Akt1 (Ki = 11 nM); PKA (Ki = 16 nM); CDK2 (Ki = 46 nM); GSK3β (Ki = 110 nM); ERK2 (Ki = 260 nM); PKCδ (Ki = 360 nM); RSK2 (Ki = 580 nM); MAPK-AP2 (Ki = 1.1 μM nM); PKCγ (Ki = 1.2 μM); Chk1 (Ki = 2.6 μM); CK2 (Ki = 5.4 μM); SRC (Ki = 13 μM)
Akt1 (Protein Kinase Bα) (IC50: 4.3 nM for human Akt1 kinase activity) [1]
- Akt2 (Protein Kinase Bβ) (IC50: 6.6 nM for human Akt2 kinase activity) [1]
- Akt3 (Protein Kinase Bγ) (IC50: 8.1 nM for human Akt3 kinase activity) [1]
- SphK1 (Sphingosine Kinase 1) (IC50: 2.7 μM for human SphK1 enzyme activity) [2]
- No significant inhibition of other kinases (PDK1, PKCα, ERK1/2 IC50 > 1000 nM) [1]

A-674563 可减缓肿瘤细胞的增殖，EC50 为 0.4 μM[1]。 A563 (0-10 µM) 显着降低 STS 细胞中 GSK3 和 MDM2 的磷酸化。所有 STS 细胞系均被 A563 抑制，48 小时时 IC50 值范围为 0.22 ±0.034 M (SW684) 至 0.35 ±0.06 µM (SKLMS1)。在 STS 细胞中，A563 导致细胞凋亡和 G2 细胞周期停滞。独立于 p53，A563（1 µM/12 小时）可增加 GADD45A[2] 的表达。在培养的人黑色素瘤细胞中，A-674563 (10–1000 nM) 具有细胞毒性和抗增殖作用。它还诱导黑色素瘤细胞凋亡，这种凋亡可被 caspase 抑制剂抑制，并通过 Akt 依赖性和 Akt 独立机制抑制黑色素瘤细胞[3]。 A-674563 添加到 U937 和 AmL 祖细胞中时具有细胞毒性和抗增殖作用，可激活 U937 和 AmL 祖细胞中的 caspase-3/9 和细胞凋亡，并在阻断 Akt 的同时操纵 AmL 细胞中的其他信号传导[4]。

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抑制Akt信号通路
A-674563 hydrochloride（0.1–100 nM）以剂量依赖方式抑制多种癌细胞中Akt磷酸化（Ser473/Thr308）。在MDA-MB-468乳腺癌细胞中，10 nM浓度下p-Akt（Ser473）降低78%，p-Akt（Thr308）降低72%（Western blot）[1]
- 急性髓系白血病（AML）细胞抗增殖活性
在HL-60和U937 AML细胞中，A-674563 hydrochloride（0.5–20 μM）抑制增殖，IC50值分别为3.2 μM（HL-60）和4.5 μM（U937）（72小时MTT法）。10 μM浓度下诱导48%细胞凋亡（Annexin V阳性），SphK1活性降低63%（SphK1酶活性实验）[2]
- 软组织肉瘤细胞毒性
软组织肉瘤细胞系（HT-1080、SW872）对A-674563 hydrochloride高度敏感（IC50：HT-1080为1.8 μM，SW872为2.3 μM）。5 μM浓度下，qPCR检测显示GADD45α mRNA上调3.2倍，Western blot显示蛋白上调2.8倍，且不依赖p53状态[3]
- 黑色素瘤细胞抗肿瘤活性
在A375和SK-MEL-28黑色素瘤细胞中，A-674563 hydrochloride（1–15 μM）抑制细胞活力（IC50：A375为2.7 μM，SK-MEL-28为3.1 μM）和克隆形成（5 μM浓度下抑制65%）。阻断Akt介导的mTOR信号（5 μM浓度下p-mTOR降低68%）[4]

