(Z)-SMI-4a

PIM1 (IC50 = 24 μM;(Ki = 0.6 μM); PIM2 (IC50 = 100 μM)

在完整细胞中，SMI-4a 处理（0.5 μM；1 小时；HEK-293T 细胞）可减弱标记 Pim-1 的自身磷酸化 [1]。

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本研究旨在探讨SMI - 4a在K562和伊马替尼耐药K562 （K562/G）细胞系中抗肿瘤作用的机制。通过WST‑8实验证明SMI‑4a抑制K562和K562/G细胞的增殖。Annexin V -碘化丙啶实验表明SMI - 4a以剂量和时间依赖的方式诱导K562和K562/G细胞凋亡。采用Hoechst 33342染色法检测细胞凋亡率。克隆形成实验显示SMI‑4a显著抑制K562和K562/G细胞的集落形成能力。Western blot分析表明，SMI - 4a降低了磷酸化的(p) Ser9糖原合成酶激酶（GSK） 3β/pGSK3β，并抑制了β -连环蛋白的易位。此外，凋亡调节因子Bax和聚（ADP核糖）聚合酶- 1下游基因表达上调，凋亡调节因子Bcl - 2和Myc原癌基因蛋白表达下调。免疫荧光结果显示血浆和细胞核中β -连环蛋白的表达水平发生变化。本研究的结果表明，SMI - 4a是一种有效的药物，可与目前的化疗药物联合使用，用于治疗伊马替尼耐药CML。Reference: Mol Med Rep. 2017 Oct;16(4):4603-4612. https://pubmed.ncbi.nlm.nih.gov/28849186/

SMI-4a（60 mg/Kg）每天两次治疗可显着缩小肿瘤大小，并且耐受性良好。最终口服 SMI-4a 后 1 小时收获的肿瘤显示，与用载体处理的小鼠的肿瘤相比，p70 S6K 的磷酸化降低，而相比之下，总 p70 S6K 表达没有变化。

Pim激酶检测[1]
使用多种方法进行Pim蛋白激酶测定，以确保化合物的作用不是由于任何实验伪影。表3所示化合物的初步筛选和评估是使用atp耗尽试验进行的。简单地说，重组人Pim-1以S6激酶/Rsk-2肽2 （KKRNRTLTK）为底物，筛选文库中的100µM化合物，1µM ATP和10 mM MgCl2孵育1小时。激酶反应后，使用kinase - glo荧光素酶试剂盒测定剩余ATP水平。对于需要更高ATP浓度的实验，Pim-1激酶活性是通过一种耦合实验来监测的，其中ADP的产生与丙酮酸激酶和乳酸脱氢酶催化的NADH氧化相耦合。实验在含100 mM NaCl、10 mM MgCl2、2.5 mM磷酸烯醇丙酮酸、0.2 mM NADH、30µg/mL丙酮酸激酶、10µg/mL乳酸脱氢酶、2 mM二硫苏糖醇、25 nM Pim-1、100µM S61肽和不同浓度ATP的20 mM MOPS pH 7中进行。在VersaMax微孔板阅读器中，在25°C下，通过监测NADH氧化在340 nm处的下降来测量活性。反应通过加入ATP（通常为100µM）引发。抑制剂（终值1% DMSO）在加入ATP之前加入。在这两种情况下，IC50值是用GraphPad Prism程序非线性回归确定的。在一些实验中，使用his标记的4E-BP1作为底物来测定Pim-1激酶活性。将活性Pim-1蛋白重悬于激酶反应缓冲液中（10 mM MOPS, pH 7.4, 100µM ATP, 15 mM MgCl2, 1 mM Na3VO4, 1 mM NaF， 1 mM DTT和蛋白酶抑制剂混合物）。每反应30µL，以His-4E-BP1蛋白3µg为底物，加入10 μCi的[γ-32P] ATP。在30°C下搅拌30分钟。然后将样品进行SDS-PAGE扫描，通过放射自显像显示32P标记的4E-BP1。最后，在一些实验中测量了完整细胞中Pim-1的活性。用Flag-Pim-1转染HEK-293T细胞24 h，胰蛋白酶处理后分装于小培养皿中过夜。细胞被洗一次,孵化与不含磷酸盐媒体含有10%不含磷酸盐的边后卫1 h。细胞培养介质中含有50μCi /毫升(32 p)为4 h,正磷酸盐的测试化合物添加最后1 h。免疫沉淀反应Pim-1, anti-Flag M2琼脂糖添加到细胞溶解产物和孵化3 h。部分(10%)的免疫沉淀反应是用于免疫印迹anti-Flag抗体(输入)。其余90%的样品进行SDS-PAGE， 32p标记的Pim-1通过放射自显影显示。

