BAY-876   中文名称: BAY-876

Glucose transporter 1/GLUT1 (IC50 = 2 nM); GLUT2/3/4 (IC50 = 10~ 0.3 μM)

生长抑制性 BAY-876（25–75 nM；24 和 72 小时）以剂量依赖性方式减少 SKOV-3 和 OVCAR-3 细胞的数量 [2]。

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为了了解GLUT1失调在肿瘤发生中的生物学结果，我们询问癌症卵巢细胞是否依赖GLUT1来支持糖酵解代谢。正如我们和其他人之前所证明的，卵巢癌症细胞在常规培养条件下表现出高的基础糖酵解活性（图1A）。尽管BAY-876已被证明可以抑制葡萄糖摄取，但其对下游糖酵解代谢的影响尚不清楚。因此，我们检测了BAY-876对卵巢癌症细胞系中糖酵解和乳酸产生的影响，包括已知缺乏功能性GLUT1的A2780作为阴性对照。如图1A所示，与BAY-876一起孵育会剂量依赖性地降低SKOV-3、OVCAR-3和HEY细胞的糖酵解速率。同样，BAY-876降低了这些细胞培养上清液中的乳酸水平（图1B）。尽管BAY-876的这种抗糖酵解作用在单个数字纳摩尔浓度下是可以检测到的，但在25-50nM的化合物下实现了半最大抑制。我们还在其他常用的卵巢癌症细胞系如OVCR-429和OVCA-432中观察到BAY-876类似的抗糖酵解活性。 据报道，在A2780卵巢癌症细胞系中，GLUT1与突变PTEN共同定位于细胞核而非质膜。与BAY-876通过特异性抑制GLUT1而非其他GLUT1成员或脱靶蛋白抑制糖酵解一致，BAY-876不影响A2780细胞中的糖酵解或乳酸产生（图1A、B）。BAY-876对GLUT1介导的糖酵解的特异性作用进一步通过GLUT1的siRNA沉默得到证实。分子方法降低了SKOV-3和OVCAR-3细胞的糖酵解速率和乳酸产量（图1C）。 [2]

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GLUT1介导应激条件下糖酵解的上调[2]
与正常细胞相比，癌症和其他恶性细胞的基础有氧糖酵解通常较高。癌症细胞的糖酵解活性也随着肿瘤微环境提示的变化而波动，以满足快速生长的肿瘤细胞的生物能量和生物合成需求。特别是，糖酵解在缺氧条件下会增强。已知GLUT1是缺氧靶基因之一，由HIF上调[18]。我们使用氯化钴（CoCl2）作为稳定HIF的手段来检测卵巢癌症细胞中GLUT1的诱导性。如图2A所示，在所有卵巢癌症细胞系中，CoCl2高度诱导GLUT1。GLUT1的诱导与细胞糖酵解的增加有关（图2A）BAY-876降低了SKOV-3、OVCAR-3和HEY细胞中基础和CoCl2刺激的糖酵解。尽管CoCl2增加了A2780细胞中GLUT1的表达和糖酵解，但BAY-876对基础糖酵解或CoCl2驱动的糖酵解都没有影响，这表明GLUT1非依赖性机制涉及A2780细胞的HIF驱动糖酵解。

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抑制GLUT1会导致生物能量损失和AMP激活蛋白激酶（AMPK）的激活[2]
与使用氧化磷酸化作为主要生物能量源的正常细胞不同，癌症细胞严重依赖糖酵解对ADP进行底物磷酸化以形成ATP。接下来，我们确定GLUT1抑制是否足以减少卵巢癌症细胞中ATP的产生。如图3A所示，用BAY-876处理SKOV-3和OVCAR-3后，细胞ATP水平显著降低。与ATP丰度降低一致，治疗导致5′-腺苷酸活化蛋白激酶α（AMPKα）激活，这反映在AMPKα在Thr-172处的磷酸化增加（图3B）。BAY-876再次对A2780细胞中的ATP水平或AMPKα磷酸化没有影响。总之，这些结果支持了BAY-876通过特异性抑制GLUT1来损害糖酵解相关ATP生成的结论。

