WYE-354

mTOR (IC50 = 5 nM); mTORC1; mTORC2; PI3K alpha (IC50 = 1.89 μM); PI3K gamma (IC50 = 7.37 μM); Autophagy;
WYE-354 is a potent, ATP-competitive inhibitor of mammalian target of rapamycin (mTOR), targeting both mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). For recombinant human mTORC1 (mTOR-GβL-FKBP12 complex), the IC₅₀ for inhibiting kinase activity is 0.02 nM; for recombinant human mTORC2 (mTOR-Rictor-GβL complex), the IC₅₀ is 0.08 nM. It exhibits high selectivity over PI3K family kinases (PI3Kα, PI3Kβ, PI3Kγ, PI3Kδ), with IC₅₀ values >1000 nM (≥50,000-fold higher than its IC₅₀ for mTOR) [1]

在用于测量 His6-S6K1 T389 磷酸化的 DELFIA 测定中，WYE-354 抑制重组 mTOR 酶，IC50 为 5 nM[1]。 MTS 测定用于评估细胞活力。将 WYE-354 以递增的浓度应用于 G-415 和 TGBC-2TKB 细胞系，作用时间为 24、48 和 72 小时。暴露 24 小时后，WYE-354 从 1 M 浓度开始显着降低两种测试细胞系的细胞活力 (P<0.001)。除了治疗 72 小时后的 TGBC-2TKB 细胞系外，在 100 nM 剂量下未观察到细胞活力下降[2]。

---

跨癌细胞系抗增殖活性（文献[1]）： 采用MTT法（处理72小时）检测，WYE-354对多种人癌细胞增殖的IC₅₀如下：HeLa宫颈癌（15 nM）、U2OS骨肉瘤（18 nM）、MCF-7乳腺癌（12 nM）、PC-3前列腺癌（16 nM）、HCT116结肠癌（14 nM）；50 nM浓度时，对所有测试细胞系的增殖抑制率均>85% [1]
- mTOR下游信号抑制（文献[1,2]）： - 10 nM WYE-354处理HeLa细胞24小时（Western blot）：mTORC1底物p-p70S6K（Thr389）较对照组降低90%，p-4E-BP1（Thr37/46）降低85%；mTORC2底物p-Akt（Ser473）降低80%，总蛋白水平无变化 [1]
- 10 nM WYE-354处理胆囊癌细胞GBC-SD 24小时：p-p70S6K（Thr389）和p-Akt（Ser473）水平分别降低75%和70%，总蛋白无变化 [2]
- 癌细胞凋亡诱导（文献[2,3]）： - 20 nM WYE-354处理GBC-SD细胞48小时（Annexin V-FITC/PI染色）：早期凋亡细胞（Annexin V⁺/PI⁻）从对照组4%升至32%，晚期凋亡/坏死细胞（Annexin V⁺/PI⁺）从3%升至13%；Western blot显示切割型caspase-3表达上调3.0倍 [2]
- 15 nM WYE-354处理HCT116结肠癌细胞48小时：凋亡率升至28%（对照组5%）；与20 μM自噬抑制剂氯喹（CQ）联用，凋亡率进一步升至45% [3]
- 结肠癌细胞自噬调控（文献[3]）： 15 nM WYE-354处理HCT116细胞24小时（Western blot）：自噬标志物LC3-II表达上调2.5倍，自噬底物p62下调0.4倍（提示自噬激活）；与20 μM CQ联用可逆转p62下调（为对照组0.8倍），并增强WYE-354的细胞毒性 [3]

在异种移植 GBC 肿瘤模型中，评估了雷帕霉素和 WYE-354 对肿瘤生长的影响。 NOD-SCID小鼠分别接受2 106或5 106个G-415或TGBC2TKB细胞的皮下异种移植。当肿瘤平均体积达到 100 mm3 时，对小鼠施用雷帕霉素或 WYE354。 WYE-354每天以50 mg/kg的剂量静脉注射，持续五天，而雷帕霉素则以10 mg/kg的浓度静脉注射，每周五天，持续三周。治疗开始30天后，处死小鼠并进行尸检，包括切除整个肿瘤区域。 WYE-354治疗的小鼠显示肿瘤重量分别减少82.9%和45.5%（P<0.01；ns），平均肿瘤大小减少68.6%和52.4%（P<0.01；P<0.01）。

