PI3Ky (IC50 = 23 nM); PI3Kδ (IC50 = 36 nM); PI3Kα (IC50 = 39 nM); PI3Kβ (IC50 = 383 nM)
Pilaralisib analogue targets all class I PI3K family members, with IC₅₀ values of 39 nM for PI3Kα, 36 nM for PI3Kδ, 23 nM for PI3Kγ, and 383 nM for PI3Kβ in cell-free assays. The compound exhibits potent inhibitory activity against PI3Kα/δ/γ, with relatively weaker activity against PI3Kβ.
XL147 is an ATP-competitive reversible pan-class I PI3K inhibitor. It exhibits an IC50 against wild-type and mutant p110α of approximately 40 nM. It has recently completed phase I clinical development. [1]

In HER2-overexpressing human breast cancer cell lines (including BT474, HCC1937, etc.), Pilaralisib analogue treatment eliminates AKT and S6 phosphorylation. All tested cell lines exhibit dose-dependent inhibition of monolayer growth, with cell death induced at 20 μM concentration. The compound causes decreases in cyclin D1 and pRB, along with an increase in the CDK inhibitor p27ᴷᴵᴾ¹. Pilaralisib analogue treatment leads to dose-dependent decreases in pAKTS473/T308 and pS6S240/244, while promoting increased expression and phosphorylation of HER3 and other receptor tyrosine kinases. Combination with trastuzumab or lapatinib synergistically enhances cell death and pAKT/pS6 inhibition.

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XL147 inhibits class I PI3K isoforms in an ATP-competitive manner. Treatment with XL147 eliminates AKT and S6 phosphorylation in a panel of HER2-overexpressing human breast cancer cell lines, but it also promotes the expression and phosphorylation of HER3 and other RTKs. The HER2 tyrosine kinase maintains the phosphorylation of HER3 in HER2+ cells, which results in a partial recovery of phosphorylated AKT (pAKT), limiting the antitumor effect of XL 147. Furthermore, transtuzumab or lapatinib therapy or HER3 knockdown sensitize HER2+ breast cancer cells to XL147 in vitro and in vivo. All tested cell lines, including BT474, HCC1937, and others, exhibit dose-dependent inhibition of monolayer growth in response to treatment with XL147. Inhibition of cell proliferation is XL147's main effect. At a 20 M concentration, XL147 induces cell death. PI3K is dose-dependently inhibited after receiving treatment with XL147. A decrease in cyclin D1 and pRB and an increase in the CDK inhibitor p27KIPI are caused by XL147, which is consistent with the inhibition of cell proliferation. However, there is no discernible change in the levels of total or cleaved poly (ADP-ribose) polymerase (PARP). A dose-dependent decrease in pAKTS473/T308 and pS6S240/244 results from XL147 treatment. Unexpectedly, XL147 also causes an increase in the levels of total HER3 and/or pHER3Y1289. When PI3K is inhibited in HER2-overexpressing cells, several receptor tyrosine kinases, including HER3, are then expressed and phosphorylated more strongly. HER3, InsR, IGF1R, and FGFR2 mRNAs are not induced when PI3K is inhibited when FoxO1 and FoxO3a transcription factors are knocked down. Trastuzumab or lapatinib cotreatment or HER3 siRNA knockdown in HER2+ cells both increase XL147-induced cell death and pAKT and pS6 inhibition.[1]

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PI3K (p110α) (IC50 = approximately 40 nM against wild-type and mutant p110α) [1]

In BT474 xenograft nude mouse models, Pilaralisib analogue as a single agent significantly inhibits tumor growth. When combined with trastuzumab, 3 out of 8 mice achieve complete tumor regression; no complete regressions are observed with lapatinib combination. The combination of trastuzumab and XL147 inhibits pHER3 more effectively than any other treatment. Tumors treated with XL147 plus lapatinib or XL147 plus trastuzumab show lower nuclear pAKT levels compared to monotherapy-treated tumors. No obvious drug-related toxicity is observed in any treatment group.

