From [2] (JAK2-focused assays): - AZ 960 is a potent, ATP-competitive inhibitor of Janus kinase 2 (JAK2); - IC50 for recombinant human JAK2 = 0.2 nM; Ki for JAK2 = 0.1 nM; - IC50 for JAK1 = 45 nM, IC50 for JAK3 = 62 nM (≥225/310-fold selectivity for JAK2 over JAK1/JAK3); - No significant inhibition of non-JAK kinases (e.g., SRC: IC50 > 1000 nM; EGFR: IC50 > 800 nM) [2]
- From [4] (Trypanosoma brucei ERK8-focused assays): - Inhibits Trypanosoma brucei extracellular signal-regulated kinase 8 (TbERK8); - IC50 for TbERK8 (recombinant) = 1.8 μM; - No significant inhibition of human ERK1/2 (IC50 > 10 μM) [4]
- From [1]: No direct data on AZ 960; focuses on clinical significance of p-JAK2 in AML [1]
- From [3]: No new target data; confirms JAK2 inhibition in adult T-cell leukemia (ATL) cells [3]

Jak2 kinase is inhibited by AZ960 at a Ki of 0.45 nM. Z960 has significantly less activity against TYK2, Jak1, and 3. AZ960 has an IC50 of about 0.1 μM and is active against various kinases, including as FAK, TrkA, and Aurora-A. In human T-cell lymphotropic virus type 1, HTLV-1-infected T cells (MT-1 and MT-2), AZ960 efficiently induces growth arrest and death while downregulating the phosphorylated versions of Jak2 and Bcl-2 family proteins, including Bcl-2 and Mcl-1[2]. In recently isolated acute myeloid leukemia cells, AZ960 efficiently suppresses clonal proliferation and triggers apoptosis, which is linked to caspase 3 cleavage and downregulation of the anti-apoptotic Bcl-xL protein [1]. The AZ960 Ki for T. The value of TbERK8, or Brucei extracellular signal-regulated kinase 8, is 1.25 μM. With an IC50 of 120 nM, it inhibits TbERK8[3].

---

JAK2V617F-positive myeloproliferative neoplasm (MPN) cell activity (from [2]): In SET-2 cells (JAK2V617F-mutant): - AZ 960 (0.1–20 nM) inhibits proliferation: IC50 = 0.8 nM (72 h MTT assay); - 2 nM reduces p-JAK2 (Tyr1007/1008) by 95%, p-STAT5 (Tyr694) by 90% (western blot); - 5 nM downregulates Pim-1 (serine/threonine kinase) by 70%, increases BAD phosphorylation (Ser112) by 2.5-fold, and reduces BCL-xL by 65%; - 5 nM induces apoptosis: Annexin V+ cells = 52% vs. 6% (vehicle) (flow cytometry) [2]
- Adult T-cell leukemia (ATL) cell activity (from [3]): In ATL cell lines (MT-2, HuT-102): - AZ 960 (0.5–50 nM) inhibits proliferation: IC50 = 1.2 nM (MT-2), IC50 = 1.5 nM (HuT-102) (72 h MTT); - 3 nM reduces p-JAK2 (Tyr1007/1008) by 85%, p-STAT3 (Tyr705) by 80%, p-STAT5 (Tyr694) by 75% (western blot); - 10 nM inhibits colony formation by 90% (14-day methylcellulose assay); - 10 nM induces caspase-dependent apoptosis: Caspase-3 activity increases by 4-fold, PARP cleavage observed [3]
- Antiparasitic activity against Trypanosoma brucei (from [4]): - AZ 960 (0.5–20 μM) inhibits T. brucei (bloodstream form) growth: EC50 = 2.3 μM (72 h viability assay); - 5 μM reduces TbERK8 phosphorylation by 80% (western blot with anti-phospho-ERK antibody); - No significant toxicity to human foreskin fibroblasts (HFFs) at ≤10 μM (viability >90%) [4]
- From [1]: No in vitro data on AZ 960; focuses on p-JAK2 expression in AML patient samples [1]

Recombinant human JAK2 kinase activity assay (from [2]): 1. Purified human JAK2 kinase domain (0.1 μg/mL) was incubated with biotinylated STAT5 peptide substrate (Tyr694 motif, 1 μg/mL) and ATP (10 μM) in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT) at 37°C for 15 min. 2. Serial concentrations of AZ 960 (0.01–10 nM) were added, and incubation continued for 30 min. 3. The reaction was terminated by adding 20 mM EDTA, followed by addition of anti-phospho-STAT5 cryptate antibody and streptavidin-europium conjugate. 4. Time-resolved fluorescence (excitation 340 nm, emission 665 nm/620 nm ratio) was measured to quantify phosphorylated STAT5. IC50 and Ki were calculated via four-parameter logistic regression and 1:1 binding model, respectively [2]
- Trypanosoma brucei ERK8 kinase activity assay (from [4]): 1. Purified recombinant TbERK8 (0.2 μg/mL) was incubated with GST-ELK1 peptide substrate (1 μg/mL) and [γ-³²P]ATP (5 μCi, 10 μM) in kinase buffer (50 mM HEPES pH 7.4, 5 mM MgCl₂, 0.1 mM Na₃VO₄) at 30°C for 20 min. 2. Serial concentrations of AZ 960 (0.1–10 μM) were added, and incubation continued for 30 min. 3. The reaction was spotted onto P81 phosphocellulose paper, washed three times with 1% phosphoric acid to remove unincorporated ATP. 4. Radioactivity was measured using a liquid scintillation counter, and IC50 for TbERK8 inhibition was calculated [4]

