EIDD-1931 (Beta-d-N4-hydroxycytidine; NHC)

Alias: EIDD-1931; EIDD 1931; EIDD1931; N4-Hydroxycytidine; β-D-N4-hydroxycytidine; Uridine, 4-oxime; NHC; EIDD-2801-metabolite; Molnupiravir-,etabolite

Cat No.:V39191 Purity: ≥98%

COA Product Data Instructions for use SDS

EIDD-1931(EIDD1931; Beta-d-N4-hydroxycytidine; NHC) is anovel and potentribonucleoside analog with antiviral activity.

EIDD-1931 (Beta-d-N4-hydroxycytidine; NHC) Chemical Structure CAS No.: 3258-02-4
Product category: SARS-CoV

This product is for research use only, not for human use. We do not sell to patients.

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Top Publications Citing lnvivochem Products

Nature 2021, 597(7874):119-125.

Cell 2020,183:1202-1218.e25.

Science Adv 2022: 8, eabm9427.

Nature 597, 119–125 (2021)

Cell Stem Cell 2020,26(6):845-861.e12.

Science Trans Med 2023,15(701):eadd7872.

STTT 2023,8(1):91.

Cell Reports 2023,42(4):112313

Science Trans Med 2023, 15: eadd7872.

Cancer Cell 2021,39(4):529-547.e7.

Cancer Discov 2022,12(4):1106-1127.

Immunity 2023,56(3):620-634.e11.

J Am Chem Soc 2022,144:21831-21836.

Cell 2020,183:1202-1218.e25.

Science Adv 2022:8,eabm9427.

Nature 2021,597(7874):119-125.

Cell 2020,183:1202-1218.e25.

PNAS Nexus 2022,1(3):pgac063.

ACS Nano 2023,17(10):9110-9125.

Biomaterial 2023 Sep16:302:122333.

Purity & Quality Control Documentation

Current batch：

Purity: ≥98%

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MSDS

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Instructions

Product Description
Bioactivity & Protocols
Physicochemical Properties
Solubility Data
Calculators
Biological Data

Product Description

EIDD-1931 (EIDD1931; Beta-d-N4-hydroxycytidine; NHC) is a novel and potent ribonucleoside analog with antiviral activity. EIDD-1931 is an active metabolite of the UK-approved anti-COVID-19 drug Molnupiravir (EIDD2801; prodrug-EIDD1931; MK-4482; Lagevrio), thus has the potential to be used as an anti-COVID-19 drug. EIDD-1931 has a broad spectrum antiviral activity and can inhibit replication of severe acute respiratory syndrome coronavirus (SARS-CoV) in Vero 76 cells, Middle East respiratory syndrome coronavirus (MERS-CoV) in Calu-3 2B4 cells, and SARS-CoV-2 in Vero cells (IC 50 s =0.1, 0.15 and 0.3 μM, respectively. It has increased potency against a coronavirus bearing resistance mutations to the nucleoside analog inhibitor remdesivir.

Biological Activity I Assay Protocols (From Reference)

ln Vitro	The anti-VEEV (venezuelan equine encephalitis virus) agent beta-d-N4-hydroxycytidine has EC50, EC90, and EC99 values of 0.426, 1.036, and 2.5 μM, respectively[1]. In the Huh-7–CHIKV replicon cell line, beta-d-N4-hydroxycytidine inhibits CHIKV replicon activity and the 50% effective concentration (EC50) is 0.8 μM. Comparable outcomes have been reported regarding the replicon in BHK-21 cells (EC50=1.8 μM).According to MTT assays, NHC does not cause any cytotoxicity in the Huh-7 cell culture system up to 100 μM. For peripheral blood mononuclear (PBM), Vero, and CEM cells, the 50% cytotoxic concentration (CCsub>50) values for NHC are found to be 30.6 μM, 7.7 μM, and 2.5 μM, respectively.NHC functions as a pyrimidine analog; exogenous nucleosides, such as pyrimidines C and U, can reverse NHC-mediated inhibition of the CHIKV replicon; however, the replicon is unaffected by dA, dC, dG, dU, or T. Pyrimidines A and G have a role in replicon inhibition both with and without NHC [2].
References	[1]. β-d-N4-Hydroxycytidine Is a Potent Anti-alphavirus Compound That Induces a High Level of Mutations in the Viral Genome. J Virol. 2018 Jan 17;92(3). pii: e01965-17. [2]. Metabolism of the anti-hepatitis C virus nucleoside beta-D-N4-hydroxycytidine in different liver cells. Antimicrob Agents Chemother. 2004 Dec;48(12):4636-42 [3]. Characterization of β-d- N4-Hydroxycytidine as a Novel Inhibitor of Chikungunya Virus.

