S1P2 (Sphingosine-1-Phosphate 2; EDG-5); human S1P2 (IC50 = 17 nM); rat S1P2 (IC50 = 22 nM)

JTE-013（50-200 μM；1-3 天）会降低细胞活力[1]。 JTE-013（10-1000 nM；30 分钟）可逆转 S1P 诱导的 Akt 抑制并抑制 S1P 诱导的 ERK 激活[1]。 JTE-013 对 S1P3 具有 4.2% 的抑制作用，并且在浓度高达 10 μM 时不会拮抗 S1P1[1]。hr> 细胞活力测定[1]
细胞系：SK-N-AS 细胞
浓度：50、100、150、200 μM
孵育时间：1-3 天
结果：细胞活力降低。

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Western Blot 分析[1]
细胞系：SK-N-AS 细胞
浓度：10、100、1000 nM
孵育时间：30 分钟
结果：逆转 S1P 诱导的 Akt 抑制并抑制 S1P 诱导的 ERK 激活。

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JTE-013对GB细胞迁移的影响[1]
S1P2信号转导的一个很好的生物学后果是抑制细胞迁移（Lepley等，2005）。为了比较JTE-013和AB1作为S1P2拮抗剂的效率，进行了细胞迁移实验。使用GB细胞系U118和U87，因为众所周知，S1P抑制S1P2高表达的U118细胞的细胞迁移，而S1P诱导S1P2低表达的U87细胞的细胞迁移(Lepley et al., 2005；图3，A和C)。在逆转U118细胞中s1p介导的细胞迁移抑制和通过阻断S1P2信号传导增强U87细胞中s1p刺激的细胞迁移方面，AB1处理比JTE-013更有效（图3，B和D）。

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AB1与JTE-013对SK-N-AS细胞S1P2下游分子水平的影响[1]
S1P2通过调控不同的下游效应分子发挥多种细胞功能。我们之前的研究以及其他人的研究表明，这些分子包括细胞内信号介质（p-Akt, p-ERK），以及生长和分化调节剂，如CTGF (Sanchez et al., 2007；Li et al., 2008a)。为了进一步比较JTE-013和AB1的体外效率，我们进行了Western blot分析。与JTE-013类似，AB1能够逆转s1p诱导的Akt抑制，并抑制s1p诱导的ERK激活，浓度在100 nM至1 μM之间（图4A）。实时定量聚合酶链反应进一步显示AB1比JTE-013在抑制s1p诱导的CTGF mRNA表达方面相对更有效（图4B）。

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AB1与JTE-013对SK-N-AS细胞活力的影响[1]
为了研究AB1抑制肿瘤作用的潜在机制，我们对JTE-013或AB1处理的SK-N-AS细胞的细胞活力进行了评估。MTT实验显示，在SK-N-AS细胞中，浓度高于50 μM时，AB1对细胞活力的抑制作用不如JTE-013，而在较低浓度时，AB1的抑制作用相似（图6），这表明AB1引起的抑制效果的改善不是由直接抑制癌细胞的细胞存活引起的。

JTE-013（灌胃；每天 30 mg/kg，连续 14 天）可减小肿瘤大小和肿瘤重量[1]。
动物模型：六周龄雌性无胸腺NCr-nu/nu裸鼠[1]
剂量：30 mg/kg
给药方式：灌胃；连续 14 天每天一次
结果：肿瘤大小和重量减小。

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JTE-013的对比[1]
在之前的研究中，JTE-013被证明可以显著抑制NB异种移植物的生长（Li et al., 2011）。在这里，发现AB1在抑制NB异种移植物生长方面再次比JTE-013更有效，无论是肿瘤大小（图5A）还是治疗后14天的肿瘤重量（图5B）。综上所述，上述数据有力地表明，与JTE-013相比，AB1可能具有更强的体内抗肿瘤活性。

