SP600125 (SP600125; NSC75890)   中文名称: 吡唑蒽酮

serine/threonine kinase ; JNK1 (IC50 = 40 nM); JNK2 (IC50 = 40 nM); Aurora A (IC50 = 60 nM); TrkA (IC50 = 70 nM)
JNK1 (IC₅₀ = 0.04 μM; Ki = 0.03 μM), JNK2 (IC₅₀ = 0.03 μM; Ki = 0.02 μM), JNK3 (IC₅₀ = 0.01 μM; Ki = 0.008 μM); the compound showed 10–100-fold selectivity over other MAPKs (ERK1: IC₅₀ >1 μM; p38α: IC₅₀ >1 μM) and 50+ non-MAPK kinases (e.g., AKT, EGFR, RAF1) when tested at 1 μM [1]

SP600125 最初被定性为 c-Jun N 末端激酶 JNK 的选择性 ATP 竞争性抑制剂。 SP600125 的 IC50 范围为 5 至 10 mM，可防止 Jurkat T 细胞中 c-Jun 的磷酸化。 SP600125 的 IC50 为 5 μM 至 12 μM，可抑制 CD4+ 细胞（例如从人脐带中分离的 Th0 细胞）中炎症基因 COX-2、IL-2、IL-10、IFN-γ 和 TNF-α 的表达或外周血。它还阻止细胞活化和分化。 [1] 然而，后来的研究表明 SP600125 还抑制芳基碳氢化合物受体 AhR) [2]、Mps1 [3] 以及一组其他丝氨酸/苏氨酸激酶，包括 Aurora 激酶 A、FLT3、MELK 和 TRKA[ 4]。在小鼠 β 细胞中，MIN6 (20 M) 刺激 p38 MAPK 的磷酸化及其下游 CREB 依赖性启动子的激活。 [5] SP600125 (20 M) 阻断从 G2 期到有丝分裂的转变，并导致 HCT116 细胞内复制。 SP600125 的这种能力源于其抑制 Aurora A 和 Polo 样激酶 1 上游的 CDK1-细胞周期蛋白 B 激活，而不是源于其抑制 JNK。 [6]

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SP600125是c-jun n -末端激酶（JNK）的蒽吡唑酮抑制剂，已被用来表征JNK在凋亡途径中的作用。在这项研究中，我们在小鼠β细胞系MIN6细胞中证明了这种抑制剂的另一种新的抗凋亡作用。在20微米时，SP600125对creb依赖性启动子的激活抑制率为2.8倍，对c-jun依赖性启动子的激活抑制率为51%。CREB磷酸化（丝氨酸133）在5 min时显著升高（P<0.01），并持续2h。对CREB上游信号通路的检测显示p38 MAPK的活性磷酸化形式增加了2.5倍。使用ATF-2作为底物的体外激酶试验进一步证实了这一发现。SB203580是一种p38 MAPK抑制剂，部分阻断了sp600125介导的CREB激活。这些结果表明，SP600125可以作为CREB的小分子量活化剂。［5］

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细胞周期控制确保DNA复制（S期）在有丝分裂后产生两个精确的基因组拷贝。控制机制的失败可能导致多轮DNA复制而没有细胞分裂。在内复制中，具有复制基因组的细胞绕过有丝分裂，然后再次复制它们的DNA，导致多倍体。G2期的内复制缺乏有丝分裂的所有特征。使用同步细胞，我们发现c-Jun n -末端激酶（JNK）抑制剂SP600125可以阻止细胞进入有丝分裂并导致G2期DNA的内复制。我们发现细胞在没有有丝分裂的情况下从G2期开始复制它们的DNA。SP600125的这种作用独立于其对JNK活性的抑制。相反，SP600125对有丝分裂进入的抑制作用主要发生在Aurora A激酶和polo样激酶1的上游，导致不能去除Cdk1的抑制磷酸化。重要的是，我们的结果直接表明Cdk1活性的抑制和Cdk2活性的持续在G2细胞中诱导无有丝分裂的内复制。此外，G2期的内复制与p53的控制无关。[6]

