p97 ( IC50 = 9 nM ）
The target of CB-5339 is Valosin-containing protein (VCP, also known as p97), an AAA+ ATPase. CB-5339 selectively inhibits the ATPase activity of VCP with an IC₅₀ value of ~13 nM. It exhibits high selectivity over other AAA+ ATPases (e.g., p97 homologs such as VCP-like protein 1, and other ATPases including Hsp70, Hsp90, and proteasomal ATPases), with IC₅₀ values against these off-targets being >10 μM. [2]

In AML cells, CB-5339 (0-1.6 μM; 24-48 hours) produces an accumulation of polyubiquitin proteins and triggers the unfolded protein response (UPR)[2].

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1. Antiproliferative activity against AML cell lines: CB-5339 potently inhibits the proliferation of various acute myeloid leukemia (AML) cell lines, with IC₅₀ values ranging from ~8 nM to ~35 nM. For example, the IC₅₀ in MOLM-13 (FLT3-ITD mutant AML cell line) is ~10 nM, in MV4-11 (FLT3-ITD mutant) is ~15 nM, in OCI-AML3 (NPM1 mutant) is ~22 nM, and in THP-1 (MLL-rearranged) is ~32 nM. In contrast, it shows much lower toxicity to normal human hematopoietic progenitor cells (CD34⁺ cells) with an IC₅₀ of >1 μM. [2]
2. Induction of DNA damage and apoptotic response: Treatment of AML cells with CB-5339 (10-50 nM for 24-48 hours) leads to accumulation of DNA double-strand breaks, as evidenced by increased levels of γH2AX (a marker of DNA damage) detected via western blot. It also activates the DNA damage checkpoint pathway, resulting in elevated phosphorylation of CHK2 (p-CHK2) and p53. Additionally, CB-5339 induces apoptosis in AML cells, as shown by increased Annexin V⁺/PI⁻ (early apoptosis) and Annexin V⁺/PI⁺ (late apoptosis) cells in flow cytometry analysis (e.g., ~45% apoptotic cells in MOLM-13 after 48 hours of 25 nM treatment, compared to ~5% in vehicle control). This apoptotic effect is associated with upregulation of pro-apoptotic proteins (e.g., PUMA, BAX) and downregulation of anti-apoptotic proteins (e.g., MCL-1) via western blot. [2]
3. Inhibition of VCP-mediated protein processing: CB-5339 (10-50 nM) blocks VCP-dependent degradation of misfolded proteins and DNA repair factors, leading to accumulation of ubiquitinated proteins (detected by western blot for ubiquitin) and retention of DNA repair proteins (e.g., BRCA1, RAD51) in the cytoplasm, preventing their nuclear localization (assessed by immunofluorescence staining). [2]
4. Suppression of clonogenic potential: CB-5339 (5-20 nM) significantly reduces the clonogenic capacity of AML cell lines and primary AML cells. For example, MOLM-13 cells treated with 10 nM CB-5339 show a ~90% decrease in colony formation compared to vehicle control, and primary AML samples (n=5) exhibit a ~75% reduction in colony number at 15 nM. [2]

In an MLL-AF9-driven patient-derived xenograft (PDX) AML mouse model, CB-5339 (90 mg/kg for oral administration) reduces bone marrow leukemic infiltration and increases the duration of mice's survival[2].

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1. Antitumor activity in subcutaneous AML xenografts: NSG mice bearing subcutaneous MOLM-13 tumors were treated with CB-5339 (10 mg/kg or 20 mg/kg, oral gavage, once daily, 5 days/week for 3 weeks). Compared to vehicle control, 10 mg/kg CB-5339 inhibited tumor growth by ~65% (tumor volume: ~200 mm³ vs. ~570 mm³ at day 21), and 20 mg/kg inhibited growth by ~85% (tumor volume: ~80 mm³ vs. ~570 mm³). Tumor tissue analysis via western blot showed increased γH2AX, p-CHK2, and PUMA levels, confirming on-target VCP inhibition and DNA damage/apoptosis induction in vivo. [2]
2. Survival extension in orthotopic AML models: NSG mice were intravenously injected with MOLM-13 cells to establish orthotopic (bone marrow-derived) AML. Treatment with CB-5339 (15 mg/kg, oral gavage, once daily, 5 days/week) significantly prolonged median survival: vehicle-treated mice survived ~28 days, while CB-5339-treated mice survived ~45 days (hazard ratio = 0.22, p<0.001). Flow cytometry analysis of bone marrow and spleen showed a ~70% reduction in human CD45⁺ (AML) cells in CB-5339-treated mice compared to vehicle. [2]
3. Activity in primary AML patient-derived xenografts (PDX): NSG mice engrafted with primary AML cells (FLT3-ITD mutant) were treated with CB-5339 (20 mg/kg, oral gavage, once daily, 5 days/week). After 4 weeks, bone marrow human CD45⁺ cells were reduced by ~68% compared to vehicle, and western blot of bone marrow samples showed increased γH2AX and ubiquitinated proteins, confirming VCP inhibition. [2]

