CCT245737 is an orally active, selective Chk1 inhibitor with an IC50 of 1.3 nM, and is >1,000-fold selective over CHK2 and CDK1.

Chk1:1.4 nM (Cell-free)

方法： CCT245737（0.01,0.05,0.1,0.5，1,2,5,24小时） 处理 K562 细胞，WseternBlot检测CHK1总表达的蛋白；用 VP16 （5 μM）、CCT245737 （5 μM） 或 VP16 和 CCT245737（0.01,0.05,0.1,0.5，1,2,5,24小时）的组合处理的K562 细胞，进行的蛋白质印迹分析。
结果：增加 CCT245737 浓度并未显着降低 CHK1 总蛋白水平，并且 CHK1 仅在高浓度 CCT245737 （5 μM） 下被抑制；CCT245737可以通过抑制VP16诱导的CHK1在Ser296自磷酸化并促进VP16处理的K562细胞中DNA损伤的积累来有效抑制CHK1的激活。[3]

方法：CCT245737（10mg/kg，静脉注射/口服）给药小鼠后，检测体内的药代动力学。
结果：静脉注射CCT245737血药峰浓度为4μmol/，半衰期为2.86h，AUC为9.96μmol.h/L，血浆清除率为2.1L/h/kg，分布容积大（0.19L）；等效的口服剂量与 AUC 几乎相同，显示完全口服生物利用度（F = 105%）。[1]

Commercial in vitro 33P radiometric kinase assays is carried out against 124 human kinases using 10 μM CCT245737 at ATP concentrations corresponding to the kinase Km, ATP [2].

Cytotoxicity is determined as the drug concentration that gives 50% inhibition of tumour cell proliferation (GI50) using a 96 h Sulforhodamine B (SRB) assay. Inhibition of intracellular CHK1 activity is measured using a cell-based ELISA for the abrogation of an etoposide-induced G2 checkpoint (mitosis induction assay, MIA). The IC50 for G2 checkpoint abrogation (MIA) is determined in the presence of nocodazole using UCN01 as a positive control. The activity index (AI) is used as a measure of the compounds ability to induce mitosis relative to its toxicity (i.e., ratio of MIA IC50: 96 h SRB GI50). Routine potentiation studies are carried out using a fixed concentration (GI50) of either gemcitabine or SN38 in combination with a range of CCT245737 concentrations to determine the combination GI50 of CCT245737. The ability of CCT245737 to enhance gemcitabine or SN38 cell killing is expressed as a potentiation index (PI) equal to the ratio of the GI50 for CCT245737 alone versus the combination GI50 for CCT245737. PI values > 1 indicate the potentiation of the genotoxic activity. In addition, a series of experiments is carried out using fixed, non- or minimally toxic concentrations of CCT245737 (≤GI20) with a range of different concentrations of gemcitabine or SN38 to determine the extent to which CCT245737 enhances drug cytotoxicity compared with the genotoxic agent alone, i.e. conventional PI (ratio GI50 genotoxic alone: GI50 genotoxic combined with non-toxic CCT245737 concentration, Con PI)[2].

Human HT29 colorectal carcinoma cells are injected s.c into the flanks of female NCr athymic mice 6-8 weeks of age. Dosing commenced 5 days after transplantation when tumours reach a mean diameter of 5.5 mm. Gemcitabine (100 mg/kg i.v.) is dosed in saline on days 0, 7 and 14 and compounds 4 (CCT245737) and 41 (150 mg/kg p.o.) in 10% DMSO 20% PEG 400, 5% Tween 80, 65% water, 24 and 48 h after each dose of gemcitabine. Tumours are measured and body weights recorded three times weekly. Animals are culled on an individual basis when tumours reach a predetermined humane endpoint (mean diameter <15 mm)[1].