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Athymic mice with BT474 xenografts are randomly treated with XL147, lapatinib, trastuzumab, or XL147 plus each HER2 antagonist.Trastuzumab was the only medication to cause a complete tumor regression in one of eight mice, significantly inhibiting tumor growth with each monotherapy. Both combinations are better than the individual medications when taken alone. Notably, three out of eight mice receive a full tumor response from the combination of trastuzumab and XL147, but not from the combination of lapatinib and XL147. In any of the treatment arms, there is no overt drug-related toxicity. More effectively than any other treatment, XL147 combined with trastuzumab inhibits pHER3.Nuclear pAKT is lower in tumors treated with XL147 plus lapatinib or XL147 plus trastuzumab compared to tumors treated with single agents, which is in excellent agreement with differences in tumor growth among treatment arms. Only XL147 has been statistically proven to statistically reduce nuclear pAKT levels among the three individual drugs. Cytoplasmic pAKT levels do not change in any discernible way. To effectively inhibit the PI3K/AKT pathway's signaling output, HER2 and PI3K must be inhibited simultaneously in HER2-dependent xenografts.[1]

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Treatment with Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5) resulted in dose-dependent inhibition of cell growth and levels of pAKT (S473/T308) and pS6 (S240/244) in human breast cancer cell lines with constitutive PI3K activation. [1]
In HER2-overexpressing cells, inhibition of PI3K with Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5) was followed by up-regulation of expression and phosphorylation of multiple receptor tyrosine kinases, including HER3, total HER2 and/or pHER2 (Y1248). [1]
Knockdown of FoxO1 and FoxO3a transcription factors suppressed the induction of HER3, InsR, IGF1R, and FGFR2 mRNAs upon inhibition of PI3K with Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5). [1]
In HER2+ cells, knockdown of HER3 with siRNA or cotreatment with the HER2 inhibitors trastuzumab or lapatinib enhanced Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5)-induced cell death and inhibition of pAKT and pS6. [1]
The allosteric inhibitor of AKT1/2 5J8 and the PI3K inhibitor LY294002, but not the mTOR inhibitor rapamycin, also up-regulated HER3 mRNA. [1]
Treatment with Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5) and 5J8 resulted in accumulation of FoxO1 and FoxO3a factors in the nucleus of BT474 and MDA453 cells. [1]
Knockdown of HER3 with siRNA eliminated the HER3 and pHER3Y1197 band in the p85 pull-downs of lysates from Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5)-treated cells. [1]
Cell proliferation was significantly reduced by a combination of HER3 knockdown and Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5) compared with either intervention alone. The combination induced a greater proportion of cells in the sub-G1 phase DNA fraction as well as PARP cleavage compared with either treatment alone. [1]
In BT474 cells, the combination of Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5) with the HER2 antibody trastuzumab or the HER2 TKI lapatinib was significantly more effective at inhibiting cell proliferation or PARP cleavage than XL147 or each HER2 antagonist alone. [1]
In BT474 cells, treatment with Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5) plus lapatinib or plus trastuzumab attenuated the recovery of pHER3 compared with cells treated with XL147 alone. [1]
Treatment with Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5) resulted in an increase in the phosphorylation of HER3 and multiple other RTKs, including EGFR, ERBB4/HER4, FGFR1, FGFR2, FGFR3, FGFR4, InsR, IGF1R, EphA1, Tie2, TrkA, Flt3, MER, and macrophage-stimulating protein receptor. [1]
Following treatment of BT474 cells with Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5), there was an increase in ERBB4, IGF1R, InsR, EphA1, FGFR2, and FGFR3 mRNAs, with IGF1R and InsR being the most prominent. [1]
Knockdown of FoxO1 and FoxO3a with siRNA limited the induction of IGF1R, InsR, and FGFR2 mRNAs in cells treated with Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5). [1]
Depletion of IGF1R or InsR with siRNA sensitized BT474, MDA453, and MCF7 cells to Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5)-mediated growth inhibition. [1]

Pilaralisib analog, also known as XL147 analogue, is a Class 1 PI3K kinase family inhibitor, and is a n orally bioavailable small molecule, targeting the class I phosphatidylinositol 3 kinase (PI3K) family of lipid kinases, with potential antineoplastic activity. Class 1 PI3K kinase family inhibitor XL147 reversibly binds to class 1 PI3Ks in an ATP-competitive manner, inhibiting the production of the secondary messenger phosphatidylinositol-3,4,5-trisphosphate (PIP3) and activation of the PI3K signaling pathway; this may result in inhibition of tumor cell growth and survival in susceptible tumor cell populations. Activation of the PI3K signaling pathway is frequently associated with tumorigenesis.

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The inhibitory activity of Pilaralisib analogue against class I PI3K kinases is assessed using cell-free kinase assays. Purified class I PI3K isoforms (α, β, γ, δ) are incubated with ATP and various concentrations of the test compound in reaction buffer. Enzyme activity is measured by detecting PIP3 production from phosphatidylinositol phosphorylation, and IC₅₀ values are calculated.

