Ruxolitinib (INCB18424) |JAK1/2 Inhibitor | CAS 941678-49-5

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Quality Control

Batch: Purity: 99.98%

99.98

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Related Targets	EGFR STAT Pim
Other JAK Inhibitors	AZD1480 WP1066 Momelotinib (CYT387) Filgotinib (GLPG0634) AT9283 Gandotinib (LY2784544) TG101209 Cerdulatinib (PRT062070) hydrochloride Pacritinib NVP-BSK805 2HCl

Cell Culture, Treatment & Working Concentration

Cell Lines	Assay Type	Concentration	Incubation Time	Formulation	Activity Description	PMID
SNU423	Function Assay	50 μM	24 h	DMSO	Inhibition of STAT1 and STAT3 phosphorylation significantly	23941832
SNU182	Function Assay	50 μM	24 h	DMSO	Inhibition of STAT1 and STAT3 phosphorylation significantly	23941832
HuH7	Function Assay	50 μM	24 h	DMSO	Inhibition of STAT1 and STAT3 phosphorylation significantly	23941832
SNU423	Growth Inhibition Assay	50 μM	48 h	DMSO	>81% reduction	23941832
SNU182	Growth Inhibition Assay	50 μM	48 h	DMSO	>64% reduction	23941832
HuH7	Growth Inhibition Assay	50 μM	48 h	DMSO	>82% reduction	23941832
RKO	Apoptosis Assay	25 μM	48 h	DMSO	Induces apoptosis by activating caspase 3	24050550
DLD-1	Apoptosis Assay	25 μM	48 h	DMSO	Induces apoptosis by activating caspase 3	24050550
DLD-1	Growth Inhibition Assay	50 μM	48 h	DMSO	IC50=15.51 μM	24050550
RKO	Growth Inhibition Assay	50 μM	48 h	DMSO	IC50=14.76 μM	24050550
RKO	Kinase Assay	25 μM	48 h	DMSO	does not inhibit JAK1 phosphorylation	24050550
DLD-1	Kinase Assay	25 μM	48 h	DMSO	Inhibition of JAK2 phosphorylation	24050550
RKO	Kinase Assay	25 μM	48 h	DMSO	Inhibition of JAK1 phosphorylation	24050550
DLD-1	Kinase Assay	25 μM	48 h	DMSO	Inhibition of JAK1 phosphorylation	24050550
BaF3	Kinase Assay	80 nM	6 h	DMSO	Reduces the phosphorylation of STAT5 in JAK2V617F-mutated BAF3-EPOR cell	24237791
Huh7	Function Assay	1 μM	16 h	DMSO	Impaires the capacity of IHCA-associated gp130 mutants to signal to STAT3	24501689
HepG2	Function Assay	1 μM	16 h	DMSO	Impaires the capacity of IHCA-associated gp130 mutants to signal to STAT3	24501689
Hep3B	Function Assay	1 μM	16 h	DMSO	Impaires the capacity of IHCA-associated gp130 mutants to active STAT3 with IC50 of ~50 μM	24501689
NCI-H2347	Apoptosis Assay	30 nM	48 h	DMSO	Induction of apoptosis	25213670
NCI-H1299	Apoptosis Assay	30 nM	48 h	DMSO	Induction of apoptosis	25213670
A549/DDP	Apoptosis Assay	30 nM	48 h	DMSO	Induction of apoptosis	25213670
NCI-H1299	Function Assay	30 nM	48 h	DMSO	Down-regulation of STAT3 phosphorylation	25213670
NCI-H2347	Function Assay	30 nM	48 h	DMSO	Decrease in Bcl2 expression	25213670
A549/DDP	Function Assay	30 nM	48 h	DMSO	Down-regulation of STAT3 phosphorylation	25213670
NCI-H2347	Growth Inhibition Assay			DMSO	IC50=0.17 μM	25213670
NCI-H1299	Growth Inhibition Assay			DMSO	IC50=0.28 μM	25213670
A549/DDP	Growth Inhibition Assay			DMSO	IC50=0.22 μM	25213670
A549	Growth Inhibition Assay			DMSO	IC50=0.04 μM	25213670
