PI3Kα (IC50 = 16 nM); PI3Kγ (IC50 = 25 nM); PI3Kδ (IC50 = 42 nM); PI3Kβ (IC50 = 68 nM); Vps34 (IC50 = 7470 nM); mTOR (IC50 = 37 nM); DNA-PK (IC50 = 1270 nM); mTORC1; mTORC2

VS-5584 is an ATP-competitive inhibitor which selectively inhibits PI3K/mTOR signaling with equivalent low nanomolar potency against all human Class I PI3K isoforms and mTOR kinase. With an EC50 of 15 nM in HMLE breast cancer cells, VS-5584 is roughly 10-fold selective for cancer stem cells. In a HMLER immortalized mammary cancer cell line, VS-5584 preferentially reduces CD44Hi/CD24Lo cells. VS-5584 successfully eradicates the cancer stem cell side population in SUM159 cells. Wide-ranging antiproliferative sensitivity is found in a large human cancer cell line panel screen (436 lines), and cells with PI3KCA mutations are generally more responsive to VS-5584 treatment.

In mice bearing triple negative breast cancer tumors, oral dosing of VS-5584 decreases tumor cancer stem cells and induces tumor regression in taxane-resistant models. Treatment with VS-5584 significantly inhibits tumor growth (TGI) in a PTENnull human prostate PC3 xenograft model by 79% and 113% for 11 and 25 mg/kg, respectively. A dose-dependent inhibition of tumor growth is brought about by VS-5584 treatment in a FLT3-ITD AML xenograft model (28% at 3.7 mg/kg and 76% at 11 mg/kg). [1]

The reaction mixture consisted of the following components in 10 μL assay buffer (50 mM Hepes pH 7.5, 10 mM MgCl 2, 3 mM MnCl 2, 1 mM EGTA, 2 mM DTT, 0.01%Tween-20): 10 μM ATP, 0.05 μM ULight-eIF4E-binding protein 1 (Thr37/46) peptide, and 0.10 μg/mL of house-made mTOR enzyme. At room temperature, the mixture is incubated for 60 minutes. Then, 1X LANCE® Detection Buffer and 10 μL of the detection mixture, which included 16 mM EDTA, 0.004 mM Eu-W1024-labeled Anti-Phospho-eIF4E-binding protein 1-(Thr37/46) antibody, are added. The mixture is then incubated for 60 min.

For proliferation assays in 96-well plates, SET-2, SNU-478, SNU-1196, SNU-245, SNU-1079, SNU-308, SNU-869, and MKN7 cells are used. The multiple myeloma cells (H929, MM1.S, MM1.R, R8226, U266) and nasopharyngeal cells (CNE-1, CNE-2, HONE1, HK1) are used. The following day, cells are treated with VS-5584 (in triplicates) at concentrations up to 10 μMfor 48 hours after being seeded at 30% to 50% confluency for adherent cells or 2,000 to 6,000 cells for suspension cells. The CellTiter-Glo assay is used to track cell viability. Using the XL-fit program, dose-response curves were plotted to determine the IC50 values for the compounds.

Mice: Athymic BALB/c nude mice (BALB/cOlaHsd-Foxn1nu) are used. Fox-Chase severe combined immunodeficient (SCID) mice (CB17/Icr-Prkdcscid/CrlBltw) are used. The right flank of male (PC3 and COLO 205), female (MV4-11 and HuH7), or male (NCI-N87) BALB/c nude mice is intradermally implanted with 5106 (PC3, COLO205, HuH7, NCI-N87), or 1107 (MV4-11) cells. Using a 27.5-gauge needle, cells are resuspended in 70% (v/v; only for COLO205 and HuH7) or 50% (v/v) serum-free growth medium/Matrigel and injected in a total volume of 100 L. Dosing began seven to fourteen days after tumor implantation. Oral doses of VS-5584 (11 and 25 mg/kg) are administered daily[1].

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Dosing started 7 to 14 days after tumor implantation. VS-5584 was dosed daily orally. The reference compounds everolimus and gefitinib were dosed p.o. at 5 and 150 mg/kg, respectively, with everolimus dosed daily and gefitinib dosed for 5 day-on and 2 day-off in cycles. 5-FU was administered intraperitoneally, at 25 mg/kg, every Tuesday, Thursday, and Saturday. All statistics conducted were done using GraphPad Prism (v5).

Pharmacokinetic and Pharmacodynamic Characteristics of VS-5584 To investigate the efficacy of VS-5584 in disease models, we determined its pharmacokinetic and pharmacodynamic characteristics to select the optimal dosing regimen. Following a single oral administration of VS-5584, the drug was rapidly absorbed, with a time to peak concentration (tmax) of 0.9 hours and an elimination half-life of 10 hours (Supplementary Figure S3). To determine the pharmacokinetic and pharmacodynamic relationship of VS-5584 in tumors, we treated PC3 tumor-bearing mice with a single dose of VS-5584 and collected plasma and tumor tissues 6 hours later to analyze the concentration of VS-5584 and its effect on targeted efficacy biomarkers. The concentration of VS-5584 in plasma increased in a dose-dependent manner (Figure 2A). Plasma pharmacokinetics did not differ significantly from tumor pharmacokinetics. Starting from 3.7 mg/kg, the drug concentration exceeded the IC50 value of the target kinase inhibition in both enzymatic and cytological assays. Dose-dependent inhibition of pAkt (S473) and pS6 (S240/244) was observed in tumor tissue, with complete inhibition achieved at 33 mg/kg (EC50 values of 4.2 and 1.7 mg/kg, respectively; Figure 2B). To investigate the time progression of drug concentrations in plasma and tumor and the inhibition of the target kinase signaling pathway, we administered a single oral dose of 33 mg/kg VS-5584 to PC3-bearing mice and collected tissue samples at 1, 6, and 24 hours post-administration. Following administration of the 33 mg/kg dose of VS-5584, plasma concentrations reached their peak at 1 hour post-administration (1221 ng/mL or 3.55 μmol/L) and remained above the concentrations required to block the target in vitro (15 ng/mL or 43 nmol/L; Figure 2C) at 24 hours. Within 1 hour of treatment with VS-5584, the inhibition rates of pAkt(S473) and pS6(S240/244) reached 90% or more, and remained at 60% to 70% after 24 hours (Figure 2D). [1] In summary, VS-5584 has good oral bioavailability, dose-linear pharmacokinetics, and produces a significant and durable pharmacodynamic response in tumor tissues after a single oral administration in tumor-bearing mice. [1]

