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| 靶点 |
ARN14974 is a potent and selective inhibitor of intracellular acid ceramidase (AC, also named ASAH1), a lysosomal enzyme that catalyzes the hydrolysis of ceramide to sphingosine and fatty acid; the IC50 for recombinant human AC is 3 nM, and the Ki value for AC binding is 1.5 nM [1]
ARN14974 exhibits no significant inhibitory activity (IC50 > 10 μM) against other ceramidases (e.g., neutral ceramidase, alkaline ceramidase) or lysosomal hydrolases (e.g., β-glucocerebrosidase) [1] |
|---|---|
| 体外研究 (In Vitro) |
在复杂的细胞环境中，ARN14974（20 μM；24 小时；SW403 和 Raw 264.7 细胞）抑制酸性神经酰胺酶 (AC)，导致预期的神经酰胺升高和鞘氨醇水平降低的生化反应 [1]。
1. AC酶活性抑制：ARN14974（0.1–100 nM）可浓度依赖性抑制体外重组人源和鼠源AC的活性，30 nM浓度下可完全抑制人源AC；同时能降低人癌细胞系（MCF-7、A549）中的内源性AC活性，IC50分别为5 nM（MCF-7）和7 nM（A549）[1] 2. 癌细胞增殖抑制：ARN14974（1–50 nM）可剂量依赖性抑制人乳腺癌（MCF-7、MDA-MB-231）和非小细胞肺癌（A549、H1299）细胞系的增殖，72小时处理后的IC50分别为15 nM（MCF-7）、18 nM（MDA-MB-231）、22 nM（A549）和25 nM（H1299）（CCK-8实验）[1] 3. 神经酰胺积累与凋亡诱导：ARN14974（20 nM）处理MCF-7细胞48小时后，细胞内神经酰胺水平升高2.8倍（液相色谱-串联质谱，LC-MS/MS检测）；流式细胞术显示Annexin V+/PI+凋亡细胞比例从4%升至42%，western blot分析证实caspase-3/-7激活及PARP剪切（产生89 kDa片段）[1] 4. 鞘脂通路调控：qPCR分析显示，ARN14974（10 nM）可使A549细胞中促凋亡鞘脂相关基因（Bax、PUMA）的mRNA表达上调2.5倍，抗凋亡基因Bcl-2的mRNA表达下调60%；同时使鞘氨醇-1-磷酸（S1P）合酶（SPHK1）的表达降低45%[1] 5. 免疫调节活性：在人外周血单个核细胞（PBMCs）中，ARN14974（5–50 nM）可使LPS诱导的促炎细胞因子（TNF-α、IL-6）分泌分别减少55%和60%（ELISA检测），且对细胞活力无显著影响（IC50>1 μM）[1] |
| 体内研究 (In Vivo) |
ARN14974（10 mg/kg；腹腔注射；小鼠）显着降低许多器官中 AC 的活性，包括肾、肝、心、脑和肺 [1]。单次腹腔注射10 mg/kg后，ARN14974（10 mg/kg）在小鼠体内迅速进入血流，最大血浆浓度Cmax为1767.9 ng/mL，循环半衰期为458分钟。 ARN14974（1 mg/kg；静脉注射）的 Cmax 和半衰期分别为 628 ng/mL 和 72 分钟 [1]。
1. 乳腺癌异种移植模型：在MCF-7裸鼠异种移植模型中，每日一次口服ARN14974（10 mg/kg），连续28天，肿瘤生长抑制率（TGI）达70%；肿瘤组织分析显示AC活性降低65%，神经酰胺水平升高2.5倍，TUNEL阳性凋亡细胞比例增加40%[1] 2. 肺癌异种移植模型：在A549裸鼠异种移植模型中，每日两次腹腔注射ARN14974（5 mg/kg），连续21天，肿瘤体积减少65%，小鼠中位生存期从溶媒组的35天延长至58天；肿瘤组织western blot检测证实caspase-3激活及Bcl-2下调[1] 3. 小鼠急性炎症模型：在LPS诱导的小鼠腹腔炎症模型中，LPS攻击前1小时口服ARN14974（3 mg/kg），可使腹腔渗出液中TNF-α和IL-6水平分别降低50%和55%，中性粒细胞浸润减少45%（流式细胞术检测）[1] 4. 体内药效学效应：口服ARN14974（10 mg/kg）后4小时，小鼠肝脏（1.8倍）、脾脏（2.1倍）和肿瘤组织（2.5倍）中的神经酰胺水平显著升高，而正常组织中的鞘氨醇和S1P水平无明显变化[1] |
| 酶活实验 |
1. 酸性神经酰胺酶（AC）荧光活性实验：将重组人AC与系列浓度的ARN14974（0.1 nM–1 μM）及荧光底物C6-NBD-神经酰胺共同孵育于96孔板中，在pH 4.5（溶酶体pH）、37°C条件下孵育1小时；检测产物C6-NBD-脂肪酸的荧光强度（激发光460 nm，发射光530 nm）以定量AC活性，绘制剂量-反应曲线并计算IC50和Ki值[1]
2. 神经酰胺酶选择性实验：将重组人中性神经酰胺酶（NC）、碱性神经酰胺酶（ACER1）和溶酶体β-葡萄糖脑苷脂酶与ARN14974（100 nM）及各自的荧光底物共同孵育；采用相同的荧光法检测酶活性，评估ARN14974对酸性神经酰胺酶的选择性[1] 3. 等温滴定量热法（ITC）结合实验：将纯化的人AC蛋白透析后，在25°C等温滴定量热仪中与ARN14974（0.1–10 μM）进行滴定；记录结合反应中的热量变化，确定ARN14974与AC的结合亲和力（Ki）和结合化学计量比[1] |
| 细胞实验 |
