NHC-triphosphate tetrasodium directly targets and inhibits viral RNA-dependent RNA polymerases (RdRp). It acts as a weak alternative substrate, meaning the viral polymerase can mistake it for a natural cytidine triphosphate (CTP) and incorporate it into the growing viral RNA chain. Upon incorporation, the compound exhibits a dual mechanism of action: it can act as a non-obligate chain terminator, which prevents the addition of further nucleotides and halts RNA synthesis, or it can cause lethal mutagenesis, where the incorporated analog leads to an accumulation of mutations in the viral genome, rendering the virus non-infectious. This mechanism targets the viral replication machinery and is distinct from that of many other antiviral drugs, making it effective against a wide range of RNA viruses. NHC-TP shows high selectivity for viral polymerases over host DNA polymerases.

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 event that NHC-triphosphate (NHC-TP)(5–40 μM) is absent, full-length polymerization products result; it may serve as a feeble 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 continues to be the most prevalent metabolite, trace levels of NHC-monophosphate (MP) and NHC-diphosphate (DP) are also visible[2]. The NHC-triphosphate (NHC-TP) metabolite has the ability to act as a nonobligate chain terminator by directly targeting the viral polymerase. It is important because it inhibits early negative-strand RNA synthesis via mutagenesis or chain termination, which can obstruct the proper assembly of replicase complexes.

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In an intracellular metabolism assay using HCV replicon cells treated with 10 uM 3H-labeled NHC, it was shown that NHC is rapidly converted into its mono-, di-, and triphosphate forms. NHC-triphosphate (NHC-TP) reached up to 71.12 pM after 8 hours of incubation, demonstrating its efficient intracellular generation. In a cell-free HCV NS5B polymerase activity assay, NHC-TP (5-40 uM) is incorporated into the growing RNA chain instead of CTP. This incorporation increases the molecular weight of the polymerization product by 16 daltons (one extra oxygen) per event and causes an obvious electrophoretic mobility shift, confirming its role as a weak alternative substrate. Huh-7 cells incubated with NHC (10-50 uM for 4 hours) show that NHC-triphosphate remains the most abundant phosphorylated metabolite within the cell, as measured by LC-MS/MS.

No specific in vivo data is available for the direct administration of this tetrasodium salt in animals. Its in vivo activity is known from studies using its parent prodrugs, which are cell-permeable. For instance, molnupiravir, the prodrug of NHC, has demonstrated potent in vivo antiviral efficacy in mouse models of influenza, SARS-CoV-2, and other RNA viruses. The in vivo mechanism involves the conversion of the prodrug to NHC, which then enters cells and is phosphorylated to the active triphosphate metabolite (NHC-TP). NHC-TP is believed to play a prominent role in inhibiting early negative-strand RNA synthesis, either through chain termination or lethal mutagenesis, which in turn interferes with the formation of the correct replicase complex. The tetrasodium salt form is primarily for biochemical assays rather than direct in vivo dosing, as the charged molecule is membrane-impermeable.

Cell-free polymerase assays are used to evaluate the direct inhibitory activity of NHC-triphosphate tetrasodium. The assay is performed with purified viral RNA-dependent RNA polymerase (RdRp). The reaction mixture (50 uL) contains 50 mM Tris-HCl (pH 7.5), 10 mM NaCl, 1 mM MnCl2 or MgCl2, 0.5 uM of RNA template-primer (e.g., a 5‘-biotinylated RNA template), 100 uM each of ATP, GTP, UTP, and varying concentrations of CTP and the competitor NHC-triphosphate (0.1-100 uM). The reaction is initiated by adding 100 ng of purified RdRp and incubating at 30degC for 90-120 minutes. The reaction products can be captured on a Streptavidin-coated plate and detected using a colorimetric or chemiluminescent detection system for biotin-labeled UTP incorporation. For more detailed analysis, the RNA products can be separated on a polyacrylamide gel to observe chain termination or a shift in electrophoretic mobility. The IC50 is determined by plotting the percent inhibition of RNA synthesis as a function of the NHC-triphosphate concentration.

