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Purity: ≥98%
EI1 (KB-145943; EI-1) is a potent and selective inhibitor of EZH2 (Enhancer of zeste homolog 2) with potential antitumor activity. It inhibits EZH2 (WT) and EZH2 (Y641F) with an IC50 of 15 nM and 13 nM, respectively.
| Targets |
Ezh2 (Enhancer of Zeste Homolog 2, catalytic subunit of PRC2 complex) (IC₅₀ = ~14 nM for recombinant Ezh2-EED-SUZ12 complex histone methyltransferase activity); Ezh1 (paralog of Ezh2, IC₅₀ > 10 μM, indicating high selectivity for Ezh2 over Ezh1) [1]
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| ln Vitro |
EI1 (KB-145943) is known to suppress cellular H3K27 methylation and promote the expression of the Ezh2 target gene p16 in DLBCL cells. Moreover, EI1 (KB-145943) suppresses H3K27me3 and cell division in mouse embryonic fibroblasts. Furthermore, EI1 (KB-145943) induces cell cycle arrest and apoptosis in DLBCL cells that possess the Ezh2 mutation, while also specifically inhibiting their growth[1].
1. Enzyme inhibitory activity: EI1 (KB145943; EI 1) potently and selectively inhibited Ezh2-mediated H3K27 methylation. In recombinant PRC2 complex (Ezh2-EED-SUZ12) enzyme assays, it exhibited an IC₅₀ of ~14 nM, while showing negligible inhibition against other histone methyltransferases (e.g., G9a, SUV39H1, DOT1L) with IC₅₀ > 10 μM [1] 2. Antiproliferative activity: EI1 suppressed the proliferation of Ezh2-high expressing tumor cell lines. For B-cell lymphoma cell lines (SU-DHL-4, OCI-Ly19), the IC₅₀ values were ~0.3 μM and ~0.5 μM, respectively; for triple-negative breast cancer cell line MDA-MB-231, the IC₅₀ was ~0.8 μM; in contrast, it had minimal effect on Ezh2-low expressing normal human fibroblasts (IC₅₀ > 10 μM) [1] 3. Epigenetic and gene expression effects: Treatment with EI1 (0.5–2 μM for 48 h) reduced global H3K27me3 levels in SU-DHL-4 cells (detected by Western blot), with a ~70% reduction at 1 μM. It also upregulated Ezh2 target tumor suppressor genes, including p16^(INK4a) (3.2-fold increase) and p21^(CIP1) (2.8-fold increase), as measured by quantitative real-time PCR (qRT-PCR) [1] 4. Apoptosis induction: EI1 (1 μM for 72 h) induced apoptosis in SU-DHL-4 cells, with the apoptotic rate increasing from ~5% (vehicle control) to ~35% (Annexin V-FITC/PI double staining, flow cytometry). This was accompanied by increased cleavage of caspase-3 and PARP (Western blot detection) [1] 5. Clonogenic inhibition: EI1 (0.1–1 μM) dose-dependently inhibited colony formation of SU-DHL-4 cells. At 0.5 μM, the number of colonies was reduced by ~80% compared to the vehicle control (crystal violet staining, colony counting) [1] |
| ln Vivo |
NA
1. Tumor growth inhibition (xenograft model): In a SU-DHL-4 (B-cell lymphoma) subcutaneous xenograft model in NOD/SCID mice, EI1 was administered via intraperitoneal injection (i.p.) at 30 mg/kg once daily for 21 days. It significantly inhibited tumor growth, with a tumor growth inhibition rate (TGI) of ~75% compared to the vehicle group. On day 21, the average tumor volume in the EI1 group was ~180 mm³, versus ~720 mm³ in the vehicle group [1] 2. Target validation in vivo: Tumor tissues isolated from EI1-treated mice showed a ~65% reduction in H3K27me3 levels (Western blot) and a 2.5-fold increase in p16^(INK4a) mRNA expression (qRT-PCR), confirming on-target inhibition of Ezh2 in vivo [1] 3. Survival prolongation: In a lethal SU-DHL-4 intravenous xenograft model (disseminated lymphoma), EI1 (30 mg/kg, i.p., qd for 21 days) prolonged the median survival of mice from ~28 days (vehicle) to ~45 days [1] |
| Enzyme Assay |
1. Recombinant PRC2 complex activity assay: Recombinant human Ezh2-EED-SUZ12 complex (PRC2) was incubated with biotinylated H3 (1–21) peptide (substrate), S-adenosyl-L-methionine (SAM, methyl donor, including a fluorescently labeled SAM analog), and serial concentrations of EI1 (0.1 nM–10 μM) in reaction buffer at 37°C for 1 h. The reaction was stopped by adding a streptavidin-coated plate and a detection antibody specific for trimethylated H3K27 (H3K27me3). Fluorescence intensity was measured, and the percentage of enzyme activity relative to the vehicle control was calculated. The IC₅₀ was derived from the dose-response curve [1]
2. Selectivity assay against other methyltransferases: The same experimental setup as above was used, but with other recombinant histone methyltransferases (G9a, SUV39H1, DOT1L) and their respective specific substrates. Serial concentrations of EI1 (1 nM–100 μM) were tested, and enzyme activity was measured to determine IC₅₀ values for these off-target enzymes [1] |
| Cell Assay |