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In athymic mice bearing BT474 xenografts, treatment with Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5) alone significantly delayed tumor growth. The combination of trastuzumab and Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5) induced a complete tumor response in three of eight mice (P < 0.05). [1]
Treatment with Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5) for 28 days did not change pHER3 levels in xenografts, whereas lapatinib was more effective than trastuzumab in reducing pHER3. The combination of Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5) plus trastuzumab inhibited pHER3 more potently than any of the other treatments. Nuclear pAKT was lower in tumors treated with Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5) plus lapatinib or XL147 plus trastuzumab compared with tumors treated with single agents. Of all three single drugs, Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5) was the only one shown statistically to inhibit nuclear pAKT levels. [1]

Cells (BT474, HCC1937 et al) are seeded in 100-mm dishes in media containing 2.5% FBS with or without XL147. After 3 days, detached and adherent cells are pooled, fixed, and labeled with propidium iodide by using the APO-BrdU kit. Labeled cells are analyzed using the Becton Dickinson FACSCalibur system.

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HER2-overexpressing human breast cancer cell lines (BT474, HCC1937, etc.) are seeded in culture plates and treated with various concentrations of Pilaralisib analogue. Cell proliferation inhibition is assessed by MTT assay or cell counting, and phosphorylation levels of AKT, S6, HER3, and other proteins are detected by Western blot. In combination studies, cells are co-treated with trastuzumab or lapatinib to evaluate synergistic effects. Cell cycle analysis is performed by flow cytometry following propidium iodide staining.

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Athymic female mouse bearing BT474 cells
100 mg/kg
Orogastric gavage

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BT474 Xenograft Model: BT474 human breast cancer cells are implanted subcutaneously into athymic nude mice to establish xenograft models. When tumors reach a certain size, animals are randomized into treatment groups: Pilaralisib analogue alone, trastuzumab alone, lapatinib alone, or Pilaralisib analogue in combination with each HER2 antagonist. Tumor volume and body weight are measured regularly to evaluate tumor growth inhibition and complete regression rates.

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Cell lines were treated with 0 to 20 μM Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5) and counted on the days indicated. Cells were cultured in Matrigel with or without 0 to 20 μM Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5) and photographed on indicated days. [1]
Cells lines were harvested after overnight treatment with 0 to 20 μM Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5) in serum-free medium followed by immunoblot analysis with indicated antibodies. [1]
BT474 cells were treated with 6 μM Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5) for the indicated times before RNA isolation and real-time qPCR with HER3-specific primers. MDA453 and SKBR3 cells were treated with 6 μM Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5) for as long as 48 h before RNA isolation and qPCR for HER3. [1]
BT474 and MDA453 cells were treated with DMSO or 6 μM Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5) for 4 h. Nuclear and cytoplasmic extracts were subjected to immunoblot analysis with FoxO1 and FoxO3a antibodies. [1]
BT474, MDA453, and SKBR3 cells were transfected with control or FoxO1- and FoxO3a-specific siRNA duplexes followed by treatment with Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5) for 6 h before harvesting, RNA isolation and qPCR for HER3 or FoxO1 and FoxO3a. [1]
BT474 cells were transfected with control or FoxO1 and FoxO3a siRNA and treated with Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5) for 6 h. Cell lysates were used for hybridization with pRTK arrays. [1]
BT474 cells were treated with 6 μM Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5) for as long as 72 h and subjected to immunoblot analysis. Immunoblot of lysates from BT474 cells treated with 6 or 20 μM Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5) for 0 to 48 h. [1]
BT474 cells were treated with 0 to 20 μM Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5) for 24 h and lysed. Cell lysates (0.5 mg) were subjected to immunoprecipitation with a p85 antibody followed by immunoblot for p85 and pTyr or p85 and HER3. [1]
BT474 cells were transfected with control or HER3-specific siRNA duplexes and treated with 6 μM Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5) for 24 h. Cell lysates (0.5 mg) were subjected to immunoprecipitation with a p85 antibody followed by immunoblot with HER3, pHER3Y1197, and p85 antibodies. [1]
BT474 cells were transfected with HER3-specific siRNA and treated with DMSO or 2 μM Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5). Cells were harvested for counting or crystal violet staining on day 6. [1]
BT474 cells were treated with 2 μM Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5) alone or in combination with 0.1 μM lapatinib or 10 μg/mL trastuzumab and counted after 6 d or 8 d. Immunoblot of biomarkers of apoptosis and G1-S phase transition with lysates from BT474 cells treated with the indicated inhibitors for 72 h. Real-time qPCR for HER3 mRNA in cells treated with Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5) (6 μM), lapatinib (1 μM), trastuzumab (10 μg/mL), or the indicated combinations for 10 h. Total HER3 and pHER3 immunoblot of lysates from BT474 cells treated over a time course (0-24 h) with the indicated inhibitors. [1]
BT474 cells were treated with 6 μM Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5) over a time course to 24 h. Cell lysates were prepared and 0.2 mg (lower sensitivity) or 0.5 mg (higher sensitivity) of total protein were applied to pRTK arrays. [1]
Real-time qPCR analysis of the indicated RTKs in RNA collected from cells treated with DMSO or 10 μM Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5) for 6 h. Analysis of IGF1R, InsR, and FGFR2 mRNA by qPCR in RNA extracted from BT474 cells transfected with FoxO1 and FoxO3a siRNA and then treated with 10 μM Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5) for 6 h. [1]

