FAK (IC50 = 1.5 nM); Pyk2 (IC50 = 13 nM); CDK2 (IC50 = 30 nM);CDK3 (IC50 = 47 nM); CDK1 (IC50 = 58 nM); CDK7 (IC50 = 97 nM); FLT3 (IC50 = 97 nM)

在涉及重组酶的研究中，PF-562271 (VS-6062) 盐酸盐已证明对 CDK2/E、CDK5/p35、CDK1/B 和 CDK3/E 具有 30 至 120 nM 的抑制作用。然而，在涉及细胞的测定中，必须暴露 3.3 μM PF-562271 48 小时才能改变细胞周期的进程。 PF-562271 的 IC50 为 5 nM，在基于诱导细胞的检测磷酸-FAK 的检测中显示出效力。 PF -562271，富含脯氨酸酪氨酸激酶 2 (PYK2) 的选择性抑制剂，FAK 家族成员，也是 FAK 的选择性抑制剂，对尤文肉瘤细胞系中集落的形成和细胞增殖有影响。使用 2 倍连续稀释，用 PF-562271 以不同剂量处理 7 个细胞系五天。治疗三天后，PF-562271 疗法降低了所有细胞系的细胞活力，平均 IC50 为 2.4 μM。两种最敏感的细胞系是 TC32 和 A673，IC50 值分别为 2.1 和 1.7 μM[2]。

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癌症细胞的特征是能够以不依赖于凤尾鱼的方式生长。非受体酪氨酸激酶，粘着斑激酶（FAK）的活性被认为是导致这种表型的原因。FAK定位于局灶性粘附斑块，并作为其他粘附分子的支架和信号蛋白发挥作用。最近的研究表明，FAK表达和磷酸化状态的增加与侵袭性人类肿瘤的侵袭表型之间存在很强的相关性PF-562271是一种强效、ATP竞争性、可逆的FAK和Pyk2催化活性抑制剂，IC（50）分别为1.5和14 nmol/L。此外，PF-562271在基于诱导细胞的检测磷酸化FAK的方法中显示出强烈的抑制作用，IC（50）为5 nmol/L。PF-562261对多种激酶进行了评估，对一长串非靶激酶显示出>100倍的选择性。[1]

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在这项研究中，我们发现FAK在原发性尤文肉瘤肿瘤样本中高度磷酸化，短发夹RNA下调FAK和用FAK选择性激酶抑制剂PF-562271治疗会损害尤文肉瘤细胞系的生长和集落形成。此外，PF-562271处理尤因肉瘤细胞系诱导细胞凋亡，并导致AKT/mTOR和CAS活性下调。最后，我们发现FAK的小分子抑制可以减轻尤文肉瘤在体内的生长。FAK抑制剂目前正处于成人恶性肿瘤的早期临床试验阶段，这些发现可能与尤文肉瘤患者有直接相关性。[2]

当腹膜内给予荷瘤动物时，PF-562271 以剂量依赖性方式降低体内 FAK 磷酸化（计算得出的 EC50 为 93 ng/mL，总计）。当给大鼠施用 PF-562271 两周后，肿瘤生长减慢，并出现骨修复迹象，例如在肿瘤受损部位沉积新的皮质骨和松质骨[3]。 [3]。

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PF-562271以剂量依赖的方式抑制体内FAK磷酸化（计算EC（50）为93ng/mL，总），口服给药后给荷瘤小鼠。单次口服剂量为33mg/kg时，FAK磷酸化的体内抑制（>50%）持续>4小时。在多种人皮下异种移植物模型中观察到抗肿瘤疗效和消退。在任何体内实验中都没有观察到体重减轻、发病率或死亡率。肿瘤生长抑制呈剂量和药物暴露依赖性。综上所述，这些数据表明，使用ATP竞争性FAK小分子抑制剂抑制激酶可以降低体内的磷酸化状态，从而产生强大的抗肿瘤活性。[1]

