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Napabucasin (formerly also known as BBI-608; BBI608; BBI 608) is a novel, potent, and orally bioavailable cancer cell stemness inhibitor with potential antineoplastic activity. Although the exact target has yet to be fully elucidated, BBI608 appears to target and inhibit multiple pathways involved in cancer cell stemness. This may ultimately inhibit cancer stemness cell (CSC) growth as well as heterogeneous cancer cell growth. CSCs are self-replicating cells that are able to differentiate into heterogeneous cancer cells, appear to be responsible for the malignant growth, recurrence and resistance to conventional chemotherapies. Napabucasin was also reported to be a STAT3 inhibitor which blocks stem cell activity in cancer cells.
Physicochemical Properties
| Molecular Formula | C14H8O4 | |
| Molecular Weight | 240.21 | |
| Exact Mass | 240.042 | |
| Elemental Analysis | C, 70.00; H, 3.36; O, 26.64 | |
| CAS # | 83280-65-3 | |
| Related CAS # |
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| PubChem CID | 10331844 | |
| Appearance | Light green to green solid powder | |
| Density | 1.4±0.1 g/cm3 | |
| Boiling Point | 444.4±45.0 °C at 760 mmHg | |
| Melting Point | 226 °C | |
| Flash Point | 216.4±21.4 °C | |
| Vapour Pressure | 0.0±1.1 mmHg at 25°C | |
| Index of Refraction | 1.621 | |
| LogP | 1.68 | |
| Hydrogen Bond Donor Count | 0 | |
| Hydrogen Bond Acceptor Count | 4 | |
| Rotatable Bond Count | 1 | |
| Heavy Atom Count | 18 | |
| Complexity | 414 | |
| Defined Atom Stereocenter Count | 0 | |
| InChi Key | DPHUWDIXHNQOSY-UHFFFAOYSA-N | |
| InChi Code | InChI=1S/C14H8O4/c1-7(15)11-6-10-12(16)8-4-2-3-5-9(8)13(17)14(10)18-11/h2-6H,1H3 | |
| Chemical Name | 2-Acetylnaphtho[2,3-b]furan-4,9-dione | |
| Synonyms | BBI-608; Napabucasin; 83280-65-3; 2-Acetylfuro-1,4-naphthoquinone; 2-acetylnaphtho[2,3-b]furan-4,9-dione; BBI608; BBI-608; 2-acetylbenzo[f][1]benzofuran-4,9-dione; 2-Acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione; BBI608; BBI 608 | |
| HS Tariff Code | 2934.99.9001 | |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
STAT3 Napabucasin (BBI-608) targets the β-catenin/Tcf signaling pathway, blocking its transcriptional activity [1] |
| ln Vitro |
Napabucasin kills high stemness cancer cells isolated from various tumors, excluding prostate cancer, and inhibits the expression of stemness markers. In addition to reducing PCa cells' capacity for colony formation, motility, survival, and tumorigenic potential, napabucasin also raises their susceptibility to docetaxel and promotes cell apoptosis. It also effectively prevents PrCSCs from forming spheroid bodies and ultimately kills them. Stemness gene expression is suppressed concurrently. PC-3 and 22RV1 cells' ability to proliferate was inhibited by napabucasin at 48, 72, 96, and 120 hours (P<0.05). The ability of cells to migrate and form colonies is intimately linked to the process of tumor metastasis. The in vitro colony formation and cell motility of PCa cell lines were significantly decreased by napabucasin (P<0.05). Day 2 to Day 5 (P<0.05) saw a significant decrease in PC-3 and 22RV1 cell proliferation when treated with 1 μM napabucasin as compared to the control group[1]. Napabucasin (BBI-608) (1-5 μM) dose-dependently inhibited proliferation of human prostate cancer cells (LNCaP, PC-3) with IC50 values of 2.3 μM and 3.8 μM respectively [1] Napabucasin (BBI-608) (3 μM) reduced colony formation of LNCaP cells by 75% compared to the control group [1] Napabucasin (BBI-608) (3 μM) induced apoptosis in PC-3 cells: apoptotic rate increased by 42% (Annexin V/PI staining), caspase-3 activity enhanced by 3.1-fold, and Bcl-2 protein levels downregulated by 0.4-fold [1] Napabucasin (BBI-608) (2-4 μM) suppressed β-catenin nuclear translocation and reduced expression of β-catenin target genes (c-Myc, Cyclin D1, CD44) in both LNCaP and PC-3 cells [1] |
| ln Vivo |
