CH5132799

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Biological Activity

Description	CH5132799 inhibits class I PI3Ks, particularly PI3Kα with IC50 of 14 nM; less potent to PI3Kβδγ, while sensitive in PIK3CA mutations cell lines. Phase 1.
Targets	PI3Kα	PI3Kβ	PI3Kδ	PI3Kγ
IC50	0.014 μM	0.12 μM	0.50 μM	0.036 μM [1]
In vitro	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. CH5132799 exhibits more inhibitory activities against PI3Kα with oncogenic mutations E542K (IC50 = 6.7 nM), E545K (IC50 = 6.7 nM) and H1047R (IC50 = 5.6 nM) than wild-type PI3Kα. 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 harboring PIK3CA mutations are significantly sensitive to CH5132799 [1] In human tumor cell lines with PI3K pathway activation by mutation, CH5132799 shows potent antiproliferative activity [HCT116(CRC): IC50 = 0.20 lM, KPL-4(BC):13 IC50 = 0.032 lM, T-47D(BC): IC50 = 0.056 lM, SK-OV-3(Ovarian): IC50 = 0.12 lM]. CH5132799 effectively suppresses phosphorylation of AKT in KPL-4 cells. [2]
In vivo	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] CH5132799 as a clinical candidate that shows excellent oral bioavailability (BA) (101% in mouse), human liver microsomal stability and in vivo antitumor activity in the PC-3 xenograft model (TGI: 101% at 25 mg/kg, po, q.d. × 11 days). CH5132799 exhibits good oral BA in mouse, rat, monkey and dog (F: 54.2-101%). In a human breast cancer (KPL-4: PI3Ka H1047R) xenograft model in mice, oral treatment with CH5132799 (12.5 mg/kg, q.d.) shows strong tumor regression. The strong regression is maintained during the 6 week administration, even in the intermittent dosing schedule (q.d., 2 weeks on/1 week off; q.d., 5 days on/2 days off), suggesting that a flexible administration schedule can be applicable in the clinic.[2]
Features

Protocol(Only for Reference)

Kinase Assay: ^[1]

PI3K Assay

The E542K, E545K, and H1047R mutants of PI3Kα are prepared with an overlapped extension-polymerase chain reaction. Glutathione S-transferase-tagged PI3Kα mutants and His-tagged p85α are co-expressed with BAC-TO-BAC Baculovirus Expression System. The inhibitory activities of CH5132799 on PI3Kα (p110α/p85α), PI3Kβ(p110β/p85α), PI3Kδ (p110δ/p85α), PI3Kγ (p110γ), PI3KC2α, PI3KC2β, Vps34, and PI3Kα mutants are determined by Adapta Universal Kinase Assay Kit. Time-resolved fluorescence is measured with an EnVision HTS microplate reader. IC50 values are calculated using XLfit.

Cell Assay: ^[1]

Cell lines	various cancer cell lines belonging to 4 types of cancer—breast, ovarian, prostate, and endometrial cancer—in which the PIK3CA mutation and PTEN deficiency are frequently found and in which RAS/RAF is rarely mutated
Concentrations	0 - 10μM
Incubation Time	4 days
Method	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 200

Animal Study: ^[1]

Animal Models	A total of 4 × 106 to 1.2 × 107 cells are injected subcutaneously into the right flank of female BALB-nu/nu mice.
Dosages	0.39, 0.78, 1.56, 3.13, 6.25, 12.5 and 25 mg/kg
Administration	Orally administered once a day.
Solubility	1% DMSO/30% polyethylene glycol/1% Tween 80, 30 mg/mL

1

References

Clinical Trial Information( data from http://clinicaltrials.gov)

NCT Number	Recruitment	Conditions	Sponsor /Collaborators	Start Date	Phases
NCT01222546	Active, not recruiting	Solid Tumors	Chugai Pharma Europe Ltd.	2010-08	Phase 1

Chemical Information

Download CH5132799 SDF

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

Preparing Stock Solutions

Stock Solution (1ml DMSO)	1mM	10mM	20mM	30mM
Mass(mg)	0.37742	3.7742	7.5484	11.3226

Customer Reviews (1)

Click to enlarge	Rating
	Source	CH5132799 purchased from Selleck
	Method	Western blot
	Cell Lines	A549 cells
	Concentrations	1-20 uM
	Incubation Time	24 h
	Results	This PI3K inhibitor dramatically reduced AKT phosphorylation at 10uM.