Akt1 (IC50 = 5 nM); Akt3 (IC50 = 8 nM); Akt2 (IC50 = 18 nM); PKA (IC50 = 3100 nM)

与密切相关的 PKA 和 p70S6K 相比，imatasertib diHClide 对 Akt1 的选择性分别高出 600 倍和 100 倍以上，IC50 值更高。在 230 种蛋白质缀合物（包括人类 AGC 家族的 36 名成员）中，Ipatasertib diHClide 在 1 μM 浓度下仅抑制三种蛋白质缀合物（PRKG1α、PRKG1β 和 p70S6K）超过 70%。 ..对于这三个物种，定期测量的 IC50 值依次为 98、69 和 860 nM。因此，与第二个最有效的非 Akt 抑制剂（上面的 p70S6K Multiples 面板）相比，ipatasertib 二盐酸盐对筛选中的 Akt1 选择性高出 100 倍，但 PKG1 除外（其中 ipatasertib 二盐酸盐对 Akt1 的选择性高出 10 倍以上） 。使用展示药物治疗剂量依赖性反应的三种异种移植模型来研究二盐酸ipatasertib：MCF7-neo的药代动力学（PK）和药效学（PD）之间的关系。 /HER2、TOV-21G.x1 和 LNCaP Ipatasertib diHClide 在这三种细胞系中的平均细胞活力 IC50 分别为 2.56、0.44 和 0.11 μM [2]。

在 Akt 由 PTEN 缺失、PIK3CA 突变或突变或 HER2 过度表达等遗传变化触发的移植模型中，二盐酸伊马替尼通常会成功。这些模型中的肿瘤在 100 mg/kg 或以下（在免疫功能低下的小鼠中观察到的最大耐受剂量）缓慢发育、生成或恢复。在体内检查中，每天将 RP-56976 与 Ipatasertib diHClide 组合可在 PC-3 和 MCF7-neo/HER2 异种移植小鼠中产生镇痛和肿瘤消退，但毒素本身要么无效，要么只是导致肿瘤发展。逐渐增加剂量。同样，当 Ipatasertib diHClide 和 NSC 241240 偶联时，在 OVCAR3 卵巢癌异种移植模型中观察到 TGI 升高。与额外化疗相比，Ipatasertib diHClide 联合 RP-56976 或 NSC 241240 单独治疗导致体重减轻不到 5% [2]。

Enzymatic Assays/酶实验[1]
用于测定Akt1/2/3和PKA激酶活性的测定采用IMAP荧光偏振（FP）磷酸化检测试剂来检测已被相应激酶磷酸化的荧光标记肽底物。这些研究中使用的Akt酶由重组杆状病毒表达、氨基末端、聚组氨酸标记、全长、野生型人类形式组成，并从商业上获得。这些研究中使用的PKA酶由商业上获得的大肠杆菌中表达的重组未标记的PKA人分离催化亚基组成。在环境温度下，将抑制剂、酶（9 nM Akt1或100 pM PKA）和底物（100 nM Crosstide）与5μM ATP在测定缓冲液（10 mM Tris-HCl（pH 7.2）、10 mM MgCl2、0.1%BSA（w/v）、最终DMSO 2%（v/v））中在5μL反应体积中孵育60分钟。通过向ATP溶液中加入酶+肽底物引发反应。加入IMAP结合试剂（15μL）终止反应，并将停止的反应在室温下孵育至少30分钟。

384 孔板每孔接种 2,000 个细胞，体积为 54 L，然后在 37°C、5% CO2 下孵育过夜（大约 16 小时）。为了产生所需的库存浓度，化合物（如 Ipatasertib）在 DMSO 中稀释，然后以每孔 6 L 的体积添加。每种处理均测试四次。孵育四天后，在 Wallac Multilabel Reader 上测量总发光，并使用 CellTiter-Glo 估计相对活力。 4 参数曲线分析 (XLfit) 用于计算产生 IC50 的药物浓度，并且基于至少三个实验的结果。列出了未达到 IC50 的细胞系的最高测试浓度 (10 M)[2]。

