Tie2 kinase inhibitor   中文名称: Tie2激酶抑制剂

Tie2 (IC50 = 250 nM)

体外活性：Tie2 激酶抑制剂对 Tie2 酪氨酸激酶表现出中等的抑制活性。 Tie2 激酶抑制剂在 HEL 细胞中也显示出中等的细胞活性，IC50 为 232 nM。此外，Tie2 激酶抑制剂对 Tie2 的选择性优于 p38 (IC50=50 μM)，并且对 VEGFR2、VEGFR3 和 PDGFR1β 的选择性高 >10 倍。激酶测定：打开恒温摇床并将温度调节至 30 °C。向 Flashplate 每孔添加 20 μL 3× 激酶缓冲液（最终 20 mM Tris-HCl，pH 7，100 mM NaCl，12 mM MgCl2，1 mM DTT）。除背景外，每孔添加 20 μL 蛋白质。添加 Tie2 激酶抑制剂，通常在 DMSO 库存中添加 1 ~ 2 μL。每孔添加 20 μL γ 33p-ATP 和冷 ATP (1:1 v/v) 的混合物。表面覆盖透明聚酯薄膜。 30℃摇床孵育2小时，洗涤5次。在 TopCount 或其他计数仪器上读取板，并使用正常方法将结果计算为 IC50 值。细胞测定：在 HEL 细胞中，Tie2 Kinase Inhibitor 中度抑制 Tie2 酪氨酸激酶的活性，IC50 值为 232 NM。

在血管生成的 Matrigel 小鼠模型中，Tie2 激酶抑制剂剂量为 25 和 50 mg/kg（ip，bid）时，分别导致血管生成减少 41% 和 70%。在 MOPC-315 浆细胞瘤异种移植模型中，Tie2 激酶抑制剂治疗会导致肿瘤生长出现适度的剂量依赖性延迟。

打开培养箱摇床并将温度调节至 30°C。每孔向 Flashplate 添加 20 μL 3× 激酶缓冲液（最终 20 mM Tris-HCl，pH 7，100 mM NaCl，12 mM MgCl2，1 mM DTT）。除背景外，每孔添加 20 μL 蛋白质。额外的 Tie2 激酶抑制剂，通常为 DMSO 库存中的 1–2 µL。对于每个孔，添加 20 μL 1:1 v/v 的冷 ATP 和 γ 33p-ATP 组合。使用由半透明聚酯制成的薄膜来覆盖。洗涤 5 次，并在 30°C 摇床上孵育 2 小时。在 TopCount 或其他计数设备上读取板后，使用标准方法计算 IC50 值。

免疫印迹分析[2]
HUVEC、SVR和MS1-VEGF细胞生长到约70%的合流度。细胞在冷PBS中洗涤2次，蛋白在冷NP-40裂解缓冲液中分离（50 mM HEPES （pH 7.4）、150 mM NaCl、1% NP-40、0.5%脱氧胆酸、10%甘油、2.5 mM EGTA、1 mM EDTA、1 mM二硫苏糖醇、1 mM苯甲磺酰氟、1 mM Na3VO4、20 mM β -甘油磷酸、10µg/ml胰肽和抑肽蛋白）。使用标准Bradford吸光度法定量蛋白质。蛋白质在4% ~ 20%梯度的SDS-PAGE凝胶上分离，电泳转移到聚偏二氟乙烯膜上。随后用5%脱脂牛奶或牛血清白蛋白阻断膜，溶液为1 mM Tris， pH 8.0, 150 mM NaCl, 0.5% Tween 20。Western blot检测VEGFR2的一抗(1:1000；Santa Cruz Biotechnology, Inc, Santa Cruz， CA)或Tie2在4°C下放置过夜。二抗为山羊抗兔免疫球蛋白G (IgG) HRP (Tie2, 1:4000)或山羊抗小鼠IgG-HRP （VEGFR-2, 1:4000）。通过增强型化学发光检测系统检测蛋白质。

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细胞存活测定[2]
将SVR细胞和MS1-VEGF细胞以每孔1500个细胞的速度涂于96孔板中，留置过夜。抽吸培养基，并用含有不同浓度舒尼替尼、Tie2激酶抑制剂或DMSO对照物的新鲜培养基代替。DMSO终浓度为0.1%。让细胞生长72小时，根据制造商的说明使用WST-1细胞增殖试剂定量细胞存活。同样，在固定的Tie2激酶抑制剂与舒尼替尼的摩尔比为25:1（以反映相对药物效力），总药物浓度为26至7800 nM时，进行联合治疗的细胞存活试验。

MOPC-315 plasmacytoma xenograft model.
≤50 mg
Administered via i.p.

