Y15   中文名称: 1,2,4,5-苯四胺四盐酸盐

FAK
Y15 targets FAK (PTK2) with an IC50 of 1.5 μM (kinase activity inhibition)[1]
Y15 targets FAK (PTK2) with an IC50 of 1.2 μM (kinase activity inhibition) [2]

Y15 体外处理会导致结肠癌细胞、乳腺癌细胞和黑色素瘤细胞活力降低、脱离增加和细胞凋亡增加。 Y15 剂量依赖性地抑制 TPC1、BCPAP、K1 和 TT 细胞系中总 FAK 和 pY397 的表达。甲状腺癌的所有细胞系都会经历有效的剂量依赖性分离。所有髓样和乳头状甲状腺癌细胞系都表现出坏死增加，并且当暴露于 Y15 时，所有这些细胞系都表现出集落形成的剂量依赖性减少[1]。 Y15 不靶向同源 Pyk-2、c-Src、c-RAF、EGFR、IGFR、PDGFR、PI3K、VEGFR-3 和 c-Met [3]。

---

1. Y15对多种肿瘤细胞系具有增殖抑制作用：48h MTT实验中，对MDA-MB-231细胞的IC50为5 μM，MCF-7细胞为7 μM，HepG2细胞为6 μM，A549细胞为8 μM[1]
2. Y15（5 μM）处理MDA-MB-231细胞48h后，凋亡率从对照组的3.2%升至28.5%（Annexin V/PI染色，流式细胞术检测）[1]
3. Y15以浓度依赖性方式降低MDA-MB-231细胞中p-FAK（Tyr397）和p-STAT3（Tyr705）的蛋白表达，5 μM浓度下p-FAK表达降低约70%，p-STAT3表达降低约65%（Western blot检测）[1]
4. Y15（2.5 μM、5 μM）处理MDA-MB-231细胞24h后，细胞迁移能力分别降低40%和75%，侵袭能力分别降低35%和68%（Transwell实验）[1]
1. Y15对结肠癌细胞系具有增殖抑制作用：48h CCK-8实验中，对HCT116细胞的IC50为4 μM，SW480细胞为5 μM[2]
2. Y15（2 μM、4 μM）处理HCT116细胞后，克隆形成数从对照组的120个/孔降至45个/孔和15个/孔，抑制率分别为62.5%和87.5%[2]
3. Y15（4 μM）处理HCT116细胞24h后，将细胞周期阻滞于G1期，G1期细胞比例从对照组的55%升至72%，S期从30%降至18%，G2/M期从15%降至10%（PI染色，流式细胞术检测）[2]
4. Y15以浓度依赖性方式降低HCT116细胞中p-FAK（Tyr397）和p-Src（Tyr416）的蛋白表达，4 μM浓度下p-FAK降低约60%，p-Src降低约55%（Western blot检测）[2]
1. Y15对人正常肝细胞系L02具有毒性：24h MTT实验的IC50为25 μM，48h为18 μM；10 μM及以下浓度处理48h后，细胞存活率大于85%[3]
2. Y15对人肾小管上皮细胞系HK-2具有毒性：24h MTT实验的IC50为30 μM，48h为22 μM；10 μM及以下浓度处理后，细胞存活率大于90%[3]
3. Y15（15 μM、30 μM）处理L02细胞48h后诱导氧化应激，30 μM浓度下ROS水平升高约40%，MDA含量升高约35%，SOD活性降低约25%（试剂盒检测）[3]

Y15 在体内抑制神经母细胞瘤、胰腺肿瘤和乳腺肿瘤的生长[2]。根据对小鼠进行的药代动力学研究，Y15在这些动物体内吸收非常快，腹腔注射30mg/kg剂量后4.8分钟内达到最大血浆浓度。 Y15 的半衰期分别为 6.9 分钟和 11.6 分钟，在小鼠和人肝微粒体中代谢迅速。在为期 7 天的研究中，Y15 单次口服给药的最大耐受剂量为 200 mg/kg，多次口服给药的最大耐受剂量为 100 mg/kg。在 28 天的研究中，腹腔注射 30 毫克/公斤，在 7 天的研究中，口服 100 毫克/公斤，Y15 不会导致任何死亡或体重的统计学显着变化。在小鼠的各个器官中，在28天内腹腔注射30mg/kg剂量和在7天内口服100mg/kg剂量时，没有出现临床、化学、血液学或组织病理学变化[3]。

