Z3 selectively inhibits Jak2 tyrosine kinase. It inhibits Jak2-WT autophosphorylation with an IC50 of ~15 μmol/L, and Jak2-V617F mutant autophosphorylation with an IC50 of ~28 μmol/L. It shows no inhibitory effect on Tyk2 or c-Src kinase activity at concentrations effective against Jak2.[1]

Tyk2 and c-Src kinase function are unaffected by NSC 42834 (JAK2 inhibitor V), which specifically suppresses Jak2 kinase activity. The proliferation of the human erythroleukemia cell line HEL 92.1.7, which expresses Jak2-V617F, is greatly inhibited by NSC 42834. Significant cell cycle arrest and lower levels of Jak2 and STAT3 tyrosine phosphorylation were linked to the NSC 42834-mediated reduction in cell proliferation. Lastly, hematopoietic progenitor cells derived from the bone marrow of individuals with polycythemia vera who have the Jak2-F537I mutation and essential thrombocythemia who harbor the Jak2-V617F mutation are inhibited in growth by NSC 42834.

---

Z3 dose-dependently inhibits autophosphorylation of both wild-type (Jak2-WT) and mutant (Jak2-V617F) Jak2 at the critical activation loop residue Tyr1007 in transfected BSC-40 cells, with greater potency against Jak2-WT.[1]
Z3 (25 μmol/L) significantly inhibits the proliferation of the human erythroleukemia cell line HEL 92.1.7, which is homozygous for the Jak2-V617F mutation, over a 72-hour period compared to DMSO control. This inhibition is more pronounced in HEL cells than in Raji cells (a Burkitt's lymphoma cell line driven by c-Myc translocation), indicating selectivity for Jak2-V617F-dependent growth.[1]
The anti-proliferative effect of Z3 in HEL cells correlates with reduced levels of total tyrosine-phosphorylated Jak2, reduced phosphorylation of Jak2 at Tyr1007, and reduced phosphorylation of the downstream effector STAT3.[1]
Z3 (25 μmol/L) induces cell cycle arrest in HEL cells, significantly increasing the percentage of cells in G1 phase and decreasing the percentage in S phase after 72 hours of treatment, without inducing apoptosis.[1]
In ex vivo colony formation assays, Z3 (25 μmol/L) inhibits the growth of hematopoietic progenitor cells isolated from the bone marrow of an essential thrombocythemia patient harboring the Jak2-V617F mutation and a polycythemia vera patient carrying a Jak2-F537I mutation. This inhibition was observed both in the presence and absence of the relevant hematopoietic cytokines (thrombopoietin or erythropoietin).[1]
Z3 is not cytotoxic to BSC-40 cells at concentrations that effectively inhibit Jak2 autophosphorylation (25-100 μmol/L). Cytotoxicity, as indicated by propidium iodide staining, was observed only at a higher concentration of 250 μmol/L.[1]

An in vitro kinase assay was performed to test the effect of Z3 on c-Src activity. Catalytically active recombinant c-Src protein was incubated in a kinase reaction buffer containing either DMSO, 25 μmol/L Z3, or 25 μmol/L of the known Src kinase inhibitor PP2 as a control. The reactions were carried out at room temperature for 20 minutes and then terminated by adding SDS-containing sample buffer. The samples were analyzed by Western blotting using an antibody specific for the active, phosphorylated form of c-Src (pY418) to determine relative kinase activity.[1]

