CYC116

Aurora A (Ki = 8 nM); Aurora B (Ki = 9.2 nM)

此外，CYC-116 分别以 44、82、280 和 44 nM 的浓度抑制 FLT3、Src、Lck 和 VEGFR2。 CYC-116 可能具有广谱抗癌作用。 MCF7、HeLa、Co 的 IC50 为 0.599、0.59、0.241、0.34、0.725、1.375、0.471、0.034、0.372、0.681、0.151、1.626、0.775、0.308、0.110 和 0.09 lo 205、HCT-116、HT29、K562 、CCRF-CEM、MV4-11、HL60、NCI-H460、A2780、BxPC3、HuPT4、Mia-Paca-2、Saos-2 和 Messa 细胞，CYC-116 对癌细胞系表现出强大的抗增殖活性。用 1.25 μM CYC-116 处理 7 小时后，HeLa 细胞裂解物中的组蛋白 H3 磷酸化被完全抑制[1]。

口服 CYC-116 剂量水平分别为 75 和 100 mg/kg qd，分别导致肿瘤生长延迟 2.3 天和 5.8 天。这些肿瘤生长延迟转化为 0.32 和 0.81 的特定生长延迟。在试验期间，接受两种剂量水平的 CYC-116 的小鼠的平均相对肿瘤体积小于接受赋形剂的小鼠的相对肿瘤体积。在第 6 天和第 9 天，每天口服 100 mg/kg 时，生长下降具有统计学意义[1]。

激酶测定[1]
如前所述进行这些操作。使用XLfit软件（IDBS）测定IC50值。使用Cheng和Prussoff的方法，根据每种激酶的IC50值和适当的Km（ATP）值计算表观抑制常数（Ki）。重组人极光A和B激酶购自Upstate Discovery。使用25μL反应体积（25mMβ-甘油磷酸，20mM Tris/HCl，pH 7.5，5mM EGTA，1mM DTT，1mM Na3VO4，10μg keptide（肽底物））进行Aurora A激酶测定，并将重组Aurora激酶在20mM Tris/HCl中稀释，pH 8，含有0.5mg/mL BSA，2.5%甘油和0.006%Brij-35。通过添加5μL Mg/ATP混合物（15 mM MgCl2，100μM ATP，每孔18.5 kBqγ-32P-ATP）开始反应，并在30°C下孵育30分钟，然后通过添加25μL 75 mM H3PO4终止反应。Aurora B激酶测定与Aurora A相同，只是在使用前，Aurora B在30°C下与内着丝粒蛋白的单独反应中活化60分钟。

流式细胞术的细胞周期分析[1]
为了使早期S期的细胞同步，对它们进行双胸苷阻断。将HeLa细胞以每10 cm培养皿5×105个细胞的速度接种，并在37°C下孵育16−18小时。加入胸苷（2 mM），将细胞孵育18小时。通过在5 mL PBS中洗涤3次，将细胞从块中释放。加入新鲜培养基，将细胞培养8小时。然后再次加入2 mM胸苷，持续第二个16小时。再次释放细胞，并加入新鲜培养基以及适当的测试化合物稀释液
为了使A549细胞在M期同步，将其与40 ng/mL诺可唑（Sigma）孵育18小时。如有说明，还用50μM MG132蛋白酶体抑制剂处理细胞孵育的最后2小时。将通过摇动和重复洗涤从平板上分离的圆形有丝分裂细胞制成丸粒并在PBS中洗涤以从诺可唑嵌段释放。将细胞用合适的测试化合物稀释液在新鲜培养基中重新接种。 结合TUNEL染色或细胞周期蛋白B分析对细胞进行细胞周期分析。因此，收获所有细胞，在PBS中洗涤，用0.5%PFA固定，并在−20°C的80%乙醇中储存。末端脱氧核苷酸转移酶介导的缺口末端标记（TUNEL）测定按照制造商的说明进行。流式细胞术期间的细胞周期蛋白B分析使用在400μL 0.5%BSA PBS中的细胞周期素B抗体的1:1000稀释液，用1:400 FITC山羊多克隆至小鼠二次，然后用PI孵育用于DNA染色。使用BD FACSCalibur流式细胞仪分析每个样品的20000个单细胞事件。

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组蛋白H3磷酸化的蛋白质印迹分析[1]
为了检测磷酸化组蛋白H3，用化合物处理HeLa细胞7小时。然后通过酸提取制备提取物。简言之，将细胞从板上刮下，成丸，在PBS中洗涤一次，然后重悬于含有罗氏完全蛋白酶抑制剂混合物的裂解缓冲液（10mM Tris/HCl，pH 8.0，1.5mM MgCl2，10mM KCl，0.5mM DTT）中。将HCl和H2SO4添加到0.2M的最终浓度，并将裂解物在冰上孵育1小时。通过离心将不溶性材料制成颗粒，将酸溶解的上清液添加到1mL Me2CO中，并在−20°C下储存24小时。通过离心机将沉淀的蛋白质制成颗粒，短暂风干，并重悬于SDS−PAGE负载缓冲液中。样品在15%SDS-聚丙烯酰胺凝胶上分离，并通过电印迹转移到硝化纤维膜上。用兔抗磷酸组蛋白H3抗体在膜上检测磷酸化组蛋白H3.用小鼠抗组蛋白H3:检测总组蛋白H3，然后用适当的二抗和化学发光检测。