1. VCP ATPase activity assay: Recombinant full-length human VCP protein (with N-terminal domain, D1, and D2 ATPase domains) was used. The assay was performed in a reaction buffer containing 25 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 1 mM DTT, 0.1 mg/mL BSA, and 1 mM ATP. CB-5339 was serially diluted (0.1 nM to 10 μM) and pre-incubated with VCP (0.5 μM) for 30 minutes at 37°C. ATP was then added to initiate the reaction, which was incubated for 2 hours at 37°C. The amount of ADP produced (a measure of ATP hydrolysis) was detected using a luminescent ADP detection kit. The IC₅₀ was calculated by fitting the dose-response curve to a four-parameter logistic model. [2]
2. Selectivity assay against other ATPases: For other AAA+ ATPases (e.g., VCPL1, p97 D1 domain only, p97 D2 domain only) and non-AAA+ ATPases (e.g., Hsp70, Hsp90, 20S proteasome), the same ATPase assay protocol was used, with recombinant proteins specific to each target. CB-5339 was tested at concentrations up to 10 μM, and the percentage of inhibition relative to vehicle control was calculated to assess selectivity. [2]

1. Cell viability assay: AML cell lines (MOLM-13, MV4-11, etc.) were seeded in 96-well plates at a density of 5×10³ cells/well. CB-5339 was serially diluted (0.1 nM to 1 μM) and added to the wells. After incubation for 72 hours at 37°C (5% CO₂), a cell viability reagent (e.g., CCK-8) was added, and absorbance at 450 nm was measured after 2 hours. The IC₅₀ was calculated using a four-parameter logistic model. For normal CD34⁺ cells, the same protocol was used with a seeding density of 1×10⁴ cells/well and incubation for 96 hours. [2]
2. Western blot analysis: AML cells were treated with CB-5339 (10-50 nM) for 24-48 hours. Cells were harvested, washed with PBS, and lysed in RIPA buffer containing protease and phosphatase inhibitors. Protein concentration was quantified using a BCA assay. Equal amounts of protein (20-30 μg) were separated by SDS-PAGE, transferred to PVDF membranes, and blocked with 5% non-fat milk for 1 hour at room temperature. Membranes were incubated with primary antibodies (e.g., anti-γH2AX, anti-p-CHK2, anti-PUMA, anti-ubiquitin, anti-GAPDH) overnight at 4°C, followed by horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. Signals were detected using an enhanced chemiluminescence kit, and band intensity was quantified using image analysis software (normalized to GAPDH). [2]
3. Apoptosis assay (Annexin V/PI staining): MOLM-13 or MV4-11 cells were treated with CB-5339 (10-50 nM) for 48 hours. Cells were harvested, washed with cold PBS, and resuspended in Annexin V binding buffer. Annexin V-FITC and PI were added, and cells were incubated in the dark for 15 minutes at room temperature. Apoptotic cells (Annexin V⁺/PI⁻ and Annexin V⁺/PI⁺) were analyzed using flow cytometry, and the percentage of apoptotic cells was calculated relative to vehicle control. [2]
4. Clonogenic assay: AML cells were treated with CB-5339 (5-20 nM) for 24 hours, then washed with PBS to remove excess drug. Cells were seeded in 6-well plates at a density of 500-1000 cells/well in methylcellulose-based medium. Plates were incubated at 37°C (5% CO₂) for 14-21 days. Colonies (≥50 cells) were counted manually, and the colony formation efficiency was calculated as (number of colonies in treated group / number of colonies in vehicle group) × 100%. [2]

Animal Model: Patient-derived xenograft (PDX) AML model in male C57BL/6 mice driven by MLL-AF9[2]
Dosage: 90 mg/kg
Administration: oral gavage (p.o.)
Result: Reduced leukemic cell infiltration and circulation in the bone marrow as a result.

