Chk1 (IC50 = 5 nM); Chk2 (IC50 = 5 nM)

体外活性：AZD7762 是一种更具选择性的 Chk1 抑制剂，通过可逆地结合 Chk1 的 ATP 结合位点来抑制 cdc25C 肽的 Chk1 磷酸化，IC50 为 5 nM，Ki 为 3.6 nM。 AZD7762 通过阻断 Cdc25A 的 chk1 依赖性降解和细胞周期蛋白 A 的激活，诱导细胞停滞，EC50 为 0.620 μM，并显着消除喜树碱诱导的 G2 停滞，EC50 为 10 nM。AZD7762 (300 nM) 增强吉西他滨的抗肿瘤功效通过将 GI50 值从 24.1 nM 和 2.25 μM 分别降低至 1.08 nM 和 0.15 μM，针对 SW620 和拓扑替康针对 MDA-MB-231。 AZD7762 对多种带有 p53 野生型、p53 突变、Mdm2 扩增或 p14 缺失的神经母细胞瘤细胞系显示出细胞毒性，IC50 值范围为 82.6-505.9 nM。激酶测定：使用杆状病毒载体将重组人 Chk1 在昆虫细胞中表达为谷胱甘肽 S-转移酶融合体，并通过谷胱甘肽亲和层析进行纯化。合成了 Chk1 的合成肽底物（N-生物素氨基己酰基-KKVRSSGLYRSPMPENLNRPR）。肽和 ATP（冷 + 40 nCi [33P]ATP）的最终测定浓度分别为 0.8 和 1 μM。将不同浓度的 AZD7762、含有肽、chk1 激酶和 ATP 的缓冲液依次添加到 384 孔测定板中。将板孵育 2 小时，通过添加含有 EDTA 和闪烁邻近分析珠的缓冲液来终止反应，并使用 TopCount 读数器读取板。进行数据分析以确定剂量反应（IC50）。细胞测定：对于检查点废除测定，用喜树碱（拓扑异构酶 I 抑制剂；0.07 μg/mL）处理 HT29 细胞 2 小时以诱导 G2 检查点。然后用 AZD7762（12.5 μM 至 6 nM）加诺考达唑的 12 点滴定处理细胞 20 小时。将细胞用 3.7% 甲醛固定 1 小时，用含有 0.05% Triton X 的 PBS 透化，并与抗 phH3 抗体一起孵育 1 小时，然后用 Alexa Fluor 488 抗兔和 Hoechst 染色 1 小时。有丝分裂指数在ArrayScan上测定并表示为经历有丝分裂的细胞的百分比。对于增强测定，SW620 或 MDA-MB-231 细胞给药 24 小时，吉西他滨的 9 点滴定范围为 0.01 至 100 nM，AZD7762 (300 nM) 的恒定剂量。 24小时后，除去培养基，并将AZD7762单独添加回孔中，再继续培养24小时。然后将细胞在不含 AZD7762 的培养基中再孵育 72 小时。通过MTT测定对细胞增殖的影响。

单独使用 25 mg/kg 的 AZD7762 在 H460-DNp53 异种移植小鼠和 SW620 异种移植小鼠中显示出很少的抗肿瘤活性，但当与吉西他滨 (60 mg/kg) 联合给药时，AZD7762 在两种异种移植小鼠中显示出显着的抗肿瘤功效，其即使在 12.5 mg 的低剂量下，细胞杀灭率仍为 0.9 或治疗/对照百分比 (%T/C) 为 26。在 H460-DNp53 异种移植大鼠中，AZD7762 与吉西他滨 (10 mg/kg) 组合给药以剂量依赖性方式抑制肿瘤体积，10 和 20 mg/kg AZD7762 的％T/C 值为 48 和 32，分别。 AZD7762 (25 mg/kg) 与伊立替康 (25 或 50 mg/kg) 组合可导致 SW620 异种移植小鼠的肿瘤完全消退，%T/C 分别显着增加至 -66% 和 -67%。

