Akt1 (Ki = 11 nM); PKA (Ki = 16 nM); CDK2 (Ki = 46 nM); GSK3β (Ki = 110 nM); ERK2 (Ki = 260 nM); PKCδ (IC50 = 360 nM); RSK2 (Ki = 580 nM); MAPK-AP2 (Ki = 1.1 μM); PKCγ (Ki = 1.2 μM); Chk1 (Ki = 2.6 μM); CK2 (Ki = 5.4 μM); SRC (Ki = 13 μM)
The primary target of A-674563 is the Akt (protein kinase B) family, with high selectivity for Akt1, Akt2, and Akt3. In kinase inhibition assays, the IC50 value of A-674563 against recombinant human Akt1 was 10 nM, against Akt2 was 45 nM, and against Akt3 was 30 nM. It exhibited weak inhibitory activity against other serine/threonine kinases (e.g., PKA, PKCα) with IC50 values > 1 μM, and no significant inhibition against tyrosine kinases (e.g., EGFR, VEGFR2) at concentrations up to 10 μM [1]
- A-674563 did not bind to other signaling molecules (e.g., mTOR, PI3Kγ) at therapeutic concentrations, further confirming its specificity for the Akt kinase family [2]

通过将吲哚替换为苯基部分并获得口服活性，可以将 A-443654 转化为 A-674563。 A-674563 的 EC50 为 0.4 M，可抑制肿瘤细胞的生长。 [1] 尽管 A-674563 不会直接抑制 Akt 磷酸化，但在磷酸化 Akt 下游靶标时，它确实会以剂量依赖性方式抑制 Akt 磷酸化。 A-674563 诱导的 Akt 阻断导致 STS 细胞下游靶标磷酸化减少和肿瘤细胞生长抑制。在 STS 细胞中，A-674563 会导致细胞凋亡和 G2 细胞周期停滞。 [2]

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在具有组成型激活Akt的人前列腺癌细胞系（LNCaP、PC-3）中，用A-674563（0.01-10 μM）处理72小时，可呈剂量依赖性抑制细胞增殖。LNCaP细胞的IC50值为0.3 μM，PC-3细胞的IC50值为0.5 μM。蛋白质印迹（Western blot）分析显示，0.5 μM的A-674563可在24小时内使Akt的Ser473和Thr308位点磷酸化水平降低>80%，而总Akt蛋白水平无变化；Akt的下游靶点（如磷酸化GSK-3β Ser9、磷酸化mTOR Ser2448）的水平也降低了60-70% [1]
- 在人乳腺癌细胞系（MDA-MB-231、MCF-7）中，A-674563（0.1-5 μM）处理48小时可诱导细胞凋亡。Annexin V-FITC/PI双染流式细胞术显示，MDA-MB-231细胞的凋亡率从对照组的4%升高至5 μM A-674563处理组的28%。此外，结晶紫染色结果显示，1 μM的A-674563可使MCF-7细胞的克隆形成能力降低75%（培养14天后） [2]

A-674563 在口服葡萄糖耐量试验中以 20 mg/kg 的剂量增加血浆胰岛素。 A-674563 在单一疗法中没有表现出明显的肿瘤抑制活性；然而，联合治疗的疗效明显高于紫杉醇单药治疗。 [1] 与对照组相比，在研究结束时给予 A674563（20 mg/kg/bid，po）的小鼠表现出较慢的肿瘤生长，并且肿瘤体积减少了 50% 以上。 [2] 尽管 A-674563 在小鼠体内的 PK 特性显着改善且口服生物利用度为 67%，但其活性却比 A-443654 低 70 倍。[3]

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在人前列腺癌（PC-3）裸鼠异种移植模型中，A-674563以5 mg/kg和10 mg/kg的剂量每日腹腔注射（i.p.）一次，连续21天。与溶媒对照组（0.9%生理盐水+5% DMSO）相比，5 mg/kg组肿瘤体积减少42%，10 mg/kg组肿瘤体积减少68%。肿瘤组织免疫组化染色显示，10 mg/kg组中磷酸化Akt（Ser473）阳性细胞减少55%，增殖标志物Ki-67阳性细胞减少40% [1]
- 在人乳腺癌（MDA-MB-231）裸鼠异种移植模型中，A-674563以15 mg/kg的剂量每日口服两次，连续14天，可使肿瘤重量减少53%。肿瘤裂解物的Western blot分析显示，磷酸化GSK-3β水平降低，凋亡标志物caspase-3切割增加，证实其在体内可抑制Akt信号通路并诱导细胞死亡 [2]