ln Vitro

The anti-VEEV (venezuelan equine encephalitis virus) agent beta-d-N4-hydroxycytidine has EC50, EC90, and EC99 values of 0.426, 1.036, and 2.5 μM, respectively[1].
In the Huh-7–CHIKV replicon cell line, beta-d-N4-hydroxycytidine inhibits CHIKV replicon activity and the 50% effective concentration (EC50) is 0.8 μM. Comparable outcomes have been reported regarding the replicon in BHK-21 cells (EC50=1.8 μM).According to MTT assays, NHC does not cause any cytotoxicity in the Huh-7 cell culture system up to 100 μM. For peripheral blood mononuclear (PBM), Vero, and CEM cells, the 50% cytotoxic concentration (CCsub>50) values for NHC are found to be 30.6 μM, 7.7 μM, and 2.5 μM, respectively.NHC functions as a pyrimidine analog; exogenous nucleosides, such as pyrimidines C and U, can reverse NHC-mediated inhibition of the CHIKV replicon; however, the replicon is unaffected by dA, dC, dG, dU, or T. Pyrimidines A and G have a role in replicon inhibition both with and without NHC [2].

References

[1]. β-d-N4-Hydroxycytidine Is a Potent Anti-alphavirus Compound That Induces a High Level of Mutations in the Viral Genome. J Virol. 2018 Jan 17;92(3). pii: e01965-17.

[2]. Metabolism of the anti-hepatitis C virus nucleoside beta-D-N4-hydroxycytidine in different liver cells. Antimicrob Agents Chemother. 2004 Dec;48(12):4636-42

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

These protocols are for reference only. InvivoChem does not independently validate these methods.

Physicochemical Properties

Molecular Formula	C9H13N3O6
Molecular Weight	259.21602
Exact Mass	259.08
Elemental Analysis	C, 41.70; H, 5.06; N, 16.21; O, 37.03
CAS #	3258-02-4
Appearance	Solid powder
SMILES	C1(N2C=C/C(=N\O)/NC2=O)OC(CO)C(O)C1O
InChi Key	XCUAIINAJCDIPM-XVFCMESISA-N
InChi Code	InChI=1S/C9H13N3O6/c13-3-4-6(14)7(15)8(18-4)12-2-1-5(11-17)10-9(12)16/h1-2,4,6-8,13-15,17H,3H2,(H,10,11,16)/t4-,6-,7-,8-/m1/s1
Chemical Name	N4-Hydroxycytidine
Synonyms	EIDD-1931; EIDD 1931; EIDD1931; N4-Hydroxycytidine; β-D-N4-hydroxycytidine; Uridine, 4-oxime; NHC; EIDD-2801-metabolite; Molnupiravir-,etabolite
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 Note: This product is not stable in solution, please use freshly prepared working solution for optimal results.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility Data

Solubility (In Vitro)	DMSO : ~100 mg/mL (~385.77 mM) H2O : ≥ 25 mg/mL (~96.44 mM)
Solubility (In Vivo)	Solubility in Formulation 1: ≥ 2.08 mg/mL (8.02 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 (8.02 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 (8.02 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.)

Solubility (In Vitro)

DMSO : ~100 mg/mL (~385.77 mM)
H2O : ≥ 25 mg/mL (~96.44 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.08 mg/mL (8.02 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 (8.02 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 (8.02 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

(Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	3.8577 mL	19.2886 mL	38.5773 mL
	5 mM	0.7715 mL	3.8577 mL	7.7155 mL
	10 mM	0.3858 mL	1.9289 mL	3.8577 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.

