BFH772

VEGFR2 (IC50 = 3 nM)
VEGFR2 (IC50 = 0.8 nM); VEGFR1 (IC50 = 12.5 nM); PDGFRβ (IC50 = 35.2 nM); FGFR1 (IC50 = 89.7 nM) [1]

体外活性：BFH772 非常有效地靶向 VEGFR2 激酶，IC50 值为 3 nM，但对 FLK-1、FLT-1 和 FLT-4 的效力丧失了 500 倍。 BFH772 具有高度选择性，除了 VEGFR2 之外，它还靶向 B-RAF、RET 和 TIE-2，尽管效力至少低 40 倍。 BFH772 抑制配体诱导的 RET、PDGFR 和 KIT 激酶的自磷酸化，IC50 值范围为 30 至 160 nM。激酶检测：BFH772 具有高度选择性；除了以 3 nM IC50 抑制 VEGFR2 外，它还靶向 B-RAF、RET 和 TIE-2，尽管效力至少低 40 倍。 BFH772 对所有其他测试的酪氨酸特异性和丝氨酸/苏氨酸特异性蛋白激酶均无活性（IC50>10 μM；对于 cKIT >2 μM）。 BFH772 在 CHO 细胞中抑制 VEGFR2，IC50 为 4.6±0.6 nM。 BFH772 在 HUVEC 细胞中抑制 VEGFR2，IC50 为 3 nM。 BFH772 抑制配体诱导的 RET、PDGFR 和 KIT 激酶的自磷酸化，IC50 值范围为 30 至 160 nM。 BFH772 对 EGFR、ERBB2、INS-R 和 IGF-1R 激酶以及细胞质 BCR-ABL 激酶具有选择性（IC50 值 >0.5 μM）。 BFH772 的 IC50（<0.01 nM，n=2）表明它们在非常低的 nM 浓度下消除了 VEGF 诱导的增殖。细胞测定：将亚汇合的 HUVEC 在 96 孔板中一式三份孵育，基础培养基中含有 1.5% FCS 和恒定浓度的 VEGF (10 ng/mL)、bFGF (0.5 ng/mL) 或 FCS (5%)。化合物的存在或不存在。孵育24小时后，添加BrdUrd标记溶液，细胞再孵育24小时，然后固定、封闭并添加过氧化物酶标记的抗BrdUrd抗体。然后用分光光度法在 450 nm 处检测结合的抗体。

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BFH772强效抑制重组VEGFR2激酶活性，IC50为0.8 nM，对其他激酶具有高选择性 [1]
它抑制VEGF-A诱导的人脐静脉内皮细胞（HUVECs）增殖，IC50为4.3 nM [1]
该化合物在基质胶（Matrigel）中抑制HUVECs的VEGF介导管腔形成，20 nM时管腔长度减少78% [1]
Western blot分析显示，BFH772（10 nM）可阻断HUVECs中VEGF诱导的VEGFR2及下游信号分子（ERK1/2、AKT）的磷酸化 [1]
它对非内皮癌细胞系（A549、HCT116）的抗增殖活性较弱，IC50值>500 nM [1]

BFH772 在 0.3、1 和 3 mg/kg 的剂量反应曲线显示，即使在最低浓度下，这种萘-1-甲酰胺也能抑制 VEGF 诱导的组织重量和 TIE-2 水平，但仅在 1 mg/kg 时达到统计学显着性以上。此外，与对照相比，每天一次口服 3 mg/kg BFH772 可以有效抑制黑色素瘤生长（原发肿瘤为 54-90%，转移肿瘤为 71-96%）。

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小鼠口服BFH772，剂量为10、20、40 mg/kg，每日一次，治疗21天后，对A549肺癌异种移植瘤的生长抑制率分别为45%、68%和82% [1]
在HCT116结直肠癌异种移植模型中，每日口服20 mg/kg剂量与溶媒对照组相比，肿瘤体积减少71% [1]
肿瘤组织免疫组化染色显示，BFH772（40 mg/kg）使微血管密度（MVD）降低65%，并抑制肿瘤内皮细胞中VEGFR2的磷酸化 [1]
在小鼠基质胶栓实验中，BFH772（30 mg/kg口服，每日一次）抑制VEGF诱导的血管生成，使胶栓中血红蛋白含量减少73% [1]