1. 癌细胞增殖实验：将MCF-7、MDA-MB-231、A549和H1299细胞以5×10³个/孔接种于96孔板，加入ARN14974（0.1 nM–1 μM）处理72小时；加入CCK-8试剂孵育2小时，检测450 nm处吸光度，计算细胞活力及增殖抑制的IC50值[1]
2. 凋亡检测实验：用ARN14974（0–50 nM）处理MCF-7和A549细胞48小时，经Annexin V-FITC和碘化丙啶（PI）染色后，通过流式细胞术定量凋亡细胞；提取总蛋白，通过western blot检测剪切型caspase-3、剪切型PARP、Bax和Bcl-2的表达（以β-肌动蛋白为内参）[1] 3. 细胞内神经酰胺定量实验：用ARN14974（10–50 nM）处理MCF-7细胞24–48小时；采用氯仿-甲醇（2:1，v/v）提取细胞脂质，以鞘氨醇-1-磷酸-d7为内标，通过LC-MS/MS定量神经酰胺水平[1] 4. PBMC细胞因子分泌实验：从健康供体血液中分离人PBMCs，用ARN14974（5–50 nM）和LPS（1 μg/mL）处理24小时；收集培养上清，通过ELISA检测TNF-α/IL-6浓度，台盼蓝排斥实验评估细胞活力[1] |
| 动物实验 |
1. MCF-7 breast cancer xenograft model: Female BALB/c nude mice (6–8 weeks old) were subcutaneously inoculated with MCF-7 cells (2×10⁶) into the right flank; when tumors reached ~100 mm³, mice were randomized into vehicle and ARN14974 groups (n=8 per group); ARN14974 was dissolved in a vehicle of 10% DMSO, 30% PEG400, and 60% normal saline, and administered by oral gavage at 10 mg/kg once daily for 28 days; tumor volume (length × width² / 2) and body weight were measured twice weekly; at study end, tumors were excised for AC activity, ceramide quantification, and TUNEL staining [1]
2. A549 lung cancer xenograft model: Nude mice were subcutaneously injected with A549 cells (1×10⁷) into the flank; after tumor establishment (150 mm³), mice received ARN14974 (5 mg/kg, intraperitoneal injection, twice daily) or vehicle for 21 days; tumor growth was monitored every 3 days, and survival was tracked for 80 days; tumor tissues were collected for western blot analysis of apoptotic markers [1] 3. LPS-induced acute inflammation model: C57BL/6 mice (8–10 weeks old) were randomly divided into vehicle and ARN14974 groups (n=6 per group); ARN14974 was administered orally at 3 mg/kg 1 hour before intraperitoneal injection of LPS (5 mg/kg); 6 hours post-LPS, peritoneal exudate was collected for cytokine ELISA, and leukocyte subsets were analyzed by flow cytometry [1] |
| 药代性质 (ADME/PK) |
1. Oral bioavailability: ARN14974 has an oral bioavailability of 42% in mice and 48% in rats after oral administration of 10 mg/kg [1]
2. Plasma pharmacokinetics: In mice, oral ARN14974 (10 mg/kg) reached a maximum plasma concentration (Cmax) of 1.8 μM at 1 hour post-administration, with a plasma half-life (t1/2) of 5.2 hours; the area under the curve (AUC0-24h) was 10.5 μM·h [1] 3. Tissue distribution: ARN14974 exhibits high accumulation in tumor tissues (3.5 μM at 1 hour post-oral 10 mg/kg), liver (2.8 μM), and spleen (2.1 μM), with a tumor/plasma ratio of 1.9; brain penetration is low (brain/plasma ratio = 0.06) [1] 4. Metabolism and excretion: ARN14974 is primarily metabolized in the liver by CYP3A4-mediated hydroxylation at the benzoxazolone ring; approximately 65% of the drug is excreted via feces and 25% via urine within 48 hours, with unchanged drug accounting for 12% of total excretion [1] |
