Lufotrelvir (PF-07304814) is a phosphate prodrug of PF-00835231 which acts as a 3CLpro protease (Mpro) inhibitor with anti-SARS-CoV-2 activity. It is being developed by Pfizer As an antiviral drug. As a prodrug, Lufotrelvir has to be metabolized (with the phosphate group being cleaved) in vivo to yield the active agent PF-00835231.

Physicochemical Properties

Molecular Formula	C24H33N4O9P
Molecular Weight	552.51398730278
Exact Mass	552.198
CAS #	2468015-78-1
PubChem CID	154699467
Appearance	White to off-white solid powder
LogP	0.5
Hydrogen Bond Donor Count	6
Hydrogen Bond Acceptor Count	9
Rotatable Bond Count	13
Heavy Atom Count	38
Complexity	927
Defined Atom Stereocenter Count	3
SMILES	P(=O)(O)(O)OCC([C@H](C[C@H]1C(NCC1)=O)NC([C@H](CC(C)C)NC(C1=CC2C(=CC=CC=2N1)OC)=O)=O)=O
InChi Key	FQKALOFOWPDTED-WBAXXEDZSA-N
InChi Code	InChI=1S/C24H33N4O9P/c1-13(2)9-18(28-24(32)19-11-15-16(26-19)5-4-6-21(15)36-3)23(31)27-17(10-14-7-8-25-22(14)30)20(29)12-37-38(33,34)35/h4-6,11,13-14,17-18,26H,7-10,12H2,1-3H3,(H,25,30)(H,27,31)(H,28,32)(H2,33,34,35)/t14-,17-,18-/m0/s1
Chemical Name	(S)-3-((S)-2-(4-methoxy-1H-indole-2-carboxamido)-4-methylpentanamido)-2-oxo-4-((S)-2-oxopyrrolidin-3-yl)butyl dihydrogen phosphate
Synonyms	PF-07304814Lufotrelvir PF 07304814 Lufotrelvirum PF07304814
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
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

ln Vivo	Lufotrelvir is cleaved into PF-00835231 when given intravenously, which then goes on to work as an antiviral agent. The cardiovascular safety profile of lufotrelvir is favorable [1]. Intravenous lofetrelvir is administered to rats, dogs, and monkeys. It formed 68%, 81%, and 76% of PF-00835231 in rats, dogs, and monkeys, respectively, compared with the systemic exposure obtained by intravenous injection of PF00835231, indicating higher systemic clearance and shorter half-life [1].
References	[1]. Considerations for the discovery and development of 3-chymotrypsin-like cysteine protease inhibitors targeting SARS-CoV-2 infection. Curr Opin Virol. 2021 Apr 27;49:36-40. [2]. Title: Discovery of a Novel Inhibitor of Coronavirus 3CL Protease as a Clinical Candidate for the Potential Treatment of COVID-19 Short Title: Novel 3CL Protease Inhibitor for COVID-19.
Additional Infomation	PF-07304814 is an indolecarboxamide resulting from the formal condensation of the carboxy group of 4-methoxy-1H-indole-2-carboxylic acid with the primary amino group of N-[(2S)-3-oxo-1-[(3S)-2-oxopyrrolidin-3-yl]-4-(phosphonooxy)butan-2-yl]-L-leucinamide. It is the phosphate prodrug of PF-00835231, an anticoronaviral agent. It has a role as a prodrug, an EC 3.4.22.69 (SARS coronavirus main proteinase) inhibitor and an anticoronaviral agent. It is an aromatic ether, an indolecarboxamide, a secondary carboxamide, a member of pyrrolidin-2-ones, a L-leucine derivative and a phosphate monoester. It is functionally related to a PF-00835231. PF-07304814 is a small molecule prodrug that targets the 3CLpro protease (Mpro), which is used by viruses like SARS-CoV-2 to assemble and multiply. Once administered through intravenous infusion, the compound is cleaved into PF-00835231 to exert its anti-viral effects. PF-07304814, developed by Pfizer, was first identified during the SARS outbreak in 2002-2003; it was subsequently shelved as the outbreak was controlled. Now, it is being tested regarding the novel SARS-CoV-2 virus, including studies in conjunction with [Remdesivir] to treat COVID-19 infection; in vitro data showed synergistic effects on articles published on preprint servers. Phase 1b studies are also exploring the safety and tolerability of PF-07304814 in COVID-19 patients who are hospitalized (NCT04535167). Lufotrelvir is a phosphate prodrug of PF-00835231, a broad-spectrum inhibitor of coronavirus 3CL protease (3CLpro), with potential antiviral activity against coronaviruses including severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) and SARS coronavirus-2 (SARS-CoV-2). Upon administration, lufotrelvir is converted to its active moiety, PF-00835231, which selectively targets and inhibits the activity of coronavirus 3CLpro. This inhibits the proteolytic cleavage of viral polyproteins and 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. Mechanism of Action The structure of PF-07304814 contains a phosphate group, allowing the compound to be soluble and then subsequently cleaved in tissue by alkaline phosphatase. This step allows for the release of PF-00835231, which is the compound that exerts anti-viral activity against the 3CL protease of SARS-CoV-2. The active compound, PF-00835231, was shown to have potent and broad-spectrum inhibitory activity against numerous coronavirus 3CL proteases.

Solubility Data

Solubility (In Vitro)

DMSO : ~130 mg/mL (~235.29 mM) H2O : ~50 mg/mL (~90.50 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 3.25 mg/mL (5.88 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 32.5 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: ≥ 3.25 mg/mL (5.88 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 32.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 3: 3.25 mg/mL (5.88 mM) in 10% DMSO + 90% Corn Oil (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 32.5 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	1.8099 mL	9.0496 mL	18.0992 mL
	5 mM	0.3620 mL	1.8099 mL	3.6198 mL
	10 mM	0.1810 mL	0.9050 mL	1.8099 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.