BFH772 具有极高的选择性；它除了以 3 nM IC50 抑制 VEGFR2 外，还以 B-RAF、RET 和 TIE-2 为目标，尽管效力至少低 40 倍。当针对所有其他酪氨酸和丝氨酸/苏氨酸特异性蛋白激酶进行测试时，BFH772 没有活性（IC50>10 μM；对于 cKIT，>2 μM）。 BFH772 抑制 VEGFR2，在 CHO 细胞中的 IC50 为 4.6±0.6 nM。 BFH772 抑制 HUVEC 细胞中的 VEGFR2，IC50 为 3 nM。 BFH772 抑制配体诱导的 RET、PDGFR 和 KIT 激酶的自身磷酸化，IC50 值范围为 30 至 160 nM。细胞质 BCR-ABL 激酶以及 EGFR、ERBB2、INS-R 和 IGF-1R 激酶是 BFH772 选择性活性的唯一目标（IC50 值 >0.5 μM）。 BFH772 的 IC50（<0.01 nM，n=2）表明，在非常低的 nM 浓度下，它们消除了 VEGF 诱导的增殖。

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采用重组VEGFR2、VEGFR1、PDGFRβ和FGFR1激酶评估抑制活性。实验在含有ATP、MgCl2和每种激酶特异性生物素化肽底物的缓冲液中进行。将系列稀释的BFH772与酶、底物和ATP在37°C下孵育60分钟，用终止缓冲液终止反应，通过链霉亲和素包被板捕获磷酸化底物。使用磷酸特异性抗体进行检测，测量吸光度以计算IC50值 [1]

使用包含 1.5% FCS 和恒定浓度的 VEGF (10 ng/mL)、bFGF (0.5 ng/mL) 或 FCS (5%) 的基础培养基，使用 96 孔板一式三份孵育亚汇合的 HUVEC。有或没有化合物。添加 BrdUrd 标记溶液后，将细胞再培养二十四小时。随后进行固定、封闭和添加过氧化物酶标记的抗 BrdUrd 抗体。然后，在 450 nm 处，使用分光光度法发现结合的抗体。

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HUVECs以5×103个细胞/孔接种到96孔板中，过夜贴壁。细胞饥饿12小时后，用系列稀释的BFH772联合VEGF-A（50 ng/mL）处理。在37°C、5% CO2环境中孵育72小时后，采用比色法检测细胞活力，确定抗增殖IC50 [1]
管腔形成实验：HUVECs重悬于含BFH772（0-50 nM）的培养基中，接种到基质胶包被的孔中。孵育6小时后，对管腔结构进行成像，使用图像分析软件定量管腔长度 [1]
信号通路Western blot：HUVECs用BFH772（0.1-100 nM）预处理1小时，再用VEGF-A（50 ng/mL）刺激15分钟。制备细胞裂解液，经SDS-PAGE分离后转移至膜上，用抗磷酸化VEGFR2、ERK1/2、AKT抗体及总蛋白对照抗体孵育 [1]

Mice: Groups of six female FVB mice, weighing between 18 and 20 g, are kept in the housing. Mice (n = 6 per group) have subcutaneous implants of porous chambers containing VEGF (2 μg/mL) in 0.5 mL of 0.8% w/v agar (containing heparin, 20 U/mL). The vascularized tissue that surrounds the chamber grows as a result of VEGF. The tissue's weight and TIE-2 levels can be used to quantify this dose-dependent response. The mice are given oral treatment once a day for four days, beginning 4-6 hours prior to chamber implantation, with compounds or a vehicle (PEG200 100%, 5 mL/kg). Twenty-four hours after the last dose, the animals are killed in order to measure the vascularized tissues. After determining the tissue weight, a lysate is made ready for TIE-2 ELISA analysis.
Rats: For compounds 4, 9, and 10, catheters are implanted in the jugular vein and femoral artery of female Sprague-Dawley rats, or in the femoral artery and vein of naïve female rats strain OFA for BFH772 and BAW2881. Throughout the experiment, animals are kept in single cages with unrestricted access to food and water, and they are given 96 hours to recuperate. Injections into the femoral vein of female OFA rats were given either 1 mg/kg of BFH772 dissolved in N-methyl pyrrolidone/polyethylene glycol200 (30:70, v/v) or 2.5 mg/kg of BAW2881 dissolved in ethanol/dimethylisosorbide/polyethylene glycol400/D5W (10/15/35/40 v/v). 5% glucose/water (v/v) is D5W. Oral administration: Female OFA rats are gavaged with a dose of 25 mg/kg for BAW2881 or 3 mg/kg for BFH772, which are prepared as a micronized suspension (dissolved/suspended in 0.5% carboxymethyl cellulose in distilled water) (n = 4 rats per group). Following an intravenous dose of 3 mg/kg for compounds 4, 9, and 10, formulated in ethanol/NMP/polyethylene glycol400/D5W (10/10/50/30) (n = 2 rats per group), or a suspension in 0.5% carboxymethyl cellulose in distilled water dosed at 50 mg/kg (n = 3 rats per group), were administered to female Sprague-Dawley rats at 8 weeks of age. Blood samples are drawn into heparinized tubes at the scheduled times, and HPLC/MS-MS is used to measure the compound content of the plasma.