| 毒性/毒理 (Toxicokinetics/TK) |
1. Acute toxicity: ARN14974 was well tolerated in mice at oral doses up to 200 mg/kg and intraperitoneal doses up to 100 mg/kg, with no mortality or severe clinical signs (weight loss, lethargy, abnormal behavior) observed [1]
2. Subchronic toxicity: In a 28-day rat study, oral ARN14974 (10, 30, 100 mg/kg/day) caused mild elevation of serum ALT/AST (1.5-fold) only at 100 mg/kg, which reversed 7 days after drug withdrawal; no significant changes in hematological parameters (RBC, WBC, platelets) or renal function indices (creatinine, urea) were found [1] 3. Plasma protein binding: ARN14974 has a plasma protein binding rate of 93% in human plasma, 91% in mouse plasma, and 89% in rat plasma (measured by ultrafiltration) [1] 4. Organ toxicity: Histological analysis of liver, kidney, heart, and lung tissues from ARN14974-treated mice showed no signs of inflammation, necrosis, or fibrosis; no nephrotoxicity or cardiotoxicity was detected even at the highest dose (100 mg/kg/day) [1] 5. Drug-drug interactions: In vitro studies showed that ARN14974 does not inhibit or induce major CYP450 isoforms (CYP3A4, CYP2C9, CYP2D6) at therapeutic concentrations (up to 1 μM) [1] |
| 参考文献 | |
| 其他信息 |
1. ARN14974 is a novel benzoxazolone carboxamide derivative and the first systemically active small-molecule inhibitor of intracellular acid ceramidase (AC), developed to target the sphingolipid metabolic pathway [1]
2. The mechanism of action of ARN14974 involves competitive binding to the catalytic pocket of lysosomal AC, inhibiting its ceramidase activity and leading to intracellular ceramide accumulation, which triggers mitochondrial apoptosis in cancer cells and suppresses pro-inflammatory cytokine secretion in immune cells [1] 3. ARN14974 is being investigated for the treatment of solid tumors (breast cancer, lung cancer) and inflammatory diseases; it is in preclinical development with no clinical trials or FDA warning information reported as of the literature publication [1] 4. ARN14974 exhibits tumor-selective ceramide accumulation with minimal disruption of sphingolipid homeostasis in normal tissues, indicating a favorable therapeutic index [1] |
| 分子式 |
C24H21FN2O3
|
|---|---|
| 分子量 |
404.433549642563
|
| 精确质量 |
404.153
|
| 元素分析 |
C, 71.27; H, 5.23; F, 4.70; N, 6.93; O, 11.87
|
| CAS号 |
1644158-57-5
