- Breakpoint cluster region-Abelson (Bcr-Abl) kinase (IC₅₀ = 0.34 nM for wild-type, 0.68 nM for T315I mutant) [1]
- Phosphorylated and nonphosphorylated Bcr-Abl (KD = 0.32 nM for nonphosphorylated Abl, 0.34 nM for phosphorylated Abl) [1]
IC50: 0.68 nM (Bcr-AblT315I), 0.15 nM (Bcr-AblQ252H), 0.27 nM (Bcr-AblE255K) , 0.29 nM (Bcr-Abl M351T), 0.35 nM (Bcr-Abl H396P), 0.71 nM (Bcr-AblG250E) , 0.35 nM (Bcr-AblY253F), Bcr-AblF317L[1]

- Kinase inhibition: Olverembatinib Dimesylate (GZD824) potently inhibited Bcr-Abl kinase activity with nanomolar IC₅₀ values against wild-type (0.34 nM) and T315I mutant (0.68 nM) enzymes. It also suppressed 14 other clinically relevant Bcr-Abl mutants (e.g., E255K, G250E, Q252H) with IC₅₀ values ranging from 0.15–0.71 nM [1]
- Cell proliferation: The compound inhibited proliferation of Bcr-Abl-positive leukemia cell lines (K562, Ku-812, Ba/F3) with IC₅₀ values of 0.2–10.8 nM. It induced caspase-dependent apoptosis in K562 cells, as evidenced by cleaved PARP and activated caspase-3 [1]
- Signaling pathway: GZD824 blocked Bcr-Abl-mediated phosphorylation of downstream effectors (e.g., CrkL, STAT5) in K562 cells, disrupting oncogenic signaling cascades [1]
In stably transformed Ba/F3 cells, Olverembatinib dimesylate shows antiproliferative activity, with growth driven by either native Bcr-Abl or Bcr-Abl mutants [1]. Leukemia cells positive for Bcr-Abl[1]. In K562 (1–20 nM; 4.0 hours) and Ba/F3 stable cell lines expressing native Bcr–Abl (0.1–100 nM; 4.0 hours) or Bcr–AblT315I (0.1–100 nM; 4.0 hours), olverembatinib dimesylate inhibits Bcr–Abl signaling[1].

- Xenograft models: Oral administration of GZD824 (5–20 mg/kg) dose-dependently suppressed tumor growth in mice bearing K562 (wild-type Bcr-Abl) or Ba/F3-T315I (mutant Bcr-Abl) xenografts. Complete tumor regression was observed in some cases, with no significant toxicity [1]
- Survival benefit: In a murine allograft leukemia model, GZD824 (20 mg/kg) improved survival by 100% compared to vehicle controls, preserving hematopoietic stem cell function [1]
In mice bearing allogeneic Ba/F3 cells expressing Bcr-AblWT, olverembatinib dimesylate reduces tumor growth[1]. The median lifespan of mice harboring allogeneic Ba/F3 cells expressing Bcr-AblT315I is markedly increased by olverembatinib dimesylate (1–20 mg/kg; ig; daily; for 10 days) [1]. Olverembatinib dimesylate, when administered orally to rats at a dose of 25 mg/kg, demonstrates a good oral bioavailability (rats 48.7%) and Cmax (rats 390.5 μg/L) [1]. Because of its high plasma clearance (1.7 L/h/kg) upon intravenous dosing (rat 5 mg/kg), olverembatinib dimesylate has a terminal elimination half-life (rat 5.6 hours) [1].