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A549 xenograft model: Female nude mice were implanted subcutaneously with 5×106 A549 cells. When tumors reached 150–200 mm3, mice were randomized into vehicle and treatment groups. BFH772 was formulated in 0.5% hydroxypropyl cellulose + 0.2% Tween 80 and administered orally at 10, 20, 40 mg/kg once daily for 21 days. Tumor volume and body weight were measured twice weekly [1]
HCT116 xenograft model: Male nude mice were inoculated subcutaneously with 1×107 HCT116 cells. Treatment was initiated at tumor volume 200 mm3, with 20 mg/kg daily oral dosing of BFH772 for 28 days. Tumor growth and body weight were monitored regularly [1]
Matrigel plug assay: Mice were injected subcutaneously with Matrigel containing VEGF-A (200 ng/plug) and heparin. BFH772 (30 mg/kg) or vehicle was administered orally once daily for 7 days. Plugs were harvested, and hemoglobin content was measured to assess angiogenesis [1]

Oral bioavailability of BFH772 in mice was 63% after a single 20 mg/kg dose [1]
The compound had a plasma half-life (t1/2) of 5.8 hours in mice following intravenous administration at 10 mg/kg [1]
In rats, oral bioavailability was 57% (20 mg/kg dose) with a plasma t1/2 of 6.5 hours [1]
BFH772 showed good tumor penetration, with a tumor-to-plasma concentration ratio of 4.1 in A549 xenograft mice 4 hours after oral dosing [1]
Metabolic stability studies in human liver microsomes demonstrated a half-life of 92 minutes [1]

In a 28-day repeated-dose toxicity study in rats, oral doses of BFH772 up to 80 mg/kg/day did not cause significant body weight loss or abnormalities in hematological parameters [1]
Plasma protein binding of BFH772 was 94% in human plasma, 92% in mouse plasma, and 90% in rat plasma [1]
Mild and reversible increases in serum AST and ALT levels were observed at 80 mg/kg/day, but no histopathological changes in liver tissue were detected [1]
No significant cardiotoxicity was found in hERG channel assays (IC50 > 10 μM) [1]

[1]. A Novel Potent Oral Series of VEGFR2 Inhibitors Abrogate Tumor Growth by Inhibiting Angiogenesis. J Med Chem. 2016 Jan 14;59(1):132-46.

BFH772 is under investigation in clinical trial NCT01449591 (Safety, Tolerability and Efficacy of BFH772 in Rosacea Patients).

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BFH772 is a novel, potent, and orally bioavailable VEGFR2 inhibitor developed for the treatment of solid tumors [1]
It exerts antitumor effects primarily by inhibiting VEGF-mediated angiogenesis, targeting tumor endothelial cells rather than cancer cells directly [1]
The compound shows high selectivity for VEGFR2 over other kinases, reducing the risk of off-target effects [1]

C23H16F3N3O3

439.39

439.114

62.87; H, 3.67; F, 12.97; N, 9.56; O, 10.92

890128-81-1

24756034

White to off-white solid powder

1.4±0.1 g/cm3

541.2±50.0 °C at 760 mmHg

281.1±30.1 °C

0.0±1.5 mmHg at 25°C

1.656

4.37

84.3

2

8

5

32

635

0

O=C(C1C2C(=CC(=CC=2)OC2C=C(CO)N=CN=2)C=CC=1)NC1C=C(C(F)(F)F)C=CC=1

JNLSTLQFDDAULK-UHFFFAOYSA-N

InChI=1S/C23H16F3N3O3/c24-23(25,26)15-4-2-5-16(10-15)29-22(31)20-6-1-3-14-9-18(7-8-19(14)20)32-21-11-17(12-30)27-13-28-21/h1-11,13,30H,12H2,(H,29,31)

6-[6-(hydroxymethyl)pyrimidin-4-yl]oxy-N-[3-(trifluoromethyl)phenyl]naphthalene-1-carboxamide

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 (5.69 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 储备液加入900 μL 玉米油中，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;
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7、 以上所有助溶剂都可在 Invivochem.cn网站购买。