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Ralimetinib dimysylate (also known as LY-2228820; LY2228820) is a novel, potent and selective ATP-competitive inhibitor of p38 MAPK with potential anti-inflammatory activity. In a cell-free assay, it inhibits p38 MAPK with an IC50 of 7 nM and does not affect p38 MAPK activation. In cell-based assays, LY2228820 potently and selectively inhibited phosphorylation of MK2 (Thr334) in anisomycin-stimulated HeLa cells (at 9.8 nmol/L by Western blot analysis) and anisomycin-induced mouse RAW264.7 macrophages (IC(50) = 35.3 nmol/L) with no changes in phosphorylation of p38α MAPK, JNK, ERK1/2, c-Jun, ATF2, or c-Myc ≤ 10 μmol/L.
Physicochemical Properties
| Molecular Formula | C24H29FN6.2CH4O3S |
| Molecular Weight | 612.74 |
| Exact Mass | 612.219 |
| Elemental Analysis | C, 50.97; H, 6.09; F, 3.10; N, 13.72; O, 15.67; S, 10.46 |
| CAS # | 862507-23-1 |
| Related CAS # | Ralimetinib;862505-00-8 |
| PubChem CID | 11570805 |
| Appearance | White to yellow solid powder |
| Boiling Point | 634.4ºC at 760 mmHg |
| Flash Point | 337.5ºC |
| LogP | 6.653 |
| Hydrogen Bond Donor Count | 4 |
| Hydrogen Bond Acceptor Count | 11 |
| Rotatable Bond Count | 5 |
| Heavy Atom Count | 41 |
| Complexity | 701 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | O=S(C)(O)=O.FC1C=CC(C2=C(C3C=CC4N=C(N(C=4N=3)CC(C)(C)C)N)NC(C(C)(C)C)=N2)=CC=1 |
| InChi Key | NARMJPIBAXVUIE-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C24H29FN6.2CH4O3S/c1-23(2,3)13-31-20-17(28-22(31)26)12-11-16(27-20)19-18(14-7-9-15(25)10-8-14)29-21(30-19)24(4,5)6;2*1-5(2,3)4/h7-12H,13H2,1-6H3,(H2,26,28)(H,29,30);2*1H3,(H,2,3,4) |
| Chemical Name | 5-[2-tert-butyl-4-(4-fluorophenyl)-1H-imidazol-5-yl]-3-(2,2-dimethylpropyl)imidazo[4,5-b]pyridin-2-amine;methanesulfonic acid |
| Synonyms | LY-2228820; LY 2228820; Ralimetinib; LY2228820; LY2228820 dimesylate; Ralimetinib dimesylate |
| 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: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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 | p38β MAPK (IC50 = 3.2 nM); p38α MAPK (IC50 = 5.8 nM) |
| ln Vitro | Ralimetinib dimesylate inhibits p38α and the quantity of phosphoMAPKAPK-2 (pMK2) in RAW 264.7 cells with IC50 values of 7 nM and 34.3 nM, respectively. Additionally, Ralimetinib dimesylate inhibits lipopolysaccharide (LPS)-induced TNFα synthesis in murine peritoneal macrophages with an IC50 of 5.2 nM[1]. Ralimetinib dimesylate (LY2228820) (200 nM-800 nM) significantly inhibits p38MAPK signaling in multiple myeloma (MM) cells, including INA6, RPMI-8226, U266 and RPMI-Dox40, as shown by its inhibition on phosphorylation of HSP27, a downstream target of p38MAPK, without changing the expression level of HSP27. Ralimetinib dimesylate (200 nM–400 nM) increases bortezomib-induced cytotoxicity and apoptosis, but Ralimetinib dimesylate alone does not inhibit the growth of MM.1S cells. Additionally, ralimetinib dimesylate (200 nM-800 nM) inhibits the release of IL-6 and MIP-1 from long-term BM stromal cells (LT-BMSCs), BM mononuclear cells (BMMNCs), and peripheral blood (PB) CD138+, CD138-, or PB CD14+ cells. Additionally, osteoclastogenesis from CD14+ cells is prevented by ralimetinib dimesylate (400 nM–800 nM)[2]. |
| ln Vivo | Ralimetinib dimesylate inhibits the production of TNFα in LPS-induced mice with a threshold minimum 50% effective dose (TMED50) of less than 1 mg/kg. Ralimetinib dimesylate exhibits strong effects on paw swelling, bone deterioration, and cartilage destruction in a rat model of collagen-induced arthritis (CIA), with a threshold minimum 50% effective dose (TMED50) of 1.5 mg/kg[1]. In mice implanted with B16-F10 melanoma, ralimetinib dimesylate inhibits tumor phospho-MK2 in a dose-dependent manner (TED50=1.95 mg/kg, TED70=11.17 mg/kg). Mouse in vivo TED50=1.01 mg/kg (compound exposure approximately 100 nM) and human ex vivo IC50=0.12 μM with either mouse or human PBMC[3] are the values for ralimetinib dimesylate's ability to inhibit MK2 phosphorylation. |
