AZ20 is a potent and selective inhibitor of ATR with an IC₅₀ of 5 nM, and has 8-fold selectivity against mTOR (IC₅₀=38 nM).

AZ20 inhibits ATR immunoprecipitated from HeLa nuclear extracts with an IC₅₀ of 5 nM and ATR mediated phosphorylation of Chk1 in HT29 colorectal adenocarcinoma tumor cells with an IC₅₀ of 50 nM^[1].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

AZ20 (25, 50 mg/kg, p.o.) has high permeability combined with good stability to rat hepatocytes and, despite the lack of progress in achieving markedly higher solubility, has respectable bioavailability in a low dose rat PK study. AZ20 (25, 50 mg/kg, p.o.) leads to significant tumor growth inhibition in female nude mice bearing LoVo tumors^[1].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

412.51

C₂₁H₂₄N₄O₃S

1233339-22-4

Room temperature in continental US; may vary elsewhere.

Powder	-20°C	3 years
	4°C	2 years
In solvent	-80°C	6 months
	-20°C	1 month

In Vitro: 

DMSO : ≥ 100 mg/mL (242.42 mM)

* “≥” means soluble, but saturation unknown.

*

请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。
储备液的保存方式和期限：-80°C, 6 months; -20°C, 1 month。-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。

In Vivo:

请根据您的实验动物和给药方式选择适当的溶解方案。以下溶解方案都请先按照 In Vitro 方式配制澄清的储备液，再依次添加助溶剂：

——为保证实验结果的可靠性，澄清的储备液可以根据储存条件，适当保存；体内实验的工作液，建议您现用现配，当天使用； 以下溶剂前显示的百
分比是指该溶剂在您配制终溶液中的体积占比；如在配制过程中出现沉淀、析出现象，可以通过加热和/或超声的方式助溶

• 1.

  请依序添加每种溶剂： 10% DMSO    40% PEG300    5% Tween-80    45% saline

  Solubility: ≥ 2.5 mg/mL (6.06 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (6.06 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加入50 μL Tween-80，混合均匀；然后继续加入 450 μL生理盐水定容至 1 mL。

  将 0.9 g 氯化钠，完全溶解于 100 mL ddH₂O 中，得到澄清透明的生理盐水溶液

• 2.

  请依序添加每种溶剂： 10% DMSO    90% (20% SBE-β-CD in saline)

  Solubility: ≥ 2.5 mg/mL (6.06 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (6.06 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 900 μL 20% 的 SBE-β-CD 生理盐水水溶液中，混合均匀。

  将 2 g 磺丁基醚 β-环糊精加入 5 mL 生理盐水中，再用生理盐水定容至 10 mL，完全溶解，澄清透明

• [1]. Foote KM, et al. Discovery of 4-{4-[(3R)-3-Methylmorpholin-4-yl]-6-[1-(methylsulfonyl)cyclopropyl]pyrimidin-2-yl}-1H-indole (AZ20): a potent and selective inhibitor of ATR protein kinase with monotherapy in vivo antitumor activity. J Med Chem. 2013 Mar 14

Compound dose ranges are created by diluting in 100% DMSO and then further into assay medium (EMEM, 10% FCS, 1% glutamine) using a Labcyte Echo acoustic dispensing instrument. Cells are plated in 384-well Costar plates at 9×10⁴ cells per mL in 40 μL of EMEM, 10% FCS, 1% glutamine and grown for 24 h. Following addition of compound the cells are incubated for 60 min. A final concentration of 3 μM 4NQO (prepared in 100% DMSO) is then added using the Labcyte Echo, and the cells are incubated for a further 60 min. The cells are fixed by adding 40 μL of 3.7% v/v formaldehyde solution for 20 min. After removal of fix, cells are washed with PBS and permeabilized in 40 μL of PBS containing 0.1% Triton X-100. The cells are then washed, and 15 μL primary antibody solution (pChk1 Ser345) is added. The plates are incubated at 4°C overnight. The primary antibody is then washed off, and 20 μL of secondary antibody solution and 1 μM Hoechst 33258 added for 90 min at room temperature. The plates are washed and left in 40 μL of PBS. Plates are then read on an ArrayScan VTI instrument to determine staining intensities, and dose responses are obtained and used to determine the IC₅₀ values for the compounds.

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

Female Swiss nu/nu mice are housed in negative pressure isolators. LoVo tumor xenografts are established in 8- to 12-week-old mice by injecting 1×10⁷ tumor cells subcutaneously (100 μL in serum free medium) on the left dorsal flank. Animals are randomized into treatment groups when tumors become palpable. AZ20 is prepared in 10% DMSO/40% propylene glycol/50% water and administered orally. Tumors are measured up to three times per week with calipers. Tumor volumes are calculated and the data plotted using the geometric mean for each group versus time.

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Foote KM, et al. Discovery of 4-{4-[(3R)-3-Methylmorpholin-4-yl]-6-[1-(methylsulfonyl)cyclopropyl]pyrimidin-2-yl}-1H-indole (AZ20): a potent and selective inhibitor of ATR protein kinase with monotherapy in vivo antitumor activity. J Med Chem. 2013 Mar 14