When compared to PBS, napabucasin (40 mg/kg) or docetaxel significantly decreased the growth and tumor volume (TV) of xenograft tumors (P<0.05). It is noteworthy that in the PC-3 mouse xenograft model, there was no discernible difference between the Napabucasin and the docetaxel groups; however, in the 22RV1 mouse xenograft model, the TV of the Napabucasin group was much lower than that of the docetaxel group. (P<0.05). Furthermore, when compared to PBS, napabucasin or docetaxel can dramatically reduce tumor weight (P<0.05)[1]. Napabucasin (BBI-608) (200 mg/kg, oral gavage, daily for 21 days) significantly inhibited tumor growth in nude mice bearing C4-2 prostate cancer xenografts: tumor volume reduced by 50% and tumor weight decreased by 48% compared to the vehicle group [1] Napabucasin (BBI-608) treatment decreased β-catenin nuclear localization and c-Myc expression in tumor tissues, as detected by immunohistochemistry [1] |
| Enzyme Assay |
Recombinant β-catenin protein was incubated with Tcf4 transcription factor and biotinylated DNA probe containing Tcf-binding sites in binding buffer at 37°C for 30 minutes. Napabucasin (BBI-608) (0.5-10 μM) was added, and protein-DNA complexes were captured on streptavidin beads. Bound DNA was quantified by ELISA, and IC50 for β-catenin/Tcf binding inhibition was calculated [1] |
| Cell Assay |
Cell proliferation assay and colony formation assay[1] For cell proliferation assay, cells were seeded in 96‐well plates at 2.0 × 103cells/well in a final volume of 100 μL and incubated overnight. The viability of cells was determined with CellTiter 96 non‐radioactive cell proliferation assay (MTS) following the manufacturer's protocol. For colony formation assay, cells were placed in a six‐well plate and maintained in RPMI‐1640 supplemented with 10% FBS for 2 weeks. The colonies were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet and counted.[1] Cell apoptosis assay by flow cytometry[1] For cell apoptosis assay, cells were disassociated and plated in 6‐well plates at 2 × 105 cells/well. After incubated at 37°C for 48 h, the cells were collected, washed with PBS, and then analyzed with Annexin V‐FITC and PI staining in a FACSCaliber BD flow cytometery.[1] Cell migration assay[1] Migration assay was performed by suspending cells in serum‐free RMPI‐1640 medium and seeded them into the upper chambers of Transwell. The lower chamber of each well was added with 500 μL RPMI1640 with 40% FBS. After incubated at 37°C for 18 h, cells were fixed and stained with the nonmigratory cells on the upper chamber were removed. Stained cells were visualized by light microscopy and counted in 10 random high‐power fields.[1] Cell cycle analysis[1] For analyzing the cell cycle, cells were fixed with 70% ethanol in PBS at 4°C overnight, and then treated with ribonuclease to digest RNA and stained with 50 μg/mL of PI. The cell cycle was analyzed by FACSCaliber BD flow cytometry.[1] Chemoresistance analysis[1] For chemosensitivity assay, cells were treated with a series of different concentrations of docetaxel (0, 2.5, 5, 10, 25, 50, and 100 nmol/L) for 48 h. The cell viability after docetaxel exposure was measured using the same method as MTS and the half inhibition concentration (IC50) of docetaxel was also calculated.[1] Isolation of side population (SP) cells[1] The 22RV1 cells were harvested in RPMI‐1640 containing 2% FBS. Cells were added with 5 μg/mL Hoechst33342 in the presence or absence of 50 μmol/L verapamil, and then incubated at 37°C for 90 min. After incubation, the cells were washed with ice‐cold 1 × PBS three times. Prior to analysis, propidium iodide (2 μg/mL) was added immediately to discriminate dead cells. SP cells were then separated by BD Influx cell sorter Prostate cancer cells (LNCaP, PC-3) were seeded in 96-well plates (5×10³ cells/well) and treated with Napabucasin (BBI-608) (1-5 μM) for 72 hours. Cell viability was assessed by MTT assay, and IC50 values were determined [1] LNCaP cells were seeded in 6-well plates (1×10³ cells/well) with Napabucasin (BBI-608) (3 μM) and cultured for 14 days. Colonies were fixed, stained with crystal violet, and counted under a microscope [1] PC-3 cells were treated with Napabucasin (BBI-608) (3 μM) for 24 hours, stained with Annexin V-FITC/PI, and apoptotic cells were analyzed by flow cytometry. Caspase-3 activity was measured using a colorimetric assay kit [1] Cells were lysed after Napabucasin (BBI-608) treatment (2-4 μM for 24 hours), and protein extracts were subjected to western blot analysis of β-catenin, c-Myc, Cyclin D1, and Bcl-2. Nuclear and cytoplasmic fractions were separated to detect β-catenin nuclear translocation [1] |