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为了进一步了解GLUT1抑制的代谢结果，我们通过跟踪用或不用BAY-876处理的SKOV-3、OVCAR-3和A2780细胞的耗氧率（OCR）来测量氧化磷酸化。与对糖酵解的影响相反，BAY-876增加了这些细胞的OCR，这是使用Seahorse XF24分析仪测定的（图3C）。在SKOV-3细胞中观察到基础线粒体呼吸（基础OCR）、ATP连锁OCR和最大呼吸能力的显著增加。在OVCAR-3中，检测到基础OCR和ATP相关OCR的增加。BAY-876治疗后呼吸增加的机制尚不完全清楚。由于它在A2780细胞中不存在，BAY-876对SKOV-3和OVCAR-3细胞OCR的这种影响也是GLUT1依赖性的，可能是对GLUT1糖酵解ATP级联抑制的补偿反应。 [2]

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抑制GLUT1抑制卵巢癌症细胞的增殖、活力和锚定依赖性生长[2]
鉴于过度活跃的糖酵解在支持癌症恶性特征方面的重要性，我们接下来评估了BAY-876对卵巢癌症细胞生长和生存能力的影响。首先，我们用<100 nM浓度的BAY-876处理卵巢癌症细胞系，其显著降低糖酵解，细胞毒性很小，如图1所示。用这些浓度的BAY-876治疗一天导致SKOV-3和OVCAR-3细胞数量的剂量依赖性减少（图4A）。在75 nM BAY-876存在下的三天生长曲线进一步证实了SKOV-3、OVCAR-3和HEY的生长抑制作用，但在A2780细胞中没有（图4B）。为了评估BAY-876对细胞生长和细胞毒性的联合作用，这些细胞系用高达10µM的更大剂量范围的BAY-877处理3天。用MTT法测定活细胞的线粒体活性。图4C的结果显示，OVCAR-3对BAY-876最敏感，IC50值约为60 nM。SKOV-3和HEY的IC50值分别为188和1002 nM。与缺乏功能性GLUT1和抗糖酵解作用一致，A2780细胞即使在高达2µM的浓度下也对BAY-876的处理无效（图4C）。

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从中等效力和代谢不稳定的HTS hit 1开始，在喹啉的2位携带呋喃基部分，总共携带四个甲基，我们通过替换呋喃并去除两个喹啉甲基，迅速提高了效力和代谢稳定性。使用化合物3的喹啉核心，我们发现一个未取代的亚甲基是苯基和吡唑环之间的最佳间隔物。在成功地将一个吡唑甲基替换为代谢上更稳定的CF3基团（12）后，对喹啉核心的进一步SAR探索表明，2位的未取代酰胺和7位的氟（19，BAY-876）对GLUT1的效力和对其他GLUTs的选择性非常有益。 在吡唑核心，发现位置3和5的双取代对于优异的效力/选择性特征至关重要，尤其是对GLUT3。关于苄基环上的取代基，对位比邻位或间位产生了更有前景的化合物。化合物54具有对氰基和额外的环氮，也表现出有趣的GLUT谱。 N-（1H-吡唑-4-基）喹啉-4-甲酰胺的合成很简单，如BAY-876所示（19）。体外PK数据显示，BAY‐876（19）和54在肝微粒体和肝细胞中都非常稳定，尽管54的流出率很高，约为16[1]。

口服 BAY-876（1.5–4.5 mg/kg/天，持续 28 天）的小鼠表现出显着的剂量依赖性致癌性降低 [2]。

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BAY-876抑制卵巢癌症细胞系和卵巢癌症PDXs的致瘤性[2]
GLUT1在调节癌症卵巢细胞糖酵解、能量代谢和凤尾藻依赖性和非依赖性生长中的作用表明，GLUT1是一个有前途的抗癌靶点。由于BAY-876尚未在体内被彻底评估为抗癌剂，我们评估了其在携带SKOV-3皮下（皮下）异种移植物的雌性NOD-cid IL2rgull（NSG）小鼠中的抗肿瘤潜力和安全性。本研究中的所有动物实验均按照VCU IACUC的政策和规定进行。四组肿瘤大小范围相似（~100 mm3）的小鼠口服0、1.5、3.0和4.5 mg/kg/天，持续4周。监测肿瘤生长曲线和体重变化，如图5所示。BAY-876对致瘤性有明显的剂量依赖性抑制作用。在4.5mg/kg/天治疗组中观察到最大效果。治疗2周后，肿瘤明显缩小。在终点，与赋形剂对照组的这些参数相比，最终的平均肿瘤体积和肿瘤重量分别下降了68%和66%。然而，4.5 mg/kg/天的剂量对NSG小鼠有一定的毒性。在治疗的最后一周，小鼠的体重开始下降。在4周结束时，与对照组或其他两个较低剂量组相比，该组的体重减轻平均达到18%。然而，除了体重减轻，这些小鼠没有其他明显的健康状况