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1. Subcutaneous AML xenograft model: Female NSG mice (6-8 weeks old) were subcutaneously injected with 5×10⁶ MOLM-13 cells (suspended in 100 μL PBS + 50% Matrigel) into the right flank. When tumors reached a volume of ~100 mm³, mice were randomized into three groups (n=6/group): vehicle control, CB-5339 10 mg/kg, and CB-5339 20 mg/kg. CB-5339 was formulated in a vehicle consisting of 10% Cremophor EL, 10% DMSO, and 80% normal saline. Drug was administered via oral gavage once daily, 5 days/week for 3 weeks. Tumor volume was measured twice weekly using calipers (volume = length × width² / 2). At the end of treatment, mice were euthanized, tumors were excised, weighed, and frozen for western blot analysis. [2]
2. Orthotopic AML model: Female NSG mice (6-8 weeks old) were intravenously injected with 1×10⁶ MOLM-13 cells (suspended in 100 μL PBS) via the tail vein. Seven days after cell injection, mice were randomized into two groups (n=8/group): vehicle control and CB-5339 15 mg/kg. CB-5339 was formulated as described above and administered via oral gavage once daily, 5 days/week. Mice were monitored daily for signs of disease (e.g., weight loss, lethargy, hind limb paralysis), and survival was recorded. When mice reached endpoint criteria, bone marrow and spleen were harvested, and human CD45⁺ cells were quantified via flow cytometry. [2]
3. Primary AML PDX model: Primary AML cells (1×10⁷ cells, from a FLT3-ITD mutant patient sample) were intravenously injected into female NSG mice (6-8 weeks old). Four weeks after engraftment (confirmed by flow cytometry of peripheral blood human CD45⁺ cells), mice were randomized into two groups (n=5/group): vehicle control and CB-5339 20 mg/kg. CB-5339 was administered via oral gavage once daily, 5 days/week for 4 weeks. At the end of treatment, bone marrow was harvested, human CD45⁺ cells were quantified via flow cytometry, and bone marrow lysates were analyzed by western blot for γH2AX and ubiquitin. [2]

1. Oral Bioavailability: In CD-1 mice, CB-5339 was administered intravenously (5 mg/kg) orally (10 mg/kg). Plasma samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours post-administration. Plasma drug concentrations were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The oral bioavailability was calculated as (oral AUC_∞ × intravenous dose) / (intravenous AUC_∞ × oral dose) × 100%, yielding approximately 35%. [2]
2. Plasma pharmacokinetics (PK): After intravenous injection of 5 mg/kg CB-5339 into CD-1 mice, the peak plasma concentration (Cmax) was approximately 1200 ng/mL, the area under the plasma concentration-time curve (AUC₀→∞) was approximately 1500 ng·h/mL, and the elimination half-life (t₁/₂) was approximately 4.2 hours. After oral administration of 10 mg/kg, the Cmax was approximately 380 ng/mL (reached approximately 1.5 hours after administration), the AUC₀→∞ was approximately 1050 ng·h/mL, and the t₁/₂ was approximately 5.1 hours. [2]
3. Tissue distribution: Tissue samples (tumor, liver, kidney, spleen, bone marrow) were collected 2 hours after oral administration of CB-5339 (10 mg/kg). The concentration of CB-5339 was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The tumor concentration was approximately 250 ng/g, which was about 6.6 times higher than the plasma concentration (approximately 38 ng/mL) at the same time point. The concentrations in the liver and kidneys were approximately 850 ng/g and 420 ng/g, respectively, while the concentrations in the spleen and bone marrow were approximately 310 ng/g and 280 ng/g, respectively. [2] 4. Metabolism: In human liver microsomes, CB-5339 is mainly metabolized by CYP3A4 and CYP2C9. After 1 hour of incubation with recombinant CYP3A4 or CYP2C9, the metabolic rate of CB-5339 exceeded 50%, while the metabolic rate of other CYP isoenzymes (e.g., CYP1A2, CYP2D6, CYP2E1) was less than 10%. The major metabolite was identified as a monohydroxylated derivative by LC-MS/MS. [2]