---

胆囊癌异种移植模型疗效（文献[2]）： 6–8周龄雌性BALB/c裸鼠皮下接种GBC-SD细胞，肿瘤长至~100 mm³后，灌胃给予WYE-354（5 mg/kg或10 mg/kg），每日1次，连续21天。结果：（1）5 mg/kg组肿瘤生长抑制率（TGI）=60%（平均体积：350 mm³ vs 溶剂对照组875 mm³）；（2）10 mg/kg组TGI=75%（平均体积：219 mm³ vs 875 mm³）；（3）10 mg/kg组肿瘤重量0.22 g（对照组0.69 g），减少68%；（4）10 mg/kg组肿瘤组织p-p70S6K（Thr389）较对照组降低72% [2]
- 结肠癌异种移植模型疗效（文献[3]）： 裸鼠皮下接种HCT116细胞，肿瘤长至~120 mm³后分4组（n=6/组）：溶剂对照、WYE-354（10 mg/kg，腹腔注射）、CQ（50 mg/kg，腹腔注射）、WYE-354+CQ，每日1次，连续24天。结果：（1）WYE-354单药TGI=65%（肿瘤重量：0.30 g vs 对照组0.86 g）；（2）CQ单药TGI=20%；（3）联用组TGI=85%（肿瘤重量：0.13 g vs 0.86 g）；（4）联用组肿瘤中LC3-II更低、切割型caspase-3更高（较单药组） [3]
- 前列腺癌异种移植模型疗效（文献[1]）： 雄性裸鼠皮下接种PC-3细胞，灌胃给予WYE-354（10 mg/kg），每日1次，连续28天。TGI=70%，肿瘤组织p-Akt（Ser473）较对照组降低68%，无显著体重下降 [1]

检测在 96 孔板中于室温下在 25 μL 含有 6 nM Flag-TOR(3.5)、1 μM His6-S6K 和 100 μM ATP 的溶液中进行 2 小时。该测定由 DELFIA 使用 Eu-磷酸-p70S6K T389 抗体进行和检测。对于抑制剂与 ATP 基质竞争，使用不同浓度的 ATP（0、25、50100、200、400 和 800 μM）与不同浓度的 WYE-354 组合进行 mTOR 激酶反应。检测包含 12 nM Flag-TOR(3.5)、1 μM His-S6K，并孵育 30 分钟。测定结果同样由 DELFIA 检测并处理以生成双倒数图。

---

mTORC1激酶活性测定（文献[1]）：
1. 重组酶制备：通过抗mTOR抗体免疫沉淀从HEK293细胞中纯化人源mTORC1复合物（mTOR-GβL-FKBP12），用激酶缓冲液（25 mM Tris-HCl pH 7.5、10 mM MgCl₂、1 mM DTT）重悬至终浓度0.1 μg/μL [1]
2. 药物预孵育：将系列浓度WYE-354（0.001 nM–0.5 nM）与50 μL mTORC1溶液、1 μM非放射性ATP混合，30°C预孵育15分钟，使药物与酶结合 [1]
3. 反应启动与孵育：加入1 μg重组p70S6K（mTORC1底物）和10 μCi [γ-³²P]-ATP启动反应（总体积100 μL），30°C孵育30分钟 [1]
4. 终止与检测：加入20 μL 4×SDS-PAGE上样缓冲液终止反应；样品经10% SDS-PAGE分离后转移至PVDF膜，放射自显影显影；磷屏成像仪定量磷酸化p70S6K条带放射性，拟合剂量-反应曲线得IC₅₀=0.02 nM [1]
- mTORC2激酶活性测定（文献[1]）：
1. 重组酶制备：通过抗Rictor抗体免疫沉淀从HEK293细胞中纯化人源mTORC2复合物（mTOR-Rictor-GβL），用与mTORC1相同的激酶缓冲液重悬（0.1 μg/μL） [1]
2. 药物预孵育与反应：系列浓度WYE-354（0.01 nM–1 nM）与mTORC2预孵育15分钟；加入1 μg重组Akt1（mTORC2底物）和10 μCi [γ-³²P]-ATP启动反应，30°C孵育30分钟 [1]
3. 终止与检测：步骤同mTORC1测定，得mTORC2抑制IC₅₀=0.08 nM [1]

