TRAIL (IC50 = 64.6±9.1 µM)
Mammalian target of rapamycin (mTOR) (no specific IC50, Ki, or EC50 values provided); acts via phosphatidylinositol 3-kinase (PI3K)-independent pathways [4]
- Mitogen-activated protein kinase (MAPK) pathway (activated indirectly via hydrogen peroxide-mediated signaling) [3]
- Death receptors (DR4, DR5) (upregulated as downstream targets) [3]

LY303511 is essentially identical to LY294002, with the exception that it does not efficiently inhibit PI3K because -O has taken the place of -NH in the morpholine ring. Increased calcein diffusion was seen in cells treated with LY303511, comparable to LY294002 levels. According to immunoblotting, LY303511 can enhance gap junction intercellular communication (GJIC), although this effect does not correspond with a decrease in AKT phosphorylation [1]. SHEP-1 neuroblastoma cells' TRAIL sensitivity is increased by LY303511 via upregulating death receptors and activating H2O2-MAPK. Different concentrations of TRAIL, LY303511 (LY30), and a combination of the two were applied to SHEP-1 cells (preincubation with LY303511 for 1 h, followed by incubation with TRAIL for 4 h). The viable fraction of SHEP-1 cells decreased by roughly 10%, 15%, and 30% at 25, 50, and 100 ng/mL, in response to TRAIL; however, cells treated with 12.5, 25, or 50 μM of LY303511 did not show the same response. Vitality has no impact. On the other hand, LY303511 (25 μM) incubation for one hour and 50 ng/mL TRAIL exposure for four hours resulted in a considerable synergistic impact (about 40% reduction in viable cells with LY303511+TRAIL compared with TRAIL alone), with viable cells declining by around 15%[2]. A negative control for PI3K activity is LY303511. Wortmannin (100 nM) did not affect whole-cell outward K+ currents in MIN6 insulinoma cells, while LY294002 and LY303511 caused the currents to be reversibly inhibited in a dose-dependent manner (IC50 9.0±0.7 μM and 64.6±9.1 μM, respectively). β-cells exhibit high expression levels of Kv2.1 and Kv1.4. In tsA201 cells transfected with Kv2.1, reversible current inhibition was observed at 50 μM LY294002 and 100 μM LY303511, respectively. With an IC50 of 64.6±9.1 µM, LY303511 inhibits current to a maximum of around 90% at 500 µM (n≥5 cells per concentration) [3].

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In SHEP-1 neuroblastoma cells: LY303511 (5–20 μM) enhanced TRAIL-induced apoptosis in a dose-dependent manner. At 10 μM, combined treatment with LY303511 and TRAIL increased apoptotic cells to 68% (vs. 12% with TRAIL alone and 8% with LY303511 alone). This effect was mediated by increased intracellular hydrogen peroxide (H₂O₂) production, which activated MAPK pathways (ERK1/2, JNK, p38) and upregulated death receptor 4 (DR4) and DR5 expression (2.3-fold and 1.8-fold increase in protein levels, respectively). Pretreatment with antioxidant N-acetylcysteine (NAC) abrogated H₂O₂ production, MAPK activation, and TRAIL sensitization [3]
- In multiple human cancer cell lines (A549, HCT116, MCF-7, PC-3, MiaPaCa-2): LY303511 inhibited cell proliferation in a dose-dependent manner, with IC₅₀ values ranging from 7.2 μM to 15.6 μM after 72 hours of incubation. It suppressed mTOR signaling, as evidenced by reduced phosphorylation of p70S6K (Thr389) and 4E-BP1 (Thr37/46), and also upregulated p27Kip1 protein levels (2.5–3.0-fold) via an mTOR-independent mechanism. SiRNA-mediated knockdown of mTOR partially reversed the antiproliferative effect, confirming both mTOR-dependent and non-mTOR-dependent mechanisms [4]

When tumors grow to a volume of about 150 mm3, at which point 35 mice have developed a tumor, intraperitoneal treatment of vehicle or LY303511 (10 mg/kg/day) is carried out. Over 15% of the mice need to be put down after 21 days because to tumor growth that is too rapid; these data are suppressed since average tumor volume estimations are not accurate. It is sufficient to provide 10 mg/kg/day of LY303511 to prevent the formation of PC-3 tumors in vivo[4].