A standard protocol for studying the intracellular metabolism of NHC involves the use of Huh-7 cells. For an intracellular conversion assay, 1×10⁶ Huh-7 cells are seeded in 6-well plates and allowed to adhere overnight. The cells are then incubated with the parent compound, NHC, at a concentration of 10-50 uM for 4 hours. After incubation, the media is removed, and the cells are washed twice with ice-cold PBS. Intracellular metabolites are extracted by adding 500 uL of ice-cold 70% methanol/30% water containing an internal standard (e.g., ¹3C-labeled NHC-triphosphate). The cells are scraped, and the lysate is centrifuged at 15,000 rpm for 10 minutes at 4degC. The supernatant is collected and dried under a stream of nitrogen. The dried extract is reconstituted in an appropriate mobile phase and analyzed by LC-MS/MS. The mass spectrometer is operated in multiple reaction monitoring (MRM) mode to simultaneously quantify the levels of NHC-monophosphate (MP), NHC-diphosphate (DP), and NHC-triphosphate (TP). The tetrasodium salt serves as the primary reference standard for the quantification of NHC-TP in these experiments.

While the charged NHC-triphosphate tetrasodium is not used directly in vivo, its parent prodrug (e.g., molnupiravir) is used extensively. In a mouse efficacy model, 6-8 week old BALB/c mice are infected intranasally with a lethal dose of mouse-adapted influenza virus or SARS-CoV-2 (e.g., 10⁵ PFU per mouse). Starting 4-24 hours post-infection, the mice are treated with the prodrug (e.g., 100-500 mg/kg) administered orally twice daily for 5 days. For the analysis of the active metabolite in the lung, lung tissue is collected from infected mice at various time points after the final dose. The tissue is homogenized, and the active metabolites (including NHC-TP) are extracted. The concentrations of NHC-TP in the lung homogenates are then quantified by LC-MS/MS using the tetrasodium salt as a standard. A reduction in viral titers (measured by TCID50 or plaque assay) and improvement in survival rates (e.g., weight loss, survival %) are the primary endpoints. This protocol demonstrates the link between prodrug administration, active metabolite generation, and antiviral efficacy.

This compound is a tetrasodium salt with a molecular formula of C9H12N3Na4O15P3 and a molecular weight of 587.08 g/mol. It is provided as a white powder and should be stored at -20degC in a sealed container, protected from light and moisture. Under these conditions, it is stable for up to 3 years. The compound is highly soluble in water (up to 100 mg/mL, 170.33 mM) and DMSO. For biological assays, stock solutions can be stored at -80degC for up to 6 months. It is not orally bioavailable due to its high polarity and negative charge, as it lacks the necessary lipophilicity to cross cell membranes. It is designed for use as a reference standard in analytical chemistry and as a substrate in cell-free biochemical assays.

This product is for research use only and is not intended for diagnostic or therapeutic applications in humans. No specific toxicity data is available for this tetrasodium salt. The primary potential hazard is the presence of the sodium salt, which is generally recognized as safe (GRAS). However, standard chemical hygiene practices should be followed when handling the powder to avoid inhalation or ingestion. The compound is an active metabolite of a known antiviral drug, and as such, it possesses biological activity. It should be handled with care in the laboratory to avoid unintended exposure. The salt itself is not considered acutely toxic, but as a nucleotide analog, it could be incorporated into DNA in rapidly dividing cells; however, its design shows high selectivity for viral polymerases.

[1]. Ribonucleoside analogue that blocks replication of bovine viral diarrhea and hepatitis C viruses in culture.Antimicrob Agents Chemother. 2003 Jan;47(1):244-54.

[2]. Characterization of β-d- N4-Hydroxycytidine as a Novel Inhibitor of Chikungunya Virus.

This compound is the active triphosphate form of the nucleoside analog N4-hydroxycytidine (NHC), which is the active metabolite of the prodrug molnupiravir (MK-4482/EIDD-2801), an antiviral agent used to treat COVID-19. This tetrasodium salt is a crucial research standard for studying the pharmacology and mechanism of action of this important class of antiviral drugs. It is instrumental in resistance studies, allowing researchers to examine how mutations in the viral RdRp (e.g., in SARS-CoV-2) affect the incorporation efficiency of the active metabolite. It is also used to screen for novel inhibitors of viral RNA polymerases in high-throughput biochemical assays. The CAS number for the related free acid is 39023-73-9.

C9H12N3NA4O15P3

587.08

NHC-diphosphate;39023-73-9;NHC-triphosphate tetraammonium;NHC-triphosphate;34973-27-8

White to off-white solid powder

2934.99.9001

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 and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O :~100 mg/mL (~170.33 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.)