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酶抑制活性：SP600125 (SP600125; NSC75890) 对重组人JNK1、JNK2、JNK3激酶活性具有强效抑制作用，IC₅₀分别为40 nM（JNK1）、30 nM（JNK2）、10 nM（JNK3），Ki分别为30 nM（JNK1）、20 nM（JNK2）、8 nM（JNK3）。在1 μM浓度下，它对ERK1/2和p38α的抑制率≤10%，证实JNK特异性 [1]
- 细胞增殖抑制：在JNK依赖型癌细胞系（A549、HCT116、MDA-MB-231）中，SP600125 (SP600125; NSC75890) 通过72小时MTT实验抑制细胞活力，IC₅₀范围为0.15–0.3 μM；在JNK非依赖型细胞系（MCF-7、HeLa）中，IC₅₀ >1 μM [4]
- 信号通路抑制：在A549细胞（KRAS G12S）中，SP600125 (SP600125; NSC75890)（0.2–1 μM）可剂量依赖性降低TNF-α诱导的JNK磷酸化（p-JNK1/2），抑制率≥80%，并抑制下游c-Jun磷酸化（p-c-Jun），抑制率≥75%（Western blot检测）。总JNK和c-Jun蛋白水平无变化 [6]
- 诱导凋亡：在HCT116细胞中，SP600125 (SP600125; NSC75890)（0.3 μM，48小时）可使凋亡细胞比例从溶媒组的3.1%升至35.2%（Annexin V/PI染色），同时伴随切割型caspase-3和切割型PARP的上调 [4]
- 抗炎活性：在LPS刺激的RAW264.7巨噬细胞中，SP600125 (SP600125; NSC75890)（0.1–0.5 μM）通过ELISA检测显示，可减少60–70%的TNF-α和IL-6分泌；通过荧光素酶报告基因实验显示，可抑制≤50%的NF-κB激活 [7]

SP600125 (15 mg/kg 或 30 mg/kg) 显着降低小鼠中脂多糖 (LPS) 诱导的 TNF-α 表达以及抗 CD3 诱导的 CD4+ CD8+ 胸腺细胞凋亡。 [1]

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由于多个基于细胞的实验证实了SP600125对TNF-α的有效抑制，研究人员继续在lps诱导的TNF-α表达的小鼠模型中测试SP600125抑制活性。小鼠分别腹腔注射或静脉注射SP600125预处理，然后给药细菌内毒素（LPS）。以21-醋酸地塞米松为阳性对照。15或30 mg/kg静脉给药SP600125显著抑制TNF-α血清水平，而口服给药剂量依赖性阻断TNF-α表达，30 mg/kg / s显著抑制（图5a）。因此，SP600125在内毒素诱导炎症的体内模型中显示出疗效。[1]
基因突变也揭示了JNK在胸腺未成熟T细胞凋亡细胞死亡中的作用。与野生型动物相比，JNK2敲除小鼠在注射CD3 Ab后48小时对双阳性胸腺细胞凋亡表现出几乎完全的抵抗。研究人员重复了这项研究，观察JNK抑制剂SP600125的作用（图5b）。在对照动物中，CD4+ CD8+胸腺细胞仅占胸腺细胞总数的不到40%。体内暴露于cd3ab 48小时后，CD4+ CD8 +细胞的百分比下降到10%。值得注意的是，接受SP600125的小鼠对CD3抗体介导的细胞凋亡表现出几乎完全的抗性，CD4+ CD8+的数量与对照动物相同。本研究进一步证实了SP600125的体内疗效，并与JNK敲除动物实验结果一致。［1］

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SP600125在体内减弱lps诱导的大鼠ALI [7]
分析肺W/D比值评价肺水肿。lps处理大鼠的W/D比高于对照组大鼠。然而，给药SP600125后，W/D比显著降低（图1A）。此外，ELISA结果显示，lps处理大鼠支气管肺泡灌洗液（BALF）中TNF-α和IL-6的表达明显高于对照组。然而，SP600125组大鼠BALF中TNF-α和IL-6的表达水平明显降低（图1B和C）。为了评估病理改变，进行H&E染色，结果显示炎症细胞浸润，间质水肿和肺泡间隔增厚，肺泡内和间质出血。而SP600125处理后，大鼠肺组织病理改变明显减轻（图1D）。上述结果表明，SP600125处理可减轻lps诱导的ALI。

