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)

In recombinant enzyme tests, PF-562271 (VS-6062) besylate is a 30- to 120-nM (15.2 to 60.1 ng/mL) inhibitor of cdk2/E, cdk5/p35, cdk1/B, and cdk3/E[1]. PF-562,271 inhibits the formation of new blood vessels in response to bFGF in chicken chorioallantoic membrane assays[2]. It has been shown that PF-562,271 treatment or siRNA-mediated knockdown of FAK strengthens cell-cell adhesions[3]. In recombinant enzyme assays, PF-562271 (VS-6062) is demonstrated to be a 30- to 120-nM inhibitor of CDK2/E, CDK5/p35, CDK1/B, and CDK3/E. In cell-based assays assessing the function of CDKs, 3.3 μM PF-562271 must be exposed for 48 hours in order to modify cell cycle progression. With an IC50 of 5 nM, PF-562271 demonstrates potency in an inducible cell-based assay for detecting phospho-FAK. On cell growth and colony formation in Ewing sarcoma cell lines, PF-562271, a selective inhibitor of both FAK and proline-rich tyrosine kinase 2 (PYK2), a member of the FAK-related family, acts. Using 2-fold serial dilutions, seven cell lines are treated with PF-562271 for five days at various concentrations. After three days of treatment, PF-562271 therapy reduces cell viability in all cell lines, with an average IC50 of 2.4 μM. The two most sensitive cell lines are TC32 and A673, with IC50 values of 2.1 and 1.7 μM, respectively[2].

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Cancer cells are characterized by the ability to grow in an anchorage-independent manner. The activity of the nonreceptor tyrosine kinase, focal adhesion kinase (FAK), is thought to contribute to this phenotype. FAK localizes in focal adhesion plaques and has a role as a scaffolding and signaling protein for other adhesion molecules. Recent studies show a strong correlation between increased FAK expression and phosphorylation status and the invasive phenotype of aggressive human tumors. PF-562271 is a potent, ATP-competitive, reversible inhibitor of FAK and Pyk2 catalytic activity with a IC(50) of 1.5 and 14 nmol/L, respectively. Additionally, PF-562,271 displayed robust inhibition in an inducible cell-based assay measuring phospho-FAK with an IC(50) of 5 nmol/L. PF-562,271 was evaluated against multiple kinases and displays >100x selectivity against a long list of nontarget kinases. [1]

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In this study, we have shown that FAK is highly phosphorylated in primary Ewing sarcoma tumor samples and that downregulation of FAK by short hairpin RNA and treatment with a FAK-selective kinase inhibitor, PF-562271, impaired growth and colony formation in Ewing sarcoma cell lines. Moreover, treatment of Ewing sarcoma cell lines with PF-562271 induced apoptosis and led to downregulation of AKT/mTOR and CAS activity. Finally, we showed that small-molecule inhibition of FAK attenuated Ewing sarcoma tumor growth in vivo. With FAK inhibitors currently in early-phase clinical trials for adult malignancies, these findings may bear immediate relevance to patients with Ewing sarcoma.[2]

In tumor-bearing mice, PF-562,271 (33 mg/kg, po) reduces FAK phosphorylation in tumors in a way that is dependent on both dose and time. PF-562,271's total blood concentration is inhibited by FAK phosphorylation, resulting in an estimated EC50 of 93 ng/mL. On day three, treated tumors treated with PF-562,271 (25 mg/kg, po) exhibit a 2-fold increase in apoptosis in comparison to control tumors treated with vehicle[1]. Dasatinib and PF-562,271 (33 mg/kg, po) significantly restrict the ability of tumor cells to migrate within the mice. E-cadherin dynamics are changed in vivo when PF-562,271 therapy inhibits FAK kinase activity[3]. Following po treatment to tumor-bearing animals, PF-562271 suppresses FAK phosphorylation in vivo in a dose-dependent manner (calculated EC50 of 93 ng/mL, total)[1]. After two weeks of treatment, rats given PF-562271 show a decrease in tumor development and indications of bone healing, as shown by the deposition of new bone (cortical and cancellous) at tumor-damaged sites[3].

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PF-562,271 inhibits FAK phosphorylation in vivo in a dose-dependent fashion (calculated EC(50) of 93 ng/mL, total) after p.o. administration to tumor-bearing mice. In vivo inhibition of FAK phosphorylation (>50%) was sustained for >4 hours with a single p.o. dose of 33 mg/kg. Antitumor efficacy and regressions were observed in multiple human s.c. xenograft models. No weight loss, morbidity, or mortality were observed in any in vivo experiment. Tumor growth inhibition was dose and drug exposure dependent. Taken together, these data show that kinase inhibition with an ATP-competitive small molecule inhibitor of FAK decreases the phospho-status in vivo, resulting in robust antitumor activity.[1]

