AAL-993

VEGFR1 (IC50 = 130 nM); VEGFR2 (IC50 = 23 nM); VEGFR3 (IC50 = 18 nM)

AAL993 通过阻断 ERK 来阻止 HIF-1α 的表达，而不改变 Akt 的磷酸化 [2]。 AAL993抑制HIF-1α蛋白的积累。AAL993对VEGF表达和HIF-1转录活性的抑制。VEGFR抑制剂SU5416和KRN633抑制缺氧诱导的HIF蛋白积累。[2]

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在这项研究中，研究人员表明，AAL993抑制了缺氧诱导的HIF-1α在各种人类癌细胞中的积累，包括HeLa、A431、HCT 15、HCT 116和MCF7细胞，并且AAL993通过下调HeLa细胞中HIF-1α的转录活性来抑制缺氧介导的VEGF表达。AAL993在抑制缺氧诱导的HIF-1α积累和VEGF产生的浓度下降低了HIF-1αmRNA的表达（图5A）。此外，添加蛋白酶体抑制剂MG-132（10μM）并没有导致AAL993介导的HIF-1α积累抑制的恢复（图5C），这表明AAL993对缺氧诱导的HIF-1β积累的抑制是由于抑制了HIF-1α的表达。根据这些结果，推测AAL993对HIF-1αmRNA表达的抑制可能是通过其对上游信号转导的调节来介导的。[2]

AAL993（化合物 5）对植入模型中 VEGF 诱导的血管生成具有有效的抑制作用，ED50 值为 7 mg/kg [1]。
AAL993（24–100 mg/kg；口服；每日；用于14 天）抑制 B16 黑色素瘤异种移植模型中自发外周转移的形成以及原发肿瘤的生长[1]。
AAL993（化合物5）可以在植入模型中显着抑制VEGF诱导的血管生成，ED50值为7 mg/kg[1]。在 B16 黑色素瘤异种移植模型中，AAL993（24-100 mg/kg；口服；每日；持续 14 天）可减少原发性肿瘤生长和自发性外周转移瘤的产生 [1]。
2-[（4-吡啶基）甲基]氨基-N-[3-（三氟甲基）苯基]苯甲酰胺（AAL993（化合物5））和N-3-异喹啉基-2-[（4-吡啶基甲基）氨基]苯甲胺（7）能有效和选择性地抑制重组VEGFR-2和VEGFR-3激酶。由于其物理化学性质，这些邻氨基苯甲酰胺很容易渗透细胞，并在小鼠每天口服一次后被吸收。在植入模型中，5和7均能有效抑制VEGF诱导的血管生成，ED（50）值为7mg/kg。在小鼠黑色素瘤原位模型中，6和7能有效抑制原发性肿瘤的生长和自发外周转移的形成。邻氨基苯甲酰胺5和7代表了一种新的结构类VEGFR激酶抑制剂，具有强效的抗血管生成和抗肿瘤特性。

酶联免疫吸附试验（ELISA）[2]
通过夹心ELISA法测定VEGF的释放量。ELISA板在4°C下用100μl 2μg/ml的抗VEGF165抗体在PBS中包被12小时。用含0.1%吐温20（TPBS）的PBS洗涤平板，并在25°C下与200μl/孔的1%BSA在PBS中孵育1小时。将条件培养基或不同浓度的重组人VEGF在25°℃下孵育2小时，然后用TPBS洗涤四次。在25℃下与100μl 0.2μg/ml的生物素化抗VEGF抗体孵育2小时时，洗涤平板，然后用100μl HRP偶联的链霉抗生物素蛋白孵育45分钟。洗涤后，通过加入50μl四甲基联苯胺对板进行显影，并通过加入50µl 2 N H2SO4停止反应。用96孔板读数器测量450nm处的吸光度。

细胞培养[2]
将人宫颈癌细胞系HeLa、人上皮癌细胞系A431、人结直肠癌细胞系HCT 15和HCT 116以及人乳腺癌细胞系MCF7在37°C、5%CO2气氛下在添加了10%胎牛血清（FBS）、100 U/ml青霉素和100μg/ml链霉素的RPMI 1640培养基中培养。在后续实验中，将细胞以2×105个细胞/ml/孔的密度接种在12孔TC板中，并在37°C下孵育24小时。通过在多气体培养箱中将细胞替换为1%O2、94%N2和5%CO2来实现缺氧状态。在暴露于缺氧之前，细胞与每种药物预孵育1小时。