NCI-H358	Growth Inhibition Assay			DMSO	IC50=0.1 μM	25213670
NCI-H460	Growth Inhibition Assay			DMSO	IC50=0.13 μM	25213670
CMK	Growth Inhibition Assay				Inhibition of CMK carrying the WT JAK cell proliferation with IC50 of 0.075 μM	25352124
CMK	Growth Inhibition Assay				Inhibition of CMK carrying the JAK3A63D mutation cell proliferation with IC50 of 0.163 μM	25352124
CMK	Growth Inhibition Assay				Inhibition of CMK carrying the JAK3A572V mutation cell proliferation	25352124
HT93A	Growth Inhibition Assay	320 nM	5 d	DMSO	Inhibition of GCS-F induced granulocytic differentiation	25805962
SET-2	Cytotoxic Assay	5 μM	48 h		Cytotoxic index=18.7%	25931349
HEL	Cytotoxic Assay	5 μM	48 h		Cytotoxic index=12.2%	25931349
Human monocyte	Kinase Assay				Inhibition of JAK2/1 in human monocytes expressing CD14 assessed as inhibition of IFNgamma-stimulated STAT1 phosphorylation with IC50 of 0.031μM	23540648
Human monocyte	Kinase Assay				Inhibition of JAK2 in human monocytes expressing CD14 assessed as inhibition of GM-CSF-stimulated STAT5a phosphorylation with IC50 of 0.026μM	23540648
Human T cell	Kinase Assay				Inhibition of JAK3/1 in human T cells expressing CD3 assessed as inhibition of IL2-stimulated STAT5a phosphorylation with IC50 of 0.023μM	23540648
TF1	Kinase Assay		20 min	DMSO	Inhibition of JAK1 in human TF1 cells assessed as inhibition of IL6-induced STAT3 phosphorylation with IC50 of 0.024μM	22698084
TF1	Kinase Assay		20 min	DMSO	Inhibition of JAK2 in human TF1 cells assessed as inhibition of EPO-induced STAT5 phosphorylation with IC50 of 0.012μM	22698084
Sf9 cells	JAK inhibition assay		1 h		Ki = 0.0001 μM	23668484
Sf9 cells	JAK inhibition assay		1 h		Ki = 0.0002 μM	23668484
Sf9 cells	JAK inhibition assay		1 h		Ki = 0.0005 μM	23668484
SET2 cells	JAK inhibition assay				IC50 = 0.00184 μM	23061660
Sf21 cells	JAK inhibition assay		1 h		IC50 = 0.0028 μM	22591402
Sf21 cells	JAK inhibition assay		60 min		IC50 = 0.003 μM	27137359
Sf9 cells	JAK inhibition assay		1 h		Ki = 0.0032 μM	23668484
Sf21 cells	JAK inhibition assay		1 h		IC50 = 0.0033 μM	22591402
TF1 cells	JAK inhibition assay		30 min		IC50 = 0.00685 μM	23061660
CD34+ cells	JAK inhibition assay				IC50 = 0.008 μM	26927423
TF1 cells	JAK inhibition assay		20 min		EC50 = 0.012 μM	22698084
Sf21 cells	TYK2 inhibition assay		1 h		IC50 = 0.019 μM	22591402
T cells	JAK inhibition assay				IC50 = 0.023 μM	23540648
T cells	JAK inhibition assay				IC50 = 0.023 μM	23540648
TF1 cells	JAK inhibition assay		20 min		EC50 = 0.024 μM	22698084
T cells	JAK inhibition assay				IC50 = 0.031 μM	23540648
T cells	JAK inhibition assay				IC50 = 0.031 μM	23540648
PBMC cells	JAK inhibition assay				IC50 = 0.04 μM	26927423
Sf21 cells	JAK inhibition assay		1 h		IC50 = 0.428 μM	22591402
PBMC cells	STAT5 inhibition assay				IC50 = 0.448 μM	26927423
CD34+ cells	JAK inhibition assay		45 min		IC50 = 0.677 μM	24417533
Click to View More Cell Line Experimental Data

