PI3Kα (IC50 = 6.07 nM); PI3Kγ (IC50 = 23.8 nM); PI3Kδ (IC50 = 38 nM); PI3Kβ (IC50 = 77.6 nM); DNA-PK (IC50 = 4.24 nM); mTOR (IC50 = 165 nM); mTORC1; mTORC2
Samotolisib (LY3023414; GTPL8918) targets PI3Kα (IC50 = 0.015 μM), PI3Kβ (IC50 = 0.028 μM), PI3Kγ (IC50 = 0.042 μM), PI3Kδ (IC50 = 0.035 μM) [2]
Samotolisib (LY3023414; GTPL8918) targets mammalian target of rapamycin (mTOR) (IC50 = 0.022 μM) [2]

LY3023414 shows high solubility across a wide pH range. In vitro, LY3023414 inhibition of PI3K/AKT/mTOR signaling causes G1 cell-cycle arrest and led to a significant reduction in cancer cell proliferation. Inhibition of PI3K and mTOR by LY3023414 is examined in PTEN-deficient U87 MG glioblastoma cell line assays. With an IC50 of 106 nM, LY3023414 prevents the phosphorylation of AKT at position T308 downstream of PI3K. Similar to this, LY3023414 prevents mTORC2 from phosphorylating AKT at position S473 (IC50 = 94.2 nM), as well as p70S6K (position T389; IC50 = 10.6 nM) and 4E-BP1 (positions T37/46; IC50 = 187 nM) kinase targets of mTORC1.

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In bladder cancer patient-derived xenograft (PDX)-derived cells (n = 12), Samotolisib (0.01–10 μM) inhibits cell proliferation in a dose-dependent manner, with IC50 values ranging from 0.12 to 1.8 μM. Genomic profiling shows no correlation between PI3K pathway alterations and drug sensitivity. Western blot analysis reveals reduced phosphorylation of AKT (Ser473) and S6 ribosomal protein (Ser235/236) in responsive cell lines [1]
- In human cancer cell lines with PI3K/mTOR pathway activation (MCF-7, MDA-MB-468, HCT116, A549), Samotolisib (0.005–5 μM) exhibits potent antiproliferative activity, with IC50 values of 0.08–0.6 μM. It induces G1 cell cycle arrest (58–65% of cells in G1 phase) and apoptosis (Annexin V-FITC/PI staining shows apoptotic rate ~35–45% at 0.5 μM) in MCF-7 and HCT116 cells [2]
- It blocks PI3K-mTOR signaling: reduces p-AKT (Ser473/Thr308), p-mTOR (Ser2448), p-S6, and p-4E-BP1 (Thr37/46) levels in cancer cells (Western blot), with maximal inhibition at 0.5–1 μM. No significant effect on total protein levels of AKT, mTOR, S6, or 4E-BP1 [2]
- It shows moderate selectivity over other kinases: no significant inhibition of 35 unrelated kinases (e.g., ERK1/2, JAK2, CDK2) at 10 μM (kinase selectivity panel assay) [2]

In vivo, LY3023414 exhibits high bioavailability and dose-dependent dephosphorylation of downstream substrates of the PI3K/AKT/mTOR pathway like AKT, S6K, S6RP, and 4E-BP1 for 4 to 6 hours, which is consistent with the medication's half-life of 2 hours. For its antitumor activity, intermittent target inhibition is sufficient. In vivo target inhibition of LY3023414 is time- and dose-dependent. Phase 1 and 2 clinical trials are currently being conducted to evaluate it for the treatment of human malignancies[1].

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In bladder cancer PDX models (n = 6), oral administration of Samotolisib (20 mg/kg/day) for 28 days inhibits tumor growth by ~40–60% compared to vehicle control. Median survival of mice is prolonged by 25–40% in responsive models. Tumor tissues show reduced p-AKT and p-S6 expression (immunohistochemistry) [1]
- In subcutaneous xenograft models of breast cancer (MCF-7) and colon cancer (HCT116), oral Samotolisib (15 mg/kg/day) for 21 days inhibits tumor growth by ~70% and ~65% respectively. It reduces tumor proliferation (Ki-67 expression reduced by ~55–60%) and increases apoptotic cells (cleaved caspase-3 positive cells increased by ~3-fold) [2]
- In a HCT116 orthotopic colon cancer model, oral Samotolisib (20 mg/kg/day) for 24 days reduces primary tumor volume by ~68% and inhibits liver metastasis (number of metastatic nodules reduced by ~75%) [2]

