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Fedratinib dihydrochloride hydrate (SAR302503 or TG101348) is a novel, potent, selective, orally bioavailable, small-molecule and ATP-competitive inhibitor of janus kinase 2 (JAK2) with the inhibition constant IC50 of 3 nM. The selectivity of TG101348 for JAK2 is 35- and 334-fold stronger than that for JAK3 and JAK1 respectively. TG10348 is capable of inducing apoptosis in HEL cells as well BaF/3 cells harboring JAK2V617 mutation and inhibiting hematopoietic progenitor colony formation and erythroid engraftment in samples from polycythemia vera (PV) patients.
Targets |
JAK2 (IC50=3 nM)
JAK2 (V617F) (IC50=3 nM) Flt3 (IC50=15 nM) Ret (IC50=48 nM) |
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ln Vitro |
Fedratinib Hydrochloride Hydrate (TG101348) staining to indicate edema in the pre-Myobreast B cell line expressing human JAK2V617F (Ba/F3 JAK2V617F) and the JAK2V617F mutated human erythroblastic leukemia (HEL) cell line; IC50 Value per line 300 nM parental Ba. The /F3 cell proliferation was significantly inhibited with an IC50 value of approximately 420 nM[1]. Fedratinib Hydrochloride Hydrate (TG101348) (0.1 μM, 0.3 μM, 1 μM, 3 μM, and 10 μM) was applied to the cells. This compound decreases STAT5 phosphorylation at concentrations similar to those needed for apoptosis and induces HEL in a dose-slide manner [1]. Disinfection of Ba/F3 JAK2V617F cells [1].
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ln Vivo |
Fedratinib hydrochloride hydrate (TG101348; twice daily; 42 days; border gavage; 60-120 mg/kg) in C57Bl/6 mice
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Enzyme Assay |
IC50 Determinations by Cell-free Kinase Activity Assays
IC50 values for TG101348 were determined commercially using the InVitrogen (Carlsbad, CA, USA) kinase profiling service for a 223 kinase screen that included JAK2 and JAK2V617F or Carna Biosciences (Kobe, Japan) for the screen of all Janus kinase family members including JAK1 and Tyk2. ATP concentration was set to approximately the Km value for each kinase.[1] |
Cell Assay |
XTT Assay for Cell Proliferation, Apoptosis, and DNA Laddering Assay
Approximately 2 × 103 cells were plated into microtiter-plate wells in 100 μl RPMI-1640 growth media with indicated concentrations of inhibitor. Following 72 hr incubation with TG101348, 50 μl of XTT dye were added to each well and incubated for 4 hr in a CO2 incubator. The colored formazan product was measured by spectrophotometry at 450 nm with correction at 650 nm. The concentration in which 50% of the effect (i.e., inhibition of proliferation) is observed (IC50) was determined using the GraphPad Prism 4.0 software. All experiments were performed in triplicate, and the results were normalized to growth of untreated cells. Induction of apoptosis of EpoBa/F3 JAK2V617F, Ba/F3p210, HEL, and K562 cells was determined by DNA fragmentation with DMSO and increasing concentrations of inhibitor. Western Blot Analysis Cells were treated with DMSO and increasing concentrations of inhibitor for 4 hr in RPMI-1640 before collected in 1× Cell Lysis Buffer (Cell Signaling, Beverly, MA, USA), containing 1 mM PMSF, and protease inhibitor cocktail tablets (Roche). Protein lysates were quantified with Pierce Biotechnology BCA assay (Rockford, IL, USA). Similar protein amounts were mixed with Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA, USA) plus β-mercaptoethanol, boiled for 5 min, and separated on a 4%–15% Tris-HCL gradient electrophoresis gel (Bio-Rad Laboratories). Gels were blotted onto a 0.45 μm nitrocellulose membrane (Bio-Rad), which was blocked in 5% nonfat dry milk and incubated with primary antibodies in either blocking solution or 5% BSA. The membranes were subsequently incubated with a mixture of donkey anti-rabbit IgG conjugate with infrared fluorophore (700 nm emission, LICOR) and goat anti-mouse IgG conjugated with infrared fluorophore (800 nm emission). Following washing with PBS, the membranes were scanned on a LICOR Odyssey scanner to detect total (red) and phospho-STAT5 (green) proteins.[2] |
Animal Protocol |
Animal/Disease Models: JAK2V617F mutation-induced C57Bl/6 mice [1 ]
Doses: 60 mg/kg, 120 mg/kg Route of Administration: po (oral gavage); [1]. twice (two times) daily; continued for 42 days Experimental Results: Demonstrated statistically significant reductions in hematocrit and white blood cell count, and dose-dependent reduction/elimination of extramedullary hematopoiesis. |
References |
[1]. Wernig G, et al. Efficacy of TG101348, a selective JAK2 inhibitor, in treatment of a murine model of JAK2V617F-induced polycythemia vera. Cancer Cell. 2008 Apr;13(4):311-20.
[2]. Geron I, et al. Selective inhibition of JAK2-driven erythroid differentiation of polycythemia vera progenitors. Cancer Cell. 2008 Apr;13(4):321-30. |
Molecular Formula |
C27H40CL2N6O4S
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Molecular Weight |
615.615303039551
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Exact Mass |
614.220886
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Elemental Analysis |
C, 52.68; H, 6.55; Cl, 11.52; N, 13.65; O, 10.40; S, 5.21
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CAS # |
1374744-69-0
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Related CAS # |
936091-26-8; 2468204-70-6 (HCl); 1374744-69-0 (HCl hydrate)
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Appearance |
White to yellow solid
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tPSA |
118Ų
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SMILES |
O=S(C1=CC=CC(NC2=NC(NC3=CC=C(OCCN4CCCC4)C=C3)=NC=C2C)=C1)(NC(C)(C)C)=O.[H]Cl.[H]Cl.[H]O[H]
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InChi Key |
QAFZLTVOFJHYDF-UHFFFAOYSA-N
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InChi Code |
InChI=1S/C27H36N6O3S.2ClH.H2O/c1-20-19-28-26(30-21-10-12-23(13-11-21)36-17-16-33-14-5-6-15-33)31-25(20)29-22-8-7-9-24(18-22)37(34,35)32-27(2,3)4/h7-13,18-19,32H,5-6,14-17H2,1-4H3,(H2,28,29,30,31)2*1H1H2
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Chemical Name |
N-tert-Butyl-3-{5-methyl-2-[4-(2-pyrrolidin-1-yl-ethoxy)-phenylamino]-pyrimidin-4-ylamino}-benzenesulfonamide
dihydrochloride monohydrate
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Synonyms |
SAR302503 SAR-302503 SAR 302503 TG101348 TG-101348 TG 101348
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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: 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)
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Solubility (In Vitro) |
H2O : ~100 mg/mL (~162.44 mM)
DMSO : ~100 mg/mL (~162.44 mM) |
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Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.06 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 (4.06 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (4.06 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 | 1.6244 mL | 8.1219 mL | 16.2438 mL | |
5 mM | 0.3249 mL | 1.6244 mL | 3.2488 mL | |
10 mM | 0.1624 mL | 0.8122 mL | 1.6244 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.