| Size | Price | Stock | Qty |
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| Targets |
Methylstat targets Jumonji C domain-containing histone demethylases (JHDMs), including JMJD2A (H3K9me3 and H3K36me3 demethylase), JMJD2E (H3K9me3 demethylase), and JMJD3 (H3K27me3 demethylase). It is a methyl ester prodrug of a JMJD inhibitor.
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| ln Vitro |
Mmethylstat is non-cytotoxic to HUVEC at 1-2 µM and exhibits anti-proliferative action at 0-5 µM over 48 and 72 hours [1]. In the G0/G1 phase, mmethylstat (0, 1, 2 µM; 48 hours) dose-dependently promotes cell cycle arrest [1]. Mmethylstat (0, 1, 2 µM; 48 hours) inhibits the protein levels of cyclinD1, but enhances the expression of p53 mRNA, H3K27 methylation, and the accumulation of p53 and p21 [1]. Mmethylstat (0, 1, 2 µM) suppresses capillary formation during CAM (chick chorioallantoic membrane) growth and shows anti-angiogenic activity produced by VEGF, bFGF, and TNF-α in HUVEC cells. No symptoms of bleeding or thrombosis were seen. 1]. Mmethylstat dramatically promoted apoptosis in ARH77 and U266 cells (2.1, 4.2 mM for ARH77 cells, and 1.1, 2.2 mM for U266 cells; 72 hours) [2].
In vitro, Methylstat inhibits JHDM activity. It is non-cytotoxic to HUVEC at 1-2 µM and exhibits anti-proliferative action at 0-5 µM over 48 and 72 hours. It dose-dependently promotes cell cycle arrest at G0/G1 phase, inhibits cyclinD1 protein levels, and enhances p53 mRNA, H3K27 methylation, and p53/p21 accumulation. It shows anti-angiogenic activity in HUVEC cells. It promotes apoptosis in ARH77 and U266 cells. |
| ln Vivo |
In vivo, Methylstat is a cell-permeable prodrug. It has shown anti-proliferative activity and low cytotoxicity. Detailed in vivo efficacy data including tumor growth inhibition and biomarker modulation are available in preclinical literature. Its prodrug nature suggests it is converted to the active form in cells.
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| Enzyme Assay |
In vitro enzyme assays for Methylstat typically involve measuring the inhibition of JHDM activity using demethylase assays. The enzyme is incubated with a substrate (methylated histone peptide) and varying concentrations of the compound. Demethylation is measured by mass spectrometry or antibody-based detection. IC50 values are calculated from dose-response curves. Selectivity over other demethylases is assessed.
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| Cell Assay |
Cytotoxicity assay [1]
Cell Types: HUVEC Cell Tested Concentrations: 0-5 µM Incubation Duration: 48, 72 hrs (hours) Experimental Results: No cytotoxicity was shown to HUVEC at a concentration of 1-2 µM. Cell viability assay [1] Cell Types: HUVEC, HepG2, HeLa, CHANG Cell Tested Concentrations: 0-5 µM Incubation Duration: 72 hrs (hours) Experimental Results: demonstrated anti-proliferative activity against HUVEC, HepG2, IC50 is 4, 10, 5, 7.5 µM , HeLa, CHANG cells. Cell cycle analysis[1] Cell Types: HUVEC Cell Tested Concentrations: 0, 1, 2 µM Incubation Duration: 48 hrs (hours) Experimental Results: G0/G1 phase increased by 16.8%, while S and G2/M phases diminished by 5.5 compared to untreated cells %, respectively 6.1%. Western Blot Analysis[1] Cell Types: HUVEC Cell Tested Concentrations: 0, 1, 2 µM Incubation Duration: 0-48 hrs (hours) Experimental Results: Causes accumulation of p53 and p21 protein levels in a time- and dose-dependent manner and increases H3K27 methylation levels , but inhibited the protein level of cyclinD1. Apoptosis analysis [2] Cell Types: U266, ARH77 Cell Tested Concentrations: 1.1, 2.2 mM for U26 Cell-based assays for Methylstat involve culturing cancer cell lines or HUVEC cells in appropriate media. Cells are treated with Methylstat at concentrations ranging from 0.1 µM to 10 µM for 24-72 hours. Cell viability is assessed by MTT or CellTiter-Glo assays. Cell cycle analysis is performed by flow cytometry. Histone methylation levels are measured by Western blot using methylation-specific antibodies. Apoptosis is evaluated by flow cytometry. |
| Animal Protocol |
In vivo animal experiments for Methylstat typically involve administration to tumor-bearing mice via oral gavage or intraperitoneal injection. Tumor growth inhibition is monitored. Histone methylation levels in tumor tissues are assessed by Western blot or immunohistochemistry. Pharmacokinetic parameters are evaluated by measuring compound levels in blood and tissues. Toxicity is assessed by monitoring body weight and organ histology.
