PPARβ/δ (IC50 = 22.9 μM)

BS-181 diHClide（0–40 μM；72 小时）可抑制癌细胞的生长，同时还可抑制乳腺癌细胞系（IC50 值：15.1 μM–20 μM）和结直肠癌细胞系（IC50 值）的生长。 ：11.5 μM–20 μM）。 15.3 μM，对肺癌、骨肉瘤、前列腺癌和肝癌细胞系的IC50值分别为11.5 μM至37.3 μM [1]。 RNA 聚合酶 II 的 C 末端结构域 (CTD) 丝氨酸 5 (P-Ser5) 的磷酸化可被 BS-181 二盐酸盐（0-50 μM；4 小时）抑制。虽然它对其他 CDK 或细胞周期蛋白没有影响，但它会下调 CDK4 和细胞周期蛋白 D1 的表达 [1]。在低浓度下，当暴露于 BS-181 二盐酸盐（0-50 μM；24 小时）时，处于 G1 期的细胞数量增加，而处于 S 和 G2/M 期的细胞数量减少。然而，较高浓度会导致细胞积聚在亚 G1 期，这表明细胞凋亡 [1]。

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BS-181的合成和体外激酶抑制[1]
BS-181由二氯吡唑并[1,5-a]嘧啶2合成，通过使用苄胺依次选择性取代C-7氯化物，Boc保护，在Buchwald-Hartwig反应条件下使用二-Boc-1,6-己二胺钯催化取代C-5氯化物，并在酸性甲醇中脱保护（补充图S1）。通过将越来越多的BS-181与纯化的重组CDK7/CycH/MAT1复合物孵育来测量CDK7活性的抑制，然后使用萤光素酶测定法（PkLight，Cambrex，UK）测量反应中剩余的游离ATP，萤光素酶活性因此提供了CDK7活性抑制的衡量标准。BS-181抑制CDK7活性，IC50=21 nM（表1；补充数据图S2），而用罗斯科维汀达到的IC50为510 nM，与之前关于罗斯科维廷抑制CDK7的报告一致（13）。BS-181对CDK1/cycB、CDK4/cycD1、CDK5/p35NCK、CDK6/cycD1和CDK9/cycT的抑制IC50值远高于1μM，对CDK2/cycE的抑制IC50=880 nM，比CDK7的IC50高约40倍。

来自许多不同类别的70种蛋白激酶被BS-181测试了抑制作用。使用高浓度（10μM）的BS-181观察到对几种激酶活性的一些抑制（补充表5）。对表现出最大抑制作用的激酶CDK2/cycA、CK1和DYRK1A进行IC50测量，结果显示IC50分别为730 nM、7.36μM和2.3μM。这些数据证实BS-181是CDK7活性的高选择性抑制剂。

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BS-181促进细胞周期阻滞并抑制癌症细胞生长[1]
为了评估BS-181的抗增殖活性，一组代表一系列肿瘤类型的细胞系，包括乳腺癌、肺癌、前列腺癌和结直肠癌癌症，用浓度增加的BS-181治疗72小时。使用磺基罗丹明B测定法测定增殖表明，所有受试细胞系的生长都受到抑制，IC50值范围为11.5至37μM（表2）。对BS-181观察到的生长抑制与对Roscovitine观察到的相似，其IC50值在8至33.5μM的范围内。

BS-181或Roscovitine治疗4小时后，CDKs和细胞周期蛋白的免疫印迹显示CDK4和细胞周期素D1下调（图2C），其他CDKs和周期蛋白的水平不受影响。此外，BS-181降低了抗凋亡蛋白XIAP和Bcl-xL的水平，而Bcl-2的水平没有变化。

用低浓度BS-181处理24小时后，G1期细胞数量增加，同时S期和G2/M期细胞数量减少（图3A；图S3）。然而，在较高浓度下，细胞在亚G1期积聚，表明细胞凋亡。BS-181处理24小时后，细胞的Annexin V染色证实了这一点，其中30%和83%的细胞分别用25μM和50μM BS-181对AnnexinⅤ染色呈阳性（图3B）。roscovitine未观察到明显的细胞凋亡。