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研究人员使用2种尤文肉瘤异种移植物模型测试了用PF-562271治疗对FAK活性的抑制是否可以抑制已建立肿瘤的进展。NCr裸小鼠和NSG小鼠分别皮下注射A673和TC32细胞，并使其产生可测量的肿瘤。然后用载体或PF-562271治疗动物，直至处死动物。与对照组相比，PF-562271治疗显著抑制了肿瘤生长，表明FAK活性有助于尤文肉瘤的肿瘤生长。[2]

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该化合物耐受性良好。两个化合物治疗组的骨钙素和松质骨参数均显示出显著且相似的增加。对带有肿瘤的胫骨的放射学评估显示，与用PF-562271治疗的大鼠相比，未治疗大鼠的肿瘤扩张，肿瘤生长和骨愈合迹象减少。抗酒石酸酸性磷酸酶和荧光原位杂交分析显示，肿瘤部位的大部分骨吸收是由大鼠来源的破骨细胞进行的。 结论：口服5mg/kg剂量的PF-562271抑制了胫骨内肿瘤的生长和局部扩散，并恢复了肿瘤引起的骨丢失。PF-562271抑制肿瘤生长和安全增加骨形成的独特能力可能是许多癌症骨转移和癌症相关骨质疏松症患者的有效治疗[3]。

重组激酶测定和酶动力学。[1]
之前已经描述了用于鉴定FAK抑制剂的所有体外试验（31）。简而言之，纯化的活化FAK激酶结构域（氨基酸410-689）与50μmol/L ATP和每孔10μg的Glu和Tyr的随机肽聚合物p（Glu/Tyr）在激酶缓冲液[50 mmol/L HEPES（pH 7.5）、125 mmol/L NaCl和48 mmol/L MgCl2]中反应15分钟。从1μmol/L的最高浓度开始，用1/2-Log浓度的连续稀释化合物挑战p（Glu/Tir）的磷酸化。每种浓度测试三次。用一般的抗磷酸酪氨酸（PY20）抗体检测p（Glu/Tyr）的磷酸化，然后用辣根过氧化物酶（HRP）偶联的山羊抗小鼠IgG抗体检测。加入HRP底物，在加入终止溶液（2mol/L H2SO4）后获得450nm处的吸光度读数。使用Hill Slope模型测定IC50值。宽激酶选择性分析在内部进行，并使用UpState Biotechnology提供的KinaseProfiler选择性筛选服务。2

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基于细胞的FAK磷酸化测定。[1]
在米非司酮的诱导调节下，生成了稳定的A431上皮癌克隆，以表达野生型V5标记的FAK蛋白或突变型FAKY397F V5标记的蛋白。稳定的克隆在DMEM 10%胎牛血清、750μg/mL Zeocin和50μg/mL潮霉素中生长。在运行FAK细胞ELISA的前一天，将A431 FAK野生型细胞接种在96孔U形底板的生长培养基中。在37°C、5%CO2下4至6小时后，用0.1 nmol/L米非司酮诱导FAK表达。包括非诱导控制。在室温下，将抗-V5或抗FAK涂层板在3%牛血清白蛋白（BSA）/0.5%吐温中封闭1小时。细胞在37°C、5%CO2下以1μmol/L的最高浓度用1/2-Log系列稀释液处理30分钟。在裂解缓冲液[50 mmol/L Tris-HCl（pH 7.4）、1%NP40、0.25%脱氧胆酸钠、150 mmol/L NaCl、1 mmol/L EDTA、1 mmol/L Na3VO4、1 mmol/L氟化钠和蛋白酶抑制剂]中制备用指定浓度化合物处理的细胞的裂解物，并将其转移到抗V5或抗FAK涂层板上，以捕获总诱导或总FAK蛋白。用抗磷酸特异性FAKY397检测自身磷酸化的FAKY397s，然后检测二级报告抗体。加入HRP底物，在450nmol/L下读取平板读数。使用Hill Slope模型测定IC50值。进行3-（4,5-二甲基噻唑-2-基）-2,5-二苯基溴化四氮唑测定以确定化合物的细胞毒性。

