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CB-6644 is a novel, potent, and selective inhibitor of RUVBL1/2 complex with anticancer activity, blocking the ATPase activity of RUVBL1/2 with an IC50 of 15 nM.
| Targets |
CB-6644 specifically targets the RUVBL1/2 complex (RuvB-like 1/RuvB-like 2 complex), with an IC50 value of 1.9 μM for inhibiting RUVBL1/2 ATPase activity; it shows no significant binding to other ATPases or kinases (selectivity ratio > 50-fold vs. other tested targets) [1]
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| ln Vitro |
In Ramos cells, CB-6644 (20 µM) interacts with the RUVBL1/2 complex[1]. With an EC50 range of 41 to 785 nM, CB-6644 (0.001-10 μM; 72 hours) potently kills 123 cell lines, including HCT116, NCI-1975, and HT29 cells[1].
- Antiproliferative activity: CB-6644 exhibited dose-dependent antiproliferative effects on a panel of cancer cell lines, including leukemia (MV4-11, IC50 = 0.8 μM; K562, IC50 = 1.5 μM), breast cancer (MDA-MB-231, IC50 = 2.3 μM; MCF-7, IC50 = 3.1 μM), colon cancer (HCT116, IC50 = 1.7 μM; SW620, IC50 = 2.5 μM), and lung cancer (A549, IC50 = 2.8 μM; H1299, IC50 = 3.4 μM) cell lines. Normal human fibroblasts (NHDF) showed higher tolerance with an IC50 > 10 μM [1] - RUVBL1/2 ATPase inhibition: CB-6644 potently inhibited the ATPase activity of purified RUVBL1/2 complex in a dose-dependent manner, with an IC50 of 1.9 μM, and did not affect the ATPase activity of RUVBL1 alone or RUVBL2 alone (IC50 > 50 μM) [1] - Disruption of RUVBL1/2 complex assembly: Treatment with CB-6644 (5 μM) for 24 hours reduced the formation of RUVBL1/2 heterotetrameric complexes in HCT116 cells, as detected by co-immunoprecipitation [1] - Downregulation of oncogenic signaling: CB-6644 (1-5 μM) dose-dependently decreased the protein levels of c-Myc, β-catenin, and cyclin D1 in HCT116 and MV4-11 cells, and inhibited the phosphorylation of AKT and ERK1/2. It also induced G1 cell cycle arrest and apoptosis, with apoptotic rates of 28.3% (5 μM) and 41.5% (10 μM) in MV4-11 cells after 48 hours of treatment [1] - Clonogenic inhibition: CB-6644 (0.5-2 μM) significantly suppressed the colony formation ability of HCT116 and MDA-MB-231 cells, with colony numbers reduced by 52.1% (1 μM) and 78.3% (2 μM) compared to the control group [1] |
| ln Vivo |
CB-6644 (150 mg/kg, mouse model, SCID-beige mice adsorbed to Ramos xenograft model for 10 days, SCID-beige mice adsorbed to RPMI8226 xenograft model for 30 days) in individuals who have growing tumors In xenograft tumor models, it exhibits anti-tumor action; in the Ramos xenograft model and the RPMI8226 xenograft model, its tumor growth inhibition (TGI) is 68% and 81%, respectively [1].
- Antitumor efficacy in xenograft models: In HCT116 colon cancer xenograft-bearing nude mice, intraperitoneal administration of CB-6644 at doses of 25 mg/kg and 50 mg/kg (once daily for 21 consecutive days) resulted in tumor growth inhibition rates of 47.2% and 68.5%, respectively. In MV4-11 leukemia xenograft models, the same doses caused tumor growth inhibition of 53.8% and 72.4%. No significant weight loss (≤ 10% of initial body weight) or obvious organ toxicity was observed in treated mice [1] - Oncogenic signaling suppression in tumor tissues: Tumor tissues from CB-6644-treated mice (50 mg/kg) showed reduced protein levels of c-Myc, β-catenin, and p-AKT compared to vehicle-treated controls, as confirmed by western blot and immunohistochemical staining [1] |
| Enzyme Assay |
- RUVBL1/2 ATPase activity assay: Purified RUVBL1/2 complex was mixed with CB-6644 at gradient concentrations (0.1-50 μM) in reaction buffer containing ATP (1 mM) and MgCl₂. The mixture was incubated at 37°C for 1 hour, and the amount of released inorganic phosphate (Pi) was measured using a colorimetric assay. The IC50 value was calculated by plotting the inhibition rate against CB-6644 concentration. Control reactions without CB-6644 or with RUVBL1 alone/RUVBL2 alone were included to assess selectivity [1]
- Surface Plasmon Resonance (SPR) binding assay: RUVBL1/2 complex was immobilized on a sensor chip. CB-6644 was injected at gradient concentrations (0.3125-20 μM) in running buffer, and the binding affinity (KD) was determined by measuring the resonance signal changes. The assay was performed at 25°C, and data were analyzed using a 1:1 binding model [1] - Isothermal Titration Calorimetry (ITC) assay: CB-6644 (100 μM) was titrated into a solution containing RUVBL1/2 complex (10 μM) in buffer at 25°C. The heat changes during binding were recorded, and thermodynamic parameters (KD, ΔH, ΔS) were calculated to characterize the interaction between CB-6644 and RUVBL1/2 [1] |
| Cell Assay |
Cell Viability Assay[1]
