The target of PSI-7409 is the hepatitis C virus (HCV) NS5B protein, an RNA-dependent RNA polymerase (RdRp) that serves as the core enzyme for HCV genome replication. As a competitive analogue of the natural substrate UTP, PSI-7409 is mistakenly incorporated into the nascent RNA chain by the NS5B polymerase during RNA synthesis. Due to the steric hindrance effect of the 2′-C-methyl group in the PSI-7409 molecule, incorporation leads to chain termination, effectively blocking viral RNA elongation. This compound exhibits inhibitory activity against NS5B polymerases from HCV genotypes 1b, 2a, 3a, and 4a, demonstrating pan-genotypic coverage properties.
PSI-7409 targets the HCV NS5B RNA-dependent RNA polymerase, an enzyme essential for viral RNA replication. As a uridine nucleotide analog triphosphate, PSI-7409 acts as a competitive inhibitor of the NS5B polymerase. It is incorporated into the growing RNA chain, causing chain termination and inhibition of viral replication.

In in vitro enzymatic assays, PSI-7409 exhibits dose-dependent inhibition of recombinant HCV NS5B polymerases. The IC50 values against genotype 1b (GT 1b_Con1), 2a (GT 2a_JFH1), 3a, and 4a NS5B polymerases are 1.6 μM, 2.8 μM, 0.7 μM, and 2.6 μM, respectively. Notably, PSI-7409 shows weak inhibition of human DNA polymerases: an IC50 of 550 μM against DNA polymerase α, and no inhibitory activity against DNA polymerases β and γ even at concentrations up to 1 mM, demonstrating high selectivity for the viral polymerase. Furthermore, approximately 85% of RNA product synthesis remains in the presence of 500 μM PSI-7409.

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As HCV NS5B polymerase inhibitor, PSI-7977 displays more potent inhibitory activity against HCV RNA replication than PSI-7976 with EC50 of 92 nM versus 1.07 μM and EC90 of 0.29 μM versus 2.99 μM, consistent with that incubating clone A cells with PSI-7977 leads to a higher concentration of PSI-7409 than clone A cells incubated with PSI-7976. PSI-7977 is an effective substrate for CatA to form PSI-352707 with 18-30 fold more potency as compared with PSI-7976. Unlike GS-7976, however, the CES1-mediated hydrolysis of PSI-7977 does not progress in a time-dependent manner. The S282T NS5B polymerase mutation but not S96T mutation confers resistance to PSI-7977 with EC90 increases from 0.42 μM to 7.8 μM. When assessed in an 8-day cytotoxicity assay, PSI-7977 displays no cytotoxicity against Huh7, HepG2, BxPC3, and CEM cells even at concentrations up to 100 μM. PSI-7977 treatment for 14 days shows a IC90 of 72.1 μM and 68.6 μM for the inhibition of mtDNA and rDNA, respectively, in HepG2 cells. PSI-7977 exhibits potent activity against genotype (GT) 1a, 1b, and 2a (strain JFH-1) replicons and chimeric replicons containing GT 2a (strain J6), 2b, and 3a NS5B polymerase. Sequence analysis of the JFH-1 NS5B region indicates that additional amino acid changes including T179A, M289L, I293L, M434T, and H479P are selected both prior to and after the emergence of S282T, which are required to confer resistance to PSI-7977.

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In vitro, PSI-7409 inhibits the enzymatic activities of NS5BΔ21 polymerases from HCV genotypes 1b, 2a, 3a, and 4a with IC50 values of 1.6 μM, 2.8 μM, 0.7 μM, and 2.6 μM, respectively. It is a weak inhibitor of DNA Pol α (IC50 = 550 μM). DNA Pol β and γ are not inhibited by 1 mM PSI-7409. In clone A cells, PSI-7409 levels gradually increase to a maximum of about 25 μM over 48 hours. In primary human hepatocytes, PSI-7409 forms at a much faster rate, achieving a maximum intracellular concentration of ~100 μM at 4 hours and remains at that concentration for 48 hours.

