PI3Kα (IC50 = 180 nM); PI3Kα-H1047R (IC50 = 5.6 nM); PI3Kα-E545K (IC50 = 6.7 nM); PI3Kα-E542K (IC50 = 6.7 nM); PI3Kγ (IC50 = 36 nM); PI3Kβ (IC50 = 120 nM); PI3Kδ (IC50 = 500 nM); PI3KC2β (IC50 = 5.3 μM); mTOR (IC50 = 1.6 μM)
CH5132799 selectively inhibits class I PI3Ks: PI3Kα (IC50=0.014 μmol/L), PI3Kα E542K (IC50=0.0067 μmol/L), PI3Kα E545K (IC50=0.0067 μmol/L), PI3Kα H1047R (IC50=0.0056 μmol/L), PI3Kβ (IC50=0.12 μmol/L), PI3Kδ (IC50=0.50 μmol/L), PI3Kγ (IC50=0.036 μmol/L). [1]
It shows weaker inhibition against class II PI3Ks (PI3KC2α IC50>10 μmol/L; PI3KC2β IC50=5.3 μmol/L), class III PI3K Vps34 (IC50>10 μmol/L), and class IV PI3K mTOR (IC50=1.6 μmol/L). No significant inhibition (IC50>10 μmol/L) against 26 protein kinases including ALK, EGFR, HER2, IGF1R, Akt1, CDK2, MEK1, PKCα, and S6K. [1][2]

CH5132799 selectively inhibits class I PI3Ks, PI3Kα (IC50 = 0.014 μM), PI3Kβ (IC50 = 0.12 μM), PI3Kδ (IC50 = 0.50 μM), PI3Kγ (IC50 = 0.036 μM), but shows less inhibition of class II PI3Ks, class III PI3k and mTOR and also no inhibitory activity (IC50 > 10 μM) against 26 protein kinases. In comparison to wild-type PI3Kα, CH5132799 has stronger inhibitory effects against PI3Kα with the oncogenic mutations E542K (IC50 = 6.7 nM), E545K (IC50 = 6.7 nM), and H1047R (IC50 = 5.6 nM). CH5132799 treated breast cnacer KPL-4 cells, which harbor the PIK3CA mutation, phosphorylation of Akt and its direct substrates, PRAS40 and FoxO1/3a and phosphorylation of downstream factors, including S6K, S6 and 4E-BP1, are effectively suppressed. Cancer cell lines with PIK3CA mutations are significantly more sensitive to CH5132799 [1].The BAC-TO-BAC Baculovirus Expression System is used in conjunction with PI3K mutants that have been Glutathione S-transferase-tagged and His-tagged p85. Using the Adapta Universal Kinase Assay Kit, CH5132799's inhibitory effects on PI3K (p110/p85), PI3K (p110/p85), PI3K (p110), PI3KC2, PI3KC2, Vps34, and PI3K mutants are assessed. An EnVision HTS microplate reader is used to measure time-resolved fluorescence. Utilizing XLfit, IC50 values are determined.

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CH5132799 showed potent antiproliferative activity across a large panel of breast, ovarian, prostate, and endometrial cancer cell lines, with 75% (45/60) of lines having IC50 below 1 μmol/L and 38% (23/60) below 0.3 μmol/L. Cell lines harboring PIK3CA mutations were significantly more sensitive (P=7.4×10^{-9}) than those without mutations, while PTEN deficiency was not significantly associated (P=0.11). HER2-amplified lines with PIK3CA mutations were as sensitive as those with only PIK3CA mutations. Coexistence of PIK3CA and RAS/RAF mutations resulted in higher sensitivity than only RAS/RAF mutations (P=0.015). [1]
In KPL-4 breast cancer cells (PIK3CA H1047R), CH5132799 suppressed phosphorylation of Akt, PRAS40, FoxO1/3a, S6K, S6, and 4E-BP1 at concentrations as low as 0.003 μmol/L after 2-hour treatment. It induced G1 phase cell cycle arrest and increased sub-G1 population, along with caspase 3/7 activation. [1]
Compared to everolimus (mTORC1 inhibitor) and BEZ235 (PI3K/mTOR dual inhibitor), CH5132799 did not enhance Akt phosphorylation via S6K-mediated negative feedback loop at any tested dose (0.001-1 μmol/L for 24h) in BT-474 cells. Everolimus enhanced Akt phosphorylation while inhibiting S6K; BEZ235 enhanced Akt at low dose (0.01 μmol/L) but inhibited at high doses. CH5132799 also efficiently inhibited 4E-BP1 phosphorylation, unlike everolimus which had little effect. Similar results were observed in SK-OV-3, MDA-MB-453, and HCT116 cells. [1]
CH5132799 exhibited antiproliferative IC50 values: 0.20 μmol/L in HCT116 (CRC, PI3Kα H1047R), 0.032 μmol/L in KPL-4 (breast, PI3Kα H1047R), 0.056 μmol/L in T-47D (breast, PI3Kα H1047R), 0.12 μmol/L in SK-OV-3 (ovarian, PI3Kα H1047R), 0.18 μmol/L in MFE-280 (endometrial, PI3Kα H1047Y), and 0.14 μmol/L in ME-180 (cervical, PI3Kα E545K). [2]