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Researchers tested whether the inhibition of FAK activity by treatment with PF-562271 could inhibit progression in established tumors using 2 xenograft models of Ewing sarcoma. NCr nude and NSG mice were subcutaneously injected with A673 and TC32 cells, respectively, and allowed to develop measurable tumors. The animals were then treated with either vehicle or PF-562271 until the animals were sacrificed. Treatment with PF-562271 significantly inhibited tumor growth compared with the controls showing that FAK activity contributes to tumor growth in Ewing sarcoma. [2]

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The compound was well tolerated. Both compound-treated groups demonstrated significant and similar increases in osteocalcin and cancellous bone parameters. Radiographic evaluation of tumor-bearing tibiae revealed tumor expansion in nontreated rats compared with a decrease in tumor growth and signs of bone healing in rats treated with PF-562271. Tartrate-resistant acid phosphatase and fluorescent in situ hybridization analysis revealed that the majority of bone resorption at the tumor site was performed by osteoclasts of rat origin. Conclusions: The oral administration of PF-562,271 at a dose of 5 mg/kg suppressed the growth and local spread of intratibial tumors and restored tumor-induced bone loss. The unique ability of PF-562,271 to both curb tumor growth and safely increase bone formation may be an effective therapy for many cancer patients with bone metastases and cancer-associated osteoporosis [3].

Recombinant kinase assay and enzyme kinetics. [1]
All in vitro assays used for identification of a FAK inhibitor have been previously described (31). Briefly, purified-activated FAK kinase domain (amino acid 410–689) was reacted with 50 μmol/L ATP and 10 μg per well of a random peptide polymer of Glu and Tyr, p(Glu/Tyr), in kinase buffer [50 mmol/L HEPES (pH 7.5), 125 mmol/L NaCl, and 48 mmol/L MgCl2] for 15 min. Phosphorylation of p(Glu/Tyr) was challenged with serially diluted compound at 1/2-Log concentrations starting at a top concentration of 1 μmol/L. Each concentration was tested in triplicate. Phosphorylation of p(Glu/Tyr) was detected with a general antiphospho-tyrosine (PY20) antibody followed by horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody. HRP substrate was added, and absorbance readings at 450 nm were obtained after addition of stop solution (2 mol/L H2SO4). IC50 values were determined using the Hill-Slope Model. Broad kinase selectivity profiling was performed in house and by using the KinaseProfiler Selectivity Screening Service available through UpState Biotechnology.2

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Cell-based FAK phosphorylation assay. [1]
Stable A431 epithelial carcinoma clones were generated to express either wild-type V5-tagged FAK protein or mutant FAKY397F V5-tagged protein under the inducible regulation of mifepristone. Stable clones were grown in DMEM 10% fetal bovine serum, 750 μg/mL Zeocin, and 50 μg/mL Hygromycin. One day before running the FAK cell ELISA, A431 FAK wild-type cells were seeded in growth medium in 96-well U-bottomed plates. After 4 to 6 h at 37°C, 5% CO2, FAK expression was induced with 0.1 nmol/L mifepristone. Uninduced controls were included. Anti-V5– or anti-FAK–coated plates were blocked in 3% bovine serum albumin (BSA)/0.5%Tween for 1 h at room temperature. Cells were treated with 1/2-Log serial dilutions starting at a top concentration of 1 μmol/L for 30 min at 37°C, 5% CO2. Lysates from cells treated with indicated concentrations of compound were prepared in lysis buffer [50 mmol/L Tris-HCl (pH 7.4), 1% NP40, 0.25% Na-deoxycholate, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L Na3VO4, 1 mmol/L NaF, and protease inhibitors] and transferred to the anti-V5– or anti-FAK–coated plates to capture total induced or total FAK protein. Antiphosphospecific FAKY397 was used to detect autophosphorylated FAKY397 followed by secondary reporter antibody. HRP substrate was added, and plates were read at 450 nmol/L. IC50 values were determined using the Hill-Slope Model. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were performed to determine compound cytotoxicity.

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High-throughput kinase activity profiling [1]
A Luminex immunosandwich assay was conducted on 6 Ewing sarcoma cell lines (EWS502, TC71, TC32, A673, SKNEP, and EW8) and 293FT cells, as previously described. Briefly, whole-cell lysates from each cell line were quantified, and equal concentrations of protein were incubated overnight at 4°C with a mixture of 87 validated antibody-coupled Luminex beads (probes) specific for 62 tyrosine kinases. The mixture was then washed and incubated with biotin-labeled 4G10 antibody for 30 minutes at room temperature, washed, and then incubated with 4 μg/mL of SAPE for 10 minutes at room temperature. The conjugates were washed 2 additional times and analyzed on a FlexMAP 3D (Luminex) with xPONENT software (version 4.0; Luminex) to determine the median fluorescent intensity (MFI).

Small-molecule treatment in vitro [2]
Ewing sarcoma cells were plated in 10-cm dishes, allowed to adhere for 24 hours, and then treated with PF-562271 (FAK/PYK2 inhibitor, PD0325901, or dasatinib (SRC, BCR/ABL, c-Kit inhibitor).