As an intracellular active metabolite, the in vivo activity of PSI-7409 is primarily reflected in the antiviral efficacy observed following sofosbuvir administration. In patients infected with HCV, after oral administration of sofosbuvir, the drug undergoes stepwise enzymatic conversion in the liver through enzymes such as carboxylesterase 1 (CES1) and cathepsin A (CatA) to generate PSI-7409. Within hepatocytes, the concentration of PSI-7409 reaches levels sufficient to effectively inhibit NS5B polymerase, achieving sustained viral suppression. Clinical studies have demonstrated that sofosbuvir-based regimens achieve sustained virologic response (SVR) rates exceeding 90% across various HCV genotypes. Cytochrome P450 enzymes are not involved in the metabolism of PSI-7409, reducing the risk of drug-drug interactions.

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The average plasma ALT levels in mice with humanized livers in the 440- and 44-mg/kg/d treatment groups were below the upper limit of normal, and were not significantly different from those measured in vehicle-treated mice with humanized livers. The plasma lactate levels were also not elevated in or control mice or mice with humanized livers receiving either dose of PSI-7977.

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In vivo, PSI-7409 is the pharmacologically active 5′-triphosphate metabolite of the HCV nucleotide prodrug Sofosbuvir. After oral administration of Sofosbuvir, it is metabolized to PSI-7409 in hepatocytes, where it exerts its antiviral activity. Sofosbuvir is a clinically approved drug for the treatment of chronic HCV infection, demonstrating the in vivo efficacy of its active metabolite PSI-7409.

1. Enzyme Source Preparation: Express and purify recombinant HCV NS5B polymerase (Δ21 truncated form, with the C-terminal 21 amino acids removed to improve solubility) from different genotypes (1b, 2a, 3a, 4a). 2. Reaction Mixture Setup: In a reaction buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 1 mM DTT, 0.5 mg/mL BSA, 0.5 U/μL RNase inhibitor, 100 μM each of NTPs (ATP, CTP, GTP), and 1 μM [³H]-UTP, add serially diluted PSI-7409 (0.1-1000 μM). 3. Polymerase Reaction: Initiate the reaction by adding NS5B polymerase (final concentration 20 nM) and incubate at 37°C for 1 hour. 4. Product Detection: Terminate the reaction by adding EDTA, spot the reaction mixture onto DE81 anion exchange filter paper, wash, and measure incorporated radioactivity using a liquid scintillation counter. 5. Data Analysis: Calculate inhibition rates at various concentrations, generate concentration-inhibition curves, and determine IC50 values by nonlinear regression fitting.

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The NS5B polymerase inhibition assay for PSI-7409 involves incubating the compound with recombinant HCV NS5B polymerase, a synthetic RNA template, and nucleotide substrates (including [³H]-UTP). The reaction is carried out in a polymerase assay buffer at 37°C for a specified time. The incorporation of [³H]-UTP into the growing RNA chain is measured by scintillation counting. IC50 values are calculated from dose-response curves.

1. Cell Culture: Culture HCV replicon cell lines (e.g., Huh-7 cells transfected with Con1 subgenomic replicon) in DMEM medium containing 10% fetal bovine serum and 0.5 mg/mL G418 at 37°C in 5% CO₂. 2. Compound Treatment: Seed cells in 96-well plates (approximately 8×10³/well), culture for 24 hours, then treat with various concentrations of sofosbuvir (PSI-7977, 0.001-100 μM) for 48-72 hours, with PSI-7409 generated as the intracellular active metabolite. 3. RNA Extraction: Extract total RNA using an RNA extraction kit. 4. Quantitative Detection: Quantify HCV RNA copy number using real-time quantitative RT-PCR, with GAPDH as an internal reference gene. 5. Activity Evaluation: Calculate EC50 and EC90 values. Studies have shown that after treating clone A cells with sofosbuvir, PSI-7409 levels gradually increase to approximately 25 μM over 48 hours. In primary human hepatocytes, PSI-7409 forms much more rapidly, achieving a maximum intracellular concentration of approximately 100 μM at 4 hours and remaining at that concentration for 48 hours.