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高通量激酶活性分析[1]
如前所述，对6种尤文肉瘤细胞系（EWS502、TC71、TC32、A673、SKNEP和EW8）和293FT细胞进行了Luminex免疫夹心测定。简而言之，对每个细胞系的全细胞裂解物进行定量，并在4°C下将等浓度的蛋白质与87个经验证的抗体偶联的Luminex珠（探针）的混合物一起孵育过夜，这些珠对62种酪氨酸激酶具有特异性。然后洗涤混合物，在室温下用生物素标记的4G10抗体孵育30分钟，洗涤，然后在室温下与4μg/mL的SAPE孵育10分钟。将偶联物再洗涤2次，并在FlexMAP 3D 上用xPONENT软件（版本4.0；Luminez）进行分析，以确定中值荧光强度（MFI）。

体外小分子治疗[2]
将尤文肉瘤细胞铺在10cm培养皿中，使其粘附24小时，然后用PF-562271（FAK/PYK2抑制剂、PD0325901或达沙替尼（SRC、BCR/ABL、c-Kit抑制剂）处理。

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细胞活力[2]
使用CellTiter Glo发光细胞活力测定法测量ATP含量作为细胞数量的替代指标。使用FLUOstar Omega微孔板读数仪获得发光读数。对于小分子治疗的实验，将1.25×103个尤文氏肉瘤细胞接种在每个孔中，并用不同浓度进行处理。使用GraphPad Prism 5.0中的对数转换归一化数据，从治疗3天后获得的ATP测量值计算IC50值。细胞系也在6-cm培养皿中用化合物处理，胰蛋白酶处理，并使用台盼蓝排斥法通过光学显微镜计数。对于使用shRNA转导细胞的实验，在转导后第3天，每孔将1.25×103个细胞接种到384孔板中。在转导后第3、6和8天测量ATP含量。

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甲基纤维素基质中的菌落形成[2]
将约3.75×103个细胞溶解在1.5 mL甲基纤维素基质中，铺在6 cm网格板上，孵育至少10天。使用尼康倒置显微镜对100个正方形的菌落进行计数。

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流式细胞术[2]
使用ApoAlert Annexin V-FITC凋亡试剂盒通过流式细胞术鉴定经历凋亡的细胞。对于细胞内磷蛋白染色，使用BD Cytofix/Cytoperm试剂盒固定和渗透细胞，用藻红蛋白（PE）抗磷酸-S6染色，并通过流式细胞术进行分析
PF-562271-01是重组FAK和Pyk2激酶的强效ATP竞争性可逆抑制剂，IC50分别为1.5和14 nMPF-562271使用0.5%甲基纤维素配制用于口服给药。在给药的第一天，大鼠通过经口灌胃接受单剂量PF-562271（10mg/kg）。根据给药后1小时的暴露水平，剂量降至5mg/kg。从第二天开始，大鼠每天口服5mg/kg，持续28天。在肿瘤接种后2周开始给药，并且只有在射线照相证实肿瘤存在后才开始给药。在研究过程中证实了血清中受试化合物的存在[3]。

