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| Targets |
The primary biochemical role of NHC-diphosphate is as an intermediate in the intracellular activation of NHC. The enzyme nucleoside diphosphate kinase (NDPK) converts NHC-diphosphate to the active NHC-triphosphate. As such, it does not directly bind to or inhibit viral targets like the RdRp with high affinity. However, it can act as a weak alternative substrate for viral polymerases in cell-free systems, though with much lower efficiency than the triphosphate. The compound is an essential tool for mapping the complete metabolic pathway of NHC and for studying the kinetics of its phosphorylation by cellular kinases. Understanding the kinetics of NHC-DP conversion to NHC-TP is critical for optimizing the pharmacology of this class of antiviral agents.
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| ln Vitro |
HCV replicon cells are treated with 10 μM 3H-labeled NHC in an intracellular metabolism test. After 1, 2, and 8 hours of incubation, intracellular nucleotide levels are measured. NHC is quickly transformed into the mono-, di-, and triphosphate forms; within 8 hours, NHC-TP can reach 71.12 pM[1]. In the absence of NHC-triphosphate triammonium (NHC-TP) (3-5 μM), full-length polymerization products result, and it may be a poor substitute substrate. Furthermore, in cell-free HCV NS5B polymerization events, an evident electrophoretic shift is seen, and the molecular weight of the polymerization product increases by 16 (one extra oxygen) upon insertion of NHC-TP rather than CTP[1]. NHC (10–50 μM; 4 h) or a McGuigan phosphoramidate prodrug of NHC are cultured in Huh-7 cells. LC-MS/MS is used to evaluate the intracellular levels of the primary chemicals and phosphorylated metabolites. While NHC-triphosphate triammonium continues to be the most prevalent metabolite, trace levels of NHC-monophosphate (MP) and NHC-diphosphate (DP) are also visible[2]. The metabolite NHC-triphosphate triammonium (NHC-TP) may act as a nonobligate chain terminator and directly target the viral polymerase. Through chain termination or mutagenesis, it inhibits early negative-strand RNA synthesis in a significant way. This can potentially obstruct the proper assembly of replicase complexes.
In an intracellular metabolism assay using HCV replicon cells, NHC-diphosphate is observed as one of the phosphorylated metabolites following treatment with 10 uM 3H-labeled NHC. In cell-free HCV NS5B polymerase assays, the incorporation of the triphosphate form is the primary mechanism of action, while the diphosphate form has minimal effect. Studies have shown that NHC is rapidly converted into its mono-, di-, and triphosphate forms in cells. The conversion of NHC-diphosphate to NHC-triphosphate is a rate-limiting step in some cellular contexts, and the diphosphate form can accumulate to measurable levels as an intermediate. The triammonium salt is used primarily as an analytical standard for the quantification of NHC-DP in cell lysates and tissue samples via LC-MS/MS. |
| ln Vivo |
No in vivo data is available for the direct administration of this salt. The in vivo activity of the parent NHC prodrug is well-characterized. After oral administration of the prodrug, it is rapidly de-esterified to NHC, which then enters cells. Inside the cell, NHC is sequentially phosphorylated to NHC-monophosphate (NHC-MP), NHC-diphosphate (NHC-DP), and finally to the active NHC-triphosphate (NHC-TP). The diphosphate is a crucial intermediate, and its concentration is a biomarker for the metabolic capacity of cells to activate the drug. The triammonium salt is used to accurately measure the levels of this intermediate in pharmacokinetic and pharmacodynamic studies to understand the rate and extent of prodrug activation in different tissues, such as the liver and lungs.
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| Enzyme Assay |
Cell-free assays can be performed to measure the substrate activity of NHC-diphosphate for NDPK or its direct (weak) inhibitory activity against RdRp. For a NDPK activity assay, a 96-well plate is prepared with the reaction mixture: 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 10% glycerol, and the substrate NHC-diphosphate at various concentrations (0-500 uM). The reaction is initiated by the addition of 10 ng of recombinant human NDPK-A, 1 mM ATP, and an ATP-regeneration system (phosphoenolpyruvate and pyruvate kinase). The plate is incubated at 37degC. After 30 minutes, the reaction is stopped by heating to 95degC for 5 minutes. The product, NHC-triphosphate, can be quantified by HPLC-UV or LC-MS/MS. This allows for the determination of the Km and catalytic efficiency of NDPK towards NHC-DP. For an RdRp assay, the same protocol as for the triphosphate is used, but the diphosphate is added to assess its direct competitive inhibition against CTP.