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BS-181-抑制癌症细胞增殖、迁移和侵袭[2]
为了评估BS-181在GC中的抗增殖能力，将包括MKN28、SGC-7901、AGS和BGC823在内的几种不同细胞系用越来越高浓度的BS-181处理48小时。CCK-8试验表明，BS-181抑制GC细胞生长，抑制浓度（IC50）范围为17至22μM。对于正常胃上皮RGM-1细胞系，IC50为6.5μM（表1）。此外，我们还研究了BS-181对细胞迁移和侵袭能力的影响。正如预期的那样，BS-181以剂量依赖的方式显著抑制细胞迁移和侵袭能力（分别为P<0.05；图1）。

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BS-181诱导癌症细胞凋亡和细胞周期阻滞[2]
使用流式细胞术测定细胞凋亡。与对照组相比，在BS-181处理的BGC823细胞中观察到凋亡细胞的显著增加（分别为P<0.05；图2A）。我们的研究结果还表明，BS-181以剂量和时间依赖的方式诱导细胞凋亡。此外，与对照组相比，经BS-181处理的细胞中caspase-3和Bax的表达显著增加，而Bcl-2水平降低（分别为P<0.05）（图2B）。这些结果表明，BS-181可以诱导GC细胞凋亡。此外，CDK7活性的抑制导致关键抗凋亡蛋白XIAP和细胞周期调节因子细胞周期蛋白D1的显著减少（P<0.05）（图2C）。因此，BS-181可能通过下调BGC823细胞中XIAP和细胞周期蛋白的表达来调节细胞凋亡和细胞周期进程。在本研究中，通过流式细胞术分析了细胞周期分布（图3）。BS-181治疗显示G0/G1期细胞增加，同时S期和G2/M期细胞数量减少。这些结果表明，BS-181诱导细胞周期阻滞在G0/G1期，并延迟了细胞周期的进展。

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BS-181抑制癌症细胞CDK7活性[2]
BS-181是一种特异性CDK7抑制剂。在这项研究中，我们还证实了BS-181是CDK7的特异性抑制剂。如表2所示，BS-181抑制CDK7活性，IC50=0.019μM，而用罗斯科维汀达到的IC50为0.48μM。此外，BS-181对其他CDKs的抑制IC50值>1μM，远高于CDK7。此外，免疫印迹显示，BS-181抑制了RNA聚合酶II CTD在成熟的CDK7磷酸化位点丝氨酸5（P-Ser5）的磷酸化。

用 BS-181 二盐酸盐（10 mg/kg、20 mg/kg；单剂量）治疗的小鼠对药物反应良好，体重没有显着变化 [1]。 BS-181 二盐酸盐以剂量依赖性方式抑制肿瘤生长（腹腔注射；5 mg/kg 或 10 mg/kg，每日两次；每日总剂量 10 mg/kg 或 20 mg/kg；14 天）。与对照组相比，每天 10 mg/kg 和 20 mg/kg/天的肿瘤生长速度分别减慢了 25% 和 50% [1]。 BS-181 体内稳定，小鼠腹腔注射 10 mg/kg 后血浆消除半衰期为 405 分钟。 BS-181 以剂量依赖性方式抑制裸鼠模型中 MCF-7 异种移植物的生长，10 mg/kg/天和 20 mg/kg 治疗 2 周后，肿瘤生长减少 25% 和 50% /天，分别无明显毒性。

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体内肿瘤生长抑制[1]
腹腔注射（i.p）的BS-181的最大耐受单剂量为30mg/kg，其中10mg/kg和20mg/kg耐受良好（数据未显示）。因此，对于异种移植物肿瘤生长抑制研究，在14天内，每天两次向动物腹腔注射5mg/kg或10mg/kg，以给予10mg/kg或20mg/kg的总日剂量。与对照组相比，BS-181的10mg/kg/天和20mg/kg/天剂量分别以剂量依赖的方式抑制了肿瘤生长，肿瘤生长减少了25%和50%（图4A）。根据对动物体重没有明显不良影响的判断，在这些剂量下没有明显的毒性（图4B）。