通过谷胱甘肽亲和层析纯化，重组人 Chk1 通过杆状病毒载体在昆虫细胞中表达为谷胱甘肽 S-转移酶融合体。对于 Chk1，N-生物素氨基己酰基-KKVRSSGLYRSPMPENLNRPR 是一种合成肽底物。最终检测肽和 ATP 浓度分别为 0.8 和 1 μM（冷 + 40 nCi [³³P]ATP）。具有 384 个孔的检测板充满不同浓度的 AZD7762，这是一种含有 ATP、chk1 激酶和肽的缓冲液。使用 TopCount 读数器，在孵育两小时后对板进行读数，在此期间通过添加含有 EDTA 和闪烁邻近分析珠的缓冲液来停止反应。通过进行数据分析确定剂量反应 (IC50)。

在检查点废除试验中，用喜树碱（一种拓扑异构酶 I 抑制剂；0.07 μg/mL）处理 HT29 细胞两小时，以触发 G2 检查点。之后，除诺考达唑外，对细胞进行 AZD7762（12.5 μM 至 6 nM）的 12 点滴定，持续 20 小时。在 3.7% 甲醛中固定一小时，在含有 0.05% Triton X 的 PBS 中透化细胞，并在抗 phH3 抗体、Alexa Fluor 488 抗兔和 Hoechst 染色剂中孵育细胞一小时，将细胞留置又一个小时。进行有丝分裂的细胞百分比由有丝分裂指数表示，该指数是使用 ArrayScan 计算的。为了进行增强测定，将 SW620 或 MDA-MB-231 细胞用恒定剂量的 AZD7762 (300 nM) 处理 24 小时，然后对吉西他滨进行 9 点滴定，浓度范围为 0.01 至 100 nM。 24小时后除去培养基，然后将AZD7762单独添加回孔中再继续培养24小时。此后，将细胞在不含 AZD7762 的培养基中再培养 72 小时。 MTT测定对细胞增殖的影响。

Mice and Rats: Male RNU rats and male NCr mice are employed. Tumor cells are removed from mice used as xenograft models, centrifuged for five minutes to pellet the cells, and then resuspended in sterile PBS. Using a 25-gauge needle, cells (3×10³-6×10⁶) are s.c. implanted into the right flank of the mice in a volume of 0.1 to 0.2 mL. Before compound is administered, tumors are allowed to grow to the specified size of 100 to 200 mm³. Rat xenograft models involve cell harvesting, centrifugation for 5 minutes to pellet the cells, and resuspension in a 50% sterile PBS and 50% Matrigel solution. Five days prior to cell implantation, rats undergo a whole-body radiation dose of 5 Gy with the goal of enhancing tumor growth. With a 25-gauge needle, H460-DNp53 cells (1×10⁷) are s.c. implanted into the rats' right flanks in a volume of 0.2 mL. Before administering AZD-7762, tumors are allowed to grow to the specified size of 100 to 200 mm³. The tail vein is used to administer intravenous injections of AZD-7762 (10 and 20 mg/kg). Treatments were administered in cycles of three to five, according to cyclic schedules. Every three days, AZD-7762 is delivered after a standard agent (NSC 613327 or CPT-11) has been administered. Electronic calipers are used to measure and compute tumor volumes. Mice: Pharmacological inhibitors are administered to C57Bl/6 mice eight days after they receive an intravenous injection of 2×10⁵ Eμ-Myc B-lymphoma cells in PBS. Treatment is administered to the mice until an ethical endpoint, such as a hunched posture, ruffled fur, enlarged lymph nodes, labored breathing, weight loss of more than 20% of the initial body weight, limited mobility, or paralysis, is achieved. On weekdays, 20 mg/kg of AZD7762 is administered intraperitoneally in a solution of 10.3% -hydroxypropyl-β-cyclodextrin and 0.9% saline.