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| ADME/Pharmacokinetics |
Methylstat (molecular weight 505.57, formula C28H31N3O6) is a methyl ester prodrug. It is cell-permeable. It is soluble in DMSO. Detailed pharmacokinetic parameters including absorption, distribution, metabolism, and excretion are available in preclinical literature. Its prodrug nature influences its bioavailability and cellular uptake.
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| Toxicity/Toxicokinetics |
Methylstat has low cytotoxicity. It is non-cytotoxic to HUVEC at 1-2 µM. Comprehensive toxicological evaluation including acute, subchronic, and genotoxicity studies has likely been conducted in preclinical development. The compound is for research use only. Standard laboratory safety precautions should be followed.
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| References | |
| Additional Infomation |
4-[hydroxy-[4-[[4-[[(1-naphthylamino)-oxomethoxy]methyl]phenyl]methylamino]butyl]amino]-4-oxo-2-butenoic acid methyl ester is a naphthalene compound.
Methylstat (CAS#: 1310877-95-2) is a novel and potent inhibitor of Jumonji C domain-containing histone trimethyl demethylases (JHDMs). It is a methyl ester prodrug with anti-proliferative activity and low cytotoxicity. It induces apoptosis and G0/G1 cell cycle arrest. Molecular weight: 505.57, formula: C28H31N3O6. |
| Molecular Formula |
C28H31N3O6
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| Molecular Weight |
505.562247514725
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| Exact Mass |
505.221
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| CAS # |
1310877-95-2
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| PubChem CID |
53392493
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.3±0.1 g/cm3
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| Index of Refraction |
1.635
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| LogP |
3.3
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
14
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| Heavy Atom Count |
37
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| Complexity |
752
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| Defined Atom Stereocenter Count |
0
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| SMILES |
COC(=O)/C=C/C(=O)N(CCCCNCC1=CC=C(C=C1)COC(=O)NC2=CC=CC3=CC=CC=C32)O
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| InChi Key |
MUJOCHRZXRZONW-FOCLMDBBSA-N
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| InChi Code |
InChI=1S/C28H31N3O6/c1-36-27(33)16-15-26(32)31(35)18-5-4-17-29-19-21-11-13-22(14-12-21)20-37-28(34)30-25-10-6-8-23-7-2-3-9-24(23)25/h2-3,6-16,29,35H,4-5,17-20H2,1H3,(H,30,34)/b16-15+
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| Chemical Name |
methyl (E)-4-[hydroxy-[4-[[4-(naphthalen-1-ylcarbamoyloxymethyl)phenyl]methylamino]butyl]amino]-4-oxobut-2-enoate
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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 Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| 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) |
DMSO : ~100 mg/mL (~197.80 mM)
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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 | 1.9780 mL | 9.8900 mL | 19.7800 mL | |
| 5 mM | 0.3956 mL | 1.9780 mL | 3.9560 mL | |
| 10 mM | 0.1978 mL | 0.9890 mL | 1.9780 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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