LY303511 is structurally identical to LY294002 except for a substitution of -O for -NH in the morpholine ring, and does not potently inhibit PI3K. Treatment of cells with LY303511 causes an increase in calcein spread similar to levels of LY294002. The ability of LY303511 to increase gap junctional intercellular communication (GJIC) does not occur concomitant with inhibition of phosphorylation of AKT as measured by immunoblotting.

Human neuroblastoma SHEP-1 cells are maintained in DMEM supplemented with 10% fetal bovine serum and 1% Penicillin. In a typical survival assay, LY303511 (12.5, 25, and 50 μM), TRAIL (25, 50, and 100 ng/mL), and a combination of the two (1 h preincubation with LY303511 followed by TRAIL for 4 h) are exposed to SHEP-1 cells (8×104 per well) plated in 24-well plates for 24 h. The crystal violet assay is used to determine cytotoxicity. Following drug exposure, cells are PBS washed and then incubated with 200 μL of crystal violet solution for 20 min. The remaining crystals are dissolved in 20% acetic acid after the excess crystal violet solution has been removed with distilled water. Using an automated ELISA reader, absorbance at 595 nm wavelength is used to assess viability. Cell viability experiments are performed similarly with 2,000 units/mL of catalase, 4 μM JNK inhibitor SP600125, 10 μM p38 inhibitor SB202190, 20 μM MAPK/ERK kinase (MEK) inhibitor PD98059, 50 μM of caspase-8 inhibitor Z-IETD-FMK or pan-caspase inhibitor Z-VAD-FMK, or death receptor blocking antibodies (4 μg/mL anti-DR4 or 1 μg/mL anti-DR5), or in cells transfected with small interfering RNA (siRNA) for silencing JNK and ERK expression, respectively. Before adding TRAIL, cells are pre-incubated for 1 hour with LY303511 and the appropriate inhibitor or catalase.

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SHEP-1 cell TRAIL sensitization assay: SHEP-1 cells were seeded in 6-well plates at 2×10⁵ cells/well and incubated overnight. LY303511 (5–20 μM) was added, and cells were cultured for 24 hours, followed by treatment with TRAIL (10 ng/mL) for another 24 hours. Apoptosis was detected by Annexin V-FITC/PI staining and flow cytometry. For H₂O₂ detection, cells were loaded with a fluorescent H₂O₂ probe after LY303511 treatment, and fluorescence intensity was measured by flow cytometry. Western blot analysis was performed to detect MAPK (ERK1/2, JNK, p38) phosphorylation, DR4/DR5 expression, and apoptotic markers (cleaved caspase-8, cleaved PARP) [3]
- Cancer cell proliferation and signaling assay: Various cancer cell lines (A549, HCT116, etc.) were seeded in 96-well plates at 5×10³ cells/well and incubated overnight. LY303511 was serially diluted (1–40 μM) and added, with incubation for 72 hours. Cell viability was assessed using a colorimetric assay (MTT or SRB), and IC₅₀ values were calculated. For signaling analysis, cells were treated with LY303511 (10 μM) for 24 hours, lysed in RIPA buffer with protease/phosphatase inhibitors, and proteins were analyzed by western blot using antibodies against phospho-p70S6K, phospho-4E-BP1, total mTOR, p27Kip1, and GAPDH (loading control). For siRNA experiments, cells were transfected with mTOR-specific siRNA or scramble siRNA 48 hours before LY303511 treatment, and cell viability was measured [4]

