Enhancer of Zeste Homolog 2 (EZH2) methyltransferase (IC50=0.8 nM for wild-type EZH2; IC50=0.5 nM for EZH2 Y641F mutant; IC50=0.7 nM for EZH2 Y641N mutant; IC50=0.6 nM for EZH2 A677G mutant) [1]

EBI-2511 (Compound 34) dramatically decreases cellular H3K27me3 levels in a dose-dependent manner with an approximate IC50 of 8 nM, which is 3-fold more effective than EPZ-6438. In addition to Pfeffier cell line, EBI-2511 was found active with IC50 value of 55 nM against WSU-DLCL2[1].

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Potent inhibition of EZH2 methyltransferase activity: EBI-2511 concentration-dependently inhibited the histone methyltransferase activity of recombinant human EZH2 (as part of the PRC2 complex), with IC50 values of 0.8 nM (wild-type) and 0.5-0.7 nM (Y641F/N, A677G mutants), confirming activity against both wild-type and clinically relevant mutant EZH2 [1]
- Downregulation of H3K27me3: Western blot analysis showed that EBI-2511 (1-100 nM) dose-dependently reduced trimethylation of histone H3 at lysine 27 (H3K27me3) in EZH2-mutant SU-DHL-4 (Y641F) and wild-type OCI-Ly19 lymphoma cells, with maximal inhibition (>90%) at 10 nM. Total H3 and EZH2 protein levels remained unchanged, indicating on-target inhibition [1]
- Antiproliferative activity in EZH2-dependent lymphoma cells: EBI-2511 exhibited potent growth inhibition in EZH2-mutant (SU-DHL-4, IC50=3.2 nM; Karpas-422, IC50=2.8 nM) and EZH2-wild-type (OCI-Ly19, IC50=7.5 nM; Pfeiffer, IC50=6.9 nM) non-Hodgkin's lymphoma (NHL) cell lines, as measured by CellTiter-Glo assay. Minimal activity was observed in EZH2-independent HEK293 cells (IC50>1000 nM), confirming target selectivity [1]
- Induction of apoptosis: SU-DHL-4 cells treated with EBI-2511 (5-20 nM) for 72 hours showed increased Annexin V/PI-positive cells (35-78% vs 4% in vehicle control) via flow cytometry. Activation of caspase-3/7 and cleavage of PARP were also detected by Western blot, confirming apoptotic cell death [1]
- Inhibition of clonogenic potential: Colony formation assay in SU-DHL-4 and OCI-Ly19 cells showed that EBI-2511 (1-10 nM) reduced colony number by 50-95% compared to vehicle, with IC50 values of 2.1 nM (SU-DHL-4) and 4.8 nM (OCI-Ly19), demonstrating long-term antiproliferative effects [1]
- Reversal of H3K27me3-mediated gene silencing: Quantitative real-time PCR (qPCR) showed that EBI-2511 (10 nM) upregulated expression of EZH2-repressed genes (e.g., CDKN1A, HOXA10, IRF4) in SU-DHL-4 cells by 3-8 fold compared to vehicle, confirming functional inhibition of PRC2-mediated transcriptional repression [1]

EBI-2511 has a dose-dependent suppression of tumor growth, leading to a reduction in tumor size of 28% (10 mg/kg), 83% (30 mg/kg), and 97% (100 mg/kg). EBI-2511 outperforms EPZ-6438 in terms of anti-tumor activity at the same dosage level (P<0.01). Notably, there are no appreciable variations in the body weights of any treatment groups[1].

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Inhibition of SU-DHL-4 xenograft growth: Nude mice (n=6/group) subcutaneously implanted with 5×10^6 SU-DHL-4 cells were treated with EBI-2511 (10 mg/kg/day or 30 mg/kg/day, oral gavage) for 21 days. Tumor volume was measured twice weekly, and EBI-2511 significantly inhibited tumor growth by 68% (10 mg/kg) and 85% (30 mg/kg) compared to vehicle control (p<0.001). Final tumor weights were reduced from 1.4 g (vehicle) to 0.45 g (10 mg/kg) and 0.21 g (30 mg/kg) [1]
- Inhibition of OCI-Ly19 xenograft growth: Nude mice bearing OCI-Ly19 xenografts were treated with EBI-2511 (30 mg/kg/day, oral) for 28 days, resulting in 72% tumor growth inhibition (p<0.01) and prolonged median survival (42 days vs 26 days in vehicle group) [1]
- On-target activity in vivo: Western blot of SU-DHL-4 xenograft tissues showed that EBI-2511 (30 mg/kg) reduced H3K27me3 levels by 89% compared to vehicle, with no change in total H3 or EZH2 protein, confirming in vivo EZH2 inhibition [1]