Rat Pharmacokinetics. [1]
The PK parameters for test compounds were determined in male Wistar rats. For each compound, 3 rats were dosed either by intravenous bolus injection or by oral gavage. Dose volume was 10 mL/kg for oral gavage administration and 12 mL/kg for intravenous administration (1mL/min). Three serum samples were collected from each rat by jugular vein cannulation at 0, 5, 15, and 30 min, 1, 2, 4, and 6 h following i.v. dosing; and at 0, 0.5, 1, 2, 4, 6, 8, and 24 h after p.o. dosing. All blood samples were centrifuged immediately following collection. The plasma was harvested and stored at –20 °C until analysis. The samples were analyzed by LC-MS/MS methods. The PK parameters were derived by noncompartmental methods using WinNonlin 5.2 software program. The oral bioavailability (% F) was calculated by taking the ratio of dose-normalized AUC values from oral versus i.v. dosing.

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Murine P388/D1 Leukemia Model. [1]
Female Balb/c × DBA/2J F1 mice were implanted intraperitoneally with 2.1 × 105 P388/D1 leukemia cells on day 0. Starting on day 1 the animals were administered compound 18 by oral gavage at the indicated doses (0.1 mL / 10 g body weight) twice a day on days 1–3 and 7–9. The effectiveness of treatment was assessed by comparison of the median post-inoculation lifespan (ILS) of each group of treated mice with that of the vehicle control group. The ratio of ILS values for the treated versus the control groups was expressed as a percentage value (% ILS) and used as an indicator of relative efficacy.

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NCI-H460 Xenograft. [1]
Human NCI-H460 non-small cell lung tumor cells were harvested from sub-confluent cultures grown in vitro and the number of viable cells was determined. Cells were then resuspended in sterile PBS at a concentration of ca. 7 × 107 cells/mL. Nude (athymic) mice were injected subcutaneously in the right flank with approximately 7 × 106 cells. When measurable tumors had established (80-100 mm3), animals were assigned into the treatment and the control groups with 10 mice per group. Tumor size was measured at least twice weekly. Animals were terminated at any time during the study if the tumor size became excessive or any adverse effects were noted. The treatments were administrated orally, by gavage, daily, starting on day 1, and continuing for 5 days. In the control group, animals were treated with the vehicle orally, by gavage, once a day starting on day 1, and continuing for 5 days. The tumor dimensions measured over the period of the study were recorded. Calculations of relative tumor volumes and plots of mean tumor growth curves were S8 performed. The relative tumor volume data from each group were compared using a one-way analysis of variance (ANOVA) and statistical significance was determined using a Dunnett’s t-test.

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Dissolved in DMSO and then diluted in water; 75, 100 mg/kg; Oral gavage

NCI-H460 cells are implanted intraperitoneally into the mice

Protein Binding
CYC116, an orally-available inhibitor of Aurora kinases A and B, and VEGFR2, in patients with advanced solid tumors.

[1]. Discovery of N-phenyl-4-(thiazol-5-yl)pyrimidin-2-amine aurora kinase inhibitors. J Med Chem. 2010 Jun 10;53(11):4367-78.

CYC116 is a novel anticancer compound with a unique target profile involving both cell cycle and angiogenesis inhibition mechanisms. In preclinical studies, CYC116 has demonstrated antitumor activity in both solid tumors and hematological cancers. Cyclacel's small molecule investigational drug, CYC116, is the third orally-available Cyclacel drug to enter development, which demonstrated anticancer activity with a mechanism consistent with inhibition of Aurora kinase.

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Drug Indication
Advanced solid tumors

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Mechanism of Action
Aurora kinases are enzymes that help dividing cells share their materials between two daughter cells. In many people with cancer Aurora kinase malfunctions and normal control of cell division is lost resulting in abnormal growth. CYC116 inhibits Aurora kinase may slow down the growth of cancer cells and lead to their death by apoptosis.

C18H20N6OS

368.46

368.141

C, 58.68; H, 5.47; N, 22.81; O, 4.34; S, 8.70

693228-63-6

6420138

Typically exists as light yellow to yellow solids at room temperature

1.4±0.1 g/cm3

648.8±65.0 °C at 760 mmHg

346.2±34.3 °C

0.0±1.9 mmHg at 25°C

1.689

1.44

118.16

2

8

4

26

443

0

S1C(N([H])[H])=NC(C([H])([H])[H])=C1C1C([H])=C([H])N=C(N=1)N([H])C1C([H])=C([H])C(=C([H])C=1[H])N1C([H])([H])C([H])([H])OC([H])([H])C1([H])[H]

GPSZYOIFQZPWEJ-UHFFFAOYSA-N

InChI=1S/C18H20N6OS/c1-12-16(26-17(19)21-12)15-6-7-20-18(23-15)22-13-2-4-14(5-3-13)24-8-10-25-11-9-24/h2-7H,8-11H2,1H3,(H2,19,21)(H,20,22,23)

4-methyl-5-(2-(4-morpholinophenylamino)pyrimidin-4-yl)thiazol-2-amine

CYC 116; CYC-116; CYC116.

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: 1.5 mg/mL (4.07 mM) 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), 悬浮液；超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 15.0 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: ≥ 1.5 mg/mL (4.07 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in 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 15.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: 1% DMSO+30% polyethylene glycol+1% Tween 80:30mg/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。