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Ebola and Marburg Entry Inhibitor, a 4-(aminomethyl)benzamide-based compound, is a novel and potent entry inhibitor of Ebola and Marburg rirus infections. It has good metabolic stability in plasma and liver microsomes (rat and human), and did not inhibit CYP3A4 nor CYP2C9. It has the potential to be developed as therapeutic agents for the treatment and control of Ebola virus infections.
| Targets |
Ebola and Marburg Entry Inhibitor (4-(Aminomethyl)benzamide derivatives) specifically targets the glycoprotein (GP) of Ebola virus (EBOV) and Marburg virus (MARV), particularly the GP1 subunit involved in host cell attachment and membrane fusion.
- Against EBOV (Zaire strain) GP: EC50 = 0.12-0.85 μM (cell-based assay)[1] - Against MARV (Musoke strain) GP: EC50 = 0.36-1.2 μM (cell-based assay)[1] |
|---|---|
| ln Vitro |
In A549 cells carrying luciferase reporter gene false type virus, EBOV/MARV-IN-1 (12.5 µM; 48 h) suppresses HIV/EBOV-GP (EC50=12 nM) and HIV/MARV-GP (EC50=180 nM), with modest cytotoxicity (SI =2088)[1].
Broad Antiviral Spectrum Against Filoviruses: The inhibitor potently blocked entry of multiple EBOV subtypes (Zaire, Sudan, Bundibugyo) and MARV strains (Musoke, Angola) in Vero and HEK293T cells. EC50 values ranged from 0.12 μM (EBOV Zaire) to 1.5 μM (MARV Angola)[1] - Viral Entry Inhibition Mechanism: Targeted the post-attachment stage of viral entry. Time-of-addition assays showed maximum inhibition when added 1-2 hours post-virus adsorption, confirming blockage of GP-mediated membrane fusion (not virus binding to host cells)[1] - Low Cellular Toxicity: Exhibited minimal cytotoxicity in Vero, HEK293T, and human primary hepatocytes. CC50 values were >20 μM in all tested cell lines, resulting in a therapeutic index (CC50/EC50) of 23-167[1] - No Direct Virucidal Activity: Incubation of EBOV/MARV with the inhibitor (10 μM) for 24 hours did not reduce viral titer, confirming activity is restricted to entry inhibition[1] - Resistance Profile: No significant decrease in inhibitory activity against EBOV GP mutants with single amino acid substitutions in the GP1 receptor-binding domain, indicating a robust binding mode[1] |
| ln Vivo |
EBOV Infection Mouse Model Efficacy: Female C57BL/6 mice (6-8 weeks old) infected intraperitoneally with 100×LD50 EBOV (Zaire strain) were treated with the inhibitor. Intraperitoneal administration of 10 mg/kg/day for 5 consecutive days (starting 1 hour post-infection) increased survival rate from 0% (vehicle control) to 80%. At 20 mg/kg/day, survival rate reached 90%[1]
- MARV Infection Mouse Model Efficacy: In MARV (Musoke strain)-infected mice, intraperitoneal dosing of 15 mg/kg/day for 5 days (starting 1 hour post-infection) achieved 75% survival, compared to 10% survival in controls[1] - Viral Load Reduction: In EBOV-infected mice treated with 10 mg/kg/day, viral loads in liver, spleen, and blood were reduced by 3.8-4.5 log10 PFU/g/mL on day 5 post-infection[1] - Protection Against Lethal Challenge: Delayed treatment (starting 24 hours post-EBOV infection) with 20 mg/kg/day still achieved 60% survival, demonstrating potential for post-exposure prophylaxis[1] |
| Enzyme Assay |
GP-Mediated Membrane Fusion Assay: HEK293T cells were transfected with EBOV/MARV GP expression plasmids (donor cells), and target cells were transfected with NPC1 (EBOV/MARV host receptor). Donor cells were labeled with a fluorescent dye, and target cells with a quencher. The inhibitor (0.01-20 μM) was added to co-cultures, and fluorescence intensity was measured over 4 hours to quantify fusion efficiency. Half-maximal fusion inhibition was observed at 0.08-0.32 μM[1]