我们开发了来自高级别浆液性卵巢癌患者的PDX。这些PDX的H&E染色证实了乳头状腺癌的组织学表现，与原始患者肿瘤组织相似（补充图S2）。我们研究了在表达GLUT1蛋白的两个PDX OVC-PDX2和OVC-PDX3中BAY-876的作用（图6D，H）。携带PDX的雌性NSG小鼠接受4.0mg/kg/天的治疗，该剂量是根据早期SKOV-3异种移植物实验确定的。在30天内用这种剂量的BAY-876治疗显著降低了肿瘤生长，体重没有明显减轻（图6B，F）。在两个PDX中，平均终点肿瘤体积减少了60%以上（图6A，E）。OVC-PDX2和OVC-PDX3的最终肿瘤重量分别降低了50%和71%（图6C，G）。

这些结果表明，当以4.0mg/kg/天或更低的剂量口服时，BAY-876是一种有效且安全的抗癌剂。我们接下来问，在较短的时间内使用更高剂量的急性治疗是否可以获得更好的治疗效果。因此，携带OVC-PDX2的雌性NSG小鼠接受7.5mg/kg/天的治疗，并密切监测肿瘤生长和健康状况。不幸的是，这些小鼠不能耐受这种剂量，在18天的治疗后全部死亡。补充图S3显示了7.5 mg/kg/天的BAY-876对对照组和治疗组的肿瘤生长、体重和Kaplan-Meier生存率的影响。

人GLUT1超高通量筛选（uHTS）：[1]
众所周知，线粒体电子传递链和葡萄糖分解代谢的小分子抑制剂的组合协同抑制ATP的产生。对于uHTS，CHO-K1细胞用人GLUT1和组成型表达萤光素酶稳定转染，如前所述。 将细胞接种在1536个密度为每孔1000个细胞的微量滴定板中，并在1%FCS存在的无葡萄糖DMEM中饥饿24小时。在测量之前，细胞在37°C下在10μm鱼藤酮的存在下孵育30分钟，以完全阻断氧化磷酸化。试验化合物和笼状荧光素同时装载。在应用0.5mm葡萄糖并相应激活GLUT1之前，通过萤光素酶活性间接测量基础ATP，以确定对细胞ATP水平的影响，而与葡萄糖无关；施加500μm葡萄糖后10分钟的萤光素酶动力学记录允许研究化合物诱导的GLUT1抑制。

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GLUT亚型特异性检测：[1]
为了进行GLUT1、GLUT2、GLUT3和GLUT4之间的特异性测试，我们使用了DLD1（用于GLUT1）、DLD1GLUT1-/-（用于GLUT3）、CHO-hGLUT2和CHO-hGRUT4（GLUT2与4）细胞，并结合氧化磷酸化抑制剂（鱼藤酮1μm）。在标准条件下，细胞系在添加了10%FCS、1%青霉素-链霉素溶液和2%谷氨酸的DMEM培养基中维持。用胰蛋白酶处理细胞，并以每孔4000个细胞的密度接种到384个板中。然后将细胞在含有1%FCS的无葡萄糖培养基中培养过夜，以降低细胞内ATP水平。对于GLUT1/2/3，16小时后将细胞与适当的葡萄糖浓度或GLUT2果糖浓度（GLUT1为0.1m，GLUT3为0.3 m，GLUT2为30 mm果糖），含或不含化合物和1μm鱼藤酮，持续15分钟。然后使用CellTiter‐Glo®发光细胞活力测定法测量ATP水平。将测定标准化为对照细胞松弛素B（IC50 GLUT1:0.1μm GLUT2：2.8μm，GLUT3：0.12μm，GLUT4：0.28μm），测定方差：9 %, IC50计算R2>0.9。对于GLUT4，16小时后去除无葡萄糖培养基，将细胞适应无KCl的酪德缓冲液3小时。加入化合物和鱼藤酮，20分钟后将细胞与葡萄糖（终浓度0.1m）孵育15分钟 然后使用Promega的CellTiter‐Glo®发光细胞活力测定法测量ATP水平。

