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Entinostat (formerly known as MS-275; SNDX-275) is a potent, benzamide-based and class-selective but generally pan-HDAC (histone deacetylase) inhibitor with potential anticancer activity. It is more selective for HDAC1/2/3 over HDACs 4, 6, 8, and 10, and it strongly inhibits HDAC1, HDAC2, and HDAC3 with IC50 values of 0.24, 0.45, and 0.25 μM in cell-free experiments, respectively. By attaching to and inhibiting histone deacetylase, an enzyme that controls chromatin structure and gene transcription, entinostat may have anticancer properties. In human leukemia cells, this agent appears to have dose-dependent effects, including, at low drug concentrations, cyclin-dependent kinase inhibitor 1A (p21/CIP1/WAF1)-dependent growth arrest and differentiation.
Physicochemical Properties
| Molecular Formula | C21H20N4O3 | |
| Molecular Weight | 376.41 | |
| Exact Mass | 376.153 | |
| Elemental Analysis | C, 67.01; H, 5.36; N, 14.88; O, 12.75 | |
| CAS # | 209783-80-2 | |
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| PubChem CID | 4261 | |
| Appearance | White off white solid powder | |
| Density | 1.3±0.1 g/cm3 | |
| Boiling Point | 566.7±50.0 °C at 760 mmHg | |
| Melting Point | 159-160ºC | |
| Flash Point | 296.6±30.1 °C | |
| Vapour Pressure | 0.0±1.6 mmHg at 25°C | |
| Index of Refraction | 1.672 | |
| LogP | 1.46 | |
| Hydrogen Bond Donor Count | 3 | |
| Hydrogen Bond Acceptor Count | 5 | |
| Rotatable Bond Count | 7 | |
| Heavy Atom Count | 28 | |
| Complexity | 508 | |
| Defined Atom Stereocenter Count | 0 | |
| SMILES | O=C(NCC1=CC=C(C=C1)C(NC2=CC=CC=C2N)=O)OCC3=CC=CN=C3 |
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| InChi Key | INVTYAOGFAGBOE-UHFFFAOYSA-N | |
| InChi Code | InChI=1S/C21H20N4O3/c22-18-5-1-2-6-19(18)25-20(26)17-9-7-15(8-10-17)13-24-21(27)28-14-16-4-3-11-23-12-16/h1-12H,13-14,22H2,(H,24,27)(H,25,26) | |
| Chemical Name | pyridin-3-ylmethyl N-[[4-[(2-aminophenyl)carbamoyl]phenyl]methyl]carbamate | |
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| 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 |
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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) |
Biological Activity
| Targets | HDAC1 ( IC50 = 243 nM ); HDAC3 ( IC50 = 248 nM ); HDAC2 ( IC50 = 453 nM ) | |
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| ln Vivo |
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| Enzyme Assay | HDAC activity biochemical assays are performed by Nanosyn in 384-well microplates with a reaction volume of 10 μL. Five microliters of a 2× HDAC inhibitor (such as Entinostat), four microliters of 2.5× enzyme, and one microliter of 10× substrate are combined with assay buffer (100 mM HEPES, pH 7.5, 25 mM KCl, 0.1% BSA, 0.01% Triton X-100, 1% DMSO) in a typical enzymatic reaction. In the enzymatic assays, the final concentration of each HDAC ranges from 0.5 to 5 nM. In every experiment, a final substrate concentration of 1 μM FAM-RHKK(Ac)-NH2 or FAM-RHKK(trifluoroacetyl)-NH2 is employed, and it is discovered to be lower than the calculated Km,app for every enzyme[1]. | |
| Cell Assay | SH-SY5Y cells are split twice a week and kept in a humidified incubator with 5% CO2 at 37°C under standard culture conditions. After plating cells at a density of 2500 cells per well in a 20-μL volume of DMEM/F-12 culture media supplemented with 10% FBS, the cells are left to adhere for the entire night in black 384-well plates. After being serially diluted in 100% DMSO the next day, HDAC inhibitors (such as Entinostat) are then cross-diluted into culture media. To achieve the desired inhibitor final concentration (e.g., 0.1% DMSO), 5 μL of the compound (e.g., Entinostat) diluted in media is added to the appropriate well of the cell plate. Cellular ATP levels are quantified using CellTiter-Glo reagents after treated cells are incubated for 6, 24, 48, 72, or 96 hours under standard tissue culture conditions. Similarly, media from different cell plates are aspirated after 6 hours of incubation with HDAC inhibitors (such as Entinostat), and cells are once again washed with media free of inhibitors. After 24, 48, 72, or 96 hours of incubation, the cells are given 25 μL of media supplemented with 10% FBS and 0.1% DMSO (no inhibitors), and the levels of cellular ATP are measured using CellTiter-Glo. An Envision Instrument with a 0.1 s count time is used to measure luminosity at each time point[1]. | |
| Animal Protocol |
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| References |
[1]. Histone deacetylase (HDAC) inhibitor kinetic rate constants correlate with cellular histone acetylation but not transcription and cell viability. J Biol Chem. 2013 Sep 13;288(37):26926-43. [2]. The histone deacetylase inhibitor MS-275 promotes differentiation or apoptosis in human leukemia cells through a process regulated by generation of reactive oxygen species and induction of p21CIP1/WAF1 1. Cancer Res. 2003 Jul 1;63(13):3637-45. [3].A synthetic inhibitor of histone deacetylase, MS-27-275, with marked in vivo antitumor activity against human tumors. Proc Natl Acad Sci U S A, 1999, 96(8), 4592-4597. [4]. MS-275, an histone deacetylase inhibitor, reduces the inflammatory reaction in rat experimental autoimmune neuritis. Neurosci, 2010, 169, 370-377. |
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| Additional Infomation |