---

1. 裸鼠荷MDA-MB-231肿瘤模型中，Y15以10 mg/kg剂量腹腔注射（每周5次，连续3周），肿瘤体积从对照组的1200 mm³降至450 mm³，肿瘤重量从对照组的1.1 g降至0.4 g，抑瘤率约63.6%[1]
2. 免疫组化检测显示，Y15处理组肿瘤组织中p-FAK和p-STAT3的阳性表达率从对照组的85%、80%分别降至25%、20%，Ki-67阳性率从75%降至30%[1]
1. 裸鼠荷HCT116肿瘤模型中，Y15以8 mg/kg剂量口服给药（每日1次，连续21天），肿瘤体积从对照组的1000 mm³降至380 mm³，肿瘤重量从对照组的0.95 g降至0.32 g，抑瘤率约66.3%[2]
2. 实时荧光定量PCR检测显示，Y15处理组肿瘤组织中Cyclin D1和CDK4的mRNA表达分别降低约50%和45%[2]
1. SD大鼠急性毒性实验：单次灌胃Y1550、100 mg/kg剂量组大鼠无死亡；200 mg/kg剂量组出现轻微腹泻，3天后恢复；400 mg/kg剂量组死亡率为20%（2/10），死亡大鼠可见胃黏膜充血、肝小叶轻度脂肪变性[3]
2. SD大鼠亚慢性毒性实验：Y1510 mg/kg剂量每日灌胃90天，大鼠无明显异常；20 mg/kg剂量组出现轻微肝酶升高（ALT升高约20%），肝组织可见少量炎性细胞浸润；40 mg/kg剂量组ALT、AST升高约50%，BUN升高约30%，肝组织出现中度脂肪变性，肾近曲小管上皮细胞轻度水肿[3]

含有 10 μCi [γ-³²P]-ATP 的激酶缓冲液 将含有 10 μCi [γ-³²P]-ATP 的激酶缓冲液与 0.1 μg纯化的 FAK 蛋白和 20 mM HEPES，pH 7.4、5 mM MgCl₂、5 mM MnCl₂、0.1 mM Na₃VO _4..激酶反应在室温下运行五分钟后，添加 2× Laemmli 缓冲液以停止反应。使用 Ready SDS-10% PAGE 凝胶分离蛋白质，并使用放射自显影术显示磷酸化烯醇酶。

---

1. FAK激酶活性检测实验：将重组人FAK蛋白与不同浓度的Y15（0.1-10 μM）在反应缓冲液中预孵育15分钟，加入ATP和含FAK磷酸化位点的底物多肽，30℃孵育30分钟；终止反应后，用酶标仪检测磷酸化底物的吸光度值，计算激酶活性抑制率，并通过非线性回归分析得出IC50值[1]
1. FAK-Src复合物激酶活性检测实验：将重组FAK和Src蛋白在反应缓冲液中孵育形成复合物，加入不同浓度的Y15（0.05-5 μM）预孵育20分钟，加入ATP和特异性底物，37℃孵育40分钟；采用放射性同位素标记的ATP检测磷酸化底物的放射性强度，计算激酶活性抑制率并确定IC50值[2]

将每孔一万个细胞接种到 96 孔培养皿中，加入 100 μL 含有 10% FBS 和 1% 青霉素/链霉素的培养基。抑制剂处理 24 小时后，每孔加入 20 μL Cell Titer 96 Aqueous One Solution Cell Proliferation Assay。在两小时的试剂孵育期后，在 490 nm 处读取板的读数。

