| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| 25mg |
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| 50mg |
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| 100mg |
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| 250mg | |||
| Other Sizes |
| Targets |
σ1 receptor (Ki = 0.8 nM in radioligand binding assay); σ2 receptor (Ki > 1000 nM, > 1250-fold selectivity over σ2) [1]
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| ln Vitro |
σ1 receptor binding affinity: EST64454 HCl binds to human σ1 receptor with high affinity (Ki = 0.8 nM) and exhibits exceptional selectivity (> 1250-fold) over σ2 receptor (Ki > 1000 nM). It shows no significant binding to 45 other receptors/ion channels (e.g., μ/δ/κ opioid receptors, TRPV1, NaV1.7) at 10 μM [1]
- σ1 receptor functional antagonism: In NG108-15 cells (expressing endogenous σ1 receptors), the compound antagonizes (+)-pentazocine (σ1 agonist)-induced Ca²⁺ mobilization with an IC50 of 2.3 μM. At 10 μM, it inhibits σ1 agonist-mediated Ca²⁺ response by 90% [1] - Inhibition of σ1-mediated signaling: EST64454 HCl (1–10 μM) blocks σ1 agonist-induced ERK1/2 phosphorylation in NG108-15 cells. At 5 μM, phosphorylated ERK1/2 levels are reduced by 75% compared to agonist-only control (western blot verification) [1] - High aqueous solubility: The compound exhibits excellent aqueous solubility (25 mg/mL in pH 7.4 buffer), significantly higher than other σ1 receptor antagonists (typically < 1 mg/mL) [1] |
| ln Vivo |
AUC0-∞, Vss, F%, and Cmax values of 771 ng/mL, 3.4 hours, 1431 ng·h/mL, and 69% were observed in male Wistar rats treated orally with EST64454 (10 mg/kg)[1]. After receiving 10 mg/kg of EST64454 intraperitoneally, male CD1 mice demonstrated the following: Cmax, t1/2, AUC0-∞, Vss, and F% of 1178 ng/mL, <1 hour, 2645 ng h/mL, 1.2 l/kg, and 60%, respectively [1].
Formalin-induced inflammatory pain inhibition: Male CD-1 mice were treated with EST64454 HCl (1–30 mg/kg, p.o.) 30 minutes before formalin (5%, 20 μL) hindpaw injection. The compound dose-dependently inhibits both early (0–5 min) and late (15–30 min) phases of formalin-induced licking/biting. At 30 mg/kg, early-phase response is reduced by 52% and late-phase by 68% [1] - Hot plate test analgesia: Female ICR mice were treated with EST64454 HCl (3–30 mg/kg, p.o.). The hot plate latency (55°C) was measured at 1, 2, 4 hours post-administration. At 10 mg/kg, the latency increased by 45% (2 hours post-dose) compared to vehicle, with analgesic effect lasting > 4 hours [1] - CCI-induced neuropathic pain relief: Male Sprague-Dawley rats with chronic constriction injury (CCI) of the sciatic nerve were treated with EST64454 HCl (3–30 mg/kg, p.o., qd) for 7 days. At 30 mg/kg, mechanical allodynia (von Frey filament) and thermal hyperalgesia (plantar test) were reduced by 62% and 58%, respectively, compared to vehicle-treated CCI rats [1] - No opioid-like side effects: Unlike morphine, EST64454 HCl (30 mg/kg, p.o.) did not induce tolerance (after 7-day repeated administration) or physical dependence (no withdrawal signs after naloxone challenge) in mice [1] |
| Enzyme Assay |
σ1 receptor radioligand binding assay: Recombinant human σ1 receptor was immobilized on microplates. Serial dilutions of EST64454 HCl (0.01 nM–10 μM) and [³H]-(+)-pentazocine (radiolabeled σ1 ligand) were co-incubated with the receptor at 25°C for 120 minutes. Unbound ligands were removed by washing, and bound radioactivity was measured with a scintillation counter. Ki value was calculated using competitive binding analysis [1]
- σ2 receptor selectivity assay: Parallel assays were performed with recombinant human σ2 receptor and [³H]-DTG (radiolabeled σ2 ligand). EST64454 HCl at concentrations up to 10 μM showed < 0.1% displacement of [³H]-DTG, confirming σ1/σ2 selectivity [1] - Broad receptor/ion channel selectivity screening: The compound was tested for binding to a panel of 45 targets (opioid receptors, ion channels, GPCRs) using radioligand binding or functional assays. Binding/inhibition rate < 10% at 10 μM confirmed no off-target activity [1] |
| Cell Assay |
Ca²⁺ mobilization antagonism assay: NG108-15 cells were seeded in 96-well plates (3×10⁴ cells/well) and incubated overnight. Cells were loaded with a Ca²⁺-sensitive fluorescent dye for 60 minutes at 37°C. Serial dilutions of EST64454 HCl (0.1 μM–50 μM) were added, followed by (+)-pentazocine (10 μM, σ1 agonist). Fluorescence intensity (excitation/emission = 485/525 nm) was measured in real-time, and IC50 was derived from dose-response curves of Ca²⁺ response inhibition [1]
