TRAIL (IC50 = 64.6±9.1 µM)

LY303511 本质上与 LY294002 相同，但它不能有效抑制 PI3K，因为吗啉环中的 -O 已取代 -NH。在用 LY303511 处理的细胞中观察到钙黄绿素扩散增加，与 LY294002 水平相当。根据免疫印迹，LY303511 可以增强间隙连接细胞间通讯 (GJIC)，尽管这种作用与 AKT 磷酸化的降低并不对应 [1]。 LY303511 通过上调死亡受体和激活 H2O2-MAPK 来增加 SHEP-1 神经母细胞瘤细胞的 TRAIL 敏感性。将不同浓度的 TRAIL、LY303511 (LY30) 以及两者的组合应用于 SHEP-1 细胞（与 LY303511 预孵育 1 小时，然后与 TRAIL 孵育 4 小时）。 TRAIL 在 25、50 和 100 ng/mL 浓度下，SHEP-1 细胞的活细胞分数大约下降 10%、15% 和 30%；然而，用 12.5、25 或 50 μM LY303511 处理的细胞没有表现出相同的反应。活力没有影响。另一方面，LY303511 (25 μM) 孵育一小时和 50 ng/mL TRAIL 暴露四小时产生了相当大的协同效应（与单独使用 TRAIL 相比，使用 LY303511+TRAIL 的活细胞减少了约 40%），细胞减少约 15%[2]。 PI3K 活性的阴性对照是 LY303511。 Wortmannin (100 nM) 不会影响 MIN6 胰岛素瘤细胞中的全细胞外向 K+ 电流，而 LY294002 和 LY303511 会导致电流以剂量依赖性方式被可逆抑制（IC50 分别为 9.0±0.7 μM 和 64.6±9.1 μM） 。 β 细胞表现出高水平的 Kv2.1 和 Kv1.4 表达。在转染 Kv2.1 的 tsA201 细胞中，分别在 50 μM LY294002 和 100 μM LY303511 下观察到可逆电流抑制。 LY303511 的 IC50 为 64.6±9.1 µM，在 500 µM 浓度下最大可抑制约 90% 的电流（每个浓度 n≥5 个细胞）[3]。

当肿瘤生长至约150mm3的体积时，此时35只小鼠已形成肿瘤，进行载体或LY303511（10mg/kg/天）的腹膜内治疗。超过15%的小鼠在21天后需要被处死，因为肿瘤生长太快；由于平均肿瘤体积估计不准确，这些数据被抑制。提供10 mg/kg/天的LY303511足以预防体内PC-3肿瘤的形成[4]。

LY303511 在结构上与 LY294002 相同，只是吗啉环中的 -NH 被 -O 取代，并且不会有效抑制 PI3K。用 LY303511 处理细胞会导致钙黄绿素扩散增加，与 LY294002 的水平相似。通过免疫印迹测量，LY303511 增加间隙连接细胞间通讯 (GJIC) 的能力不会与 AKT 磷酸化的抑制同时发生。

人神经母细胞瘤 SHEP-1 细胞保存在补充有 10% 胎牛血清和 1% 青霉素的 DMEM 中。在典型的生存测定中，LY303511（12.5、25 和 50 μM）、TRAIL（25、50 和 100 ng/mL）以及两者的组合（用 LY303511 预孵育 1 小时，然后用 TRAIL 孵育 4 小时）暴露于铺在24孔板中的SHEP-1细胞（每孔8×104）24小时。结晶紫测定用于测定细胞毒性。药物暴露后，用 PBS 清洗细胞，然后与 200 μL 结晶紫溶液一起孵育 20 分钟。用蒸馏水除去过量的结晶紫溶液后，将剩余的晶体溶解在20%乙酸中。使用自动 ELISA 读数器，使用 595 nm 波长处的吸光度来评估活力。使用 2,000 单位/mL 过氧化氢酶、4 μM JNK 抑制剂 SP600125、10 μM p38 抑制剂 SB202190、20 μM MAPK/ERK 激酶 (MEK) 抑制剂 PD98059、50 μM caspase-8 抑制剂 Z-IETD-FMK 进行类似的细胞活力实验或泛半胱天冬酶抑制剂 Z-VAD-FMK，或死亡受体阻断抗体（4 μg/mL 抗 DR4 或 1 μg/mL 抗 DR5），或在用小干扰 RNA (siRNA) 转染的细胞中用于沉默 JNK 和 ERK分别表达。在添加 TRAIL 之前，将细胞与 LY303511 和适当的抑制剂或过氧化氢酶预孵育 1 小时。

