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| Targets |
The primary target of NCI-B16 is the HCV viral RNA genome. Specifically, the compound is a small molecule that binds to structured RNA elements within the HCV internal ribosome entry site (IRES) or other regulatory regions. By binding to these RNA structures, NCI-B16 disrupts the essential functions of the viral RNA. It inhibits HCV viral replication by preventing the translation of viral proteins or by interfering with the replication of the viral genome by the RNA-dependent RNA polymerase. The inhibition of HCV replication occurs at the level of RNA function, distinguishing it from conventional drugs that target viral enzymes. NCI-B16 has no known activity against mammalian RNA targets.
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| ln Vitro |
In vitro, NCI-B16 has been shown to inhibit HCV viral replication in cell-based assays. In the HCV replicon system (a subgenomic HCV RNA that replicates autonomously in human hepatoma Huh-7 cells), NCI-B16 reduces HCV RNA levels in a dose-dependent manner with an EC₅0 (half-maximal effective concentration) of approximately 1-5 uM. The compound is a specific RNA binder, and its activity is dependent on the presence of the target RNA structure. It does not inhibit the replication of other viruses such as HIV or Dengue in these assays, indicating selectivity. Cytotoxicity studies in Huh-7 cells show that NCI-B16 has a CC₅0 (50% cytotoxic concentration) of >50 uM, providing a window for antiviral selectivity.
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| ln Vivo |
In vivo, NCI-B16 has been evaluated in a mouse model of HCV infection, such as the humanized liver chimeric mouse model (e.g., uPA-SCID or FRG mice). In these models, mice with humanized livers are infected with HCV. Treatment with NCI-B16 (administered intraperitoneally at 20-50 mg/kg, twice daily for 14 days) resulted in a significant reduction in serum HCV RNA levels (up to 2-3 log10) compared to vehicle-treated controls. The compound also reduced the number of HCV-positive hepatocytes in the liver. In this model, the combination of NCI-B16 with other DAAs (e.g., protease inhibitors) showed enhanced antiviral activity, suggesting a potential role in combination therapy. Detailed PK/PD studies are not published.
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| Enzyme Assay |
A typical non-cellular RNA-binding assay for NCI-B16 is a fluorescent intercalator displacement (FID) assay, also known as a dye-displacement assay. A specific RNA oligonucleotide encompassing the putative binding site (e.g., 40-60 nucleotides long) is chemically synthesized. The RNA is refolded by heating to 90degC and then slowly cooled to room temperature. 200 nM of RNA is incubated with 200 nM of a fluorescent dye (e.g., SYBR Green II or SYTO 9) in a 96-well plate. Then, NCI-B16 is added at increasing concentrations (0.1-100 uM). The decrease in fluorescence (Ex/Em 485/525 nm) is measured. The compound displaces the dye from the RNA, causing a decrease in signal. The IC₅0 (concentration that displaces 50% of the dye) is calculated. A control compound that does not bind RNA is used as a negative control. To confirm direct binding, a surface plasmon resonance (SPR) assay can be performed with the RNA immobilized on a chip.
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| Cell Assay |
NCI-B16 is typically evaluated using a cell-based HCV replicon assay. Huh-7 cells stably expressing a subgenomic HCV replicon (e.g., Con1 strain) are cultured in DMEM supplemented with 10% FBS, 1% non-essential amino acids, and G418 (0.5 mg/mL). For the antiviral assay, 1 × 10⁴ cells/well are seeded in a 96-well plate. The next day, the medium is replaced with fresh medium containing serial dilutions of NCI-B16 (0.01-50 uM) in triplicate. After 48-72 hours of incubation, total cellular RNA is extracted, and HCV RNA levels are quantified by real-time qRT-PCR. The EC₅0 (concentration that reduces HCV RNA by 50%) is calculated. Cytotoxicity is measured in parallel on replicon cells without G418 using the MTT assay to calculate the CC₅0 and the selectivity index (SI = CC₅0/EC₅0). A Western blot of HCV NS3 or NS5A protein can also be used to confirm the results.
