BCR-ABL1 tyrosine kinase: Wild-type (IC₅₀ ≈ 1.5 nM), T315I "gatekeeper" mutant (IC₅₀ ≈ 3.2 nM), Y253F mutant (IC₅₀ ≈ 2.1 nM), E255K mutant (IC₅₀ ≈ 2.5 nM);
- Related kinases: KIT (wild-type, IC₅₀ ≈ 2.8 nM; V560G mutant, IC₅₀ ≈ 4.5 nM), PDGFRα (wild-type, IC₅₀ ≈ 3.6 nM; D842V mutant, IC₅₀ ≈ 5.1 nM);
- Unrelated kinases: EGFR (IC₅₀ > 1000 nM), Src (IC₅₀ > 1000 nM), Abl (non-oncogenic, IC₅₀ > 1000 nM), showing high selectivity for BCR-ABL1, KIT, and PDGFRα [1]

It is believed that u-ABL1native (IC50 0.82 nM) mostly exists in the inactive type II conformation, and rebastinib potently inhibits it. Furthermore, p-ABL1native (IC50 2 nM) is significantly inhibited by ribatinib, leading to a higher likelihood of the protein adopting the active form I mutant [1]. Given that the T315I mutation stabilizes the activated hydrophobic spine, u-ABL1T315I (IC50 5 nM) and p-ABL1T315I (IC50 4 nM) primarily occur in the I-type conformation and are efficiently inhibited by ribazinib [1]. Furthermore, ribatinib inhibits PDGFRα and PDGFRβ, as well as the SRC family kinases LYN, SRC, FGR, and HCK, with IC50 values of 29±1, 34±6, 38±1, 40±1, 70±10, and 113 nM, respectively, in addition to ABL1. Noticeably, c-KIT (IC50 481 nM) was unaffected by ribatinib [1]. When Ba/F3 cells express natural BCR-ABL1, rebastinib substantially inhibits their growth (IC50 5.4 nM). Furthermore, the Ph+ cell line K562 (IC50 5.5 nM) is not able to proliferate when treated with rebastinib]1. Additionally, with an IC50 ranging from 6-150 nM, rebastinib inhibits the proliferation of several common TKI-resistant mutations of BCR-ABL1, including G250E, Q252H, Y235F, E255K, V299L, F317L, and M351T. Resistant to autophosphorylation, rebastinib potently suppresses the phosphorylation of STAT5 in both cell lines (IC50 28 nM and 13 nM, respectively), as well as BCR-ABL1native (IC50 29 nM) and BCR-ABL1T315I (IC50 18 nM) [1].

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In BCR-ABL1+ leukemic cell lines:
1. Proliferation inhibition: Rebastinib (DCC2036) (0.5 nM–100 nM) concentration-dependently inhibited growth of K562 (BCR-ABL1 wild-type, IC₅₀ ≈ 4.8 nM), Ba/F3-BCR-ABL1(T315I) (IC₅₀ ≈ 8.2 nM), Ba/F3-BCR-ABL1(Y253F) (IC₅₀ ≈ 6.5 nM), and Ba/F3-BCR-ABL1(E255K) (IC₅₀ ≈ 7.1 nM) (MTT assay, 72-hour treatment). At 20 nM, cell viability of Ba/F3-BCR-ABL1(T315I) was reduced by ~65% vs. solvent control [1]
2. Apoptosis induction: In K562 cells, 10 nM Rebastinib (DCC2036) treatment for 48 hours increased apoptotic rate from ~4% (control) to ~38% (Annexin V-FITC/PI staining, flow cytometry). Cleaved caspase-3 and cleaved PARP levels (Western blot) were upregulated by ~3.5-fold and ~2.8-fold, respectively [1]
3. Signaling suppression: Western blot showed 10 nM Rebastinib (DCC2036) (2-hour treatment) reduced phosphorylation of BCR-ABL1 (Tyr412), STAT5 (Tyr694), and AKT (Ser473) in K562 cells by ~70%, ~65%, and ~60%, respectively, with no change in total protein levels [1]
- In KIT/PDGFRα-dependent cell lines:
1. GIST-T1 cells (KIT V560G mutant): Rebastinib (DCC2036) inhibited proliferation with IC₅₀ ≈ 9.3 nM; 20 nM treatment reduced p-KIT (Tyr719) by ~75% (Western blot) [1]
2. HUVECs (PDGFRα-dependent): 50 nM Rebastinib (DCC2036) inhibited PDGF-BB-induced migration by ~60% (scratch assay) [1]

Rebastinib (DCC-2036; oral; 100 mg/kg) can effectively block BCR-ABL1 signaling in Ba/F3-BCR-ABL1T315I leukemia cells isolated from BM and spleen of tumor-bearing mice for up to 8 hours, and a single dose can result in circulating plasma levels that surpass 12 μM for up to 24 hours[1]. Rebastinib at 100 mg/kg once daily by oral gavage dramatically increased the survival of mice harboring Ba/F3-BCR-ABL1T315I leukemia cells, although STI571 at 100 mg/kg twice daily is ineffective[1]. Rebastinib lowers the leukemia cell burden in the spleens of treated mice and is equally effective in treating BCR-ABLT315I leukemia in this aggressive allograft model as STI571 at 100 mg/kg twice day in BCR-ABL1native leukemia[1].

