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

在体外，AZD-7762 盐酸盐也能纠正 Chk1 和 Chk2。 AZD-7762 盐酸盐可提高 NSC 613327 和 SKF 104864A 的有效性，抑制 DNA 损伤诱导的 S 和 G2 检查点，并修改下游检查点稀释蛋白。通过使用闪烁出租车测定，发现 AZD-7762 盐可有效阻断 cdc25C 肽的 Chk1 磷酸化，IC50 为 5 nM。发现盐 AZD-7762 的 Ki 为 3.6 nM。根据动力学表征，AZD-7762 盐与 Chk1 的 ATP 结合位点结合，假定它参与直接、可逆的 ATP 结合竞争。事实证明，AZD-7762 盐酸盐可以完全消除喜树碱诱导的 G2 阻断，最大消除范围为 100 nM，平均 EC50 为 10 nM (n=12) [1]。

在 H460-DNp53 异种移植实验中，AZD-7762 盐酸盐（10 mg/kg 和 20 mg/kg）剂量依赖性地增加 NSC 613327 的抗癌功效。换句话说，随着剂量的增加，抑制率 (%T /C) 分别下降至 48% 和 32%。当在小鼠异种移植研究中与 CPT-11 结合使用时，AZD-7762 盐酸盐将 CPT-11 活性和 %T/C 分别显着提高至 -66% 和 -66%。 -67% [1]。当与 CX-5461 联合使用时，AZD7762 盐酸盐会导致 Tp53-null (Tp53-/-) Eμ-Myc 的内部和外部表观细胞死亡 [2]。

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)。

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.

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]

C17H20CLFN4O2S

398.8827

398.098

C, 51.19; H, 5.05; Cl, 8.89; F, 4.76; N, 14.05; O, 8.02; S, 8.04

1246094-78-9

AZD-7762;860352-01-8

56972142

Typically exists as solid at room temperature

4.821

124.49

5

5

4

26

495

1

Cl[H].S1C(C2C([H])=C([H])C([H])=C(C=2[H])F)=C([H])C(=C1C(N([H])[C@]1([H])C([H])([H])N([H])C([H])([H])C([H])([H])C1([H])[H])=O)N([H])C(N([H])[H])=O

WFZBLOIXZRZEDG-YDALLXLXSA-N

InChI=1S/C17H19FN4O2S.ClH/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);1H/t12-;/m0./s1

(S)-5-(3-Fluorophenyl)-N-(piperidin-3-yl)-3-ureidothiophene-2-carboxamide Hydrochloride

AZD-7762 Hydrochloride; AZD-7762 HCl; AZD7762; AZD 7762; AZD-7762.

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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。