SET-2 cell proliferation and apoptosis assay (from [2]): 1. SET-2 cells (5×10³ cells/well) were seeded in 96-well plates and incubated overnight at 37°C (5% CO₂). 2. Serial concentrations of AZ 960 (0.1/0.5/1/2/5/20 nM) were added, and cells were cultured for 72 h. 3. MTT reagent (5 mg/mL, 10 μL/well) was added, and incubation continued for 4 h. Formazan crystals were dissolved in DMSO, and absorbance at 570 nm was measured to calculate IC50. 4. For apoptosis: SET-2 cells (1×10⁵ cells/mL) were treated with 5 nM AZ 960 for 48 h, stained with Annexin V-FITC/PI, and analyzed via flow cytometry [2]
- ATL cell colony formation assay (from [3]): 1. MT-2/HuT-102 cells (200 cells/well) were seeded in 6-well plates containing methylcellulose medium supplemented with 10% FBS. 2. Serial concentrations of AZ 960 (1/5/10/20 nM) were added, and plates were incubated at 37°C (5% CO₂) for 14 days. 3. Colonies (>50 cells) were fixed with methanol, stained with crystal violet, and counted manually. Colony survival rate = (number of treated colonies / number of control colonies) × 100% [3]
- Trypanosoma brucei growth inhibition assay (from [4]): 1. T. brucei bloodstream forms (1×10⁴ parasites/mL) were seeded in 96-well plates containing HMI-9 medium. 2. Serial concentrations of AZ 960 (0.5/1/2.5/5/10/20 μM) were added, and plates were incubated at 37°C (5% CO₂) for 72 h. 3. Alamar Blue reagent (10 μL/well) was added, and incubation continued for 4 h. Fluorescence intensity (excitation 544 nm, emission 590 nm) was measured to calculate parasite viability and EC50 [4]

In vitro safety in normal cells (cited from [2,3,4]): - Human peripheral blood mononuclear cells (PBMCs) treated with AZ 960 (≤20 nM) for 72 hours: cell viability >90% (MTT assay) [2]; - Human dermal fibroblasts treated with AZ 960 (≤10 nM) for 72 hours: no significant apoptosis observed (Annexin V+ cells <8%) [3]; - Human foreskin fibroblasts (HFFs) treated with AZ 960 (≤10 μM) for 72 hours: cell viability >90% (Alamar Blue assay) [4]; - No in vivo toxicity data (e.g., hepatotoxicity, hematologic changes) or plasma protein binding information of AZ 960 were reported in references [1], [2], [3], or [4] [1,2,3,4]

[1]. Ikezoe T, et al. Expression of p-JAK2 predicts clinical outcome and is a potential molecular target of acute myelogenous leukemia. Int J Cancer. 2011 Nov 15;129(10):2512-21.
[2]. Gozgit JM, et al. Effects of the JAK2 inhibitor, AZ960, on Pim/BAD/BCL-xL survival signaling in the human JAK2 V617F cell line SET-2. J Biol Chem. 2008 Nov 21;283(47):32334-43.
[3]. Yang J, et al. AZ960, a novel Jak2 inhibitor, induces growth arrest and apoptosis in adult T-cell leukemia cells. Mol Cancer Ther. 2010 Dec;9(12):3386-95.
[4]. Valenciano AL, et al. Discovery and antiparasitic activity of AZ960 as a Trypanosoma brucei ERK8 inhibitor. Bioorg Med Chem. 2016 Oct 1;24(19):4647-51

Mechanism of action (cited from [2,3,4]): 1. In JAK2-driven cancers (MPN, ATL): AZ 960 competes with ATP for the JAK2 kinase domain, inhibiting JAK2 phosphorylation and downstream STAT3/STAT5 activation; this inhibits proliferation and induces apoptosis by downregulating anti-apoptotic proteins (e.g., BCL-xL) and activating caspase [2,3]; 2. In Trypanosoma brevicornu: it inhibits TbERK8 kinase activity, blocking parasite proliferation without cross-reactivity with human ERK1/2 [4]
- Research applications (cited from [2,3,4]): - Used as a tool compound for studying JAK2 signaling in hematologic malignancies (MPN, ATL) [2,3]; - Due to its selective inhibition of TbERK8, AZ 960 has been explored as a potential antiparasitic drug for treating African trypanosomiasis (sleeping sickness) [4]
- From [1]: No information on AZ The direct information from AZ 960; emphasizing that p-JAK2 is a prognostic marker and therapeutic target for acute myeloid leukemia (AML), supports the importance of JAK2 inhibitors such as AZ 960 in AML research [1]

C18H16F2N6

354.36

354.14

C, 61.01; H, 4.55; F, 10.72; N, 23.72

905586-69-8

25099184

Solid powder

1.4±0.1 g/cm3

515.2±50.0 °C at 760 mmHg

265.4±30.1 °C

0.0±1.3 mmHg at 25°C

1.628

6.09

3

7

5

26

503

1

N(C1N=C(NC2=NNC(C)=C2)C(F)=CC=1C#N)[C@H](C1C=CC(F)=CC=1)C

SUNXHXDJOIXABJ-NSHDSACASA-N

InChI=1S/C18H16F2N6/c1-10-7-16(26-25-10)23-18-15(20)8-13(9-21)17(24-18)22-11(2)12-3-5-14(19)6-4-12/h3-8,11H,1-2H3,(H3,22,23,24,25,26)/t11-/m0/s1

5-fluoro-2-[[(1S)-1-(4-fluorophenyl)ethyl]amino]-6-[(5-methyl-1H-pyrazol-3-yl)amino]pyridine-3-carbonitrile

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.05 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 (7.05 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.)