ZM 447439

Aurora A (IC50 = 110 nM); Aurora B (IC50 = 130 nM)

ZM-447439 处理的细胞经历间期，正确进入有丝分裂，并形成双极纺锤体。另一方面，胞质分裂、分离和染色体排列都失败了。 ZM-447439 抑制有丝分裂期间组蛋白 H3 的磷酸化并限制细胞分裂。 ZM-447439 抑制染色体排列和分离。 ZM-447439 损害了主轴检查点的功能。与基因抑制 Aurora-B 的细胞类似，这些经过 ZM 处理的 G2/M 停滞细胞积累 4N/8N DNA 并产生多极纺锤体。 ZM-447439 治疗会导致细胞死亡。 Akt Ser473 磷酸化及其底物 GSK3α/β Ser21 和 Ser9 磷酸化的减少与 ZM-447439 对 Aurora 激酶的抑制密切相关[2]。

体外激酶测定[1]
根据制造商的说明，使用杆状病毒表达系统将重组Aurora A和B表达为NH2末端His6标记的融合蛋白。Aurora A通过Ni-NTA琼脂糖亲和层析纯化，Aurora B通过CM-Sepharose Fast Flow离子交换层析纯化。将1 ng纯化的重组酶加入到含有25 mM Tris-HCl、pH 7.5、12.5 mM KCl、2.5 mM NaF、0.6 mM DTT、6.25 mM MnCl2、10μM肽底物（Biotinyl Ahx-tetra（LRRWSLG））、10μM Aurora a或5μM Aurora B ATP和0.2μCiγ[33P]ATP（比活度≥2500 Ci/mmol）的反应混合物中，然后在室温下孵育60分钟。通过加入20%磷酸停止反应，产物在P30硝化纤维过滤器上捕获，并用Betaplate™计数器检测33P的掺入情况。不使用酶和化合物对照值来确定ZM447439的浓度，该浓度对酶活性的抑制率为50%。

细胞培养[1]
ZM447439溶解在10 mM的DMSO中，在-20°C下储存长达9个月，以避免冻融循环，然后在培养基中新鲜稀释。Aurora A和B的IC50值（约100 nM）在ATP的Km处测定（见上文）。然而，由于细胞ATP浓度高出约200倍，并且ZM447439是ATP的竞争对手，除非另有说明，否则我们在所有细胞检测中都使用浓度为2μM的ZM447439。将DMSO添加到无药物培养物中以考虑溶剂。

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细胞周期分析和克隆分析[1]
如前所述（Taylor和McKeon，1997），使用双胸苷块对TA HeLa细胞在G1/S期进行DNA含量和有丝分裂指数测量以及同步。为了确定克隆效率，将MCF7细胞接种在无酚红的DME加5%剥离血清中，然后用或不用1μM的抗雌激素ICI 182780处理48小时。然后以指定浓度加入ZM44743972小时。收获细胞，洗涤，并在不含ZM447439的完整培养基中，将约400个细胞接种在6孔板的每个孔中。10天后，固定菌落，用结晶紫染色并计数。克隆效率表示与DMSO处理的对照相比，经ZM447439处理的平板上的菌落数量。

[1]. Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores. J Cell Biol. 2003 Apr 28;161(2):267-80.

[2]. ZM 447439 inhibition of aurora kinase induces Hep2 cancer cell apoptosis in three-dimensionalculture. Cell Cycle. 2008 May 15;7(10):1473-9.

ZM447439 is a member of the class of quinazolines that is quinazoline which is substituted at positions 4, 6 and 7 by a (4-benzamidophenyl)nitrilo group, methoxy group and a 3-(morpholin-4-yl)propoxy group, respectively. It is an ATP-competitive inhibitor of Aurora A and Aurora B kinases with IC50 of 110 nM and 130 nM, respectively. It has a role as an Aurora kinase inhibitor, an antineoplastic agent and an apoptosis inducer. It is a member of benzamides, a member of quinazolines, an aromatic ether, a member of morpholines, a polyether, a secondary amino compound and a tertiary amino compound.

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Mitotic Aurora kinases are essential for accurate chromosome segregation during cell division. Forced overexpression of Aurora kinase results in centrosome amplification and multipolar spindles, causing aneuploidy, a hallmark of cancer. ZM447439 (ZM), an Aurora selective ATP-competitive inhibitor, interferes with the spindle integrity checkpoint and chromosome segregation. Here, we showed that inhibition of Aurora kinase by ZM reduced histone H3 phosphorylation at Ser10 in Hep2 carcinoma cells. Multipolar spindles were induced in these ZM-treated G(2)/M-arrested cells with accumulation of 4N/8N DNA, similar to cells with genetically suppressed Aurora-B. Cells subsequently underwent apoptosis, as assessed by cleavage of critical apoptotic associated protein PARP. Hep2 cells formed a tumor-like cell mass in 3-dimensional matrix culture; inhibition of Aurora kinase by ZM either destructed the preformed cell mass or prevented its formation, by inducing apoptotic cell death as stained for cleaved caspase-3. Lastly, ZM inhibition of Aurora kinase was potently in association with decrease of Akt phosphorylation at Ser473 and its substrates GSK3alpha/beta phosphorylation at Ser21 and Ser9. Together, we demonstrated that Aurora kinase served as a potential molecular target of ZM for more selective therapeutic cancer treatment.[2]

C29H31N5O4

513.59

513.237

C, 67.82; H, 6.08; N, 13.64; O, 12.46

331771-20-1

9914412

White to gray solid powder

1.3±0.1 g/cm3

639.7±55.0 °C at 760 mmHg

117-120ºC

340.7±31.5 °C

0.0±1.9 mmHg at 25°C

1.664

2.66

97.84

2

8

10

38

709

0

OGNYUTNQZVRGMN-UHFFFAOYSA-N

InChI=1S/C29H31N5O4/c1-36-26-18-24-25(19-27(26)38-15-5-12-34-13-16-37-17-14-34)30-20-31-28(24)32-22-8-10-23(11-9-22)33-29(35)21-6-3-2-4-7-21/h2-4,6-11,18-20H,5,12-17H2,1H3,(H,33,35)(H,30,31,32)

N-(4-((6-methoxy-7-(3-morpholinopropoxy)quinazolin-4-yl)amino)phenyl)benzamide

ZM-447439; ZM 447439; N-[4-[[6-METHOXY-7-[3-(4-MORPHOLINYL)PROPOXY]-4-QUINAZOLINYL]AMINO]PHENYL]BENZAMIDE; N-(4-((6-methoxy-7-(3-morpholinopropoxy)quinazolin-4-yl)amino)phenyl)benzamide; TCMDC-125873; C29H31N5O4; ZM447439.

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 (4.87 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 25.0 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: ≥ 2.5 mg/mL (4.87 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 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 3 中的溶解度: 30% PEG400+0.5% Tween80+5% propylene glycol:30mg/mL

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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。