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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.
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 |
| PubChem CID | 197020 |
| Appearance | White to off-white solid powder |
| LogP | -2.2 |
| Hydrogen Bond Donor Count | 5 |
| Hydrogen Bond Acceptor Count | 6 |
| Rotatable Bond Count | 3 |
| Heavy Atom Count | 18 |
| Complexity | 398 |
| Defined Atom Stereocenter Count | 4 |
| 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; N(4)-Hydroxycytidine; 3258-02-4; EIDD-1931; Beta-D-N4-hydroxycytidine; Uridine, 4-oxime; N-hydroxycytidine; 4-N-Hydroxycytidine; 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) |
Biological Activity
| Targets | P2Y12 Receptor |
| 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]. |
| ln Vivo |
A ribonucleoside analog EIDD-1931 (β-D-N4-hydroxycytidine), an inhibitor of VEEV, has been characterized in mouse model.[4] • EIDD-1931 is orally available with successful delivery to brain tissue and conversion to the active 5′-triphosphate form.[4] • EIDD-1931 demonstrated good tolerability in 7-day dose ranging toxicology study up to 1000 mg/kg/day.[4] • EIDD-1931 protects mice against lethal intranasal challenge with VEEV when dosed prophylactically or therapeutically.[4] |
| Enzyme Assay |
Determination of intracellular NHC-TP t1/2s. [2] Huh-7 cells (2.5 × 106 per well of a six-well plate) were incubated with 10 μM [3H]NHC (β-d-N4-hydroxycytidine (NHC), 500 dpm/pmol) for a period of 24 h at 37°C in a 5% CO2 atmosphere. The cells were then washed three times with drug-free medium to remove extracellular NHC and incubated with regular culture medium for specific time periods (0, 1, 2, 4, 8, and 24 h). Intracellular metabolites were extracted as described below. Determination of intracellular metabolites. [2] At selected times for NHC-TP accumulation or for the determination of NHC-TP t1/2 study time points, extracellular medium was removed and the cell layer was washed with cold phosphate-buffered saline. After cell scraping with 60% methanol (1 ml), NHC and its respective metabolites were extracted by incubation overnight at −20°C, and then samples were centrifuged at 14,000 rpm for 5 min and supernatant was collected. This was followed by extraction for 1 h on ice the next day (200 μl with 60% methanol), and samples were centrifuged again at 14,000 rpm (Eppendorf centrifuge model 5415C) for 5 min. Extracts were combined, dried under a gentle filtered airflow, and then stored at −20°C until they were analyzed by HPLC. Residues were resuspended in 200 μl of water, and aliquots were injected into the HPLC column. Stability study of NHC in monkey and human whole blood. [2] Ten micromolar [3H]NHC (1,000 dpm/pmol) was incubated in either monkey or human blood for different periods of time (0, 0.08, 0.16, 1, 2, 4, and 24 h). At a selected time point, an aliquot of 200 μl was taken and centrifuged at 14,000 rpm for 5 min. The supernatant was collected, and 500 μl of acetonitrile was added and mixed. The sample was recentrifuged at 14,000 rpm for 5 min, and the supernatant was dried using a DNA speed vacuum. Residues were resuspended in 200 μl of water, and aliquots were injected into the HPLC column. |
| Cell Assay |
Analysis of viral replication. A total of 5 × 105 Vero cells were seeded into six-well Costar plates and infected at MOIs indicated in the figure legends. β-d-N4-hydroxycytidine (NHC) was added to the cells at the indicated times, media were harvested, and viral titers in the samples were determined by a plaque assay on Vero cells as described elsewhere[1] β-d-N4-hydroxycytidine (NHC) was found to have selective anti-hepatitis C virus (HCV) activity in the HCV replicon system. The intracellular metabolism of tritiated NHC was investigated in the HCV replicon system, Huh-7 cells, HepG2 cells, and primary human hepatocytes. Incubation of cells with 10 microM radiolabeled NHC demonstrated extensive and rapid phosphorylation in all liver cells. Besides the 5'-mono, -di-, and -triphosphate metabolites of NHC, other metabolites were characterized. These included cytidine and uridine mono-, di-, and triphosphates. UTP was the predominant early metabolite in Huh-7 cells and primary human hepatocytes, suggesting deamination of NHC as the primary catabolic pathway. The intracellular half-lives of radiolabeled NHC-triphosphate and of CTP and UTP derived from NHC incubation in Huh-7 cells were calculated to be 3.0 +/- 1.3, 10.4 +/- 3.3, and 13.2 +/- 3.5 h (means +/- standard deviations), respectively. Studies using monkey and human whole blood demonstrated more-rapid deamination and oxidation in monkey cells than in human cells, suggesting that NHC may not persist long enough in plasma to be delivered to liver cells.[2] |
| Animal Protocol |