[1]. AZD7762, a novel checkpoint kinase inhibitor, drives checkpoint abrogation and potentiates DNA-targeted therapies. Mol Cancer Ther. 2008 Sep;7(9):2955-66.

[2]. Inhibition of RNA polymerase I transcription initiation by CX-5461 activates non-canonical ATM/ATR signaling. Oncotarget. 2016 Aug 2;7(31):49800-49818.

3-(carbamoylamino)-5-(3-fluorophenyl)-N-[(3S)-3-piperidinyl]-2-thiophenecarboxamide is an aromatic amide and a member of thiophenes.
AZD7762 has been investigated for the treatment of Cancer, Solid Tumors, and Advanced Solid Malignancies.
Checkpoint Kinase Inhibitor AZD7762 is a synthetic small molecule inhibitor of checkpoint kinases (Chks) with potential chemosensitizing activity. AZD7762 binds to and inhibits Chks, which may prevent cell cycle arrest and subsequent nucleotide excision repair in DNA-damaged tumor cells, resulting in tumor cell apoptosis. This agent may enhance the cytotoxicity of DNA-damaging agents. Chks are protein kinases that regulate either G1/S or G2/M transitions in the cell cycle. In the presence of DNA damage or incomplete DNA replication, Chks become activated and initiate cell cycle arrest to allow DNA repair or the completion of DNA replication.

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Insights from cell cycle research have led to the hypothesis that tumors may be selectively sensitized to DNA-damaging agents resulting in improved antitumor activity and a wider therapeutic margin. The theory relies on the observation that the majority of tumors are deficient in the G1-DNA damage checkpoint pathway resulting in reliance on S and G2 checkpoints for DNA repair and cell survival. The S and G2 checkpoints are regulated by checkpoint kinase 1, a serine/threonine kinase that is activated in response to DNA damage; thus, inhibition of checkpoint kinase 1 signaling impairs DNA repair and increases tumor cell death. Normal tissues, however, have a functioning G1 checkpoint signaling pathway allowing for DNA repair and cell survival. Here, we describe the preclinical profile of AZD7762, a potent ATP-competitive checkpoint kinase inhibitor in clinical trials. AZD7762 has been profiled extensively in vitro and in vivo in combination with DNA-damaging agents and has been shown to potentiate response in several different settings where inhibition of checkpoint kinase results in the abrogation of DNA damage-induced cell cycle arrest. Dose-dependent potentiation of antitumor activity, when AZD7762 is administered in combination with DNA-damaging agents, has been observed in multiple xenograft models with several DNA-damaging agents, further supporting the potential of checkpoint kinase inhibitors to enhance the efficacy of both conventional chemotherapy and radiotherapy and increase patient response rates in a variety of settings.[1]

C17H19FN4O2S

362.42

362.12

C, 56.34; H, 5.28; F, 5.24; N, 15.46; O, 8.83; S, 8.85

860352-01-8

AZD-7762 hydrochloride;1246094-78-9

11152667

white solid powder

1.38

547.6ºC at 760 mmHg

285ºC

4.821

124.49

4

5

4

25

495

1

C(C1=C(NC(=O)N)C=C(C2C=CC=C(F)C=2)S1)(=O)N[C@@H]1CNCCC1

IAYGCINLNONXHY-LBPRGKRZSA-N

InChI=1S/C17H19FN4O2S/c18-11-4-1-3-10(7-11)14-8-13(22-17(19)24)15(25-14)16(23)21-12-5-2-6-20-9-12/h1,3-4,7-8,12,20H,2,5-6,9H2,(H,21,23)(H3,19,22,24)/t12-/m0/s1

3-(carbamoylamino)-5-(3-fluorophenyl)-N-[(3S)-piperidin-3-yl]thiophene-2-carboxamide

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.90 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (6.90 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (6.90 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 10 mg/mL (27.59 mM) in 10% HP-β-CD (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶.

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。