| Size | Price | Stock | Qty |
|---|---|---|---|
| 5mg |
|
||
| 10mg |
|
||
| 25mg |
|
||
| 50mg |
|
||
| 100mg |
|
||
| 250mg | |||
| 500mg | |||
| Other Sizes |
Purity: ≥98%
PF-06726304 is a novel, potent and selective SAM-competitive EZH2 (enhancer of zeste homolog 2) inhibitor with a Ki of 0.7 nM. PF-06726304 displayed improved LipE and on-target potency in both biochemical and cellular readouts relative to compound 18. PF-06726304 also displayed robust in vivo antitumor growth activity and dose-dependent de-repression of EZH2 target genes.
| Targets |
PF-06726304 (Compound 31) inhibits Karpas-422 H3K27me3 with an IC50 of 15 nM [1]. PF-06726304, with an IC50 of 25 nM, suppresses the growth of Karpas-422 cells carrying wild-type EZH2[1].
|
|---|---|
| ln Vitro |
PF-06726304 (Compound 31) inhibits Karpas-422 H3K27me3 with an IC50 of 15 nM [1]. PF-06726304, with an IC50 of 25 nM, suppresses the growth of Karpas-422 cells carrying wild-type EZH2[1].
PF-06726304 potently inhibits EZH2 enzymatic activity in biochemical assays using PRC2 complexes containing either wild-type or Y641N mutant EZH2, showing competitive inhibition with SAM. [1] PF-06726304 selectively inhibits EZH2/EZH1 over a panel of other histone methyltransferases (e.g., G9a, SETD7, PRMTs) and a diverse kinase panel (e.g., ABL1, AKT1, EGFR). The highest off-target inhibition in the kinase screen was observed for MAP4K4 (~30% inhibition). [1] In KARPAS-422 cells (harboring EZH2 Y641N mutation) and OCI-LY19 cells (EZH2 wild-type), treatment with PF-06726304 induced dose-dependent upregulation of genes PRDM1 and TNFRSF21, which are repressed by PRC2, as measured by qRT-PCR. [1] In cell-based models, PF-06726304 treatment reduced global levels of the histone mark H3K27me3, as measured by ELISA and Western blot analysis. [1] |
| ln Vivo |
In a subcutaneous Karpas-422 xenograft model, PF-06726304 (200 and 300 mg/kg; BID 20 days) suppresses tumor development and causes a strong modification of downstream indicators [1].
|
| Enzyme Assay |
The enzymatic activity of the PRC2 four-protein complex (containing wild-type or Y641N mutant EZH2) was monitored by measuring the incorporation of a radioactive methyl group from tritiated SAM into oligonucleosome substrates isolated from HeLa cells. The reaction mixture contained assay buffer, 5 nM enzyme complex, 25 µg/ml oligonucleosomes, and was initiated by adding tritiated SAM. After incubation, the reaction was stopped with trichloroacetic acid, filtered, washed, and the incorporated radioactivity was measured by scintillation counting. IC50 values were determined by fitting data to a standard four-parameter equation. For Ki determination and mechanistic studies, assays were performed at a fixed SAM concentration, and data were fitted to models for competitive inhibition. [1]
Isothermal titration calorimetry (ITC) experiments were performed to measure direct binding. Protein samples were dialyzed into a suitable buffer. In a typical experiment, multiple injections of the compound solution were made into a solution containing the PRC2 complex. The heat changes were recorded and analyzed using software to fit a 1:1 binding model, yielding binding constants. [1] |
| Cell Assay |
For gene expression analysis (qRT-PCR), cells treated with PF-06726304 were harvested, and RNA was isolated using commercial kits according to standard protocols. One-step quantitative reverse transcription PCR was performed using specific TaqMan gene expression assays for target genes (PRDM1, TNFRSF21) and a normalizer gene (RPN1). Experiments were performed in triplicate. [1]
