BMS 599626 2HCl (873837-23-1(HCl)) (AC480 dihydrochloride) is a selective inhibitor of HER1 and HER2 (IC50s: 20 nM and 30 nM), ~8-fold less potent to HER4, >100-fold to Lck, VEGFR2, c-Kit, MET, etc.

HER2:30 nM (cell free), HER1:20 nM (cell free), HER3:190 nM (cell free)

BMS-599626在抑制HER1和HER2活性方面展现出了显著效果，IC50分别为20和30 nmol/L，并且在针对广泛多样蛋白激酶的测试中显示出高度选择性。该化合物能够有效中断HER1和HER2的信号传导，并抑制依赖这些受体的肿瘤细胞系的增殖，IC50范围在0.24到1 micromol/L之间。BMS-599626对依赖HER1/HER2的肿瘤细胞具有高度选择性，并且对不表达这些受体的细胞系的增殖无影响。在能够形成HER1/HER2异二聚体的肿瘤细胞中，BMS-599626抑制了异二聚体形成及其下游信号传导[1]。在10 μM浓度下，BMS-599626对控制组A2780肿瘤细胞未显示出显著的非靶向效应[2]。在HN-5细胞中，BMS-599626抑制了pEGFR、pHER2、cyclins D和E、pRb、pAkt、pMAPK、pCDK1和2、CDK 6以及Ku70蛋白的表达，并导致细胞在G1周期阶段的积累、抑制了细胞生长，并增强了放射敏感性[3]。

在60 mg/kg的剂量下，BMS-599626显著延缓了治疗过程中的肿瘤生长，但一旦停止治疗，肿瘤生长即恢复。更高剂量的BMS-599626能够更持久地抑制肿瘤生长，但所有情况下，停止治疗后肿瘤生长仍会恢复。在每日一次给药方案中，连续给药14天，BMS-599626的最大耐受剂量为240 mg/kg [1]。

The entire cytoplasmic sequences of HER1, HER2, and HER4 were expressed as recombinant proteins in Sf9 insect cells. HER1 and HER4 were expressed as fusion proteins with glutathione-S-transferase and were purified by affinity chromatography on glutathione-S-Sepharose. HER2 was subcloned into the pBlueBac4 vector and expressed as an untagged protein using an internal methionine codon for translation initiation. The truncated HER2 protein was isolated by chromatography on a column of DEAE-Sepharose equilibrated in a buffer that contained 0.1 mol/L NaCl, and the recombinant protein was eluted with a buffer containing 0.3 mol/L NaCl. For the HER kinase assays, reaction volumes were 50 μL and contained 10 ng of glutathione-S-transferase fusion protein or 150 ng of partially purified HER2. The mixtures also contained 1.5 μmol/L poly(Glu/Tyr) (4:1), 1 μmol/L ATP, 0.15 μCi [γ-33P]ATP, 50 mmol/L Tris-HCl (pH 7.7), 2 mmol/L DTT, 0.1 mg/mL bovine serum albumin, and 10 mmol/L MnCl2. Reactions were allowed to proceed at 27°C for 1 hour and were terminated by the addition of 10 μL of a stop buffer (2.5 mg/mL bovine serum albumin and 0.3 mol/L EDTA), followed by a 108-μL mixture of 3.5 mmol/L ATP and 5% trichloroacetic acid. Acid-insoluble proteins were recovered on GF/C Unifilter plates with a Filtermate harvester. Incorporation of radioactive phosphate into the poly(Glu/Tyr) substrate was determined by liquid scintillation counting. Percent inhibition of kinase activity was determined by nonlinear regression analyses and data were reported as the inhibitory concentration required to achieve 50% inhibition relative to control reactions (IC50). Data are the averages of triplicate determinations. All other tyrosine kinases were also assayed using poly(Glu/Tyr) as a substrate [1].

All cell lines were maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin. Cells were plated at 1,000 per well in 96-well plates and were cultured for 24 hours before test compounds were added. Compounds were diluted in culture medium such that the final concentration of DMSO never exceeded 1%. Following the addition of compounds, the cells were cultured for an additional 72 hours before cell viability was determined by measuring the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye with the CellTiter96 kit. For some cell lines, there was a lack of a correlation between 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye metabolism and cell number, and a thymidine uptake assay was used to measure proliferation of these cell lines. Cells were plated in 96-well plates and treated with compounds as above. At the end of the 72-hour incubation, cells were pulsed with [3H]thymidine (0.4 μCi/well) for 3 hours before they were harvested. Cells were digested with 2.5% trypsin for 10 minutes at 37°C and were harvested by filtration using a Packard Filtermate Harvester and GF/C Unifilter plates. Incorporation of radioactive thymidine into nucleic acids was determined by liquid scintillation counting [1].

The following murine and human tumor models were employed in the evaluation of BMS-599626: 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. All tumors were maintained and passaged in athymic female nude mice (nu/nu, HSD). Tumors were propagated as s.c. transplants using tumor fragments obtained from donor mice. For oral administration to mice, BMS-599626 was dissolved in a mixture of propylene glycol/water (50:50). The volume of all compounds administered was 0.01 mL/g body weight. Each nude mouse was given a s.c. implant of a tumor fragment (～20 mg) with a 13-gauge trocar. Tumors were allowed to grow to ～100 to 200 mm3 and animals were evenly distributed to various treatment and control groups of eight mice each. Tumor response was determined by measurement of tumors with a caliper twice a week until the tumors reached a predetermined "target" size of 0.5 to 1.0 g. Tumor weights (mg) were estimated from the following formula: tumor weight = (length × width2) / 2 [1].