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| Cell Assay |
A typical protocol for measuring intracellular NHC-DP levels involves treating cells with the parent NHC or its prodrug. Huh-7 or primary human hepatocytes are seeded in 6-well plates at 7×10⁵ cells per well. The next day, the cells are treated with 10-50 uM of NHC for 4-24 hours. The cells are then washed three times with ice-cold PBS to remove extracellular drug. Metabolites are extracted by adding 0.5 mL of ice-cold 80% methanol containing an internal standard (e.g., ¹3C-labeled NHC-DP). The cells are scraped, and the mixture is transferred to a microcentrifuge tube. The samples are vortexed, incubated at -20degC for 20 minutes, and then centrifuged at 13,000 rpm for 10 minutes at 4degC. The supernatant is transferred to a clean vial and evaporated to dryness under a stream of nitrogen at 37degC. The residue is reconstituted in 50 uL of a 1:1 mixture of water and methanol. The reconstituted sample is injected onto a C18 column coupled to a triple-quadrupole mass spectrometer. The NHC-diphosphate triammonium salt is used as the primary standard to generate a calibration curve for quantification. The levels of NHC-DP and NHC-TP are monitored to assess the efficiency of the cellular phosphorylation cascade.
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| Animal Protocol |
As with the triphosphate, the diphosphate is not used in vivo because it is charged and membrane-impermeable. Its primary in vivo application is as an analyte to be measured in tissue samples following administration of the parent prodrug. In a typical mouse pharmacokinetic study, 6-8 week old BALB/c mice are administered the prodrug (e.g., molnupiravir, 200 mg/kg) by oral gavage. At various time points (0.5, 1, 2, 4, 6, 8, and 24 hours post-dose), groups of mice (n=3 per time point) are euthanized. Blood is collected via cardiac puncture, and tissues of interest (e.g., liver, lung) are immediately excised, snap-frozen in liquid nitrogen, and stored at -80degC. Tissues are homogenized in a cold extraction solution containing the internal standard. NHC-DP and NHC-TP levels in the homogenates are extracted and quantified using LC-MS/MS with the NHC-diphosphate triammonium salt as the calibration standard. The resulting concentration-time data are used to calculate the pharmacokinetic parameters (Cmax, Tmax, AUC, t½) of the active metabolites in the target tissues.
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| ADME/Pharmacokinetics |
This compound has a molecular formula of C9H24N6O12P2 and a molecular weight of 470.27 g/mol. It appears as a white to off-white solid. It should be stored at -20degC, protected from light and stored under an inert atmosphere (e.g., nitrogen) to prevent degradation. Under these conditions, the powder is stable for up to 3 years, and solutions stored at -80degC are stable for up to 6 months. The triammonium salt is highly polar and is readily soluble in water (typically >10 mg/mL). It is not cell-permeable and is not intended for direct therapeutic use in vivo. The compound is a necessary standard for pharmacology studies. The purity is typically >98%.
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| Toxicity/Toxicokinetics |
This product is for research use only and is not for human diagnostic or therapeutic use. No specific toxicity data is available for this triammonium salt. The primary potential hazard is the ammonium counterion, which can be an irritant, but it is present at low levels. Standard chemical hygiene protocols should be followed when handling the powder, including the use of lab coats, gloves, and safety glasses. As a nucleotide analog, researchers should exercise caution as it may be metabolized to the active triphosphate, which has potent antiviral activity, though this is unlikely via dermal exposure. The TFA salt form is not applicable; this is a triammonium salt, which is commonly used to increase the solubility of polar nucleotides.
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| References | |
| Additional Infomation |
NHC-diphosphate is a key pharmacologically active intermediate responsible for the broad-spectrum antiviral activity of molnupiravir. The diphosphate form has minimal direct activity but is rapidly converted to the active triphosphate by cellular NDPK enzymes. The triammonium salt enhances solubility and stability, making it suitable for biochemical assays and antiviral mechanism studies. It is widely used in research on viral replication and RNA virus inhibition, providing a tool for understanding the kinetics of drug activation. It serves as a critical standard in pharmacokinetic studies to distinguish between metabolic activation bottlenecks (lack of diphosphate vs. lack of triphosphate) in different cell types. The diphosphate can sometimes serve as a more stable surrogate for the triphosphate in certain biochemical contexts. This product is a specific tool for metabolic profiling and analytical chemistry.
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| Molecular Formula |
C9H24N6O12P2
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| Molecular Weight |
470.27
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| Related CAS # |
NHC-diphosphate;39023-73-9
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| Appearance |
White to off-white solid powder
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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: (1). This product requires protection from light (avoid light exposure) during transportation and storage. (2). 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) |
H2O :~160 mg/mL (~340.23 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: 50 mg/mL (106.32 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
(Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1264 mL | 10.6322 mL | 21.2644 mL | |
| 5 mM | 0.4253 mL | 2.1264 mL | 4.2529 mL | |
| 10 mM | 0.2126 mL | 1.0632 mL | 2.1264 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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