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BS-181-抑制体内肿瘤生长并提高存活率[2]
如前所述，7腹腔注射BS-181的最大耐受单剂量为30mg/kg/d，BALB/c-nu小鼠对10mg/kg/d或20mg/kg/d的剂量耐受良好。在这项研究中，小鼠接受BS-181腹腔注射，每天两次，每次5mg/kg/d或10mg/kg/d，在2周内给予10mg/kg或20mg/kg的日剂量。此外，另一组15只大鼠接受了为期14天的roscovitine（20mg/kg/d）注射。我们观察到，与对照组相比，BS-181以剂量依赖的方式显著抑制了肿瘤生长（分别为P<0.05）（图5A）。然而，在14天的观察期间，各组小鼠的体重没有显著差异（图5B）。这表明，每日剂量为10mg/kg或20mg/kg时没有明显的毒性。此外，所有动物都被再饲养30天进行存活观察。如图5C所示，在对照组中，每10只小鼠中有8只（80%）死亡，在roscovitine组中有5只（50%）死亡，而在BS-181治疗组中，有6只（60%，10mg/kg/d）和3只（30%，20mg/kg/d）死亡。BS-181治疗组和未治疗组大鼠的存活率总体差异显著（分别为P<0.05）。

将纯化的重组 CDK7/CycH/MAT1 复合物与浓度不断增加的 BS-181 一起孵育，以测量受抑制的 CDK7 活性的量。然后使用荧光素酶测定来测量反应中剩余的游离 ATP 的量，并且荧光素酶活性提供了用于 IC50 计算的 CDK7 活性抑制的量度。

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体外激酶测定[1]
纯化的重组CDK2/cycE（0050-0055-1）、CDK4/cycD1（0142-0143-1），CDK5/p35NCK（0356-0355-1）和CDK7/CycH/MAT1（0366-0360-4）和CDK9/CycT（0371-0345-1）购自Proqinase GmbH。激酶测定根据制造商的方案进行，使用购自Proqinase GmbH的底物肽，如下所述。根据制造商的方案，使用萤光素酶测定来确定激酶反应结束时剩余的ATP，这提供了激酶活性的测量。

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体外激酶测定[2]
如前所述，进行了7次激酶测定，以评估BS-181对体外CDK活性的抑制作用。使用购自ProQinase GmbH的底物肽进行激酶测定。根据制造商的方案，使用萤光素酶测定法 测定激酶反应结束时剩余的ATP。

细胞增殖实验[1]
细胞类型：乳腺癌
细胞类型： MCF-7、MDA-MB-231、T47D、ZR-75- 1等结直肠癌
细胞类型： COLO-205、HCT-116、HCT-116 (p53-/-) 肺癌
细胞类型： > A549、NCI-460 骨肉瘤
细胞类型： U2OS、SaOS2 前列腺癌
细胞类型： PC3、LNCaP
测试浓度： 0-40 μM
孵育时间：72小时
实验结果：一组具有抗增殖作用的细胞活动系统，包括乳腺癌、肺癌、前列腺癌和结直肠癌。

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蛋白质印迹分析[1]
细胞类型： 乳腺癌
细胞类型： MCF-7 细胞
测试浓度： 0 μM； 25μM； 50 μM
孵育时间：4小时
实验结果：抑制CDK7底物的磷酸化。

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细胞凋亡分析 [1]
细胞类型： 乳腺癌
细胞类型： MCF-7 细胞
已测试浓度：0μM； 25μM； 50 μM
孵育持续时间： 24 小时 (hrs (小时))
实验结果： 导致细胞 G1 期停滞和凋亡。

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细胞生长试验[1]
所有细胞均购自ATCC，并在添加了10%胎牛血清（FCS）的DMEM中常规培养。如所述，使用磺基罗丹明B（SRB）测定法评估细胞生长。