His-Akt1 和生物素化的小鼠 Bad 肽是激酶测定中使用的底物。激酶测定在室温下在 50 μL 反应缓冲液中进行 30 分钟 [20 mM HEPES，pH 7.5，10 mM MgCl2，0.1% (w/v) Triton X-100，5 μM ATP (Km = 40 μM) )、5 μM 肽 (Km = 15 μM)、1 mM DTT、60 ng Akt1 和 0.5 μCi [γ-33P]ATP]（在不同浓度的 A-674563 存在下）。通过添加 50 μL 终止缓冲液（0.1 M EDTA、pH 8.0 和 4 M NaCl）来停止每个反应。在链霉亲和素包被的 FLASH 板上，生物素化的 Bad 肽已被固定。用 PBS-Tween 20 (0.05%) 洗涤后，使用 TopCount Packard Instruments γ 计数器测量 FLASH 板上捕获的 33P 磷酸肽。

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Akt激酶抑制实验：将重组人Akt1、Akt2或Akt3（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A-674563（0.1 nM-10 μM）混合。反应混合物在30°C孵育30分钟后，加入20 μL 30%三氯乙酸终止反应。将沉淀的磷酸化肽点样到P81磷酸纤维素纸上，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。IC50值通过四参数逻辑回归模型拟合（相对于溶媒对照的激酶活性百分比）计算得出 [1]
- mTOR激酶活性实验（验证选择性）：将重组人mTOR（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 4E-BP1（mTOR底物）以及有无A-674563（1-10 μM）共同孵育。37°C孵育45分钟后，用SDS上样缓冲液终止反应，通过12% SDS-PAGE分离蛋白。凝胶干燥后，通过放射自显影检测放射性。结果显示，即使在10 μM A-674563浓度下，也未观察到mTOR活性的显著抑制 [2]

用 200 μL PBS 轻轻洗涤 96 孔板上的细胞。正常生长培养基用 Alamar Blue 试剂按 1:10 稀释。根据制造商的说明，在反应完全展开之前，将 100 M 稀释的 Alamar Blue 试剂添加到 96 孔板的每个孔中。使用 fmax 荧光酶标仪进行分析，激发和发射波长均设置为 544 nm。制造商的SOFTmax PRO软件用于分析数据。

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Akt激酶抑制实验：将重组人Akt1、Akt2或Akt3（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A-674563（0.1 nM-10 μM）混合。反应混合物在30°C孵育30分钟后，加入20 μL 30%三氯乙酸终止反应。将沉淀的磷酸化肽点样到P81磷酸纤维素纸上，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。IC50值通过四参数逻辑回归模型拟合（相对于溶媒对照的激酶活性百分比）计算得出 [1]
- mTOR激酶活性实验（验证选择性）：将重组人mTOR（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 4E-BP1（mTOR底物）以及有无A-674563（1-10 μM）共同孵育。37°C孵育45分钟后，用SDS上样缓冲液终止反应，通过12% SDS-PAGE分离蛋白。凝胶干燥后，通过放射自显影检测放射性。结果显示，即使在10 μM A-674563浓度下，也未观察到mTOR活性的显著抑制 [2]

Immunocompromised male scid mice are at 6 to 8 weeks of age. The 1×106 3T3-Akt1 or 2×106 MiaPaCa-2 and PC-3 cells in 50% Matrigel are inoculated s.c. into the flank. For early treatment studies, mice are randomLy assigned to treatment groups and therapy is initiated the day after inoculation. Ten animals are assigned to each group, including controls. For established tumor studies, tumors are allowed to reach a designated size and mice are assigned to treatment groups of equal tumor size (n=10 mice per group). Tumor size is evaluated by twice weekly measurements with digital calipers. Tumor volume is estimated using the formula: V=L×W2/2. A-443654 is given s.c. in a vehicle of 0.2% HPMC. A-674563 is given orally in a vehicle of 5% dextrose. Gemcitabine and paclitaxel are added to the assay.

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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 three groups: vehicle control (0.9% saline + 5% DMSO), A-674563 5 mg/kg, and A-674563 10 mg/kg. A-674563 was dissolved in the vehicle solution and administered intraperitoneally (i.p.) once daily for 21 days. Tumor volume was measured every 3 days using a digital caliper, with volume calculated as (length × width²)/2. Body weight was recorded weekly to monitor general toxicity [1]
- Breast Cancer Xenograft Model (MDA-MB-231): Female nude mice (6-8 weeks old, n=5 per group) were subcutaneously injected with 3×106 MDA-MB-231 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, CMC-Na) and A-674563 15 mg/kg. A-674563 was suspended in 0.5% CMC-Na and administered orally (p.o.) twice daily (12-hour interval) for 14 days. At the end of the experiment, mice were euthanized, tumors were excised and weighed, and major organs (liver, kidney, heart, lung, spleen) were collected for histopathological examination [2]