Pharmacokinetics and tissue distribution in mice[4] Female ICR (CD-1®) mice, 6–8 weeks of age, were used in the studies (to match mice used in efficacy studies). EIDD-1931 was administered by oral gavage (PO) in 240 mM citrate buffer pH 3 ± 0.3 or intraperitoneally (IP) in saline. The oral doses tested were 50, 150 and 500 mg/kg of body weight, and the IP doses were 10 and 50 mg/kg of body weight. Blood samples were collected at 0.08, 0.25, 0.5, 1, 2, 4, 8, and 24 h post IP administration, and at 0.25, 0.5, 1, 2, 3, 4, 8, and 24 h post oral administration. Plasmas were prepared within 30 min after collection by centrifugation at 2000g for 10 min at 4 °C and stored at −80 °C before processing for analysis by LC-MS/MS. Mouse organs (lung, spleen liver, kidney, heart and brain) were collected from all mice immediately following blood collection starting from 0.5 h post dose. The tissues were immediately snap-frozen in liquid nitrogen and stored at −80 °C before processing for analysis by LC-MS/MS. Dose range finding (DRF) toxicology and toxicokinetic study[4] This study was conducted in two phases: Phase A (single dose, acute toxicity) and Phase B (multiple doses). During Phase A, two groups of 6 mice (3 males and 3 females each) were administered EIDD-1931 once via oral gavage at 500 and 1000 mg/kg dose levels, and following a four-day washout period the same animals were administered 1500 and 2000 mg/kg doses. The compound was delivered at 10 ml/kg volumes in sodium citrate vehicle (0.24M sodium citrate, pH 3 ± 0.3). After dosing, the animal's weight, food consumption, general physical appearance and behavior were monitored twice daily for four days. During Phase B, EIDD-1931 was administered once daily for 7 consecutive days. Ten male and ten female mice per dose (80 mice total) were tested at dose levels of 200, 500, and 1000 mg/kg/day administered at a dose volume of 10 mL/kg. [4] Animals in the toxicokinetic (TK) arm of the study received EIDD-1931 at the same doses and dose volumes and in the same manner as the main study groups at doses of 200, 500, and 1000 mg/kg/day. Thirty six male and 36 female mice per dose level were used in the TK arm. Blood samples were collected from TK animals for determination of the plasma concentrations of EIDD-1931. Samples were collected from cohorts of 3 TK animals/sex/group/timepoint at 1, 2, 4, 6, 8, and 24 h postdose on Day 1 and at predose and 1, 2, 4, 6, and 8 h postdose on Day 7. Samples were collected in tubes containing lithium heparin as an anticoagulant and kept on ice. Murine models of intranasal VEEV infection[4] Seven to eight-week-old ICR (Crl:CD1) female mice were used in all studies. The dose dependency of EIDD-1931 was determined in a prophylaxis study. Four groups of mice were dosed via gavage with 150, 300 or 500 mg/kg EIDD-1931 in 240 mM sodium citrate buffer pH 3 ± 0.3 or mock-treated with vehicle only, all at 10 ml/kg dose volume, starting at 2 h before infection. The second treatment was delivered at +2 h post-infection (PI), and then the treatment was continued twice daily (b.i.d.) for 6 days. In a second (therapeutic) study, treatment with 500 mg/kg EIDD-1931 was initiated starting at 6, 12, 24 or 48 h post-infection and the treatment was compared to a vehicle (mock) treated group. For the +6 h group, the second treatment was performed at 12 h post-infection and then, for all groups, the treatment was continued every 12 h (b.i.d.) for 6 days. |
| ADME/Pharmacokinetics |
Absorption, Distribution and Excretion N4-hydroxycytidine is orally bioavailable in mice but poorly bioavailable in non-human primates. Metabolism / Metabolites N4-hydroxycytidine distributes into tissues where it is is phosphorylated to the 5'-triphosphate form. |
| 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. Antimicrob Agents Chemother. 2017 Mar 24;61(4):e02395-16. [4]. The prophylactic and therapeutic activity of a broadly active ribonucleoside analog in a murine model of intranasal venezuelan equine encephalitis virus infection. Antiviral Res . 2019 Nov:171:104597. |
| Additional Infomation |
N(4)-hydroxycytidine is a nucleoside analogue that is cytidine which carries a hydroxy group at the N(4)-positon. It has broad-spectrum antiviral activity against influenza, SARS-CoV , SARS-CoV-2 and MERS-CoV. It has a role as a drug metabolite, a human xenobiotic metabolite, an anticoronaviral agent and an antiviral agent. It is a nucleoside analogue and a ketoxime. It is functionally related to a cytidine. N4-Hydroxyctidine, or EIDD-1931, is a ribonucleoside analog which induces mutations in RNA virions. N4-hydroxycytidine was first described in the literature in 1980 as a potent mutagen of bacteria and phage. It has shown antiviral activity against Venezuelan equine encephalitis virus, and the human coronavirus HCoV-NL63 in vitro. N4-hydroxycytodine has been shown to inhibit SARS-CoV-2 as well as other human and bat coronaviruses in mice and human airway epithelial cells. It is orally bioavailable in mice and distributes into tissue before becoming the active 5’-triphosphate form, which is incorporated into the genome of new virions, resulting in the accumulation of inactivating mutations. In non-human primates, N4-hydroxycytidine was poorly orally bioavailable. A [remdesivir] resistant mutant mouse hepatitis virus has also been shown to have increased sensitivity to N4-hydroxycytidine. The prodrug of N4-hydroxycytidine, [EIDD-2801], is also being investigated for its broad spectrum activity against the coronavirus family of viruses. Drug Indication N4-hydroxycytidine and its prodrug [EIDD-2801] is being studied for its activity against a number of viral infections including influenza, MERS-CoV, and SARS-CoV-2. Mechanism of Action N4-hydroxycytidine is phosphorylated in tissue to the active 5’-triphosphate form, which is incorporated into the genome of new virions, resulting in the accumulation of inactivating mutations, known as viral error catastrophe. A [remdesivir] resistant mutant mouse hepatitis virus has also been shown to have increased sensitivity to N4-hydroxycytidine. |
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. 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 |