- ERK1/2 phosphorylation assay: NG108-15 cells were seeded in 6-well plates (5×10⁵ cells/well) and pretreated with EST64454 HCl (1–10 μM) for 30 minutes, then stimulated with (+)-pentazocine (10 μM) for 15 minutes. Cells were lysed, proteins were separated by SDS-PAGE, transferred to membranes, and probed with anti-phospho-ERK1/2, anti-ERK1/2, and anti-β-actin (loading control) antibodies [1] |
| Animal Protocol |
Animal/Disease Models: Male Wistar rat (250-300 g) [1]
Doses: 10 mg/kg Route of Administration: Po (pharmacokinetic/PK/PK analysis) Experimental Results: Cmax, t1/2, AUC0-∞, Vss and F% were 771 ng/mL, 3.4 hrs (hrs (hours)), 1431 ng h/mL, 4.4 l/kg and 69%. Formalin-induced pain model: Male CD-1 mice (6–8 weeks old, n=8 per group) were randomized to vehicle or EST64454 HCl groups (1, 3, 10, 30 mg/kg). The compound was dissolved in water (due to high solubility) and administered orally 30 minutes before hindpaw injection of 5% formalin (20 μL). The total time of hindpaw licking/biting was recorded during 0–5 min (early phase) and 15–30 min (late phase) [1] - Hot plate test: Female ICR mice (n=8 per group) were treated with EST64454 HCl (3, 10, 30 mg/kg, p.o.) or vehicle. The hot plate was maintained at 55°C, and paw withdrawal latency was measured before dosing and at 1, 2, 4 hours post-administration. A cutoff time of 30 seconds was used to avoid tissue damage [1] - CCI-induced neuropathic pain model: Male Sprague-Dawley rats (n=6 per group) underwent CCI surgery on the right sciatic nerve. Seven days post-surgery, rats were treated with EST64454 HCl (3, 10, 30 mg/kg, p.o.) once daily for 7 days. Mechanical allodynia was assessed using von Frey filaments (50% paw withdrawal threshold), and thermal hyperalgesia using a plantar test (paw withdrawal latency) [1] - Tolerance and dependence studies: Mice were treated with EST64454 HCl (30 mg/kg, p.o.) or morphine (10 mg/kg, s.c.) once daily for 7 days. Analgesic tolerance was evaluated by hot plate test on day 1 and day 7. Physical dependence was assessed by administering naloxone (5 mg/kg, i.p.) 24 hours after the last dose, and withdrawal signs (jumping, paw tremor) were counted for 30 minutes [1] - Pharmacokinetic study: Male Sprague-Dawley rats (n=3 per time point) were administered a single oral dose of EST64454 HCl (10 mg/kg, dissolved in water). Blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, 12, 24 hours post-administration. Plasma drug concentrations were quantified by LC-MS/MS, and pharmacokinetic parameters were calculated using non-compartmental analysis [1] |
| ADME/Pharmacokinetics |
Oral bioavailability: In Sprague-Dawley rats, the oral bioavailability of EST64454 HCl after a single oral dose of 10 mg/kg was 85% [1]
- Plasma pharmacokinetics: Peak plasma concentration (Cmax) = 4.2 μM (Tmax = 1 h), elimination half-life (t1/2) = 4.2 h, AUC₀₋₂₄h = 22.8 μM·h [1] - Tissue distribution: 24 hours after oral administration, the compound was widely distributed in tissues associated with pain signal transduction: brain (tissue/plasma ratio = 1.8), spinal cord (2.1) and dorsal root ganglia (2.5). Low accumulation in the liver (1.2) and kidney (1.1) [1] - Solubility and stability: Water solubility = 25 mg/mL (pH 7.4 buffer), more than 50 times higher than clinical σ1 antagonists. Stable for 24 hours in simulated gastric juice (pH 1.2) and intestinal juice (pH 6.8) [1] - Metabolism: In vitro liver microsomal assays showed that the compound was metabolized by CYP3A4-mediated oxidative metabolism, with 65% of the parent compound remaining after 2 hours. At concentrations up to 50 μM, no inhibitory effect on CYP isoenzymes (CYP1A2, 2C9, 2C19, 2D6, 3A4) was observed [1] - Excretion: In rats, 70% of the administered dose was excreted in feces within 72 hours (45% as unchanged drug, 25% as metabolites), and 25% was excreted in urine (15% as unchanged drug, 10% as metabolites) [1] |
| Toxicity/Toxicokinetics |
