σ1 receptor (Ki = 0.8 nM in radioligand binding assay); σ2 receptor (Ki > 1000 nM, > 1250-fold selectivity over σ2) [1]

σ1受体结合亲和力：EST64454 HCl 高亲和力结合人σ1受体（Ki = 0.8 nM），对σ2受体具有极高选择性（>1250倍，Ki > 1000 nM）。10 μM浓度下，对45种其他受体/离子通道（如μ/δ/κ阿片受体、TRPV1、NaV1.7）无显著结合 [1]
- σ1受体功能拮抗：在表达内源性σ1受体的NG108-15细胞中，该化合物拮抗σ1激动剂(+)-戊唑辛诱导的钙流，IC50为2.3 μM。10 μM浓度下，抑制σ1激动剂介导的钙响应达90% [1]
- σ1介导的信号通路抑制：EST64454 HCl（1–10 μM）阻断NG108-15细胞中σ1激动剂诱导的ERK1/2磷酸化。5 μM浓度下，磷酸化ERK1/2水平较仅激动剂处理组降低75%（Western blot验证）[1]
- 高水溶性：该化合物具有优异的水溶性（pH 7.4缓冲液中25 mg/mL），显著高于其他σ1受体拮抗剂（通常<1 mg/mL）[1]

在口服 EST64454 (10 mg/kg) 的雄性 Wistar 大鼠中观察到 AUC0-∞、Vss、F% 和 Cmax 值为 771 ng/mL、3.4 小时、1431 ng·h/mL 和 69%[1] 。腹腔注射 10 mg/kg EST64454 后，雄性 CD1 小鼠表现出以下结果：Cmax、t1/2、AUC0-∞、Vss 和 F% 为 1178 ng/mL，<1 小时，2645 ng h/mL，1.2 l /kg 和 60%，分别 [1]。

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福尔马林诱导的炎症痛抑制：雄性CD-1小鼠口服EST64454 HCl（1、3、10、30 mg/kg）30分钟后，后爪注射5%福尔马林（20 μL）。化合物呈剂量依赖性抑制福尔马林诱导的早时相（0–5分钟）和晚时相（15–30分钟）舔咬行为。30 mg/kg剂量下，早时相反应减少52%，晚时相减少68% [1]
- 热板实验镇痛效果：雌性ICR小鼠口服EST64454 HCl（3、10、30 mg/kg）后，于1、2、4小时检测55°C热板痛阈。10 mg/kg剂量下，给药2小时后痛阈提高45%，镇痛效果持续>4小时 [1]
- CCI诱导的神经病理性痛缓解：坐骨神经慢性压迫损伤（CCI）的雄性Sprague-Dawley大鼠，口服EST64454 HCl（3、10、30 mg/kg，每日一次）治疗7天。30 mg/kg剂量下，机械痛觉过敏（冯·弗雷丝）和热痛觉过敏（足底测试）分别减轻62%和58%（相较于溶剂处理的CCI大鼠）[1]
- 无阿片类副作用：与吗啡不同，EST64454 HCl（30 mg/kg，口服）连续7天给药后无耐受形成，纳洛酮激发后无戒断症状（跳跃、爪震颤）[1]

σ1受体放射性配体结合实验：重组人σ1受体固定于酶标板，系列稀释的EST64454 HCl（0.01 nM–10 μM）与[³H]-(+)-戊唑辛（放射性标记σ1配体）在25°C下与受体共孵育120分钟。洗涤去除未结合配体后，闪烁计数器检测结合放射性强度，通过竞争性结合分析计算Ki值 [1]
- σ2受体选择性实验：用重组人σ2受体和[³H]-DTG（放射性标记σ2配体）进行平行实验。EST64454 HCl浓度高达10 μM时，对[³H]-DTG的置换率<0.1%，证实σ1/σ2选择性 [1]
- 广谱受体/离子通道选择性筛选：通过放射性配体结合或功能实验，检测化合物对45种靶点（阿片受体、离子通道、GPCR）的结合/抑制活性。10 μM浓度下结合/抑制率<10%，证实无脱靶活性 [1]

钙流拮抗实验：NG108-15细胞以3×10⁴个细胞/孔接种于96孔板，过夜孵育。负载钙敏感荧光染料（37°C，60分钟）后，加入系列稀释的EST64454 HCl（0.1 μM–50 μM），随后加入(+)-戊唑辛（10 μM，σ1激动剂）。酶标仪实时检测荧光强度（激发/发射=485/525 nm），从钙响应抑制的剂量-反应曲线推导IC50值 [1]
- ERK1/2磷酸化实验：NG108-15细胞以5×10⁵个细胞/孔接种于6孔板，用EST64454 HCl（1–10 μM）预处理30分钟后，(+)-戊唑辛（10 μM）刺激15分钟。细胞裂解后，蛋白经SDS-PAGE分离、转膜，用抗磷酸化ERK1/2、ERK1/2及内参β-肌动蛋白抗体检测 [1]

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%.