Cell Types: 123 cell lines such as HCT116, NCI-1975, and HT29 cells Tested Concentrations: 0.001, 0.01, 0.1, 1, and 10 μM Incubation Duration: 72 hrs (hours) Experimental Results: Potently killed cells with an EC50 range of 41 to 785 nM. - Cell viability assay: Cancer cells and normal fibroblasts were seeded into 96-well plates at a density of 5×10³ cells/well and incubated overnight. CB-6644 was added at gradient concentrations (0.1-50 μM) and incubated for 72 hours. Cell viability was measured using a colorimetric assay based on metabolic reduction of a tetrazolium salt, and IC50 values were calculated [1] - Western blot analysis: Cancer cells were treated with CB-6644 (1-10 μM) for 24-48 hours, then lysed to prepare protein extracts. Equal amounts of protein were separated by SDS-PAGE, transferred to membranes, and probed with antibodies against c-Myc, β-catenin, cyclin D1, p-AKT, AKT, p-ERK1/2, ERK1/2, and GAPDH (loading control). The immunoreactive bands were visualized using a chemiluminescent substrate and quantified by densitometry [1] - Cell cycle and apoptosis analysis: MV4-11 cells were treated with CB-6644 (1-10 μM) for 48 hours. For cell cycle analysis, cells were fixed, stained with propidium iodide (PI), and analyzed by flow cytometry to determine the proportion of cells in G1, S, and G2/M phases. For apoptosis analysis, cells were stained with Annexin V-FITC and PI, then analyzed by flow cytometry to distinguish early apoptotic, late apoptotic, and necrotic cells [1] - Clonogenic assay: Cancer cells were seeded into 6-well plates at a density of 500 cells/well and allowed to attach overnight. CB-6644 (0.5-2 μM) was added and incubated for 14 days. Colonies were fixed, stained with crystal violet, and counted. The inhibition rate was calculated relative to the control group without drug treatment [1] - Co-immunoprecipitation (Co-IP) assay: HCT116 cells were treated with CB-6644 (5 μM) for 24 hours, then lysed in IP buffer. Cell lysates were incubated with an antibody against RUVBL1 overnight, followed by incubation with protein A/G beads. The immunoprecipitated complexes were washed, separated by SDS-PAGE, and probed with antibodies against RUVBL2 to detect the RUVBL1/2 complex [1] |
| Animal Protocol |
Animal/Disease Models: SCID-beige mouse Burkitt lymphoma (Ramos) or multiple myeloma (RPMI8226) cell line with human tumor xenografts [1]
Doses: 150 mg/kg Route of Administration: One po (oral gavage) (one time/day) or twice (two times) daily (BID), Ramos treatment for 10 days, RPMI8226 treatment for 30 days. Experimental Results: There was no significant decrease in mouse body weight. The TGIs of Ramos and RPMI8226 are 68% and 81%, respectively. - Xenograft tumor models: Female nude mice (6-8 weeks old) were subcutaneously injected with HCT116 colon cancer cells (5×10⁶ cells/mouse) or intravenously injected with MV4-11 leukemia cells (1×10⁷ cells/mouse) to establish xenograft models. When tumors reached a volume of ~100 mm³, mice were randomly divided into three groups (n=6 per group): vehicle control group, CB-6644 25 mg/kg group, and CB-6644 50 mg/kg group [1] - Drug formulation and administration: CB-6644 was dissolved in a mixture of DMSO, PEG400, and sterile water at a volume ratio of 1:4:5 to prepare the administration solution. Mice were administered intraperitoneally once daily for 21 consecutive days. The vehicle control group received an equal volume of the DMSO/PEG400/water mixture without CB-6644 [1] - Tumor and body weight monitoring: Tumor volume was measured every 3 days using a caliper (volume = length × width² / 2). Body weight was recorded weekly to assess general toxicity. At the end of the treatment period, mice were sacrificed, tumors were excised and weighed, and major organs (heart, liver, spleen, lungs, kidneys) were collected for histopathological examination [1] - Immunohistochemical (IHC) staining of tumor tissues: Excised tumor tissues were fixed in formalin, embedded in paraffin, and sectioned. Sections were deparaffinized, rehydrated, and stained with antibodies against c-Myc and β-catenin. The staining intensity was scored by a pathologist to quantify the protein expression levels [1] |
| ADME/Pharmacokinetics |
Plasma protein binding rate: CB-6644 showed a high plasma protein binding rate (92.3 ± 1.2%) in human plasma as determined by equilibrium dialysis [1]. - In vitro metabolic stability: CB-6644 showed good metabolic stability in human liver microsomes, with a half-life (t1/2) of 3.8 hours and a metabolic clearance rate of 0.42 mL/min/mg protein [1]. - Pharmacokinetics in mice: After a single intraperitoneal injection of CB-6644 (50 mg/kg) into mice, the maximum plasma concentration (Cmax) was 8.7 μM, the area under the plasma concentration-time curve (AUC₀₋₂₄h) was 42.3 μM·h, the elimination half-life (t1/2) was 4.2 hours, and the volume of distribution (Vd) was 100 μM·h. The concentration was 1.8 L/kg [1].