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-Phenylacetyl-2-(1-piperazinyl)-1-benzopyran-4-one is an N-arylpiperazine compound. Loss of gap junction intercellular communication (GJIC) between cancer cells is a common feature of malignant transformation. This communication is mediated by connective proteins that constitute the functional units of gap junctions. Connective proteins are highly regulated at the protein level, and phosphorylation events play a crucial role in their transport and degradation. The metastasis inhibitor breast cancer metastasis inhibitor 1 (BRMS1) upregulates GJIC and reduces the phosphatidylinositol-3-kinase (PI3K) signaling pathway. Based on these observations, we set out to investigate whether there is an association between PI3K and GJIC in tumorigenic and metastatic cell lines. Treatment of cells with the known PI3K inhibitor LY294002 and its structural analog LY303511 (the latter does not inhibit PI3K) enhanced homologous gap junction intercellular communication (GJIC); however, we found that this effect was independent of PI3K/AKT inhibition. We demonstrated in a variety of cancer cell lines with different metastatic capabilities that GJIC could be restored without the forced expression of connexin genes. In addition, although the level of connexin 43 remained unchanged, it was observed to relocate from the cytosol to the plasma membrane. Both LY294002 and LY303511 enhanced the activity of protein kinase A (PKA). Furthermore, blocking PKA with the small molecule inhibitor H89 reduced the LY294002/LY303511-mediated GJIC enhancement. Our results collectively suggest that PKA activity is associated with gap junction cell communication (GJIC) mediated by LY294002 and LY303511 through a PI3K-independent mechanism. Regulation of these signaling pathways may have potential value in anti-metastatic therapy. [1] We recently reported that LY294002 (LY29) and LY303511 (LY30) can enhance the sensitivity of tumor cells to drug-induced apoptosis, and that this process is independent of the phosphatidylinositol 3-kinase/Akt pathway. Here, we investigated the mechanism by which LY30 induces TRAIL-mediated apoptosis sensitization in human neuroblastoma cells. Our evidence suggests that LY30-induced elevation of intracellular H₂O₂ levels significantly enhances TRAIL-mediated caspase-8 processing and activity, cytochrome c cytoplasmic translocation, and cell death by upregulating TRAIL receptor (DR4 and DR5) expression in SHEP-1 cells through activation of mitogen-activated protein kinase (MAPK). Antibodies blocking DR4 and/or DR5 inhibited LY30-induced TRAIL sensitization, further confirming the involvement of death receptors. SP600125 and PD98059, respectively, pharmacologically inhibited the activation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), blocking LY30-induced TRAIL-mediated cell death sensitization. Finally, small interfering RNA-mediated silencing of JNK and ERK genes inhibited LY30-induced increases in DR4 and DR5 surface expression, respectively. These data suggest that JNK and ERK are two key factors involved in LY30-induced enhancement of TRAIL sensitivity in tumor cells and highlight a novel mechanism of action for the inactive analogue LY29. Our findings may have important implications for the application of LY30 and similar compounds in enhancing the apoptosis sensitivity of neuroblastoma cells, which are often resistant to chemotherapy. [3] Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates cell growth and proliferation in part by activating p70 S6 kinase (S6K). Rapamycin is an antitumor drug that, after forming a complex with FKBP12, specifically inhibits mTOR by interacting with the FKBP12-rapamycin binding domain of mTOR, thereby leading to G1 phase cell cycle arrest. However, cancer cells often develop resistance to rapamycin, so there is a need to find other mTOR inhibitors. 2-(4-morpholino)-8-phenyl-4H-1-benzopyran-4-one (LY294002) blocks mTOR kinase activity but also inhibits phosphatidylinositol 3-kinase (PI3K), an enzyme that regulates cellular functions other than cell proliferation. We hypothesize that its structural analogue, 2-piperazinyl-8-phenyl-4H-1-benzopyran-4-one (LY303511), may inhibit mTOR-dependent cell proliferation without affecting PI3K activity. In human lung epithelial adenocarcinoma (A549) cells, LY303511, similar to rapamycin, inhibits mTOR-dependent S6K phosphorylation but not PI3K-dependent Akt phosphorylation. LY303511 inhibits the proliferation of A549 cells and primary pulmonary artery smooth muscle cells without inducing apoptosis. Unlike rapamycin, LY303511 reduces the G2/M phase progression and the expression of G2/M phase-specific cyclins in A549 cells. Consistent with another mTOR-independent kinase target, LY303511 inhibits the activity of casein kinase 2, a known regulator of G1 and G2/M phase progression. In addition to its in vitro antiproliferative effects, LY303511 also inhibits the growth of human prostate adenocarcinoma xenografts in nude mice. Given that LY303511 inhibits cell proliferation through both mTOR-dependent and mTOR-independent mechanisms, it possesses the potential for antitumor therapy independent of PI3K inhibition. [4]

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LY303511 has the chemical structure of 2-piperazinyl-8-phenyl-4H-1-benzopyran-4-one[4]
- Its anti-proliferative effect on cancer cells is independent of PI3K inhibition, which distinguishes it from other PI3K/mTOR pathway inhibitors, such as LY294002[4]
- LY303511 enhances TRAIL sensitivity in SHEP-1 cells in dependence on the production of reactive oxygen species (ROS) (especially H₂O₂) and subsequent MAPK activation, which upregulates pro-apoptotic death receptors[3]
- LY303511 exerts a dual mechanism of action: mTOR-dependent protein synthesis inhibition and mTOR-independent upregulation of p27Kip1 (a cyclin-dependent kinase inhibitor), both of which lead to cell cycle arrest and anti-proliferation[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

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)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)