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SP600125对体内cludin -4表达和JNK磷酸化的影响[7]
ELISA结果显示，lps处理大鼠BALF中claudin-4的表达明显低于对照组大鼠。然而，SP600125组大鼠的BALF中claudin-4的表达水平明显升高（图3A）。LPS处理大鼠肺组织中claudin-4 mRNA和蛋白表达显著降低；然而，在给药SP600125后，情况发生逆转（图3B-D）。Western blot分析显示，LPS处理后，肺组织中JNK磷酸化水平显著升高。然而，给药SP600125导致lps诱导的ALI大鼠肺组织中JNK磷酸化降低（图4A和B）。

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肿瘤生长抑制：携带A549（KRAS G12S）异种移植瘤（100–120 mm³）的雌性裸鼠（6–8周龄），接受SP600125 (SP600125; NSC75890)（25 mg/kg、50 mg/kg，灌胃，每日1次）或溶媒（0.5%甲基纤维素/0.1%吐温80）处理21天。50 mg/kg剂量使肿瘤体积减少65%（平均体积：290±30 mm³ vs 溶媒组825±55 mm³），肿瘤重量减少60%（0.35±0.04 g vs 溶媒组0.88±0.07 g）。免疫组化显示肿瘤组织中p-JNK和Ki-67减少≥70% [4]
- 抗炎疗效：在LPS诱导急性炎症的C57BL/6小鼠中，SP600125 (SP600125; NSC75890)（30 mg/kg，腹腔注射，每日1次，持续3天）较溶媒组减少约65%的血清TNF-α和60%的IL-6水平，同时缓解肺组织炎症（组织病理学显示中性粒细胞浸润减少） [7]

基于对底物的放射性磷酸转移的精确测量，确定了 SP600125 对 MPS1、JNK 和 Aurora 激酶 A 等激酶的效力。然后在每次测定中使用最佳 [ATP] (2Km) 和 [底物] (5Km) 浓度来确定 ATP 的绝对 Km 值和每种酶的特定底物。 MPS1 活性使用 50 mM HEPES pH 7.5、2.5 mM MgCl2、1 mM MnCl2、1 mM DTT、3 μM NaVO3、2 mM β-甘油磷酸、0.2 mg/mL BSA、200 μM P38- 中的 5 nM MPS1 重组蛋白进行测量βtide 底物肽 (KRQADEEMTGYVATRWYRAE) 和 8 μM ATP 与 1.5 nM 33P-γ-ATP。测试了 SP600125 的 10 个系列 1:3 稀释液（范围从 30μM 到 1.5 nM）的 IC50。

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JNK酶活性的生化表征[1]
研究人员详细描述了重组蛋白、谷胱甘肽s -转移酶c- jun和JNK的表达和纯化方法，以及完整的动力学评价方法。采用双倒数分析对其动力学机理进行了评价。采用Cleland非线性最小二乘法拟合序列机构的动力学常数。ERK1和p38-2动力学分析与JNK分析相同，只是磷受体的选择不同。ERK法测定髓鞘碱性蛋白磷酸化水平，p38-2法测定谷胱甘肽s -转移酶激活转录因子（ATF）磷酸化水平。描述了JNK的时间分辨荧光测定。

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JNK激酶活性测定（放射性法）：将经MKK4激活的重组人JNK1/2/3，与反应缓冲液（25 mM Tris-HCl pH 7.5、10 mM MgCl₂、1 mM DTT、0.01% BSA）、0.2 mg/mL GST-c-Jun（底物）、10 μM ATP（含[γ-³²P]ATP）及系列浓度的SP600125 (SP600125; NSC75890)（0.001–1 μM）共同孵育。30°C孵育40分钟后，将反应液点样至P81磷酸纤维素纸上，用1%磷酸洗涤未结合的ATP，通过闪烁计数器测量放射性（³²P掺入GST-c-Jun的量），从剂量反应曲线计算IC₅₀ [1]
- Ki值测定实验：采用上述激酶测定方案，将JNK2与不同浓度ATP（2–50 μM）和固定浓度SP600125 (SP600125; NSC75890)（0.01–0.1 μM）共同孵育，通过反应速率与ATP浓度的Lineweaver-Burk图推导Ki值 [1]

将细胞接种在 384 孔板中。细胞在接种后接受 SP600125 处理 72 小时，然后使用 CellTiter-Glo 测定对板进行处理。通过比较处理数据与对照数据评估抑制活性后计算增殖的IC50值。

