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Ilginatinib (NS018; NS-018) HCl, the hydrochloride salt form of NS-018, is an oral and potent JAK2 inhibitor with potential anticancer 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.
| Targets |
IC50 for JAK2:0.72 nM; Tyk2:22 nM; JAK1:33 nM; JAK3:39 nM
JAK2 (IC50 = 0.72 nM) SRC (inhibition >80% at 100 nM) FYN (inhibition >80% at 100 nM) ABL (inhibition >80% at 100 nM) FLT3 (inhibition >80% at 100 nM) [1] |
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| ln Vitro |
An very potent JAK2 inhibitor, ilginatinib hydrochloride (NS-018 hydrochloride) with an IC50 of 0.72 nM and a 46-, 54-, and 31-fold selectivity for JAK2 over JAK1 (IC50, 33 nM), JAK3 (IC50, 39 nM), and Tyk2 (IC50, 22 nM). Additionally, ilginatinib hydrochloride mildly inhibits ABL and FLT3, with 45- and 90-fold selectivity for JAK2, and inhibits Src-family kinases, particularly SRC and FYN. At an IC50 of 11–120 nM, iligatinib hydrochloride exhibits strong inhibitory activity against cell lines with mutations in JAK2V617F or MPLW515L, as well as the TEL-JAK2 fusion gene, which expresses a constitutively activated JAK2. However, it is only slightly cytotoxic against the majority of other hematopoietic cell lines that lack constitutively activated JAK2[1]. The generation of colony-forming units, granulocyte/macrophages (CFU-GM) from bone marrow mononuclear cells (BMMNCs) derived from myelodysplastic syndrome (MDS) is preferentially suppressed by ilginatinib hydrochloride (0.5 μM). In CFU-GM-forming cells from MDS patients, ilginatinib hydrochloride (1 μM) decreases the phosphorylation of STAT3, the downstream kinase of JAK2[2].
NS-018 showed potent antiproliferative activity against Ba/F3-JAK2V617F cells (IC50 = 60 nM) and SET-2 cells (IC50 = 120 nM). [1] It also inhibited growth of Ba/F3-MPLW515L cells (IC50 = 61 nM) and Ba/F3-TEL-JAK2 cells (IC50 = 11 nM). [1] Ba/F3-TEL-JAK3 cells were less sensitive (IC50 = 800 nM). [1] NS-018 inhibited phosphorylation of STAT5, STAT3, and ERK in Ba/F3-JAK2V617F cells in a dose-dependent manner. [1] It induced apoptosis in Ba/F3-JAK2V617F cells, as shown by annexin V/propidium iodide staining and DNA fragmentation. [1] NS-018 inhibited erythropoietin-independent erythroid colony formation from PV patient cells (mean IC50 = 224 ± 26 nM). [1] |
| ln Vivo |
In a mouse Ba/F3-JAK2V617F illness model, iligatinib hydrochloride (NS-018 hydrochloride) (12.5, 25, 50, 100 mg/kg, po) potently prolongs mouse survival and lowers splenomegaly[1]. In JAK2V617F transgenic mice, ilginatinib hydrochloride (25, 50 mg/kg, po) prolongs longevity, improves nutritional status, and dramatically lowers leukocytosis, hepatosplenomegaly, and extramedullary hematopoiesis[1].
In a Ba/F3-JAK2V617F mouse model, NS-018 (12.5–100 mg/kg, oral, twice daily) significantly prolonged survival and reduced splenomegaly. [1] In JAK2V617F transgenic mice, NS-018 (25 or 50 mg/kg, oral, twice daily for 24 weeks) reduced leukocytosis, hepatosplenomegaly, extramedullary hematopoiesis, and improved nutritional status and survival. [1] |
| Enzyme Assay |
The kinase domains of human JAK1, JAK2, JAK3, and TYK2 were incubated with NS-018, biotinylated peptide substrate, ATP, and MgCl2 in a streptavidin-coated plate for 1 hour at 30°C. [1]
Phosphorylated substrates were detected spectrophotometrically using horseradish peroxidase-linked antibody and TMB solution. [1] IC50 values were estimated by fitting absorbance data to a logistic curve. [1] |
| 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].
Cells were seeded in 96-well plates at optimized densities and treated with serial dilutions of NS-018 for 72 hours at 37°C with 5% CO2. [1] Cell viability was measured by MTT assay. [1] For apoptosis assay, cells were treated with NS-018 for 48 hours and stained with annexin V/propidium iodide, followed by flow cytometry analysis. [1] DNA fragmentation was assessed by agarose gel electrophoresis after 29 hours of NS-018 treatment. [1] |
| 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].
In the Ba/F3-JAK2V617F mouse model, Ba/F3-JAK2V617F cells were intravenously inoculated into female BALB/c nude mice. [1] NS-018 or vehicle (0.5% methylcellulose) was administered orally twice daily starting the day after inoculation. [1] In the JAK2V617F transgenic mouse model, NS-018 was administered orally twice daily on weekdays for 24 weeks at doses of 25 or 50 mg/kg. [1] |
| References |
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| Additional Infomation |
NS-018 is a potent and selective JAK2/Src inhibitor that has shown good activity in preclinical models of myeloproliferative neoplasms. [1] X-ray cocrystal structure analysis showed that it binds to JAK2 in the “DFG-in” active conformation. [1] The selectivity of NS-018 depends primarily on the size of amino acid 993 in JAK2. [1] It is expected to enter early clinical trials in 2011. [1]
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| Molecular Formula |
C21H21CLFN7
|
|---|---|
| Molecular Weight |
425.889745473862
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| Exact Mass |
425.153
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| CAS # |
1239358-85-0
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| Related CAS # |
Ilginatinib maleate;1354799-87-3;Ilginatinib;1239358-86-1
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| PubChem CID |
89571724
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| Appearance |
Light yellow to yellow solid powder
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
30
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| Complexity |
501
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| Defined Atom Stereocenter Count |
1
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| SMILES |
C[C@@H](C1=CC=C(C=C1)F)NC2=CC(=CC(=N2)NC3=NC=CN=C3)C4=CN(N=C4)C.Cl
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| InChi Key |
XFNVGPXGVAXXSH-UQKRIMTDSA-N
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| InChi Code |
InChI=1S/C21H20FN7.ClH/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);1H/t14-;/m0./s1
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| Chemical Name |
6-N-[(1S)-1-(4-fluorophenyl)ethyl]-4-(1-methylpyrazol-4-yl)-2-N-pyrazin-2-ylpyridine-2,6-diamine;hydrochloride
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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) |
DMSO : ≥ 35 mg/mL (~82.18 mM)
H2O : ~2 mg/mL (~4.70 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.87 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3480 mL | 11.7401 mL | 23.4802 mL | |
| 5 mM | 0.4696 mL | 2.3480 mL | 4.6960 mL | |
| 10 mM | 0.2348 mL | 1.1740 mL | 2.3480 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.
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