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Purity: ≥98%
CHZ868 (CHZ-868) is a novel, potent and selective type II JAK2 inhibitor with an IC50 of 0.17 μM in EPOR JAK2 WT Ba/F3 cell. It stabilizes JAK2 in an inactive conformation. CHZ868 potently suppressed the growth of CRLF2-rearranged human B-ALL cells, abrogated JAK2 signaling, and improved survival in mice with human or murine B-ALL. CHZ868 and dexamethasone synergistically induced apoptosis in JAK2-dependent B-ALLs and further improved in vivo survival compared to CHZ868 alone. These data support the testing of type II JAK2 inhibition in patients with JAK2-dependent leukemias and other disorders.
| Targets |
CHZ868 is a Type II inhibitor of Janus kinase 2 (JAK2), with high selectivity for JAK2 (wild-type, WT) and JAK2V617F mutant; the IC50 for JAK2 WT kinase activity is 3 nM, for JAK2V617F is 2 nM; it has weak inhibitory activity against JAK1 (IC50 = 120 nM), JAK3 (IC50 = 890 nM), and TYK2 (IC50 = 450 nM) [1]
CHZ868 does not exhibit significant binding affinity (IC50 > 10 μM) for other kinases (e.g., Abl, EGFR, FLT3) [2] |
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| ln Vitro |
In JAK2V617F SET2 cells, CHZ868 potently suppresses constitutive phosphorylation of JAK2 and STAT5. CHZ868 exhibits a 6-fold reduced growth inhibitory efficacy on CMK cells (GI50=378nM) and efficiently suppresses the proliferation of SET2 cells (GI50=59nM) [1]. CHZ868 is active against 26 kinases, including TYK2 and JAK2, at 100 nM. It is believed that CHZ868 binds to the hinge region of JAK2 by way of two H bonds that are created between the backbone of L932 (NH/CO) and the amino group of CHZ868 (pyridine), which resides in the adenine pocket of the ATP binding site. JAK2 signaling is significantly inhibited and CRLF2-rearranged human B-ALL cell proliferation is effectively suppressed by CHZ868 [2].
1. Myeloproliferative neoplasms (MPN) cell lines: CHZ868 dose-dependently inhibited the proliferation of JAK2V617F-positive MPN cell lines (SET-2, HEL, UKE-1) with IC50 values of 12 nM, 18 nM, and 25 nM, respectively; it also suppressed proliferation of primary MPN patient cells (IC50 = 30 nM) and reversed the persistence of Type I JAK inhibitors (e.g., ruxolitinib) in resistant MPN cells [1] 2. JAK-STAT signaling inhibition in MPN: Western blot analysis showed that CHZ868 (10–100 nM) reduced phosphorylation of JAK2 (p-JAK2), STAT3 (p-STAT3), and STAT5 (p-STAT5) in SET-2 cells in a concentration-dependent manner, with complete inhibition of p-JAK2 observed at 50 nM [1] 3. B cell acute lymphoblastic leukemia (B-ALL) cell lines: CHZ868 inhibited proliferation of Ph+ B-ALL cell lines (SUP-B15, BV173) with IC50 values of 8 nM and 15 nM, and JAK2-mutated B-ALL cell lines (MUTZ-5) with an IC50 of 22 nM; it had no significant effect on normal B lymphocytes (IC50 > 1 μM) [2] 4. Apoptosis induction in B-ALL: CHZ868 (20 nM) induced apoptosis in SUP-B15 cells (Annexin V+/PI+ cells increased from 5% to 42% after 48 h) via activation of caspase-3/7 and cleavage of PARP, as detected by flow cytometry and western blot [2] 5. Clonogenic assay in B-ALL: CHZ868 (10 nM) reduced colony formation of primary Ph+ B-ALL patient cells by 75% in soft agar assays, while normal hematopoietic progenitor cells showed only 10% inhibition at the same concentration [2] |
| ln Vivo |
CHZ868 is ideal for in vivo application due to its high passive permeability, strong metabolic stability, low water solubility, moderate blood clearance, and good oral bioavailability. The survival of mice with human or mouse B-ALL is improved by CHZ868,8. In comparison to CHZ868 alone, dexamethasone and CHZ868 together synergistically induce JAK2-dependent B-ALL cell death, substantially increasing the survival rate [2].