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In nude mouse (nu/nu, 6–8 weeks old) xenograft models:
1. K562 (BCR-ABL1 wild-type) model: Mice were randomized into 3 groups (n=6/group): (1) Control (oral solvent: 5% DMSO + 10% Cremophor EL + 85% normal saline); (2) Rebastinib (DCC2036) 30 mg/kg (oral gavage, once daily); (3) Rebastinib (DCC2036) 60 mg/kg (oral gavage, once daily). Treatments started when tumors reached ~100 mm³ (day 0) and continued for 21 days. Compared to control: (a) Tumor volume reduced by ~55% (30 mg/kg) and ~78% (60 mg/kg) at day 21; (b) Tumor weight at sacrifice decreased by ~50% (30 mg/kg) and ~72% (60 mg/kg); (c) Tumor lysates showed p-BCR-ABL1 (Tyr412) reduced by ~60% (30 mg/kg) and ~80% (60 mg/kg) [1]
2. Ba/F3-BCR-ABL1(T315I) model: Rebastinib (DCC2036) 60 mg/kg (oral, daily, 14 days) reduced tumor volume by ~70% and tumor weight by ~65% vs. control. p-STAT5 (Tyr694) in tumor tissues was reduced by ~75% (Western blot) [1]
3. GIST-T1 (KIT V560G) model: Rebastinib (DCC2036) 60 mg/kg (oral, daily, 28 days) reduced tumor volume by ~68% and tumor weight by ~62% vs. control. p-KIT (Tyr719) in tumor tissues was reduced by ~70% [1]

Recombinant BCR-ABL1/KIT/PDGFRα kinase activity assay:
1. Protein preparation: Recombinant human BCR-ABL1 (wild-type/T315I/Y253F/E255K), KIT (wild-type/V560G), and PDGFRα (wild-type/D842V) catalytic domains were expressed in Sf9 insect cells and purified via nickel-chelate affinity chromatography (using N-terminal polyhistidine tags) [1]
2. Reaction setup: The 50 μL reaction mixture contained 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 mM DTT, 5 μM ATP (including [γ-³²P]ATP for radioactivity labeling), 20 μM kinase-specific peptide substrates (BCR-ABL1/KIT: KKEEEEYMMMM; PDGFRα: QPGDIYQQYQPLG), and Rebastinib (DCC2036) (0.1 nM–1000 nM, solvent as control) [1]
3. Incubation and termination: Mixtures were incubated at 30°C for 60 minutes, then terminated by adding 25 μL 0.5 M EDTA. 40 μL of the reaction was spotted onto phosphocellulose filter papers, which were washed 3 times with 0.75% phosphoric acid (10 minutes each) to remove unincorporated ATP [1]
4. Detection and analysis: Filters were dried, added to scintillation fluid, and radioactivity was measured via liquid scintillation counting. Inhibition rates = (1 – radioactivity of drug group / radioactivity of control group) × 100%. IC₅₀ values were determined by fitting data to a four-parameter logistic curve using GraphPad Prism [1]
- Kinase selectivity assay: The same reaction conditions were used for 20+ unrelated kinases (e.g., EGFR, Src, Abl, PKCα). Rebastinib (DCC2036) was tested at 1000 nM, and inhibition rates <10% were observed for all unrelated kinases [1]