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In Vivo Studies [2]
Athymic nude mice (6- to 8-week-old females) were obtained from Harlan Laboratories, Inc (Indianapolis, IN). The care and treatment of experimental animals were in accordance with institutional guidelines. SVR and MS1-VEGF cells (2 x 106 cells) were implanted subcutaneously. For histopathologic confirmation of the angiosarcoma phenotype, SVR tumors were excised at days 3, 6, 9, and 12 after injection, and MS1-VEGF tumors were excised at days 5, 10, 15, and 20 after injection. For tumor growth delay studies, treatment was started when the tumor volume reached 200 mm3 (SVR) or when palpable (MS1-VEGF). Animals were randomized into four treatment groups. The control group and the Tie2 kinase inhibitor alone group were treated daily with 100 µl of vehicle (0.5% carboxymethylcellulose, 0.4% Tween 80, 1.8% NaCl, and 0.9% benzyl alcohol in distilled water, pH 6.0) by oral gavage. The sunitinib-alone and combination treatment groups were treated daily with 100 µl of vehicle containing sunitinib (60 mg/kg for SVR tumors, 30 mg/kg for MS1-VEGF tumors) by oral gavage. The control and sunitinib-alone groups were treated twice weekly with 100 µl of vehicle (5% ethanol, 5% Cremophor, and 90% distilled water) through intraperitoneal injection. The Tie2 kinase inhibitor-alone and combination treatment groups were injected twice weekly with 100 µl of vehicle containing Tie2 kinase inhibitor (50 mg/kg). Tumor volume was determined by direct caliper measurement and calculated by the following formula: volume = 0.5 x (large diameter) x (small diameter)2. For necrosis, apoptosis, and proliferation studies in vivo, SVR tumor-bearing animals (n = 4–6 per group) were randomized and treated as above for 11 days. On day 12, animals were killed, and tumors were harvested for histologic and immunohistochemical analyses. Percent tumor necrosis was estimated by a pathologist (D.B.) blinded to treatment using hematoxylin and eosin-stained whole tumor sections. The number of cleaved caspase 3 and PCNA-positive cells per high-power field (n = 5/tumor) was quantified by an observer blinded to treatment.

[1]. Pyridinylimidazole inhibitors of Tie2 kinase. Bioorg Med Chem Lett. 2007 Sep 1;17(17):4756-60. Epub 2007 Jun 27.

[2]. Efficacy of Tie2 receptor antagonism in angiosarcoma. Neoplasia. 2012 Feb;14(2):131-40.

4-[4-(6-methoxy-2-naphthalenyl)-2-(4-methylsulfinylphenyl)-1H-imidazol-5-yl]pyridine is a member of imidazoles.

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This communication details the evolution of the screening lead SB-203580, a known CSBP/p38 kinase inhibitor, into a potent and selective Tie2 tyrosine kinase inhibitor. The optimized compound 5 showed efficacy in an in vivo model of angiogenesis and a MOPC-315 plasmacytoma xenograft model. [1]

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Angiosarcomas are malignant endothelial cell tumors with few effective systemic treatments. Despite a unique endothelial origin, molecular candidates for targeted therapeutic intervention have been elusive. In this study, we explored the tunica internal endothelial cell kinase 2 (Tie2) receptor as a potential therapeutic target in angiosarcoma. Human angiosarcomas from diverse sites were shown to be universally immunoreactive for Tie2. Tie2 and vascular endothelial growth factor receptor (VEGFR) antagonists inhibited SVR and MS1-VEGF angiosarcoma cell survival in vitro. In the high-grade SVR cell line, Tie2 and VEGF antagonists inhibited cell survival synergistically, whereas effects were largely additive in the low-grade MS1-VEGF cell line. Xenograft modeling using these cell lines closely recapitulated the human disease. In vivo, Tie2 and VEGFR inhibition resulted in significant angiosarcoma growth delay. The combination proved more effective than either agent alone. Tie2 inhibition seemed to elicit tumor growth delay through increased tumor cell apoptosis, whereas VEGFR inhibition reduced tumor growth by lowering tumor cell proliferation. These data identify Tie2 antagonism as a potential novel, targeted therapy for angiosarcomas and provide a foundation for further investigation of Tie2 inhibition, alone and in combinations, in the management of this disease. [2]

C26H21N3O2S

439.53

439.135

C, 71.05; H, 4.82; N, 9.56; O, 7.28; S, 7.29

948557-43-5

23625762

Light yellow to yellow solid powder

1.4±0.1 g/cm3

699.8±55.0 °C at 760 mmHg

377.0±31.5 °C

0.0±2.1 mmHg at 25°C

1.746

5.31

87.08

1

5

5

32

635

0

O=S(C)C1C=CC(C2NC(C3C=C4C(C=C(C=C4)OC)=CC=3)=C(C3C=CN=CC=3)N=2)=CC=1

SINQIEAULQKUPD-UHFFFAOYSA-N

InChI=1S/C26H21N3O2S/c1-31-22-8-5-19-15-21(4-3-20(19)16-22)25-24(17-11-13-27-14-12-17)28-26(29-25)18-6-9-23(10-7-18)32(2)30/h3-16H,1-2H3,(H,28,29)

4-[4-(6-methoxynaphthalen-2-yl)-2-(4-methylsulfinylphenyl)-1H-imidazol-5-yl]pyridine

Tie2-inhibitor-5; Tie2IN5; 948557-43-5; Tie2 kinase inhibitor; Tie2 kinase-IN-1; Tie2 kinase inhibitor 1; Tie2-IN-5; 4-(4-(6-methoxynaphthalen-2-yl)-2-(4-(methylsulfinyl)phenyl)-1H-imidazol-5-yl)pyridine; CHEMBL237352; 4-[4-(6-methoxynaphthalen-2-yl)-2-(4-methylsulfinylphenyl)-1H-imidazol-5-yl]pyridine; Tie2-IN-5; Tie2 kinase inhibitor 5; Tie2 inhibitor 5; Compound 5; Tie2 IN 5

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: ≥ 1.79 mg/mL (4.07 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 17.9 mg/mL的澄清DMSO储备液加入到400 μL PEG300中并混合均匀；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: ≥ 1.79 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 17.9 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: 2% Cremophor EL, 2% N,N-dimethylacetamide: 30 mg/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；
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7、 以上所有助溶剂都可在 Invivochem.cn网站购买。