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流式细胞术[1]
将MCF-7细胞（4×105）接种在含有10%FCS的DMEM中的6孔板中，并使其粘附24小时，然后加入化合物或DMSO并孵育24小时。将细胞胰蛋白酶化，以1100 rpm离心5分钟，然后重新悬浮在5 ml冰冷的PBS中，如上所述离心，轻轻重新悬浮在2 ml冰冷的70%乙醇中，并在4°C下孵育一小时。用5ml冰冷的PBS洗涤细胞两次，然后重新悬浮在100μl PBS中，PBS中含有100μg/ml RNase和1ml 50μg/ml碘化丙啶。在4°C的黑暗中孵育过夜，用70μm纱布过滤到FACS管中以去除细胞团块后，使用Beckman Coulter Elite ESP上的RXP细胞组学软件采集染色细胞，并使用Flow Jo v7.2.5分析数据。对于碘化丙啶和膜联蛋白V的双重标记，将细胞胰蛋白酶化，用培养基收集，以10 rpm离心5分钟，用5 ml含有2%（w/V）BSA的冰冷PBS洗涤两次。根据制造商的说明，使用膜联蛋白V-FITC凋亡检测试剂盒I用膜联蛋白V-FITC标记细胞。使用Beckman Coulter Elite ESP上的RXP细胞组学软件在1小时内采集标记细胞，并使用Flow Jo v7.2.5分析数据。对三个独立实验进行了统计分析，使用非配对学生t检验来确定p值。

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免疫印迹[1]
将1×106个细胞铺在10cm板上24小时后用化合物处理。4小时后，通过加入500μl预热至100°C的热裂解缓冲液（4%SDS（w/v）、20%甘油（v/v）、0.1%溴酚蓝（w/v。

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细胞周期和凋亡分析[2]
将细胞接种在含有10%胎牛血清的Dulbecco改良Eagle培养基中的平板上。在0μM、1μM、10μM和20μM下用BS-181孵育12小时、24小时、48小时和72小时后，收获细胞，用冷PBS冲洗，用70%冰冷乙醇固定30分钟，然后在流式细胞术前用碘化丙啶（PI）孵育30分钟。采用Annexin V-FITC/PI双染色法通过流式细胞术测定细胞凋亡。膜联蛋白V阳性和PI阴性细胞被鉴定为凋亡细胞。使用CellQuest软件测定凋亡率。

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细胞活力测定[2]
根据供应商的介绍，使用细胞计数试剂盒检测细胞活力。简而言之，在有或没有BS-181的情况下，以每孔104个细胞的速度接种BGC823细胞48小时。然后，在每个孔中在450nm处检测吸光度（在650nm处检测参考）。

Animal/Disease Models: 7weeks old female nu/nu-BALB/c athymic nude mice, MCF-7 cells [1]
Doses: 5 mg/kg or 10 mg/kg; 10 mg/kg or 20 mg/kg
Route of Administration: intraperitoneal (ip) injection; twice a day or one time/day in total; 14-day
Experimental Results: Dramatically inhibited tumor growth.

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The mice receive a subcutaneous injection of 5×10⁶ BGC823 cells (0.1 mL) in their flanks. Tumor sizes are measured twice a week, and volumes are computed with the following formula: tumor size = (length ×width²)/2. Ultimately, thirty mice with tumor volumes ranging from 100 to 200 mm³ are chosen, and they are randomly assigned to three groups. BS-181 is made in the following conditions: 10% dimethyl sulfoxide/50 mM HCl/5% Tween 20/85% saline, as previously mentioned. For 14 days, mice are given BS-181 injection (ip) twice a day at the prescribed doses (10 mg/kg/d for BS-181 or 20 mg/kg/d for Roscovitine). Injectable vehicles are given to control mice. Daily measurements of the tumor volume and animal weights are made during the course of the 14-day treatment. To observe their survival, all rats are also held for an additional 30 days. In mice, BS-181 at a dose of 5 mg/kg or 10 mg/kg is administered intraperitoneally twice a day.

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Human tumor xenografts [1]
7-week old female nu/nu-BALB/c athymic nude mice were used. Before inoculation of animal with cells, a 0.72 mg 17β-estradiol 60-day release pellet was implanted subcutaneously. 5×106 cells MCF-7 cells were injected subcutaneously in not more than 0.1ml volume into the flank of the animals. Tumor measurements were performed twice per week, and volumes were calculated using the formula 1/2 [length (mm)] × [width (mm)]2. The animals were randomized and when tumors had reached a volume of 100–200 mm3, animals were entered into the different treatment groups and treatment with test drug or vehicle control was initiated. Animals were treated with compound twice daily by i.p. injection for a total of 14 days. The compounds were prepared in the vehicle of 10% DMSO/50mM HCl/5% Tween 20/85% Saline. Control mice were injected with the vehicle. Compounds were administered by exact body weight, with the injection volume being not more than 0.2ml. At the end of the 14-day treatment period, the mice were sacrificed. Throughout the 14-day treatment period animal weights were determined each day and tumor volumes on alternate days.