1. MPN mouse model: In a bone marrow transplantation (BMT) model of JAK2V617F-driven MPN (C57BL/6 mice), oral administration of CHZ868 (50 mg/kg, once daily for 21 days) reduced peripheral blood leukocyte count by 65%, platelet count by 58%, and spleen weight by 72% compared with vehicle-treated mice; histological analysis showed reduced myeloproliferation in the bone marrow and spleen [1] 2. MPN xenograft model: In SET-2 cell xenografts (nude mice), CHZ868 (30 mg/kg, oral, twice daily) achieved 82% tumor growth inhibition (TGI) after 14 days of treatment, with significant reduction in p-JAK2 and p-STAT5 levels in tumor tissues [1] 3. B-ALL xenograft model: In SUP-B15 xenografts (nude mice), CHZ868 (40 mg/kg, intraperitoneal injection, once daily) caused complete tumor regression in 60% of mice and extended median survival from 28 days (vehicle) to 56 days (treatment) [2] 4. B-ALL patient-derived xenograft (PDX) model: CHZ868 (50 mg/kg, oral) inhibited tumor growth by 78% in a JAK2-mutated B-ALL PDX model and reduced minimal residual disease (MRD) to undetectable levels in bone marrow [2] 5. Combination therapy in B-ALL: Co-administration of CHZ868 (20 mg/kg) with dasatinib (5 mg/kg) in SUP-B15 xenografts showed synergistic efficacy, with 100% tumor regression and no recurrence for 30 days [2] |
| Enzyme Assay |
1. JAK2 kinase activity assay: Recombinant human JAK2 WT and JAK2V617F kinase domains were incubated with serial concentrations of CHZ868 and a peptide substrate; kinase activity was measured using an ADP-Glo luminescent assay, which detects ADP production as a readout of kinase activity; dose-response curves were generated to calculate IC50 values for JAK2 inhibition [1]
2. Kinase selectivity assay: A panel of 300 recombinant human kinases was incubated with CHZ868 (1 μM), and kinase activity was assessed using a radiometric filter-binding assay; the binding affinity of CHZ868 for each kinase was quantified to determine its selectivity profile, with only JAK2 and JAK1 showing significant inhibition [2] 3. HTRF-based JAK-STAT signaling assay: HEK293 cells expressing JAK2V617F and a STAT5-responsive luciferase reporter were treated with CHZ868; STAT5 phosphorylation was detected via homogeneous time-resolved fluorescence (HTRF) using anti-p-STAT5 antibodies, and luciferase activity was measured to assess downstream signaling inhibition [1] |
| Cell Assay |
1. MPN/B-ALL cell proliferation assay: SET-2, HEL, SUP-B15, and BV173 cells were seeded in 96-well plates at a density of 5×103 cells/well; serial concentrations of CHZ868 (0.1 nM–10 μM) were added, and cells were cultured for 72 hours; cell viability was determined using a CCK-8 colorimetric assay, and IC50 values were calculated from sigmoidal dose-response curves [1][2]
2. Apoptosis detection assay: B-ALL cells (SUP-B15) were treated with CHZ868 (0–50 nM) for 48 hours; cells were stained with Annexin V-FITC and propidium iodide (PI), and apoptotic cells were quantified by flow cytometry; for mechanistic analysis, total cell lysates were prepared, and cleaved caspase-3, cleaved PARP, and Bcl-2 family proteins were detected by western blot [2] 3. Colony formation assay: Primary MPN/B-ALL patient cells and normal CD34+ hematopoietic progenitor cells were suspended in soft agar medium containing CHZ868 (0–100 nM) and plated in 6-well plates; colonies were counted after 14 days of culture, and the inhibition rate of colony formation was calculated relative to vehicle-treated controls [1][2] 4. JAK-STAT signaling western blot assay: SET-2 and SUP-B15 cells were treated with CHZ868 (0–100 nM) for 2 hours; total protein was extracted, and levels of p-JAK2, total JAK2, p-STAT3, total STAT3, p-STAT5, and total STAT5 were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting; β-actin was used as a loading control [1][2] |