| Enzyme Assay | Inhibition of p38α is determined using recombinant human p38α in a standard filter binding protocol using ATP[γ-33P] and EGFR 21-mer peptide as substrate. Functional inhibition of TNFα in murine peritoneal macrophages is determined using LPS stimulation in the presence of LY2228820. To assess p38α activity in cells more directly, RAW 264.7 cells are treated with LY2228820 and then stimulated with anisomycin. The level of p38α activity is detected using a phosphoMAPKAPK-2 (pMK2) (Thr 334) antibody which reacts with a residue specifically phosphorylated by p38α. |
| Cell Assay | Recombinant human p38α is used in a standard filter binding protocol with ATP[γ-33P] and the EGFR 21-mer peptide as substrate to measure the inhibition of p38. Using LPS stimulation and Ralimetinib, the functional inhibition of TNFα in murine peritoneal macrophages is assessed. RAW 264.7 cells are given ralimetinib treatment before being stimulated with anisomycin in order to more accurately measure p38α activity in the cells. A phosphoMAPKAPK-2 (pMK2) (Thr 334) antibody that reacts with a residue that has been specifically phosphorylated by p38 is used to measure the level of p38α activity. |
| Animal Protocol | Murine B16-F10 melanoma cells are cultured in Dulbecco's Modified Eagle Medium with l-glutamine, high glucose, and 10% FBS (GIBCO 11965-092). B16-F10 cells (2×106) are implanted into the rear flank of C57/bl6 mice, and when the tumors grow to a size of about 200 mm3, Ralimetinib dimesylate in 1% carboxymethylcellulose/0.25% Tween 80 is given orally. Tumors are removed, homogenized, and lysed two hours after the dose for Western blot analysis. Chemiluminescent detection is used to measure MK2 phosphorylation (p-Thr334), which is normalized to total glyceraldehyde-3-phosphate dehydrogenase. In xenograft models, the 50% or 70% threshold effective dose (TED50 and TED70, respectively) is calculated to approximate the effective dose ranges where significant target inhibition is seen. The TED50 or TED70 is the dose at which a statistically significant effect is obtained and there is at least 50% or 70% inhibition, in comparison to the vehicle control, respectively. |
| References |
[1]. Imidazolyl benzimidazoles and imidazo[4,5-b]pyridines as potent p38alpha MAP kinase inhibitors with excellent in vivo antiinflammatory properties. Bioorg Med Chem Lett, 2008, 18(1), 179-183. [2]. p38 mitogen-activated protein kinase inhibitor LY2228820 enhances bortezomib-induced cytotoxicity and inhibits osteoclastogenesis in multiple myeloma; therapeutic implications. Br J Haematol, 2008, 141(5), 598-606. [3]. Characterization of LY2228820 dimesylate, a potent and selective inhibitor of p38 MAPK with antitumor activity. Mol Cancer Ther. 2014 Feb;13(2):364-74. |
| Additional Infomation | Ralimetinib Mesylate is the dimesylate salt form of LY2228820, a tri-substituted imidazole derivative and orally available, p38 mitogen-activated protein kinase (MAPK) inhibitor with potential anti-inflammatory and antineoplastic activities. Upon administration, ralimetinib inhibits the activity of p38, particularly the alpha and beta isoforms, thereby inhibiting MAPKAPK2 phosphorylation and preventing p38 MAPK-mediated signaling. This may inhibit the production of a variety of cytokines involved in inflammation, cellular proliferation and angiogenesis such as tumor necrosis factor alpha (TNFa), interleukin (IL)-1, -6 and -8, vascular endothelial growth factor, and macrophage inflammatory protein-1 alpha. Ultimately this induces apoptosis and reduces tumor cell proliferation. In addition, inhibition of the p38 MAPK pathway by LY2228820 increases the antineoplastic activity of certain chemotherapeutic agents. p38 MAPK, a serine/threonine protein kinase that is often upregulated in cancer cells, plays a crucial role in tumor cell proliferation, angiogenesis and metastasis. |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.08 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 25.0 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (4.08 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 25.0 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. Solubility in Formulation 3: ≥ 2.5 mg/mL (4.08 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 4: Saline: 30mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.6320 mL | 8.1601 mL | 16.3201 mL | |
| 5 mM | 0.3264 mL | 1.6320 mL | 3.2640 mL | |
| 10 mM | 0.1632 mL | 0.8160 mL | 1.6320 mL |