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葡萄糖竞争： [1]
对于葡萄糖竞争，DLD1细胞用胰蛋白酶处理，以每孔4000个细胞的密度接种到384个板中。然后将细胞在含有1%FCS的无葡萄糖培养基中培养过夜，以降低细胞内ATP水平。16小时后，将细胞与不同葡萄糖浓度（分别为0.1；1和10 mm）、化合物（30μm至1 nm）和1μm鱼藤酮一起孵育15分钟。然后使用Promega的CellTiter‐Glo®发光细胞活力测定法测量ATP水平。

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GLUT1敲除[2]
根据制造商的说法，使用Amaxa™Nucleofector™试剂盒V将GLUT1 siRNA（赛默飞世尔科技公司，siRNA ID:s12925）或非靶向对照siRNA转染到SKOV-3或OVCAR-3细胞中。用100 pmol siRNA对100万个细胞进行电穿孔。接种后24小时，转染细胞被喂食新鲜培养基并再孵育72小时，然后进行糖酵解和免疫印迹分析。

细胞增殖测定[2]
细胞类型： SKOV-3 和 OVCAR-3 细胞
测试浓度： 25、50、75 nM
孵育持续时间：24 和 72 小时
实验结果：导致 SKOV-3 和 OVCAR-3 细胞数量呈剂量依赖性减少。

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OCR测量[2]
如前所述，使用Seahorse XF24细胞外通量分析仪测量用或不用BAY-876处理的培养细胞的OCR（pMoles/min）。将卵巢癌症细胞系接种在XF24微孔板中，培养18-20小时，然后切换到补充有10 mM葡萄糖和2 mM谷氨酰胺的海马测定培养基。如前所述，计算了基础线粒体呼吸、ATP连接呼吸、最大呼吸能力和储备能力。

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细胞生长试验[2]
为了评估BAY-876（0、25、50、75 nM）对增殖的影响，在胰蛋白酶处理后，用库尔特计数器对12孔板中的细胞进行计数。为了确定BAY-876的细胞毒性作用，用更宽范围的指定浓度处理细胞3天，然后进行MTT染色并测量570nm处的吸光度。使用SigmaPlot 13.0计算IC50值。 进行锚定非依赖性生长，以评估BAY-876对细胞在半固体软琼脂中生长能力的影响。在完全培养基中用1.5 mL 0.6%软琼脂预涂六孔板。将悬浮在含有0.3%软琼脂的1.5mL生长培养基中的细胞覆盖在预涂孔上。每3-5天将含有0.3%琼脂（1.5 mL）的新鲜完整培养基添加到顶部。3周后计数直径大于100μm（SKOV-3、HEY和A2780）或50μm（OVCAR-3）的菌落。

Animal/Disease Models: Female NOD-scid IL2rgnull (NSG) mice carrying SKOV-3 subcutaneous (sc) xenografts[2]
Doses: 1.5, 3, 4.5 mg/kg
Route of Administration: Oral administration; daily; for 28 days
Experimental Results: Caused a clear dose -dependent inhibition of tumorigenicity.

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Analysis of Anti-Cancer Activity of BAY-876 in Mice [2]
The above-described PDXs and cell-line-derived xenografts were utilized to assess the anti-tumor effect of BAY-876. The cell lines in exponential growth phase were trypsinized, washed twice with PBS and resuspended in the serum-free medium. SKOV-3 cells (4 × 106) were injected s.c. on the right flank of 6–7 weeks old female NSG mice. The formation of s.c. tumors was monitored and measured with a digital caliper. The tumor volumes were calculated based on the formula lw2/2 where l is the length and w is the shortest width of the tumor. When PDXs or cell line-derived tumors reached an average volume of 100 mm3, the mice were divided into control and experimental groups (5 mice/group) with a similar range of tumor volumes. The mice were treated by gavage feeding with the indicated doses of BAY-876.

