JAK2 (IC50=3 nM)
JAK2 (V617F) (IC50=3 nM)
Flt3 (IC50=15 nM)
Ret (IC50=48 nM)

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].

Fedratinib hydrochloride hydrate (TG101348; twice daily; 42 days; border gavage; 60-120 mg/kg) in C57Bl/6 mice

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]

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/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.

[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.

C27H40CL2N6O4S

615.615303039551

614.220886

C, 52.68; H, 6.55; Cl, 11.52; N, 13.65; O, 10.40; S, 5.21

1374744-69-0

936091-26-8; 2468204-70-6 (HCl); 1374744-69-0 (HCl hydrate)

White to yellow solid

118Ų

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]

QAFZLTVOFJHYDF-UHFFFAOYSA-N

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

N-tert-Butyl-3-{5-methyl-2-[4-(2-pyrrolidin-1-yl-ethoxy)-phenylamino]-pyrimidin-4-ylamino}-benzenesulfonamide dihydrochloride monohydrate

SAR302503 SAR-302503 SAR 302503 TG101348 TG-101348 TG 101348

2934.99.9001

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 (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~100 mg/mL (~162.44 mM)
DMSO : ~100 mg/mL (~162.44 mM)

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.
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.

---

 (Please use freshly prepared in vivo formulations for optimal results.)