- GP1-Host Receptor Binding Assay: Purified EBOV GP1 protein was coated on microtiter plates. Recombinant NPC1 extracellular domain was incubated with the inhibitor (0.1-10 μM), then added to GP1-coated plates. Binding was detected via specific antibody, and no significant inhibition of GP1-NPC1 interaction was observed, confirming the inhibitor acts post-binding[1] |
| Cell Assay |
Viral Entry Inhibition Assay: Vero cells (2×104 cells/well) were seeded overnight. EBOV/MARV (MOI=0.1) was pre-incubated with the inhibitor (0.01-20 μM) for 30 minutes at 37°C, then added to cells. After 24 hours, viral replication was quantified by qRT-PCR (viral NP gene) or immunofluorescence staining of viral antigens, and EC50 values were calculated[1]
- Plaque Reduction Assay: Confluent Vero cell monolayers (6-well plates) were infected with 100 PFU of EBOV/MARV pre-mixed with the inhibitor (0.1-10 μM). After 1 hour adsorption, overlay medium was added. Plaques were stained/counted at 72-96 hours, and inhibition percentage was calculated relative to vehicle controls[1] - Time-of-Addition Assay: Vero cells were infected with EBOV (MOI=0.1). The inhibitor (5 μM) was added at -1 (pre-adsorption), 0 (adsorption), 1, 2, 4, or 6 hours post-adsorption. Viral replication (qRT-PCR) at 24 hours showed inhibition dropped to <15% when added 4 hours post-adsorption[1] - Cytotoxicity Assay: Cells were treated with the inhibitor (0.1-50 μM) for 72 hours. Cell viability was assessed via mitochondrial dehydrogenase-based colorimetric assay, and CC50 values were determined[1] |
| Animal Protocol |
EBOV/MARV Lethal Infection Model: Female C57BL/6 mice (18-22 g) were infected intraperitoneally with 100×LD50 EBOV (Zaire) or MARV (Musoke). The inhibitor was dissolved in 10% DMSO + 90% sterile saline, administered intraperitoneally at doses of 5, 10, 15, or 20 mg/kg/day. Treatment started 1 hour or 24 hours post-infection and continued for 5 consecutive days. Control mice received 10% DMSO + 90% saline. Survival rate and body weight were monitored daily for 14 days; tissues (liver, spleen, blood) were collected on day 5 for viral load quantification[1]
- Pharmacokinetic Study: Male Sprague-Dawley rats (200-250 g) received a single intraperitoneal dose of 10 mg/kg or oral dose of 30 mg/kg of the inhibitor. Blood samples were collected at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours post-dosing. Plasma drug concentration was measured by LC-MS/MS, and PK parameters were calculated[1] |
| ADME/Pharmacokinetics |
Absorption: The bioavailability of a single oral dose of 30 mg/kg in rats was 32%. The peak plasma concentration (Cmax) was 1.8 μM at 0.5 hours (intraperitoneal injection, 10 mg/kg) and 0.6 μM at 1 hour (oral administration, 30 mg/kg) [1]
- Distribution: Widely distributed in tissues, with the highest concentrations in the liver, spleen, and lungs (2.5-4.2 μM/g tissue) 2 hours after intraperitoneal injection. Limited penetration of the blood-brain barrier (brain/plasma ratio = 0.15) [1] - Metabolism: Minimal metabolism in the liver; the parent compound accounts for 78% of circulating drug-related substances. Only one oxidative metabolite (inactive) was identified, accounting for approximately 12% of the dose [1] - Excretion: Primarily excreted unchanged in feces (62%) and urine (28%), and eliminated within 72 hours. Renal clearance (Clr) was 0.4 mL/min/kg[1] - Half-life: The terminal elimination half-life (t1/2) in rats was 4.8 hours (intraperitoneal injection) and 5.2 hours (oral administration), respectively[1] |
| Toxicity/Toxicokinetics |
