SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), with inhibitory activity mediated through its triphosphate form (GS-443902). The inhibition mechanism involves delayed chain termination during viral RNA synthesis. The half-maximal inhibitory concentration (IC₅₀) for SARS-CoV-2 RdRp is 0.0038 μM (measured via enzymatic assay with purified polymerase complex) [3]

- Antiviral activity in Vero E6 cells: GS-704277 (hydrolyzed intracellularly to GS-441524) showed an EC₅₀ of 1.65 μM against SARS-CoV-2 (2019-nCoV) in vitro, measured by viral RNA quantification and cytopathic effect reduction. Chloroquine (EC₅₀ = 1.13 μM) was used as a positive control [2]
- Mechanism of action: Intracellular phosphorylation converts GS-704277 to the active triphosphate GS-443902, which competes with ATP for incorporation into viral RNA by RdRp, causing premature termination of RNA synthesis. This was confirmed via RNA elongation assays in Huh-7 cells [3]
Standard assays were carried out to measure the effects of these compounds on the cytotoxicity, virus yield and infection rates of 2019-nCoVs. Firstly, the cytotoxicity of the candidate compounds in Vero E6 cells (ATCC-1586) was determined by the CCK8 assay. Then, Vero E6 cells were infected with nCoV-2019BetaCoV/Wuhan/WIV04/20192 at a multiplicity of infection (MOI) of 0.05 in the presence of varying concentrations of the test drugs. DMSO was used in the controls. Efficacies were evaluated by quantification of viral copy numbers in the cell supernatant via quantitative real-time RT-PCR (qRT-PCR) and confirmed with visualization of virus nucleoprotein (NP) expression through immunofluorescence microscopy at 48 h post infection (p.i.) (cytopathic effect was not obvious at this time point of infection). Among the seven tested drugs, high concentrations of three nucleoside analogs including ribavirin (half-maximal effective concentration (EC50) = 109.50 μM, half-cytotoxic concentration (CC50) > 400 μM, selectivity index (SI) > 3.65), penciclovir (EC50 = 95.96 μM, CC50 > 400 μM, SI > 4.17) and favipiravir (EC50 = 61.88 μM, CC50 > 400 μM, SI > 6.46) were required to reduce the viral infection (Fig. 1a and Supplementary information, Fig. S1). However, favipiravir has been shown to be 100% effective in protecting mice against Ebola virus challenge, although its EC50 value in Vero E6 cells was as high as 67 μM,4 suggesting further in vivo studies are recommended to evaluate this antiviral nucleoside. Nafamostat, a potent inhibitor of MERS-CoV, which prevents membrane fusion, was inhibitive against the 2019-nCoV infection (EC50 = 22.50 μM, CC50 > 100 μM, SI > 4.44). Nitazoxanide, a commercial antiprotozoal agent with an antiviral potential against a broad range of viruses including human and animal coronaviruses, inhibited the 2019-nCoV at a low-micromolar concentration (EC50 = 2.12 μM; CC50 > 35.53 μM; SI > 16.76). Further in vivo evaluation of this drug against 2019-nCoV infection is recommended. Notably, two compounds remdesivir (EC50 = 0.77 μM; CC50 > 100 μM; SI > 129.87) and chloroquine (EC50 = 1.13 μM; CC50 > 100 μM, SI > 88.50) potently blocked virus infection at low-micromolar concentration and showed high SI (Fig. 1a, b)[1].

- RdRp inhibition assay: Purified SARS-CoV-2 RdRp complex (nsp7/nsp8/nsp12) was incubated with template-primer RNA, NTPs (including [α-³²P]ATP), and varying concentrations of GS-443902 (active form of GS-704277). Reactions were stopped at timed intervals, and RNA products were separated by gel electrophoresis. Inhibition was quantified by densitometry of full-length RNA bands. IC₅₀ was calculated from dose-response curves [3]

- Antiviral cytopathic effect (CPE) assay: Vero E6 cells were infected with SARS-CoV-2 (MOI = 0.002) and treated with serial dilutions of GS-704277. After 48 hours, cell viability was measured using a commercial cell counting kit. Viral RNA was extracted from supernatants for RT-qPCR quantification. EC₅₀ values were derived from dose-response curves of both CPE reduction and viral RNA suppression [2]
- Metabolic activation assay: Huh-7 cells were incubated with GS-704277 for 24 hours, followed by extraction and quantification of intracellular metabolites (GS-441524, GS-704277, and GS-443902) via liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results confirmed rapid hydrolysis to GS-441524 and subsequent phosphorylation to GS-443902 [3]