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Animal preparation and human tumor xenografts [2]
Human tumor xenografts were established as previously described.7 In total, 5×106 BGC823 cells (0.1 mL) were injected subcutaneously into the flank of the mice. Tumor measurements were performed two times per week, and volumes were calculated using the formula: tumor size = (length [mm] × width2 [mm])/2. Finally, 30 mice (tumor volume 100–200 mm3) were selected and randomly assigned into three groups. As previously described, BS-181 was prepared in 10% dimethyl sulfoxide/50 mM HCl/5% Tween 20/85% saline. Mice received BS-181 injection (ip) twice daily at indicated doses (BS-181 [10 mg/kg/d or 20 mg/kg/d] or roscovitine [20 mg/kg/d]) for a total of 14 days. Control mice were injected with vehicles. Animal weights and tumor volume were measured each day throughout the 14-day treatment. In addition, all rats were kept for another 30 days for survival observation. Mice were injected intraperitoneally twice daily with BS-181 at 5 mg/kg or 10 mg/kg.

In vivo pharmacokinetic studies and tumour growth inhibition [1]
The maximum tolerated single dose for BS-181 given intraperitoneally (i.p), was determined as 30 mg/kg, with 10 and 20 mg/kg being well tolerated (data not shown). For xenograft tumour growth inhibition studies, therefore, the animals were injected intraperitoneally twice daily with 5 mg/kg or 10 mg/kg, to give total daily doses of 10 mg/kg or 20 mg/kg, over a period of 14 days. Tumour growth was inhibited in a dose-dependent manner, with 25% and 50% reduction in tumour growth, compared with the control group, for 10 mg/kg/day and 20 mg/kg/day doses of BS-181, respectively (Fig. 4A). At these doses there was no apparent toxicity, as judged by lack of significant adverse effects on animal weights (Fig. 4B).
Intravenous (i.v) and i.p administration of 10 mg/kg BS-181 showed rapid clearance (Supplemental data Fig. S4). The terminal half-lives were 405 and 343 minutes for i.p and i.v administration, respectively, with the measured plasma concentration at 15 minutes of 1,950 (SEM = 203) and 2,530 (SEM = 269) ng/mL, respectively, and bioavailability being 37% for i.p administration of BS-181 (Tables 3 and 4, Supporting Information).

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Pharmacokinetic studies showed rapid clearance of BS-181 administered i.p. or i.v. In the case of i.p. administration, the maximal blood concentration of BS-181 was 1317 ng/mL. Further, bioavailability was only 37%, indicating a need for further refinement of the BS-181 structure to improve stability and bioavailability. As it stands the studies described here indicate that continuous i.v. infusion or repeated administration is needed for further in vivo evaluation. The observed efficacy, despite the low plasma levels (lower than the IC50 for growth inhibition in vitro), could therefore be due, at least in part to more active metabolites generated following i.p adminsitration. Elucidation of the structures of possible metabolites and their activities will be the subject of future studies.[1]

[1]. The development of a selective cyclin-dependent kinase inhibitor that shows antitumor activity. Cancer Res. 2009 Aug 1;69(15):6208-15.

[2]. Selective CDK7 inhibition with BS-181 suppresses cell proliferation and induces cell cycle arrest and apoptosis in gastric cancer. Drug Des Devel Ther. 2016 Mar 16;10:1181-9.