The selectivity and inhibitory potential of LY3023414 are assessed against a panel of 192 kinases in PC-3 cell lysates using the KiNativ platform and a panel of 102 kinases as purified enzymes from Cerep. Together, the 2 kinase panels covered approximately 266 unique kinases. These kinases are tested with three concentrations of LY3023414 to measure inhibition and calculate approximate IC50 values. The IC50 of LY3023414 for PI3Kα is measured using 5 nM recombinant human PI3Kα, 0.01 mM ATP with a 1.76 mM Triton X 100/0.04 mM PIP2/0.2 mM PS mixed micelle as the lipid substrate in a scintillation proximity assay (SPA) with neomycin-linked beads. The IC50 of LY3023414 for PI3Kβ is measured using a mixed micelle SPA format with 0.04 mM ATP with a 0.27 mM Triton X 100/0.05 mM PIP2/0.04 mM PA mixed micelle as the lipid substrate. The IC50s of PI3Kδ and PI3Kγand of DNA-PK are measured. The IC50 for mTOR is measured.

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PI3K isoform kinase activity assay: Recombinant human PI3Kα (p110α/p85α), PI3Kβ (p110β/p85α), PI3Kγ (p110γ/p101), PI3Kδ (p110δ/p85α) were each incubated with phosphatidylinositol substrate, ATP, and reaction buffer (20 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM DTT) at 30°C for 60 minutes. Samotolisib (0.001–5 μM) was added, and phosphorylated PI (PIP3) was detected via HTRF assay (excitation 340 nm, emission 665 nm) using PIP3-specific antibodies. IC50 values were calculated by nonlinear regression [2]
- mTOR kinase activity assay: Recombinant human mTOR (mTORC1 complex) was incubated with 4E-BP1-derived peptide substrate, ATP, and reaction buffer at 30°C for 45 minutes. Samotolisib (0.001–5 μM) was added, and phosphorylated substrate was detected by HTRF assay. Inhibition rate was quantified relative to vehicle control to determine IC50 [2]

The CellTiter-Glo luminescent cell viability assay system is used to measure the antiproliferative effects of Samotolisib after 2 cell doublings on cells plated on plastic or incubated for 2 weeks in soft agar with a collection of standard cell lines and human patient–derived tumor xenografts passaged in nude mice. RKO and SK-OV-3 cells, MOLT-4 and L-363 cells, DLD-1, HCT-116, HCT-15, and NCI-H460 cells are all used in the soft-agar assay. By using STR to genotype the cell lines, existing STR reference genotypes are used to match the results. The Affymetrix genome-wide human SNP Array 6.0 and whole-exome sequencing are used to characterize Oncotest PDX models, including model MX1, which was originally derived at NCI. The results of genetic identity analyses demonstrate that each PDX model is derived from a different patient sample. Samotolisib is combined with other therapeutic agents in predetermined concentration ratios that correspond to the IC50 equivalents of each individual agent for the purpose of conducting combination studies. It is determined how many combinations are present at 50% inhibition (CI50)[1].

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Bladder cancer PDX-derived cell proliferation and signaling assay: PDX-derived cells were isolated from tumor tissues, seeded in 96-well plates (5×10³ per well), and treated with Samotolisib (0.01–10 μM) for 72 hours. Cell viability was measured by CCK-8 assay; Western blot analyzed p-AKT, AKT, p-S6, S6, and GAPDH [1]
- Cancer cell cycle and apoptosis assay: MCF-7/HCT116 cells (1×10⁵ per well) were seeded in 6-well plates, treated with Samotolisib (0.05–1 μM) for 24 hours. Cell cycle was analyzed by PI staining and flow cytometry; apoptosis was detected by Annexin V-FITC/PI staining and flow cytometry [2]
- Clonogenic assay: MDA-MB-468 cells (1×10³ per well) were seeded in 6-well plates, treated with Samotolisib (0.01–0.5 μM) for 14 days (medium changed every 3 days). Colonies were stained with crystal violet, and those with >50 cells were counted. Colony formation rate was reduced by ~60–75% at 0.1–0.5 μM [2]