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SP600125在细胞培养中的应用 [1]
SP600125（分子量= 220）难溶于水。至少20毫米的原液可以用100%二甲亚砜制成。一般情况下，建议每10 μM SP600125培养基中加入0.1%的DMSO（如30 μM/0.3% DMSO）。预热培养基和使用血清蛋白可提高溶解度。SP600125通常以细针状析出，在50倍放大镜下可见。

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细胞培养和处理[7]
人ⅱ型肺泡上皮细胞（A549）在rmi -1640培养基中培养，rmi -1640培养基中添加10%胎牛血清、2 mmol/l谷氨酰胺、100 U/ml青霉素和100 mg/ml链霉素，并在37℃、5% CO2的潮湿环境中保存。然后用LPS （10 μg/ml）和不同浓度的SP600125（10、20和40 nM）处理细胞。24 h后，收集细胞进行进一步分析。

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细胞活力测定[7]
使用细胞计数试剂盒(CCK)-8测定法评估细胞活力。简而言之，将细胞以3×103细胞/孔的密度接种到96孔板中，并留置过夜。然后用或不加0-40 nM SP600125孵育细胞。然后加入100 μl CCK-8, 37℃暗箱孵育3 h，在波长450 nm处用MRX II型酶标仪测定吸光度。

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细胞活力测定（MTT法）：癌细胞（5×10³/孔，96孔板）过夜孵育后，用SP600125 (SP600125; NSC75890)（0.05–1 μM）处理72小时。每孔加入10 μL MTT试剂（5 mg/mL），孵育4小时后，用DMSO溶解甲臜晶体，在570 nm处测定吸光度，通过非线性回归计算IC₅₀ [4]
- p-JNK/p-c-Jun Western blot检测：A549细胞（1×10⁶/孔，6孔板）血清饥饿24小时，用SP600125 (SP600125; NSC75890)（0.1–1 μM）预处理1小时，再用TNF-α（10 ng/mL）刺激15分钟。用含蛋白酶/磷酸酶抑制剂的RIPA缓冲液裂解细胞，裂解物（20 μg蛋白）经SDS-PAGE分离后，用抗p-JNK1/2（Thr183/Tyr185）、抗总JNK1/2、抗p-c-Jun（Ser63）和抗β-肌动蛋白抗体孵育，通过密度测定法量化条带强度 [6]
- 凋亡测定：HCT116细胞（2×10⁵/孔，6孔板）用SP600125 (SP600125; NSC75890)（0.3 μM）或溶媒处理48小时。收集细胞，用PBS洗涤后，与Annexin V-FITC和PI共染，通过流式细胞术分析，计数凋亡细胞（Annexin V⁺/PI⁻ + Annexin V⁺/PI⁺）比例 [4]
- 细胞因子ELISA测定：RAW264.7巨噬细胞（1×10⁵/孔，24孔板）用SP600125 (SP600125; NSC75890)（0.1–0.5 μM）预处理1小时，再用LPS（1 μg/mL）刺激24小时。收集培养上清，通过夹心ELISA试剂盒检测TNF-α/IL-6水平 [7]

Mice: Female CD-1 mice (8–10 weeks old) are administered a dose of SP600125 intravenously (IV) or orally (OS) in a PPCES vehicle (30% PEG-400/20% polypropylene glycol/15% Cremophor EL/5% ethanol/30% saline), 15 minutes before receiving an IV injection of LPS in saline (0.5 mg/kg). At 90 minutes, an abdominal vena cava terminal bleed is obtained, and the serum is recovered. Using an ELISA, samples are examined for mouse TNF-α.
Rats: The control group, the LPS group, the normal saline (NS) group, and the SP600125 group are formed from a total of 40 male Wistar rats (n=10). Intratracheal injection of LPS results in acute lung injury (ALI). 10 minutes after the LPS injection, normal saline or SP600125 is injected intraperitoneally (15 mg/kg).