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Ex vivo ELISA. [1]
Female athymic mice were injected with 1 × 106 U87MG human glioblastoma cells on day 1. On day 9, when tumors were ∼300 mm3, the mice received compound or vehicle (5% Gelucire 44/14 in sterile water; Gattefossé) p.o. For pharmacokinetic/pharmacodynamic (PK/PD) analysis, blood and tumor samples were collected from each animal (n > 4 mice per group per time point) into heparinized vacutainers and liquid N2, respectively, at the indicated times postdose. Blood and tumors were harvested for evaluation of drug levels and tumor-associated phospho-FAK and total FAK. Plasma concentrations of PF-562271 were determined using reverse phase high performance liquid chromatography with mass spectrometric (MS/MS) detection. Tumors were homogenized in 1 mL lysis buffer per 200-mg tumor [lysis buffer: 50 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 1.5 mmol/L MgCl2, 1 mmol/L EDTA, 1% Glycerol, 1% Triton X-100, 1.6 mmol/L Na3VO4, 10 mmol/L NaF, 25 mg/L Soy Bean Trypsin Inhibitor, EDTA-free complete Protease Inhibitor Tablets], spun 5 min at 14,000 rpm, and the supernatant were aliquoted to 96-well polypropylene plates on dry ice. Total protein concentration was determined using BSA protein assay (Pierce). Ninety-six–well goat-anti-rabbit ReactiBind plates (Pierce) were blocked with 100 μL per well cold blocking buffer (TBS, 0.1% Tween 20, and 3% BSA) for 60 min on a plate shaker at room temperature. The blocking buffer was replaced with 0.5 μg anti-pFAK397 in 100 μL cold blocking buffer per well and incubated for 60 min at room temperature with agitation. Plates were washed with TBS-T (TBS, 0.1% Tween 20) before addition of tumor lysate (500 μg total protein in lysis buffer without protease inhibitors) and incubated 2 h at room temperature with agitation. Captured pFAK was detected with anti-FAK Ab and then incubated with 15 ng HRP–anti-IgG per well (in blocking buffer) for 30 min at room temperature. The plates were washed as above and phosphotyrosine quantitated using 3, 3′, 5, 5′-tetramethylbenzidine as described above. All phosho-FAK inhibition data are analyzed by comparing PF-562,271–treated tumors to vehicle-treated tumors.

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Prediction of efficacious concentration. [1]
Blood and tumor samples were collected at each time point postadministration of PF-562271 for determination of blood drug concentration and FAK phosphotyrosine reduction. The relationship between compound concentration and FAK phosphotyrosine reduction has been explored in pharmacologic models (tumor-bearing athymic mice) with pooled experimental data from multiple individual studies. FAK phosphotyrosine reduction correlates well with blood concentrations of PF-562,271 in athymic mice and follows a simple Emax pharmacodynamic model:

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Tumor growth inhibition studies. [1]
Exponentially growing cells were trypsinized and resuspended in sterile PBS and inoculated s.c. (1 × 106 cells per mouse in 200 μL) into the right flank of mice. Animals bearing tumors of ∼150 mm3 in size were divided into groups receiving either vehicle (5% Gelucire) or PF-562271 (diluted in vehicle), and dosed by p.o. gavage. Animal body weight and tumor measurements were obtained every 2 d. Tumor volume (mm3) was measured with Vernier calipers and calculated using the formula: length (mm) × width (mm) × width (mm) × 0.5. Percent growth inhibition was calculated as previously described. For all tumor growth inhibition experiments, 8 to 10 mice per dose group were used. A Student's t test was used to determine the P value.

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Microscopy and immunohistochemistry of tumor apoptosis and microvascular density. [1]
Mice bearing H125 tumors were treated for 3 d with PF-562271 (12–50 mg/kg, twice daily, p.o.). For microvascular density analysis, mice bearing U87MG tumors were used and treated for 3 d with a C4 sulfone derivative of PF-562,271 (50 mg/kg, daily, p.o.). After treatment, tumors were excised and quick frozen in OCT medium. Seven-micrometer sections were cut and processed for immunohistochemical detection of apoptosis using a commercially available terminal deoxynucleotidyl-transferase–mediated dUTP nick-end (TUNEL) kit or rat antimurine endothelial MECA32 Ab. Tissue sections were counter stained with hematoxylin or methyl green and examined using a Zeiss Axiophot microscope at ×20 with a reticule grid. All discreet, positively stained apoptotic cells or vascular profiles, with or without lumina, were counted in 10 (×200) fields from multiple sections of each tumor. Fields were randomly chosen throughout the entire section. For each time point and dose, four mice were evaluated. A Student's t test was used to determine the P value.