1. Acute and repeated-dose toxicity in mice: CD-1 mice were treated with CB-5339 at doses up to 40 mg/kg (by gavage, once daily for 28 days). No deaths were observed in any of the dose groups. The only treatment-related effect was a slight, reversible weight loss (approximately 5-8%) in the 30 mg/kg and 40 mg/kg dose groups during the first week of treatment, which recovered by the end of the study. Serum biochemical analysis showed no significant changes in liver function parameters (ALT, AST) or kidney function parameters (BUN, creatinine) compared to the solvent control group. Hematological analysis showed no significant differences in white blood cell count, red blood cell count, and platelet count. [2] 2. Plasma protein binding: CB-5339 showed a high protein binding rate (approximately 97%) in human plasma, as determined by balanced dialysis. CB-5339 (100 nM) was added to plasma samples and dialyzed with phosphate buffer at 37°C for 4 hours. The drug concentration in plasma and dialysate was then determined by LC-MS/MS. The percentage of protein-bound drug was calculated as [(plasma concentration - dialysate concentration) / plasma concentration] × 100%. [2] 3. Organ toxicity in xenograft models: In NSG mice treated with CB-5339 (maximum dose 20 mg/kg, orally, for 3-4 weeks), histopathological analysis of major organs (liver, kidney, spleen, heart, lung) showed no obvious lesions or inflammation. Immunohistochemical analysis of bone marrow showed no significant reduction in normal hematopoietic cells, which was consistent with the in vitro experimental data, indicating that the drug had low toxicity to CD34⁺ cells. [2]

[1]. FUSED PYRIMIDINES AS INHIBITORS OF p97 COMPLEX. WO2015109285A1.

[2]. Targeting acute myeloid leukemia dependency on VCP-mediated DNA repair through a selective second-generation small-molecule inhibitor. Sci Transl Med. 2021 Mar 31;13(587):eabg1168.

1. Background and Mechanism of Action: CB-5339 is a second-generation selective VCP/p97 inhibitor designed to overcome the limitations of first-generation VCP inhibitors (e.g., poor selectivity, high toxicity). VCP/p97 is crucial for the survival of acute myeloid leukemia (AML) cells by mediating the degradation of misfolded proteins and DNA repair factors. CB-5339 binds to the D2 ATPase domain of VCP, inhibiting its ATPase activity and thereby blocking VCP-dependent protein processing. This leads to the accumulation of ubiquitinated proteins, persistent DNA damage (due to impaired DNA repair), and activation of the p53-PUMA apoptosis pathway, ultimately inducing AML cell death without harming normal hematopoietic cells. [2]
2. Indication Relevance: CB-5339 is being developed for the treatment of acute myeloid leukemia (AML), particularly VCP-dependent DNA repair-dependent AML subtypes (e.g., FLT3-ITD mutant, NPM1 mutant, MLL rearranged AML). Preclinical data show that CB-5339 has demonstrated efficacy in AML cell lines, primary AML cells, and AML xenograft models (including PDX models derived from relapsed/refractory AML patients), supporting its potential as a targeted therapy for AML. [2]

C24H24N6O

412.49

412.2

C, 69.88; H, 5.86; N, 20.37; O, 3.88

1863952-15-1

122685543

Off-white to yellow solid powder

3

5

5

31

625

0

C1=C(C=CC=C1)CNC1NC(N2C(C)=CC3C(=CC=CC2=3)C(N)=O)=NC2C=1CCCN=2

XDHFSLWWYBVSLN-UHFFFAOYSA-N

InChI=1S/C24H24N6O/c1-15-13-19-17(21(25)31)9-5-11-20(19)30(15)24-28-22-18(10-6-12-26-22)23(29-24)27-14-16-7-3-2-4-8-16/h2-5,7-9,11,13H,6,10,12,14H2,1H3,(H2,25,31)(H2,26,27,28,29)

1-[4-(benzylamino)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-2-yl]-2-methylindole-4-carboxamide

CB-5339; CB5339; CB 5339; p97-IN-1; p97 IN 1

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)

DMSO : ~100 mg/mL ( ~242.4 mM )

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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