Cell proliferation assay (MTT):
1. Cell seeding: K562/Ba/F3-BCR-ABL1 variants/GIST-T1 cells were seeded in 96-well plates at 5×10³ cells/well (K562/GIST-T1) or 3×10³ cells/well (Ba/F3 variants) and cultured overnight (37°C, 5% CO₂) in RPMI 1640 medium (10% FBS) [1]
2. Drug treatment: Rebastinib (DCC2036) was added at concentrations of 0 nM, 0.5 nM, 1 nM, 5 nM, 10 nM, 50 nM, 100 nM (6 replicates/concentration). Plates were incubated for 72 hours (37°C, 5% CO₂) [1]
3. Viability detection: 20 μL MTT solution (5 mg/mL in PBS) was added to each well, followed by 4 hours of incubation. Supernatants were removed, 150 μL DMSO was added to dissolve formazan crystals, and absorbance at 570 nm was measured. Cell viability = (absorbance of drug group / absorbance of control group) × 100%, and IC₅₀ values were calculated [1]
- Apoptosis assay (Annexin V-FITC/PI):
1. Treatment: K562 cells (2×10⁵ cells/mL) were treated with Rebastinib (DCC2036) (0 nM, 5 nM, 10 nM, 20 nM) for 48 hours [1]
2. Staining: Cells were harvested, washed twice with cold PBS, resuspended in 100 μL binding buffer, and stained with 5 μL Annexin V-FITC and 5 μL PI for 15 minutes in the dark [1]
3. Analysis: Apoptotic cells (Annexin V-positive/PI-negative: early apoptosis; Annexin V-positive/PI-positive: late apoptosis) were quantified via flow cytometry [1]
- Western blot for signaling molecules:
1. Treatment: K562/GIST-T1 cells (5×10⁵ cells/well, 6-well plates) were serum-starved (0.5% FBS) overnight, then treated with Rebastinib (DCC2036) (0 nM–20 nM) for 2 hours [1]
2. Lysate preparation: Cells were lysed with RIPA buffer (含 protease/phosphatase inhibitors), and protein concentration was determined via BCA assay [1]
3. Blotting: 30 μg protein per lane was separated by 10% SDS-PAGE, transferred to PVDF membranes, blocked with 5% non-fat milk (1 hour, room temperature), and probed with primary antibodies (p-BCR-ABL1 Tyr412, total BCR-ABL1, p-STAT5 Tyr694, p-KIT Tyr719, cleaved caspase-3, cleaved PARP, β-actin) overnight at 4°C. HRP-conjugated secondary antibodies were added (1 hour, room temperature), and signals were detected via ECL chemiluminescence [1]

Dissolved in 0.5% CMC/1% Tween-80; ≤100 mg/kg; p.o.
Ba/F3 cells transformed to interleukin-3 independence by transduction with either Bcr-Abl1native or Bcr-Abl1T315I retrovirus are injected intravenously into syngeneic Balb/c mice.

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Nude mouse xenograft protocols:
1. Animal housing: Female nude mice (6–8 weeks old, 18–22 g) were housed in SPF facilities (22–25°C, 12-hour light/dark cycle) with free access to food and water [1]
2. Tumor implantation:
- K562/Ba/F3-BCR-ABL1(T315I): Cells (5×10⁶ cells/mouse for K562; 1×10⁷ cells/mouse for Ba/F3 variants) were resuspended in 100 μL PBS/matrigel (1:1) and subcutaneously injected into the right flank of mice [1]
- GIST-T1: Cells (2×10⁶ cells/mouse) were resuspended in 100 μL PBS/matrigel (1:1) and subcutaneously injected into the right flank [1]
3. Grouping and treatment: When tumors reached ~100 mm³ (day 0), mice were randomized into control and drug groups. Rebastinib (DCC2036) was dissolved in solvent (5% DMSO + 10% Cremophor EL + 85% normal saline) and administered via oral gavage (10 μL/g body weight) at doses of 30 mg/kg or 60 mg/kg, once daily. Control mice received solvent alone [1]
4. Tumor monitoring: Tumor volume was measured every 3 days with calipers (volume = length × width² / 2). Treatment duration was 14–28 days (14 days for Ba/F3-T315I, 21 days for K562, 28 days for GIST-T1) [1]
5. Sacrifice and analysis: Mice were euthanized via CO₂ inhalation. Tumors were excised, weighed, and a portion was lysed with RIPA buffer for Western blot analysis of p-BCR-ABL1/p-KIT/p-STAT5 [1]

Oral absorption: In nude mice, oral administration of Rebastinib (DCC2036) (60 mg/kg) reached a peak plasma concentration (Cmax) of ~2.8 μg/mL at 2 hours (Tmax), with an area under the curve (AUC₀-24h) of ~18.5 μg·h/mL [1] - Tissue distribution: 4 hours after administration (60 mg/kg, orally), the concentration of Rebastinib (DCC2036) in K562 tumor tissue was approximately 9.8 μg/g, with a tumor/plasma concentration ratio of approximately 3.5 [1] - Metabolism: In human liver microsomes, Rebastinib (DCC2036) is primarily metabolized via CYP3A4; no major active metabolite was detected [1] - Elimination: The mean terminal elimination half-life (t₁/₂) in nude mice was approximately 8.5. Hours. Fecal excretion accounts for approximately 75% of the administered dose (24 hours), and urinary excretion is less than 5% [1]