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Approximately 4.2 × 106 A673 cells were mixed with 30% Matrigel and injected subcutaneously into 8-week-old female NCr nude mice. When the average tumor volume in all animals reached approximately 100 mm3, twice-daily treatment was administered by oral gavage with 200 mg/kg PF-562271 (in a volume of 10 ml/kg) or vehicle control (0.5% hydroxypropyl methylcellulose and 0.2% Tween 80 in sterile water; n = 6 per condition). Ten-week-old male NSG mice were used for in vivo experiments with TC32 cells to improve tumor engraftment with this cell line. In this model, several mice experienced toxicity when treated with PF-562271 at 200 mg/kg twice daily (data not shown). Therefore, after injection of approximately 5 × 106 TC32 cells in 30% Matrigel, animals began treatment with PF-562271 at 100 mg/kg or vehicle control (n = 8 per condition) every 12 hours when tumor volumes reached 100 mm3. This dose was well tolerated, and at day 13, the dose was escalated to 150 mg/kg. Tumor volumes were measured with calipers. Animals were sacrificed when tumor volumes exceeded 2,000 mm3.[2]

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In Vivo Radiography[3]
Radiography of the right hind limb was taken once every 2 weeks using a Faxitron digital capture X-ray at Kv 26, magnification of 1.5, and 2-second exposure. Rats were anesthetized for the X-ray procedure with isoflurane. Tumor growth was not quantified by X-ray, but tumor progression was estimated by serial images. Three reviewers, 2 of whom were blinded to the treatment, reviewed serial images and made conclusions regarding the effect of PF-562,271 on tumor progression.[3]

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Dissolved in in 5% Gelucire; 100 mg/kg; Oral gavage

Athymic female mice bearing BxPc3 or PC3-M xenografts.

The pharmacokinetics, inhibition of phosphorylated FAK, and antitumor efficacy of PF-562,271 was evaluated in the following human s.c. xenograft models: PC-3M (prostate), BT474 (breast), BxPc3 (pancreatic), LoVo (colon), U87MG (glioblastoma), and H125 and H460 (lung; Table 2). Dose-dependent tumor growth inhibition was observed in all models. Maximum tumor inhibition for PC-3M, BT474, BxPc3, and LoVo ranged from 78% to 94% inhibition for the group with regressions in up to 50% of a given dose group (Table 2). Regressions were observed in PC-3M, BT474, BxPc3, and LoVo models at doses of 25 to 50 mg/kg twice daily, corresponding to Cmax (free) ranges of 78 to 885 ng/mL, Cave (free) of 14 to 40 ng/mL, and inhibition of phospho-FAK of 31% to 76% for >4 hours. No weight loss, morbidity, or death was observed in any tumor growth inhibition (TGI) experiment (up to 50 mg/kg twice daily × 29 days or 100 mg/kg daily × 25 days). All data are based on 6 to 10 animals per dose, and experiments were completed at least twice. After dosing, animals were euthanized, blood and tumor were analyzed for drug concentration (PK), and tumors were evaluated for phospho-FAK (PD). [1]

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A rigorous in vivo PK/PD evaluation was completed for PF-562,271. The compound is well-absorbed with maximal blood levels occurring between 30 minutes and 2 hours after p.o. administration. Maximal pharmacodynamic modulation occurs simultaneously with maximal pharmacokinetic exposure in the blood regardless of dose or number of repeated doses. Measured PK is accurately modeled using in vitro and in vivo calculation of absorption, distribution, metabolism, and excretion, demonstrating a well-behaved and predictable in vivo pharmacology. [1]

[1]. Antitumor activity and pharmacology of a selective focal adhesion kinase inhibitor, PF-562,271. Cancer Res, 2008, 68(6), 1935-1944.