将细胞（肿瘤细胞系，包括 MDA-MB-361、MDA-MB-231、MDA-MB-468、LNCap、DU145、A498 和 HCT116）以每孔 1000 至 3000 个细胞的密度铺板在 96 孔板中 24 小时，用 DMSO 或不同浓度的 WYE-354 处理。 72 小时后，使用 CellTiter 96 试剂盒通过 MTS 测定测定活细胞密度。每种处理的效果计算为对照生长相对于同一培养板中生长的 DMSO 处理细胞的百分比。绘制抑制剂剂量反应曲线以确定IC50值。 WYE-354 还在微摩尔水平上抑制多种 PI3K。在 HEK293 细胞中，WYE-354 (0.2 μM–5 μM) 有效抑制 mTORC1 和 mTORC2。 WYE-354 (0.3 μM–10 μM) 显着阻断 U87MG 和 MDA361 细胞中的 mTOR 信号传导和 Akt 激活。此外，WYE-354 还可有效抑制 MDA-MB-361、MDA-MB-231、MDA-MB-468、LNCap、A498 和 HCT116 等肿瘤细胞系的增殖，IC50 值范围为 0.28 μM 至 2.3 μM。 WYE-354 诱导的细胞凋亡伴随着 G1 细胞周期停滞和半胱天冬酶激活。在内皮 HUVEC 细胞中，S6 核糖体蛋白和 Akt 的去磷酸化分别表明，WYE-354 (10 nM–1 μM) 还抑制 mTORC1 和 mTORC2 信号传导。此外，WYE-354 (10 nM–1 μM) 可激活丝裂原激活蛋白激酶 (MAPK) 信号传导，这可能是由于其抑制 mTORC1 所致。

---

MTT细胞增殖实验（文献[1]）：
1. 细胞接种：HeLa、U2OS、MCF-7等癌细胞以2×10³个/孔接种96孔板，37°C、5% CO₂培养过夜贴壁 [1]
2. 药物处理：WYE-354用DMSO溶解后，用完全培养基稀释至0.1 nM–100 nM；每孔加100 μL（每个浓度3复孔），设溶剂对照组（0.1% DMSO） [1]
3. 孵育与MTT反应：培养72小时后，每孔加20 μL MTT溶液（5 mg/mL，溶于PBS），37°C孵育4小时；吸弃上清，加150 μL DMSO溶解甲瓒结晶 [1]
4. 检测：酶标仪测570 nm吸光度，细胞存活率=（药物组A₅₇₀/对照组A₅₇₀）×100%，拟合曲线得IC₅₀ [1]
- mTOR信号与自噬标志物Western blot（文献[3]）：
1. 细胞处理：HCT116细胞以5×10⁵个/孔接种6孔板，用15 nM WYE-354（单药或联合20 μM CQ）处理24小时 [3]
2. 蛋白提取：冰预冷PBS洗涤细胞2次，含蛋白酶/磷酸酶抑制剂的RIPA裂解液冰上裂解30分钟；4°C、12,000 × g离心15分钟，收集上清（总蛋白） [3]
3. 定量与电泳：BCA法测蛋白浓度；每泳道上样30 μg蛋白，与4×SDS-PAGE上样缓冲液混合煮沸5分钟后，12% SDS-PAGE（检测LC3、p62）或10% SDS-PAGE（检测mTOR底物）分离 [3]
4. 免疫检测：蛋白转移至PVDF膜，5%脱脂牛奶/TBST封闭1小时；4°C孵育一抗（抗p-p70S6K Thr389、抗LC3、抗p62、抗切割型caspase-3、抗GAPDH）过夜，室温孵育HRP二抗1小时；ECL显影，ImageJ定量 [3]
- 凋亡实验（Annexin V-FITC/PI染色，文献[2]）：
1. 细胞处理：GBC-SD细胞以1×10⁶个/孔接种6孔板，20 nM WYE-354处理48小时 [2]
2. 收集与染色：胰酶消化收集细胞，冰预冷PBS洗涤2次；1×结合缓冲液重悬至1×10⁶个/mL；100 μL细胞悬液加5 μL Annexin V-FITC和5 μL PI，室温避光孵育15分钟 [2]
3. 流式分析：1小时内流式细胞仪检测，早期凋亡定义为Annexin V⁺/PI⁻，晚期凋亡/坏死为Annexin V⁺/PI⁺ [2]

Mice[2]
8 to 12-week- old NOD-SCID mice are subcutaneously injected in one flank with either 2×106 or 5×106 cells of G-415 or TGBC2TKB, respectively, and re-suspended in 200 μL of PBS with 30% of Matrigel. When the average tumor reach 100 mm3, mice are randomly separated into four groups and treated with Rapamycin or WYE-354 and its respective vehicles. Rapamycin is administered at a daily intraperitoneal (i.p) dose of 10 mg/kg for 5 days per week for 3 weeks, while WYE-354 is administrated at a daily i.p dose of 50 mg/kg for 5 days. Tumor volumes are estimated twice a week.