Solubility

In vitro
Batch:

DMSO : 300 mg/mL (979.2 mM)
(Moisture-contaminated DMSO may reduce solubility. Use fresh, anhydrous DMSO.)

Ethanol : 12 mg/mL

Water : Insoluble

Molarity Calculator

Mass	Concentration	Volume	Molecular Weight
Mass value Mass unit	Concentration value Concentration unit	Volume value Volume unit	Molecular weight (g/mol)

Dilution Calculator Molecular Weight Calculator

In vivo batch selection In vivo Batch:
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.
Clear solution	5%DMSO 1 40%PEG 300 2 5%Tween80 3 50% ddH2O Validated by Selleck labs. Should you need adjustments to this formulation, contact our sales team for custom testing.	3.000mg/ml (9.79mM)	Taking the 1 mL working solution as an example, add 50 μL of 60 mg/mL clarified DMSO stock solution to 400 μL PEG300, mix evenly to clarify it; add 50 μL Tween-80 to the above system, mix evenly to clarify it; then continue Add 500 μL ddH2O to adjust the volume to 1 mL. The mixed solution should be used immediately for optimal results.
Clear solution	5%DMSO 1 95% Corn oil Validated by Selleck labs. Should you need adjustments to this formulation, contact our sales team for custom testing.	3.000mg/ml (9.79mM)	Taking the 1 mL working solution as an example, add 50 μL of 60 mg/mL clear DMSO stock solution to 950 μL of corn oil and mix evenly. The mixed solution should be used immediately for optimal results.
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.

In vivo Formulation Calculator (Clear solution)

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Working concentration: mg/ml;

Method for preparing DMSO master liquid: mg drug pre-dissolved in μL DMSO ( Master liquid 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 master liquid, next addμL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH₂O, mix and clarify.

Method for preparing in vivo formulation: Take μL DMSO master liquid, next add μL Corn oil, mix and clarify.

Note: 1. Please make sure the liquid is clear before adding the next solvent.
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Chemical Information, Storage & Stability

Molecular Weight	306.37	Formula	C₁₇H₁₈N₆	Storage (From the date of receipt)	3 years-20°Cpowder
CAS No.	941678-49-5	Download SDF		Storage of Stock Solutions	1 year-80°Cin solvent 1 month-20°Cin solvent
Synonyms	INCB018424			Smiles	C1CCC(C1)C(CC#N)N2C=C(C=N2)C3=C4C=CNC4=NC=N3