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AB1与JTE-013对NB异种移植物CCL2表达和肿瘤相关巨噬细胞浸润的影响[1]
为了阐明AB1增强抗肿瘤作用的机制，研究人员量化了AB1对处理过的NB异种移植物中几种S1P2下游分子基因表达水平的影响。CCL2就是这些基因之一（Li et al., 2014）。CCL2的表达与肿瘤相关巨噬细胞（TAM）浸润呈正相关（Zhang et al., 2010）。在我们之前的研究中，JTE-013阻断S1P2信号不仅抑制NB异种移植物的生长（Li et al., 2011），而且还降低了CCL2的表达和随后的TAM浸润（Li et al., 2014），表明抑制CCL2有利于抗癌治疗。正如预期的那样，在JTE-013处理或AB1处理的NB异种移植物中，CCL2在mRNA和蛋白水平上的表达都有降低的趋势（图7，A和B）。此外，小鼠巨噬细胞标志物F4/80的免疫组织化学染色显示，JTE-013和AB1均显著抑制TAM的浸润。然而，AB1没有表现出任何改善的抑制作用（图7C），这表明AB1抗肿瘤作用的增强不是由于影响CCL2表达和随后的TAM浸润。

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AB1与JTE-013对NB异种移植物肿瘤纤维化和凋亡的影响[1]
CTGF是纤维化的中心介质（Lipson et al., 2012）。有趣的是，在mRNA和蛋白水平上，AB1比JTE-013抑制CTGF表达的程度更大（图8），这表明AB1抗肿瘤效果的提高可能部分是由于其对肿瘤纤维化的有益作用。使用Ki67染色的组织学研究未发现三组之间Ki67阳性增殖细胞的数量有显著差异（补充图2）。然而，末端脱氧核苷酸转移酶介导的地高西根-脱氧尿苷镍端标记染色（图9A）和cleaved caspase-3检测（图9B）显示，AB1在诱导这些NB异种移植物上的肿瘤细胞凋亡方面比JTE-013更有效。综上所述，我们的数据表明，AB1引起的抗肿瘤效果的提高可能归因于对NB异种移植物肿瘤纤维化和肿瘤凋亡的影响。

荧光成像平板阅读器试验。[1]
钙通量测定由一家CRO公司在FLIPRTETRA仪器上进行[荧光成像板阅读器（flir）测定]，以分析测试化合物在S1P1-5上的剂量依赖性激动剂和拮抗剂活性。简单地说，激动剂实验是在FLIPRTETRA仪器上进行的，在荧光基线建立后，将测试化合物、对照物和参比激动剂S1P添加到检测板上。180秒的时间用来评估每种化合物激活每种S1PR的能力。激动剂实验完成后，从FLIPRTETRA仪器中取出检测板，在25°C下孵育7分钟。之后，将检测板放回FLIPRTETRA仪器中，开始拮抗剂检测。利用激动剂试验中确定的EC80效价值，在建立荧光基线后，用参比激动剂S1P的EC80浓度挑战所有预孵育的样品复合孔。另外180秒的时间用来评估每种化合物抑制每种S1PR的能力。所有分析板数据都进行了适当的基线校正。应用基线校正后，导出最大荧光值，并处理数据以计算激活百分比（相对于Emax参考激动剂S1P和载体控制值）和抑制百分比（相对于EC80和载体控制值）。所有剂量反应曲线均采用GraphPad Prism软件生成

细胞系：SK-N-AS 细胞 浓度：50、100、150、200 μM 孵育时间：1-3 天 结果：细胞活力降低。 细胞系：SK-N-AS细胞
浓度：50、100、150、200 μM
孵育时间：1-3天
结果：细胞活力降低，

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迁移试验。[1]
如前所述，迁移试验在96孔趋化微室中进行（Li等人，2009b）。简单地说，聚碳酸酯过滤器（孔径为8µm）涂覆50µg/ml纤维连接蛋白。S1P稀释后以85µl /孔加入下腔。胰蛋白酶化前对GB细胞进行血清饥饿2小时，用或不加<强JTE-013和AB1预处理10分钟。在0.39 ml培养基中，每孔5 × 104个细胞置于上隔室，在37℃下迁移5小时。然后将过滤器在4°C下固定过夜，用棉签去除未迁移的细胞。在96孔板上用0.1%结晶紫染色，10%醋酸洗脱。在595 nm处测定吸光度。