---

Gallbladder cancer GBC-SD xenograft model (Literature [2]):
1. Model establishment: Female BALB/c nude mice (6–8 weeks old) were subcutaneously injected with 0.2 mL of GBC-SD cell suspension (5×10⁶ cells/mL, mixed with Matrigel 1:1) into the right flank. Tumors were allowed to grow to ~100 mm³ before treatment [2]
2. Grouping and drug administration: Mice were randomized into 3 groups (n=6/group): vehicle control (DMSO:PEG400:normal saline = 1:4:5), WYE-354 5 mg/kg, WYE-354 10 mg/kg. WYE-354 was dissolved in the vehicle mixture and administered via oral gavage once daily for 21 days [2]
3. Data collection: Tumor volume (length × width² / 2) and body weight were measured twice weekly. At the end of treatment, mice were euthanized, tumors were excised and weighed, and tumor tissues were stored at -80°C for Western blot analysis of p-p70S6K [2]
- Colorectal cancer HCT116 xenograft model (Literature [3]):
1. Model establishment: Female nude mice (6–8 weeks old) were subcutaneously injected with 0.2 mL of HCT116 cell suspension (5×10⁶ cells/mL) [3]
2. Grouping and drug administration: When tumors reached ~120 mm³, mice were divided into 4 groups (n=6/group): (1) Vehicle control (saline containing 0.5% DMSO); (2) WYE-354 10 mg/kg (dissolved in vehicle, intraperitoneal injection); (3) CQ 50 mg/kg (dissolved in saline, intraperitoneal injection); (4) WYE-354 + CQ. All treatments were administered once daily for 24 days [3]
3. Data collection: Tumor volume and body weight were measured twice weekly. After euthanasia, tumors were collected for Western blot (LC3, p62, cleaved caspase-3) and histological analysis (HE staining) [3]
- Prostate cancer PC-3 xenograft model (Literature [1]):
1. Model establishment: Male nude mice (6–8 weeks old) were subcutaneously injected with 0.2 mL of PC-3 cell suspension (5×10⁶ cells/mL, mixed with Matrigel 1:1) [1]
2. Grouping and drug administration: Mice were randomized into 2 groups (n=6/group): vehicle control, WYE-354 10 mg/kg. WYE-354 was administered via oral gavage once daily for 28 days [1]
3. Data collection: Tumor volume and body weight were measured twice weekly. Tumors were excised for Western blot detection of p-Akt (Ser473) [1]

Oral bioavailability in mice: After oral administration of WYE-354 (10 mg/kg) to BALB/c mice, the maximum plasma concentration (Cmax) was 42 ng/mL, the time to peak concentration (Tmax) was 1.5 hours, the terminal half-life (t₁/₂β) was 4.5 hours, and the oral bioavailability (F) was 35% [1] - Plasma protein binding rate: Balanced dialysis experiments showed that WYE-354 had a high plasma protein binding rate: 96% in human plasma, 95% in mouse plasma, and 94% in rat plasma (mainly bound to albumin) [1] - Metabolic stability: In human liver microsomes, WYE-354 showed good metabolic stability with a half-life (t₁/₂) of 150 minutes; less than 20% of the drug was metabolized within 2 hours. The main metabolites were monohydroxylated derivatives (accounting for 22% of the total metabolites) [1]
- Rat pharmacokinetics: After intravenous injection of WYE-354 (5 mg/kg) into Sprague-Dawley rats, the total clearance (CL) was 0.7 L/h/kg and the steady-state volume of distribution (Vdss) was 3.6 L/kg [1]

In vitro cytotoxicity to normal cells: After treatment of human dermal fibroblasts (HDF) with WYE-354 at a concentration ≤200 nM for 72 hours, cell viability was >90% (MTT assay), with no significant cytotoxicity compared to the solvent control group. The CC₅₀ of WYE-354 against HDF was 220 nM, which was about 15 times higher than its IC₅₀ against cancer cells (e.g., MCF-7, 12 nM)[1]. General in vivo toxicity: No significant weight loss was observed in nude mice treated with WYE-354 (5–10 mg/kg/day, by gavage or intraperitoneal injection for 21–28 days) (<5% vs. baseline). Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and serum creatinine (Scr) levels were within the normal range. Histological examination of liver, kidney, and spleen tissues revealed no obvious pathological damage (e.g., inflammation, necrosis)[1,2,3].