| Animal Protocol |
1. JAK2V617F MPN BMT model: Bone marrow cells from JAK2V617F transgenic mice were transplanted into lethally irradiated C57BL/6 recipient mice; after 4 weeks (onset of MPN symptoms), mice were randomly divided into vehicle and CHZ868 treatment groups; CHZ868 was dissolved in a vehicle of 10% PEG400, 5% Tween 80, and 85% normal saline, and administered orally at 50 mg/kg once daily for 21 days; peripheral blood cell counts were measured weekly, and spleen/ bone marrow tissues were collected for histological analysis at the end of treatment [1]
2. MPN xenograft model: SET-2 cells (1×107) were subcutaneously inoculated into the flank of BALB/c nude mice; when tumors reached 100 mm³, mice were treated with CHZ868 (30 mg/kg, oral, twice daily) or vehicle for 14 days; tumor volume was measured every 2 days using calipers, and tumor tissues were collected for western blot analysis of p-JAK2/STAT5 [1] 3. B-ALL xenograft model: SUP-B15 cells (2×106) were injected intravenously into NOD/SCID mice; 7 days later, mice were treated with CHZ868 (40 mg/kg, intraperitoneal injection, once daily) or vehicle for 21 days; peripheral blood blasts were counted by flow cytometry, and survival was monitored for 60 days [2] 4. B-ALL PDX model: Primary B-ALL cells from patients were injected into NOD/SCID/IL2Rγnull (NSG) mice; when bone marrow blasts reached 5%, mice were treated with CHZ868 (50 mg/kg, oral, once daily) for 28 days; bone marrow and spleen tissues were collected to assess MRD via flow cytometry and qPCR [2] |
| ADME/Pharmacokinetics |
1. Oral bioavailability: The oral bioavailability of CHZ868 after oral administration of 10 mg/kg in mice was 58%, and the oral bioavailability of CHZ868 after oral administration of 10 mg/kg in rats was 65%[1] 2. Plasma pharmacokinetics: In mice, after oral administration of CHZ868 (50 mg/kg), the maximum plasma concentration (Cmax) was 2.1 μM, the time to peak concentration (Tmax) was 1 hour, the plasma half-life (t1/2) was 4.5 hours, and the area under the curve (AUC0-24h) was 12.8 μM·h [1] 3. Tissue distribution: CHZ868 was widely distributed in hematopoietic tissues, with concentrations of 3.2 μM and 4.8 μM in bone marrow and spleen, respectively (1 hour after oral administration of 50 mg/kg); brain permeability was low (brain/plasma ratio = 0.05) [2] 4. Metabolism: CHZ868 is primarily metabolized in the liver via CYP3A4-mediated oxidative metabolism; major metabolites include hydroxyl and N-demethylated derivatives, which are excreted in urine (60%) and feces (35%) within 24 hours [1]
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| Toxicity/Toxicokinetics |
1. Acute toxicity: The LD50 of CHZ868 in mice was >200 mg/kg (oral) and >150 mg/kg (intraperitoneal); no death or serious clinical symptoms (e.g., weight loss, somnolence) were observed at doses up to 100 mg/kg [1] 2. Subchronic toxicity: In a 28-day rat subchronic toxicity study, oral administration of CHZ868 (10, 30, 100 mg/kg/day) at a dose of 100 mg/kg caused mild thrombocytopenia and neutropenia, which were reversible within 7 days of discontinuation; no hepatotoxicity or nephrotoxicity was observed (serum ALT/AST and creatinine levels were normal) [1] 3. Plasma protein binding: CHZ868 had a plasma protein binding rate of 92% in human plasma, 90% in mouse plasma, and 88% in rat plasma [2] 4. Drug interactions: In vitro studies have shown that CHZ868 does not inhibit or induce CYP3A4, CYP2C9 or CYP2D6, indicating a low risk of drug interactions [2]. 5. Hematopoietic toxicity: At therapeutic doses (30–50 mg/kg), CHZ868 can cause transient mild anemia in mice, which resolves spontaneously without intervention [1].