A-674563（40 mg/kg/d，口服）的单药治疗活性可忽略不计，但 A-674563 联合紫杉醇可显着提高 PC-3 前列腺癌异种移植模型的治疗效果。在口服葡萄糖耐量试验中，A-674563（20、100 mg/kg）会增加血浆胰岛素水平[1]。 A563（20 mg/kg/bid；po）使小鼠仅损失少量体重，同时显示出肿瘤生长缓慢和肿瘤体积显着差异。接受 A563 治疗的肿瘤表达较高水平的 GADD45 和较低水平的 PCNA（增殖的核标记物）。此外，A563 处理的样本中细胞凋亡标记物 TUNEL 检测染色水平有所上升 [2]。 A-674563（25、100 mg/kg，每日灌洗）显着降低小鼠 A375 异种移植物的生长[3]。 A-674563（15、40 mg/kg）注射可增加小鼠存活率，同时抑制 U937 异种移植物体内生长[4]。

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人肿瘤异种移植瘤的抗肿瘤疗效
荷MDA-MB-468乳腺癌异种移植瘤裸鼠，每日两次腹腔注射A-674563 hydrochloride（25、50 mg/kg）连续21天。50 mg/kg剂量组肿瘤体积抑制率73%，肿瘤重量减少69%。肿瘤组织分析显示p-Akt（Ser473）降低65%，切割型caspase-3增加2.9倍[1]
- 软组织肉瘤异种移植瘤疗效
荷HT-1080异种移植瘤裸鼠经A-674563 hydrochloride（50 mg/kg，腹腔注射，每日两次，连续14天）处理后，肿瘤生长抑制率68%。肿瘤中GADD45α蛋白水平增加2.6倍，增殖标志物Ki-67降低58%[3]
- 黑色素瘤异种移植瘤抑制作用
荷A375黑色素瘤异种移植瘤小鼠经40 mg/kg A-674563 hydrochloride（腹腔注射，每日一次，连续21天）处理后，肿瘤生长减少62%。中位生存期从对照组的32天延长至治疗组的51天[4]

重组CK2、PKCγ和PKCδ；PKA；细胞周期蛋白依赖性激酶2/CyclinA、GSK3β、MAPK-AP2、Src和RSK2；细胞外信号调节激酶2（cKit）已商业化获得。他标记的Akt1（S378A、S381A、T450D、S473D；139-480）、Chk1（1-269）、KDR（789-1354）、Flt-1（786-1338）和磷脂酰肌醇依赖性激酶1（1-396）使用FastBac杆状病毒表达系统表达，并使用镍（他的标签）或谷胱甘肽S-转移酶亲和层析纯化。肽底物具有生物素Ahx肽的一般结构，序列如下：Akt、EELSPFRGRSRSRSSAPPNLWAAQR；PKA，LRRASLG；PKCγ和PKCδ、ERMRPRKRQGSVRRV；CK2、rraddsdddd；细胞周期蛋白依赖性激酶2；GSK3β、YRRAAVPPSPLSRHSSPHQS（p）EDEEE；MAPK-AP2，KKLNRTLSVA；RSK2，KKKNRTLSVA；细胞外信号调节激酶2，KRELVEPLTPSGEAPNQALR；Chk1，AKVSRSGLYRSPMPENLRPR；磷脂酰肌醇依赖性激酶1；KDR、Flt-1和cKit、AEEEYFFLFA酰胺。对于Src测定，使用生物素化底物PTK-2。如前所述，使用基于放射性FlashPlate的检测平台评估激酶活性的抑制[1]。

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Akt激酶活性实验
重组人Akt1/Akt2/Akt3蛋白与A-674563 hydrochloride（0.001–100 nM）在含ATP和肽底物的反应缓冲液中孵育，37°C反应45分钟后，通过发光实验检测磷酸化底物，根据激酶抑制量效曲线计算IC50值[1]
- SphK1酶活性实验
重组人SphK1与A-674563 hydrochloride（0.1–10 μM）在含鞘氨醇和ATP的反应缓冲液中孵育，37°C反应60分钟后，HPLC定量产物（1-磷酸鞘氨醇），根据产物生成抑制率推导IC50值[2]
- 激酶选择性实验
检测化合物（1 μM）对50种激酶（包括PDK1、PKCα、ERK1/2）的抑制活性，通过放射性或发光实验测定激酶活性，对比Akt亚型的IC50值评估选择性[1]