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甲基噻唑基二苯基溴化四氮唑测定[1]
用JTE-013或AB1处理的SK-N-AS细胞的活力通过甲基噻唑基二苯基溴化四唑（MTT）测定，如先前所述（Li et al., 2013）。简单地说，将SK-N-AS细胞接种于96孔板中，用不同浓度的JTE-013或AB1处理不同时间，然后在37℃下MTT孵育2小时。用二甲基亚砜溶解活细胞中形成的不溶性甲醛，用Bio-Rad微孔板仪在595 nm处测定吸光度。结果显示为细胞活力相对于非药物治疗对照的百分比。

Six-week-old female athymic NCr-nu/nu nude mice
30 mg/kg
Gavage; daily for 14 consecutive days

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Subcutaneous NB Tumor Model. [1]
Animal experiments were conducted according to our institution’s and the National Research Council’s guide for the care and use of laboratory animals. Six-week-old female athymic NCr-nu/nu nude mice (National Cancer Institute, Frederick, MD) were used in this study. Briefly, each mouse received a subcutaneous flank injection containing 1 × 107 SK-N-AS cells in 0.1 ml phosphate-buffered saline. After the tumor size reached approximately 100 mm3, the mice were randomized into three groups: vehicle control, JTE-013, and AB1. Both JTE-013 and AB1 were dissolved in dimethylsulfoxide first, diluted with 2% (2-hydroxypropyl)-β-cyclodextrin in phosphate-buffered saline, and given by gavage at 30 mg/kg daily for 14 consecutive days. Tumors were measured every other day with a caliper, and tumor volumes were calculated using the following formula: tumor volume = length × width2 × 0.52. Two weeks later, the mice were euthanized and tumor masses were collected for different assays. Intravenous Pharmacokinetic Analysis. [1]
Male CD-1 mice were used in this study. Briefly, a catheter was implanted in the carotid artery of each mouse to facilitate the subsequent repeated blood draws. Then the tested compounds were given intravenously at 1 mg/kg. A small amount of blood (30 μl) was taken from the catheter at different time points and the drug concentrations in the blood were determined by high-performance liquid chromatography analysis/mass spectrometry.

AB1 versus JTE-013 as S1P2 Antagonists and Stability In Vivo. [1]
JTE-013 is the current literature standard S1P2 antagonist but is unstable in vivo (Swenson et al., 2011). Through structural modification, a series of JTE-013 derivatives named AB compounds were synthesized and the FLIPR assay was conducted to determine their agonistic and antagonistic activities on S1P1–5. Among them, AB1 (Fig. 1) had the strongest S1P2 antagonist activity, with an IC50 of 3.5 versus 11 nM for JTE-013 (Fig. 2A). By contrast, no significant agonistic or antagonistic activities on other S1PRs were observed at micromolar levels (Supplemental Fig. 1). Furthermore, pharmacokinetic analysis after intravenous administration showed that the blood concentration of AB1 in mice remained higher than that of JTE-013 over 12 hours (Fig. 2B), indicating either better stability or slower clearance of AB1 in vivo. These data suggest that AB1 may have improved potency and stability in vivo.

[1]. Antitumor Activity of a Novel Sphingosine-1-Phosphate 2 Antagonist, AB1, in Neuroblastoma. J Pharmacol Exp Ther. 2015 Sep;354(3):261-8.