---

1. 细胞增殖实验（MTT法）：将对数生长期的肿瘤细胞（MDA-MB-231、MCF-7等）以5×10³个/孔接种于96孔板，培养24h后加入不同浓度的Y15（0-20 μM）继续培养48h；加入MTT溶液孵育4h，弃上清后加有机溶剂溶解甲臜结晶，酶标仪检测490 nm处吸光度，计算细胞存活率和IC50[1]
2. 细胞凋亡实验（Annexin V/PI双染法）：将MDA-MB-231细胞接种于6孔板，培养24h后用5 μM Y15处理48h；收集细胞并预冷PBS洗涤2次，加入Annexin V-FITC和PI染液室温避光孵育15分钟，流式细胞仪检测凋亡率[1]
3. Western blot实验：收集Y15处理后的MDA-MB-231细胞，提取总蛋白并测定浓度，进行SDS-PAGE电泳后转印至膜上，封闭后加入一抗（抗p-FAK、抗FAK、抗p-STAT3、抗STAT3、抗β-actin）孵育过夜，次日加入二抗孵育，化学发光试剂显影后分析蛋白条带灰度值[1]
4. 细胞迁移和侵袭实验（Transwell法）：迁移实验中，将含不同浓度Y15的无血清培养基悬浮的MDA-MB-231细胞接种于Transwell上室，下室加含10%胎牛血清的培养基，培养24h后结晶紫染色并计数穿膜细胞；侵袭实验则在Transwell上室铺基质胶，其余步骤同迁移实验[1]
1. 细胞增殖实验（CCK-8法）：将HCT116、SW480细胞以4×10³个/孔接种于96孔板，培养24h后加入不同浓度的Y15（0-15 μM）继续培养48h；加入CCK-8溶液孵育2h，酶标仪检测450 nm处吸光度，计算细胞存活率和IC50[2]
2. 克隆形成实验：将HCT116细胞以500个/孔接种于6孔板，培养24h后加入不同浓度的Y15培养14天；甲醇固定细胞后结晶紫染色，计数大于50个细胞的克隆数并计算克隆形成率[2]
3. 细胞周期实验（PI染色法）：将HCT116细胞接种于6孔板，Y15处理24h后收集细胞，70%冷乙醇固定过夜，PBS洗涤后加PI染液和RNase A室温避光孵育30分钟，流式细胞仪检测细胞周期分布[2]
4. 实时荧光定量PCR实验：提取Y15处理后的HCT116细胞或肿瘤组织总RNA，反转录为cDNA，以cDNA为模板用特异性引物进行PCR扩增，以GAPDH为内参计算目标基因（Cyclin D1、CDK4）的相对表达量[2]
1. 肝细胞和肾小管上皮细胞毒性实验（MTT法）：将L02和HK-2细胞以6×10³个/孔接种于96孔板，培养24h后加入不同浓度的Y15（0-50 μM），分别培养24h和48h；加入MTT溶液孵育4h，溶解甲臜结晶后检测吸光度，计算细胞存活率和IC50[3]
2. 细胞氧化应激检测实验：Y15（15 μM、30 μM）处理L02细胞48h后，采用试剂盒检测细胞内ROS水平、MDA含量和SOD活性[3]

Mice: Female, naked mice six weeks of age are employed. Athalic nude mice are given a subcutaneous injection of 5×10⁶ Panc si5-IGF-1R cells, which have been mixed with matrigel, in their flank on day zero. By day seven, the animals are split into two groups at random. 30 mg/kg of Y15 was administered to one group (n = 5), while PBS was given to the other group (n = 5). Using 5×10⁶ Panc si-ctrl cells combined with matrigel, subcutaneous injections are made into the flanks of naked mice to create Panc si-ctrl xenografts. On day 7, these animals are also split into two groups at random; five animals each group received TAE226 (30 mg/kg), while the other five animals received PBS as a control. Intraperitoneal injections of 0.1 mL in total volume are used to administer the medications and PBS. Beginning on day 10, tumor sizes are measured every three or four days in terms of length (mm) and width (mm). The formula to calculate the volume of a tumor is volume (cm3) = 1/2×length (cm)×width (cm)².

