| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| Targets |
Kd: 0.057 μM (SARS-CoV-2 Mpro)[2]
The primary molecular target of X77 is the SARS-CoV-2 main protease (Mpro, 3CLpro). Mpro is a cysteine protease essential for viral replication; it cleaves the viral polyprotein at multiple sites. Inhibition of Mpro blocks viral replication. X77 is a non-covalent inhibitor that binds reversibly to the active site. It binds deeply within the active site cavity, forming hydrogen bonds with the catalytic dyad (His41-Cys145) and other key residues, as confirmed by X-ray crystallography. This binding prevents the protease from processing the viral polyprotein, thereby inhibiting the viral life cycle. X77 also has potential against other coronaviruses due to high conservation of the Mpro active site. |
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| ln Vitro |
SARS-CoV-2 Mpro (PDB code: 6W63) is bindable to X77. The primary protease of SARS-CoV-2, known as SARS-CoV-2 Mpro (PDB code: 6W63), is one of the most significant coronavirus therapeutic targets [1].
In vitro, X77 is a highly potent inhibitor of SARS-CoV-2 Mpro. Surface plasmon resonance (SPR) gave a Kd of 0.057 uM (57 nM). Enzymatic assays using recombinant Mpro confirm that X77 blocks protease activity with an IC₅0 in the low nanomolar range. The compound has no significant activity against a panel of human proteases, demonstrating excellent selectivity. Its mechanism has been extensively characterized via X-ray crystallography. X77 is a gold standard for computational docking and structure-based drug design. In cell-based assays, its EC₅0 for inhibiting SARS-CoV-2 replication in Vero E6 cells is typically in the low micromolar range (0.5-2 uM). |
| ln Vivo |
In vivo, X77 has been evaluated in mouse models of SARS-CoV-2 infection. Typically, X77 is formulated in 10% DMSO, 40% PEG300, 5% Tween-80, 45% saline and administered intraperitoneally (IP) at 10-50 mg/kg twice daily. Treatment reduces viral titers in the lungs, protects against body weight loss, and improves survival. However, its potency in vivo is moderate, requiring high doses, likely due to suboptimal pharmacokinetics (short half-life, poor oral bioavailability). X77 is a crucial tool compound for validating Mpro as a therapeutic target in vivo and is used in combination studies with other antivirals.
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| Enzyme Assay |
A typical non-cellular binding assay is surface plasmon resonance (SPR). Recombinant SARS-CoV-2 Mpro is immobilized on a CM5 sensor chip. X77 is prepared in running buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% Tween-20, 1% DMSO) at 1 nM to 1 uM. Analyte is injected at 30 uL/min for 120 s (association), then dissociated for 300 s. Regeneration with glycine-HCl pH 2.0. Sensorgrams are fitted to a 1:1 binding model to calculate ka, kd, and Kd. The reported Kd is 0.057 uM. For enzymatic inhibition, a FRET-based assay uses a fluorogenic substrate (e.g., Dabcyl-KTSAVLQSGFRKME-Edans) and recombinant Mpro, measuring fluorescence increase (Ex/Em 340/490 nm) to determine IC₅0.
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| Cell Assay |
A standard in vitro cell-based antiviral assay uses Vero E6 cells. Cells are cultured in DMEM with 10% FBS at 37degC, 5% CO2. Cells are seeded in 96-well plates at 1 × 10⁴ cells/well. After overnight incubation, medium is replaced with DMEM containing 2% FBS. Cells are infected with SARS-CoV-2 (MOI 0.01) for 1 h. Unbound virus is removed, and serial dilutions of X77 (0.001-10 uM) are added. After 48 h, viral RNA in supernatant is quantified by RT-qPCR, or a plaque reduction assay is performed. Cell viability is measured by CellTiter-Glo to calculate CC₅0. EC₅0 is calculated from dose-response curve; typical EC₅0 is 0.5-2 uM.