Normal progression through the cell cycle requires the sequential action of cyclin-dependent kinases CDK1, CDK2, CDK4, and CDK6. Direct or indirect deregulation of CDK activity is a feature of almost all cancers and has led to the development of CDK inhibitors as anticancer agents. The CDK-activating kinase (CAK) plays a critical role in regulating cell cycle by mediating the activating phosphorylation of CDK1, CDK2, CDK4, and CDK6. As such, CDK7, which also regulates transcription as part of the TFIIH basal transcription factor, is an attractive target for the development of anticancer drugs. Computer modeling of the CDK7 structure was used to design potential potent CDK7 inhibitors. Here, we show that a pyrazolo[1,5-a]pyrimidine-derived compound, BS-181, inhibited CAK activity with an IC(50) of 21 nmol/L. Testing of other CDKs as well as another 69 kinases showed that BS-181 only inhibited CDK2 at concentrations lower than 1 micromol/L, with CDK2 being inhibited 35-fold less potently (IC(50) 880 nmol/L) than CDK7. In MCF-7 cells, BS-181 inhibited the phosphorylation of CDK7 substrates, promoted cell cycle arrest and apoptosis to inhibit the growth of cancer cell lines, and showed antitumor effects in vivo. The drug was stable in vivo with a plasma elimination half-life in mice of 405 minutes after i.p. administration of 10 mg/kg. The same dose of drug inhibited the growth of MCF-7 human xenografts in nude mice. BS-181 therefore provides the first example of a potent and selective CDK7 inhibitor with potential as an anticancer agent. [1]
In summary we have discovered the most potent CDK7-selective inhibitor to date by computer-aided drug design. BS-181 selectively exhibited nanomolar enzymatic potency and inhibited all cell lines tested at low micromolar concentrations. For the given route of administration (37% bioavailability) the drug demonstrated in vivo activity in human tumor xenografts. BS-181 warrants further pre-clinical and clinical evaluation as a candidate cancer therapeutic.[1]

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Cyclin-dependent kinase (CDK) family members have been considered as attractive therapeutic targets for cancer. In this study, we aim to investigate the anticancer effects of a selective CDK7 inhibitor, BS-181, in gastric cancer (GC) cell line. Human GC cells (BGC823) were cultured with or without BS-181 at different concentrations for 24-72 hours. BS-181 significantly reduced the activity of CDK7 with downregulation of cyclin D1 and XIAP in GC cells. Treatment with BS-181 induced cell cycle arrest and apoptosis. The expression of Bax and caspase-3 was significantly increased, while Bcl-2 expression was decreased in cells treated with BS-181. In addition, the inhibition of CDK7 with BS-181 resulted in reduced rates of proliferation, migration, and invasion of gastric cells. Those results demonstrated the anticancer activities of selective CDK7 inhibitor BS-181 in BGC823 cells, suggesting that CDK7 may serve as a novel therapeutic target or the treatment of GC.
In conclusion, our study demonstrates that BS-181, the selective inhibitor of CDK7, prevents cell growth both in vitro and in vivo, induces the G1 arrest and apoptosis, and inhibits cell migration and invasion of GC. Therefore, BS-181 provides potent and selective CDK7 inhibitor with the potential as an antigastric cancer agent.[2]

C22H34CL2N6

453.4516

452.222

C, 58.27; H, 7.56; Cl, 15.64; N, 18.53

1883548-83-1

BS-181 hydrochloride;1397219-81-6;BS-181;1092443-52-1

91826108

Typically exists as solid at room temperature

80.3

5

5

11

30

425

0

Cl[H].Cl[H].N12C(=C([H])C(=NC1=C(C([H])=N2)C([H])(C([H])([H])[H])C([H])([H])[H])N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])N([H])[H])N([H])C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H]

XYXAMTBYYTXHSO-UHFFFAOYSA-N

InChI=1S/C22H32N6.2ClH/c1-17(2)19-16-26-28-21(25-15-18-10-6-5-7-11-18)14-20(27-22(19)28)24-13-9-4-3-8-12-23;;/h5-7,10-11,14,16-17,25H,3-4,8-9,12-13,15,23H2,1-2H3,(H,24,27);2*1H

5-N-(6-aminohexyl)-7-N-benzyl-3-propan-2-ylpyrazolo[1,5-a]pyrimidine-5,7-diamine;dihydrochloride

BS 181 dihydrochloride; BS-181 (dihydrochloride); 1883548-83-1; 5-N-(6-aminohexyl)-7-N-benzyl-3-propan-2-ylpyrazolo[1,5-a]pyrimidine-5,7-diamine;dihydrochloride; BS 181 2HCl; IAD54883; AKOS025293513;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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。