Methylstat is a novel and potent inhibitor of the Jumonji C domain-containing histone trimethyl demethylases (JHDMs) with anti-proliferative activity and low cytotoxicity. It induces apoptosis and cell cycle arrest at G0/G1 phase.
Physicochemical Properties
| Molecular Formula | C28H31N3O6 |
| Molecular Weight | 505.562247514725 |
| Exact Mass | 505.221 |
| CAS # | 1310877-95-2 |
| PubChem CID | 53392493 |
| Appearance | Light yellow to yellow solid powder |
| Density | 1.3±0.1 g/cm3 |
| Index of Refraction | 1.635 |
| LogP | 3.3 |
| Hydrogen Bond Donor Count | 3 |
| Hydrogen Bond Acceptor Count | 7 |
| Rotatable Bond Count | 14 |
| Heavy Atom Count | 37 |
| Complexity | 752 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | COC(=O)/C=C/C(=O)N(CCCCNCC1=CC=C(C=C1)COC(=O)NC2=CC=CC3=CC=CC=C32)O |
| InChi Key | MUJOCHRZXRZONW-FOCLMDBBSA-N |
| 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+ |
| Chemical Name | methyl (E)-4-[hydroxy-[4-[[4-(naphthalen-1-ylcarbamoyloxymethyl)phenyl]methylamino]butyl]amino]-4-oxobut-2-enoate |
| 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 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) |
Biological Activity
| 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]. |
| 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 |
| References |
[1]. A histone demethylase inhibitor, methylstat, inhibits angiogenesis in vitro and in vivo. RSC Advances, 2014. [2]. Synergistic Apoptotic Effects of Bortezomib and Methylstat on Multiple Myeloma Cells. Arch Med Res. 2020 Apr;51(3):187-193. |
| Additional Infomation | 4-[hydroxy-[4-[[4-[[(1-naphthalenylamino)-oxomethoxy]methyl]phenyl]methylamino]butyl]amino]-4-oxo-2-butenoic acid methyl ester is a member of naphthalenes. |
Solubility Data
| Solubility (In Vitro) | DMSO : ~100 mg/mL (~197.80 mM) |
| 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 (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *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). 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)] *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. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL 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). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently. (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 |