PDGFRα (IC50 = 10 nM); PDGFRβ (IC50 = 1 nM)

CP 673451 (once-daily p.o.?0 days dosing regularly) inhibits tumor growth (ED50 < 33 mg/kg) in several human tumor xenografts grown s.c. in athymic mice, such as U87MG human glioblastoma multiforme, Colo205 and LS174T human colon carcinomas, and H460 human lung carcinoma.[1] CP 673451 inhibits tumor growth and stromal cell infiltration in RUCH2 xenograft-bearing mice.[2]

Sf-9 cells (baculovirus expression system) express a glutathione S-transferase-tagged kinase domain construct of the intracellular portion of PDGFR-β (amino acids 693-1401, accession no. J03278). The phosphorylation buffer [50 mmol/L HEPES (pH 7.3), 125 mmol/L NaCl, 24 mmol/L MgCl₂ in Nunc Immuno MaxiSorp 96-well plates previously coated with 100 μL of 100 μg/mL poly-Glu-Tyr (4:1 ratio) diluted in PBS] is used to incubate the enzyme at increasing concentrations of ATP to determine the enzyme's kinetics. The plates are incubated with anti-phosphotyrosine-horseradish peroxidase antibody for 10 minutes, followed by a 30-minute room-temperature diluting step in PBS, 0.05% Tween 20, and 3% BSA. After the previously mentioned washing, the plates are incubated with 3,3',5,5'-tetramethylbenzidine. By adding an equal volume of 0.09 NaH₂SO₄, the reaction is terminated. Next, the phosphotyrosine-dependent signal is measured at 450 nm using a plate reader. For standard enzyme tests, the enzyme is incubated for 30 minutes at room temperature with 10 μM (final) ATP in the presence of a compound diluted in DMSO (1.6% v/v DMSO assay final) in the previously mentioned plates coated with 100 μL of 6.25 μg/mL poly-Glu-Tyr. The assay is completed as previously described, and IC50 values are computed as a percentage of control's inhibition.

It is possible to generate PAE cells that express full-length PDGFR and VEGFR consistently. Full-length human PDGFR-a, PDGFR-h, or VEGFR-2 are transfected into PAE cells in order to perform cell-based selectivity assays. 50 μL of growth medium (Ham's F-12 media supplemented with 10% fetal bovine serum, 50,000 units of penicillin and streptomycin, and 500 μg/mL gentamicin) is used per well in 96-well plates to seed cells at a density of 4×10³ cells/mL. After 6 to 8 hours, the cells are incubated overnight in 50 μL of serum-depleted medium (as above, but with 0.1% fetal bovine serum) in place of the growth medium. 95 μL of serum-depleted medium was added to the medium just before the compound was added. The compounds are added to the cells at a final DMSO concentration of 0.25% v/v after being diluted in 100% DMSO, and they are then incubated for 10 minutes at 37°C. The proper ligand is used to stimulate the cells, and they are then incubated for a further eight minutes. The medium is taken out, and the cells are rinsed once with PBS before being lysed for five minutes at room temperature in 50 μL HNTG buffer (20 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 2% Triton X-100, 10% glycerol, 5 μmol/L EDTA, 2 mmol/L NaVO₄, and 1 EDTA-free complete protease inhibitor tablet per 25 mL). 50 μL of HG buffer (20 mmol/L HEPES (pH 7.5), 10% glycerol)] is then added to the lysates to dilute them. Once the diluted cell lysates are well combined, 50 μL of the supernatant is added to the ELISA capture plate and it is incubated for two hours at room temperature while being stirred. Coating 96-well ReactiBind goat-antirabbit plates with 100 μL/well of rabbit anti-human PDGFR-h, anti-PDGFR-a, or anti-VEGFR-2 antibody for 60 to 90 minutes is the process used to prepare ELISA capture plates. Following a 2-hour incubation period, the plates are cleaned in PBS with 0.1% Tween 20 and then incubated for 30 minutes at room temperature with a diluted anti-phosphotyrosine-horseradish peroxidase antibody (PBS with 0.05% Tween 20). The plates are once more cleaned, tetramethylbenzidine is added, and they are assessed as previously mentioned. Percentage inhibition of control is used to compute IC50 values.

The anticancer efficacy of CP-673451 is assessed in nude mice using a subcutaneous A549 xenograft model. In short, mice are given 2×10⁶ cells/mouse of A549 cells injected into their axillary regions. Upon reaching 70 mm³, the mice are randomized into two groups: one for control and the other for CP-673451 (n = 6 per group) at low and high doses (20 mg/kg and 40 mg/kg, respectively) (vehicle 10% 1-methyl-2-pyrrolidinone and 90% polyethylene glycol 300). CP-673451 (20 or 40 mg/kg/day) or a vehicle is injected intraperitoneally into these animals. The implanted tumors are measured blindly once a day with a calliper during the course of treatment. Simultaneously, the animal body weights are measured. Tumor volume is computed. Following therapy, the tumors are removed and examined, and the mice are euthanized.

[1]. Cancer Res . 2005 Feb 1;65(3):957-66.

[2]. Cancer Res . 2013 Apr 1;73(7):2139-49.

1-[2-[5-(2-methoxyethoxy)-1-benzimidazolyl]-8-quinolinyl]-4-piperidinamine is an aminoquinoline.

C24H27N5O2

417.5

417.216

C, 69.04; H, 6.52; N, 16.77; O, 7.66

343787-29-1

10158940

White to off-white solid powder

1.3±0.1 g/cm3

656.3±65.0 °C at 760 mmHg

350.7±34.3 °C

0.0±2.0 mmHg at 25°C

1.677

2.77

1

6

6

31

572

0

O(C([H])([H])C([H])([H])OC([H])([H])[H])C1C([H])=C([H])C2=C(C=1[H])N=C([H])N2C1C([H])=C([H])C2C([H])=C([H])C([H])=C(C=2N=1)N1C([H])([H])C([H])([H])C([H])(C([H])([H])C1([H])[H])N([H])[H]

DEEOXSOLTLIWMG-UHFFFAOYSA-N

InChI=1S/C24H27N5O2/c1-30-13-14-31-19-6-7-21-20(15-19)26-16-29(21)23-8-5-17-3-2-4-22(24(17)27-23)28-11-9-18(25)10-12-28/h2-8,15-16,18H,9-14,25H2,1H3

1-[2-[5-(2-methoxyethoxy)benzimidazol-1-yl]quinolin-8-yl]piperidin-4-amine

CP-673451; CP673451; CP 673451; CP-673,451; CP 673,451

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)

Solubility in Formulation 1: ≥ 2.75 mg/mL (6.59 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 27.5 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: ≥ 2.75 mg/mL (6.59 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 27.5 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: 30% PEG 400+5% propylene glycol+1% Tween 80+ddH2O: 30mg/mL

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