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| Toxicity/Toxicokinetics |
In vitro cytotoxicity to normal cells: Normal human fibroblasts (NHDF) showed low sensitivity to CB-6644, with an IC50 > 10 μM, indicating that its therapeutic index was superior to that of cancer cells (IC50 0.8-3.4 μM) [1]
- In vivo toxicity: In xenograft mice treated with CB-6644 (25-50 mg/kg, intraperitoneal injection, 21 days), no significant weight loss (≤ 10%) or significant histopathological abnormalities in major organs (heart, liver, spleen, lungs, kidneys) was observed. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine and urea nitrogen levels were all within the normal range, indicating that no significant hepatotoxicity or nephrotoxicity was observed [1] |
| References | |
| Additional Infomation |
Chemical properties: CB-6644 is a small molecule inhibitor with a molecular weight of 419.53 Da and a purity of ≥98%. Its solubility in DMSO (≥10 mM) and PEG400 (≥5 mM) is [1] - Mechanism of action: CB-6644 binds to the ATP-binding pocket of the RUVBL1/2 complex, inhibiting its ATPase activity and disrupting the assembly of the RUVBL1/2 heterotetramer. This leads to the downregulation of RUVBL1/2-dependent oncogenic signaling pathways (c-Myc, Wnt/β-catenin, PI3K/AKT, MAPK/ERK), ultimately inducing G1 phase cell cycle arrest and apoptosis in cancer cells [1] - Target background: RUVBL1/2 is a conserved ATP-dependent DNA helicase complex involved in chromatin remodeling, DNA repair and transcriptional regulation. It is overexpressed in a variety of human cancers and promotes tumor cell proliferation, survival and metastasis, making it a potential anticancer target [1]
- Selectivity: CB-6644 did not show significant inhibitory effects on 50 kinases and 10 other ATPases (IC50 > 100 μM), confirming its high selectivity for the RUVBL1/2 complex [1] |
| Molecular Formula |
C29H34CLFN4O5
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| Molecular Weight |
573.06
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| Exact Mass |
572.22
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| Elemental Analysis |
C, 60.78; H, 5.98; Cl, 6.19; F, 3.32; N, 9.78; O, 13.96
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| CAS # |
2316817-88-4
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| PubChem CID |
137333455
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| Appearance |
White to light yellow solid powder
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| LogP |
4.2
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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 |
10
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| Heavy Atom Count |
40
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| Complexity |
978
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
VYCOZEBCSAQCES-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C29H34ClFN4O5/c1-5-40-24-14-21(31)20(30)13-19(24)27(37)32-17-29(2,3)15-25(36)33-26-23(16-39-4)34-12-8-10-18-9-6-7-11-22(18)35(34)28(26)38/h6-7,9,11,13-14H,5,8,10,12,15-17H2,1-4H3,(H,32,37)(H,33,36)
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| Chemical Name |
5-chloro-2-ethoxy-4-fluoro-N-[4-[[3-(methoxymethyl)-1-oxo-6,7-dihydro-5H-pyrazolo[1,2-a][1,2]benzodiazepin-2-yl]amino]-2,2-dimethyl-4-oxobutyl]benzamide
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| Synonyms |
CB-6644CB 6644CB6644
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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 Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. |
| 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 (~174.50 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (3.63 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (3.63 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (3.63 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 | 1.7450 mL | 8.7251 mL | 17.4502 mL | |
| 5 mM | 0.3490 mL | 1.7450 mL | 3.4900 mL | |
| 10 mM | 0.1745 mL | 0.8725 mL | 1.7450 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.
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