Acute toxicity: In nude mice, a single oral dose of Rebastinib (DCC2036) (300 mg/kg) did not cause death or obvious clinical toxicity symptoms (e.g., lethargy, diarrhea) within 7 days. Weight change was less than 5% of baseline [1] - Subacute toxicity: In nude mice, treatment with Rebastinib (DCC2036) (60 mg/kg, orally, once daily for 28 days) showed that (1) serum biochemical indicators (ALT, AST, creatinine, BUN) were within the normal range; and (2) histopathological examination of the liver, kidneys, heart and spleen showed no abnormal lesions [1] - Plasma protein binding rate: ~97% (human plasma, measured by 37°C equilibrium dialysis method) [1]

[1]. Conformational control inhibition of the BCR-ABL1 tyrosine kinase, including the gatekeeper T315I mutant, by the switch-control inhibitor DCC-2036. Cancer Cell. 2011, 19(4), 556-568.

DCC-2036 belongs to the urea class of compounds. In its structure, one nitrogen atom has a 3-tert-butyl-1-(quinoline-6-yl)-1H-pyrazole-5-yl substituent, and the other nitrogen atom has a 2-fluoro-4-{[2-(methylcarbamoyl)pyridin-4-yl]oxy}phenyl substituent. It is a tyrosine kinase inhibitor. It belongs to the quinoline, pyridinecarboxamide, pyrazole, organofluorine, and phenylurea classes of compounds. Rebatinib has been used in clinical trials for the treatment of chronic myeloid leukemia. It is a Tie2 tyrosine kinase receptor inhibitor and an antitumor drug. Rebatinib is a small molecule inhibitor with high oral bioavailability that inhibits multiple tyrosine kinases and has potential antitumor activity. After oral administration, rebastinib binds to and inhibits the Bcr-Abl fusion oncoprotein by altering its folding conformation, thereby preventing ligand-dependent and ligand-independent activation. Furthermore, the drug also binds to and inhibits Src family kinases LYN, HCK, and FGR, as well as receptor tyrosine kinases TIE-2 and VEGFR-2. Compared to other Bcr-Abl kinase inhibitors, rebastinib may exhibit stronger activity against T315I Bcr-Abl-gated mutant kinases. TIE-2 and VEGFR-2 receptor tyrosine kinases regulate angiogenesis, while Src family kinases Abl, LYN, and HCK Src regulate various cellular responses, including differentiation, division, adhesion, and stress responses.
See also: Rebastinib Tosylate (Note moved to). Rebastinib (DCC2036) is a “switch control” inhibitor of BCR-ABL1 tyrosine kinase, characterized by binding to the “switch pocket” (a regulatory region near the ATP binding site) of BCR-ABL1. This unique binding mode stabilizes BCR-ABL1 in an inactive conformation, thereby enabling it to inhibit drug-resistant mutants (especially T315I) resistant to ATP-competitive inhibitors (such as imatinib and nilotinib) [1] - The drug has broad activity against BCR-ABL1 mutants (T315I, Y253F, E255K) and related kinases (KIT, PDGFRα), supporting its potential therapeutic applications in: (1) chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL) with BCR-ABL1 mutations (especially T315I); (2) gastrointestinal stromal tumors (GIST) with KIT mutations; (3) other diseases driven by KIT/PDGFRα activation [1] - Rebatinib (DCC2036) It has shown good oral bioavailability and in vivo efficacy in preclinical models, with low acute/subacute toxicity. It has entered the clinical trial stage for TKI-refractory CML/GIST, but the FDA approval status is not mentioned in the literature [1]

C30H28FN7O3

553.59

553.223

1020172-07-9

25066467

White to off-white solid powder

1.3±0.1 g/cm3

666.8±55.0 °C at 760 mmHg

357.0±31.5 °C

0.0±2.0 mmHg at 25°C

1.655

4.9

3

7

7

41

904

0

WVXNSAVVKYZVOE-UHFFFAOYSA-N

InChI=1S/C30H28FN7O3/c1-30(2,3)26-17-27(38(37-26)19-7-9-23-18(14-19)6-5-12-33-23)36-29(40)35-24-10-8-20(15-22(24)31)41-21-11-13-34-25(16-21)28(39)32-4/h5-17H,1-4H3,(H,32,39)(H2,35,36,40)

N-[3-tert-Butyl-1-(quinolin-6-yl)-1H-pyrazol-5-yl]-N'-[2-fluoro-4-[(2-(methylcarbamoyl)pyridin-4-yl)oxy]phenyl]urea

DCC-2036; DCC 2036; DCC2036; Rebastinib.

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: ≥ 2.08 mg/mL (3.76 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 (3.76 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: 0.5% CMC+0.25% Tween 80:16 mg/mL

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