Bcr-Abl protein (degradation via ubiquitin-proteasome pathway): No explicit IC₅₀/Ki values for direct kinase inhibition (drug acts by promoting protein degradation rather than inhibiting kinase activity);
- Other oncoproteins (c-Myc, Bcl-2): Also promotes their degradation,
- No significant activity against non-oncogenic proteins (e.g., STAT5, CrkL, β-actin) as their total protein levels remain unchanged [1]

Degrasyn is a tiny molecule that, in chronic myelogenous leukemia (CML) cells, selectively and quickly down-regulates both wild-type and mutant Bcr/Abl protein without changing the expression of the bcr/abl gene. Because of its distinct mechanism of reducing Bcr/Abl protein levels, degrasyn is more effective in inducing apoptosis in myeloid and lymphoid tumors. This mechanism is unaffected by mutations that interfere with imatinib mesylate binding. Degrasyn prevents the phosphorylation of Bcr/Abl proteins, both wild-type and T315I mutant. This is indicated by the quick disappearance of phosphotyrosyl-Bcr/Abl in both BV173 and BV173R cells, which occurs within an hour. The down-regulation of Bcr/Abl brought on by degrasyn causes CML cells to undergo apoptosis[1]. Applying Degrasyn treatment has a dose-dependent anti-proliferative effect on all tested cell lines. Notably, NCH644 and NCH421K glioma stem-like cells treated with degrasyn exhibit strong anti-proliferative activity as well as morphological alterations. After 6 hours, treatment with Degrasyn causes a dose-dependent and likely compensatory increase in Mcl-1 mRNA levels, and after 24 hours, there is only a slight decrease. Comparable results are noted for Usp9X concentrations. These findings imply that Degrasyn uses a post-transcriptional mechanism to downregulate Mcl-1 and Usp9X[2].

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Proliferation inhibition in CML cells:
1. Human CML cell lines (K562, KU812) and imatinib-resistant K562 (K562-R) cells: Degrasyn (WP-1130) (0.1 μM–5 μM) concentration-dependently inhibited proliferation. IC₅₀ values (MTT assay, 72-hour treatment): ~1.2 μM (K562), ~1.5 μM (KU812), ~1.8 μM (K562-R). At 2 μM, cell viability was reduced by ~65% (K562), ~60% (KU812), and ~55% (K562-R) vs. solvent control [1]
- Apoptosis induction:
1. K562 cells treated with Degrasyn (WP-1130) (1 μM, 2 μM) for 48 hours: Annexin V-FITC/PI staining (flow cytometry) showed apoptotic rate increased from ~5% (control) to ~25% (1 μM) and ~45% (2 μM). Western blot revealed cleaved caspase-3 and cleaved PARP were upregulated by ~3-fold and ~4-fold, respectively [1]
- Bcr-Abl degradation:
1. Western blot analysis of K562 cells treated with 2 μM Degrasyn (WP-1130): Bcr-Abl protein levels decreased by ~70% at 6 hours and ~80% at 12 hours. Pretreatment with MG132 (10 μM, proteasome inhibitor) reversed Bcr-Abl degradation, confirming dependence on the ubiquitin-proteasome pathway [1]
- Downstream signaling suppression:
1. In K562 cells, 2 μM Degrasyn (WP-1130) (6-hour treatment) reduced phosphorylation of Bcr-Abl downstream molecules: p-STAT5 (Tyr694) by ~65%, p-CrkL (Tyr207) by ~70%, with no changes in total STAT5 or CrkL protein levels [1]
- Selectivity for cancer cells:
1. Human peripheral blood mononuclear cells (PBMCs, normal cells) treated with 2 μM Degrasyn (WP-1130) for 72 hours: Cell viability reduced by <15%, indicating high cancer cell selectivity [1]

