Bemnifosbuvir hemisulfate (RG-6422; AT-527), the hemisulfate salt of AT-511, is a guanosine nucleotide prodrug and orally bioactive HCV viral replication inhibitor. It is a prodrug form of the antiviral nucleoside analog AT-9010 and being used for COVID-19 research. It reduces the infectious virus yield in cells infected with Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome CoV (SARS-CoV), SARS-CoV-2, human CoV 229E (HCoV-229E), and HCoV-OC43 (EC90s = 37, 0.47, 2.8, 1.2, and 0.5 µM, respectively).

Physicochemical Properties

Molecular Formula	C24H33FN7O7P
Molecular Weight	581.533689260483
Exact Mass	1260.4
CAS #	2241337-84-6
Related CAS #	Bemnifosbuvir;1998705-64-8;HCV-IN-31;1998705-62-6
PubChem CID	155926085
Appearance	White to off-white solid powder
Hydrogen Bond Donor Count	10
Hydrogen Bond Acceptor Count	32
Rotatable Bond Count	24
Heavy Atom Count	85
Complexity	1000
Defined Atom Stereocenter Count	12
SMILES	C[C@@H](C(=O)OC(C)C)N[P@](=O)(OC[C@@H]1[C@H]([C@@]([C@@H](O1)N2C=NC3=C(N=C(N=C32)N)NC)(C)F)O)OC4=CC=CC=C4.C[C@@H](C(=O)OC(C)C)N[P@](=O)(OC[C@@H]1[C@H]([C@@]([C@@H](O1)N2C=NC3=C(N=C(N=C32)N)NC)(C)F)O)OC4=CC=CC=C4.OS(=O)(=O)O
InChi Key	QIGYBLSWYRTXCA-NVSCJZCKSA-N
InChi Code	InChI=1S/2C24H33FN7O7P.H2O4S/c2*1-13(2)37-21(34)14(3)31-40(35,39-15-9-7-6-8-10-15)36-11-16-18(33)24(4,25)22(38-16)32-12-28-17-19(27-5)29-23(26)30-20(17)32;1-5(2,3)4/h2*6-10,12-14,16,18,22,33H,11H2,1-5H3,(H,31,35)(H3,26,27,29,30);(H2,1,2,3,4)/t2*14-,16+,18+,22+,24+,40-;/m00./s1
Chemical Name	propan-2-yl (2S)-2-[[[(2R,3R,4R,5R)-5-[2-amino-6-(methylamino)purin-9-yl]-4-fluoro-3-hydroxy-4-methyloxolan-2-yl]methoxy-phenoxyphosphoryl]amino]propanoate;sulfuric acid
Synonyms	AT 527; AT-527; AT527; RG 6422; RG-6422; RG6422; Bemnifosbuvir hemisulfate; AT-511 hemisulfate; AT511; AT-511
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	HCV(EC50: 5-28 nM);SARS-CoV-2(EC90: 0.47 μM)
ln Vitro	Pan-genotypic antiviral activities of bemnifosbuvir hemisulfate inhibit the replication of HCV genotype 1a (HCV GT1a), HCV genotype 1b, HCV GT2a, HCV GT3a, HCV GT4a, and HCV GT5a, with EC50 values of 12.8 nM, 12.5 nM, 9.2 nM, 10.3 nM, 14.7 nM, and 28.5 nM, respectively[1]. The concentration of Bemnifosbuvir hemisulfate needed to prevent SARS-CoV-2 replication by EC90 in normal human airway epithelial cells is 0.47 μM, which is comparable to the concentration of EC90 needed to prevent HCoV-229E, HCoV-OC43, and SARS-CoV in Huh-7 cells[2].
ln Vivo	High levels of AT-9010 are preferentially delivered in the liver in vivo by bemnifosbuvir hemisulfate when given orally to rats (500 mg/kg) and monkeys (30 mg/kg, 100 mg/kg, or 300 mg/kg)[1].
References	[1]. Preclinical evaluation of AT-527, a novel guanosine nucleotide prodrug with potent, pan-genotypic activity against hepatitis C virus. PLoS One. 2020 Jan 8;15(1):e0227104. [2]. AT-527, a double prodrug of a guanosine nucleotide analog, is a potent inhibitor of SARS-CoV-2 in vitro and a promising oral antiviral for treatment of COVID-19. Antimicrob Agents Chemother. 2021 Feb 8;AAC.02479-20. [3]. Safety, pharmacokinetics and antiviral activity of AT-527, a novel purine nucleotide prodrug, in HCV-infected subjects with and without cirrhosis. Antimicrob Agents Chemother. 2019 Sep 30;63(12):e01201-19.
Additional Infomation	Bemnifosbuvir Sulfate is the sulfate salt form of bemnifosbuvir, an orally bioavailable direct-acting antiviral and purine nucleotide prodrug, with potential antiviral activity against a variety of RNA viruses. Upon oral administration, bemnifosbuvir, being a prodrug, is metabolized into its active form. The active form inhibits the activity of viral RNA-dependent RNA polymerase. This results in the termination of viral RNA transcription, decreases viral RNA production and inhibits viral RNA replication. Mechanism of Action AT-527 is a prodrug of a guanosine nucleotide analog which has the potential to potently inhibit the in vitro replication of SARS-CoV-2. It also inhibits the NS5B polymerase in the Hepatitis C Virus. This drug is currently being investigated against COVID-19 and has also been investigated against Hepatitis C infections.

Solubility Data

Solubility (In Vitro)

DMSO : ~180 mg/mL (~285.45 mM) H2O : ≥ 100 mg/mL (~158.58 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 5 mg/mL (7.93 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 50.0 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: ≥ 5 mg/mL (7.93 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 50.0 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: ≥ 5 mg/mL (7.93 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 50.0 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	1.7196 mL	8.5980 mL	17.1960 mL
	5 mM	0.3439 mL	1.7196 mL	3.4392 mL
	10 mM	0.1720 mL	0.8598 mL	1.7196 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.