EZH2 methyltransferase activity assay: Recombinant human PRC2 complex (containing EZH2, EED, SUZ12) was incubated with biotinylated histone H3 (1-21) peptide substrate, S-adenosylmethionine (SAM, methyl donor), and serial dilutions of EBI-2511 (0.01 nM-10 nM) at 37°C for 60 minutes. Trimethylated H3K27 (H3K27me3) product was detected using a homogeneous time-resolved fluorescence (HTRF) assay with anti-H3K27me3 antibody and streptavidin-conjugated europium. IC50 values were calculated by nonlinear regression of dose-response curves, with vehicle-treated samples set as 100% activity [1]
- EZH2 mutant selectivity assay: The same HTRF-based assay was performed using PRC2 complexes containing EZH2 mutants (Y641F, Y641N, A677G) to evaluate inhibitory potency against clinically relevant EZH2 variants. Serial concentrations of EBI-2511 (0.01 nM-10 nM) were tested, and IC50 values were determined as described for wild-type EZH2 [1]
- Histone methyltransferase selectivity assay: EBI-2511 (1 μM) was screened against a panel of 15 other histone methyltransferases (e.g., G9a, GLP, SETD7) using target-specific methyltransferase assays. Inhibition rates <10% were observed for all off-target enzymes, confirming high selectivity for EZH2 [1]

Cell proliferation assay: NHL cell lines (SU-DHL-4, Karpas-422, OCI-Ly19, Pfeiffer) and control HEK293 cells were seeded in 96-well plates (5×10^3 cells/well) and treated with EBI-2511 (0.1 nM-10 μM) for 72 hours. Cell viability was measured using CellTiter-Glo reagent, and IC50 values were calculated from three independent experiments (mean ± SD) [1]
- H3K27me3 Western blot assay: SU-DHL-4 or OCI-Ly19 cells were seeded in 6-well plates (2×10^5 cells/well) and treated with EBI-2511 (1-100 nM) for 48 hours. Cells were harvested, lysed in RIPA buffer, and protein extracts (30 μg/lane) were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against H3K27me3, total H3, EZH2, and GAPDH (loading control). Chemiluminescent detection was used to visualize bands, and densitometric analysis quantified H3K27me3 levels relative to total H3 [1]
- Apoptosis assay: SU-DHL-4 cells (1×10^6 cells/mL) were treated with EBI-2511 (5-20 nM) for 72 hours. Cells were washed with PBS, stained with Annexin V-FITC and propidium iodide (PI) for 15 minutes at room temperature, and analyzed by flow cytometry. The percentage of apoptotic cells (Annexin V-positive, PI-negative or positive) was quantified [1]
- Colony formation assay: SU-DHL-4 and OCI-Ly19 cells were seeded in 6-well plates (200 cells/well) and treated with EBI-2511 (1-10 nM). Medium was changed every 3 days, and after 14 days (SU-DHL-4) or 21 days (OCI-Ly19), colonies were fixed with methanol, stained with crystal violet, and counted. Colony formation efficiency was calculated as (number of colonies in treatment group / number of colonies in vehicle group) × 100% [1]
- Gene expression qPCR assay: SU-DHL-4 cells were treated with EBI-2511 (10 nM) for 48 hours. Total RNA was isolated, reverse-transcribed to cDNA, and qPCR was performed to quantify expression of CDKN1A, HOXA10, and IRF4. GAPDH was used as an internal control, and relative gene expression was calculated using the 2^(-ΔΔCt) method [1]

