Mpro (IC50 = 53 nM)[1]
FB2001 (also known as compound 11a) is a drug candidate that targets the main protease (Mpro, also called 3CL protease) of SARS-CoV-2. It inhibits Mpro by covalently binding to cysteine 145 (Cys145). The IC50 value of FB2001 against SARS-CoV-2 Mpro is 0.053 ± 0.005 μM. [2]

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].

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FB2001 exhibits potent antiviral efficacy against several SARS-CoV-2 variants in Vero E6 cells, as determined by viral copy number reduction. The EC50 values are: 0.39 ± 0.01 μM for B.1.1.7 (Alpha), 0.28 ± 0.11 μM for B.1.351 (Beta), 0.27 ± 0.05 μM for B.1.617.2 (Delta), and 0.26 ± 0.06 μM for B.1.1.529 (Omicron). The corresponding EC90 values are 0.75 ± 0.01 μM (Alpha), 0.57 ± 0.21 μM (Beta), 0.81 ± 0.20 μM (Delta), and EC50 for Omicron is reported. The selectivity index (SI50) values are 704, 980, 1016, and 933, respectively. [2]
In the presence of a P-glycoprotein (P-gp) inhibitor (CP-100356), the inhibitory activity of FB2001 against the Omicron variant significantly increased, with an EC50 of 0.042 ± 0.007 μM and CC50 of 142.4 μM, suggesting that FB2001 may be a substrate of P-gp. [2]
Time-of-addition assays showed that FB2001 (2 μM, 10 μM, and 1 μM + P-gp inhibitor) exhibited potent antiviral activities under all tested conditions, particularly when administered post-entry (2-24 h) and full-time (-2 to 24 h), consistent with its mechanism as a main protease inhibitor. It also showed inhibitory effects on the virus entry process. [2]
Reversibility studies demonstrated that FB2001 is a reversible Mpro inhibitor. After incubation of SARS-CoV-2 Mpro (2 mM) with 2 μM of FB2001 for 60 min and subsequent 50-fold and 200-fold dilution, Mpro activity was recovered, similar to the reversible inhibitor PF-07321332, but not with the irreversible inhibitor N3. FB2001 was more difficult to dissociate from Mpro compared to PF-07321332. [2]
Sequence alignment of Mpro from wild-type and variants (Alpha, Beta, Delta, Omicron) revealed two mutations: K90R in Beta and P132H in Omicron. X-ray crystal structure analysis showed that these mutations are located far from the ligand binding site of FB2001, indicating they would not affect its inhibitory activity. [2]
FB2001 retained potent anti-SARS-CoV-2 activity in the presence of high concentrations of human serum. EC50 values increased from 1.10 μM (10% serum) to 2.40 μM (50% serum), indicating that human serum has no significant effect on its in vitro inhibitory activity. [2]
Combination studies with remdesivir showed an additive effect against SARS-CoV-2. The synergy score calculated by the Zero interaction potency (ZIP) model was 6.947, indicating an additive effect (score between -10 and 10). [2]

In K18-hACE2 mice challenged with the SARS-CoV-2 Delta variant, intraperitoneal administration of FB2001 at 100 mg/kg and 200 mg/kg twice daily for 4 days significantly reduced viral loads and titers in a dose-dependent manner. [2]
On day 2 post-infection, viral titers in the lung were reduced by 3.57 log10 PFU/g in the 200 mg/kg group compared to vehicle. On day 4, viral titers in the 200 mg/kg group were below the detection limit in both lung and brain. [2]
Viral RNA loads in the lung were reduced by 1.02 and 1.60 log10 copies/g (day 2) and 0.67 and 1.14 log10 copies/g (day 4) for the 100 mg/kg and 200 mg/kg groups, respectively. In the brain, viral RNA loads were significantly reduced by 2.37 and 5.26 log10 copies/g on day 4 for the 100 mg/kg and 200 mg/kg groups, respectively. [2]
Histopathological analysis showed that FB2001 treatment significantly improved lung and brain pathology. In the lungs, it reduced interstitial inflammatory cell infiltration, alveolar septal thickening, and perivascular infiltrates. In the brain, it reduced neuronal degeneration, cellular infiltration, and protected brain tissue from virus-induced damage. Immunohistochemistry staining for viral NP confirmed dose-dependent reduction of viral antigen in both lung and brain tissues. [2]

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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].