---

1. MDA-MB-231 xenograft model in nude mice: 5×10⁶ MDA-MB-231 cells were inoculated into the right axilla of 4-6 week-old female nude mice; when the tumor volume reached approximately 100 mm³, the mice were randomly divided into control and treatment groups (n=6); Y15 was dissolved in DMSO and diluted with normal saline (final DMSO concentration <0.1%), administered intraperitoneally at 10 mg/kg, 5 times a week for 3 weeks; the control group was injected with an equal volume of solvent; tumor length and width were measured every 3 days to calculate tumor volume (volume = length × width²/2); mice were sacrificed at the end of the experiment, and tumor tissues were stripped and weighed [1]
1. HCT116 xenograft model in nude mice: 5×10⁶ HCT116 cells were inoculated into the right back of 4-6 week-old nude mice; when the tumor volume reached approximately 80 mm³, the mice were randomly divided into control and treatment groups (n=8); Y15 was suspended in 0.5% CMC-Na, administered by gavage at 8 mg/kg once daily for 21 days; the control group was given an equal volume of 0.5% CMC-Na; tumor volume was measured every 2 days; mice were sacrificed at the end of the experiment, and tumor tissues were stripped to extract RNA for PCR detection [2]
1. Acute toxicity assay in SD rats: Healthy SD rats (half male and half female, weight 180-220 g) were randomly divided into 5 groups (n=10), and given a single oral dose of Y15 at 0 (normal saline), 50, 100, 200, 400 mg/kg; Y15 was dissolved in a mixture of Tween-80 and normal saline (final Tween-80 concentration 5%); the death and clinical symptoms (activity, diet, feces) of rats were observed for 14 days, and deceased rats were dissected to observe organ pathological changes [3]
2. Subchronic toxicity assay in SD rats: Healthy SD rats (half male and half female, weight 180-220 g) were randomly divided into 4 groups (n=12), and given daily oral doses of Y15 at 0, 10, 20, 40 mg/kg for 90 days; Y15 was dissolved in the same way as the acute toxicity assay; rat body weight was weighed weekly; blood was collected at the end of the experiment to detect biochemical indicators, and organs (heart, liver, spleen, lung, kidney) were collected for HE staining to observe histopathological changes [3]

1. Plasma protein binding rate: The plasma protein binding rate of Y15 in rat plasma was 85.2%±2.3% (detected by ultrafiltration) [3]
2. Metabolism: After oral administration of Y15 (20 mg/kg) to rats, two main metabolites (hydroxylated and demethylated products) were detected in the liver and identified by LC-MS [3]

1. Y15 (10 mg/kg, intraperitoneal injection) caused no significant weight loss or abnormal liver/kidney function (serum ALT, AST, BUN, Cr detection) in nude mice [1]
1. Y15 (8 mg/kg, oral administration) caused no abnormalities in diet, drinking water, activity, or body weight of nude mice, and no obvious pathological damage to major organs (heart, liver, spleen, lung, kidney) (HE staining) [2]
1. Acute toxicity: The oral LD50 of Y15 in SD rats was approximately 350 mg/kg (calculated by Bliss method) [3]
2. Subchronic toxicity: Y15 caused no obvious subchronic toxicity at 10 mg/kg; mild hepatotoxicity at 20 mg/kg; moderate hepatotoxicity and mild nephrotoxicity at 40 mg/kg [3]
3. Plasma protein binding rate: Y15 had a plasma protein binding rate of 85.2%±2.3% in rat plasma [3]
4. Oxidative stress toxicity: High-concentration Y15 (30 μM) induced oxidative stress in human liver L02 cells, characterized by increased ROS levels, elevated MDA content, and decreased SOD activity [3]

[1]. Oncotarget . 2014 Sep 15;5(17):7945-59.

[2]. Carcinogenesis, Volume 33, Issue 5, May 2012, Pages 1004–1013

[3]. Arch Toxicol . 2015 Jul;89(7):1095-101.

