p110α (IC50 = 2 nM); p110β (IC50 = 3 nM); p110δ (IC50 = 3 nM); p110γ (IC50 = 15 nM); mTORC1 (IC50 = 20 nM); mTORC2 (IC50 = 83 nM); DNA-PK (IC50 = 23 nM)

PI-103 potently inhibits both the rapamycin-sensitive (mTORC1) and rapamycin-insensitive (mTORC2) complexes of the protein kinase mTOR.[1] PI-103 blocks PI3K/Akt activation that is both intrinsically occurring and induced by growth factors. [2] In blast cells, PI-103 prevents leukemia from proliferating, prevents leukemia progenitors from cloning, and triggers mitochondrial apoptosis, particularly in the compartment housing leukemia stem cells. PI-103 inhibits p110α more effectively than p110 by a factor of >200-fold. Additionally, PI-103 effectively inhibits the synthesis of PIP3 and PI(3,4)P2 in myotubes and adipocytes, respectively. [2] With an IC95 100 times lower than that of LY294002, PI-103 inhibits Akt phosphorylation. Surprisingly, PI-103 completely shields animals from the decline in blood sugar caused by insulin. In blast cells and immature leukemic cells, PI-103 has proapoptotic effects that are additive to those of etoposide. [2]

When tumors reach 50-100 mm3, animals are randomized and treated with vehicle or PI-103. After 18 days, PI-103 shows significant activity, with an average 4-fold reduction in tumor size. [2] Premorbidly (based on body weight, amount of food and water consumed, activity level, and general exam) or at necropsy, mice treated with PI-103 show no overt toxic effects. As expected from the blockade of p110α and mTOR, treated tumors show lower levels of phosphorylated Akt and S6. Treatment with PI-103 is cytostatic to glioma xenografts. [2]

IC50 values are measured using either a standard thin-layer chromatography (TLC) assay for lipid kinase activity or a high-throughput membrane capture assay. Kinase reactions are performed by preparing a reaction mixture containing kinase, inhibitor (2% DMSO final concentration), buffer (25 mM HEPES, pH 7.4, 10 mM MgCl2), and freshly sonicated phosphatidylinositol (100 μg/mL). Reactions are initiated by the addition of ATP containing 10 μCi of γ-32P-ATP to a final concentration 10 or 100 μM, and allowed to proceed for 20 minutes at room temperature. For TLC analysis, reactions are then terminated by the addition of 105 μL 1N HCl followed by 160 μL CHCl3:MeOH (1:1). The biphasic mixture is vortexed, briefly centrifuged, and the organic phase transferred to a new tube using a gel loading pipette tip precoated with CHCl3. This extract is spotted on TLC plates and developed for 3-4 hours in a 65:35 solution of n-propanol:1M acetic acid. The TLC plates are then dried, exposed to a phosphorimager screen, and quantitated. For each compound, kinase activity is typically measured at 10-12 inhibitor concentrations representing two-fold dilutions from the highest concentration tested (100 μM). For compounds showing significant activity, IC50 determinations are repeated two to four times, and the reported value is the average of these independent measurements.

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Phosphatidylinositide 3-kinase inhibitory activity was determined using a scintillation proximity assay in the presence of 1 μmol/L ATP. A TR-FRET-based LanthaScreen technique from Invitrogen was used to determine whether mTOR protein kinase was inhibited. Using GraphPad Prism software, IC50 values were calculated for each compound at a maximum concentration of 10 mol/L in the presence of 1 mol/L ATP.[1]

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Protein Kinase Assays[1]
Abl, Abl (T315I) Inhibitors (final concentration: 10 μM) were assayed in triplicate against recombinant full-length Abl or Abl (T315I) (Upstate) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgCl2, 200 μM ATP (2.5 μCi of γ-32P-ATP), and 0.5 mg/mL BSA. The optimized Abl peptide substrate EAIYAAPFAKKK was used as phosphoacceptor (200 μM). Reactions were terminated by spotting onto phosphocellulose sheets, which were washed with 0.5% phosphoric acid (approximately 6 times, 5-10 minutes each). Sheets were dried and the transferred radioactivity quantitated by phosphorimaging. [1]

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Akt1, Akt1 (ΔPH), Akt2, Akt2 (ΔPH), Akt3 (ΔPH) [1]
Inhibitors (final concentration: 10 μM) were assayed in triplicate against recombinant full-length Akt1, Akt2, Akt3, Akt1 (ΔPH), or Akt2 (ΔPH) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgCl2, 200 μM ATP (2.5 μCi of γ-32P-ATP), and 0.5 mg/mL BSA. Myelin basic protein (0.2 mg/mL) was used as a substrate. Reactions were terminated by spotting onto nitrocellulose, which was washed with 1M NaCl/1% phosphoric acid (approximately 6 times, 5-10 minutes each). Sheets were dried and the transferred radioactivity quantitated by phosphorimaging.[1]

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Fore more kinase assay, please refer to the SI of this article (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2946820/).