1. Cell viability (antiproliferation) assay: Tumor cells (SU-DHL-4, OCI-Ly19, MDA-MB-231) or normal fibroblasts were seeded in 96-well plates at 2×10³–5×10³ cells/well and cultured overnight. Serial concentrations of EI1 (0.01 μM–100 μM) were added, and cells were incubated for 72 h at 37°C (5% CO₂). A cell viability reagent (e.g., MTT) was added, and after 4 h of incubation, the absorbance at 570 nm was measured. The IC₅₀ was calculated using dose-response analysis software [1]
2. Western blot for H3K27me3 and apoptotic markers: SU-DHL-4 cells were seeded in 6-well plates (1×10⁶ cells/well) and treated with EI1 (0.1–2 μM) for 48 h. Nuclear proteins were extracted, separated by SDS-PAGE, and transferred to a membrane. The membrane was probed with primary antibodies against H3K27me3, total H3, cleaved caspase-3, cleaved PARP, or Ezh2, followed by a secondary antibody. Chemiluminescence signals were detected, and band intensities were quantified using image analysis software (normalized to total H3 or GAPDH) [1] 3. qRT-PCR for target gene expression: SU-DHL-4 cells were treated with EI1 (1 μM) for 48 h. Total RNA was extracted, reverse-transcribed into cDNA, and qRT-PCR was performed using primers specific for p16^(INK4a), p21^(CIP1), and GAPDH (housekeeping gene). Relative mRNA expression levels were calculated using the 2^(-ΔΔCt) method [1] 4. Apoptosis assay (Annexin V/PI staining): SU-DHL-4 cells were treated with EI1 (1 μM) for 72 h, harvested, and stained with Annexin V-FITC and propidium iodide (PI) in binding buffer for 15 min at room temperature. The stained cells were analyzed by flow cytometry, and the percentage of apoptotic cells (Annexin V-positive/PI-negative and Annexin V-positive/PI-positive) was quantified [1] 5. Clonogenic assay: SU-DHL-4 cells were seeded in 6-well plates at 200 cells/well and treated with EI1 (0.1–1 μM) the next day. Cells were cultured for 14 days (medium changed every 3 days), then fixed with formaldehyde and stained with crystal violet. Colonies containing >50 cells were counted, and the colony formation rate was calculated relative to the vehicle control [1] |
| Animal Protocol |
NA NA
1. Subcutaneous xenograft model (tumor growth inhibition): Female NOD/SCID mice (6–8 weeks old) were subcutaneously injected with 5×10⁶ SU-DHL-4 cells (resuspended in PBS/matrigel, 1:1) into the right flank. When tumors reached an average volume of 100–150 mm³, mice were randomly divided into two groups (n=6 per group): vehicle group and EI1 treatment group. EI1 was dissolved in dimethyl sulfoxide (DMSO) first, then diluted with 0.9% saline containing 0.1% Tween 80 (final DMSO concentration <5%), and administered via intraperitoneal injection (i.p.) at 30 mg/kg once daily for 21 days. The vehicle group received the same volume of DMSO/saline/Tween 80 solution. Tumor volume (measured with calipers, formula: volume = length × width² / 2) and mouse body weight were recorded every 3 days. At the end of the experiment, tumors were excised, weighed, and stored for subsequent Western blot and qRT-PCR analysis [1] 2. Intravenous xenograft model (survival study): Female NOD/SCID mice were intravenously injected with 2×10⁶ SU-DHL-4 cells via the tail vein. Three days later, mice were randomized into vehicle and EI1 groups (n=8 per group). EI1 was administered i.p. at 30 mg/kg once daily for 21 days (same formulation as above). Mice were monitored daily for signs of morbidity (e.g., weight loss >20%, lethargy), and survival time was recorded. Median survival was calculated using the Kaplan-Meier method [1] |
| ADME/Pharmacokinetics |
1. Pharmacokinetics of mouse plasma: Female CD-1 mice were intraperitoneally injected with EI1 at a dose of 30 mg/kg. Blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, and 24 hours after administration. Plasma was separated by centrifugation, and the concentration of EI1 was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The pharmacokinetic parameters were calculated as follows: maximum plasma concentration (Cₘₐₓ) ≈ 1.2 μM, time to peak concentration (Tₘₐₓ) ≈ 0.5 h, plasma half-life (t₁/₂) ≈ 2.8 h, and area under the plasma concentration-time curve (AUC₀₋₂₄ₕ) ≈ 3.5 μM·h [1]
2. Tissue distribution: NOD/SCID tumor-bearing mice (SU-DHL-4 xenograft tumor) were intraperitoneally injected with EI1 (30 mg/kg). One hour after administration, the mice were sacrificed, and tissues (tumor, liver, kidney, spleen, lung) were collected, homogenized, and analyzed by LC-MS/MS. The concentration of EI1 in the tumor tissue was approximately 0.9 μM, which was approximately 75% of the plasma concentration at the same time point; the concentrations in the liver and kidney were approximately 1.5 μM and 0.7 μM, respectively [1] |
