HER1 (IC50 = 20 nM); HER2 (IC50 = 30 nM); HER4 (IC50 = 190 nM)

BMS-599626 is administered orally to a human breast tumor KPL-4 xenograft at a maximum tolerated dose of 180 mg/kg. This results in a dose-dependent inhibition of Sal2 tumor growth in vivo, and the drug exhibits potent antitumor activity. It also exhibits similar antitumor activity in other HER2 amplified xenograft models and other HER1-overexpressing xenograft models.[1]

In Sf9 insect cells, the full cytoplasmic sequences of HER1, HER2, and HER4 are expressed as recombinant proteins. Glutathione-S-transferase is used to express HER1 and HER4, which are then purified using affinity chromatography on glutathione-S-Sepharose. The pBlueBac4 vector is used to subclone HER2, which is then expressed as an untagged protein with the help of an internal methionine codon (M687) that initiates translation. By using chromatography on a column of DEAE-Sepharose that has been equilibrated in a buffer containing 0.1 M NaCl, the truncated HER2 protein is isolated, and the recombinant protein is eluted using a buffer containing 0.3 M NaCl. Reaction volumes for the HER kinase tests are 50 μL, and the contents include 150 ng of partially purified HER2 or 10 ng of glutathione-S-transferase fusion protein. The mixture additionally contains 50 mM Tris-HCl (pH 7.7), 2 mM DTT, 0.1 mg/mL bovine serum albumin, 10 mM MnCl₂, 0.15 μCi [γ-³³P]ATP, 1 μM ATP, and 1.5 μM poly(Glu/Tyr) (4:1). The reactions are allowed to continue for one hour at 27°C before being stopped with the addition of 10 μL of a stop buffer (2.5 mg/mL bovine serum albumin and 0.3 M EDTA) and 108 μL of a mixture consisting of 5% trichloroacetic acid and 3.5 mM ATP. Using a Filtermate harvester, acid-insoluble proteins are recovered on GF/C Unifilter plates. Liquid scintillation counting is used to determine whether radioactive phosphate has been incorporated into the poly(Glu/Tyr) substrate. Nonlinear regression analyses are used to calculate the percent inhibition of kinase activity. The data are presented as the inhibitory concentration needed to achieve 50% inhibition in comparison to control reactions (IC50). The averages of three duplicate determinations are called data. Poly(Glu/Tyr) is used as a substrate in the assay of all other tyrosine kinases. The inhibition kinetics of HER1 and HER2 are ascertained in reaction mixtures comprising different quantities of BMS-599626 and ATP.

In RPMI 1640 supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin, all cell lines are kept alive. Prior to adding BMS-599626, cells are plated in 96-well plates at a density of 1,000 per well and cultured for a full day. BMS-599626 is diluted in culture medium until the final DMSO concentration is less than one percent. The cells are cultured for an additional 72 hours after the addition of BMS-599626, and the CellTiter96 kit is used to measure the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye in order to determine the viability of the cells. Thymidine uptake assay is used to measure the proliferation of cell lines for which there is no correlation between the number of cells and the metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye. The aforementioned compounds are applied to cells after they have been plated in 96-well plates. After the 72-hour incubation period, cells are harvested after being pulsed with [3H]thymidine (0.4 μCi/well) for three hours. Following ten minutes of 2.5% trypsin digestion at 37 °C, the cells are collected by filtration through the use of GF/C Unifilter plates and a Packard Filtermate Harvester. Liquid scintillation counting is used to measure the amount of radioactive thymidine incorporated into nucleic acids.

Athymic female nude mice are used to maintain and pass on SAL2 murine salivary gland tumor, N87 human gastric carcinoma, BT474 human breast tumor, A549 human non-small-cell lung tumor, and GEO human colon tumor.
≤240 mg/kg
Administered via p.o.

[1]. Clin Cancer Res . 2006 Oct 15;12(20 Pt 1):6186-93.

[2]. Invest New Drugs . 2011 Aug;29(4):554-61.

[3]. Mol Cancer Ther . 2008 Sep;7(9):2589-98.

C27H27FN8O3

530.55

530.22

C, 61.12; H, 5.13; F, 3.58; N, 21.12; O, 9.05

714971-09-2

BMS-599626 Hydrochloride;873837-23-1

Solid powder

CC1=C2C(=NC=NN2C=C1NC(=O)OC[C@@H]3COCCN3)NC4=CC5=C(C=C4)N(N=C5)CC6=CC(=CC=C6)F

LUJZZYWHBDHDQX-QFIPXVFZSA-N

InChI=1S/C27H27FN8O3/c1-17-23(34-27(37)39-15-22-14-38-8-7-29-22)13-36-25(17)26(30-16-32-36)33-21-5-6-24-19(10-21)11-31-35(24)12-18-3-2-4-20(28)9-18/h2-6,9-11,13,16,22,29H,7-8,12,14-15H2,1H3,(H,34,37)(H,30,32,33)/t22-/m0/s1

[(3S)-morpholin-3-yl]methyl N-[4-[[1-[(3-fluorophenyl)methyl]indazol-5-yl]amino]-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yl]carbamate

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)