In zebrafish model, LY303511 inhibits CAL 27-xenografted tumor growth. Therefore, LY303511 displays antiproliferation potential against oral cancer cells in vitro and in vivo. https://pubmed.ncbi.nlm.nih.gov/31115172/

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Human prostate adenocarcinoma (PC-3) cells (ATCC CRL-1435) are cultured and implanted (1×10~6 cells) in 20% Matrigel per athymic NCR nude mouse by subcutaneous injection at the flank. Inoculated mice are subdivided into four groups of 10. Administration of vehicle or LY303511, 10 mg/kg/day, is begun (day 1) when tumors reach ~150 mm3 (n=35), and tumor volumes are measured for 30 days at the indicated time points.[4]

[1]. Homotypic gap junctional communication associated with metastasis suppression increases with PKA activity and is unaffected by PI3K inhibition. Cancer Res. 2010 Dec 1;70(23):10002-11.

[2]. The phosphatidylinositol 3-kinase inhibitor LY294002 potently blocks K(V) currents via a direct mechanism. FASEB J. 2003 Apr;17(6):720-2.

[3]. LY303511 enhances TRAIL sensitivity of SHEP-1 neuroblastoma cells via hydrogen peroxide-mediated mitogen-activated protein kinase activation and up-regulation of death receptors. Cancer Res. 2009 Mar 1;69(5):1941-50.

[4]. LY303511 (2-piperazinyl-8-phenyl-4H-1-benzopyran-4-one) acts via phosphatidylinositol 3-kinase-independent pathways to inhibit cell proliferation via mammalian target of rapamycin (mTOR)- and non-mTOR-dependent mechanisms. J Pharmacol E. 2005 Sep;314(3):1134-43.

8-phenyl-2-(1-piperazinyl)-1-benzopyran-4-one is a N-arylpiperazine.
Loss of gap junctional intercellular communication (GJIC) between cancer cells is a common characteristic of malignant transformation. This communication is mediated by connexin proteins that make up the functional units of gap junctions. Connexins are highly regulated at the protein level and phosphorylation events play a key role in their trafficking and degradation. The metastasis suppressor breast cancer metastasis suppressor 1 (BRMS1) upregulates GJIC and decreases phosphoinositide-3-kinase (PI3K) signaling. On the basis of these observations, we set out to determine whether there was a link between PI3K and GJIC in tumorigenic and metastatic cell lines. Treatment of cells with the well-known PI3K inhibitor LY294002, and its structural analogue LY303511, which does not inhibit PI3K, increased homotypic GJIC; however, we found the effect to be independent of PI3K/AKT inhibition. We show in multiple cancer cell lines of varying metastatic capability that GJIC can be restored without enforced expression of a connexin gene. In addition, while levels of connexin 43 remained unchanged, its relocalization from the cytosol to the plasma membrane was observed. Both LY294002 and LY303511 increased the activity of protein kinase A (PKA). Moreover, PKA blockade by the small molecule inhibitor H89 decreased the LY294002/LY303511-mediated increase in GJIC. Collectively, our findings show a connection between PKA activity and GJIC mediated by PI3K-independent mechanisms of LY294002 and LY303511. Manipulation of these signaling pathways could prove useful for antimetastatic therapy.[1]