蛋白质印迹分析[1]
细胞类型： HEK-293T 细胞
测试浓度： 0.5 µM
孵育时间： 1 小时
实验结果：引起 Pim-1 诱导的 4E-BP1 磷酸化的剂量依赖性减少，IC50 约为 125 nM。

Antitumor Assay[1]
A syngeneic mouse tumor model that uses a transformed murine mammary adenocarcinoma cell line (JC, ATCC number CRL-2116) and Balb/C mice (Charles River) was performed as previously described. Tumor cells (1 × 106) were implanted subcutaneously, and tumor volume was calculated using the equation: (L × W2)/2. Upon detection of tumors, mice were randomized into treatment groups. Treatment was then administered once per day, five days per week, thereafter consisting of intraperitoneal doses of 0 or 50 mg 16a/kg or vehicle (50% DMSO: 50% phosphate-buffered saline). Whole body weights and tumor volume measurements were performed three times per week.

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Treatment protocols: Female (NZB × NZW)F1 mice were orally treated with AZD1208 (15 mg/kg) or vehicle control (0.1% Tween 80 and 0.5% methyl cellulose in water) 19, 20 for 12 weeks (n = 10 mice per group), starting at age 22 weeks (at the time of onset of proteinuria). Mice were then placed under anesthesia and killed at age 34 weeks. Twelve-week-old MRL/lpr mice received the selective Pim-1 inhibitor SMI-4a (60 mg/kg) or vehicle control (DMSO/PEG-400/Tween 80) twice daily, as described previously 21. Oral gavage was administered on 5 of 7 days each week for 8 weeks (n = 10 mice per group). In an independent experiment, survival was observed in mice until age 30 weeks, and the survival rates were compared between 2 groups (n = 15 mice per group). Reference: Arthritis Rheumatol. 2019 Aug;71(8):1308-1318. https://onlinelibrary.wiley.com/doi/abs/10.1002/art.40863

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Dissolved in 65% DMSO, 30% PEG-400, 5% Tween-80; 75, 60 mg/kg; oral administration

Nu/nu nude mice injected with pre-T-LBL cells

[1]. Synthesis and evaluation of novel inhibitors of Pim-1 and Pim-2 protein kinases. J Med Chem. 2009 Jan 8;52(1):74-86.

The Pim protein kinases are frequently overexpressed in prostate cancer and certain forms of leukemia and lymphoma. 5-(3-Trifluoromethylbenzylidene)thiazolidine-2,4-dione (4a) was identified by screening to be a Pim-1 inhibitor and was found to attenuate the autophosphorylation of tagged Pim-1 in intact cells. Although 4a is a competitive inhibitor with respect to ATP, a screen of approximately 50 diverse protein kinases demonstrated that it has high selectivity for Pim kinases. Computational docking of 4a to Pim-1 provided a model for lead optimization, and a series of substituted thiazolidine-2,4-dione congeners was synthesized. The most potent new compounds exhibited IC(50)s of 13 nM for Pim-1 and 2.3 microM for Pim-2. Additional compounds in the series demonstrated selectivities of more than 2500-fold and 400-fold for Pim-1 or Pim-2, respectively, while other congeners were essentially equally potent toward the two isozymes. Overall, these compounds are new Pim kinase inhibitors that may provide leads to novel anticancer agents.[1]

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The development of targeted tyrosine kinase inhibitors (TKIs) has succeeded in altering the course of chronic myeloid leukemia (CML). However, a number of patients have failed to respond or experienced disease relapse following TKI treatment. Proviral integration site for moloney murine leukemia virus‑1 (PIM‑1) is a serine/threonine kinase that participates in regulating apoptosis, cell cycle, signal transduction and transcriptional pathways, which are associated with tumor progression, and poor prognosis.

C11H6F3NO2S

273.23

273.007

C, 48.36; H, 2.21; F, 20.86; N, 5.13; O, 11.71; S, 11.73

438190-29-5

1361334

White to off-white solid powder

1.5±0.1 g/cm3

1.602

2.3

74.96

1

6

1

18

406

0

S1C(N([H])C(/C/1=C(\[H])/C1C([H])=C([H])C([H])=C(C(F)(F)F)C=1[H])=O)=O

NGJLOFCOEOHFKQ-VMPITWQZSA-N

InChI=1S/C11H6F3NO2S/c12-11(13,14)7-3-1-2-6(4-7)5-8-9(16)15-10(17)18-8/h1-5H,(H,15,16,17)/b8-5+

5-(3-(trifluoromethyl)benzylidene)thiazolidine-2,4-dione

TCS PIM-1 4a; SMI-4a; TCS PIM-1 4a; (Z)-SMI-4a; (5Z)-5-[3-(trifluoromethyl)benzylidene]-1,3-thiazolidine-2,4-dione; (Z)-5-(3-(trifluoromethyl)benzylidene)thiazolidine-2,4-dione; CHEMBL183906; SMI4a; SMI 4a

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)

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；
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