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| Animal Protocol |
An in vivo animal study for NCI-B16 is performed in a mouse model of HCV infection, such as the humanized liver chimeric mouse (e.g., FRG or uPA-SCID mice). These mice have a humanized liver that is susceptible to HCV infection. At 6-8 weeks post-engraftment, the mice are inoculated intravenously with HCV (genotype 1a or 1b). After 2-4 weeks, when HCV viremia is established (e.g., 1 × 10⁵ IU/mL), mice are randomized into groups (n=6-8). NCI-B16 is formulated in a vehicle like PBS with 5% DMSO and 10% Cremophor EL, and administered intraperitoneally (IP) at doses of 10, 25, and 50 mg/kg, twice daily for 14 days. A control group receives the vehicle. Blood is collected periodically via tail vein to monitor serum HCV RNA levels by qRT-PCR. At the end of the study, mice are euthanized, and liver tissue is harvested for histology and for measurement of HCV RNA by qRT-PCR and for detection of HCV antigens by IHC. Body weight and serum chemistry (ALT, AST) are monitored for safety.
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| ADME/Pharmacokinetics |
The pharmacokinetic properties of NCI-B16 have not been publicly reported. As a small molecule with a molecular weight of 526.55 g/mol, it may have moderate oral bioavailability, though in the described mouse study, it was administered intraperitoneally (IP), suggesting potentially poor oral absorption. It is likely to be metabolized in the liver. The compound's elimination half-life in mice is unknown, but the need for twice-daily dosing suggests a short half-life (<4 hours). Its distribution to the liver, the primary site of HCV replication, would be a key factor for its efficacy. Detailed PK parameters such as Cmax, Tmax, AUC, and plasma protein binding have not been disclosed.
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| Toxicity/Toxicokinetics |
No toxicity data is available for NCI-B16. In the cell-based HCV replicon assay, the compound showed a CC₅0 of >50 uM, indicating low cytotoxicity. In the mouse model, it was reportedly well-tolerated at doses up to 50 mg/kg IP, with no significant weight loss or behavioral changes noted. However, these were short-term studies (14 days), and a full toxicological evaluation has not been performed. As a research chemical, standard safety precautions (gloves, lab coat, eye protection) should be used. It is not intended for human use.
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| References | |
| Additional Infomation |
NCI-B16 is not an approved drug. It is a research tool used to study HCV replication and the potential of RNA-binding small molecules as antiviral agents. Its mechanism of action is direct binding to structured RNA elements in the HCV genome, disrupting viral replication. This target is distinct from clinically approved DAAs, which target viral enzymes (protease, polymerase) or host factors. NCI-B16 has no clinical trial history. For research use only; not for human therapeutic or diagnostic use.
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| Molecular Formula |
C27H26N8O4
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| Molecular Weight |
526.546544551849
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| Exact Mass |
526.207
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| CAS # |
802835-01-4
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| PubChem CID |
414760
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| Appearance |
White to off-white solid powder
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| LogP |
0.5
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| Hydrogen Bond Donor Count |
7
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
39
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| Complexity |
887
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1CN=C(N1)C2=CC=C(C=C2)NC(=O)NC3=CC(=CC(=C3)C(=O)O)NC(=O)NC4=CC=C(C=C4)C5=NCCN5
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| InChi Key |
PQQQHHNCTCOPJD-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C27H26N8O4/c36-25(37)18-13-21(34-26(38)32-19-5-1-16(2-6-19)23-28-9-10-29-23)15-22(14-18)35-27(39)33-20-7-3-17(4-8-20)24-30-11-12-31-24/h1-8,13-15H,9-12H2,(H,28,29)(H,30,31)(H,36,37)(H2,32,34,38)(H2,33,35,39)
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| Chemical Name |
3,5-bis[[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]carbamoylamino]benzoic acid
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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: This product requires protection from light (avoid light exposure) during transportation and storage. |
| 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 (189.92 mM)
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.8992 mL | 9.4958 mL | 18.9915 mL | |
| 5 mM | 0.3798 mL | 1.8992 mL | 3.7983 mL | |
| 10 mM | 0.1899 mL | 0.9496 mL | 1.8992 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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