For Jak2 autophosphorylation assays, BSC-40 cells were transfected with plasmids encoding either wild-type murine Jak2 (Jak2-WT) or the V617F mutant (Jak2-V617F) under a T7 promoter. After transfection, cells were infected with a recombinant vaccinia virus (vTF7-3) to drive high-level T7 RNA polymerase expression, resulting in high-level Jak2 expression and subsequent ligand-independent tyrosine autophosphorylation. Cells were then treated with Z3 or DMSO for 16 hours. Cells were lysed, and Jak2 was immunoprecipitated from the lysates. The immunoprecipitates were analyzed by Western blotting using an anti-phosphotyrosine antibody to assess Jak2 autophosphorylation levels, and a phosphospecific antibody (pY1007/pY1008) to assess phosphorylation at the activation loop. Total Jak2 levels were also checked.[1]
For Tyk2 autophosphorylation assay, COS-7 cells were transiently transfected with an expression vector encoding wild-type human Tyk2 cDNA. After 48 hours to allow for high-level expression and autophosphorylation, cells were treated with DMSO or 25 μmol/L Z3 for 16 hours. Cells were lysed, Tyk2 was immunoprecipitated, and autophosphorylation was detected by Western blotting with an anti-phosphotyrosine antibody.[1]
For cell proliferation assays, HEL or Raji cells were plated and treated with DMSO or Z3 at indicated concentrations and times. The number of viable cells was determined by trypan blue exclusion using a hemocytometer.[1]
For cell cycle analysis, HEL cells treated with DMSO or 25 μmol/L Z3 for various times (16-72 h) were stained using a commercial DNA staining kit. The DNA content of the cells was then analyzed by flow cytometry to determine the distribution of cells in different phases of the cell cycle (G1, S, G2/M).[1]
For the ex vivo colony formation assay, mononuclear cells isolated from patient bone marrow aspirates were cultured in a semisolid methylcellulose-based medium containing specific hematopoietic cytokines (stem cell factor, interleukin-3, with or without thrombopoietin or erythropoietin). Z3 (25 μmol/L) or DMSO was added at the initiation of culture. Cultures were incubated for 14 days, after which the number of hematopoietic colonies was counted.[1]

Cytotoxicity was assessed in BSC-40 cells. Cells were treated with DMSO or Z3 at concentrations of 25, 100, or 250 μmol/L for 16 hours. Viable cells were then stained with propidium iodide, which can enter and stain necrotic/damaged cells. Cell staining and morphology were observed using fluorescence microscopy and phase contrast microscopy. Treatment with Z3 at 25 or 100 μmol/L did not increase propidium iodide staining compared to the DMSO control group, indicating that Z3 was not cytotoxic at these concentrations. A significant increase in staining was observed at 250 μmol/L, indicating cytotoxicity at this high dose. [1]

[1]. Z3, a novel Jak2 tyrosine kinase small-molecule inhibitor that suppresses Jak2-mediated pathologic cell growth . Mol Cancer Ther 2008;7(8):2308-18.

[2]. Jacqueline Sayyah, Peter P. Sayeski. Jak2 inhibitors: Rationale and role as therapeutic agents in hematologic malignancies. Current Oncology Reports. 2009, 11(2): 117-124.

[3]. Ehab Atallah , Srdan Verstovsek . Prospect of JAK2 inhibitor therapy in myeloproliferative neoplasms. Expert Review of Anticancer Therapy. 2009,9 (5):663-670.

Z3 was identified using a structure-based virtual screening method using the DOCK program. Predictions showed that it could bind to the solvent-accessible pocket near the ATP-binding site in the mouse Jak2 kinase domain. [1]
The Jak2-V617F mutation is a dominant pathogenic allele found in most patients with polycythemia vera, as well as a significant proportion of patients with essential thrombocythemia and essential myelofibrosis. It leads to constitutive activation of Jak2 and its downstream pathways (such as STAT), thereby promoting cytokine-independent cell proliferation. [1]
This study identifies Z3 as a novel Jak2 tyrosine kinase-specific inhibitor with the potential to serve as a research tool and a potential therapeutic agent for Jak2-driven myeloproliferative disorders. [1]

C23H24N2O

344.449465751648

344.188

195371-52-9

238484

Colorless to light yellow ointment

1.1±0.1 g/cm3

520.4±50.0 °C at 760 mmHg

263.0±36.5 °C

0.0±1.4 mmHg at 25°C

1.586

4.06

0

3

8

26

408

0

O=C(C1=CC=CC=C1)C(CCC2=NC=CC=C2)(C)CCC3=NC=CC=C3

SETYDCSRABYHSW-UHFFFAOYSA-N

InChI=1S/C23H24N2O/c1-23(15-13-20-11-5-7-17-24-20,16-14-21-12-6-8-18-25-21)22(26)19-9-3-2-4-10-19/h2-12,17-18H,13-16H2,1H3

2-methyl-1-phenyl-4-(pyridin-2-yl)-2-(2-(pyridin-2-yl)ethyl)butan-1-one

NSC 42834; NSC42834; Janus-Associated Kinase Inhibitor V; NSC-42834; Z3. JAK2 Inhibitor V.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO : ≥ 100 mg/mL (~290.32 mM)
Ethanol : ~100 mg/mL (~290.32 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.26 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 (7.26 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 (7.26 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.)