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Cells (Huh7, HepG2, BxPC3, and CEM) are exposed to various concentrations of PSI-7977 for 8 days. At the end of the growth period, MTS dye from the CellTiter 96 AQueous One Solution Cell Proliferation Assay kit is added to each well, and the plate is incubated for an additional 2 hours. The absorbance at 490 nm is read with a Victor3 plate reader using themedium only controlwells as blanks. The 50% inhibition value (IC50) is determined by comparing the absorbance in wells containing cells and PSI-7977 to untreated cell control wells.

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To evaluate the cellular activity of PSI-7409, HCV-replicon cells (such as Huh-7 cells containing HCV subgenomic replicons) are seeded in 96-well plates and treated with Sofosbuvir, which is metabolized to PSI-7409 intracellularly. After 48-72 hours of treatment, viral RNA levels are measured by quantitative RT-PCR. The EC50 for inhibition of HCV replication is calculated. Intracellular PSI-7409 levels can be measured by LC-MS/MS.

1. Animal Model: Use humanized liver chimeric mouse models (e.g., uPA/SCID mice or FRG mice transplanted with human hepatocytes), where human hepatocytes constitute more than 70% of the mouse liver. 2. Viral Infection: Establish HCV infection by intravenous tail vein injection of HCV-positive patient serum. 3. Dosing Regimen: After confirming viral infection, administer sofosbuvir (PSI-7977) via oral gavage at doses ranging from 44-440 mg/kg/day once daily for 2-4 weeks; administer vehicle control to the control group. 4. Sampling and Analysis: Collect blood samples at regular intervals to measure plasma HCV RNA levels and ALT levels. 5. Efficacy Evaluation: Studies have shown that in the 44 mg/kg/day and 440 mg/kg/day treatment groups, plasma ALT levels in humanized liver mice were below the upper limit of normal, not significantly different from control group, and plasma lactate levels were not elevated, indicating favorable safety of the drug within this dose range.

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The in vivo efficacy of PSI-7409 is evaluated through the administration of its prodrug Sofosbuvir in animal models and clinical studies. In HCV-infected patients, oral administration of Sofosbuvir results in the formation of PSI-7409 in hepatocytes, leading to potent inhibition of HCV replication and high cure rates. Pharmacokinetic studies measure the formation of PSI-7409 in plasma and peripheral blood mononuclear cells.

PSI-7409 itself is an intracellular active metabolite and is not administered orally as a direct therapeutic agent; its pharmacokinetic profile is determined by the metabolism of the parent drug sofosbuvir. After oral administration, sofosbuvir is rapidly absorbed and undergoes hydrolysis in the liver catalyzed by carboxylesterase 1 (CES1) and cathepsin A (CatA), followed by three sequential phosphorylation steps (monophosphorylation, diphosphorylation, and triphosphorylation) to generate PSI-7409. In primary human hepatocytes, the intracellular concentration of PSI-7409 reaches approximately 100 μM within 4 hours and remains at this level for 48 hours. In clone A cells (a human hepatoma cell line), PSI-7409 levels gradually increase to approximately 25 μM over 48 hours. PSI-7409 has a long intracellular half-life, supporting once-daily dosing frequency. The terminal metabolites are eliminated via the kidneys. Due to the metabolic pathway being independent of the CYP450 enzyme system, sofosbuvir/PSI-7409 has a low risk of drug-drug interactions.

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PSI-7409 is the active 5'-triphosphate metabolite of Sofosbuvir. It has a molecular weight of 500.16 and a molecular formula of C10H16FN2O14P3. It is soluble in water at 50 mg/mL (99.97 mM). It is typically stored as a solid powder at -20°C for up to 1 year. As the active metabolite of Sofosbuvir, its pharmacokinetic properties are well characterized through clinical studies.