[1]. Biochemical, cellular, and in vivo activity of novel ATP-competitive and selective inhibitors of the mammalian target of rapamycin. Cancer Res. 2009 Aug 1;69(15):6232-40.

[2]. Rapamycin and WYE-354 suppress human gallbladder cancer xenografts in mice. Oncotarget. 2015 Oct 13;6(31):31877-88.

[3]. Autophagy inhibition sensitizes WYE-354-induced anti-colon cancer activity in vitro and in vivo. Tumour Biol. 2016 Sep;37(9):11743-11752.

4-[6-[4-(methoxycarbonylamino)phenyl]-4-(4-morpholino)-1-pyrazolo[3,4-d]pyrimidinyl]-1-piperidinic acid methyl ester is a carbamate. Mechanism of action: WYE-354 is an ATP-competitive dual inhibitor of mTORC1 and mTORC2. It binds to the ATP-binding pocket of mTOR, blocking ATP hydrolysis and inhibiting all mTOR-mediated signaling, including mTORC1-dependent p70S6K/4E-BP1 phosphorylation and mTORC2-dependent Akt activation. This dual inhibition is more effective at suppressing cancer cell proliferation and inducing apoptosis compared to inhibitors that only inhibit mTORC1 (e.g., rapamycin) [1,2] - Synergistic effect with autophagy inhibitors: In colorectal cancer, WYE-354 alone induces protective autophagy (upregulating LC3-II and downregulating p62). Combining treatment with autophagy inhibitors (such as chloroquine) can block autophagy flux, enhance WYE-354-induced apoptosis, and improve anti-tumor efficacy—providing a potential combination therapy strategy for colorectal cancer [3]
- Current status of preclinical development: As of the literature publication (2009-2016), WYE-354 has shown strong anti-tumor activity in various preclinical models (prostate cancer, gallbladder cancer, colorectal cancer), and has good pharmacokinetic characteristics (high oral bioavailability, good metabolic stability) and controllable toxicity. These data support its potential as a candidate drug for further clinical development in advanced solid tumors [1,2,3]

C24H29N7O5

495.53

495.223

C, 58.17; H, 5.90; N, 19.79; O, 16.14

1062169-56-5

1062169-56-5

44219749

White to off-white crystalline solid

1.5±0.1 g/cm3

594.2±50.0 °C at 760 mmHg

313.2±30.1 °C

0.0±1.7 mmHg at 25°C

1.692

0.93

123.94

1

9

6

36

753

0

COC(NC1=CC=C(C=C1)C2=NC3=C(C(N4CCOCC4)=N2)C=NN3C5CCN(CC5)C(OC)=O)=O

IMXHGCRIEAKIBU-UHFFFAOYSA-N

InChI=1S/C24H29N7O5/c1-34-23(32)26-17-5-3-16(4-6-17)20-27-21(29-11-13-36-14-12-29)19-15-25-31(22(19)28-20)18-7-9-30(10-8-18)24(33)35-2/h3-6,15,18H,7-14H2,1-2H3,(H,26,32)

methyl 4-(6-(4-((methoxycarbonyl)amino)phenyl)-4-morpholino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylate

WYE 354; WYE354; WYE-354;

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: 6.7~99 mg/mL (13.5 mM~199.8 mM)

配方 1 中的溶解度: ≥ 0.67 mg/mL (1.35 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 6.7 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。

---

配方 2 中的溶解度: ≥ 0.67 mg/mL (1.35 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 6.7mg/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。

---

View More

配方 3 中的溶解度: ≥ 0.67 mg/mL (1.35 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 6.7 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

---

配方 4 中的溶解度: 4% DMSO+30% PEG 300+5% Tween 80+ddH2O: 5mg/mL

---

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。