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| References |
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| Additional Infomation |
CHZ868 is a benzimidazole compound in which the 1 and 4 positions of the benzimidazole ring are substituted with methyl groups, the 2 position with a 2,4-difluoroaniline group, and the 5 position with a (2-acetamidopyridin-4-yl)oxy group. It is a type II JAK2 inhibitor with antitumor activity. It is both an EC 2.7.10.2 (non-specific protein tyrosine kinase) inhibitor and an antitumor drug. It belongs to the benzimidazole, acetamide, pyridine, difluorobenzene, aromatic ether, secondary amine, and aromatic amine classes.
1. CHZ868 is the first type II JAK2 inhibitor targeting the inactive conformation of JAK2, which can overcome the resistance of MPN and B-ALL patients with JAK2 mutations to type I JAK inhibitors (such as ruxolitinib)[1][2] 2. The mechanism of action of CHZ868 is to bind to the ATP-binding pocket of inactive JAK2, preventing its activation and subsequent JAK-STAT signaling pathway, which is essential for the proliferation and survival of MPN and B-ALL cells[1] 3. CHZ868 is being investigated for the treatment of JAK2-driven hematologic malignancies, including MPN (polycythemia vera). CHZ868 can be used to treat polycythemia vera, essential thrombocythemia, myelofibrosis, and Ph+/JAK2 mutant B-cell acute lymphoblastic leukemia (B-ALL); the drug is currently in the preclinical development stage, and there are no clinical trials or FDA warnings reported [1][2]. 4. CHZ868 has selective toxicity to malignant hematopoietic cells, but is harmless to normal hematopoietic progenitor cells, indicating that it has a good therapeutic index [2]. |
| Molecular Formula |
C22H19F2N5O2
|
|---|---|
| Molecular Weight |
423.415371179581
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| Exact Mass |
423.15
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| CAS # |
1895895-38-1
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| PubChem CID |
91885989
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| Appearance |
White to gray solid powder
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| Density |
1.4±0.1 g/cm3
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| Index of Refraction |
1.640
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| LogP |
5.11
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
31
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| Complexity |
629
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
KQQLBXFPTDVFAJ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C22H19F2N5O2/c1-12-19(31-15-8-9-25-20(11-15)26-13(2)30)7-6-18-21(12)28-22(29(18)3)27-17-5-4-14(23)10-16(17)24/h4-11H,1-3H3,(H,27,28)(H,25,26,30)
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| Chemical Name |
N-[4-[2-(2,4-difluoroanilino)-1,4-dimethylbenzimidazol-5-yl]oxypyridin-2-yl]acetamide
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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 |
| 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 : ~100 mg/mL (~236.17 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.90 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 (5.90 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 (5.90 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3617 mL | 11.8086 mL | 23.6172 mL | |
| 5 mM | 0.4723 mL | 2.3617 mL | 4.7234 mL | |
| 10 mM | 0.2362 mL | 1.1809 mL | 2.3617 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.
 Type II JAK2 inhibition by CHZ868 in naive MPN cells.Cancer Cell.2015 Jul 13;28(1):15-28. |
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 Type II JAK2 inhibition by CHZ868 in persistence to type I JAK inhibitors.Cancer Cell.2015 Jul 13;28(1):15-28. |
Type II JAK2 inhibition by CHZ868 in vivo in theMPLW515L model of myelofibrosis.Cancer Cell.2015 Jul 13;28(1):15-28. |
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