The bioactive lipid sphingosine-1-phosphate (S1P) and its receptors (S1P1-5) play critical roles in many pathologic processes, including cancer. The S1P axis has become a bona fide therapeutic target in cancer. JTE-013 [N-​(2,​6-​dichloro-​4-​pyridinyl)-​2-​[1,​3-​dimethyl-​4-​(1-​methylethyl)-​1H-​pyrazolo[3,​4-​b]pyridin-​6-​yl]-​hydrazinecarboxamide], a known S1P2 antagonist, suffers from instability in vivo. Structurally modified, more potent, and stable S1P2 inhibitors would be desirable pharmacological tools. One of the JTE-013 derivatives, AB1 [N-(1H-4-isopropyl-1-allyl-3-methylpyrazolo[3,4-b]pyridine-6-yl)-amino-N'-(2,6-dichloropyridine-4-yl) urea], exhibited improved S1P2 antagonism compared with JTE-013. Intravenous pharmacokinetics indicated enhanced stability or slower clearance of AB1 in vivo. Migration assays in glioblastoma showed that AB1 was slightly more effective than JTE-013 in blocking S1P2-mediated inhibition of cell migration. Functional studies in the neuroblastoma (NB) cell line SK-N-AS showed that AB1 displayed potency at least equivalent to JTE-013 in affecting signaling molecules downstream of S1P2. Similarly, AB1 inhibition of the growth of SK-N-AS tumor xenografts was improved compared with JTE-013. Cell viability assays excluded that this enhanced AB1 effect is caused by inhibition of cancer cell survival. Both JTE-013 and AB1 trended to inhibit (C-C motif) ligand 2 expression and were able to significantly inhibit subsequent tumor-associated macrophage infiltration in NB xenografts. Interestingly, AB1 was more effective than JTE-013 in inhibiting the expression of the profibrotic mediator connective tissue growth factor. The terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine nick-end labeling assay and cleaved caspase-3 detection further demonstrated that apoptosis was increased in AB1-treated NB xenografts compared with JTE-013. Overall, the modification of JTE-013 to produce the AB1 compound improved potency, intravenous pharmacokinetics, cellular activity, and antitumor activity in NB and may have enhanced clinical and experimental applicability. [1]

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In summary, here we report the novel modification of the S1P2 antagonist JTE-013 to produce AB1. AB1 has moderately improved potency and intravenous pharmacokinetics that demonstrate better stability. In the context of NB, it also appears to have better cellular activity and antitumor activity. On the basis of these findings, we conclude that AB1 may have enhanced clinical and experimental applicability, overcoming some of the shortcomings of JTE-013. [1]

C17H19CL2N7O

408.2851

407.102

C, 50.01; H, 4.69; Cl, 17.37; N, 24.01; O, 3.92

383150-41-2

547756-93-4

10223146

White to off-white solid powder

1.5±0.1 g/cm3

1.697

4.42

96.76

3

5

4

27

516

0

ClC1C([H])=C(C([H])=C(N=1)Cl)N([H])C(N([H])N([H])C1C([H])=C(C2C(C([H])([H])[H])=NN(C([H])([H])[H])C=2N=1)C([H])(C([H])([H])[H])C([H])([H])[H])=O

RNSLRQNDXRSASX-UHFFFAOYSA-N

InChI=1S/C17H19Cl2N7O/c1-8(2)11-7-14(22-16-15(11)9(3)25-26(16)4)23-24-17(27)20-10-5-12(18)21-13(19)6-10/h5-8H,1-4H3,(H,22,23)(H2,20,21,24,27)

1-(2,6-dichloropyridin-4-yl)-3-[(1,3-dimethyl-4-propan-2-ylpyrazolo[3,4-b]pyridin-6-yl)amino]urea

JTE 013; JTE-013; 383150-41-2; 1-(2,6-dichloropyridin-4-yl)-3-[(1,3-dimethyl-4-propan-2-ylpyrazolo[3,4-b]pyridin-6-yl)amino]urea; CHEMBL1368758; DTXSID60436982; N-(2,6-dichloropyridin-4-yl)-2-(4-isopropyl-1,3-dimethyl-1H-pyrazolo[3,4-b]pyridin-6-yl)hydrazine-1-carboxamide; 1-[1,3-Dimethyl-4-(2-methylethyl)-1H-pyrazolo[3,4-b]pyridin-6-yl]-4-(2,6-dichloro-4-pyridinyl)-semicarbazide; JTE013

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)

DMSO : ~100 mg/mL (~244.92 mM)

配方 1 中的溶解度: ≥ 0.83 mg/mL (2.03 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 8.3 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: ≥ 0.83 mg/mL (2.03 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 8.3 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，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。