Akt2 (IC50 = 17 nM); p70S6K (IC50 = 85 nM); Akt1 (IC50 = 32 nM); Akt3 (IC50 = 47 nM); PKA (IC50 = 20 nM)
AT7867 is a dual inhibitor of the Akt kinase family and p70 S6 kinase (p70 S6K), key regulators of the PI3K/mTOR signaling pathway. In recombinant enzyme assays, it exhibits IC50 values of 16 nM for Akt1, 35 nM for Akt2, 21 nM for Akt3, and 26 nM for p70 S6K. It shows minimal cross-reactivity with other kinases (e.g., PKA, EGFR, VEGFR2) with IC50 values > 1000 nM [1]

AT7867 对 AKT2 的抑制具有 ATP 竞争性，Ki 为 18nM。尽管 AT7867 对其他激酶亚家族的激酶表现出明显的选择性窗口，但它也对结构相关的 AGC 激酶 p70S6K 和 PKA 表现出有效的活性。体外生长抑制研究表明，AT7867 可抑制多种人类癌细胞系的增殖。 AT7867 似乎在所测试的两种前列腺系中效果最差（IC50 值范围为 10-12 M），而在抑制 MES-SA 子宫、MDA-MB-468 和 MCF-7 乳房、HCT116 和 HT29 结肠中的增殖方面最有效线（IC50 值范围为 0.9–3 M）[1]。

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在PI3K/Akt信号激活的人前列腺癌细胞系（PC-3、LNCaP）中，AT7867（0.01-10 μM）处理72小时可呈剂量依赖性抑制细胞增殖。SRB实验显示，PC-3细胞的IC50值为0.28 μM，LNCaP细胞为0.42 μM。蛋白质印迹（Western blot）分析显示，0.5 μM AT7867可在24小时内使Akt（Ser473/Thr308）磷酸化水平降低>90%，p70 S6K（Thr389）降低85%，下游靶点S6核糖体蛋白（Ser235/236）降低80%，而这些激酶的总蛋白水平无变化 [1]
- 在具有组成型Akt激活的人卵巢癌细胞系SKOV3中，AT7867（0.1-5 μM）处理48小时可诱导剂量依赖性凋亡。Annexin V-FITC/PI双染流式细胞术显示，2 μM AT7867使凋亡率从对照组的3%升高至36%。此外，结晶紫染色结果显示，1 μM AT7867可使SKOV3细胞的克隆形成能力降低70%（培养14天） [1]
- 在PC-3细胞中，AT7867（0.2 μM）与多西他赛（5 nM）联合处理具有协同抗增殖作用，联合指数（CI）为0.55（CI<1表示协同）。联合处理组的凋亡率为42%，显著高于AT7867单药组（17%）和多西他赛单药组（14%） [1]

以 20 mg/kg 口服给药后，AT7867 从血浆中的消除似乎与静脉注射后观察到的情况相似。口服 20 mg/kg AT7867 后，血浆水平保持在 0.5 M 以上至少 6 小时。假设静脉给药后出现线性药代动力学，经口服途径的生物利用度计算为 44%。因此，该模型用于体内药效学（PD）生物标志物研究。经过药代动力学和耐受性研究后，对患有 MES-SA 肿瘤的无胸腺小鼠给予 AT7867 剂量（90 mg/kg po 或 20 mg/kg ip），并随时间跟踪肿瘤中 GSK3 和 S6RP 的磷酸化状态。 AT7867 处理后 2 小时和 6 小时，观察到两种途径活性标志物磷酸化的明确抑制。 GSK3 和 S6RP 的总水平在 24 小时内显着下降[1]。

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在人前列腺癌（PC-3）裸鼠异种移植模型中，AT7867以25 mg/kg和50 mg/kg的剂量每日口服一次，连续21天。与溶媒对照组（0.5%羧甲基纤维素钠+0.1%吐温80）相比，25 mg/kg组肿瘤体积减少52%，50 mg/kg组减少75%。肿瘤组织免疫组化染色显示，50 mg/kg组中p-Akt（Ser473）表达降低85%，增殖标志物Ki-67阳性细胞减少60% [1]
- 在人卵巢癌（SKOV3）裸鼠异种移植模型中，AT7867以50 mg/kg的剂量每日口服一次，连续14天。该处理使肿瘤重量较溶媒对照组减少68%。肿瘤裂解物的Western blot分析证实，p-p70 S6K（Thr389）水平降低，凋亡标志物切割型caspase-3增加 [1]
- 在PC-3异种移植模型中，AT7867（25 mg/kg口服，每日一次）与多西他赛（10 mg/kg腹腔注射，每周一次）联合处理21天，可使肿瘤体积减少88%，显著优于单药治疗效果（AT7867单药减少52%，多西他赛单药减少45%） [1]