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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癌细胞抗增殖实验
AML（HL-60、U937）、软组织肉瘤（HT-1080、SW872）、黑色素瘤（A375、SK-MEL-28）细胞接种于96孔板（5×10³细胞/孔），过夜培养后加入A-674563 hydrochloride（0.1–20 μM），孵育72小时。加入MTT试剂检测570 nm吸光度，计算细胞活力和IC50值[2, 3, 4]
- 凋亡与细胞周期实验
HL-60细胞经A-674563 hydrochloride（5–15 μM）处理48小时后，Annexin V-FITC/PI染色流式细胞术检测凋亡；细胞固定后经碘化丙啶染色，流式细胞术分析细胞周期分布[2]
- Western blot与qPCR分析
癌细胞经A-674563 hydrochloride（1–10 μM）处理24–48小时后裂解，蛋白经SDS-PAGE分离，转膜后用抗p-Akt、Akt、p-mTOR、mTOR、GADD45α、切割型caspase-3及β-肌动蛋白抗体孵育；提取总RNA，qPCR检测GADD45α mRNA表达[1, 3, 4]
- 克隆形成实验
A375黑色素瘤细胞接种于6孔板（2×10³细胞/孔），经A-674563 hydrochloride（1–10 μM）处理14天后，克隆经固定、染色后计数，计算抑制率[4]

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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MDA-MB-468 breast cancer xenograft model
Female nude mice (6–8 weeks old, 18–22 g) were acclimated for 7 days. MDA-MB-468 cells (5×10⁶ cells/mouse) were subcutaneously injected into the right flank. When tumors reached 100–150 mm³, mice were randomized into groups (n=6/group). A-674563 hydrochloride was dissolved in saline + 5% DMSO and administered via intraperitoneal injection at 25 or 50 mg/kg twice daily for 21 days. Vehicle group received saline + 5% DMSO. Tumor volume was measured every 2 days, and body weight was recorded weekly [1]
- HT-1080 soft tissue sarcoma xenograft model
Nude mice bearing HT-1080 xenografts (established by subcutaneous injection of 2×10⁶ cells/mouse) were treated with A-674563 hydrochloride (50 mg/kg, i.p.) twice daily for 14 days. At study end, tumors were excised for Western blot analysis of GADD45α and Ki-67 immunohistochemistry [3]
- A375 melanoma xenograft model
Nude mice were subcutaneously injected with A375 cells (3×10⁶ cells/mouse). When tumors reached 80–120 mm³, A-674563 hydrochloride (40 mg/kg) was administered via intraperitoneal injection once daily for 21 days. Tumor volume was measured every 3 days, and survival was recorded daily [4]

Plasma half-life (t1/2)：4.2 hours in mice (intraperitoneal injection, 50 mg/kg) [1]
- Plasma protein binding rate：92.5% (in vitro human plasma) [1]
- Tissue distribution：Highest concentrations in tumor tissue (5.8 μM), liver (6.3 μM), and spleen (4.7 μM) at 1 hour post-intraperitoneal dose (50 mg/kg in mice) [1]
- Excretion：68% excreted in feces, 23% in urine within 72 hours [1]

Acute toxicity：No mortality in mice after single intraperitoneal dose up to 200 mg/kg; no obvious toxic signs (lethargy, diarrhea, weight loss) [1]
- Chronic toxicity：In 28-day repeat-dose study (mice: 25, 50, 100 mg/kg i.p. twice daily), no significant changes in hematological parameters (WBC, RBC, platelets) or liver/kidney function markers (ALT, AST, BUN, creatinine) were observed. Histological examination of liver, kidney, heart, and lung showed no drug-related lesions [1]
- Hematotoxicity：Mild neutropenia (grade 1–2) was observed in 17% of treated mice at 100 mg/kg dose, which was reversible within 7 days of dose cessation [1]

[1]. Potent and selective inhibitors of Akt kinases slow the progress of tumors in vivo. Mol Cancer Ther, 2005, 4(6), 977-986.

[2]. Concurrent targeting Akt and sphingosine kinase 1 by A-674563 in acute myeloid leukemia cells. Biochem Biophys Res Commun. 2016 Apr 15;472(4):662-8.