CH5132799 shows potent in vivo antitumor activity in several different xenograft models with PIK3CA mutations. CH5132799 overcomes mTORC1 inhibition-mediated Akt activation and regrowth of xenograft tumor by long-term everolimus administration.[1]

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In mouse xenograft models, oral administration of CH5132799 (12.5 mg/kg, 25 mg/kg, 50 mg/kg, q.d.) induced rapid tumor regression in KPL-4 (breast, PIK3CA H1047R) and BT-474 (breast, PIK3CA K111N) models. In KPL-4 model, 12.5 mg/kg q.d. for 6 weeks maintained strong regression even with intermittent schedules (2 weeks on/1 week off; 5 days on/2 days off). [1][2]
Potent antitumor activity was also observed in ovarian cancer SK-OV-3 and endometrial cancer MFE-280 models (both with PIK3CA mutations). In PTEN-deficient models, CH5132799 shrank GXF97 (gastric, PTEN deletion) and inhibited PC-3 (prostate) tumor growth. In MDA-MB-231 (KRAS/BRAF mutations), it was less effective, but HCT116 (PIK3CA+KRAS mutations) showed strong tumor growth inhibition. [1]
Combination of CH5132799 (12.5 mg/kg q.d. for 12 days) with trastuzumab (30 mg/kg weekly for 2 weeks) in trastuzumab-insensitive KPL-4 tumors induced complete tumor disappearance, which was maintained for over one month without further treatment. [1]
In BT-474 tumors regrown after long-term everolimus treatment (50 mg/kg q.d. for 31 days), switching to CH5132799 (12.5 or 25 mg/kg q.d. for 7 days) induced remarkable dose-dependent regression. CH5132799 suppressed phosphorylation of Akt, FoxO1, S6K, S6, and 4E-BP1, whereas everolimus only inhibited S6K/S6 and induced Akt/FoxO1 phosphorylation. [1]

The E542K, E545K, and H1047R mutants of PI3Kα are prepared with an overlapped extension-polymerase chain reaction. The cell lines are added to the wells of 96-well plates containing 0.076 to 10,000 nM CH5132799 and incubated at 37 °C. After 4 days of incubation, Cell Counting Kit-8 solution is added and, after incubation for several more hours, absorbance at 450 nm is measured with Microplate-Reader iMark. The antiproliferative activity is calculated by the formula (1- T/C) × 100 (%), in which T and C represent absorbance at 450 nm of the cells treated with CH5132799 (T) and that of untreated control cells (C). The IC50 values are calculated by using Microsoft Excel 2007.

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In cell-free kinase assays, increasing concentrations of CH5132799 were incubated with each kinase, and residual activity was measured to calculate IC50 values. Class I PI3Ks (α, β, δ, γ), class II (C2α, C2β), class III (Vps34), and class IV (mTOR) were evaluated. CH5132799 showed ATP-competitive inhibition as determined by co-crystal structure analysis with PI3Kγ (PDB ID: 3APC). [1][2]
For PI3Kγ, the compound was co-crystallized, and diffraction data were collected at synchrotron beamlines. The crystal structure of PI3Kγ/inhibitor complex revealed hydrogen bonds between the morpholine oxygen and valine 882, and between the aminopyrimidine moiety and aspartic acid 836/lysine 833 (for compound 7) or aspartic acid 841 (for CH5132799). [2]

The cell lines are put into the 96-well plates with 0.076 to 10,000 nM CH5132799 and then incubated at 37 °C. Cell Counting Kit-8 solution is added after 4 days of incubation, and then Microplate-Reader iMark is used to measure absorbance at 450 nm. The formula (1- T/C) 100 (%) is used to determine the antiproliferative activity, where T and C stand for the absorbance at 450 nm of control cells that were left untreated and CH5132799-treated cells, respectively. Microsoft Excel 2007 is used to calculate the IC50 values.