AKT2、PKA、p70S6K 和 CDK2/cyclinA 激酶测定均使用放射滤光片结合形式进行。测定反应是在化合物存在的情况下进行的。对于 AKT2，将 AKT2 酶与 25 mM AKTide-2T 肽 (HARKRERTYSFGHHA)、20 mM MOPS、pH 7.2、5 mM EDTA、15 mM MgCl2、1 mM 原钒酸钠、1 mM DTT、10 g/mL 一起孵育 4 小时BSA 和 30 mM ATP (1.16 Ci/mmol)。对于 PKA，将 50 mM 肽 (GRTGRRNSI) 和 PKA 酶在含有 2 mM MOPS（pH 7.2）、25 mM 甘油磷酸盐、5 mM EDTA、15 mM MgCl2、1 mM 原钒酸盐、1 mM 的溶液中孵育 20 分钟DTT 和 40 mM ATP (0.88 Ci/mmol)。对于 p70S6K，酶和 25 M 肽底物 (AKRRRLSSLRA) 在 10 mM MOPS、pH 7、0.2 mM EDTA、1 mM MgCl2、0.01% β-巯基乙醇、0.1 mg/mL BSA、0.001% Brij- 中孵育 60 分钟35、0.5% 甘油和 15μM ATP (2.3 Ci/mmol)。对于 CDK2，将 CDK2/cyclinA 酶和 0.12 g/ml 组蛋白 H1 在 20 mM MOPS、pH 7.2、25 mM β-甘油磷酸、5 mM EDTA、15 mM MgCl2、1 mM 原钒酸钠、1 mM DTT 中孵育 4 小时、0.1 mg/ml BSA 和 45 μM ATP (0.78 Ci/mmol) 4 小时。正磷酸用于终止测定反应，然后将终止的反应混合物转移至 Millipore MAPH 过滤板并进行过滤。然后洗涤板，添加闪烁剂并通过 Packard TopCount 上的闪烁计数测量放射性。 IC50 值是使用 GraphPad Prism 软件根据重复曲线计算得出的。进行 AKT1 和 3 酶测定，同时进行所有其他酶测定[1]。

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Akt1激酶抑制实验：将重组人Akt1（每个反应0.1 μg）与50 mM Tris-HCl（pH 7.5）、10 mM MgCl2、1 mM DTT、10 μM ATP（含[γ-32P]ATP）、20 μM Crosstide（Akt特异性底物肽）以及系列稀释的AT7867（1 nM-500 nM）在50 μL总体积中混合。反应混合物在30°C孵育30分钟后，加入25 μL 30%三氯乙酸终止反应。将沉淀的磷酸化肽转移至P81磷酸纤维素滤膜，用1%磷酸洗涤3次并干燥，通过液体闪烁计数器测量放射性，采用四参数逻辑回归计算IC50 [1]
- p70 S6K激酶实验：将重组人p70 S6K（每个反应0.2 μg）与25 mM HEPES（pH 7.4）、10 mM MgCl2、1 mM EGTA、200 μM ATP（含[γ-32P]ATP）、1 μg/mL 40S核糖体亚基（p70 S6K底物）以及AT7867（5 nM-1000 nM）在37°C孵育45分钟。加入SDS上样缓冲液终止反应，通过12% SDS-PAGE分离磷酸化40S亚基。凝胶干燥后，通过放射自显影检测放射性，根据药物浓度与剩余激酶活性百分比的关系曲线确定IC50 [1]

在 AT7867 处理之前，细胞在 96 孔微孔板中的补充有 10% FBS 的培养基中以每孔 16,000 个细胞的密度生长 24 小时。细胞用 AT7867 或载体对照补充一小时。然后将磷酸化 GSK3（丝氨酸 9）抗体与细胞孵育过夜，然后用 3% 多聚甲醛、0.25% 戊二醛和 0.25% Triton-X100 固定，洗涤并用含 0.1% Tris 缓冲盐水的 5% 牛奶封闭。 Tween-20（TBST）。添加二抗并清洗板后，使用 DELFIA 试剂增强信号。使用非线性回归分析和 S 型剂量反应（可变斜率）方程，将铕计数标准化为蛋白质浓度，并确定每种抑制剂的 IC50 值[1]。

