Lufotrelvir targets the SARS coronavirus 3C-like protease (3CLpro), also known as the main protease (Mpro), which is an enzyme essential for viral replication. As a prodrug, it is converted to its active moiety, PF-00835231, which selectively inhibits 3CLpro activity. Lufotrelvir binds and inhibits SARS-CoV-2 3CLpro activity with a Ki of 174 nM.

In vitro, Lufotrelvir is a potent inhibitor of SARS-CoV-2 3CLpro with a Ki of 174 nM. As a prodrug of PF-00835231, it exhibits strong antiviral activity. The compound is used in protease inhibition research and pharmacodynamic evaluation.

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].
In vivo, Lufotrelvir is administered as a prodrug and is rapidly converted to the active inhibitor PF-00835231. It is a promising single antiviral agent and can also be used in combination with other antivirals that target other critical stages of the coronavirus life cycle.

The in vitro enzyme assay for Lufotrelvir involves measuring the inhibition of SARS-CoV-2 3CLpro activity. The active moiety, PF-00835231, is incubated with the enzyme and a fluorogenic substrate in the presence of varying compound concentrations. The Ki of 174 nM is determined from dose-response curves.

Cellular assays for Lufotrelvir are performed using SARS-CoV-2-infected cells. Cells are infected with the virus and treated with Lufotrelvir. Viral replication is assessed by measuring viral RNA by RT-qPCR or by plaque assays. The compound's antiviral activity is determined by calculating EC50 values.

In vivo animal studies for Lufotrelvir are not detailed in the available sources. The compound is being developed as an antiviral agent for COVID-19. Specific animal models, dosing regimens, and efficacy data have not been reported in the supplier documentation.

Lufotrelvir is a phosphate prodrug that is rapidly converted in vivo to the active inhibitor PF-00835231. It is an investigational antiviral compound. Specific PK parameters such as half-life, bioavailability, and clearance are not detailed in the available sources.

Toxicity data for Lufotrelvir are not reported in the available sources. As an investigational compound, it has been evaluated in preclinical models. Specific toxicological profiles are not provided in the supplier documentation.

[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.

PF-07304814 is an indolecarboxamide, formed by the condensation of the carboxyl group of 4-methoxy-1H-indole-2-carboxylic acid with the primary amino group of N-[(2S)-3-oxo-1-[(3S)-2-oxopyrrolidine-3-yl]-4-(phosphonooxy)but-2-yl]-L-leucine. It is a phosphate prodrug of the anticoronavirus drug PF-00835231. It is both a prodrug and an EC 3.4.22.69 (SARS coronavirus main protease) inhibitor and an anticoronavirus drug. It is an aromatic ether, indolecarboxamide, secondary formamide, pyrrolidine-2-one compound, L-leucine derivative, and phosphate monoester. It is functionally related to PF-00835231. PF-07304814 is a small molecule prodrug that targets the 3CLpro protease (Mpro), which is essential for the assembly and replication of viruses such as SARS-CoV-2. After intravenous infusion, the compound is cleaved into PF-00835231, thereby exerting its antiviral effect. PF-07304814, developed by Pfizer, was initially discovered during the 2002-2003 SARS outbreak; however, its use was subsequently suspended as the outbreak was brought under control. It is now being tested against the novel SARS-CoV-2 virus, including studies in combination with remdesivir for the treatment of COVID-19 infection; in vitro data show a synergistic effect in articles published on preprint servers. A Phase 1b study is also exploring the safety and tolerability of PF-07304814 in hospitalized COVID-19 patients (NCT04535167). Rufortrevir is the phosphate prodrug of PF-00835231, a broad-spectrum coronavirus 3CL protease (3CLpro) inhibitor with potential antiviral activity against multiple coronaviruses, including SARS-related coronavirus (SARS-CoV) and SARS-CoV-2. After administration, rufortrevir is converted to its active moiety, PF-00835231, which selectively targets and inhibits the activity of coronavirus 3CLpro. This compound inhibits the proteolytic cleavage of viral polyproteins and the formation of various viral proteins, including helicases, single-stranded RNA-binding proteins, RNA-dependent RNA polymerases, 20-O-ribose methyltransferases, ribonucleases, and exonucleases. This prevents viral transcription and replication.

---

Mechanism of Action
PF-07304814 contains a phosphate group in its structure, enabling it to dissolve and subsequently be cleaved by alkaline phosphatases in tissues.
This step releases PF-00835231, a compound with antiviral activity against the SARS-CoV-2 3CL protease. The active compound PF-00835231 has been shown to have potent and broad-spectrum inhibitory activity against the 3CL protease of various coronaviruses.
Lufotrelvir is an investigational antiviral prodrug for the treatment of SARS-CoV-2 and other coronavirus infections. It is a phosphate prodrug of PF-00835231, a potent 3CLpro inhibitor. The compound is available from commercial suppliers for research purposes.

C24H33N4O9P

552.51398730278

552.198

2468015-78-1

154699467

White to off-white solid powder

0.5

6

9

13

38

927

3

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

FQKALOFOWPDTED-WBAXXEDZSA-N

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

(S)-3-((S)-2-(4-methoxy-1H-indole-2-carboxamido)-4-methylpentanamido)-2-oxo-4-((S)-2-oxopyrrolidin-3-yl)butyl dihydrogen phosphate

PF-07304814Lufotrelvir PF 07304814 Lufotrelvirum PF07304814

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

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.

---

View More

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.)