Mechanism of Action

Targets/IC₅₀/Ki	JAK2 (Cell-free assay) 2.8 nM JAK1 (Cell-free assay) 3.3 nM
In vitro	Ruxolitinib (INCB18424) potently and selectively inhibits JAK2V617F-mediated signaling and proliferation in Ba/F3 cells and HEL cells. This compound markedly increases apoptosis in a dose dependent manner in Ba/F3 cells. It (64 nM) results in a doubling of cells with depolarized mitochondria in Ba/F3 cells. This chemical inhibits proliferating of erythroid progenitors from normal donors and polycythemia vera patients with IC50 of 407 nM and 223 nM, respectively. It demonstrates remarkable potency against erythroid colony formation with IC50 of 67nM.
Kinase Assay	Binding assay
Kinase Assay	Recombinant proteins are expressed using Sf21 cells and baculovirus vectors and purified with affinity chromatography. JAK kinase assays use a homogeneous time-resolved fluorescence assay with the peptide substrate (-EQEDEPEGDYFEWLE). Each enzyme reaction is carried out with Ruxolitinib (INCB18424) or control, JAK enzyme, 500 nM peptide, adenosine triphosphate (ATP; 1mM), and 2% dimethyl sulfoxide (DMSO) for 1 hour. The 50% inhibitory concentration (IC50) is calculated as the concentration of this compound required for inhibition of 50% of the fluorescent signal.
In vivo	INCB018424 (180 mg/kg, orally, twice a day) results in a survival rate of greater than 90% by day 22 in a JAK2V617F-driven mouse model. This compound (180 mg/kg, orally, twice a day) markedly reduces splenomegaly and circulating levels of inflammatory cytokines, and preferentially eliminates neoplastic cells, resulting in significantly prolonged survival without myelosuppressive or immunosuppressive effects in a JAK2V617F-driven mouse model. In the double-blind trial of myelofibrosis, the primary end point is reached in 41.9% of patients in the Ruxolitinib (INCB18424) group as compared with 0.7% in the placebo group. It results in maintaining reduction in spleen volume and improvement of 50% or more in the total symptom score. A total of 28% of the patients in the group receiving this compound (15 mg twice daily) has at least a 35% reduction in spleen volume at week 48 in patients with myelofibrosis, as compared with 0% in the group receiving the best available therapy. The mean palpable spleen length has decreased by 56% with it but has increased by 4% with the best available therapy at week 48. Patients in the ruxolitinib group has an improvement in overall quality-of-life measures and a reduction in symptoms associated with myelofibrosis.
References	[1] https://pubmed.ncbi.nlm.nih.gov/20130243/ [2] https://pubmed.ncbi.nlm.nih.gov/22375971/ [3] https://pubmed.ncbi.nlm.nih.gov/22375970/

Applications

Methods	Biomarkers	PMID
Western blot	cleaved PARP / cleaved caspase3 p-JAK2 / p-AKT / p-MAPK / Bcl-xl / MCL-1 c-Myc / c-Jun / Cyclin B / Cyclin D / Bcl-2 / HIF-1α p-STAT3	29849942
Growth inhibition assay	Cell viability Cell apoptosis Cell proliferation	29849942
Immunofluorescence	α-tubulin	26356819

Clinical Trial Information

(data from https://clinicaltrials.gov, updated on 2024-05-22)

NCT Number	Recruitment	Conditions	Sponsor/Collaborators	Start Date	Phases
NCT06397313	Not yet recruiting	Myelofibrosis	Ryvu Therapeutics SA	September 2024	Phase 2
NCT06388564	Not yet recruiting	Chronic Graft-versus-host-disease	Incyte Corporation	July 8 2024	Phase 2
NCT06251102	Not yet recruiting	Polycythemia Vera	Gruppo Italiano Malattie EMatologiche dell''Adulto	July 2024	--
NCT06343792	Not yet recruiting	Steroid Refractory GVHD	ReAlta Life Sciences Inc.	May 2024	Phase 2

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Frequently Asked Questions

Question 1:
What is the difference between S2902 and S1378 which seem to have same structure formula according to the product information?

Answer:
These two chemicals are the two different chiral forms of this compound. S2902 S-Ruxolitinib is the S form and S1378 Ruxolitinib is the D form. One of the carbon atoms in it is asymmetric, making the two molecules mirror images of each other. The biological activities of these two molecules can be very different because of the confirmation differences.

Question 2:
How about the half-life of this compound? How long is the duration of its inhibitory effect on JAK-STAT signaling?

Answer:
According to previous study, the half-life of this compound in body is about 2~3 hours. Generally, it is longer in vitro culture medium than in vivo. It was also used for 24 hours in paper. http://www.bloodjournal.org/cgi/pmidlookup?view=long&pmid=24711661.

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Ruxolitinib (INCB18424) JAK1/2 inhibitor

Chemical Structure

Molecular Weight: 306.37