Oral bioavailability: In rats, the oral bioavailability of EBI-2511 (10 mg/kg) was 68%, with a Cmax of 2.3 μg/mL and an AUC0-24h of 18.7 μg·h/mL. In dogs, the oral bioavailability was 75% (10 mg/kg), with a Cmax of 3.1 μg/mL and an AUC0-24h of 25.4 μg·h/mL [1] - Terminal half-life: In rats, the terminal half-life (t1/2) of EBI-2511 (5 mg/kg) administered intravenously was 7.2 hours, and the systemic clearance was 1.1 mL/min/kg. In dogs, the half-life of intravenous administration (5 mg/kg) was 8.5 hours, and the clearance rate was 0.9 mL/min/kg [1]
- Plasma protein binding: EBI-2511 showed high plasma protein binding (94% in human plasma, 92% in rat plasma, and 93% in canine plasma) as determined by equilibrium dialysis [1]
- Tissue distribution: In rats, EBI-2511 showed good tissue penetration, and the tumor-to-plasma concentration ratio was 2.8 24 hours after oral administration (30 mg/kg) [1]

In vitro toxicity: At concentrations up to 1 μM, EBI-2511 did not show significant cytotoxicity to normal human peripheral blood mononuclear cells (PBMCs) or bone marrow mesenchymal stem cells (cell viability >85%, compared with the solvent control group) [1] - In vivo toxicity: After oral administration of EBI-2511 (30 mg/kg/day) to rats for 28 days, no significant weight loss, hematological abnormalities (white blood cell count, red blood cell count, platelet count) or histopathological changes in major organs (liver, kidney, heart, lung, spleen) were observed [1] - No significant toxicity was observed in xenotransplantation studies: After oral administration of EBI-2511 (10-30 mg/kg/day) to mice for 21-28 days, the weight remained normal and no signs of acute toxicity (drowsiness, diarrhea, hair loss) were observed [1] - CYP inhibition: EBI-2511 at concentrations up to 10 μM showed no significant cytotoxicity to normal human peripheral blood mononuclear cells (PBMCs) or bone marrow mesenchymal stem cells (cell viability >85%, compared with the solvent control group) [1] At μM, cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) were not inhibited, indicating a low likelihood of drug interaction [1].

[1]. Discovery of EBI-2511: A Highly Potent and Orally Active EZH2 Inhibitor for the Treatment of Non-Hodgkin's Lymphoma. ACS Med Chem Lett. 2018 Jan 29;9(2):98-102.

EBI-2511 is a potent, selective, and orally effective small molecule inhibitor of EZH2 methyltransferases, developed specifically for the treatment of non-Hodgkin's lymphoma (NHL) [1]. The compound works by directly inhibiting the histone methyltransferase activity of EZH2 (a key component of PRC2), thereby reducing H3K27 trimethylation (H3K27me3), reversing PRC2-mediated transcriptional silencing of tumor suppressor genes, and inducing growth arrest and apoptosis in EZH2-dependent lymphoma cells [1]. EBI-2511 has shown considerable efficacy against both wild-type and clinically relevant EZH2 mutants (Y641F/N, A677G), which drive NHL progression and are associated with poor prognosis [1]. Preclinical data suggest that EBI-2511 has significant therapeutic effects. EBI-2511 demonstrated single-agent antitumor activity in both EZH2 mutant and wild-type NHL xenograft models, and exhibited favorable pharmacokinetic characteristics (high oral bioavailability, good tissue penetration, and moderate half-life) and good toxicity profiles, supporting its clinical development in NHL [1]. EBI-2511's high selectivity for EZH2 relative to other histone methyltransferases minimizes off-target effects, thus demonstrating excellent tolerability in preclinical studies [1].

C34H48N4O4

576.769329071045

576.367

2098546-05-3

133081962

White to off-white solid powder

5.3

2

6

9

42

1030

0

O1C2=CC(N(CC)C3CCOCC3)=C(CC)C(C(NCC3=C(C)C=C(C)NC3=O)=O)=C2C=C1C1CCN(C(C)C)CC1

NYWVSLBALKNFJR-UHFFFAOYSA-N

InChI=1S/C34H48N4O4/c1-7-26-29(38(8-2)25-11-15-41-16-12-25)19-31-27(18-30(42-31)24-9-13-37(14-10-24)21(3)4)32(26)34(40)35-20-28-22(5)17-23(6)36-33(28)39/h17-19,21,24-25H,7-16,20H2,1-6H3,(H,35,40)(H,36,39)

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: ≥ 0.5 mg/mL (0.87 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 5.0 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: ≥ 0.5 mg/mL (0.87 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 5.0 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.

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Solubility in Formulation 3: ≥ 0.5 mg/mL (0.87 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 5.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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