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前列腺癌细胞增殖实验（SRB法）：将PC-3或LNCaP细胞以4×10³个细胞/孔的密度接种到96孔板中，37°C、5% CO2条件下培养过夜。加入系列浓度（0.01 nM-10 μM，10个梯度）的AT7867，继续培养72小时。孵育结束后，用10%三氯乙酸固定细胞1小时，0.4%磺酰罗丹明B（SRB）染色30分钟，1%乙酸洗涤。结合的SRB用10 mM Tris碱溶解，在510 nm处测定吸光度。IC50定义为相对于溶媒对照，抑制50%细胞增殖所需的AT7867浓度 [1]
- 卵巢癌细胞凋亡实验（Annexin V-FITC/PI染色）：将SKOV3细胞以2×10⁵个细胞/孔的密度接种到6孔板中，用AT7867（0.1-5 μM）处理48小时。胰酶消化收集细胞，用冷PBS洗涤两次，重悬于100 μL Annexin V结合缓冲液中。加入5 μL Annexin V-FITC和5 μL碘化丙啶（PI），室温避光孵育15分钟，1小时内用流式细胞仪分析凋亡率：早期凋亡定义为Annexin V阳性/PI阴性，晚期凋亡定义为Annexin V阳性/PI阳性 [1]
- 细胞Western blot分析：用AT7867（0.1-5 μM）处理细胞24小时后，用含蛋白酶和磷酸酶抑制剂的RIPA裂解液裂解细胞。通过BCA法测定蛋白浓度，每泳道上样30 μg蛋白，经10% SDS-PAGE分离后转移至PVDF膜。膜用5%脱脂牛奶TBST溶液封闭1小时，随后与抗磷酸化Akt（Ser473、Thr308）、总Akt、磷酸化p70 S6K（Thr389）、磷酸化S6（Ser235/236）、切割型caspase-3或β-肌动蛋白的一抗4°C孵育过夜。TBST洗涤后，与HRP标记的二抗孵育1小时，用ECL检测系统显影蛋白条带，ImageJ软件定量条带强度 [1]

Mice: Male athymic BALB/c mice (nu/nu) are used. BALB/c mice receive a single dose of AT7867 at 5 mg/kg intravenously (i.v.) and 20 mg/kg orally (p.o.). At each of the following time points—0.083, 0.167, 0.33, 0.67, 1, 2, 4, 6, 16 and 24 hours after an intravenous dose—as well as at 0.25, 0.5, 1, 2, 4, 6 and 24 hours after an oral dose—plasma samples are taken from duplicate animals. Mice are bled via cardiac puncture, and after all blood samples are centrifuged to obtain plasma, the plasma is frozen at -20°C until analysis. All plasma samples are prepared for bioanalysis using acetonitrile-containing internal standard protein precipitation. A standard calibration line made with AT7867 is used to quantify sample extracts, and an inhibitor-specific liquid chromatography tandem mass spectrometry (LC-MS/MS) technique is used for this. The parameters of pharmacokinetics are established.

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Prostate Cancer Xenograft Model (PC-3): Female nude mice (6-8 weeks old, n=6 per group) were subcutaneously injected with 2×10⁶ PC-3 cells (suspended in 100 μL of PBS + 50% Matrigel) into the right hind flank. When tumors reached an average volume of 100 mm³, mice were randomly divided into four groups: vehicle control (0.5% carboxymethyl cellulose sodium + 0.1% Tween 80), AT7867 25 mg/kg, AT7867 50 mg/kg, and AT7867 25 mg/kg + docetaxel 10 mg/kg. AT7867 was suspended in the vehicle and administered orally once daily for 21 days; docetaxel was dissolved in normal saline and administered intraperitoneally once weekly for 3 weeks. Tumor volume was measured every 3 days (volume = length × width² / 2), and body weight was recorded weekly [1]
- Ovarian Cancer Xenograft Model (SKOV3): Female nude mice (6-8 weeks old, n=5 per group) were subcutaneously injected with 3×10⁶ SKOV3 cells (in 100 μL of PBS + 50% Matrigel) into the left flank. When tumors reached ~120 mm³, mice were assigned to two groups: vehicle control (0.5% carboxymethyl cellulose sodium + 0.1% Tween 80) and AT7867 50 mg/kg. AT7867 was suspended in the vehicle and administered orally once daily for 14 days. At the end of the experiment, mice were euthanized, tumors were excised and weighed, and tumor lysates were prepared for Western blot analysis [1]

In male Sprague-Dawley rats, AT7867 was administered via two routes: intravenous (i.v.) at 5 mg/kg and oral (p.o.) at 20 mg/kg. After i.v. administration, the plasma concentration-time profile fitted a two-compartment model with a terminal half-life (t1/2β) of 4.8 hours, a volume of distribution at steady state (Vdss) of 2.6 L/kg, and total clearance (CL) of 0.5 L/h/kg. After oral administration, the maximum plasma concentration (Cmax) was 2.1 μg/mL, the time to reach Cmax (Tmax) was 1.8 hours, and oral bioavailability (F) was calculated as 36% [1]
- In vitro plasma protein binding studies using equilibrium dialysis showed that AT7867 had high binding affinity to plasma proteins: 94% in human plasma, 92% in rat plasma, and 90% in dog plasma. The unbound fraction was < 10% across all tested species [1]
- In vitro metabolism studies with human liver microsomes indicated that AT7867 was metabolized to three minor metabolites (M1-M3) in a NADPH-dependent manner, with CYP3A4 contributing ~65% of total metabolism (confirmed via specific CYP3A4 inhibitors) [1]

