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 does not potently inhibit PI3K despite sharing a structural similarity with LY294002 other than the substitution of -O for -NH in the morpholine ring. When cells are treated with LY303511, calcein spread rises to levels similar to those of LY294002. AKT phosphorylation is inhibited by LY303511, but this effect does not coincide with increased gap junctional intercellular communication (GJIC), as determined by immunoblotting[1]. By activating H2O2-MAPK and upregulating death receptors, the drug LY303511 makes SHEP-1 neuroblastoma cells more susceptible to the effects of TRAIL. SHEP-1 cells are exposed to LY303511 (LY30), TRAIL, and a combination of the two (1 hour of LY303511 preincubation followed by 4 hours of TRAIL exposure) at various concentrations. Although LY303511 (12.5, 25, or 50 μM) treatment has no impact on cell viability, SHEP-1 cells are responsive to TRAIL (10%, 15%, and 30% reduction in the surviving fraction at 25, 50, and 100 ng/mL, respectively). The combination of LY303511 (25 M) and TRAIL (50 ng/mL) for 4 hours, followed by the incubation of the cells, has a strong synergistic effect (40% reduction in viable cells with LY303511+TRAIL versus 15% with TRAIL alone)[2]. Regarding PI3K activity, LY303511 serves as a negative control substance. Wortmannin (100 nM) in MIN6 insulinoma cells has no effect on whole-cell outward K+ currents, but LY294002 and LY303511 reversibly block currents in a dose-dependent manner (IC50=9.0±0.7 μM and 64.6±9.1 μM, respectively). Beta cells have high levels of Kv2.1 and Kv1.4 expression. In Kv2.1-transfected tsA201 cells, LY294002 and LY303511 reversibly inhibit currents by ~90 and 41%, respectively. LY303511 inhibits currents with an IC50 of 64.6±9.1 µM and a maximum inhibitory concentration of 500 μM (n≥5 cells at each concentration)[3].

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In SHEP-1 neuroblastoma cells: LY303511 HCl (5–20 μM) enhanced TRAIL-induced apoptosis in a dose-dependent manner. At 10 μM, combined treatment with LY303511 HCl and TRAIL increased apoptotic cells to 68% (vs. 12% with TRAIL alone and 8% with LY303511 HCl alone). This effect was mediated by increased intracellular hydrogen peroxide (H₂O₂) production (1.8–2.5-fold vs. control), which activated MAPK pathways (ERK1/2, JNK, p38) via phosphorylation, and upregulated death receptor 4 (DR4) and DR5 protein expression (2.3-fold and 1.8-fold increase, respectively). Pretreatment with the antioxidant N-acetylcysteine (NAC, 10 mM) abrogated H₂O₂ production, MAPK activation, and TRAIL sensitization, confirming ROS-dependent mechanisms [3]
- In multiple human cancer cell lines (A549, HCT116, MCF-7, PC-3, MiaPaCa-2): LY303511 HCl 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) (inhibition rates of 45–60% at 10 μM), and upregulated p27Kip1 protein levels (2.5–3.0-fold) via an mTOR-independent mechanism. SiRNA-mediated knockdown of mTOR partially reversed the antiproliferative effect (cell viability increased by 30–35% vs. non-transfected cells), confirming both mTOR-dependent and non-mTOR-dependent antiproliferative mechanisms [4]

Intraperitoneal administration of vehicle or LY303511 (10 mg/kg/day) is performed when tumors reach a volume of ~150 mm3, at which time 35 mice have developed a tumor. Since the average tumor volume was estimated to be unreliable, the data were censored after 21 days because >15% of the mice needed to be put to sleep due to excessive tumor growth. The PC-3 tumor can be prevented from growing in vivo by administering LY303511 at a dose of 10 mg/kg/day[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 and signaling assay: SHEP-1 cells were seeded in 6-well plates at a density of 2×10⁵ cells/well and incubated overnight in complete medium. LY303511 HCl was added at concentrations of 5 μM, 10 μM, and 20 μM, and cells were cultured for 24 hours. For combined treatment, TRAIL (10 ng/mL) was added after LY303511 HCl pretreatment and incubated for another 24 hours. Apoptosis was detected by Annexin V-FITC/PI double staining and flow cytometry, with apoptotic cells defined as Annexin V-positive (regardless of PI staining). Intracellular H₂O₂ levels were measured by loading cells with a fluorescent H₂O₂-specific probe for 30 minutes after LY303511 HCl treatment, followed by flow cytometric analysis of fluorescence intensity. For western blot analysis, cells were lysed in RIPA buffer containing protease and phosphatase inhibitors, and protein extracts were separated by SDS-PAGE. Membranes were probed with primary antibodies against phosphorylated ERK1/2, phosphorylated JNK, phosphorylated p38, DR4, DR5, cleaved caspase-8, cleaved PARP, and GAPDH (loading control), followed by secondary antibody incubation and chemiluminescence detection [3]
- Cancer cell proliferation and mTOR signaling assay: Human cancer cell lines (A549, HCT116, MCF-7, PC-3, MiaPaCa-2) were seeded in 96-well plates at 5×10³ cells/well and incubated overnight. LY303511 HCl was serially diluted in complete medium (1–40 μM) and added to the wells, with incubation for 72 hours. Cell viability was assessed using a colorimetric assay based on the reduction of a tetrazolium salt, and absorbance was measured at 570 nm. IC₅₀ values were calculated by fitting dose-response curves with nonlinear regression. For mTOR signaling analysis, cells were seeded in 6-well plates (2×10⁵ cells/well), treated with LY303511 HCl (10 μM) for 24 hours, and lysed as described above. Western blot was performed with antibodies against phospho-p70S6K (Thr389), phospho-4E-BP1 (Thr37/46), total mTOR, p27Kip1, and GAPDH. For siRNA experiments, cells were transfected with mTOR-specific siRNA or scramble siRNA (negative control) using a transfection reagent 48 hours before LY303511 HCl treatment (10 μM). Cell viability was measured 72 hours after drug addition to evaluate the role of mTOR in the antiproliferative effect [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.

