PSI-6206 13CD3 is the labeled analog of PSI-6206, the deaminated derivative of the potent and selective HCV NS5B polymerase inhibitor PSI-6130. The pharmacologically active entity is its intracellular 5'-triphosphate metabolite (RO2433-TP), which targets the HCV NS5B RNA-dependent RNA polymerase (RdRp), acting as a competitive inhibitor of native RNA synthesis with an inhibition constant (Ki) of 0.141 μM.
PSI-6206 13CD3 is a labeled analog of PSI-6206, which targets the HCV NS5B RNA-dependent RNA polymerase. As a nucleoside analog, PSI-6206 inhibits viral RNA replication by acting as a chain terminator. The stable isotope labeling (¹³C and deuterium) makes PSI-6206 13CD3 particularly useful in metabolic and pharmacokinetic studies as an internal standard or tracer.

As an isotopically labeled compound, PSI-6206 13CD3 is not typically used to measure direct in vitro activity. The activity profile is derived from its non-labeled counterpart, PSI-6206. PSI-6206 itself exhibits low potency against the HCV replicon with an EC₉₀ > 100 μM. Its antiviral activity is dependent on intracellular conversion to the 5'-triphosphate metabolite (RO2433-TP), which inhibits RNA synthesis by the native HCV replicase (IC₅₀ = 1.19 μM) and the recombinant NS5B polymerase (IC₅₀ = 0.52 μM).

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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-6206 13CD3 is expected to have similar biological activity to unlabeled PSI-6206, which is a potent inhibitor of the HCV NS5B polymerase. The stable isotope labeling does not significantly alter the compound's biological activity. Specific IC50 values for PSI-6206 13CD3 are not detailed in the available sources. The compound is used as a research tool in antiviral studies.

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-6206 13CD3 is used as a tracer or internal standard in pharmacokinetic and metabolic studies. The stable isotope labeling allows for accurate quantification of the compound and its metabolites in biological samples using mass spectrometry. Specific in vivo activity data for PSI-6206 13CD3 are not detailed in the available sources.

For its non-labeled counterpart, the active 5'-triphosphate metabolite (RO2433-TP) was evaluated for inhibition of RNA synthesis. The assay used purified recombinant HCV Con1 NS5B polymerase and a heteropolymeric RNA template derived from the 3'-end of the negative strand of the HCV genome. The reaction mixture, containing the polymerase, RNA template, ribonucleoside triphosphates (including a radiolabeled tracer), and varying concentrations of the inhibitor, was incubated. The IC₅₀ (0.52 μM) and Ki (0.141 μM) values were determined by measuring the amount of radiolabel incorporated into newly synthesized RNA.

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The HCV NS5B polymerase inhibition assay for PSI-6206 13CD3 involves incubating the compound with recombinant HCV NS5B polymerase, a synthetic RNA template, and nucleotide substrates. The reaction is carried out in a polymerase assay buffer at 37°C for a specified time. The incorporation of labeled nucleotides into the growing RNA chain is measured. IC50 values are calculated from dose-response curves.

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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For its non-labeled counterpart, the HCV subgenomic replicon assay is used. Huh-7 or other hepatoma cells harboring HCV replicons are treated with serial dilutions of the compound. After a 3-day incubation, total RNA is extracted, and HCV RNA levels are quantified by real-time RT-PCR. The EC₉₀ of PSI-6206 was determined to be >100 μM, confirming its lack of direct antiviral activity prior to intracellular phosphorylation.

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To evaluate the cellular activity of PSI-6206 13CD3, HCV-replicon cells are seeded in culture plates and treated with varying concentrations of the compound. 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 levels of PSI-6206 13CD3 and its metabolites can be measured by LC-MS/MS using the isotope label for accurate quantification.

Oral administration, 44 or 440 mg/kg
TK-NOG mice with non-humanized (control) or humanized livers

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Specific in vivo animal experiment protocols for PSI-6206 13CD3 are not detailed in the available sources. As a stable isotope-labeled compound, it is typically used in pharmacokinetic studies in rodents. Animals are administered PSI-6206 13CD3 orally or intravenously, and blood and tissue samples are collected at various time points. The concentration of the compound and its metabolites is measured by LC-MS/MS using the isotope label for accurate quantification.

PSI-6206 13CD3 is used as an internal standard to accurately measure the levels of PSI-6206 in biological matrices by LC-MS/MS. In rhesus monkeys, following oral administration of PSI-6130 (33.3 mg/kg), the total oral bioavailability of PSI-6130 plus its deaminated metabolite PSI-6206 was 64% ± 26%. The majority of drug recovered in urine was in the form of PSI-6206. In human clinical studies of Sofosbuvir, PSI-6206 (GS-331007) is the predominant circulating nucleoside metabolite, with a terminal elimination half-life (t₁/₂) ranging from 25.7 to 32.7 hours in healthy volunteers.

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PSI-6206 13CD3 has a molecular weight of 264.23 and a molecular formula of C9¹³CH10D3FN2O5. It is a stable isotope-labeled compound, which provides improved stability and consistency for high-precision scientific investigations. Specific pharmacokinetic parameters are not detailed in the available sources. The compound is typically stored as a powder at -20°C.

As a stable isotope-labeled compound intended for use as a research standard, it is considered non-hazardous when handled with standard laboratory precautions. PSI-6206, the unlabeled compound, has shown low cytotoxicity in vitro, with no significant effects observed in Huh7, HepG2, BxPC3, and CEM cells at concentrations up to 100 μM. The compound is for research use only and not for human or veterinary use

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Specific toxicity data for PSI-6206 13CD3 are not provided in the available sources. As a research compound, it is intended for laboratory use only and is not approved for human therapeutic applications. The compound is a stable isotope-labeled version of PSI-6206, and its toxicity profile is expected to be similar to the unlabeled compound. 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.

PSI-6206 13CD3 is a crucial tool for pharmaceutical research, enabling precise quantification of drug metabolites in complex biological samples. Synonyms include RO-2433 13CD3, GS-331007 13CD3, and Sofosbuvir metabolite GS-331007 13CD3. The labeled methyl group (¹³CD₃) is located at the 2' position of the ribose ring, which is the site of the 2'-C-methyl modification critical for the compound's mechanism of action as a non-obligate chain terminator when phosphorylated to its triphosphate form.

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PSI-6206 13CD3 is a stable isotope-labeled version of PSI-6206, which is the deaminated derivative of PSI-6130, a potent and selective inhibitor of HCV NS5B polymerase. It has a molecular formula of C9¹³CH10D3FN2O5 and a molecular weight of 264.23. The stable isotope labeling makes it useful for metabolic and pharmacokinetic studies.

C913CH10D3FN2O5

264.23

264.103

1256490-42-2

PSI-6206;863329-66-2

58463518

White to light yellow solid powder

-1.1

3

6

2

18

415

4

[2H][13C]([2H])([2H])[C@]1([C@@H]([C@H](O[C@H]1N2C=CC(=O)NC2=O)CO)O)F

ARKKGZQTGXJVKW-CWDGLZGLSA-N

InChI=1S/C10H13FN2O5/c1-10(11)7(16)5(4-14)18-8(10)13-3-2-6(15)12-9(13)17/h2-3,5,7-8,14,16H,4H2,1H3,(H,12,15,17)/t5-,7-,8-,10-/m1/s1/i1+1D3

1-[(2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)-3-(trideuterio(113C)methyl)oxolan-2-yl]pyrimidine-2,4-dione

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)

DMSO: ~100 mg/mL (378.5 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.)