Mpro enzymatic activity assay: SARS-CoV-2 Mpro (2 mM) was incubated with 2 μM of FB2001, the reversible inhibitor PF-07321332, or the irreversible inhibitor N3 for 60 min. The incubation mixtures were then diluted 50-fold and 200-fold, and Mpro activity was monitored. The inhibitory activity was not recovered after incubation with N3, but was recovered after incubation with FB2001 and PF-07321332, indicating that FB2001 is a reversible Mpro inhibitor. [2]
X-ray crystallography: Crystal structure analysis of SARS-CoV-2 Mpro in complex with FB2001 (PDB: 6LZE) showed that the aldehyde group of FB2001 forms a covalent bond with Cys145 of Mpro. The structure also revealed that mutations K90R and P132H found in variants are located far from the ligand binding site. [2]

Antiviral activity assay in Vero E6 cells: Vero E6 cells were seeded in 48-well plates (50,000 cells/well). The next day, cells were pre-incubated with serial dilutions of FB2001 for 1 h, then infected with SARS-CoV-2 variants at MOI of 0.01 or 0.005 for 1 h. After infection, cells were washed and fresh compound with or without P-gp inhibitor CP-100356 was added. Supernatants were collected at 24 h post-infection, viral RNA was extracted, and copy numbers were determined by real-time quantitative PCR. EC50 values were calculated from inhibition curves. [2]
Cytotoxicity assay: Vero E6 cells were seeded in 96-well plates and incubated with serial dilutions of FB2001 for 24 h. Cell viability was determined using the CCK8 assay kit, and CC50 values were calculated. [2]
Human serum protein binding effect assay: Vero E6 cells were infected with SARS-CoV-2 (MOI 0.01) for 1 h, then washed and incubated with different concentrations of FB2001 in medium containing 10%, 20%, 30%, 40%, or 50% human serum. After 24 h, supernatants were collected, viral RNA was extracted, and copy numbers were determined by RT-qPCR. EC50 values were calculated for each serum concentration. [2]
Immunofluorescence assay (IFA) for combination study: Vero E6 cells were seeded in 96-well plates (20,000 cells/well). Cells were pre-incubated with combinations of FB2001 and remdesivir for 1 h, then infected with SARS-CoV-2 (MOI 0.01). At 24 h post-infection, cells were fixed, permeabilized, and stained with primary antibody against SARS-CoV-2 NP and secondary antibody. Fluorescence intensity was measured, and inhibition rates were calculated. Synergy scores were determined using the ZIP model. [2]

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.

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Animals: 7-8 week old K18-hACE2 transgenic mice were used. [2]
Virus challenge: Mice were infected with 1 × 10³ PFU of SARS-CoV-2 Delta variant by intranasal drip on Day 0. [2]
Drug preparation: FB2001 for injection was prepared by dissolving in 0.9% sterile saline, shaking to completely dissolve, and filtering with a 0.22 μm membrane. Concentrations were 11.5 mg/mL (for 100 mg/kg dose) and 23 mg/mL (for 200 mg/kg dose). Vehicle control was 0.9% sterile saline. [2]
Dosing regimen: Mice were divided into three groups (n=7 per group): vehicle control, FB2001 100 mg/kg, and FB2001 200 mg/kg. Administration was by intraperitoneal injection, with a volume of 0.2 mL per mouse based on average body weight (23 g). Dosing was once on Day 0 (2 h post-infection), and twice daily (at 9-h intervals) on Days 1, 2, and 3. [2]
Sample collection: Four mice per group were sacrificed on Day 2, and three mice per group on Day 4. Lung and brain tissues were collected. Right lung was homogenized in DMEM for viral RNA extraction and titer determination. Left lung was fixed in 4% paraformaldehyde for histopathological examination and immunohistochemistry. [2]
Viral load determination: Viral RNA from lung and brain tissues was extracted, reverse transcribed, and absolute viral RNA copy numbers were quantified by real-time quantitative PCR. Viral titers were determined by plaque assay (PFU/g). [2]
Histopathology and immunohistochemistry: Fixed lung and brain tissues were embedded in paraffin, sectioned, and stained with hematoxylin-eosin for histopathological analysis. Immunohistochemistry was performed using an antibody against SARS-CoV-2 nucleocapsid protein to detect viral antigen. [2]