Entinostat is a member of the class of benzamides resulting from the formal condensation of the carboxy group of the pyridin-3-ylmethyl carbamate derivative of p-(aminomethyl)benzoic acid with one of the amino groups of benzene-1,2-diamine. It is an inhibitor of histone deacetylase isoform 1 (HDAC1) and isoform 3 (HDAC3). It has a role as an EC 3.5.1.98 (histone deacetylase) inhibitor, an antineoplastic agent and an apoptosis inducer. It is a member of pyridines, a carbamate ester, a substituted aniline, a primary amino compound and a member of benzamides. It is functionally related to a 1,2-phenylenediamine. Entinostat is under investigation for the treatment and other of Volunteers, Breast Cancer, Human Volunteers, and Normal Volunteers. Entinostat has been investigated for the treatment of Non-Small Lung Cancer, Epigenetic Therapy. Entinostat is a synthetic benzamide derivative with potential antineoplastic activity. Entinostat binds to and inhibits histone deacetylase, an enzyme that regulates chromatin structure and gene transcription. This agent appears to exert dose-dependent effects in human leukemia cells including cyclin-dependent kinase inhibitor 1A (p21/CIP1/WAF1)-dependent growth arrest and differentiation at low drug concentrations; a marked induction of reactive oxygen species (ROS); mitochondrial damage; caspase activation; and, at higher concentrations, apoptosis. In normal cells, cyclin-dependent kinase inhibitor 1A expression has been associated with cell-cycle exit and differentiation. Effects of the histone deacetylase (HDAC) inhibitor MS-275 have been examined in human leukemia and lymphoma cells (U937, HL-60, K562, and Jurkat) as well as in primary acute myelogenous leukemia blasts in relation to differentiation and apoptosis. MS-275 displayed dose-dependent effects in each of the cell lines. When administered at a low concentration (e.g., 1 micro M), MS-275 exhibited potent antiproliferative activity, inducing p21(CIP1/WAF1)-mediated growth arrest and expression of differentiation markers (CD11b) in U937 cells. These events were accompanied by an increase in hypophosphorylated retinoblastoma protein and down-regulation of cell cycle-related proteins including cyclin D1. However, at higher concentrations (e.g., 5 micro M), MS-275 potently induced cell death, triggering apoptosis in approximately 70% of cells at 48 h. In contrast to other HDAC inhibitors such as apicidin, the extrinsic, receptor-mediated pathway played a minimal role in MS-275 lethality. However, MS-275 potently induced a very early (e.g., within 2 h) increase in reactive oxygen species (ROS), followed by the loss of mitochondrial membrane potential (Delta psi(m)) and cytosolic release of cytochrome c. These events culminated in activation of the caspase cascade, manifested by poly(ADP-ribose) polymerase, p21(CIP1/WAF1), p27(KIP), Bcl-2, and retinoblastoma protein degradation. MS-275 exposure also resulted in diminished expression of cyclin D1 and the antiapoptotic proteins Mcl-1 and XIAP. Administration of the free radical scavenger L-N-acetylcysteine blocked MS-275-mediated mitochondrial injury and apoptosis, suggesting a primary role for ROS generation in MS-275-associated lethality. Lastly, U937 cells stably expressing a p21(CIP1/WAF1) antisense construct were significantly more sensitive to MS-275-mediated apoptosis than controls, but they were impaired in their differentiation response. Together, these findings demonstrate that MS-275 exerts dose-dependent effects in human leukemia cells, i.e., p21(CIP1/WAF1)-dependent growth arrest and differentiation at low drug concentrations and a marked induction of ROS, mitochondrial damage, caspase activation, and apoptosis at higher concentrations.[2] Synthetic benzamide derivatives were investigated for their ability to inhibit histone deacetylase (HDA). In this study, one of the most active benzamide derivatives, MS-27-275, was examined with regard to its biological properties and antitumor efficacy. MS-27-275 inhibited partially purified human HDA and caused hyperacetylation of nuclear histones in various tumor cell lines. It behaved in a manner similar to other HDA inhibitors, such as sodium butyrate and trichostatin A; MS-27-275 induced p21(WAF1/CIP1) and gelsolin and changed the cell cycle distribution, decrease of S-phase cells, and increase of G1-phase cells. The in vitro sensitivity spectrum of MS-27-275 against various human tumor cell lines showed a pattern different than that of a commonly used antitumor agent, 5-fluorouracil, and, of interest, the accumulation of p21(WAF1/CIP1) tended to be faster and greater in the cell lines sensitive to MS-27-275. MS-27-275 administered orally strongly inhibited the growth in seven of eight tumor lines implanted into nude mice, although most of these did not respond to 5-fluorouracil. A structurally analogous compound to MS-27-275 without HDA-inhibiting activity showed neither the biological effects in cell culture nor the in vivo therapeutic efficacy. These results suggest that MS-27-275 acts as an antitumor agent through HDA inhibition and may provide a novel chemotherapeutic strategy for cancers insensitive to traditional antitumor agents.[3] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.64 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.08 mg/mL (5.53 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 20.8 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. Solubility in Formulation 3: ≥ 2.08 mg/mL (5.53 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 20.8 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. Solubility in Formulation 4: ≥ 2.08 mg/mL (5.53 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 20.8 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly. Solubility in Formulation 5: 2% DMSO+30% PEG 300: 10mg/mL Solubility in Formulation 6: 3% DMSO + 22% Castor oil + 75% Saline (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.6567 mL | 13.2834 mL | 26.5668 mL | |
| 5 mM | 0.5313 mL | 2.6567 mL | 5.3134 mL | |
| 10 mM | 0.2657 mL | 1.3283 mL | 2.6567 mL |