Regarding further characterization we first examined BAY-876 (19) and other candidates in metabolic stability assays using liver microsomes and hepatocytes of different species. Furthermore, the most promising compounds were checked in the Caco‐2 permeability assay. Starting from compounds 2, 3, and 12 with moderate to high metabolic clearance in rat hepatocytes (Figures 1 and 2), installation of a 2‐carboxamide at the quinoline and a CF3 at the pyrazole, like in compound 13, already resulted in compounds with low metabolic clearance in human, dog and mouse liver microsomes as well as dog hepatocytes. However, rat hepatocyte clearance was higher giving only average maximal bioavailability according to the well‐stirred model. Comparing the Caco‐2 data of 2, 3, 12, and 13, the permeability from low (3.5 nm s−1) to high (200 nm s−1) (apical to basolateral) showed the high variation possible within the N‐(1H‐pyrazol‐4‐yl)quinoline compound class. [1]

BAY-876 (19) showed low metabolic in vitro clearance in all tested species except for monkey hepatocytes displaying moderate clearance (Table 6). The Caco‐2 permeability was high and the efflux ratio not considered critical. Compared with the moderate stability of triazole 33 in mouse liver microsomes and in rat hepatocytes, dimethylpyrazole 39 revealed good bioavailability of 88 % in mouse liver microsomes and 75 % in rat hepatocytes. Its stability in human microsomes was slightly lower with 66 %. In comparison with 39 the corresponding isopropylpyrazole 43 showed double the clearance in human microsomes. This metabolic liability was not only observed for 43 but also for other isopropylpyrazole compounds within the project. Due to this finding no isopropylpyrazole was selected for advanced in vivo studies. [1]

Despite the sub‐nanomolar potency of compound 52 its metabolic liability discouraged further in vivo characterization. Cyanopyridine 54 did not only show promising potency and selectivity in the GLUT assays but also very good metabolic stability in liver microsomes and hepatocytes across all tested species. However, the strong efflux ratio in the Caco‐2 assay appeared to be a major drawback of this compound relative to BAY-876 (19). The pyrazine 57 and the pyrimidine 58 showed a two‐ to threefold higher clearance in rat hepatocytes than BAY‐876 (19). [1]

Taking into account GLUT inhibition, metabolic stability, and Caco‐2 performance we selected BAY-876 (19) as candidate for in vivo pharmacokinetic studies that were conducted in two different species (Table 7). In good agreement with the in vitro hepatocyte data BAY‐876 displayed low clearance also in vivo in rat and in dog. The volume of distribution in steady state (V ss) was moderate in both species. Terminal half life was intermediate in rat and long in dog due to the very low clearance. As would be expected from the low blood clearance oral bioavailability was high at the given doses and formulations. Overall, the preliminary data of BAY‐876 demonstrate a favorable in vivo PK profile.

[1]. Identification and Optimization of the First Highly Selective GLUT1 Inhibitor BAY-876. ChemMedChem. 2016 Aug 23.

[2]. Ovarian Cancer Relies on Glucose Transporter 1 to Fuel Glycolysis and Growth: Anti-Tumor Activity of BAY-876. Cancers (Basel). 2018 Dec 31;11(1).

Despite the long-known fact that the facilitative glucose transporter GLUT1 is one of the key players safeguarding the increase in glucose consumption of many tumor entities even under conditions of normal oxygen supply (known as the Warburg effect), only few endeavors have been undertaken to find a GLUT1-selective small-molecule inhibitor. Because other transporters of the GLUT1 family are involved in crucial processes, these transporters should not be addressed by such an inhibitor. A high-throughput screen against a library of ∼3 million compounds was performed to find a small molecule with this challenging potency and selectivity profile. The N-(1H-pyrazol-4-yl)quinoline-4-carboxamides were identified as an excellent starting point for further compound optimization. After extensive structure-activity relationship explorations, single-digit nanomolar inhibitors with a selectivity factor of >100 against GLUT2, GLUT3, and GLUT4 were obtained. The most promising compound, BAY-876 [N4 -[1-(4-cyanobenzyl)-5-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl]-7-fluoroquinoline-2,4-dicarboxamide], showed good metabolic stability in vitro and high oral bioavailability in vivo. [1]