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免疫印迹[2]
在药物处理指定时间后，用PBS（无钙/镁）洗涤细胞三次，浸入100μl冰冷的裂解缓冲液（20 mM HEPES，pH 7.4，1%Triton X-100，10%甘油，1 mM EDTA，5 mM氟化钠，2.5 mM对硝基苯磷酸酯，10μg/ml苯甲磺酰氟，1 mM钒酸钠和10μg/ml亮肽）中15分钟，用Handy Sonic Disrupter破碎，裂解液在样品缓冲液（50 mM Tris，pH 7.4、4%SDS、10%甘油、4%2-硫代乙醇和0.05 Mg/ml溴酚蓝），比例为4:1。将细胞裂解物进行SDS-聚丙烯酰胺凝胶电泳（PAGE），转移到聚偏二氟乙烯（PVDF）膜上，用抗HIF-1α抗体、抗HIF-1β抗体、抗α-微管蛋白抗体、抗磷酸化ERK抗体、抗ERK抗体，抗磷酸化Akt抗体、抗Akt抗体和抗磷酸化EGFR抗体进行免疫印迹。用辣根过氧化物酶（HRP）偶联的二抗进一步孵育后，用ECL试剂盒处理印迹，用分子成像仪ChemiDoc XRS系统观察蛋白质表达。

Growth Factor Implant Model of Angiogenesis. [1]
Porous Teflon chambers containing VEGF (2 μg/mL) or bFGF (0.3 μg/mL) in 0.8% w/v agar containing heparin (20 U/mL) were implanted subcutaneously in the flank of female mice. Mice were treated orally, with the exception of SU5416 which was administered intraperitoneally, once daily with compound [AAL993 (compound 5)] or vehicle (PEG 300) from 1 day before implantation of the chambers, and the animals were sacrificed for measurement of the vascularized tissues after 5 days of treatment. The chambers were recovered from the animal and the vascularized tissue formed around the implant was carefully removed. Tissue samples were treated with water (2 mL), homogenized (1 min at 24000 rpm), and centrifuged (1 h at 7000 rpm). The supernatant was filtered to avoid fat contamination and the haemoglobin content was determined spectrophotometrically at 540 nm. Haemoglobin measurements were converted into blood volumes using a calibration curve obtained with whole blood samples from a donor mouse. Data were quantified as the percentage inhibition of the increase in blood content with respect to that in vehicle-treated control animals (Table 4). ED50 values were calculated from the dose−response curve, n = number of animals at the specified dose (Figure 1). In cases where full inhibition was not achieved at doses up to 100 mg/kg, results are expressed as percentage inhibition at the highest dose tested.

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Murine Melanoma Model of Tumor Growth and Metastasis. [1]
B16/BL6 cells (5 × 10~4), suspended in Hanks buffer containing 10% FCS, were injected intradermally into the dorsal pinna of both ears of anaesthetized syngeneic C57BL/6 mice. One week later, treatment with either drug substance [AAL993 (compound 5)] or vehicle (PEG 300) was initiated. The size of the primary tumors was monitored under a light microscope, recorded via a low-light color video camera, and quantified with a computerized imaging system. After two weeks of daily treatment, the animals were sacrificed and the cervical lymph nodes collected and weighed. Effects on primary tumor growth are plotted as relative tumor mean area (mm2) quantified on day 14 (A14) and day 21 (A21) compared to the tumor mean area (A7) at the start of drug treatment (day 7 after tumor cell injection). Effects on cervical lymph node metastases were quantified by the relative wet weight (mg) of the collected tissue of animals treated with drug compared to that of vehicle-treated animals.

AAL993 (compound 5) and 7 are stable crystalline solids. Although they have relatively low aqueous solubilities (2 μg/mL) at pH 6.8 in phosphate buffer, as a consequence of the basicity of both compound 5 (pKa 5.2) and compound 7 (pKa 5.1, 2.7), 15 their solubilities markedly increase with decreasing pH (solubility at pH 1.0 > 200 μg/mL). Furthermore, the topological polar surface areas of the compounds (TPSA 45.0 and 52.8 Å2 for 5 and 7, respectively), 16 together with their high permeabilities (passive diffusion) across Caco-2 cell monolayers (intrinsic permeability, Pm = 45.2 and 21.7 × 10-5 cm/min for 5 and 7, respectively), are predictive of intestinal absorption following oral administration.[1]