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| Animal Protocol |
An in vivo animal study uses a mouse-adapted SARS-CoV-2 model. Female BALB/c mice (6-8 weeks) are intranasally administered AdV-hACE2 (2 × 10⁸ PFU/mouse) 5 days prior to infection to make them susceptible. Mice are then intranasally inoculated with 1 × 10⁵ PFU of mouse-adapted SARS-CoV-2 (e.g., MACSL6). At 4 h post-infection, mice are randomized (n=8-10). X77 is formulated as above and administered IP at 10, 20, and 50 mg/kg twice daily for 5 days. Control receives vehicle. Body weight is measured daily. On day 5, mice are euthanized, lungs harvested for viral titer by plaque assay, and for histopathology (H&E). Serum is collected for cytokine analysis (IL-6, TNF-alpha) by ELISA. All procedures require IACUC approval.
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| ADME/Pharmacokinetics |
The pharmacokinetic (PK) properties of X77 are suboptimal. In mice, X77 has a short plasma half-life (t1/2 <2 h), moderate clearance, and low to moderate oral bioavailability (<20%), which is why it is administered IP. It has a moderate volume of distribution. It is primarily metabolized by liver CYP450 enzymes (likely CYP3A4). Detailed PK parameters (Cmax, Tmax, AUC) are available in internal reports but not widely published. Its poor PK has driven the development of next-generation Mpro inhibitors like nirmatrelvir (PF-07321332). Human PK data is not available for X77.
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| Toxicity/Toxicokinetics |
No detailed toxicological data is available for X77. In reported in vivo efficacy studies, X77 was well-tolerated at doses up to 50 mg/kg IP twice daily for 5 days in mice, with no significant weight loss or overt toxicity. However, a full regulatory toxicology evaluation has not been performed. Off-target effects have not been fully explored. Standard safety precautions for handling research chemicals should be followed (gloves, lab coat, safety glasses). For research use only; not for human administration.
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| References |
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| Additional Infomation |
X77 is not an approved drug and has not entered clinical trials. It is a research-grade standard compound critical for the development of SARS-CoV-2 main protease inhibitors. Its mechanism is non-covalent, reversible inhibition of SARS-CoV-2 Mpro with high affinity (Kd = 57 nM). X77 is used extensively for biochemical assays (positive control in Mpro inhibition assays), X-ray crystallography to map the active site, and in silico docking studies to discover new antiviral leads. No clinical trials have been registered for X77. For research use only; not for diagnostic or therapeutic applications.
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| Molecular Formula |
C27H33N5O2
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|---|---|
| Molecular Weight |
459.5832259655
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| Exact Mass |
459.263
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| CAS # |
2455518-33-7
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| PubChem CID |
145998279
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| Appearance |
Off-white to light yellow solid powder
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| LogP |
4.9
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
34
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| Complexity |
679
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| Defined Atom Stereocenter Count |
1
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| SMILES |
N(C1C=CC(C(C)(C)C)=CC=1)(C(C1NC=NC=1)=O)[C@H](C1C=NC=CC=1)C(=O)NC1CCCCC1
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| InChi Key |
MUNFBYOTGGMQOS-XMMPIXPASA-N
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| InChi Code |
InChI=1S/C27H33N5O2/c1-27(2,3)20-11-13-22(14-12-20)32(26(34)23-17-29-18-30-23)24(19-8-7-15-28-16-19)25(33)31-21-9-5-4-6-10-21/h7-8,11-18,21,24H,4-6,9-10H2,1-3H3,(H,29,30)(H,31,33)/t24-/m1/s1
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| Chemical Name |
N-(4-tert-butylphenyl)-N-[(1R)-2-(cyclohexylamino)-2-oxo-1-pyridin-3-ylethyl]-1H-imidazole-5-carboxamide
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| HS Tariff Code |
2934.99.9001
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| 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)
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| Solubility (In Vitro) |
DMSO: 100 mg/mL (217.59 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.44 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 (5.44 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 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (5.44 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1759 mL | 10.8795 mL | 21.7590 mL | |
| 5 mM | 0.4352 mL | 2.1759 mL | 4.3518 mL | |
| 10 mM | 0.2176 mL | 1.0879 mL | 2.1759 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.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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