An Invitrogen TR-FRET-based LanthaScreen technique was used to assess the inhibition of the mTOR protein kinase. In order to calculate the IC50 values, compounds were tested at a maximum concentration of 10 mol/L in the presence of 1 μmol/L ATP. PI-103 is applied to U87MG cells for 24 hours. Using a cytotoxicity detection kit and a colorimetric determination of LDH activity, cell death is measured. Calculating the percentage of cell death (mean of three 12-well plates per experimental point) is calculated [(experimental value- low control)/(high control -low control) × 100], where the low control is treated with DMSO and the high control with Triton (1% Triton X-100, 30 min, 37 °).

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The human tumor cell lines U87MG, PC3, SKOV-3, IGROV-1, Detroit 562, HCT116, SNUC2B, and LoVo were obtained from the American Type Culture Collection. All cancer cell lines were grown in DMEM containing 2 mmol/L glutamine, with 100 U/mL penicillin and 100 μg/mL streptomycin, and supplemented with 10% fetal bovine serum in 5% CO2 in air at 37°C. Human umbilical vein endothelial cells (pooled) and their appropriate growth medium and supplements were obtained from TCS CellWorks. Cells were cultured according to the supplier’s instructions and used at passages 3 to 8. Cell viability was routinely >90%, as judged by trypan blue exclusion. All cell lines routinely tested negative for mycoplasma by PCR. GI50 values (concentrations) causing 50% inhibition of proliferation for tumor cells were determined using an Alamar Blue or a sulforhodamine B assay and, for human umbilical vascular endothelial cells, by an alkaline phosphatase assay following 96 h continuous exposure to compounds. Antibiotics were removed before this assay. [3]

PTEN status was verified in most cell lines, including U87MG and IGROV-1, by protein expression using Western blotting in-house, and in addition, PIK3CA status was determined or verified experimentally by sequencing. In the case of the colon cancer cell lines, PTEN status was again confirmed experimentally in-house by Western blotting. In addition, single-nucleotide polymorphism (SNP) profiling was used to confirm that the genotypes matched those provided in the Cancer Genome Project Cosmic database3 from which data on KRAS and PIK3CA mutation status was subsequently obtained. [3]

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Translocation, Immunoblotting, and Phosphoprotein Immunoassay on Cell Lines [3]
Forkhead translocation assays were done as described previously. Immunoblotting was done as follows. Cells were seeded in six-well plates at 3 to 5 × 105 cells/well in 2 mL medium, allowed to attach overnight, and treated with phosphatidylinositide 3-kinase inhibitors for the times indicated. After the incubation period, the medium was carefully removed from wells, and ~150 μL ice-cold Cell Extraction Buffer supplemented with Protease Inhibitor and Phosphatase Inhibitor was added to each well. Cell supernatant was collected after centrifugation at 14,000 × g at 4°C for 10 min, and its protein concentration was quantified using the BCA Protein Assay Kit.

For Western blotting, 30 μg of each lysate was separated by SDS-PAGE, electrotransferred onto nitrocellulose membranes, and probed with specific primary antibody and horseradish peroxidase-conjugated secondary antibody. Signal was detected with enhanced chemiluminescence reagent. Glyceraldehyde-3-phosphate dehydrogenase was used as the loading control.

Mice: Males aged five to six months are subcutaneously injected with one million cells in PBS, either from the FVB/N strain or the nude BALB/c strain. Mice are given daily injections of 50 mg/kg sorafenib and/or 10 mg/kg or 70 mg/kg of PI-103 when the tumor has grown to between 50 and 100 mm3. The same amount of DMSO is administered to control mice. Every two days, mice weight and tumor size are measured. Tumors are removed from mice after they have died and processed.

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Xenografts [2]
U87MG:ΔEGFR cells (106) were injected subcutaneously just caudal to the left forelimb in 6- to 12-week-old Balbc nu/nu mice (Harlan Sprague-Dawley). Mice with established tumors (50–100 mm3) were randomly allocated to IP treatment with 5 mg/kg PI-103 in 50% DMSO, or 50% DMSO alone (control). Tumor diameters were measured with calipers at 4 day intervals, and volumes were calculated from five mice per data point (mm3 = width2 × length/2). At necropsy, gross and microscopic analyses of all organs were performed to assess toxicity.