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We recently reported that LY294002 (LY29) and LY303511 (LY30) sensitized tumor cells to drug-induced apoptosis independent of the phosphoinositide 3-kinase/Akt pathway. Here, we investigated the mechanism of LY30-induced sensitization of human neuroblastoma cells to TRAIL-mediated apoptosis. We provide evidence that LY30-induced increase in intracellular H(2)O(2) up-regulates the expression of TRAIL receptors (DR4 and DR5) in SHEP-1 cells by activating mitogen-activated protein kinases, resulting in a significant amplification of TRAIL-mediated caspase-8 processing and activity, cytosolic translocation of cytochrome c, and cell death. Involvement of the death receptors was further confirmed by the ability of blocking antibodies against DR4 and/or DR5 to inhibit LY30-induced TRAIL sensitization. Pharmacologic inhibition of c-Jun NH(2) terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) activation by SP600125 and PD98059, respectively, blocked LY30-induced increase in sensitization to TRAIL-mediated death. Finally, small interfering RNA-mediated gene silencing of JNK and ERK inhibited LY30-induced increase in surface expression of DR4 and DR5, respectively. These data show that JNK and ERK are two crucial players involved in H(2)O(2)-mediated increase in TRAIL sensitization of tumor cells upon exposure to LY30 and underscore a novel mode of action of this inactive analogue of LY29. Our findings could have implications for the use of LY30 and similar compounds for enhancing the apoptotic sensitivity of neuroblastoma cells that often become refractory to chemotherapy.[3]

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Mammalian target of rapamycin (mTOR), a serine/threonine kinase, regulates cell growth and proliferation in part via the activation of p70 S6 kinase (S6K). Rapamycin is an antineo-plastic agent that, in complex with FKBP12, is a specific inhibitor of mTOR through interaction with its FKBP12-rapamycin binding domain, thereby causing G(1) cell cycle arrest. However, cancer cells often develop resistance to rapamycin, and alternative inhibitors of mTOR are desired. 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) blocks mTOR kinase activity, but it also inhibits phosphatidylinositol 3-kinase (PI3K), an enzyme that regulates cellular functions other than proliferation. We hypothesized that a close structural analog, 2-piperazinyl-8-phenyl-4H-1-benzopyran-4-one (LY303511) might inhibit mTOR-dependent cell proliferation without unwanted effects on PI3K. In human lung epithelial adenocarcinoma (A549) cells, LY303511, like rapamycin, inhibited mTOR-dependent phosphorylation of S6K, but not PI3K-dependent phosphorylation of Akt. LY303511 blocked proliferation in A549 as well as in primary pulmonary artery smooth muscle cells, without causing apoptosis. In contrast to rapamycin, LY303511 reduced G(2)/M progression as well as G(2)/M-specific cyclins in A549 cells. Consistent with an additional mTOR-independent kinase target, LY303511 inhibited casein kinase 2 activity, a known regulator of G(1) and G(2)/M progression. In addition to its antiproliferative effect in vitro, LY303511 inhibited the growth of human prostate adenocarcinoma tumor implants in athymic mice. Given its inhibition of cell proliferation via mTOR-dependent and independent mechanisms, LY303511 has therapeutic potential with antineoplastic actions that are independent of PI3K inhibition.[4]

C19H18N2O2

306.36

306.136

C, 74.49; H, 5.92; N, 9.14; O, 10.44

154447-38-8

LY 303511 hydrochloride;2070014-90-1; LY 303511;154447-38-8; 854127-90-5 (2HCl)

3971

Typically exists as solid at room temperature

1.2±0.1 g/cm3

496.1±45.0 °C at 760 mmHg

253.8±28.7 °C

0.0±1.3 mmHg at 25°C

1.627

3.22

45.48

1

4

2

23

464

0

O=C1C=C(N2CCNCC2)OC3=C(C4=CC=CC=C4)C=CC=C13

InChI=1S/C19H18N2O2/c22-17-13-18(21-11-9-20-10-12-21)23-19-15(7-4-8-16(17)19)14-5-2-1-3-6-14/h1-8,13,20H,9-12H2

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)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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；
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