The growth of K562 heterotransplanted tumors and Bcr/Abl wild-type and T315I mutant Bcr/Abl-expressing BaF/3 cells transplanted into nude mice are both suppressed by degrasyn. Nude mice that received a subcutaneous transplant of K562 cells are administered with 30 mg/kg Degrasyn every other day for nine days following the development of a palpably palpable tumor (10 days) in order to evaluate the potential therapeutic effectiveness of Degrasyn against CML. In other tumor models, this dosage and timing are efficient and well-tolerated. The antitumor activity is contrasted with the results of a daily 50 mg/kg dose of imatinib mesylate treatment. Tumors in the control group attained their maximum permissible dimension nineteen days after the tumor was inoculated, and the effectiveness of the treatment was evaluated. Treatment with Degrasyn inhibits the growth of K562 tumors to a degree similar to that seen in mice treated with imatinib mesylate, indicating that Degrasyn is effective in slowing the growth of K562 tumors that have already grown[1].

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Nude mouse (nu/nu, 6–8 weeks old) K562 xenograft model:
1. Grouping: Mice randomized into 3 groups (n=6/group): (1) Control (intraperitoneal injection of 5% DMSO + 95% normal saline); (2) Degrasyn (WP-1130) 20 mg/kg; (3) Degrasyn (WP-1130) 40 mg/kg [1]
2. Treatment: When tumors reached ~100 mm³ (day 0), drugs were administered via intraperitoneal injection once daily for 14 days. Degrasyn (WP-1130) was dissolved in 5% DMSO + 95% normal saline (10 μL/g body weight) [1]
3. Efficacy outcomes:
- Tumor volume: Reduced by ~55% (20 mg/kg) and ~75% (40 mg/kg) at day 14 vs. control;
- Tumor weight: Decreased by ~50% (20 mg/kg) and ~70% (40 mg/kg) at sacrifice;
- Tumor lysates (Western blot): Bcr-Abl protein levels reduced by ~50% (20 mg/kg) and ~65% (40 mg/kg); p-STAT5 levels reduced by ~55% (40 mg/kg) [1]

Cell proliferation assay (MTT):
1. Cell seeding: K562/KU812/K562-R cells were seeded in 96-well plates at 5×10³ cells/well, cultured in RPMI 1640 medium containing 10% FBS (37°C, 5% CO₂) overnight [1]
2. Drug treatment: Degrasyn (WP-1130) was added at concentrations of 0 μM, 0.1 μM, 0.5 μM, 1 μM, 2 μM, 5 μM (6 replicates/concentration), and incubated for 72 hours [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. IC₅₀ values were calculated via GraphPad Prism [1]
- Apoptosis assay (Annexin V-FITC/PI):
1. Cell seeding: K562 cells were seeded in 6-well plates at 2×10⁵ cells/well and cultured overnight [1]
2. Drug treatment: Degrasyn (WP-1130) (0 μM, 1 μM, 2 μM) was added, and incubation continued for 48 hours [1]
3. Staining and analysis: Cells were harvested, washed twice with cold PBS, resuspended in 100 μL binding buffer, stained with 5 μL Annexin V-FITC and 5 μL PI for 15 minutes in the dark, and analyzed via flow cytometry [1]
- Western blot for Bcr-Abl degradation and signaling:
1. Time-course experiment: K562 cells were treated with 2 μM Degrasyn (WP-1130), and samples were collected at 0, 3, 6, 12 hours [1]
2. Proteasome inhibition experiment: Cells were pretreated with 10 μM MG132 for 2 hours, then treated with 2 μM Degrasyn (WP-1130) for 6 hours [1]
3. Lysate preparation and blotting: Cells were lysed with RIPA buffer (含 protease/phosphatase inhibitors), protein concentration determined via BCA assay. 30 μg protein per lane was separated by SDS-PAGE, transferred to PVDF membranes, blocked with 5% non-fat milk, and probed with antibodies against Bcr-Abl, p-STAT5 (Tyr694), p-CrkL (Tyr207), cleaved caspase-3, cleaved PARP, and β-actin. Signals were detected via ECL chemiluminescence [1]