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Xenograft Tumor Efficacy and Pharmacodynamic Studies [3]
Two million U87MG human glioblastoma cells were injected s.c., bilaterally, into female 6- to 8-wk-old CrTac: Ncr-Fox1(nu) [Ncr] athymic mice bred in-house. PI-540 was prepared in sterile saline, PI-620 in sterile water, and GDC-0941 in 10% DMSO, 5% Tween 20, and 85% sterile saline. Compounds were dosed in 0.1 mL/10 g body weight of vehicle once or twice daily (PI-540 and PI-620 i.p.; GDC-0941 p.o.). Control animals received an equivalent volume of appropriate vehicle. Dosing for therapy studies commenced when solid tumors were well established (~5 mm mean diameter) and continued according to the schedule indicated in the figure legends. Tumors were measured across two perpendicular diameters, and volumes were calculated according to the formula: V = 4/3π [(d1 + d2) / 4]3 (29). Animals were weighed regularly and observed for adverse effects. When the experiment was terminated, mice were bled, plasma samples prepared, and tumors excised and weighed. Values of the percentage treated/control (T/C) were calculated from the treated versus control final tumor weights. Tumor samples were snap frozen for pharmacokinetic and/or pharmacodynamic analysis at intervals after the last dose. For dedicated pharmacodynamic studies, animals were dosed for 4 d and samples obtained as before. Plasma and tumor samples were analyzed for compound concentrations and tumor samples assessed for evidence of biomarker modulation by Meso Scale Discovery electrochemiluminescence immunoassay and/or immunoblot, as previously described (29, 34). In some experiments, IGROV-1 human ovarian cancer xenografts were studied using similar methods to those for U87MG.

Pharmacokinetics of GDC-0941 [3] The rapid plasma and tissue clearance of PI-103 is due to the rapid glucuronidation of phenolic groups. Although the metabolism of PI-540 and PI-620 in mouse and human microsomes was reduced compared to PI-103, significant in vivo glucuronidation was still observed. This explains the rapid clearance phenomenon described above. To eliminate this metabolic defect, we synthesized and tested a variety of phenolic isosteres. The indazole derivative GDC-0941 containing the solubilizer sulfadiazine showed limited microsomal metabolism, resulting in an oral bioavailability of 78%. In addition, it also had potent inhibitory activity on the phosphatidylinositol 3-kinase pathway (Figures 1B and E). Figure 6A shows the pharmacokinetics of GDC-0941 administered orally at a dose of 75 mg/kg to athymic mice carrying U87MG glioblastoma xenografts. GDC-0941 is rapidly absorbed, reaching peak plasma concentration (Cmax) within 30 minutes of administration. Tumor distribution is also rapid, with Cmax reached at the same time. Although the tumor-to-plasma concentration ratio is approximately 0.8, these characteristics result in tumor concentrations significantly higher than GI50 6 hours after administration.

[1]. Cell. 2006 May 19;125(4):733-47.

[2]. Cancer Cell. 2006 May;9(5):341-9.

[3]. Mol Cancer Ther. 2009 Jul;8(7):1725-38.

PI-103 is an organic heterotricyclic compound with the structure pyrido[3',2':4,5]furano[3,2-d]pyrimidine, substituted at positions 2 and 4 with 3-hydroxyphenyl and morpholino-4-yl groups, respectively. It is a dual kinase inhibitor with anticancer properties. PI-103 can be used as an EC 2.7.1.137 (phosphatidylinositol 3-kinase) inhibitor, mTOR inhibitor, and antitumor drug. It belongs to the morpholine, phenol, organic heterotricyclic, tertiary amine, and aromatic amine classes. PI-103 is a p110α inhibitor of class I PI3K. Phosphatidylinositol 3-kinase (PI3-K) is an important drug target, but the unique role of PI3-K isoenzymes remains unclear. This article introduces a pharmacological method for studying the PI3-K family. We synthesized a series of chemically diverse PI3-K inhibitors and enumerated their target selectivity using biochemical methods, revealing hidden homology between different targets and chemical types. The crystal structures of three inhibitors bound to p110γ showed that p110γ has a conformationally flexible region that can be specifically utilized by selective compounds. Subsequently, we used this array of compounds to identify the PI3-K isoforms required for insulin signaling. We found that p110α is the major insulin-responsive PI3-K in cultured cells, while p110β is not essential but sets the phenotypic threshold for p110α activity. Compounds targeting p110α were able to block the acute effects of insulin therapy in vivo, while p110β inhibitors did not. These results demonstrate a method for systematic target validation using an inhibitor matrix covering a protein family. [1]