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| PubChem CID |
101913546
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| 外观&性状 |
Off-white to pink solid powder
|
| 密度 |
1.3±0.1 g/cm3
|
| 折射率 |
1.613
|
| LogP |
5.4
|
| tPSA |
58.6
|
| 氢键供体(HBD)数目 |
1
|
| 氢键受体(HBA)数目 |
4
|
| 可旋转键数目(RBC) |
6
|
| 重原子数目 |
30
|
| 分子复杂度/Complexity |
587
|
| 定义原子立体中心数目 |
0
|
| SMILES |
FC1C=CC(=CC=1)C1C=CC2=C(C=1)OC(N2C(NCCCCC1C=CC=CC=1)=O)=O
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| InChi Key |
DPYVAZSLMQCVOG-UHFFFAOYSA-N
|
| InChi Code |
InChI=1S/C24H21FN2O3/c25-20-12-9-18(10-13-20)19-11-14-21-22(16-19)30-24(29)27(21)23(28)26-15-5-4-8-17-6-2-1-3-7-17/h1-3,6-7,9-14,16H,4-5,8,15H2,(H,26,28)
|
| 化学名 |
6-(4-fluorophenyl)-2-oxo-N-(4-phenylbutyl)-3(2H)-benzoxazolecarboxamide
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| 别名 |
ARN-14974; ARN14974; ARN 14974; Acid Ceramidase Inhibitor 17a;
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| HS Tariff Code |
2934.99.9001
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| 存储方式 |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month 注意： 本产品在运输和储存过程中需避光。 |
| 运输条件 |
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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| 溶解度 (体外实验) |
DMSO : ~50 mg/mL (~123.63 mM)
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|---|---|
| 溶解度 (体内实验) |
配方 1 中的溶解度: 1 mg/mL (2.47 mM) 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), 悬浮液；超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 10.0 mg/mL澄清DMSO储备液加入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。 配方 2 中的溶解度: ≥ 1 mg/mL (2.47 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 10.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。 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。 |
| 制备储备液 | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.4726 mL | 12.3631 mL | 24.7262 mL | |
| 5 mM | 0.4945 mL | 2.4726 mL | 4.9452 mL | |
| 10 mM | 0.2473 mL | 1.2363 mL | 2.4726 mL |
1、根据实验需要选择合适的溶剂配制储备液 (母液)：对于大多数产品，InvivoChem推荐用DMSO配置母液 (比如：5、10、20mM或者10、20、50 mg/mL浓度），个别水溶性高的产品可直接溶于水。产品在DMSO 、水或其他溶剂中的具体溶解度详见上”溶解度 (体外)”部分;
2、如果您找不到您想要的溶解度信息，或者很难将产品溶解在溶液中，请联系我们;
3、建议使用下列计算器进行相关计算（摩尔浓度计算器、稀释计算器、分子量计算器、重组计算器等）;
4、母液配好之后，将其分装到常规用量，并储存在-20°C或-80°C，尽量减少反复冻融循环。
计算结果:
工作液浓度： mg/mL；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL)。如该浓度超过该批次药物DMSO溶解度，请首先与我们联系。
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL ddH2O，混匀澄清。
(1) 请确保溶液澄清之后，再加入下一种溶剂 (助溶剂) 。可利用涡旋、超声或水浴加热等方法助溶；
(2) 一定要按顺序加入溶剂 (助溶剂) 。
SOBRAC
NAAA-IN-6
W000113414_I13
p-Aminophenylmercuric acetate
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