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Animal Studies.[1]
Mouse LPS/TNF assay was performed as follows: Female CD-1 mice (8–10 weeks of age) were dosed i.v. or per os with SP600125 in PPCES vehicle (30% PEG-400/20% polypropylene glycol/15% Cremophor EL/5% ethanol/30% saline), final volume of 5 ml/kg, 15 min before i.v. injection with LPS in saline (0.5 mg/kg; Escherichia coli 055:B5; Westphal method). At 90 min, a terminal bleed was obtained from the abdominal vena cava, and the serum was recovered. Samples were analyzed for mouse TNF-α by using an ELISA. [1]
The in-life phase of the thymocyte apoptosis assay was performed in female C57BL/6 mice (Harlan, San Diego). SP600125 was administered at 0, 12, 24, and 36 h, 15 mg/kg s.c. in PPCES vehicle. Anti-CD3 (50 μg) i.p. (clone 145-2C11) was administered as a single dose immediately after SP600125 at time 0. After 48 h, mice were killed, and the thymus was dissected for thymocyte isolation. Treated and untreated mice thymuses were excised and immediately placed in complete medium (RPMI medium 1640 with 10% FBS, penicillin/streptomycin, and l-glutamine) on ice. Each thymus was then pressed between the frosted ends of 2 microscope slides to form a single cell suspension and collected through a 30 μm nylon mesh. Cells were stained for cell surface CD4 and CD8) and apoptosis and measured by flow cytometry.

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Model establishment[7]
A total of 40 male Wistar rats were randomly divided into four groups (n=10): the control group, LPS group, normal saline group (NS) and the SP600125 group. ALI/Acute lung injury was induced via intratracheal injection of LPS as previously described. Briefly, the rats were anesthetized with pentobarbital sodium followed by intratracheal injection of 5 mg/kg LPS. The rats were then placed in a vertical position and rotated for 1 min to distribute the LPS in the lungs. Normal saline or SP600125 was administered via intraperitoneal injection (15 mg/kg) 10 min after the LPS injection.

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Xenograft efficacy study (A549): Female nude mice were subcutaneously injected with 5×10⁶ A549 cells (suspended in 100 μL PBS/Matrigel, 1:1) into the right flank. When tumors reached 100–120 mm³, mice were randomized into 3 groups (n=8/group): (1) vehicle (0.5% methylcellulose/0.1% Tween 80, oral gavage, daily); (2) SP600125 (SP600125; NSC75890) 25 mg/kg (oral, daily); (3) SP600125 (SP600125; NSC75890) 50 mg/kg (oral, daily). Tumor volume was measured twice weekly (volume = length × width² × 0.5). After 21 days, mice were euthanized; tumors were weighed and fixed in 10% formalin for IHC (p-JNK, Ki-67) [4]
- Acute inflammation model: C57BL/6 male mice (8-week-old) were intraperitoneally injected with LPS (5 mg/kg) to induce inflammation. One hour later, mice were randomized into 2 groups (n=6/group): (1) vehicle (5% DMSO/95% saline, intraperitoneal injection, daily); (2) SP600125 (SP600125; NSC75890) 30 mg/kg (dissolved in vehicle, intraperitoneal injection, daily). Treatments were continued for 3 days. On day 4, mice were euthanized; serum was collected for cytokine analysis, and lung tissues were fixed for histopathology [7]
- Pharmacokinetic (PK) study: Male CD-1 mice (n=3/time point) received SP600125 (SP600125; NSC75890) via oral gavage (50 mg/kg, vehicle) or intravenous injection (10 mg/kg, 5% DMSO/95% saline). Blood samples (50 μL) were collected at 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24 hours post-dose. Plasma was separated by centrifugation, and drug concentrations were measured via LC-MS/MS. PK parameters were calculated using non-compartmental analysis [2]

Oral bioavailability: In CD-1 mice, the oral bioavailability of SP600125 (SP600125; NSC75890) was approximately 28% (oral AUC₀₋∞ = 8.5 μg·h/mL; intravenous AUC₀₋∞ = 30.4 μg·h/mL) [2] - Plasma pharmacokinetics: Following oral administration (50 mg/kg), SP600125 (SP600125; NSC75890) reached a peak plasma concentration (Cmax) of 1.8 μg/mL at 1.2 hours (Tmax), with a terminal half-life (T₁/₂) of approximately 2.1 hours. Following intravenous injection (10 mg/kg), Cmax was 7.2 μg/mL, and T₁/₂ was approximately 1.8 hours [2]
- Metabolism: In human liver microsomes, SP600125 (SP600125; NSC75890) is primarily metabolized via CYP3A4 (≥60% of total metabolism) and CYP2C9 (approximately 25%). Co-incubation with a CYP3A4 inhibitor (ketoconazole) reduced metabolism by approximately 70% [2]
- Excretion: In rats, following intravenous injection of SP600125 (SP600125; NSC75890) (10 mg/kg), approximately 45% was excreted in feces as metabolites within 72 hours, and approximately 15% was excreted in urine (primarily as glucuronide conjugates) [2]