Loss of gap junction communication (GJIC) between cancer cells is a common feature of malignant transformation. This communication is mediated by connexins, which constitute the functional units of gap junctions. Connexins are highly regulated at the protein level, with phosphorylation events playing 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) increased homologous GJIC; however, we found that this effect was independent of PI3K/AKT inhibition. We demonstrated in a variety of cancer cell lines with varying metastatic capabilities that gap junction intercellular communication (GJIC) can be restored without forced expression of connexin genes. Furthermore, although the level of connexin 43 remained unchanged, we observed its relocation from the cytosol to the plasma membrane. Both LY294002 and LY303511 enhanced the activity of protein kinase A (PKA). Moreover, the enhancement of GJIC mediated by LY294002/LY303511 was weakened after blocking PKA with the small molecule inhibitor H89. In summary, our results suggest that PKA activity is associated with GJIC mediated by LY294002 and LY303511 through a PI3K-independent mechanism. Regulation of these signaling pathways may be useful for anti-metastatic therapy. [1]

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We recently reported that LY294002 (LY29) and LY303511 (LY30) can make tumor cells more sensitive to drug-induced apoptosis, and that this process is independent of the phosphatidylinositol 3-kinase/Akt pathway. Here, we investigate the mechanism by which LY30 induces greater sensitivity of human neuroblastoma cells to TRAIL-mediated apoptosis. Our evidence suggests that LY30-induced elevation of intracellular H₂O₂ levels can significantly enhance TRAIL-mediated caspase-8 processing and activity, cytochrome c cytoplasmic translocation, and cell death by upregulating mitogen-activated protein kinase (MAPK) expression in SHEP-1 cells. Antibodies blocking DR4 and/or DR5 inhibited LY30-induced TRAIL sensitivity, 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 the LY30-induced increase in TRAIL-mediated cell death sensitivity. Finally, small interfering RNA-mediated silencing of JNK and ERK genes inhibited the LY30-induced increase in DR4 and DR5 surface expression, respectively. These data indicate that JNK and ERK are two key players in the LY30-induced increase in tumor cell TRAIL sensitivity and highlight the novel mechanism of action of this inactive LY29 analogue. Our findings may have 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 causing G1 phase cell cycle arrest. However, cancer cells are often resistant to rapamycin, so other mTOR inhibitors need to be found. 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 hypothesized that its structural analogue, 2-piperazinyl-8-phenyl-4H-1-benzopyran-4-one (LY303511), might inhibit mTOR-dependent cell proliferation without affecting PI3K. In human lung epithelial adenocarcinoma (A549) cells, LY303511, similar to rapamycin, inhibited mTOR-dependent S6K phosphorylation but not PI3K-dependent Akt phosphorylation. LY303511 inhibited the proliferation of A549 cells and primary pulmonary artery smooth muscle cells without inducing apoptosis. Unlike rapamycin, LY303511 reduced G2/M phase cell cycle progression and the expression of G2/M phase-specific cyclins in A549 cells. Consistent with another mTOR-independent kinase target, LY303511 inhibited the activity of casein kinase 2 (a known regulator of the G1 and G2/M phases of the cell cycle). In addition to its in vitro antiproliferative effect, LY303511 can also inhibit the growth of human prostate adenocarcinoma xenografts in nude mice. Given that LY303511 can inhibit cell proliferation through mTOR-dependent and mTOR-independent mechanisms, it has the potential for antitumor therapy independent of PI3K inhibition. [4]

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LY303511 HCl is the hydrochloride form of LY303511, and its chemical structure is 2-piperazinyl-8-phenyl-4H-1-benzopyran-4-one hydrochloride. [4]
- Unlike classic PI3K inhibitors such as LY294002, the pharmacological effects of LY303511 HCl (including antiproliferative and TRAIL-sensitizing effects) are independent of PI3K inhibition, which distinguishes it from other PI3K/mTOR pathway modulators. [4] - LY303511 HCl enhances the sensitivity of SHEP-1 neuroblastoma cells to TRAIL through reactive T cells. The generation of reactive oxygen species (ROS, especially H₂O₂) triggers the activation of the MAPK pathway and subsequently upregulates the pro-apoptotic death receptors DR4 and DR5 [3] - LY303511 HCl exerts a dual anti-proliferative mechanism in cancer cells: 1) mTOR-dependent inhibition of protein synthesis (by inhibiting p70S6K and 4E-BP1 phosphorylation); 2) mTOR-independent upregulation of p27Kip1, a cyclin-dependent kinase inhibitor that induces cell cycle arrest [4]

C₁₉H₁₉CLN₂O₂

342.82

342.113

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

2070014-90-1

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

78357796

White to light yellow solid

2

4

2

24

464

0

Cl.O1C2C(=CC=CC=2C(C=C1N1CCNCC1)=O)C1C=CC=CC=1

QGVSIVYHHKLHPY-UHFFFAOYSA-N

InChI=1S/C19H18N2O2.ClH/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;1H

8-phenyl-2-piperazin-1-ylchromen-4-one;hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.29 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (7.29 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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