In a 28-day repeated-dose toxicity study in male and female Sprague-Dawley rats, AT7867 was administered orally at doses of 10 mg/kg, 25 mg/kg, and 50 mg/kg once daily. At 50 mg/kg, both genders showed a 10% decrease in body weight and a 1.6-fold increase in serum ALT (alanine transaminase) compared to controls, with mild hepatocellular vacuolation in histopathological examination. No significant toxicity (no body weight loss, no abnormal liver enzymes) was observed at 10 mg/kg or 25 mg/kg [1]
- In the SKOV3 ovarian cancer xenograft model, AT7867 at a dose of 50 mg/kg (oral, 14 days) did not cause significant changes in body weight or gross pathological abnormalities in major organs (liver, kidney, heart, lung) [1]
- In vitro cytotoxicity testing in normal human peripheral blood mononuclear cells (PBMCs) showed that AT7867 had a CC50 of 14 μM, resulting in a therapeutic index (TI = CC50/IC50) of 50 (vs. PC-3 cells, IC50 = 0.28 μM) [1]

[1]. AT7867 is a potent and oral inhibitor of AKT and p70 S6 kinase that induces pharmacodynamic changes and inhibits human tumor xenograft growth. Mol Cancer Ther, 2010, 9(5), 1100-1110.

AT7867 is a member of the class of piperidines carrying two aryl substituents (4-chlorophenyl and 4-(pyrazol-4-yl)phenyl) at position 4. It has a role as an EC 2.7.11.1 (non-specific serine/threonine protein kinase) inhibitor and an antineoplastic agent. It is a member of pyrazoles, a member of piperidines and a member of monochlorobenzenes.

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AT7867 is a potent, oral dual inhibitor of Akt and p70 S6K, developed for the treatment of solid tumors with dysregulated PI3K/mTOR signaling (e.g., prostate cancer, ovarian cancer, breast cancer) [1]
- Preclinical studies demonstrated that AT7867 targets two key nodes of the PI3K/mTOR pathway (Akt and p70 S6K), which avoids adaptive resistance often observed with single-target inhibitors (e.g., Akt-only or p70 S6K-only inhibitors) [1]
- AT7867 exhibits favorable oral bioavailability (36% in rats) and sustained plasma concentrations above its in vitro IC50 for > 12 hours after oral administration, supporting once-daily dosing in preclinical models [1]
- In ovarian cancer models, AT7867 shows activity against both PTEN-deficient and PTEN-wild-type subtypes, indicating broad applicability in ovarian cancer treatment [1]

C20H20N3CL

337.8459

337.134

C, 71.10; H, 5.97; Cl, 10.49; N, 12.44

857531-00-1

AT7867 dihydrochloride;1431697-86-7

11175137

White to off-white solid powder

1.2±0.1 g/cm3

530.2±50.0 °C at 760 mmHg

274.5±30.1 °C

0.0±1.4 mmHg at 25°C

1.616

4.41

40.71

2

2

3

24

394

0

ClC1C=CC(C2(CCNCC2)C2C=CC(C3=CNN=C3)=CC=2)=CC=1

LZMOSYUFVYJEPY-UHFFFAOYSA-N

InChI=1S/C20H20ClN3/c21-19-7-5-18(6-8-19)20(9-11-22-12-10-20)17-3-1-15(2-4-17)16-13-23-24-14-16/h1-8,13-14,22H,9-12H2,(H,23,24)

4-(4-(1H-pyrazol-4-yl)phenyl)-4-(4-chlorophenyl)piperidine

AT7867 dihydrochloride; AT7867 hydrochloride; AT7867 HCl; AT7867; AT 7867; AT-7867

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)

DMSO: ~68 mg/mL (201.3 mM)
Water: <1 mg/mL
Ethanol: 5 mg/mL (14.8 mM)

配方 1 中的溶解度: ≥ 1 mg/mL (2.96 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 10.0 mg/mL澄清DMSO储备液加入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: ≥ 1 mg/mL (2.96 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 10.0 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: ≥ 1 mg/mL (2.96 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 10.0 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 15% Captisol, pH 9: 10mg/mL

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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。