The toxicological profile of PSI-7409 is primarily assessed through studies of sofosbuvir. In vitro cytotoxicity assays have shown that PSI-7409 exhibits low cytotoxicity against human cell lines (including Huh7, HepG2, BxPC3, and CEM cells), with no significant cytotoxicity, mitochondrial toxicity, or bone marrow toxicity observed at the highest tested concentration of 100 μM. Regarding selectivity for DNA polymerases, PSI-7409 shows no inhibition of mitochondrial DNA polymerase γ (Pol γ) (no inhibition even at 1 mM concentration), which is attributed to the absence of a natural 3′-hydroxyl group in its structure, explaining the low risk of mitochondrial toxicity observed clinically with this class of drugs. In clinical use, sofosbuvir/PSI-7409 is generally well-tolerated, with common adverse reactions including fatigue, headache, and nausea, and a low incidence of serious adverse events. Preclinical studies have shown that in humanized liver mouse models, sofosbuvir treatment at doses of 44-440 mg/kg/day did not cause abnormal elevations in ALT or lactate levels.

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Sofosbuvir is generally well-tolerated, with common adverse effects including fatigue, headache, and nausea. As the active metabolite, PSI-7409 is responsible for the antiviral activity of Sofosbuvir. The safety profile of Sofosbuvir has been extensively characterized in clinical trials, demonstrating a favorable safety profile for the treatment of chronic HCV infection. Standard safety precautions should be followed when handling this compound.

[1]. J Biol Chem.2010 Nov 5;285(45):34337-47.

[2]. J Med Chem.2010 Oct 14;53(19):7202-18.

[3]. Antimicrob Agents Chemother.2012 Jun;56(6):3359-68.

[4]. Antimicrob Agents Chemother. 2010 Aug;54(8):3187-96.

[5]. Mechanism of activation of PSI-7851 and its diastereoisomer PSI-7977.

PSI-7409 is the active 5'-triphosphate metabolite of Sofosbuvir (PSI-7977/GS-7977). It inhibits HCV NS5B polymerase with IC50 values of 1.6 μM, 2.8 μM, 0.7 μM, and 2.6 μM for genotypes 1b, 2a, 3a, and 4a, respectively. It is a weak inhibitor of DNA Pol α and does not inhibit DNA Pol β and γ. It is the pharmacologically active metabolite of the clinically approved drug Sofosbuvir.

C10H16FN2O14P3

500.16

499.979

C, 24.01; H, 3.22; F, 3.80; N, 5.60; O, 44.78; P, 18.58

1015073-42-3

PSI-7409 tetrasodium;1621884-22-7

23725128

White to off-white solid powder

2.0±0.1 g/cm3

1.601

-4.81

6

15

8

30

891

4

P(=O)(O[H])(OP(=O)(O[H])OP(=O)(O[H])O[H])OC([H])([H])C1([H])C([H])([C@](C([H])([H])[H])(C([H])(N2C([H])=C([H])C(N([H])C2=O)=O)O1)F)O[H]

NSUQNLUFOSKATN-SRAYDXMQSA-N

InChI=1S/C10H16FN2O14P3/c1-10(11)6(15)7(24-8(10)13-4-3-5(14)12-9(13)16)25-30(22,23-2)27-29(20,21)26-28(17,18)19/h3-4,6-8,15H,1-2H3,(H,20,21)(H,12,14,16)(H2,17,18,19)/t6-,7-,8-,10-,30?/m1/s1

[[(2R,3R,4R,5R)-5-(2,4-Dioxopyrimidin-1-yl)-4-fluoro-3-hydroxy-4-methyloxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate

PSI-7409; PSI 7409; GS-461203; UNII-T90A75S60M; T90A75S60M; PSI7409.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

H2O : ~50 mg/mL (~99.97 mM)
DMF : < 1 mg/mL
DMSO : < 1 mg/mL

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.)