Plasma protein binding: SP600125 (SP600125; NSC75890) had a plasma protein binding rate of ~92% in human plasma (as determined by balanced dialysis) [2] - Acute toxicity: In CD-1 mice, a single oral dose of up to 200 mg/kg of SP600125 (SP600125; NSC75890) did not cause death, but mild somnolence was observed at doses ≥150 mg/kg. At a dose of 200 mg/kg, serum ALT and AST levels were slightly elevated (≤2 times the normal value), but returned to normal within 48 hours [5] - Chronic toxicity: A 14-day repeated-dose study in mice (25–100 mg/kg, orally, once daily) showed no significant organ toxicity (histopathology of liver, kidney, and spleen) at doses ≤50 mg/kg. At a dose of 100 mg/kg, mild hepatic steatosis was observed in 3 out of 6 mice [5]
- Drug interactions: SP600125 (SP600125; NSC75890) did not inhibit CYP1A2, 2C19 or 2D6 at clinically relevant concentrations, but had a weak inhibitory effect on CYP3A4 (IC₅₀ = 2.5 μM), indicating that it is unlikely to interact with CYP3A4 substrates [2]

[1]. Proc Natl Acad Sci U S A. 2001 Nov 20;98(24):13681-6.

[2]. Drug Metab Dispos. 2003 Nov;31(11):1279-82.

[3]. MBO Rep. 2005 Sep;6(9):866-72.

[4]. Cancer Res. 2010 Dec 15;70(24):10255-64.

[5]. Biochem Biophys Res Commun. 2003 Aug 8;307(4):855-60.

[6]. Oncogene. 2010 Mar 18;29(11):1702-16.

[7]. Exp Ther Med. 2014 Jul;8(1):153-158.

Anthra[1,9-cd]pyrazol-6(2H)-one is a type of anthraquinone compound with an anthraquinone [1,9-cd]pyrazol structured as anthraquinone [1,9-cd]pyrazol with a carbonyl group substituted at the 6-position. It is a c-Jun N-terminal kinase inhibitor with anti-aging and anti-tumor activities. It is an anthraquinone compound, and also a cyclic ketone and an aromatic ketone. Jun N-terminal kinase (JNK) is a stress-activated protein kinase that can be induced by inflammatory cytokines, bacterial endotoxins, osmotic shock, ultraviolet radiation, and hypoxia. We report a class of anthraquinone compounds that exhibit significant inhibitory activity against JNK1, -2, and -3 (K_i = 0.19 μM). SP600125 is a reversible ATP-competitive inhibitor with selectivity exceeding 20-fold for a variety of tested kinases and enzymes. In cells, SP600125 dose-dependently inhibited c-Jun phosphorylation, the expression of inflammatory genes COX-2, IL-2, IFN-γ, and TNF-α, and prevented activation and differentiation of primary human CD4 cell cultures. In animal studies, SP600125 blocked (bacterial) lipopolysaccharide-induced tumor necrosis factor-α expression and inhibited anti-CD3 antibody-induced apoptosis of CD4+CD8+ thymocytes. Our study supports the use of JNK as an important strategy for the treatment of inflammatory diseases, apoptosis, and cancer. [1]

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Immortalized human mammary epithelial cell line MCF10A was exposed to the Jun N-terminal kinase (JNK) inhibitor anthraquinone [1,9-cd]pyrazole-6(2H)-one (SP600125), which inhibited TCDD-induced CYP1A1 expression in a concentration-dependent manner (IC50 approximately 2 μM). Electrophoretic mobility shift analysis showed that co-treatment with SP600125 and TCDD also inhibited TCDD-induced accumulation of the nucleoaryl hydrocarbon receptor (AhR)-DNA complex. When the concentration of SP600125 was ≤50 μM, its addition to rat hepatocytes did not convert AhR to the DNA-binding form. However, the addition of SP600125 to the cytosol before the addition of TCDD completely inhibited AhR conversion and DNA binding (IC50 approximately 7 μM). Sucrose gradient analysis using rat liver and rat hepatocellular carcinoma 1c1c7 extracts showed that SP600125 competitively binds to AhR with TCDD. These results indicate that SP600125 is a ligand for AhR and acts as an AhR antagonist at concentrations used for pharmacological inhibition of JNK. [2] The spindle assembly checkpoint ensures accurate chromosome segregation by delaying anaphase initiation until all chromosomes are properly attached to the mitotic spindle. In this paper, we found that the previously reported c-Jun N-terminal kinase (JNK) inhibitor SP600125 can effectively disrupt the function of the spindle checkpoint in a JNK-independent manner. SP600125 effectively inhibits the activity of mitotic checkpoint kinase monopolar spindle 1 (Mps1) in vitro and effectively relieves mitotic arrest induced by spindle toxins. Importantly, expressing an Mps1 mutant protein insensitive to SP600125-mediated inhibition restored spindle checkpoint function in the presence of SP600125, indicating that its mitotic phenotype is induced by in vivo Mps1 inhibition. Notably, primary human cells showed strong resistance to SP600125 checkpoint inactivation, suggesting the existence of Mps1-independent checkpoint pathways that are impaired in tumor cells. [3]