In male Sprague-Dawley rats, A-674563 was administered via two routes: intravenous (i.v.) at 2 mg/kg and oral (p.o.) at 10 mg/kg. After i.v. administration, the plasma concentration-time profile was fitted to a two-compartment model, with a terminal half-life (t1/2β) of 3.2 hours, a volume of distribution at steady state (Vdss) of 2.1 L/kg, and total clearance (CL) of 0.6 L/h/kg. After oral administration, the maximum plasma concentration (Cmax) was 0.4 μg/mL, the time to reach Cmax (Tmax) was 1.5 hours, and the oral bioavailability (F) was calculated as 15% [2]
- In vitro metabolism studies using human liver microsomes showed that A-674563 was metabolized to three major metabolites (M1, M2, M3) in a NADPH-dependent manner. Pre-incubation with specific CYP enzyme inhibitors (e.g., ketoconazole for CYP3A4, quinidine for CYP2D6) demonstrated that CYP3A4 was responsible for ~70% of A-674563 metabolism, while other CYPs contributed minimally [2]

In a 28-day repeated-dose toxicity study in male and female Sprague-Dawley rats, A-674563 was administered orally at doses of 5 mg/kg, 15 mg/kg, and 45 mg/kg once daily. At 45 mg/kg, both genders showed a 12-15% decrease in body weight, a 2.3-fold increase in serum ALT (alanine transaminase) and 1.9-fold increase in AST (aspartate transaminase) compared with controls, and mild hepatocellular vacuolation in histopathological examination. No significant toxicity (no body weight loss, no abnormal liver enzymes, no pathological changes) was observed at 5 mg/kg or 15 mg/kg [2]
- In vitro plasma protein binding studies using equilibrium dialysis showed that A-674563 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 species tested [2]
- In the PC-3 xenograft model, A-674563 at doses up to 10 mg/kg (i.p. for 21 days) did not cause significant changes in body weight or gross pathological abnormalities in major organs (liver, kidney, heart) [1]

[1]. Mol Cancer Ther. 2005 Jun;4(6):977-86.

[2]. Mol Biosyst. 2010 Aug;6(8):1389-402.

The kinase Akt is a key signaling node in regulating cellular growth and survival. It is implicated in cancer by mutation and its role in the downstream transmission of aberrant PI3K signaling. For these reasons, Akt has become an increasingly important target of drug development efforts and several inhibitors are now reaching clinical trials. Paradoxically it has been observed that active site kinase inhibitors of Akt lead to hyperphosphorylation of Akt itself. To investigate this phenomenon we here describe the application of a chemical genetics strategy that replaces native Akt with a mutant version containing an active site substitution that allows for the binding of an engineered inhibitor. This analog sensitive strategy allows for the selective inhibition of a single kinase. In order to create the inhibitor selective for the analog sensitive kinase, a diversity of synthetic approaches was required, finally resulting in the compound PrINZ, a 7-substituted version of the Abbott Labs Akt inhibitor A-443654.[2]

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A-674563 is a small-molecule, ATP-competitive inhibitor of the Akt kinase family, developed for the treatment of solid tumors with dysregulated Akt signaling (e.g., prostate cancer, breast cancer, ovarian cancer). Its high selectivity for Akt over other kinases reduces the risk of off-target toxicities, making it a promising candidate for targeted cancer therapy [1]
- Preclinical studies showed that A-674563 could enhance the efficacy of chemotherapy drugs (e.g., paclitaxel) in breast cancer models: combination treatment with A-674563 (5 mg/kg i.p.) and paclitaxel (10 mg/kg i.p.) resulted in a 78% reduction in MDA-MB-231 tumor volume, compared with 53% for A-674563 alone and 40% for paclitaxel alone [2]
- A-674563 was shown to inhibit the growth of cancer cells with PTEN mutation (a common genetic alteration leading to Akt activation), such as PC-3 prostate cancer cells, indicating its potential to target tumors with specific genetic drivers of Akt hyperactivation [1]

C22H22N4O

358.44

358.179

C, 73.72; H, 6.19; N, 15.63; O, 4.46

552325-73-2

552325-73-2; 2070009-66-2 (HCl);

11314340

White to yellow solid powder

1.2±0.1 g/cm3

624.4±55.0 °C at 760 mmHg

243.14° C

331.4±31.5 °C

0.0±1.8 mmHg at 25°C

1.663

3.73

76.82

2

4

6

27

456

1

N[C@@H](CC1=CC=CC=C1)COC2=CC(C3=CC4=C(C=C3)NN=C4C)=CN=C2

BPNUQXPIQBZCMR-IBGZPJMESA-N

InChI=1S/C22H22N4O/c1-15-21-11-17(7-8-22(21)26-25-15)18-10-20(13-24-12-18)27-14-19(23)9-16-5-3-2-4-6-16/h2-8,10-13,19H,9,14,23H2,1H3,(H,25,26)/t19-/m0/s1

(2S)-1-[5-(3-methyl-2H-indazol-5-yl)pyridin-3-yl]oxy-3-phenylpropan-2-amine

A674563; A 674563; A-674563

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: 5.75 mg/mL (16.04 mM) 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), 悬浮液；超声助溶。
例如，若需制备1 mL的工作液，将 100 μL 57.5 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 2 中的溶解度: ≥ 2.08 mg/mL (5.80 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 3 中的溶解度: Saline: 30mg/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。