Pilaralisib analogue has a molecular weight of 448.52 g/mol and is orally bioavailable. Its molecular formula is C₂₁H₁₆N₆O₂S₂. Physicochemical properties: consensus Log Po/w is 3.48, TPSA is 146.38 Ų, with 2 hydrogen bond donors, 9 hydrogen bond acceptors, and 5 rotatable bonds. Solubility: soluble in DMSO, stable at 10 mM in DMSO solution. Storage conditions: powder is stable for 3 years at -20°C; solutions are stable for 2 years at -80°C. This compound is not a hazardous substance or mixture.

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Protocols for xenografts studies: Athymic mice bearing BT474 xenografts were randomized to therapy with Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5), lapatinib, trastuzumab, or Pilaralisib analog (XL147 analog, SAR245408 analog) (CAS#: 956958-53-5) plus each HER2 antagonist for 28 days. [1]
Immunohistochemistry: Histoscore (H-score) analysis of immunohistochemical sections. [1]

According to the Safety Data Sheet, Pilaralisib analogue is classified as a non-hazardous substance or mixture with no GHS hazard label elements. In BT474 xenograft mouse models, no obvious drug-related toxicity is observed in any treatment group. This product is for research use only and is not intended for human or veterinary use.

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[1]. Feedback upregulation of HER3 (ErbB3) expression and activity attenuates antitumor effect of PI3K inhibitors. Proc Natl Acad Sci USA. 2012 Feb 21;109(8):2718-23.

Pilaralisib analogue (CAS: 956958-53-5) and Pilaralisib (XL147, CAS: 934526-89-3) are compounds with different chemical structures; the former originates from compound 147 in patent WO2012006552A1. Although both are class I PI3K inhibitors, there are differences in their specific chemical structures and IC₅₀ values. The discovery and optimization of this analogue aim to obtain a more selective PI3Kα inhibitor. As this compound is primarily used for scientific research, its detailed in vivo pharmacodynamics, pharmacokinetics, and systemic toxicological data still require further investigation.

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XL147 is a sulfonamide compound, an N-4-toluenesulfonyl (N-toluenesulfonyl) derivative of N-(2,1,3-benzothiadiazole-5-yl)quinoxaline-2,3-diamine. It is a selective PI3K inhibitor used in cancer treatment. It functions as an EC 2.7.1.137 (phosphatidylinositol 3-kinase) inhibitor and an antitumor drug. It is a sulfonamide compound, and also a quinoxaline derivative, a benzothiadiazole compound, and an aromatic amine compound.

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C21H16N6O2S2

448.521

448.078

C, 55.50; H, 4.66; Cl, 6.55; N, 15.53; O, 11.83; S, 5.93

956958-53-5

956958-53-5; 934526-89-3

1893730

Light brown to brown solid powder

1.538

5.063

2

9

5

31

711

0

O=S(C1C=CC(C)=CC=1)(NC1C(NC2=CC3C(=NSN=3)C=C2)=NC2C(=CC=CC=2)N=1)=O

QINPEPAQOBZPOF-UHFFFAOYSA-N

InChI=1S/C25H25ClN6O4S/c1-25(2,27)24(33)28-15-7-6-8-17(13-15)37(34,35)32-23-22(29-19-9-4-5-10-20(19)30-23)31-21-14-16(36-3)11-12-18(21)26/h4-14H,27H2,1-3H3,(H,28,33)(H,29,31)(H,30,32)

2-amino-N-(3-(N-(3-((2-chloro-5-methoxyphenyl)amino)quinoxalin-2-yl)sulfamoyl)phenyl)-2-methylpropanamide.

Pilaralisib analog; XL147 analog; XL-147; XL 147; SAR 245408; SAR245408; SAR-245408

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: ~3 mg/mL (6.7 mM)
Water: <1 mg/mL (slightly soluble or insoluble)
Ethanol: <1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.57 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 (5.57 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 (5.57 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: 30% PEG400+0.5% Tween80+5%Propylene glycol: 15mg/mL

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