Mice：Numerous patient-derived xenograft models and tumor cell line models are used to evaluate in vivo efficacy. Immunocompromised mice have cells or tumor fragments implanted subcutaneously into their flanks. Mice that are severely combined immunodeficient (SCID) or beige, or both, are used. Male mice are castrated prior to the implantation of tumor fragments, and the LuCaP35V patient-derived primary tumors are obtained. When tumor cells or fragments are implanted into mice, the tumors are watched until they reach mean tumor volumes of 180 to 350 mm3 and are then divided into groups of 8 to 10 animals each. Ipatasertib is administered every day (QD) via oral (per os; PO) gavage and is formulated in 0.5% methylcellulose/0.2% Tween-80 (MCT). Every week (QW), 2.5 or 7.5 mg/kg of RP-56976 is administered intravenously (IV) in a solution of 3% EtOH/97% saline. Saline-based NSC 241240 is administered intraperitoneally (IP) once a week at a dose of 50 mg/kg.

[1]. Discovery and preclinical pharmacology of a selective ATP-competitive Akt inhibitor (GDC-0068) for the treatment of human tumors. J Med Chem. 2012 Sep 27;55(18):8110-27.

[2]. Targeting activated Akt with GDC-0068, a novel selective Akt inhibitor that is efficacious in multiple tumor models. Clin Cancer Res. 2013 Apr 1;19(7):1760-72.

(2S)-2-(4-chlorophenyl)-1-[4-[(5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl]-1-piperazinyl]-3-(propan-2-ylamino)-1-propanone is a N-arylpiperazine.
Ipatasertib has been used in trials studying the treatment of Cancer, Neoplasms, Solid Cancers, Breast Cancer, and Gastric Cancer, among others.

Ipatasertib is an orally bioavailable inhibitor of the serine/threonine protein kinase Akt (protein kinase B) with potential antineoplastic activity. Ipatasertib binds to and inhibits the activity of Akt in a non-ATP-competitive manner, which may result in the inhibition of the PI3K/Akt signaling pathway and tumor cell proliferation and the induction of tumor cell apoptosis. Activation of the PI3K/Akt signaling pathway is frequently associated with tumorigenesis and dysregulated PI3K/Akt signaling may contribute to tumor resistance to a variety of antineoplastic agents.
Drug Indication: Treatment of breast cancer , Treatment of prostate cancer.

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The discovery and optimization of a series of 6,7-dihydro-5H-cyclopenta[d]pyrimidine compounds that are ATP-competitive, selective inhibitors of protein kinase B/Akt is reported. The initial design and optimization was guided by the use of X-ray structures of inhibitors in complex with Akt1 and the closely related protein kinase A. The resulting compounds demonstrate potent inhibition of all three Akt isoforms in biochemical assays and poor inhibition of other members of the cAMP-dependent protein kinase/protein kinase G/protein kinase C extended family and block the phosphorylation of multiple downstream targets of Akt in human cancer cell lines. Biological studies with one such compound, 28 (GDC-0068), demonstrate good oral exposure resulting in dose-dependent pharmacodynamic effects on downstream biomarkers and a robust antitumor response in xenograft models in which the phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin pathway is activated. 28 is currently being evaluated in human clinical trials for the treatment of cancer.[1]

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Purpose: We describe the preclinical pharmacology and antitumor activity of GDC-0068, a novel highly selective ATP-competitive pan-Akt inhibitor currently in clinical trials for the treatment of human cancers. Experimental design: The effect of GDC-0068 on Akt signaling was characterized using specific biomarkers of the Akt pathway, and response to GDC-0068 was evaluated in human cancer cell lines and xenograft models with various genetic backgrounds, either as a single agent or in combination with chemotherapeutic agents. Results: GDC-0068 blocked Akt signaling both in cultured human cancer cell lines and in tumor xenograft models as evidenced by dose-dependent decrease in phosphorylation of downstream targets. Inhibition of Akt activity by GDC-0068 resulted in blockade of cell-cycle progression and reduced viability of cancer cell lines. Markers of Akt activation, including high-basal phospho-Akt levels, PTEN loss, and PIK3CA kinase domain mutations, correlate with sensitivity to GDC-0068. Isogenic PTEN knockout also sensitized MCF10A cells to GDC-0068. In multiple tumor xenograft models, oral administration of GDC-0068 resulted in antitumor activity ranging from tumor growth delay to regression. Consistent with the role of Akt in a survival pathway, GDC-0068 also enhanced antitumor activity of classic chemotherapeutic agents. Conclusions: GDC-0068 is a highly selective, orally bioavailable Akt kinase inhibitor that shows pharmacodynamic inhibition of Akt signaling and robust antitumor activity in human cancer cells in vitro and in vivo. Our preclinical data provide a strong mechanistic rationale to evaluate GDC-0068 in cancers with activated Akt signaling.[2]