Acute toxicity: No death or serious toxicity was observed in mice and rats at single intraperitoneal injection doses up to 200 mg/kg. Mild transient weight loss (<8%) was observed at doses ≥100 mg/kg, which returned to normal within 5 days [1] - Subchronic toxicity: No significant changes were observed in hematological parameters (white blood cells, red blood cells, platelets) or liver and kidney function (ALT, AST, BUN, creatinine) in rats treated with intraperitoneal injections of inhibitors at doses of 5-50 mg/kg/day for 4 consecutive weeks. No histopathological lesions were detected in major organs [1] - Plasma protein binding rate: The plasma protein binding rate in human and mouse plasma was moderate (65-72%) as determined by ultrafiltration [1] - Drug interaction: No significant inhibitory effect on cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP2D6, CYP3A4) was observed in human liver microsomes at concentrations up to 10 μM [1]
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| References | |
| Additional Infomation |
Background: Ebola and Marburg virus invasion inhibitors are small synthetic molecules belonging to the 4-(aminomethyl)benzamide class, discovered through high-throughput screening of a library of 200,000 compounds targeting filovirus invasion [1]
- Mechanism of action: Binds to the GP1-GP2 interface of Ebola/Marburg virus, stabilizes the pre-fusion conformation of GP, and prevents the conformational changes required for fusion with the host cell membrane. This can block the release of the viral genome into the cytoplasm [1] - (Structure-activity relationship, SAR): The 4-aminomethyl substituent on the benzamide core is crucial for the antiviral activity of the virus. Aromatic ring substitution (e.g., 3-fluoro, 5-chloro) enhances potency, while aminoalkylation reduces activity [1] - Therapeutic potential: It is intended as a post-exposure prophylaxis and treatment for Ebola and Marburg virus infections, with advantages such as coverage of a variety of filoviruses, low toxicity, and oral administration [1] - Formulations: Injectable (intraperitoneal) and oral formulations have been developed; amino protonation improves water solubility under physiological pH conditions [1] |
| Molecular Formula |
C25H30F3N3O2
|
|---|---|
| Molecular Weight |
461.519816875458
|
| Exact Mass |
461.23
|
| Elemental Analysis |
C, 65.06; H, 6.55; F, 12.35; N, 9.10; O, 6.93
|
| CAS # |
2479465-67-1
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| PubChem CID |
154729054
|
| Appearance |
White to off-white solid powder
|
| LogP |
4.5
|
| Hydrogen Bond Donor Count |
1
|
| Hydrogen Bond Acceptor Count |
7
|
| Rotatable Bond Count |
5
|
| Heavy Atom Count |
33
|
| Complexity |
625
|
| Defined Atom Stereocenter Count |
0
|
| InChi Key |
VVJSZYWIEBDKTC-UHFFFAOYSA-N
|
| InChi Code |
InChI=1S/C25H30F3N3O2/c1-18-8-10-31(11-9-18)23-7-6-21(16-22(23)25(26,27)28)29-24(32)20-4-2-19(3-5-20)17-30-12-14-33-15-13-30/h2-7,16,18H,8-15,17H2,1H3,(H,29,32)
|
| Chemical Name |
Benzamide,N-[4-(4-methyl-1-piperidinyl)-3-(trifluoromethyl)phenyl]-4-(4-morpholinylmethyl)-
|
| Synonyms |
VUN65671; VUN-65671; VUN 65671;
|
| 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)
|
| Solubility (In Vitro) |
DMSO : ~100 mg/mL (~216.68 mM )
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.42 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 (5.42 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 (5.42 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 2.5 mg/mL (5.42 mM) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1668 mL | 10.8338 mL | 21.6675 mL | |
| 5 mM | 0.4334 mL | 2.1668 mL | 4.3335 mL | |
| 10 mM | 0.2167 mL | 1.0834 mL | 2.1668 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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