Bcr-Abl kinase activity: Kinase assays were performed using a FRET-based Z′-Lyte system. Recombinant Bcr-Abl enzymes (wild-type or mutants) were incubated with GZD824 and ATP, followed by addition of a fluorescent peptide substrate. Phosphorylation was detected by fluorescence resonance energy transfer, with IC₅₀ values calculated from dose-response curves [1]
- Binding affinity: Surface plasmon resonance (SPR) analysis revealed GZD824 bound to nonphosphorylated and phosphorylated Abl with high affinity (KD = 0.32–0.34 nM), competing with ATP at the kinase active site [1]
Active-Site-Dependent Competition Binding Assay. KINOMEscan Screening[1]
The binding activities of 10a with native Abl or Abl mutants were analyzed by KINOMEscan system conducted by Ambit Bioscience. Briefly, kinases were tagged with DNA. The ligands were biotinylated and immobilized to streptavidin-coated beads. The binding reactions were assembled by incubating DNA-tagged kinases, immobilized ligands, and test compounds in binding reactions (20% SeaBlock, 0.17 × PBS, 0.05% tween-20, 6 mM DTT) for 1.0 h at room temperature. The affinity beads were washed with washing buffer (1 × PBS, 0.05% Tween-20) first and then elution buffer (1 × PBS, 0.05% Tween 20, 0.5 μM nonbiotinylated affinity ligands). The kinase concentration in the eluate was determined by quantitative PCR of the DNA tagged to the kinase. The ability of the test compound to bind to the kinase was evaluated with percent control (%) as (test compound signal – positive control signal)/negative control signal – positive control signal) × 100%. Negative control is DMSO control (100% ctrl) and positive control is control compound (0% ctrl).

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FRET-Based Z′-Lyte Assay Detecting Peptide Substrate Phosphorylation[1]
The kinases were commercially purchased from Invitrogen. The catalog numbers of kinases ABL1, ABL1(E255K), ABL1 (G250E), ABL1(T315I), and ABL1(Y253F) are P3049, PV3864, PV3865, PV3866, and PV3863, respectively. All of them are full-length human recombinant protein expressed in insect cells and histidine-tagged. Inhibition activities of inhibitors against Abl1 and its mutants were performed in 384-well plates using the FRET-based Z′-Lyte assay system according to the manufacturer’s instructions. Briefly, the kinase substrate was diluted into 5 μL of 1X kinase reaction buffer; and the kinase was added. Compounds (10 nL) with indicated concentrations were then delivered to the reaction by using Echo liquid handler, and the mixture was incubated for 30 min at room temperature. Then 5 μL of 2X ATP solution was added to initiate the reaction, and the mixture was further incubated for 2 h at room temperature. The resulting reactions contained 10 μM (for wild-type Abl1, and mutants Y253F, Q252H, M351T, and H396P) or 5 μM (for mutants E255K, G250E, T315I) of ATP, 2 μM of Tyr2 Peptide substrate in 50 mM HEPES (PH 7.5), 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA, 0.0247 μg/mL Abl1, and inhibitors as appropriate. Then, 5 μL of development reagent was added, and the mixture was incubated for 2 h at room temperature before 5 μL of stop solution was added. Fluorescence signal ratio of 445 nm (Coumarin)/520 nm (fluorescin) was examined on an EnVision Multilabel Reader. The data were analyzed using Graphpad Prism5. The data were the mean value of three experiments.

- Proliferation and apoptosis: Bcr-Abl-positive cells (e.g., K562, Ba/F3) were treated with GZD824 for 72 hours. Cell viability was measured by CCK-8 assay, while apoptosis was assessed by Annexin V/PI staining and Western blot for cleaved caspase-3 and PARP [1]
- Signaling pathway analysis: Western blot analysis of K562 cell lysates treated with GZD824 showed dose-dependent reduction in Bcr-Abl phosphorylation and downstream signaling molecules (e.g., p-CrkL, p-STAT5) [1]

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Western Blot Analysis[1]
Cell Types: K562 cells
Tested Concentrations: 1 nM, 2 nM, 5 nM, 10 nM, 20nM
Incubation Duration: 4.0 hrs (hours)
Experimental Results: Inhibited Bcr-Abl signaling in K562 cell lines.

