PSI-7409 tetrasodium is a novel and active 5'-triphosphate metabolite of Sofosbuvir (PSI-7977). Sofosbuvir (also known as PSI-7977, GS-7977; trade names Sovaldi and Virunon) is a HCV NS5B polymerase inhibitor that is used for the treatment of chronic hepatitis C virus (HCV) infection. Sofosbuvir acts by inhibiting the RNA polymerase that the hepatitis C virus uses to replicate its RNA. It is a component of the first all-oral, interferon-free regimen approved for treating chronic Hepatitis C. In 2013, the FDA approved sofosbuvir in combination with ribavirin (RBV) for oral dual therapy of HCV genotypes 2 and 3, and for triple therapy with injected pegylated interferon (pegIFN) and RBV for treatment-naive patients with HCV genotypes 1 and 4.

Physicochemical Properties

Molecular Formula	C11H18FN2O13P3
Molecular Weight	498.16
Exact Mass	587.907
CAS #	1621884-22-7
Related CAS #	PSI-7409;1015073-42-3
PubChem CID	146673025
Appearance	White to off-white solid powder
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	15
Rotatable Bond Count	8
Heavy Atom Count	34
Complexity	909
Defined Atom Stereocenter Count	4
SMILES	O[C@@H]([C@@](C)(F)[C@H](N1C(NC(C=C1)=O)=O)O2)[C@H]2COP(O)(OP(OP(O)(O)=O)(O)=O)=O.[4Na]
InChi Key	MEVRFPOAFPAGDY-DLULJLDFSA-J
InChi Code	InChI=1S/C10H16FN2O14P3.4Na/c1-10(11)7(15)5(25-8(10)13-3-2-6(14)12-9(13)16)4-24-29(20,21)27-30(22,23)26-28(17,18)19;;;;/h2-3,5,7-8,15H,4H2,1H3,(H,20,21)(H,22,23)(H,12,14,16)(H2,17,18,19);;;;/q;4*+1/p-4/t5-,7-,8-,10-;;;;/m1..../s1
Chemical Name	tetrasodium;[[[(2R,3R,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-4-fluoro-3-hydroxy-4-methyloxolan-2-yl]methoxy-oxidophosphoryl]oxy-oxidophosphoryl] phosphate
Synonyms	PSI-7409 tetrasodium; PSI 7409 tetrasodium; PSI7409 tetrasodium;
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: Please store this product in a sealed and protected environment, avoid exposure to moisture.
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	GT 1b_Con1(IC50= 1.6 μM);GT 2a_JFH1(IC50= 2.8 μM);GT 3a(IC50= 0.7 μM);GT 4a(IC50= 2.6 μM)
ln Vitro	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.
ln Vivo	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.
Enzyme Assay	A 10-μL reaction mixture containing 50 mM Tris (pH 7.5), 50 mM NaCl, 3 mU/μL activated calf thymus DNA, 20 μM concentration of all four natural deoxynucleoside triphosphates, 4 μCi [α-32P]dCTP, 5 mM MgCl2, and increasing concentrations of PSI-7409 (up to 1 mM), D-ddFCTP, or aphidicolin is used to assay human DNA polymerase β, β, or γ. The reaction mixture is supplemented with DNA polymerase α, β, or γ to obtain final concentrations of 20, 18, and 50 μg/mL, respectively. Run at 37°C, each reaction is quenched after 30 minutes by mixing with 1 μL of 0.5 M EDTA.The items that have been radiolabeled are measured. The IC50 is found by performing a nonlinear fit. A 25-μL in vitro transcription reaction mixture containing 100 ng of the immediate-early promoter DNA of the cytomegalovirus (CMV), 400 μM ATP, CTP, and UTP, 16 μM GTP, 10 μCi [α-32P]GTP, 3 mM MgCl2, and varying concentrations of PSI-7409 (up to 1 mM), 3'-dCTP, or α-amanitin in transcription buffer (20 mM HEPES [pH 7.9], 100 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, and 20% glycerol) are used to measure the activity of RNA polymerase II. After 60 minutes, all reactions are quenched by mixing with 125 μL of the stop solution, which consists of 0.3 M Tris-HCl [pH 7.4], 0.3 M sodium acetate, 0.5% SDS, 2 mM EDTA, and 3 μg/mL tRNA. All reactions are run at 30°C. The resultant RNA is purified. After that, 12 μL of gel loading dye (consisting of 98% formamide, 10 mM EDTA, 0.1% xylene cyanol, and 0.1% bromophenol blue) is added. The samples are placed onto a 6% polyacrylamide sequencing gel after being heated to 90°C for five minutes. The gel is exposed to a phosphorscreen after running, and a phosphorimager is used to see and measure the result[1].
Cell Assay	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.
Animal Protocol	Oral administration, 44 or 440 mg/kg TK-NOG mice with non-humanized (control) or humanized livers
References	[1]. Antimicrob Agents Chemother. 2010 Aug;54(8):3187-96. [2]. J Biol Chem. 2010 Nov 5;285(45):34337-47. [3]. J Biol Chem.2010 Nov 5;285(45):34337-47. [4]. J Med Chem.2010 Oct 14;53(19):7202-18 [5]. Antimicrob Agents Chemother.2012 Jun;56(6):3359-68.

Solubility Data

Solubility (In Vitro)	H2O : ~150 mg/mL (~255.06 mM)
Solubility (In Vivo)	Solubility in Formulation 1: 100 mg/mL (170.04 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.0074 mL	10.0369 mL	20.0739 mL
	5 mM	0.4015 mL	2.0074 mL	4.0148 mL
	10 mM	0.2007 mL	1.0037 mL	2.0074 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.