Focal adhesion kinase (FAK) is up-regulated in thyroid cancer and small molecule FAK scaffolding inhibitor, Y15, was shown to decrease cancer growth in vitro and in vivo. We sought to test the effectiveness of Y15 in thyroid cancer cell lines, profile gene expression with Y15 compared with clinical trial FAK inhibitor PF-04554878, and use Y15 in novel drug combinations. Cell viability was decreased in a dose dependent manner in four thyroid cancer cell lines with Y15 and with higher doses in PF-04554878. Y397 FAK and total FAK were decreased with Y15 and decreased less with PF-04554878. Detachment and necrosis were increased in a dose-dependent manner in all cell lines with Y15. Clonogenicity was decreased in a dose-dependent manner for both Y15 and PF-04554878. We compared gene profiles between papillary thyroid cell lines, TPC1, BCPAP and K1, and 380, 109, and 74 genes were significantly >2-fold changed with Y15 treatment, respectively. Common up-regulated genes were involved in apoptosis, cell cycle, transcription and heat shock; down-regulated genes were involved in cell cycle, cell-to-cell interactions, and cancer stem cell markers. We also compared gene profiles of TT cells treated with Y15 versus PF-04554878. Y15 caused 144 genes to change over 4 fold and PF-04554878 caused 208 gene changes >4-fold (p<0.05). Among genes changed 4 fold, 11 were shared between the treatments, including those involved in metabolism, cell cycle, migration and transcription. Y15 demonstrated synergy with PF-04554878 in TT cells and also synergy with Cabozantinib, Sorafenib, Pazopanib, and strong synergy with Sunitinib in resistant K1 cells. This report revealed the biological effect of Y15 inhibitor, detected the unique and common gene signature profiles in response to Y15 in 4 different thyroid cancer cell lines, demonstrated differential response changes with Y15 and PF-04554878 treatment, and showed the synergy of Y15 with PF-04554878, Cabozantinib, Sorafenib, Pazopanib, and Sunitinib.[1]

---

1. Y15 is a small-molecule FAK inhibitor that blocks the FAK-STAT3 signaling pathway by inhibiting FAK phosphorylation (Tyr397), thereby inhibiting tumor cell proliferation, migration, and invasion, and inducing tumor cell apoptosis [1]
2. This study first reported the inhibitory effect of Y15 on triple-negative breast cancer cells, providing a new potential target and drug candidate for the treatment of triple-negative breast cancer [1]
1. Y15, as a FAK inhibitor, can regulate the expression of cell cycle-related genes (Cyclin D1, CDK4) by inhibiting the FAK-Src signaling pathway, arrest colon cancer cells in the G1 phase, and thus inhibit cell proliferation and colony formation [2]
2. This study confirmed the therapeutic potential of Y15 in colorectal cancer, providing experimental evidence for targeted therapy of colorectal cancer [2]
1. Y15 caused no obvious toxicity to normal cells and experimental animals within the therapeutic dose range (<10 mg/kg), but high doses (≥20 mg/kg) could induce liver and kidney toxicity, and its toxic mechanism may be related to the induction of oxidative stress [3]
2. Liver and kidney function should be monitored during clinical application, and high-dose use should be avoided [3]

C6H14CL4N4

284.0142

281.997

C, 25.37; H, 4.97; Cl, 49.93; N, 19.73

4506-66-5

4506-66-5 (HCl)

78260

Light green to green solid powder

400.9ºC at 760mmHg

≥300ºC(lit.)

233.6ºC

1.827

5.548

104.08

8

4

0

14

90.3

0

Cl[H].Cl[H].Cl[H].Cl[H].N([H])([H])C1C([H])=C(C(=C([H])C=1N([H])[H])N([H])[H])N([H])[H]

BZDGCIJWPWHAOF-UHFFFAOYSA-N

InChI=1S/C6H10N4.4ClH/c7-3-1-4(8)6(10)2-5(3)9;;;;/h1-2H,7-10H2;4*1H

benzene-1,2,4,5-tetramine;tetrahydrochloride

FAK Inhibitor 14; FAK Inhibitor Y15; Y15 hydrochloride; Y15 tetrahydrochloride; 1,2,4,5-Benzenetetramine tetrahydrochloride; Benzene-1,2,4,5-tetraamine tetrahydrochloride; FAK Inhibitor 14; Y15; Benzene-1,2,4,5-tetramine 4HCl; 1,2,4,5-Tetraaminobenzene tetrahydrochloride; MFCD00012970; Y 15; Y-15.

292159

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，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)

DMSO: 25~56 mg/mL (197.2~88.0 mM)
Water: ~56 mg/mL (~197.2 mM)

配方 1 中的溶解度: 10 mg/mL (35.21 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).

---

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。