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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]

Oral bioavailability: In Sprague-Dawley rats, the oral bioavailability of EST64454 HCl is 85% following a single 10 mg/kg oral dose [1]
- Plasma pharmacokinetics: Peak plasma concentration (Cmax) = 4.2 μM (Tmax = 1 hour), elimination half-life (t1/2) = 4.2 hours, AUC₀₋₂₄h = 22.8 μM·h [1]
- Tissue distribution: Twenty-four hours after oral administration, the compound distributes widely to tissues relevant to pain signaling: brain (tissue/plasma ratio = 1.8), spinal cord (2.1), and dorsal root ganglia (2.5). Low accumulation in liver (1.2) and kidneys (1.1) [1]
- Solubility and stability: Aqueous solubility = 25 mg/mL (pH 7.4 buffer), > 50-fold higher than clinical σ1 antagonists. Stable in simulated gastric fluid (pH 1.2) and intestinal fluid (pH 6.8) for 24 hours [1]
- Metabolism: In vitro liver microsome assays show metabolism via CYP3A4-mediated oxidation, with 65% of parent compound remaining after 2 hours. No inhibition of CYP isoforms (CYP1A2, 2C9, 2C19, 2D6, 3A4) at concentrations up to 50 μM [1]
- Excretion: In rats, 70% of the administered dose is excreted in feces (45% parent, 25% metabolites) and 25% in urine (15% parent, 10% metabolites) within 72 hours [1]

Acute toxicity: Single oral doses of EST64454 HCl up to 300 mg/kg in rats and mice show no mortality or acute toxicity signs (lethargy, ataxia, loss of appetite). LD50 > 300 mg/kg in both species [1]
- Repeat-dose toxicity: Rats treated with EST64454 HCl (10, 50, 200 mg/kg, p.o., qd) for 28 days show no significant changes in body weight, hematological/biochemical parameters (ALT, AST, creatinine, BUN), or histopathology of major organs (brain, liver, kidney, heart, spinal cord) [1]
- Plasma protein binding: In vitro assay shows EST64454 HCl binds to human plasma proteins at a rate of 92% [1]
- CNS safety: No sedative or motor-impairing effects in mice at doses up to 30 mg/kg (rotarod test: no significant reduction in fall latency vs. vehicle) [1]

[1]. EST64454: a Highly Soluble σ1 Receptor Antagonist Clinical Candidate for Pain Management. J Med Chem. 2020;63(23):14979-14988.

Background: The σ1 receptor is a chaperone protein involved in pain signaling pathways, overactivated in inflammatory and neuropathic pain. σ1 antagonists offer a non-opioid approach to pain management, avoiding opioid-related tolerance, dependence, and overdose risks [1]
- Mechanism of action: EST64454 HCl binds to the σ1 receptor’s allosteric site, blocking its interaction with downstream signaling partners (e.g., IP3 receptors, ERK). This inhibits σ1-mediated amplification of pain signals in the CNS and peripheral nervous system [1]
- Therapeutic potential: As a clinical candidate for pain management, the compound effectively treats inflammatory (formalin) and neuropathic (CCI) pain with a favorable safety profile. Its high aqueous solubility enables oral administration and consistent bioavailability [1]
- Advantages over existing agents: Compared to other σ1 antagonists, EST64454 HCl has superior solubility (> 25 mg/mL vs. < 1 mg/mL), higher oral bioavailability (85% vs. 40–60%), and longer analgesic duration (> 4 hours) [1]
- Indication: Developed for the treatment of acute and chronic pain (e.g., inflammatory pain, neuropathic pain) as a non-opioid alternative [1]

C18H23CLF2N4O2

400.8548

400.147

C, 53.93; H, 5.78; Cl, 8.84; F, 9.48; N, 13.98; O, 7.98

1950569-11-5

1351438-26-0;1950569-11-5 (HCl);

121418768

White to off-white solid powder

50.6

1

6

6

27

463

0

Cl.FC1=C(C=CC(=C1)N1C=CC(COCCN2CCN(C(C)=O)CC2)=N1)F

YJZGDOPAALDWAT-UHFFFAOYSA-N

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

1-(4-(2-((1-(3,4-difluorophenyl)-1H-pyrazol-3-yl)methoxy)ethyl)piperazin-1-yl)ethan-1-one hydrochloride

EST64454 hydrochlorideEST-64454EST64454 EST 64454

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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

H2O : ≥ 100 mg/mL (~249.47 mM)
DMSO : ~100 mg/mL (~249.47 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.24 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (6.24 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (6.24 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。