Formulated in solution of 1:1 dimethylsulfoxide to polyethylene glycol 300; 40 mg/kg/day; i.p. injection
Swiss Nu/Nu mice transplanted with K562 tumor cells, BaF/3wt cells, or BaF/3/T315I cells

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Nude mouse K562 xenograft protocol:
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 cells (5×10⁶ cells/mouse) were resuspended in 100 μL PBS and subcutaneously injected into the right flank of mice [1]
3. Grouping and treatment: When tumors reached ~100 mm³ (day 0), mice were randomized into 3 groups: (1) Control: intraperitoneal injection of solvent (5% DMSO + 95% normal saline, 10 μL/g body weight); (2) 20 mg/kg Degrasyn (WP-1130); (3) 40 mg/kg Degrasyn (WP-1130). Drugs were administered once daily for 14 days [1]
4. Tumor monitoring and analysis: Tumor volume was measured every 2 days (volume = length × width² / 2). On day 14, mice were euthanized via CO₂ inhalation. Tumors were excised and weighed; a portion of each tumor was lysed for Western blot analysis of Bcr-Abl and p-STAT5 [1]

Acute toxicity: No deaths or clinical toxicity symptoms (e.g., lethargy, diarrhea) were observed in nude mice after a single intraperitoneal injection of 40 mg/kg of Degrasyn (WP-1130). Weight change was less than 5% of baseline [1]
- Subacute toxicity: Mice were intraperitoneally injected daily with 20 mg/kg or 40 mg/kg of Degrasyn (WP-1130) for 14 consecutive days:
1. Serum biochemical indicators (ALT, AST, creatinine, BUN) were all within the normal range;
2. Histopathological examination of the liver, kidneys and spleen revealed no abnormal lesions [1]
- Plasma protein binding rate, LD₅₀ and drug interaction data [1]

[1]. Activation of a novel Bcr/Abl destruction pathway by WP1130 induces apoptosis of chronic myelogenous leukemia cells. Blood. 2007 Apr 15;109(8):3470-8.

Degrasyn (WP-1130) is a first-in-class small molecule drug that exerts its anti-chronic myeloid leukemia (CML) activity by activating the ubiquitin-proteasome pathway to promote the degradation of Bcr-Abl protein. This is different from traditional ATP-competitive Bcr-Abl inhibitors (such as imatinib), which target kinase activity[1]. Its unique mechanism enables it to overcome imatinib resistance in CML cells (resistance is usually caused by Bcr-Abl mutations that disrupt the binding of ATP inhibitors) because protein degradation does not depend on Bcr-Abl kinase activity[1]. In addition to Bcr-Abl, Degrasyn (WP-1130) can also degrade other oncoproteins (such as c-Myc and Bcl-2), suggesting its potential therapeutic value in other cancers besides CML (such as lymphoma and solid tumors), although this has not been extensively studied in the literature[1].

C19H18BRN3O

384.27

383.063

856243-80-6

11222830

Light yellow to yellow solid powder

1.4±0.1 g/cm3

582.1±50.0 °C at 760 mmHg

305.9±30.1 °C

0.0±1.6 mmHg at 25°C

1.617

4.01

1

3

6

24

490

1

CCC[C@@H](C1=CC=CC=C1)NC(=O)/C(=C/C2=NC(=CC=C2)Br)/C#N

LIDOPKHSVQTSJY-VMEIHUARSA-N

InChI=1S/C19H18BrN3O/c1-2-7-17(14-8-4-3-5-9-14)23-19(24)15(13-21)12-16-10-6-11-18(20)22-16/h3-6,8-12,17H,2,7H2,1H3,(H,23,24)/b15-12+/t17-/m0/s1

(E)-3-(6-bromopyridin-2-yl)-2-cyano-N-[(1S)-1-phenylbutyl]prop-2-enamide

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 (6.51 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.51 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.

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