For histone extraction from cells or tissue samples, a lysis buffer-based method was used. Frozen samples were homogenized, lysed, and acid-soluble proteins (including histones) were collected by centrifugation. The supernatant was neutralized and stored. Protein concentration was quantified. [1] For H3K27me3 ELISA, histone extracts were diluted and coated onto microplates overnight. Plates were washed, blocked, and then incubated with primary antibodies specific for H3K27me3 or total histone H3. After washing, a horseradish peroxidase-conjugated secondary antibody was added, followed by colorimetric detection using a substrate. Absorbance was measured. [1] For Western blot analysis of histone modifications, purified histone extracts were prepared with loading buffer and reducing agent, separated by gel electrophoresis, and transferred to membranes. Membranes were probed with antibodies against total histone H3 and H3K27me3. [1] |
| Animal Protocol |
Animal/Disease Models: Female Scid beige mice (6-8 weeks old) using Karpas-422 xenograft model [1]
Doses: 200 and 300 mg/kg Route of Administration: BID for 20 days Experimental Results: Inhibited tumor growth and induced downstream organisms Robust modulation of markers in a subcutaneousKarpas-422 xenograft model. |
| References | |
| Additional Infomation |
PF-06726304 is a pyridone-containing 3,4-dihydroisoquinoline-1(2H)-one derivative, developed as a novel, highly effective, and selective EZH2 inhibitor. Its inhibitory mechanism is competitive with the cofactor SAM. It exhibits targeted cell activity by reducing H3K27me3 levels and relieving the inhibition of the PRC2 silencing gene. Computational methods, including DFT calculation and conformational analysis, were used in the drug design and structure-activity relationship study. [1]
|
| Molecular Formula |
C22H21CL2N3O3
|
|---|---|
| Molecular Weight |
446.326443433762
|
| Exact Mass |
445.095
|
| CAS # |
1616287-82-1
|
| Related CAS # |
PF-06726304 acetate;2080306-28-9
|
| PubChem CID |
86723730
|
| Appearance |
White to off-white solid powder
|
| LogP |
3.6
|
| Hydrogen Bond Donor Count |
1
|
| Hydrogen Bond Acceptor Count |
4
|
| Rotatable Bond Count |
3
|
| Heavy Atom Count |
30
|
| Complexity |
799
|
| Defined Atom Stereocenter Count |
0
|
| SMILES |
C1(=O)C2=C(C(Cl)=CC(C3=C(C)ON=C3C)=C2Cl)CCN1CC1=C(C)C=C(C)NC1=O
|
| InChi Key |
PDKDOPJQPKXNCT-UHFFFAOYSA-N
|
| InChi Code |
InChI=1S/C22H21Cl2N3O3/c1-10-7-11(2)25-21(28)16(10)9-27-6-5-14-17(23)8-15(20(24)19(14)22(27)29)18-12(3)26-30-13(18)4/h7-8H,5-6,9H2,1-4H3,(H,25,28)
|
| Chemical Name |
5,8-Dichloro-2-[(1,2-dihydro-4,6-dimethyl-2-oxo-3-pyridinyl)methyl]-7-(3,5-dimethyl-4-isoxazolyl)-3,4-dihydro-1(2H)-isoquinolinone
|
| Synonyms |
PF-6726304; PF 6726304; PF6726304; PF-06726304; PF 06726304; PF06726304;
|
| HS Tariff Code |
2934.99.9001
|
| 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)
|
| Solubility (In Vitro) |
DMSO : ≥ 15 mg/mL (~33.61 mM)
|
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1 mg/mL (2.24 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 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: ≥ 1 mg/mL (2.24 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 10.0 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: ≥ 1 mg/mL (2.24 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 | 2.2405 mL | 11.2025 mL | 22.4049 mL | |
| 5 mM | 0.4481 mL | 2.2405 mL | 4.4810 mL | |
| 10 mM | 0.2240 mL | 1.1202 mL | 2.2405 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.
Products are for research use only; We do not sell to patients
Copyright 2020 InvivoChem LLC | All Rights Reserved
COA