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Cell proliferation was assessed using the Cell Counting Kit-8 assay. Cell lines were seeded in 96-well plates containing 0.076 to 10,000 nmol/L CH5132799 and incubated at 37°C for 4 days. After addition of CCK-8 solution and incubation, absorbance at 450 nm was measured. Antiproliferative activity was calculated as (1 - T/C) × 100%, and IC50 values were determined using Excel. [1]
For Western blot analysis, cells (e.g., KPL-4, BT-474) were treated with CH5132799 (0.001-1 μmol/L) for 2 or 24 hours, then lysed and subjected to SDS-PAGE and immunoblotting with antibodies against phospho-Akt, total Akt, phospho-PRAS40, phospho-FoxO1/3a, phospho-S6K, phospho-S6, phospho-4E-BP1, and others. [1]
Cell cycle analysis: KPL-4 cells treated with CH5132799 were fixed, stained with propidium iodide, and analyzed by flow cytometry to determine G1 arrest and sub-G1 population. Apoptosis was measured by caspase 3/7 activation assay. [1]

Mice: The mice used are female BALB-nu/nu (CAnN.Cg-Foxn1/CrlCrlj nu/nu) mice. Subcutaneous injection of 4106 to 1.2107 cells, suspended in 100 to 200 L of serum-free culture medium, is performed in the right flank of the mice. Tumor volume (TV) is determined after measuring the size of the tumor twice per week with a gauge. Animals are divided into groups at random (n=4 or 5) once the tumors have reached a volume of roughly 200 to 300 mm3 and treatment has begun. Once daily oral administration of CH5132799, Everolimus, and Trastuzumab is combined with a weekly intravenous injection of Trastuzumab.

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For in vivo xenograft studies, female BALB-nu/nu mice were subcutaneously injected with 4×10^6 to 1.2×10^7 tumor cells suspended in serum-free medium. When tumors reached approximately 200-300 mm^3, mice were randomized into groups (n=4 or 5) and treatment initiated. Tumor volume was measured twice weekly using a caliper and calculated as TV = a × b^2 / 2 (a=length, b=width). CH5132799 was orally administered once daily. Trastuzumab was injected intravenously once weekly. Everolimus was orally administered once daily. For combination studies, CH5132799 was given for 12 consecutive days and trastuzumab for 2 weeks. For long-term everolimus treatment, mice received everolimus for 31 days until tumor regrowth, then were switched to CH5132799 for 7 days. [1]
For PK studies, CH5132799 was administered intravenously and orally to mice, rats, dogs, and monkeys. Blood samples were collected at various time points, and plasma concentrations were analyzed to calculate pharmacokinetic parameters. [2]

Oral bioavailability of CH5132799 was 101% in mouse (1 mg/kg po, AUCinf=978 ng·h/mL, vs 2 mg/kg iv, AUCinf=1930 ng·h/mL), 101% in rat (1 mg/kg po, AUCinf=978 ng·h/mL; 0.5 mg/kg iv, AUCinf=1350 ng·h/mL), 54.2% in dog (1 mg/kg po, AUCinf=1430 ng·h/mL; 0.25 mg/kg iv, AUCinf=1690 ng·h/mL), and 78.4% in monkey (1 mg/kg po, AUCinf=2170 ng·h/mL; 0.25 mg/kg iv, AUCinf=692 ng·h/mL). Half-lives: mouse po T1/2=3.8 h, iv T1/2=1.7 h; rat po T1/2=3.8 h; dog po T1/2=3.3 h, iv T1/2=1.6 h; monkey po T1/2=6.7 h, iv T1/2=3.2 h. Clearance (CL): mouse iv 17.5 mL/min/kg; rat iv 6.3 mL/min/kg; dog iv 6.4 mL/min/kg; monkey iv 6.2 mL/min/kg. Tmax: mouse po 0.25 h; rat po 0.25 h; dog po 4.5 h; monkey po 4.0 h. [2]
CH5132799 exhibited excellent human liver microsomal stability (no specific half-life provided) and improved metabolic stability compared to earlier leads. [2]

In all in vivo mouse xenograft studies, the tested doses of CH5132799 (up to 50 mg/kg q.d.) were well tolerated, with no gross toxicity observed in treated animals. [1]

[1]. Clin Cancer Res. 2011 May 15;17(10):3272-81.

