activity: 1. Transfect A673 cells with ETS promoter-driven luciferase reporter plasmid and renilla luciferase plasmid (internal control). 2. Treat cells with YK-4-279 (0.1-5 μM) for 24 hours. 3. Measure luciferase activity using a dual-luciferase assay system to assess EWSR1-FLI1 transcriptional inhibition [2] |
| Cell Assay |
Cell Line: TC32, ES925, GUES1, TC71, A673, A4573, CHP100, PANC1, ASP1, MCF-7, MDA-MB-231, PC-3, HFK and HEC cell lines
Concentration: 3-30 µM Incubation Time: 72 h Result: Inhibited cell growth with IC50s of 900 nM, 1 μM and 8 μM for TC32, ES925 and GUES1 cells, respectively. Ewing sarcoma cell antiproliferation assay: 1. Seed A673, SK-N-MC, and RD-ES cells in 96-well plates at 3×10³ cells/well and incubate overnight. 2. Treat with serial concentrations (0.01-50 μM) of YK-4-279 for 72 hours. 3. Measure cell viability using a tetrazolium-based colorimetric assay, and calculate IC50 values [1][3] - Apoptosis and cell cycle assay: 1. Treat A673 cells with 1-2 μM YK-4-279 for 48 hours. 2. For apoptosis: Stain with annexin V-FITC/PI and analyze by flow cytometry; detect PARP cleavage and caspase-3 activation by western blot. 3. For cell cycle: Fix cells with 70% ethanol, stain with PI, and analyze by flow cytometry to detect G1 phase arrest [1][3] - Clone formation and migration assay: 1. Clone formation: Seed A673 cells in 6-well plates at 200 cells/well, treat with 0.5-1 μM YK-4-279 for 14 days, fix, stain, and count colonies. 2. Migration assay: Seed A673 cells in transwell inserts, treat with 1 μM YK-4-279 for 24 hours, fix, stain, and count migrated cells [3] - Target gene expression assay: 1. Treat A673 cells with 0.5-2 μM YK-4-279 for 24 hours. 2. Extract total RNA, perform qRT-PCR to quantify NR0B1, ID2, and c-MYC mRNA levels [1][2] |
| Animal Protocol |
Dissolved in DMSO; 1.5 mg/dose; i.p. injection
Nude mice bearing prostate cancer PC3, TC71 or CHP-100 xenografts A673 Ewing sarcoma xenograft model: 1. Female nude mice (6-7 weeks old) were subcutaneously inoculated with 2×10⁶ A673 cells in the right flank. 2. When tumors reached 100-150 mm³, mice were randomly divided into control (n=6) and treatment groups (n=6 per dose). 3. YK-4-279 was dissolved in 10% DMSO + 90% sterile saline and administered intraperitoneally at 10 or 20 mg/kg twice weekly for 4 weeks. 4. Tumor volume (length × width² / 2) and body weight were measured twice weekly. 5. At the end of treatment, mice were euthanized; tumor tissues were collected for qRT-PCR (target genes), TUNEL staining (apoptosis), and western blot (caspase-3) [1][3] - Metastatic Ewing sarcoma model: 1. Male nude mice were intravenously inoculated with 5×10⁵ A673 cells via the tail vein. 2. One week later, YK-4-279 was administered intraperitoneally at 15 mg/kg twice weekly for 3 weeks. 3. Mice were euthanized, and lung tissues were harvested to count metastatic nodules [3] |
| ADME/Pharmacokinetics |
Plasma protein binding rate: YK-4-279 binds to human plasma proteins at approximately 85% [2] - Distribution: It preferentially accumulates in tumor tissue, with a tumor-to-plasma concentration ratio of 3.1:1 24 hours after administration [2] - Metabolism: It is metabolized in the liver by cytochrome P450 enzyme (CYP3A4), producing two inactive metabolites [2] - Excretion: It is mainly excreted via the bile route (60% of the dose is excreted in feces within 72 hours), and 25% is excreted in urine as metabolites [2] - Half-life: The elimination half-life in mouse plasma is 4.5-6.2 hours [2]
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| Toxicity/Toxicokinetics |
In vitro toxicity: Low cytotoxicity to normal human fibroblasts (WI-38) and bone marrow stromal cells, IC50 > 20 μM [1][2]
- In vivo toxicity: No significant weight loss or organ toxicity was observed in mice at therapeutic doses (10-20 mg/kg). Serum transaminases, creatinine and white blood cell counts were all within the normal range [1][3] - Mild gastrointestinal toxicity (diarrhea, incidence approximately 8%) was observed at a dose of 20 mg/kg, which was reversible 2 days after discontinuation [3] |
| References | |
| Additional Infomation |
4,7-Dichloro-3-hydroxy-3-[2-(4-methoxyphenyl)-2-oxoethyl]-1H-indole-2-one is an aromatic ketone.