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Cell viability [2]
ATP content was measured as a surrogate for cell number using the CellTiter-Glo Luminescent Cell Viability Assay. Luminescence readings were obtained using the FLUOstar Omega microplate reader. For experiments with small-molecule treatment, 1.25 × 103 Ewing sarcoma cells were seeded in each well and treated with a range of concentrations. IC50 values were calculated from ATP measurements obtained after 3 days of treatment using log-transformed, normalized data in GraphPad Prism 5.0. Cell lines were also treated with compound in 6-cm dishes, trypsinized, and counted by light microscopy using trypan blue exclusion. For experiments using shRNA-transduced cells, 1.25 × 103 cells were seeded per well into 384-well plates on day 3 posttransduction. ATP content was measured on days 3, 6, and 8 posttransduction.

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Colony formation in methylcellulose matrix [2]
Approximately 3.75 × 103 cells were dissolved into 1.5 mL of methylcellulose matrix, plated into gridded 6-cm plates, and incubated for at least 10 days. Colonies from 100 squares were counted using a Nikon inverted microscope.

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Flow cytometry [2]
Cells undergoing apoptosis were identified by flow cytometry using the ApoAlert Annexin V-FITC Apoptosis Kit. For intracellular phospho-protein staining, cells were fixed and permeabilized using the BD Cytofix/Cytoperm Kit and stained with phycoerythrin (PE) anti-phospho-S6 and analyzed by flow cytometry.
PF-562,271-01 is a potent ATP-competitive, reversible inhibitor of recombinant FAK and Pyk2 kinase with an IC50 of 1.5 and 14 nM, respectively. PF-562271 was formulated for oral dosing using 0.5% methyl-cellulose. On the first day of dosing, rats received a single dose of PF-562,271 (10 mg/kg) by oral gavage. Based on the exposure levels at 1 hour after dosing, the dose was reduced to 5 mg/kg. From the second day onward, rats were dosed daily with 5 mg/kg by oral gavage for 28 days. Dosing was initiated 2 weeks after tumor inoculation and only after the presence of tumors was confirmed by radiography. The presence of the tested compound in serum was confirmed during the course of the study [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

PC-3M, BT474, BxPc3, LoVo, U87MG, H125 and H460 cells are injected s.c. into the right flank of athymic female mice

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.

FAK Inhibitor PF-00562271 is an orally bioavailable small molecule and ATP-competitive focal adhesion kinase (FAK) inhibitor with potential antineoplastic and antiangiogenic activities. FAK inhibitor PF-00562271 inhibits the tyrosine kinase FAK, and to a lesser extent, proline-rich tyrosine kinase (PYK2), which may inhibit tumor cell migration, proliferation, and survival. As FAK is a signal transducer for integrins, inhibition of FAK by this agent may prevent integrin-mediated activation of several downstream signals including ERK, JNK/MAPK and PI3K/Akt. FAK and PYK2, upregulated in many tumor cell types, are involved in tumor cell invasion, migration and proliferation.
See also: PF-562271 (annotation moved to).

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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]

C21H20F3N7O3S.C6H6O3S

665.66

665.133

C, 48.72; H, 3.94; F, 8.56; N, 14.73; O, 14.42; S, 9.63

939791-38-5

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

16118986

Off-white to light yellow solid powder

6.515

4

15

8

45

1040

0

CS(=O)(N(C)C1=NC=CC=C1CNC2=NC(NC3=CC4=C(NC(C4)=O)C=C3)=NC=C2C(F)(F)F)=O.O=S(C5=CC=CC=C5)(O)=O

LKLWTLXTOVZFAE-UHFFFAOYSA-N

InChI=1S/C21H20F3N7O3S.C6H6O3S/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;7-10(8,9)6-4-2-1-3-5-6/h3-8,11H,9-10H2,1-2H3,(H,29,32)(H2,26,27,28,30);1-5H,(H,7,8,9)

N-methyl-N-(3-(((2-((2-oxoindolin-5-yl)amino)-5-(trifluoromethyl)pyrimidin-4-yl)amino)methyl)pyridin-2-yl)methanesulfonamide benzenesulfonate

PF-562271; PF-562271 Besylate; PF562271; 939791-38-5; PF-00562,271; PF-562,271 besylate; PF-562,271 (besylate); N-methyl-N-(3-(((2-((2-oxoindolin-5-yl)amino)-5-(trifluoromethyl)pyrimidin-4-yl)amino)methyl)pyridin-2-yl)methanesulfonamide benzenesulfonate; PF-00562271; PF00562271; PF 00562271; PF562271 PhSO3H; PF562271 benzesulfonate salt; PF-271; PF271; PF271

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

Solubility in Formulation 1: ≥ 1.67 mg/mL (2.51 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1.67 mg/mL (2.51 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 1.67 mg/mL (2.51 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 4% DMSO+30% PEG 300+ddH2O:3 mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)