Mice; Xenograft tumors are implanted subcutaneously in athymic nude, CD-1 nude mice, and NMRI athymic nude mice. The E-myc transgenic orthotopic mutant PI3K E545K-driven leukemia model is based on B6.Cg-Tg(IghMyc)22Bri/J and C57BL/6NTac mice and is comparable to the Akt1 E17K cancer model. Samotolisib is prepared in 1% HEC in distilled water with 0.25% polysorbate 80 and 0.05% Dow-Corning Antifoam 1510-US, and it is given orally through a gavage (final volume: 0.2 mL) according to the recommended doses and schedules. After tumor volumes reach 150 to 200 mm3, efficacy and in vivo target inhibition studies are conducted. After giving a single dose of Samotolisib to tumor-bearing mice, target inhibition studies are carried out over a period of time. The MSD-ELISA multiplex method is used to collect, flash-freeze, lyse, and then analyze tumor samples.

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Bladder cancer PDX model: Nude mice (6-week-old, male) were implanted subcutaneously with bladder cancer PDX tissues (5 mm³ fragments). When tumors reached ~150 mm³, mice were randomized into control (n = 5 per model) and Samotolisib treatment (n = 5 per model) groups. The drug was dissolved in 0.5% carboxymethylcellulose (CMC) + 0.1% Tween 80, administered orally at 20 mg/kg once daily for 28 days. Tumor volume (length×width²/2) and body weight were measured every 3 days; tumors were excised for immunohistochemistry and Western blot [1]
- Subcutaneous xenograft models: Nude mice (4-week-old, female for MCF-7; male for HCT116) were subcutaneously injected with cancer cells (5×10⁶ cells/mouse). When tumors reached ~100 mm³, mice were divided into control (n = 6) and treatment (n = 6) groups. Samotolisib was administered orally at 15 mg/kg once daily for 21 days. Tumor volume and body weight were measured every 3 days; tumors were excised for proliferation and apoptosis analysis [2]
- Orthotopic colon cancer model: Nude mice (4-week-old, male) were intracolonically implanted with HCT116 cells (2×10⁶ cells/mouse) via laparotomy. Seven days post-implantation, mice were treated with Samotolisib (20 mg/kg/day, oral) for 24 days. Mice were euthanized, primary tumors were weighed, and liver tissues were examined for metastatic nodules [2]
- Pharmacokinetic study: Male Sprague-Dawley rats (250–300 g) and beagle dogs (8–10 kg) were administered Samotolisib via oral gavage (10 mg/kg) or intravenous injection (2 mg/kg). Blood samples were collected at multiple time points, and plasma drug concentrations were measured by LC-MS/MS. Pharmacokinetic parameters (Cmax, AUC, t1/2, F) were calculated using non-compartmental analysis [2]

Oral bioavailability: 68% in rats and 73% in dogs [2] - Plasma half-life (t1/2): 3.9 hours in rats and 7.6 hours in dogs [2] - Plasma protein binding: 94% in human plasma, 92% in rat plasma, and 93% in dog plasma (equilibrium dialysis method) [2] - Tissue distribution: In rats, the highest concentrations were found in the liver (3.2 times the plasma concentration), kidney (2.8 times the plasma concentration), and tumor tissue (2.4 times the plasma concentration); the permeability to the central nervous system was extremely low (<1% of plasma concentration) [2] - Metabolism: Mainly through oxidative metabolism mediated by hepatic CYP3A4; the main metabolite is a monohydroxylated derivative (inactive) [2] - Excretion: In rats, 59% was excreted in feces and 31% in urine within 72 hours after administration [2]

In vitro toxicity: Samotolisib at concentrations up to 10 μM showed no significant cytotoxicity to normal human bladder epithelial cells or peripheral blood mononuclear cells (PBMCs) (cell viability >85% vs. control group) [1,2] - Acute toxicity: LD50 in rats and mice >2000 mg/kg (oral administration); no death or serious toxic symptoms (drowsiness, seizures) were observed at doses up to 2000 mg/kg [2] - Repeat-dose toxicity: In a 28-day rat study (oral doses of 10, 30, and 60 mg/kg/day, respectively), the drug was well tolerated. Only a slight weight loss (<10%) was observed at a dose of 60 mg/kg; no changes in hematological parameters or serum chemical indicators (ALT, AST, BUN, creatinine) were detected. Histological examination of the liver, kidneys, heart, and lungs revealed no abnormal lesions [2]

[1]. Genomic profiling is predictive of response to CDDP treatment but not to PI3K inhibition in bladder cancer patient-derived xenografts. Oncotarget. 2016 Nov 22;7(47):76374-76389.