[1]. VS-5584, a novel and highly selective PI3K/mTOR kinase inhibitor for the treatment of cancer. Mol Cancer Ther, 2013, 12(2), 151-161.

VS-5584 has been investigated for the treatment of lymphoma, metastatic cancers, and non-hematologic malignancies. The PI3K/mTOR kinase inhibitor VS-5584 is a potent and selective inhibitor of phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) kinases, located in the PI3K/mTOR signaling pathway, and possesses potential antitumor activity. The PI3K/mTOR kinase inhibitor VS-5584 inhibits mTOR kinase and all class I PI3K isoforms. Therefore, this disrupts the phosphorylation of downstream substrates of PI3K and mTOR, potentially leading to apoptosis and growth inhibition in susceptible tumor cells. Activation of the PI3K/mTOR pathway promotes cell growth, survival, and resistance to chemotherapy and radiotherapy. mTOR is a serine/threonine kinase downstream of PI3K and also possesses PI3K-independent activity. Therefore, this drug may be more effective than drugs that inhibit either PI3K or mTOR kinases.

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PI3K/mTOR pathway dysregulation, whether due to amplification, deletion, or direct mutation, is closely associated with the development and progression of various cancers. Furthermore, activation of this pathway is a marker of poor prognosis in many tumor types and confers resistance to various cancer therapies. This article describes a novel small molecule compound, VS-5584, which exhibits equivalent potent activity against mTOR (IC50 = 37 nmol/L) and all class I phosphatidylinositol 3-kinase (PI3K) isoforms (IC50: PI3Kα = 16 nmol/L; PI3Kβ = 68 nmol/L; PI3Kγ = 25 nmol/L; PI3Kδ = 42 nmol/L). At nmol/L, VS-5584 showed no significant activity against 400 lipid and protein kinases. It significantly modulated the intracellular PI3K/mTOR pathway, inhibiting phosphorylation of PI3K and mTORC1/2 downstream substrates. A large-scale screening of human cancer cell lines (436 lines) showed that VS-5584 exhibited broad anti-proliferative sensitivity, with cells carrying PI3KCA mutations generally being more sensitive to VS-5584 treatment. In mice, VS-5584 demonstrated favorable pharmacokinetic properties and good tolerability after oral administration. VS-5584 induced durable and dose-dependent inhibition of the PI3K/mTOR signaling pathway in tumor tissues, thereby inhibiting tumor growth in various rapamycin-sensitive and resistant human xenograft tumor models. Furthermore, in a gastric tumor model, VS-5584 showed synergistic effects with EGF receptor inhibitors. The unique target selectivity, favorable pharmacological and pharmaceutical properties, and efficacy in a variety of human tumor models support further investigation of VS-5584 in clinical trials. [1] In summary, the favorable target selectivity, pharmacokinetic and pharmacodynamic properties of VS-5584, and the resulting efficacy against a variety of tumors resistant to rapamycin analogues and standard therapies, provide a strong theoretical basis for clinical evaluation of this drug in a variety of hematologic malignancies and solid tumor indications. The genetic markers of sensitivity and resistance identified in large-scale cell screening provide a theoretical basis for patient selection for monotherapy and combination therapy. [1]

C17H22N8O

354.40958

354.19166

C, 57.61; H, 6.26; N, 31.62; O, 4.51

1246560-33-7

1246560-33-7

46912230

White to off-white crystalline solid

1.7±0.1 g/cm3

1.796

2.41

1

8

3

26

466

0

NC1=NC=C(C2=C3N=C(C)N(C(C)C)C3=NC(N4CCOCC4)=N2)C=N1

QYBGBLQCOOISAR-UHFFFAOYSA-N

InChI=1S/C17H22N8O/c1-10(2)25-11(3)21-14-13(12-8-19-16(18)20-9-12)22-17(23-15(14)25)24-4-6-26-7-5-24/h8-10H,4-7H2,1-3H3,(H2,18,19,20)

5-(8-methyl-2-morpholin-4-yl-9-propan-2-ylpurin-6-yl)pyrimidin-2-amine

VS5584; VS5584; VS 5584; 1246560-33-7; VS-5584; 5-(9-Isopropyl-8-methyl-2-morpholino-9H-purin-6-yl)pyrimidin-2-amine; VS-5584 (SB2343); 5-(8-methyl-2-morpholin-4-yl-9-propan-2-ylpurin-6-yl)pyrimidin-2-amine; UNII-W71J4X250V; SB2343; SB2343; SB 2343

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)

DMSO: ~71 mg/mL (~200.3 mM)
Water: <1 mg/mL
Ethanol: ~3 mg/mL (~8.5 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.05 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 (7.05 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 (7.05 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: 0.5% methylcellulose+0.2%Tween 80: 30 mg/mL

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