Ilginatinib (NS018; NS-018) is an orally bioavailable, small molecule inhibitor of Janus-associated kinase 2 (JAK2) and Src-family kinases, with potential antineoplastic activity. It inhibits JAK2 with an IC50 of 0.72 nM, and exhibits 46-, 54-, and 31-fold selectivity for JAK2 over JAK1 (IC50, 33 nM), JAK3 (IC50, 39 nM), and Tyk2 (IC50, 22 nM). JAK2/Src inhibitor NS-018 competes with ATP for binding to JAK2 as well as the mutated form JAK2V617F, thereby inhibiting the activation of JAK2 and downstream molecules in the JAK2/STAT3 (signal transducer and activator of transcription 3) signaling pathway that plays an important role in normal development, particularly hematopoiesis. In addition, NS-018 inhibits the Src family tyrosine kinases. This eventually leads to the induction of tumor cell apoptosis.

Physicochemical Properties

Molecular Formula	C21H20FN7
Molecular Weight	389.4288
Exact Mass	389.176
CAS #	1239358-86-1
Related CAS #	Ilginatinib maleate;1354799-87-3;Ilginatinib hydrochloride;1239358-85-0
PubChem CID	46866319
Appearance	Light yellow to yellow solid powder
Density	1.3±0.1 g/cm3
Boiling Point	586.8±60.0 °C at 760 mmHg
Flash Point	308.7±32.9 °C
Vapour Pressure	0.0±1.6 mmHg at 25°C
Index of Refraction	1.671
LogP	2.66
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	6
Heavy Atom Count	29
Complexity	501
Defined Atom Stereocenter Count	1
SMILES	C[C@@H](C1=CC=C(C=C1)F)NC2=CC(=CC(=N2)NC3=NC=CN=C3)C4=CN(N=C4)C
InChi Key	UQTPDWDAYHAZNT-AWEZNQCLSA-N
InChi Code	InChI=1S/C21H20FN7/c1-14(15-3-5-18(22)6-4-15)26-19-9-16(17-11-25-29(2)13-17)10-20(27-19)28-21-12-23-7-8-24-21/h3-14H,1-2H3,(H2,24,26,27,28)/t14-/m0/s1
Chemical Name	6-N-[(1S)-1-(4-fluorophenyl)ethyl]-4-(1-methylpyrazol-4-yl)-2-N-pyrazin-2-ylpyridine-2,6-diamine
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
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	IC50 for JAK2:0.72 nM; Tyk2:22 nM; JAK1:33 nM; JAK3:39 nM
ln Vitro	Ilginatinib (NS-018) is a very potent JAK2 inhibitor, showing 46, 54, and 31-fold selectivity for JAK2 over JAK1 (IC50, 33 nM), JAK3 (IC50, 39 nM), and Tyk2 (IC50, 22 nM). Its IC50 is 0.72 nM. Along with mildly inhibiting ABL and FLT3 with 45- and 90-fold selectivity for JAK2, ignitinib (NS-018) also inhibits Src-family kinases, namely SRC and FYN. When it comes to cell lines with mutations in JAK2V617F or MPLW515L, or the TEL-JAK2 fusion gene that expresses a constitutively activated JAK2, NS-018 exhibits potent inhibitory activity with an IC50 of 11-120 nM. However, its cytotoxicity against the majority of other hematopoietic cell lines that lack constitutively activated JAK2 is minimal[1]. When obtained from bone marrow mononuclear cells (BMMNCs) of patients with myelodysplastic syndrome (MDS), ilginatinib (NS-018) (0.5 μM) preferentially reduces the development of colony-forming units, granulocyte/macrophages (CFU-GM). MDS patients' CFU-GM-forming cells show suppression of the phosphorylation of STAT3, the downstream kinase of JAK2, when exposed to ignitinib (NS-018) at a concentration of 1 μM.
ln Vivo	In a mouse Ba/F3-JAK2V617F illness model, ilginatinib (NS-018) (12.5, 25, 50, 100 mg/kg, po) potently prolongs animal survival and decreases splenomegaly[1]. In JAK2V617F transgenic mice, ilginatinib (NS-018) (25, 50 mg/kg, po) dramatically lowers leukocytosis, hepatosplenomegaly, and extramedullary hematopoiesis, enhances nutritional status, and increases survival time.
Cell Assay	Bone marrow mononuclear cells (BMMNCs) from healthy volunteers and myelodysplastic syndrome (MDS) patients are incubated in MethoCult GF H4434 methylcellulose medium containing various hematopoietic cytokines at 1.0 × 105 cells/mL with or without NS-018 at 37°C in a humidified atmosphere of 5% CO2. Commercially available purified normal human CD34-positive (CD34+) BM cells are used as a control. Burst-forming unit-erythroid (BFU-E) and colonyforming unit-granulocyte/macrophage (CFU-GM) colonies are counted under an inverted microscope on day 14 of culture[2].
Animal Protocol	Mice[1] Female BALB/c nude mice are placed in blanket cages in an environment maintained at 21-25°C and 45-65% relative humidity, with artificial illumination for 12 h and a ventilation frequency of at least 15 times/h. They are allowed free access to food pellets and tap water. Ba/F3-JAK2V617F cells (106 per mouse) are inoculated intravenously into 7-week-old mice. Administration of vehicle (0.5% methylcellulose) or NS-018 twice daily by oral gavage begins the day after cell inoculation. Survival is monitored daily, and moribund mice are humanely killed and their time of death is recorded for purposes of survival analysis. In a parallel study, all mice are humanely killed after 8 days of administration, and their spleens are removed and weighed[1].
References	[1]. Efficacy of NS-018, a potent and selective JAK2/Src inhibitor, in primary cells and mouse models of myeloproliferative neoplasms. Blood Cancer J. 2011 Jul;1(7):e29. [2]. NS-018, a selective JAK2 inhibitor, preferentially inhibits CFU-GM colony formation by bone marrow mononuclear cells from high-risk myelodysplastic syndrome patients. Leuk Res. 2014 May;38(5):619-24.
Additional Infomation	NS-018 has been used in trials studying the treatment of Primary Myelofibrosis, Post-Polycythemia Vera Myelofibrosis, and Post-Essential Thrombocythemia Myelofibrosis. Ilginatinib is an orally bioavailable, small molecule inhibitor of Janus-associated kinase 2 (JAK2) and Src-family kinases, with potential antineoplastic activity. Ilginatinib competes with ATP for binding to JAK2 as well as the mutated form JAK2V617F, thereby inhibiting the activation of JAK2 and downstream molecules in the JAK2/STAT3 (signal transducer and activator of transcription 3) signaling pathway that plays an important role in normal development, particularly hematopoiesis. In addition, ilginatinib inhibits the Src family tyrosine kinases. This eventually leads to the induction of tumor cell apoptosis. JAK2 is the most common mutated gene in bcr-abl-negative myeloproliferative disorders (MPDs); JAK2V617F is a constitutively activated kinase that activates the JAK/STAT signaling pathway and dysregulates cell growth and function, and its expression transforms hematopoietic cells to cytokine-independent growth.

Solubility Data

Solubility (In Vitro)

DMSO : ~100 mg/mL (~256.79 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.42 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 (6.42 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 (6.42 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.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.5679 mL	12.8393 mL	25.6786 mL
	5 mM	0.5136 mL	2.5679 mL	5.1357 mL
	10 mM	0.2568 mL	1.2839 mL	2.5679 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.