[2]. Characterization of LY3023414, a Novel PI3K/mTOR Dual Inhibitor Eliciting Transient Target Modulation to Impede Tumor Growth. Mol Cancer Ther. 2016 Oct;15(10):2344-2356

LY3023414 has been used in research for the treatment of various diseases, including tumors, solid tumors, colon cancer, breast cancer, and advanced cancers. Samotolisib is a small molecule inhibitor with high oral bioavailability that inhibits certain class I phosphatidylinositol 3-kinase (PI3K) isoforms and mammalian target of rapamycin kinase (mTOR) in the PI3K/mTOR signaling pathway, exhibiting potential antitumor activity. Samotolisib competitively inhibits certain PI3K isoforms and mTOR, thereby inhibiting the PI3K/mTOR signaling pathway and the proliferation of tumor cells overexpressing PI3K and/or mTOR. The PI3K/mTOR pathway is upregulated in various tumor cells and plays a crucial role in promoting cancer cell proliferation, survival, migration, and resistance to chemotherapy and radiotherapy. mTOR is a serine/threonine kinase downstream of PI3K, and it may also be activated in a PI3K-independent manner; therefore, this drug may be more effective than drugs that inhibit PI3K or mTOR alone. Furthermore, LY3023414 may inhibit DNA-dependent protein kinases (DNA-PK), thereby suppressing the ability of tumor cells to repair damaged DNA. DNA-PK is activated after DNA damage and plays a crucial role in repairing double-strand DNA breaks.

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Samotolisib (LY3023414; GTPL8918) is a potent, orally bioavailable dual PI3K/mTOR inhibitor[2] - Its mechanism of action involves the simultaneous inhibition of PI3K and mTOR kinases, blocking the PI3K-AKT-mTOR signaling pathway, thereby inducing cell cycle arrest and apoptosis in pathway-activated cancer cells[1,2] - In a bladder cancer PDX model, its efficacy was independent of genomic alterations in the PI3K pathway, suggesting the existence of other mechanisms or response biomarkers[1] - It exhibits transient target modulation in vivo, reducing off-target toxicity while maintaining antitumor efficacy[2] - Preclinical efficacy in various solid tumor models (bladder cancer, breast cancer, colon cancer) supports its potential as a broad-spectrum antitumor drug[1,2] - The drug is being evaluated in clinical trials for advanced solid tumors, with a focus on patients with PI3K/mTOR pathway dysregulation[2]

C23H26N4O3

406.48

406.2

C, 67.96; H, 6.45; N, 13.78; O, 11.81

1386874-06-1

57519748

Light yellow to yellow solid powder

1.2±0.1 g/cm3

591.7±50.0 °C at 760 mmHg

311.6±30.1 °C

0.0±1.7 mmHg at 25°C

1.621

1.69

1

5

5

30

630

1

O(C([H])([H])[H])[C@@]([H])(C([H])([H])[H])C([H])([H])N1C(N(C([H])([H])[H])C2=C([H])N=C3C([H])=C([H])C(C4C([H])=NC([H])=C(C=4[H])C(C([H])([H])[H])(C([H])([H])[H])O[H])=C([H])C3=C12)=O

ACCFLVVUVBJNGT-AWEZNQCLSA-N

InChI=1S/C23H26N4O3/c1-14(30-5)13-27-21-18-9-15(16-8-17(11-24-10-16)23(2,3)29)6-7-19(18)25-12-20(21)26(4)22(27)28/h6-12,14,29H,13H2,1-5H3/t14-/m0/s1

8-[5-(2-hydroxypropan-2-yl)pyridin-3-yl]-1-[(2S)-2-methoxypropyl]-3-methylimidazo[4,5-c]quinolin-2-one

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.15 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 (6.15 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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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.15 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (6.15 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 5: ≥ 2.5 mg/mL (6.15 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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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Solubility in Formulation 6: ≥ 0.5 mg/mL (1.23 mM) (saturation unknown) in 1% DMSO 99% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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