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The PI3 kinase family is a class of lipid kinases that promote cell growth and survival by generating the second messenger phosphatidylinositol-3,4,5-triphosphate (PI3K). In order to identify key targets for PI3K activation-driven cancer, we screened a series of highly efficient and structurally diverse drug-like molecules targeting this enzyme family. Surprisingly, although many compounds were able to block PI3K signaling through their downstream effector molecule Akt, only one compound (PI-103) was able to inhibit the proliferation of glioma cells. The unique cellular activity of PI-103 is directly derived from its ability to simultaneously inhibit PI3Kα and mTOR. PI-103 showed significant activity in xenograft tumors and no toxicity was observed. These data suggest that the combined inhibition of mTOR and PI3 kinase α has significant therapeutic efficacy in malignant gliomas. [2] The phosphatidylinositol 3-kinase pathway is often dysregulated in human cancers, and its inhibitors have great therapeutic potential. We previously reported a promising tricyclic pyridinium-furan pyrimidine lead compound and chemical tool compound, PI-103. We now report the properties of pharmaceutically optimized bicyclic thiophene-pyrimidine derivatives PI-540 and PI-620, and the resulting clinical development candidate GDC-0941. All four compounds inhibited phosphatidylinositol 3-kinase p110α with an IC50 ≤ 10 nmol/L. Despite some differences in subtype selectivity, these compounds exhibited similar antiproliferative properties to PI-103 in vitro against various human cancer cell lines, demonstrating submicromolar potency in PTEN-negative U87MG human glioblastoma cells and similar regulatory effects on the phosphatidylinositol 3-kinase pathway. PI-540 and PI-620 showed improved solubility and metabolism, and high tissue distribution in mice. In a thymic mouse model of U87MG glioblastoma xenograft, intraperitoneal injection of PI-540 and PI-620 demonstrated superior antitumor efficacy compared to PI-103. The treatment/control ratios of PI-540 (50 mg/kg, twice daily) and PI-620 (25 mg/kg, twice daily) were 34% (66% inhibition) and 27% (73% inhibition), respectively. GDC-0941 exhibited in vitro antitumor activity comparable to PI-103, PI-540, and PI-620, with an oral bioavailability of 78% in mice. Following oral administration of 150 mg/kg, the tumor exposure concentration remained above the 50% antiproliferative concentration for 8 hours and persistently inhibited the phosphatidylinositol 3-kinase pathway. These properties demonstrate excellent dose-dependent oral antitumor activity; daily oral administration of 150 mg/kg inhibited the growth of U87MG glioblastoma and IGROWV-1 ovarian cancer xenografts by 98% and 80%, respectively. In conclusion, these data support the development of GDC-0941 as a highly effective, orally bioavailable phosphatidylinositol 3-kinase inhibitor. GDC-0941 has recently entered Phase I clinical trials. [3]

C₁₉H₁₇CLN₄O₃

384.82

384.099

C, 59.30; H, 4.45; Cl, 9.21; N, 14.56; O, 12.47

371935-79-4

PI-103;371935-74-9; 371935-79-4 (HCl)

16739368

Light yellow to yellow solid

3.847

2

7

2

27

489

0

Cl.O1CCN(C2=C3C(C4=CC=CN=C4O3)=NC(C3C=CC=C(C=3)O)=N2)CC1

XSQMYBFFYPTMFE-UHFFFAOYSA-N

InChI=1S/C19H16N4O3.ClH/c24-13-4-1-3-12(11-13)17-21-15-14-5-2-6-20-19(14)26-16(15)18(22-17)23-7-9-25-10-8-23;/h1-6,11,24H,7-10H2;1H

3-(6-morpholin-4-yl-8-oxa-3,5,10-triazatricyclo[7.4.0.02,7]trideca-1(9),2(7),3,5,10,12-hexaen-4-yl)phenol;hydrochloride

PI-103 hydrochloride; PI-103 HCl; PI 103 HYDROCHLORIDE; PI-103 Hydrochloride; 3-(6-morpholin-4-yl-8-oxa-3,5,10-triazatricyclo[7.4.0.02,7]trideca-1(9),2(7),3,5,10,12-hexaen-4-yl)phenol;hydrochloride; CHEMBL538346; PI 103 HCl; PI103 HCl

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)

DMSO: 4.1~24 mg/mL (10.7~68.9 mM)
Ethanol: ~19.7 mg/mL (~60.2 mM)

5%DMSO+40%PEG300+5%Tween80+50%ddH2O: 1 mg/mL (Please use freshly prepared in vivo formulations for optimal results.)