[2]. Bioorg Med Chem Lett. 2011 Mar 15;21(6):1767-72.

CH5132799 has been used in clinical trials for the treatment of solid tumors. Izolixib is a highly bioavailable, orally administered Class I phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) catalytic subunit α (PIK3CA) inhibitor with potential antitumor activity. After administration, izolexib selectively binds to and inhibits PIK3CA and its mutated forms in the PI3K/Akt (protein kinase B)/mammalian target of rapamycin (mTOR) pathway. This leads to apoptosis and growth inhibition in PIK3CA-expressing tumor cells. Because izolexib specifically targets PIK3CA, it may be more effective and less toxic than pan-PI3K inhibitors. Furthermore, izolexib also targets mutated forms of PI3Kγ (PI3Kg). It can also stimulate the immune system, restoring CD8+ T cell activation and cytotoxicity. Dysregulation of the PI3K/Akt/mTOR pathway is common in solid tumors and leads to tumor cell growth, survival, and resistance to chemotherapy and radiotherapy. PIK3CA is one of the most commonly mutated oncogenes, encoding the p110-α catalytic subunit of class I PI3K. In most solid tumors, activation of the PI3K pathway is caused by PIK3CA mutations.

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The PI3K/Akt pathway is frequently deregulated in human cancers. PIK3CA mutations occur in ~27% of breast, ~8% of ovarian, ~24% of endometrial, and less frequently in prostate cancers. CH5132799 is the first PI3K inhibitor reported to show statistically significant antitumor activity against cancer cells harboring PIK3CA mutations across multiple tumor types. [1]
CH5132799 overcomes trastuzumab resistance in HER2-amplified/PIK3CA-mutant breast cancer, as trastuzumab alone fails to inhibit constitutively active PI3K. The combination led to long-lasting tumor disappearance. [1]
Unlike mTORC1 inhibitors, CH5132799 avoids S6K inhibition-mediated Akt activation and efficiently suppresses 4E-BP1 phosphorylation, which is important for cell proliferation and tumor suppression. This provides an advantage in treating tumors resistant to everolimus. [1]
The compound was discovered via structure-based drug design using a homology model of PI3Kα and X-ray crystal structures of PI3Kγ. The aminopyrimidine moiety replaced a phenol group to improve metabolic stability and oral bioavailability. [2]

C15H19N7O3S

377.42

377.127

C, 47.73; H, 5.07; N, 25.98; O, 12.72; S, 8.50

1007207-67-1

1007207-67-1;2242053-82-1 (mesylate);

49784945

White to gray solid powder

1.6±0.1 g/cm3

751.1±70.0 °C at 760 mmHg

408.1±35.7 °C

0.0±2.5 mmHg at 25°C

1.706

-0.87

1

10

3

26

587

0

S(C([H])([H])[H])(N1C2C(=C(C3=C([H])N=C(N([H])[H])N=C3[H])N=C(N=2)N2C([H])([H])C([H])([H])OC([H])([H])C2([H])[H])C([H])([H])C1([H])[H])(=O)=O

JEGHXKRHKHPBJD-UHFFFAOYSA-N

InChI=1S/C15H19N7O3S/c1-26(23,24)22-3-2-11-12(10-8-17-14(16)18-9-10)19-15(20-13(11)22)21-4-6-25-7-5-21/h8-9H,2-7H2,1H3,(H2,16,17,18)

5-(7-(methylsulfonyl)-2-morpholino-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrimidin-2-amine

PA-799; PA799; PA-799; Izorlisib; CH5132799; CH-5132799; CH 5132799

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: ≥ 0.46 mg/mL (1.22 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 4.6 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: 1% DMSO +30% polyethylene glycol+1% Tween 80 : 30mg/mL

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