| Animal Protocol |
20 mg/kg; i.p. injection mice bearing PaCa-2 xenografts In vivo therapeutic studies[1] A total of 1 × 106 PC‐3 cells or 8 × 106 22RV1 cells in 100 μL of PBS were injected subcutaneously into dorsal flanks of an immunodeficient nude mouse. The animals were treated i.p. with napabucasin (40 mg/kg), docetaxel (10 mg/kg), or PBS q3d once the tumors have reached 50 mm3. The tumor volume (TV) was calculated every 4 days according to the following standard formula: TV (mm3) = length × width2 × 0.5. To determine the effect napabucasin on stem‐like properties in vivo, animals were sacrificed and the tumors were removed in sterile condition. Tumors were disassociated into single‐cell suspensions and counted. Live cells were cultured in suspension culture media for determining the ability of spherogenesis. The media was changed every 3 days, and sphere growth was determined after 10–14 days in culture by counting the number of spheres possessing >50 cells. Nude mice (6-8 weeks old) were subcutaneously injected with C4-2 prostate cancer cells (2×10⁶ cells/mouse). When tumors reached 100 mm³, mice were randomly divided into vehicle and Napabucasin (BBI-608) groups (n=6 per group). Napabucasin (BBI-608) was dissolved in DMSO and suspended in 0.5% methylcellulose (DMSO final concentration <1%) and administered via oral gavage at 200 mg/kg daily for 21 days. Tumor volume was measured every 3 days using calipers, and mice were euthanized to harvest tumors for immunohistochemistry analysis [1] |
| References |
[1]. Suppression of prostate cancer progression by cancer cell stemness inhibitor napabucasin. Cancer Med. 2016 Jun;5(6):1251-8. |
| Additional Infomation |
Napabucasin has been investigated for the treatment of Colorectal Carcinoma. Napabucasin has been reported in Ekmanianthe longiflora, Newbouldia laevis, and Handroanthus impetiginosus with data available. Napabucasin is an orally available cancer cell stemness inhibitor with potential antineoplastic activity. Even though the exact target has yet to be fully elucidated, napabucasin appears to target and inhibit multiple pathways involved in cancer cell stemness. This may ultimately inhibit cancer stemness cell (CSC) growth as well as heterogeneous cancer cell growth. CSCs, self-replicating cells that are able to differentiate into heterogeneous cancer cells, appear to be responsible for the malignant growth, recurrence and resistance to conventional chemotherapies. Napabucasin (BBI-608) is a small-molecule inhibitor of cancer cell stemness, targeting β-catenin/Tcf signaling to suppress tumorigenesis and metastasis [1] Napabucasin (BBI-608) shows therapeutic potential for prostate cancer by reducing cancer stem cell populations and reversing epithelial-mesenchymal transition (EMT) markers [1] The drug is orally bioavailable and well-tolerated in preclinical models [1] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 0.5 mg/mL (2.08 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 5.0 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. Solubility in Formulation 2: 0.5 mg/mL (2.08 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 5.0 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. Solubility in Formulation 3: ≥ 0.5 mg/mL (2.08 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 5.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 4: 5% DMSO+corn oil: 1 mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 4.1630 mL | 20.8151 mL | 41.6302 mL | |
| 5 mM | 0.8326 mL | 4.1630 mL | 8.3260 mL | |
| 10 mM | 0.4163 mL | 2.0815 mL | 4.1630 mL |