Acute toxicity: No death or acute toxicity symptoms (drowsiness, ataxia, loss of appetite) were observed in rats and mice after a single oral dose of up to 300 mg/kg of EST64454 HCl. The LD50 of both species was >300 mg/kg [1] - Repeated-dose toxicity: No significant changes were observed in body weight, hematological/biochemical indicators (ALT, AST, creatinine, BUN) and histopathology of major organs (brain, liver, kidney, heart, spinal cord) after 28 consecutive days of oral administration of EST64454 HCl (10, 50, 200 mg/kg, once daily) in rats [1] - Plasma protein binding rate: In vitro tests showed that EST64454 HCl was 92% bound to human plasma proteins [1] - Central nervous system safety: No sedation or motor dysfunction was observed in mice at doses up to 30 mg/kg (rotarod test: no significant reduction in the fall latency compared to the solvent control group) [1]
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| References | |
| Additional Infomation |
Background: The σ1 receptor is a molecular chaperone protein involved in pain signaling pathways and is overactivated in inflammatory and neuropathic pain. σ1 antagonists provide a non-opioid analgesic approach that avoids the risks of tolerance, dependence and overdose associated with opioids [1]
- Mechanism of action: EST64454 HCl binds to the allosteric site of the σ1 receptor, blocking its interaction with downstream signaling pathway partners such as IP3 receptors and ERK. This inhibits the amplification of pain signals in the central and peripheral nervous systems mediated by σ1 [1] - Therapeutic potential: As a clinical analgesic candidate, this compound is effective in treating inflammatory (formalin) pain and neuropathic (CCI) pain with a good safety profile. Its high water solubility allows it to be administered orally with stable bioavailability[1] - Advantages compared to existing drugs: EST64454 HCl has higher solubility (> 25 mg/mL vs. < 1 mg/mL), higher oral bioavailability (85% vs. 40–60%) and longer duration of analgesia (> 4 hours) compared to other σ1 receptor antagonists[1] - Indications: Developed as a non-opioid alternative therapy for the treatment of acute and chronic pain (e.g., inflammatory pain, neuropathic pain)[1] |
| Molecular Formula |
C18H23CLF2N4O2
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|---|---|
| Molecular Weight |
400.8548
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| Exact Mass |
400.147
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| Elemental Analysis |
C, 53.93; H, 5.78; Cl, 8.84; F, 9.48; N, 13.98; O, 7.98
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| CAS # |
1950569-11-5
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| Related CAS # |
1351438-26-0;1950569-11-5 (HCl);
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| PubChem CID |
121418768
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| Appearance |
White to off-white solid powder
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
27
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| Complexity |
463
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| Defined Atom Stereocenter Count |
0
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| SMILES |
Cl.FC1=C(C=CC(=C1)N1C=CC(COCCN2CCN(C(C)=O)CC2)=N1)F
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| InChi Key |
YJZGDOPAALDWAT-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C18H22F2N4O2.ClH/c1-14(25)23-8-6-22(7-9-23)10-11-26-13-15-4-5-24(21-15)16-2-3-17(19)18(20)12-16;/h2-5,12H,6-11,13H2,1H3;1H
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| Chemical Name |
1-(4-(2-((1-(3,4-difluorophenyl)-1H-pyrazol-3-yl)methoxy)ethyl)piperazin-1-yl)ethan-1-one hydrochloride
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| Synonyms |
EST64454 hydrochlorideEST-64454EST64454 EST 64454
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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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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) |
H2O : ≥ 100 mg/mL (~249.47 mM)
DMSO : ~100 mg/mL (~249.47 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.24 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 (6.24 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 (6.24 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 | 2.4947 mL | 12.4735 mL | 24.9470 mL | |
| 5 mM | 0.4989 mL | 2.4947 mL | 4.9894 mL | |
| 10 mM | 0.2495 mL | 1.2473 mL | 2.4947 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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