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Prior to evaluating their effects in vivo, the compounds [e.g. AAL993 (compound 5)] were first tested to ascertain whether they were absorbed following oral administration. Plasma concentrations of drug substance were determined by HPLC/UV in normal mice after a single oral dose of 50 mg/kg, dissolved in a mixture of 5% DMSO and 0.5% Tween 80 in water, and administered by gavage. The pharmacokinetic parameters derived from these data are summarized in Table 3.[1]

[1]. Anthranilic acid amides: a novel class of antiangiogenic VEGF receptor kinase inhibitors. J Med Chem. 2002 Dec 19;45(26):5687-93.

[2]. Suppression of hypoxia-induced HIF-1alpha accumulation by VEGFR inhibitors: Different profiles of AAL993 versus SU5416 and KRN633. Cancer Lett. 2010 Oct 1;296(1):17-26.

The hypoxia-inducible factor (HIF) is a heterodimeric basic helix-loop-helix transcriptional factor and the activated HIF plays pivotal roles in various pathological conditions, including inflammation and cancer. HIF-1alpha overexpression has been observed in many common human cancers, including brain, breast, colon, lung, ovary, and prostate, and HIF-mediated genes, such as vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS), and insulin-like growth factor (IGF)-1, are associated with tumor angiogenesis, metastasis, and invasion. Therefore, the pro-oncogenic protein HIF is a novel target of cancer therapy. We examined the effects of VEGFR inhibitors, AAL993, SU5416, and KRN633, on suppression of HIF-1alpha accumulation under the hypoxic condition. We found that VEGFR tyrosine kinase inhibitors, AAL993, SU5416, and KRN633, possess dual functions: inhibition of VEGFR signaling and HIF-1alpha expression under the hypoxic condition. The detailed mechanistic study indicated that SU5416 and KRN633 suppressed HIF-1alpha expression through inhibition of both Akt and ERK phosphorylation signaling pathways, whereas AAL993 suppressed HIF-1alpha expression through ERK inhibition without affecting Akt phosphorylation.[2]

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Two readily synthesized anthranilamide, VEGF receptor tyrosine kinase inhibitors have been prepared and evaluated as angiogenesis inhibitors. 2-[(4-Pyridyl)methyl]amino-N-[3-(trifluoromethyl)phenyl]benzamide (AAL993 (compound 5)) and N-3-isoquinolinyl-2-[(4-pyridinylmethyl)amino]benzamide (7) potently and selectively inhibit recombinant VEGFR-2 and VEGFR-3 kinases. As a consequence of their physicochemical properties, these anthranilamides readily penetrate cells and are absorbed following once daily oral administration to mice. Both 5 and 7 potently inhibit VEGF-induced angiogenesis in an implant model, with ED(50) values of 7 mg/kg. In a mouse orthotopic model of melanoma, 5 and 7 potently inhibited both the growth of the primary tumor as well as the formation of spontaneous peripheral metastases. The anthranilamides 5 and 7 represent a new structural class of VEGFR kinase inhibitors, which possess potent antiangiogenic and antitumor properties.[1]

C20H16N3OF3

371.35574

371.124

C, 64.69; H, 4.34; F, 15.35; N, 11.32; O, 4.31

269390-77-4

269390-77-4

6398883

White to off-white solid powder

1.3±0.1 g/cm3

441.3±45.0 °C at 760 mmHg

220.7±28.7 °C

0.0±1.1 mmHg at 25°C

1.631

4.51

57.51

2

6

5

27

480

0

0

BLAFVGLBBOPRLP-UHFFFAOYSA-N

InChI=1S/C20H16F3N3O/c21-20(22,23)15-4-3-5-16(12-15)26-19(27)17-6-1-2-7-18(17)25-13-14-8-10-24-11-9-14/h1-12,25H,13H2,(H,26,27)

2-(pyridin-4-ylmethylamino)-N-[3-(trifluoromethyl)phenyl]benzamide

AAL 993; AA-L993; AAL993

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 本产品在运输和储存过程中需避光。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~125 mg/mL (~336.6 mM)

配方 1 中的溶解度: ≥ 2.08 mg/mL (5.60 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 20.8 mg/mL澄清DMSO储备液加入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: ≥ 2.08 mg/mL (5.60 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 20.8 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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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。