Bofutrelvir (FB2001) is an inhibitor (blocker/antagonist) of Mpro, the main protease of SARS-CoV-2, with IC50 of 53 nM and EC50 of 0.53 μM. Bofutrelvir has antiviral effects against several SARS-CoV-2 variants, with EC50s of 0.26-0.42 μM. Bofutrelvir combined with Remdesivir has additive antiviral effects.

Physicochemical Properties

Molecular Formula	C25H32N4O4
Molecular Weight	452.55
Exact Mass	452.242
CAS #	2103278-86-8
PubChem CID	145343771
Appearance	White to light yellow solid powder
Density	1.2±0.1 g/cm3
Boiling Point	836.8±55.0 °C at 760 mmHg
Flash Point	459.9±31.5 °C
Vapour Pressure	0.0±3.1 mmHg at 25°C
Index of Refraction	1.592
LogP	2.7
Hydrogen Bond Donor Count	4
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	9
Heavy Atom Count	33
Complexity	723
Defined Atom Stereocenter Count	3
SMILES	C1CCC(CC1)C[C@@H](C(=O)N[C@@H](C[C@@H]2CCNC2=O)C=O)NC(=O)C3=CC4=CC=CC=C4N3
InChi Key	HPKJGHVHQWJOOT-ZJOUEHCJSA-N
InChi Code	InChI=1S/C25H32N4O4/c30-15-19(13-18-10-11-26-23(18)31)27-24(32)21(12-16-6-2-1-3-7-16)29-25(33)22-14-17-8-4-5-9-20(17)28-22/h4-5,8-9,14-16,18-19,21,28H,1-3,6-7,10-13H2,(H,26,31)(H,27,32)(H,29,33)/t18-,19-,21-/m0/s1
Chemical Name	N-[(2S)-3-cyclohexyl-1-oxo-1-[[(2S)-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl]amino]propan-2-yl]-1H-indole-2-carboxamide
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: (1). This product requires protection from light (avoid light exposure) during transportation and storage.(2). Please store this product in a sealed and protected environment (e.g. under nitrogen), 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	IC50: 53 nM (Mpro)[1]
ln Vitro	Bofutrelvir (24 hours) demonstrates in vitro action against SARS-CoV-2 and its variations, with EC50 values for SARS-CoV-2, SARS-CoV-2 (Alpha), SARS-CoV-2 (Beta), SARS-CoV-2 (Delta), and SARS-CoV-2 (Omicron) respectively of 0.42, 0.39, 0.28, 0.27, and 0.26 μM[2]. In vero E6 cells, bofutrelvir suppresses SARS-CoV-2 replication even at EC50 values when human blood is present (1.1–2.4 μM)[2]. In vitro, bofutrelvir and remdesivi therapy together show an additive impact against SARS-CoV-2[2].
ln Vivo	Effective against SARS-CoV-2 delta variant infection in vivo is bofutrelvir (100 and 200 mg/kg; ip once daily on day 0 and twice daily on days 1, 2, and 3 for 4 consecutive days)[2].
Animal Protocol	Animal/Disease Models: K18-hACE2 mice with SARS-CoV-2 delta variant infection[2] Doses: 100 and 200 mg/kg Route of Administration: intraperitoneal (ip)injection; 100 and 200 mg/ kg one time/day on day 0 and twice (two times) daily on day 1, 2 and 3 for 4 days Experimental Results: demonstrated a dose-dependent efficacy to virus titers of lung. Effectively reduces the lung viral loads. Dose-dependently demonstrated antiviral activity against the SARS -CoV-2 Delta variant and Dramatically diminished viral load in mouse lung and brain.
References	[1]. Ullrich S, Nitsche C. The SARS-CoV-2 main protease as drug target. Bioorg Med Chem Lett. 2020 Sep 1;30(17):127377. [2]. In vitro and in vivo evaluation of the main protease inhibitor FB2001 against SARS-CoV-2. Antiviral Res. 2022 Dec;208:105450.
Additional Infomation	N-[(2S)-3-cyclohexyl-1-oxo-1-({(2S)-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}amino)propan-2-yl]-1H-indole-2-carboxamide is a secondary carboxamide resulting from the formal condensation of the carboxy group of 1H-indole-2-carboxylic acid with the primary amino group of 3-cyclohexyl-N-{(2S)-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}-L-alaninamide. It is an inhibitor of SARS coronavirus main proteinase and inhibits SARS-CoV-2 replication in cell culture (EC50 = 0.53 muM). It has a role as an EC 3.4.22.69 (SARS coronavirus main proteinase) inhibitor and an anticoronaviral agent. It is an indolecarboxamide, a member of pyrrolidin-2-ones, an aldehyde, a secondary carboxamide and an oligopeptide. Bofutrelvir is a small molecule inhibitor of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) main protease (Mpro; 3C-like protease; 3CL protease; 3CLpro; nsp5 protease), with potential antiviral activity against SARS-CoV-2. Upon intravenous administration or inhalation into the lungs, bofutrelvir selectively targets, binds to, and inhibits the activity of SARS-CoV-2 Mpro. This inhibits the proteolytic cleavage of viral polyproteins, thereby inhibiting the formation of viral proteins including helicase, single-stranded-RNA-binding protein, RNA-dependent RNA polymerase, 20-O-ribose methyltransferase, endoribonuclease and exoribonuclease. This prevents viral transcription and replication. Bofutrelvir may have antiviral activity in the brain.

Solubility Data

Solubility (In Vitro)

DMSO : 100 mg/mL (220.97 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.52 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 25.0 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: 2.5 mg/mL (5.52 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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: ≥ 2.5 mg/mL (5.52 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 25.0 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	2.2097 mL	11.0485 mL	22.0970 mL
	5 mM	0.4419 mL	2.2097 mL	4.4194 mL
	10 mM	0.2210 mL	1.1049 mL	2.2097 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.