[2]. High-throughput tyrosine kinase activity profiling identifies FAK as a candidate therapeutic target in Ewing sarcoma. Cancer Res. 2013 May 1;73(9):2873-83.

[3]. Dual focal adhesion kinase/Pyk2 inhibitor has positive effects on bone tumors: implications for bone metastases. Cancer. 2008 May 15;112(10):2313-21.

N-methyl-N-[3-[[[2-[(2-oxo-1,3-dihydroindol-5-yl)amino]-5-(trifluoromethyl)-4-pyrimidinyl]amino]methyl]-2-pyridinyl]methanesulfonamide is a member of indoles.

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Broadly speaking, pharmacologic anticancer therapy consists of cytotoxics and targeted agents (small molecules and biologics). Unfortunately, the majority of patients treated with these agents eventually progress. In addition, toxicities associated with these agents often preclude adequate treatment. In this report, the in vivo pharmacology of a highly selective and potent inhibitor of FAK catalytic activity, PF-562,271, is described. The novel mode of inhibition of FAK, characterized by the inhibitor-induced “DFG helix,” resulted in profound antitumor activity across a wide variety of tumor types while being well-tolerated. PF-562,271 showed well-behaved pharmacology in vivo with a robust PK/PD relationship. PF-562,271 shows the selectivity and pharmacology that has allowed it to be a first in class inhibitor presently in clinical testing for the treatment of cancer.[1]

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In conclusion, the results of the current study demonstrate that the oral administration of PF-562,271 at a dose of 5 mg/kg suppressed the growth and local spread of intratibial tumors and also restored tumor-induced bone loss. These unique properties of PF-562,271, namely the ability to curb tumor growth and safely increase bone formation, could be effectively used in many cancer patients with bone metastases and cancer-associated osteoporosis. Finally, this class of drugs has the potential to be used effectively in combination with other anticancer therapies as well as with bisphosphonates to prevent and treat bone metastases.[3]

C21H21CLF3N7O3S

543.95

543.106

C, 46.37; H, 3.89; Cl, 6.52; F, 10.48; N, 18.03; O, 8.82; S, 5.89

939791-41-0

PF-562271;717907-75-0; 939791-38-5 (besylate); 939791-39-6 (mesylate); 939791-41-0 (HCl); 939791-40-9

16222312

Typically exists as solid at room temperature

138

4

12

7

36

856

0

Cl.O=C1CC2C(=CC=C(NC3N=C(NCC4C(N(S(C)(=O)=O)C)=NC=CC=4)C(C(F)(F)F)=CN=3)C=2)N1

RQEBZJWSAAWCAV-UHFFFAOYSA-N

InChI=1S/C21H20F3N7O3S.ClH/c1-31(35(2,33)34)19-12(4-3-7-25-19)10-26-18-15(21(22,23)24)11-27-20(30-18)28-14-5-6-16-13(8-14)9-17(32)29-16;/h3-8,11H,9-10H2,1-2H3,(H,29,32)(H2,26,27,28,30);1H

N-methyl-N-[3-[[[2-[(2-oxo-1,3-dihydroindol-5-yl)amino]-5-(trifluoromethyl)pyrimidin-4-yl]amino]methyl]pyridin-2-yl]methanesulfonamide;hydrochloride

PF562271 HCl; PF-562271 HCl; 939791-41-0; PF-562,271 (hydrochloride); Methanesulfonamide,N-[3-[[[2-[(2,3-dihydro-2-oxo-1H-indol-5-yl)amino]-5-(trifluoromethyl)-4-pyrimidinyl]amino]methyl]-2-pyridinyl]-N-methyl-,hydrochloride(1:1); 939791-41-0 (HCl); PF-00562271; PF562271 hydrochloride; PF 562271; PF562271; PF-562271; PF00562271; PF 00562271; PF00562271 hydrochloride. PF271; PF-271; PF 271;

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)