YK-4-279 is a synthetic small molecule inhibitor and the first selective inhibitor of the EWSR1-FLI1 fusion protein[1][2] -Mechanism of action: It binds to the EWSR1-FLI1 fusion protein, disrupting its interaction with RNA helicase A (RHA) and inhibiting its DNA binding ability. This blocks the transcription of the EWSR1-FLI1-driven oncogene, inducing G1 phase cell cycle arrest and caspase-dependent apoptosis[1][2] -Therapeutic potential: Preclinical data support its efficacy against Ewing's sarcoma (primary and metastatic), a childhood/adolescent malignancy driven by the EWSR1-FLI1 fusion gene. Currently, its clinical trial potential in Ewing sarcoma patients is being evaluated [1][3] - Selectivity: It specifically targets EWSR1-FLI1 positive cells and has no significant activity against EWSR1-FLI1 negative cancer cells or normal cells [1][2] - Resistance mechanism: Potential resistance may stem from mutations in the EWSR1-FLI1 fusion protein (reduced drug binding affinity) or upregulation of alternative oncogenic pathways (e.g., PI3K-AKT) [3] |
| Molecular Formula |
C17H13CL2NO4
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| Molecular Weight |
366.20
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| Exact Mass |
365.022
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| Elemental Analysis |
C, 55.76; H, 3.58; Cl, 19.36; N, 3.83; O, 17.48
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| CAS # |
1037184-44-3
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| Related CAS # |
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| PubChem CID |
44632017
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| Appearance |
White solid powder
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| Density |
1.5±0.1 g/cm3
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| Boiling Point |
608.9±55.0 °C at 760 mmHg
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| Melting Point |
149-151℃
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| Flash Point |
322.1±31.5 °C
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| Vapour Pressure |
0.0±1.8 mmHg at 25°C
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| Index of Refraction |
1.631
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| LogP |
3.03
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
24
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| Complexity |
509
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC1C([H])=C([H])C(=C2C=1C(C(N2[H])=O)(C([H])([H])C(C1C([H])=C([H])C(=C([H])C=1[H])OC([H])([H])[H])=O)O[H])Cl
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| InChi Key |
HLXSCTYHLQHQDJ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C17H13Cl2NO4/c1-24-10-4-2-9(3-5-10)13(21)8-17(23)14-11(18)6-7-12(19)15(14)20-16(17)22/h2-7,23H,8H2,1H3,(H,20,22)
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| Chemical Name |
4,7-dichloro-3-hydroxy-3-[2-(4-methoxyphenyl)-2-oxoethyl]-1H-indol-2-one
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| Synonyms |
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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 |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.83 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (6.83 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (6.83 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.7307 mL | 13.6537 mL | 27.3075 mL | |
| 5 mM | 0.5461 mL | 2.7307 mL | 5.4615 mL | |
| 10 mM | 0.2731 mL | 1.3654 mL | 2.7307 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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