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Colon cancer is one of the three common malignant tumors, with a lower survival rate. Ipatasertib, a novel highly selective ATP-competitive pan-Akt inhibitor, shows a strong antitumor effect in a variety of carcinoma, including colon cancer. However, there is a lack of knowledge about the precise underlying mechanism of clinical therapy for colon cancer. We conducted this study to determine that ipatasertib prevented colon cancer growth through PUMA-dependent apoptosis. Ipatasertib led to p53-independent PUMA activation by inhibiting Akt, thereby activating both FoxO3a and NF-κB synchronously that will directly bind to PUMA promoter, up-regulating PUMA transcription and Bax-mediated intrinsic mitochondrial apoptosis. Remarkably, Akt/FoxO3a/PUMA is the major pathway while Akt/NF-κB/PUMA is the secondary pathway of PUMA activation induced by ipatasertib in colon cancer. Knocking out PUMA eliminated ipatasertib-induced apoptosis both in vitro and in vivo (xenografts). Furthermore, PUMA is also indispensable in combinational therapies of ipatasertib with some conventional or novel drugs. Collectively, our study demonstrated that PUMA induction by FoxO3a and NF-κB is a critical step to suppress the growth of colon cancer under the therapy with ipatasertib, which provides some theoretical basis for clinical assessment.[3]

C₂₄H₃₄CL₃N₅O₂

530.9181

529.178

C, 54.30; H, 6.46; Cl, 20.03; N, 13.19; O, 6.03

1396257-94-5

Ipatasertib;1001264-89-6; 1489263-16-2 (HCl); 1491138-24-9; 1396257-94-5 (2HCl); 1491138-23-8 (besylate)

62707512

Light yellow to yellow solid powder

5.098

81.59

4

6

6

34

622

3

ClC1C([H])=C([H])C(=C([H])C=1[H])[C@@]([H])(C([H])([H])N([H])C([H])(C([H])([H])[H])C([H])([H])[H])C(N1C([H])([H])C([H])([H])N(C([H])([H])C1([H])[H])C1C2=C([C@@]([H])(C([H])([H])[C@@]2([H])C([H])([H])[H])O[H])N=C([H])N=1)=O.Cl[H].Cl[H]

SRKVNRNRVFDUTG-VISIQVHMSA-N

InChI=1S/C24H32ClN5O2.2ClH/c1-15(2)26-13-19(17-4-6-18(25)7-5-17)24(32)30-10-8-29(9-11-30)23-21-16(3)12-20(31)22(21)27-14-28-23/h4-7,14-16,19-20,26,31H,8-13H2,1-3H32*1H/t16-,19-,20-/m1../s1

(2S)-2-(4-Chlorophenyl)-1-[4-[(5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl]piperazin-1-yl]-3-(propan-2-ylamino)propan-1-onedihydrochloride

Ipatasertib dihydrochloride; Ipatasertib; GDC 0068; GDC-0068; GDC0068; RG7440; 1396257-94-5; GDC-0068 (dihydrochloride); UNII-M2JCB5A8EV; M2JCB5A8EV; Ipatasertib (dihydrochloride); (2S)-2-(4-Chlorophenyl)-1-[4-[(5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl]piperazin-1-yl]-3-(propan-2-ylamino)propan-1-one;dihydrochloride; RG 7440; RG-7440

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~100 mg/mL (188.35 mM; Need ultrasonic)
H2O: ≥41 mg/mL (77.22 mM)

配方 1 中的溶解度: ≥ 3.88 mg/mL (7.31 mM) (饱和度未知) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*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: ≥ 3.88 mg/mL (7.31 mM) (饱和度未知) in 5% DMSO + 95% (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。
*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: ≥ 2.08 mg/mL (3.92 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 20.8 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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 4 中的溶解度: ≥ 2.08 mg/mL (3.92 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100μL 20.8mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，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 5 中的溶解度: ≥ 2.08 mg/mL (3.92 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 20.8 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

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配方 6 中的溶解度: 16.67 mg/mL (31.40 mM) in PBS (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。