AZD3759 (hydrochloride) is an effective, orally active and central nervous system-penetrant EGFR inhibitor. At Km ATP concentrations, the IC50s are 0.2/0.3/0.2 nM for EGFR (L858R)/EGFR (wt)/EGFR (exon 19Del).

EGFR (exon 19Del):0.2 nM, EGFR (WT):0.3nM, EGFR (L858R):0.2nM

在2 mM的ATP浓度下，EGFR (wt)/EGFR (L858R)/EGFR (exon 19Del)的IC50分别为102/7.6/2.4 nM。AZD3759还可针对pEGFR进行抑制，在H838wt、H3255L858R、PC-9exon 19Del中的IC50分别为64.5 nM、7.2 nM和7.4 nM。在细胞磷酸化研究中，AZD3759还展示了对激活突变EGFR细胞株相比于EGFR野生型细胞株（H838细胞系）的9倍选择性抑制作用[1]。

在大鼠中，AZD3759（2 mg/kg，口服）的吸收迅速，血液Cmax达到0.58 μM，于1.0小时实现。随后，AZD3759的血液浓度以平均消除半衰期4.3小时单指数下降，与通过静脉注射得到的4.1小时的参数相近。大鼠口服后的生物利用度为91%。在狗进行静脉注射后，AZD3759的血液清除率为14 mL/min/kg，分布容积为6.4 L/kg，其消除半衰期为6.2小时。AZD3759的吸收迅速，血液Cmax（698 nM）在0.5至1.5小时之间出现。AZD3759的口服生物利用度极佳，为90%。AZD3759表现出显著的剂量依赖性抗肿瘤效能（在治疗后4周，7.5 mg/kg/天和15 mg/kg/天分别实现了78%的肿瘤生长抑制和肿瘤退缩），且体重损失<20%。AZD3759（7.5/15 mg/kg）能显著减少肿瘤面积。此外，在服用单剂量AZD3759（15 mg/kg）1小时后，通过检测到pEGFR的调节[1]。

AZD3759 is tested at a single 1 μM concentration across each of 124 kinases from Millipore kinase panel at an ATP concentration that is within 15 μM of their corresponding apparent Km values. In brief, recombinant kinases are incubated within an appropriate buffer containing peptide substrate and radiolabelled γ-33P-ATP together with presence or absence of required inhibitor concentration. The reaction is initiated by adding ATP/Mg2+ mix. After incubation for 40 minutes at room temperature, the reaction is stopped by adding 3% phosphoric acid solution. A portion of reaction mix is spotted onto P30 filter mat to trap peptide and washed three times for 5 minutes with phosphoric acid to remove non-specific γ-33P-ATP. The phosphorylated substrate is then measured by scintillation counting, which determined the level of kinase activity inhibition compared to control reactions[1].

AZD3759 is prepared in DMSO and stored,and then diluted with the appropriate medium before use[1].Cell proliferation assay is determined by MTS methods.Briefly,cells are seeded in 96-well plates (at a density to allow for logarithmic growth during the 72-hour assay) and incubated overnight at 37°C and 5% CO2.Cells are then exposed to concentrations of compounds (e.g.,AZD3759) ranging from 30 mM to 0.3 μM for 72 hours.For the MTS endpoint,cell proliferation is measured by the CellTiter AQueous Non-Radioactive Cell Proliferation Assay reagent.Absorbance is measured with a Tecan Ultra instrument.Predose measurements are made,and concentration needed to reduce the growth of treated cells to half that of untreated cells (GI50) values are determined using absorbance readings[1].

AZD3759 is prepared in 1% methylcellulose (Rat)[1].Male Han Wistar rats are orally dosed with the AZD3759 at 2 mg/kg in 1% methylcellulose. At 0.25, 0.5, 1, 2, 4 and 7 hour post-dose, cerebral spinal fluid (CSF) is collected from cisterna magna, and blood samples (>60 μL/time point/each site) are collected via cardiac puncture, into separate EDTA coagulated tubes, and then immediately diluted with 3-fold volume of water. Brain tissue is harvested and homogenized in 3x volume of 100 mM phosphate buffered saline (pH7.4). All samples are stored at -70°C prior to LC/MS/MS analysis.