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Starting from moderately potent and metabolically labile HTS hit 1 carrying a furanyl moiety at position 2 of the quinoline and four methyl groups in total, we quickly improved both, potency and metabolic stability by substituting the furan and taking out the two quinoline methyl groups. Using the quinoline core of compound 3 we found an unsubstituted methylene to be the optimal spacer between the phenyl and pyrazole ring. After successful replacement of one of the pyrazole methyl groups for the metabolically more stable CF3 group (12), further SAR exploration of the quinoline core demonstrated an unsubstituted amide at position 2 and a fluorine at position 7 (19, BAY-876) to be very beneficial regarding GLUT1 potency and selectivity against the other GLUTs. At the pyrazole core a double substitution at positions 3 and 5 was found to be crucial for the excellent potency/selectivity profile especially against GLUT3. Regarding a substituent at the benzyl ring, the para position yielded more promising compounds than the ortho or meta position. With a para‐cyano group and an additional ring nitrogen, compound 54 demonstrated also an interesting GLUT profile. The synthesis of the N‐(1H‐pyrazol‐4‐yl)quinoline‐4‐carboxamides was straightforward as exemplified for BAY-876 (19). In vitro PK data showed that both BAY-876 (19) and 54 were very stable in liver microsomes and hepatocytes, although 54 had a strong efflux ratio of around 16. Preliminary in vivo PK studies of BAY‐876 (19) demonstrated that a good oral bioavailability and long terminal half‐life is attainable making it an excellent chemical probe to further evaluate the hypothesis of cancer treatment with a very selective GLUT1 inhibitor. [1]

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The recent progresses in understanding of cancer glycolytic phenotype have offered new strategies to manage ovarian cancer and other malignancies. However, therapeutic targeting of glycolysis to treat cancer remains unsuccessful due to complex mechanisms of tumor glycolysis and the lack of selective, potent and safe glycolytic inhibitors. Recently, BAY-876 was identified as a new-generation inhibitor of glucose transporter 1 (GLUT1), a GLUT isoform commonly overexpressed but functionally poorly defined in ovarian cancer. Notably, BAY-876 has not been evaluated in any cell or preclinical animal models since its discovery. We herein took advantage of BAY-876 and molecular approaches to study GLUT1 regulation, targetability, and functional relevance to cancer glycolysis. The anti-tumor activity of BAY-876 was evaluated with ovarian cancer cell line- and patient-derived xenograft (PDX) models. Our results show that inhibition of GLUT1 is sufficient to block basal and stress-regulated glycolysis, and anchorage-dependent and independent growth of ovarian cancer cells. BAY-876 dramatically inhibits tumorigenicity of both cell line-derived xenografts and PDXs. These studies provide direct evidence that GLUT1 is causally linked to the glycolytic phenotype in ovarian cancer. BAY-876 is a potent blocker of GLUT1 activity, glycolytic metabolism and ovarian cancer growth, holding promise as a novel glycolysis-targeted anti-cancer agent.
In conclusion, our findings provide direct evidence that GLUT1 is causally linked to the glycolytic phenotype in ovarian cancer. Selective targeting of GLUT1 with the newly developed candidate inhibitor BAY-876 is sufficient to suppress glycolytic metabolism and in vitro and in vivo growth of ovarian cancer. Therefore BAY-876 is an ideal glycolysis-targeted anti-cancer agent.[2]

C24H16F4N6O2

496.4165

496.127

C, 58.07; H, 3.25; F, 15.31; N, 16.93; O, 6.45

1799753-84-6

1799753-84-6(BAY-876)

118191391

White to off-white solid powder

1.5±0.1 g/cm3

632.3±55.0 °C at 760 mmHg

336.2±31.5 °C

0.0±1.9 mmHg at 25°C

1.649

3.89

127

2

9

5

36

870

0

FC(C1C(=C(C)N(CC2C=CC(C#N)=CC=2)N=1)NC(C1=CC(C(N)=O)=NC2C=C(C=CC1=2)F)=O)(F)F

BKLJDIJJOOQUFG-UHFFFAOYSA-N

InChI=1S/C24H16F4N6O2/c1-12-20(21(24(26,27)28)33-34(12)11-14-4-2-13(10-29)3-5-14)32-23(36)17-9-19(22(30)35)31-18-8-15(25)6-7-16(17)18/h2-9H,11H2,1H3,(H2,30,35)(H,32,36)

4-N-[1-[(4-cyanophenyl)methyl]-5-methyl-3-(trifluoromethyl)pyrazol-4-yl]-7-fluoroquinoline-2,4-dicarboxamide

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: ≥ 2.5 mg/mL (5.04 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900 μL 玉米油中，混合均匀。

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配方 2 中的溶解度: 5 mg/mL (10.07 mM) in 50% PEG300 50% Saline (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。