A physiologically based pharmacokinetic (PBPK) model was established and validated using plasma and lung concentration data from SD rats and Beagle dogs to predict human lung concentrations of FB2001. [2]
For a simulated human dosing regimen of 200 mg FB2001 twice daily (BID) for 5 consecutive days, the observed plasma trough concentration at steady state (Ctrough,ss) was 0.163 μg/mL, and the predicted total lung Ctrough,ss was 2.5 μg/mL. [2]
Compared to the EC50 of FB2001 against the Omicron variant in the presence of P-gp inhibitor (0.019 μg/mL), the plasma Ctrough,ss was approximately 9-fold higher, and the predicted lung Ctrough,ss was approximately 132-fold higher. [2]
The model predicted lung concentrations for other dosing regimens: 150 mg BID, 300 mg QD, and 400 mg QD, with corresponding Ctrough,ss values of 1.8, 1.7, and 2.2 μg/mL, respectively. [2]

In the K18-hACE2 mouse efficacy study, no significant changes in body weight were observed in either the vehicle control group or FB2001 treatment groups (100 mg/kg and 200 mg/kg), indicating good tolerability at the tested doses. [2]
No specific toxicology data (e.g., LD50, organ toxicity) were reported in this study. The study mentions that FB2001 has shown good safety and tolerability in healthy human subjects in clinical trials (NCT05197179 and NCT04766931). [2]

[1]. 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.

N-[(2S)-3-cyclohexyl-1-oxo-1-({(2S)-1-oxo-3-[(3S)-2-oxopyrrolidone-3-yl]propyl-2-yl}amino)propyl-2-yl]-1H-indole-2-carboxamide is a secondary formamide formed by the condensation of the carboxyl group of 1H-indole-2-carboxylic acid with the primary amino group of 3-cyclohexyl-N-{(2S)-1-oxo-3-[(3S)-2-oxopyrrolidone-3-yl]propyl-2-yl}-L-alanaminoamide. It is an inhibitor of the SARS coronavirus main protease and inhibits the replication of SARS-CoV-2 in cell culture (EC50 = 0.53 μM). It is an EC 3.4.22.69 (SARS coronavirus main protease) inhibitor and an anti-coronavirus drug. It is an indolecarboxamide compound, belonging to the pyrrolidine-2-one class, and is also an aldehyde, sec-formamide, and oligopeptide compound. Bovterrevir is a small molecule inhibitor that inhibits the main protease (Mpro; 3C-like protease; 3CL protease; 3CLpro; nsp5 protease) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), possessing potential anti-SARS-CoV-2 viral activity. After intravenous injection or inhalation into the lungs, borveterrevir selectively targets, binds to, and inhibits the activity of SARS-CoV-2 Mpro. This drug inhibits the proteolytic cleavage of viral polyproteins, thereby inhibiting 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. Bovterrevir may have antiviral activity in the brain.

C25H32N4O4

452.55

452.242

C, 66.35; H, 7.13; N, 12.38; O, 14.14

2103278-86-8

145343771

White to light yellow solid powder

1.2±0.1 g/cm3

836.8±55.0 °C at 760 mmHg

459.9±31.5 °C

0.0±3.1 mmHg at 25°C

1.592

2.7

4

4

9

33

723

3

C1CCC(CC1)C[C@@H](C(=O)N[C@@H](C[C@@H]2CCNC2=O)C=O)NC(=O)C3=CC4=CC=CC=C4N3

HPKJGHVHQWJOOT-ZJOUEHCJSA-N

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

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

Bofutrelvir; 2103278-86-8; Mpro inhibitor 11A; T5UX5SKK2S; MPI-10;

2934.99.9001

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.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 100 mg/mL (220.97 mM)

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.

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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.

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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.

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 (Please use freshly prepared in vivo formulations for optimal results.)