Calculator

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Molarity Calculator allows you to calculate the mass, volume, and/or concentration required for a solution, as detailed below:

Calculate the Mass of a compound required to prepare a solution of known volume and concentration
Calculate the Volume of solution required to dissolve a compound of known mass to a desired concentration
Calculate the Concentration of a solution resulting from a known mass of compound in a specific volume

See Example

An example of molarity calculation using the molarity calculator is shown below:
What is the mass of compound required to make a 10 mM stock solution in 5 ml of DMSO given that the molecular weight of the compound is 350.26 g/mol?

Enter 350.26 in the Molecular Weight (MW) box
Enter 10 in the Concentration box and choose the correct unit (mM)
Enter 5 in the Volume box and choose the correct unit (mL)
Click the “Calculate” button
The answer of 17.513 mg appears in the Mass box. In a similar way, you may calculate the volume and concentration.

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Dilution Calculator allows you to calculate how to dilute a stock solution of known concentrations. For example, you may Enter C1, C2 & V2 to calculate V1, as detailed below:

What volume of a given 10 mM stock solution is required to make 25 ml of a 25 μM solution?
Using the equation C1V1 = C2V2, where C1=10 mM, C2=25 μM, V2=25 ml and V1 is the unknown:

Enter 10 into the Concentration (Start) box and choose the correct unit (mM)
Enter 25 into the Concentration (End) box and select the correct unit (mM)
Enter 25 into the Volume (End) box and choose the correct unit (mL)
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The answer of 62.5 μL (0.1 ml) appears in the Volume (Start) box

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Molecular Weight Calculator allows you to calculate the molar mass and elemental composition of a compound, as detailed below:

Note: Chemical formula is case sensitive: C12H18N3O4 c12h18n3o4
Instructions to calculate molar mass (molecular weight) of a chemical compound:

To calculate molar mass of a chemical compound, please enter the chemical/molecular formula and click the “Calculate’ button.

Definitions of molecular mass, molecular weight, molar mass and molar weight:

Molecular mass (or molecular weight) is the mass of one molecule of a substance and is expressed in the unified atomic mass units (u). (1 u is equal to 1/12 the mass of one atom of carbon-12)
Molar mass (molar weight) is the mass of one mole of a substance and is expressed in g/mol.

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Reconstitution Calculator allows you to calculate the volume of solvent required to reconstitute your vial.

Enter the mass of the reagent and the desired reconstitution concentration as well as the correct units
Click the “Calculate” button
The answer appears in the Volume (to add to vial) box

In vivo Formulation Calculator (Clear solution)

Step 1: Enter information below (Recommended: An additional animal to make allowance for loss during the experiment)

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Step 2: Enter in vivo formulation (This is only a calculator, not the exact formulation for a specific product. Please contact us first if there is no in vivo formulation in the solubility section.)

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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.

Note： (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.

Biological Data

NHC has a strong negative effect on VEEV TC-83 replication. [1].J Virol. 2018 Jan 17;92(3).
The antiviral effect of NHC depends on its application time. [1].J Virol. 2018 Jan 17;92(3).
The first 4 h p.i. are a critical time for the antiviral effect of NHC. [1].J Virol. 2018 Jan 17;92(3).
NHC is a potent anti-VEEV compound with low cytotoxicity. [1].J Virol. 2018 Jan 17;92(3).
VEEV TC-83 accumulates a large number of mutations when exposed to NHC. [1].J Virol. 2018 Jan 17;92(3).
NHC has stronger negative effects on the release and infectivity of VEEV TC-83 and PREV1 particles than those of the PP2 mutant. [1].J Virol. 2018 Jan 17;92(3).
Drug-resistant VEEV isolate PP2 replicates more efficiently than parental VEEV TC-83 and pseudorevertant PREV1 in the presence, but not in the absence of NHC. [1].J Virol. 2018 Jan 17;92(3).
VEEV TC-83 passaging in the presence of increasing concentrations of NHC leads to rapid accumulation of mutations in viral pool.[1].J Virol. 2018 Jan 17;92(3).
The mutations that lead to NHC-resistant and NHC-sensitive phenotypes of VEEV TC-83 are closely located in the 3D structure of the catalytic domain of VEEV nsP4. [1].J Virol. 2018 Jan 17;92(3).