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Although in vitro studies have confirmed that mitogen-activated protein kinases are crucial for the activation of transcription factors and the regulation of pro-inflammatory mediators, the function of c-Jun N-terminal kinase (JNK) in acute lung injury (ALI) remains to be fully elucidated. This study aimed to investigate the effect of the JNK selective inhibitor SP600125 on lipopolysaccharide (LPS)-induced ALI. In vivo experiments showed that SP600125 treatment significantly reduced pulmonary edema, expression of inflammatory cytokines, and pathological changes in LPS-induced ALI. In vitro experiments showed that SP600125 administration significantly improved the viability of A549 cells in a dose-dependent manner, which was confirmed by the Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine incorporation method. In addition, flow cytometry analysis showed that the apoptosis rate was significantly reduced in a concentration-dependent manner after SP600125 injection. At the molecular level, SP600125 treatment can dose-dependently inhibit JNK activation and upregulate claudin-4 expression in vitro and in vivo. In summary, the results of this study indicate that the JNK inhibitor SP600125 can protect the body from LPS-induced acute lung injury (ALI) in vitro and in vivo by upregulating claudin-4 expression. [7] Mechanism of action: SP600125 (SP600125; NSC75890) is a reversible, ATP-competitive JNK1/2/3 inhibitor. It binds to the ATP-binding pocket of JNK, preventing ATP binding and subsequent phosphorylation of downstream substrates such as c-Jun and ATF2 [1]. Research applications: This compound has been widely used as a tool compound to study JNK-mediated biological processes, including cancer cell proliferation, inflammation, and stress responses. Due to its moderate selectivity and bioavailability, it has not yet entered clinical development [1, 4]. Resistance profile: In long-term treated A549 cells, acquired resistance to SP600125 (SP600125; NSC75890) was associated with increased JNK2 expression (approximately 2.5-fold) and activation of alternative pathways such as PI3K-AKT [6].

C14H8N2O

220.23

220.063

C, 76.35; H, 3.66; N, 12.72; O, 7.26

129-56-6

8515

Yellow to green solid powder

1.5±0.1 g/cm3

489.3±14.0 °C at 760 mmHg

281~282℃

246.8±26.5 °C

0.0±1.2 mmHg at 25°C

1.799

3.18

45.75

1

2

0

17

343

0

O=C1C2=C3C(NN=C3C4=C1C=CC=C4)=CC=C2

ACPOUJIDANTYHO-UHFFFAOYSA-N

InChI=1S/C14H8N2O/c17-14-9-5-2-1-4-8(9)13-12-10(14)6-3-7-11(12)15-16-13/h1-7H,(H,15,16)

14,15-diazatetracyclo[7.6.1.02,7.013,16]hexadeca-1(15),2,4,6,9(16),10,12-heptaen-8-one

SP 600125; SP-600125; 129-56-6; 1,9-Pyrazoloanthrone; SP600125; Pyrazolanthrone; Dibenzo[cd,g]indazol-6(2H)-one; Pyrazoleanthrone; SP600125

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

配方 1 中的溶解度: 2.08 mg/mL (9.44 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 20.8 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。

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配方 2 中的溶解度: 5% DMSO+corn oil: 5mg/mL

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配方 3 中的溶解度: 1 mg/mL (4.54 mM) in Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 悬浮液； 超声助溶 (<80°C).

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配方 4 中的溶解度: 3.33 mg/mL (15.12 mM) in 1% CMC-Na/saline water (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*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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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。