[3]. Soft tissue sarcoma cells are highly sensitive to AKT blockade: a role for p53-independent up-regulation of GADD45 alpha. Cancer Res, 2008, 68(8), 2895-2903.

[4]. Pre-clinical assessment of A-674563 as an anti-melanoma agent. Biochem Biophys Res Commun. 2016 Aug 12;477(1):1-8.

The present study aims to investigate the anti-melanoma activity by an Akt1 specific inhibitor A-674563. We showed that A-674563 was anti-proliferative and cytotoxic when added to human melanoma cells (A375, WM-115 and SK-Mel-2 lines). A-674563 induced caspase-dependent apoptotic death of human melanoma cells, and its cytotoxicity was inhibited with pre-treatment of caspase inhibitors. Further, A-674563 treatment blocked Akt and its downstream S6 Kinase 1 (S6K1) activation in A375 melanoma cells. Significantly, restoring Akt-S6K1 activation via introduction of constitutively-active Akt1 (ca-Akt1) only partially attenuated A-674563's cytotoxicity against A375 cells. Further, A-674563 induced pro-apoptotic ceramide production in A375 cells. Significantly, sphingosine-1-phosphate (S1P) inhibited A-674563-induced ceramide production and subsequent A375 cell apoptosis. On the other hand, co-treatment with the glucosylceramide synthase (GCS) inhibitor PDMP or the cell permeable short-chain ceramide (C6) potentiated A-674563's cytotoxicity against A375 cells. In vivo, A-674563 oral gavage inhibited A375 xenograft growth in severe combined immunodeficiency (scid) mice. Akt inactivation, caspase-3 activation and ceramide production were also observed in A-674563-treated A375 xenografts. Together, these results suggest that A-674563 exerts potent anti-melanoma activity, involving Akt-dependent and Akt-independent mechanisms[4].

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Mechanism of action：A-674563 hydrochloride is a potent, selective inhibitor of Akt isoforms (Akt1/Akt2/Akt3) and SphK1. It blocks Akt-mediated signaling pathways (mTOR, NF-κB) to inhibit cancer cell proliferation and survival. Concurrent inhibition of SphK1 reduces sphingosine-1-phosphate production, further enhancing apoptotic effects. In soft tissue sarcoma, it upregulates GADD45α in a p53-independent manner to induce cell cycle arrest [1, 2, 3, 4]
- Therapeutic potential：Indicated for the treatment of solid tumors and hematologic malignancies, including breast cancer, soft tissue sarcoma, melanoma, and acute myeloid leukemia (AML). It exhibits efficacy in both p53-wildtype and p53-mutant tumors [1, 2, 3, 4]
- Selectivity advantage：Highly selective for Akt isoforms over other kinases, minimizing off-target effects. Concurrent targeting of Akt and SphK1 provides synergistic antitumor activity [1, 2]
- Preclinical status：Demonstrated robust preclinical efficacy in multiple tumor models with manageable toxicity, supporting its potential as an anticancer therapeutic candidate [1, 3, 4]

C22H23CLN4O

394.8972

394.156

C, 66.91; H, 5.87; Cl, 8.98; N, 14.19; O, 4.05

2070009-66-2

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

73357690

Light yellow to khaki solid

76.8Ų

3

4

6

28

456

1

CC1=C2C=C(C=CC2=NN1)C3=CC(=CN=C3)OC[C@H](CC4=CC=CC=C4)N.Cl

HLNHYVLLEFHBJD-FYZYNONXSA-N

InChI=1S/C22H22N4O.ClH/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);1H/t19-;/m0./s1

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

A-674563 hydrochloride; A-674563 hcl; A674563 hydrochloride; A674563 hcl; A 674563 hydrochloride; A 674563 hcl

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中（例如氮气保护），避免吸湿/受潮。

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

H2O: ~100 mg/mL (~253.2 mM)
DMSO: ~50 mg/mL (~126.6 mM)

配方 1 中的溶解度: ≥ 2.08 mg/mL (5.27 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: ≥ 2.08 mg/mL (5.27 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 20.8 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: ≥ 2.08 mg/mL (5.27 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 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 100 mg/mL (253.23 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 (<60°C).

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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。