| Toxicity/Toxicokinetics |
1. Acute toxicity in mice: Female CD-1 mice were intraperitoneally injected with EI1 at doses of 10, 30, 60, and 100 mg/kg. No deaths were observed at doses up to 60 mg/kg; at a dose of 100 mg/kg, 2 out of 6 mice died within 48 hours. The estimated approximate lethal dose (LD₅₀) was greater than 60 mg/kg and less than 100 mg/kg [1]
2. Chronic toxicity in xenograft models: In a 21-day subcutaneous xenograft study (30 mg/kg, intraperitoneally, once daily), mice in the EI1 treatment group did not show significant weight loss (maximum weight change: 8% compared to the vector group, within acceptable limits). Hematological analysis (peripheral blood) showed no significant changes in white blood cell count, red blood cell count, and platelet count compared to the vector group. Serum biochemical indicators (ALT, AST, creatinine, urea nitrogen) were all within the normal range, indicating no obvious liver and kidney toxicity [1] 3. Plasma protein binding rate: EI1 was incubated with mouse plasma (1:1, v/v) at 37℃ for 1 hour. The unbound fraction was separated by ultrafiltration and measured by LC-MS/MS. The plasma protein binding rate of EI1 was approximately 92% [1] |
| References | |
| Additional Infomation |
1. Mechanism of action: EI1 (KB145943; EI 1) is a competitive inhibitor of Ezh2. It binds to the catalytic SET domain of Ezh2 and blocks the interaction between Ezh2 and its substrate (H3K27) or cofactor (SAM). This inhibits PRC2-mediated H3K27 trimethylation, which is a key epigenetic marker for gene silencing. Decreased H3K27me3 levels lead to the reactivation of tumor suppressor genes (e.g., p16INK4a, p21CIP1), which are usually silenced in Ezh2-overexpressing tumors, thereby inhibiting cell proliferation and inducing apoptosis [1]. 2. Therapeutic potential: Studies have shown that EI1 is a highly effective and selective Ezh2 inhibitor with preclinical efficacy in Ezh2-overexpressing tumors (e.g., B-cell lymphoma, triple-negative breast cancer). Its good in vivo safety and target-specific activity support its potential as a therapeutic agent for Ezh2-driven malignant tumors [1]
3. Background: Ezh2 is a core component of polycomb inhibitory complex 2 (PRC2) and is frequently overexpressed or mutated in a variety of cancers, including lymphoma, breast cancer and prostate cancer. Overexpression of Ezh2 leads to over-modification of H3K27me3 and silencing of tumor suppressor genes, thereby promoting tumorigenesis. Therefore, Ezh2 has become a promising target in cancer treatment [1] |
| Molecular Formula |
C23H26N4O2
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| Molecular Weight |
390.48
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| Exact Mass |
390.205
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| CAS # |
1418308-27-6
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| Related CAS # |
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| PubChem CID |
72199293
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| Appearance |
White to off-white solid powder
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| Density |
1.2±0.1 g/cm3
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| Boiling Point |
675.9±55.0 °C at 760 mmHg
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| Flash Point |
362.6±31.5 °C
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| Vapour Pressure |
0.0±2.1 mmHg at 25°C
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| Index of Refraction |
1.617
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| LogP |
2.92
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
29
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| Complexity |
771
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
PFHDWRIVDDIFRP-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C23H26N4O2/c1-5-17(6-2)27-8-7-18-19(10-16(12-24)11-21(18)27)22(28)25-13-20-14(3)9-15(4)26-23(20)29/h7-11,17H,5-6,13H2,1-4H3,(H,25,28)(H,26,29)
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| Chemical Name |
6-cyano-N-[(4,6-dimethyl-2-oxo-1H-pyridin-3-yl)methyl]-1-pentan-3-ylindole-4-carboxamide
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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) |
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| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5610 mL | 12.8048 mL | 25.6095 mL | |
| 5 mM | 0.5122 mL | 2.5610 mL | 5.1219 mL | |
| 10 mM | 0.2561 mL | 1.2805 mL | 2.5610 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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