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 fast plasma and tissue clearance of PI-103 was the result of rapid glucuronidation of the phenol group. Despite decreases in mouse and human microsomal metabolism of PI-540 and PI-620 when compared with PI-103, significant in vivo glucuronidation was still observed. This accounts for the rapid clearance described in the previous section. To remove this metabolic liability, various phenol isosteres were synthesized and tested. The indazole derivative GDC-0941, which also contained the solubilizing sulfonyl piperazine, showed limited microsomal metabolism, resulting in 78% oral bioavailability, in addition to its potent inhibitory activity on the phosphatidylinositide 3-kinase pathway (Fig. 1B and E). Figure 6A shows the pharmacokinetics of GDC-0941 administered p.o. at 75 mg/kg to athymic mice bearing U87MG glioblastoma xenografts. GDC-0941 was very rapidly absorbed with Cmax achieved 30 minutes postadministration. Tumor distribution was equally rapid with Cmax reached at the same time. Although the tumor to plasma ratio was around 0.8, these properties resulted in tumor concentrations of compound well above the GI50 at 6 hours postadministration.

[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 that is pyrido[3',2':4,5]furo[3,2-d]pyrimidine substituted at positions 2 and 4 by 3-hydroxyphenyl and morpholin-4-yl groups respectively. A dual-kinase inhibitor with anti-cancer properties. It has a role as an EC 2.7.1.137 (phosphatidylinositol 3-kinase) inhibitor, a mTOR inhibitor and an antineoplastic agent. It is a member of morpholines, a member of phenols, an organic heterotricyclic compound, a tertiary amino compound and an aromatic amine. PI-103 is an inhibitor of p110α of class I PI3K.

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Phosphoinositide 3-kinases (PI3-Ks) are an important emerging class of drug targets, but the unique roles of PI3-K isoforms remain poorly defined. We describe here an approach to pharmacologically interrogate the PI3-K family. A chemically diverse panel of PI3-K inhibitors was synthesized, and their target selectivity was biochemically enumerated, revealing cryptic homologies across targets and chemotypes. Crystal structures of three inhibitors bound to p110gamma identify a conformationally mobile region that is uniquely exploited by selective compounds. This chemical array was then used to define the PI3-K isoforms required for insulin signaling. We find that p110alpha is the primary insulin-responsive PI3-K in cultured cells, whereas p110beta is dispensable but sets a phenotypic threshold for p110alpha activity. Compounds targeting p110alpha block the acute effects of insulin treatment in vivo, whereas a p110beta inhibitor has no effect. These results illustrate systematic target validation using a matrix of inhibitors that span a protein family.[1]

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The PI3 kinase family of lipid kinases promotes cell growth and survival by generating the second messenger phosphatidylinositol-3,4,5-trisphosphate. To define targets critical for cancers driven by activation of PI3 kinase, we screened a panel of potent and structurally diverse drug-like molecules that target this enzyme family. Surprisingly, a single agent (PI-103) effected proliferative arrest in glioma cells, despite the ability of many compounds to block PI3 kinase signaling through its downstream effector, Akt. The unique cellular activity of PI-103 was traced directly to its ability to inhibit both PI3 kinase alpha and mTOR. PI-103 showed significant activity in xenografted tumors with no observable toxicity. These data demonstrate an emergent efficacy due to combinatorial inhibition of mTOR and PI3 kinase alpha in malignant glioma.[2]

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The phosphatidylinositide 3-kinase pathway is frequently deregulated in human cancers and inhibitors offer considerable therapeutic potential. We previously described the promising tricyclic pyridofuropyrimidine lead and chemical tool compound PI-103. We now report the properties of the pharmaceutically optimized bicyclic thienopyrimidine derivatives PI-540 and PI-620 and the resulting clinical development candidate GDC-0941. All four compounds inhibited phosphatidylinositide 3-kinase p110alpha with IC(50) < or = 10 nmol/L. Despite some differences in isoform selectivity, these agents exhibited similar in vitro antiproliferative properties to PI-103 in a panel of human cancer cell lines, with submicromolar potency in PTEN-negative U87MG human glioblastoma cells and comparable phosphatidylinositide 3-kinase pathway modulation. PI-540 and PI-620 exhibited improvements in solubility and metabolism with high tissue distribution in mice. Both compounds gave improved antitumor efficacy over PI-103, following i.p. dosing in U87MG glioblastoma tumor xenografts in athymic mice, with treated/control values of 34% (66% inhibition) and 27% (73% inhibition) for PI-540 (50 mg/kg b.i.d.) and PI-620 (25 mg/kg b.i.d.), respectively. GDC-0941 showed comparable in vitro antitumor activity to PI-103, PI-540, and PI-620 and exhibited 78% oral bioavailability in mice, with tumor exposure above 50% antiproliferative concentrations for >8 hours following 150 mg/kg p.o. and sustained phosphatidylinositide 3-kinase pathway inhibition. These properties led to excellent dose-dependent oral antitumor activity, with daily p.o. dosing at 150 mg/kg achieving 98% and 80% growth inhibition of U87MG glioblastoma and IGROV-1 ovarian cancer xenografts, respectively. Together, these data support the development of GDC-0941 as a potent, orally bioavailable inhibitor of phosphatidylinositide 3-kinase. 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.)