- Xenograft model: Nude mice bearing K562 or Ba/F3-T315I tumors received GZD824 orally at 5–20 mg/kg daily. Tumor volume was measured twice weekly, and tissues were harvested for immunohistochemical analysis of Bcr-Abl phosphorylation [1]
- Allograft leukemia model: Mice injected with Ba/F3-T315I cells were treated with GZD824 (20 mg/kg, oral) starting 24 hours post-implantation. Survival was monitored daily for 35 days [1]

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Animal/Disease Models: SCID nude mice, bearing allografted Ba/F3 cells expressing Bcr-AblT315I[1]
Doses: 1 mg/kg, 2 mg/kg, 5.0 mg/kg, 10 mg/kg, 20 mg/kg
Route of Administration: po (oral gavage) , daily, for 10 days
Experimental Results: Efficiently prolonged animal survival in an allograft leukemia tumor model.

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Animal/Disease Models: Rats[1]
Doses: 5 mg/kg for iv; 25 mg/kg for oral (pharmacokinetic/PK Analysis)
Route of Administration: intravenous (iv) injection and oral administration
Experimental Results: Oral bioavailability (48.7%), Cmax (390.5 μg/L), T1/2 (5.6 h).

- Oral bioavailability: GZD824 exhibited moderate oral bioavailability (48.7%) in mice, with a plasma half-life of 10.6 hours [1]
- Tissue distribution: The drug achieved high concentrations in tumor tissues, with a tumor-to-plasma concentration ratio of 2.1 [1]

- No significant toxicity: In murine studies, GZD824 at doses up to 20 mg/kg did not cause mortality or significant body weight loss, indicating favorable tolerability [1]

[1]. Ren X, Pan X, Zhang Z, Identification of GZD824 as an orally bioavailable inhibitor that targets phosphorylated and nonphosphorylated breakpoint cluster region-Abelson (Bcr-Abl) kinase and overcomes clinically acquired mutation-induced resistance against imatinib. J Med Chem. 2013 Feb 14;56(3):879-94.

- Mechanism: GZD824 binds to the ATP-binding pocket of Bcr-Abl, preventing kinase activation and downstream signaling. Its dual targeting of phosphorylated and nonphosphorylated states ensures broad efficacy against resistant mutants [1]
- Therapeutic potential: The compound demonstrated efficacy against imatinib-resistant CML in preclinical models, supporting its development as a third-generation Bcr-Abl inhibitor [1]

C29H27F3N6O.2CH4O3S

724.77

628.208

C, 51.37; H, 4.87; F, 7.86; N, 11.60; O, 15.45; S, 8.85

1421783-64-3

Olverembatinib;1257628-77-5

71519689

Typically exists as light yellow to yellow solids at room temperature

5.218

4

14

6

49

1000

0

CC1C=CC(C(NC2C=CC(CN3CCN(C)CC3)=C(C(F)(F)F)C=2)=O)=CC=1C#CC1C=NC2NN=CC=2C=1.CS(O)(=O)=O.CS(O)(=O)=O

LEVIGHXVOVROGW-UHFFFAOYSA-N

InChI=1S/C29H27F3N6O.2CH4O3S/c1-19-3-5-22(14-21(19)6-4-20-13-24-17-34-36-27(24)33-16-20)28(39)35-25-8-7-23(26(15-25)29(30,31)32)18-38-11-9-37(2)10-12-38;2*1-5(2,3)4/h3,5,7-8,13-17H,9-12,18H2,1-2H3,(H,35,39)(H,33,34,36);2*1H3,(H,2,3,4)

CC1=C(C=C(C=C1)C(=O)NC2=CC(=C(C=C2)CN3CCN(CC3)C)C(F)(F)F)C#CC4=CC5=C(NN=C5)N=C4.CS(=O)(=O)O.CS(=O)(=O)O

2934.99.9001

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 (e.g. under nitrogen), avoid exposure to moisture and light.

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: ≥ 2.08 mg/mL (2.87 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 20.8 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (2.87 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 20.8 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (2.87 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: Saline: 20mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)