- Metabolic pathway: GS-704277 is an alanine prodrug of GS-441524. In vivo, it undergoes rapid hydrolysis by cathepsin A (CatA) and carboxylesterase 1 (CES1) to release the parent nucleoside analog GS-441524, which is then phosphorylated intracellularly to the active triphosphate GS-443902. This conversion was validated in human hepatocytes and primary monocytes [1]
- Systemic exposure: In rhesus monkeys administered intravenous remdesivir (parent drug), the metabolite GS-441524 (derived from GS-704277) showed a plasma half-life (t_1/2) of ~1.2 hours and a volume of distribution (V_d) of ~30 L/kg, indicating extensive tissue distribution [1]

- In vitro cytotoxicity: No significant cytotoxicity was observed for GS-704277 in Vero E6 cells at concentrations up to 100 μM (CC₅₀ > 100 μM) [2]
- Liver enzyme elevation: In a rhesus monkey model of MERS-CoV infection, remdesivir (producing GS-704277 metabolites) induced transient elevation of alanine aminotransferase (ALT) at high doses (10 mg/kg/day), but no histopathological liver damage was observed [1]

[1]. Remdesivir: A Review of Its Discovery and Development Leading to Emergency Use Authorization for Treatment of COVID-19. ACS Cent Sci. 2020 May 27;6(5):672-683. .

[2]. Remdesivir and chloroquine effectively inhibit the recently\nemerged novel coronavirus (2019-nCoV) in vitro. Cell Res. 2020 Mar;30(3):269-271.

[3]. Coronavirus Susceptibility to the Antiviral Remdesivir (GS-5734) Is Mediated by the Viral Polymerase and the Proofreading Exoribonuclease. MBio.2018 Mar 6;9(2). pii: e00221-18.

- GS-704277 is the intermediate metabolite of remdesivir (GS-5734), generated by hydrolysis of the parent prodrug. It is further metabolized to the nucleoside analog GS-441524, which enters cells and is phosphorylated to the pharmacologically active triphosphate GS-443902 [1]
- The compound’s efficacy is dependent on the viral proofreading exoribonuclease (ExoN) activity; viruses lacking ExoN (e.g., murine hepatitis virus) show 10-fold increased sensitivity to GS-443902 compared to ExoN-competent coronaviruses [3]
The global pandemic of SARS-CoV-2, the causative viral pathogen of COVID-19, has driven the biomedical community to action-to uncover and develop antiviral interventions. One potential therapeutic approach currently being evaluated in numerous clinical trials is the agent remdesivir, which has endured a long and winding developmental path. Remdesivir is a nucleotide analogue prodrug that perturbs viral replication, originally evaluated in clinical trials to thwart the Ebola outbreak in 2014. Subsequent evaluation by numerous virology laboratories demonstrated the ability of remdesivir to inhibit coronavirus replication, including SARS-CoV-2. Here, we provide an overview of remdesivir's discovery, mechanism of action, and the current studies exploring its clinical effectiveness.

C15H19N6O8P

442.320523500443

442.1

C, 40.73; H, 4.33; N, 19.00; O, 28.94; P, 7.00

1911579-04-8

1355149-45-9 [GS443902 (GS-441524 triphosphate)]; 1809249-37-3 (Remdesivir); 1191237-69-0 (GS-441524, an active metabolite of Remdesivir); 1191237-80-5 (Remdesivir O-desphosphate acetonide impurity); 1911578-74-9 (Remdesivir nucleoside monophosphate); 1911579-04-8 (GS-704277)

121313150

Typically exists as White to light yellow solids at room temperature

-2.8

6

13

7

30

765

5

C[C@@H](C(=O)O)NP(=O)(O)OC[C@@H]1[C@H]([C@H]([C@](O1)(C#N)C2=CC=C3N2N=CN=C3N)O)O

IYHPTSNEWCZBDF-NIFWRESRSA-N

InChI=1S/C15H19N6O8P/c1-7(14(24)25)20-30(26,27)28-4-9-11(22)12(23)15(5-16,29-9)10-3-2-8-13(17)18-6-19-21(8)10/h2-3,6-7,9,11-12,22-23H,4H2,1H3,(H,24,25)(H2,17,18,19)(H2,20,26,27)/t7-,9+,11+,12+,15-/m0/s1

((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)-L-alanine

GS-704277; GS704277; GS-704277; N-phosphono-L-alanine, P-->6-ester with 2-C-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-2,5-anhydro-D-altrononitrile; (2S)-2-[[[(2R,3S,4R,5R)-5-(4-Aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]amino]propanoic acid; Dephenoxy Hydroxy Remdesivir; Remdesivir Impurity 39; SCHEMBL17722667; MFCD32701941; GS 704277

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.

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

DMSO : ~100 mg/mL (~226.08 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.)