PI3Kα (IC50 = 39 nM); PI3Kβ (IC50 = 383 nM); PI3Kδ (IC50 = 36 nM); PI3Kγ (IC50 = 23 nM); Vps34 (IC50 = 6974 nM); DNA-PK (IC50 = 4750 nM)
Pilaralisib targets all four members of the class I PI3K family, with IC₅₀ values of 39 nM for PI3Kα, 383 nM for PI3Kβ, 23 nM for PI3Kγ, and 36 nM for PI3Kδ in cell-free assays . The compound exhibits potent inhibitory activity against PI3Kα/δ/γ, with relatively weaker activity against PI3Kβ .

Pilaralisib exhibits cytotoxic activity in Pediatric Preclinical Testing Program (PPTP) cell lines, with a median relative IC50 of 10.9 mM (interquartile range, 2.7 to 24.5 mM).[2]

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In serum-free medium, pilaralisib inhibits EGF-induced PIP3 production with IC₅₀ values of 220 nM in PC-3 cells and 347 nM in MCF7 cells . In vitro cytotoxicity assays show a median relative IC₅₀ value of 10.9 µM (range 2.7-24.5 µM) for pilaralisib . In HER2-positive cells, combination of pilaralisib with HER2 inhibitors trastuzumab or lapatinib synergistically enhances cell death and inhibits pAKT and pS6 expression .

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In cellular assays, XL147 (SAR245408) inhibits PIP3 production in PC-3 cells with IC50 of 220 nmol/L and in MCF7 cells with IC50 of 347 nmol/L.
It inhibits EGF-stimulated AKT phosphorylation (IC50 477 nmol/L) and non-stimulated S6 phosphorylation (IC50 776 nmol/L) in PC-3 cells by cell-based ELISA.
Western blot analysis shows that XL147 (SAR245408) inhibits AKT phosphorylation at T308 and S473, as well as phosphorylation of PRAS40, GSK3β, p70S6K, S6, and 4EBP1, and induces a decrease in cyclin D1 protein levels in MCF7 and PC-3 cells.
In a panel of tumor cell lines, XL147 (SAR245408) inhibits proliferation with IC50 values ranging from approximately 1200 nmol/L to >30,000 nmol/L; PIK3CA-mutant and PTEN-mutant lines tend to be more sensitive, while RAS- or BRAF-mutant lines tend to be less sensitive.
In soft agar anchorage-independent growth assays, XL147 (SAR245408) inhibits colony growth with IC50 of 3996 nmol/L in PC-3 cells and 2730 nmol/L in MCF7 cells.
XL147 (SAR245408) inhibits HGF-stimulated B16 melanoma cell migration with IC50 of 899 nmol/L (cytotoxicity IC50 4494 nmol/L).
In a scratch assay, it inhibits EGF-stimulated PC-3 cell migration with IC50 of 394 nmol/L (cytotoxicity IC50 >3333 nmol/L).
XL147 (SAR245408) inhibits VEGF-induced HMVEC tubule formation with IC50 of 529 nmol/L.
In MCF7 cells, the antiproliferative effects are associated with a G1 phase block and increase of sub-G1 population, without acute cytotoxicity or induction of caspases-3/7.
It does not reduce cellular ATP levels after 24-hour incubation, indicating lack of cytotoxicity.[1]

Pilaralisib (100 mg/kg, p.o.) inhibits the growth of solid glioma xenografts in BALB/c nu/nu mice. With a toxicity rate of only 0.7% in the treated groups, pilaralisib is well tolerated and is comparable to what was seen in the control animal population. [2] Pilaralisib (100 mg/kg, p.o.) significantly slows tumor growth in athymic female mice while posing minimal drug-related side effects. [3]

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Among 44 tested xenograft models, pilaralisib induced tumor growth inhibition meeting criteria for intermediate EFS T/C activity in 4 (11%) solid tumor xenografts . In BT474 xenograft models, pilaralisib combined with trastuzumab or lapatinib synergistically inhibits pAKT and tumor growth . In clinical trials, single-agent pilaralisib (600 mg once daily) achieved a partial response rate of 50% in CLL patients, with 60% of patients showing ≥50% nodal shrinkage, and a 20% partial response rate in lymphoma patients; overall, 56% of patients had progression-free survival ≥6 months .

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Oral administration of XL147 (SAR245408) results in dose-dependent inhibition of AKT, p70S6K, and S6 phosphorylation in MCF7 and PC-3 xenograft tumors; at 300 mg/kg, maximum inhibition (65%-81%) at 4 h, with partial inhibition (25%-51%) persisting through 48 h in MCF7 tumors.
Repeat-dose administration (once daily) of XL147 (SAR245408) at 100 mg/kg results in tumor stasis or near-stasis in multiple xenograft models (MCF7, OVCAR-3, PC-3, U-87 MG, A549, A2058, WM-266-4), with reduced growth rate in KRAS- or BRAF-mutant models (Calu-6).
IHC analysis of MCF7 tumors shows dose-dependent decrease in Ki-67 staining (e.g., 32% reduction at 100 mg/kg qd) and decreased CD31-positive tumor vessels (e.g., 37% reduction at 100 mg/kg qd).
In PC-3 and Calu-6 tumors, XL147 (SAR245408) combined with paclitaxel or carboplatin enhances antitumor efficacy, increases apoptosis (TUNEL staining), and further reduces angiogenesis compared to monotherapy, and is generally well tolerated.[1]

Using luciferase-luciferin-coupled chemiluminescence, kinase activity for PI3K isoforms is quantified as the proportion of ATP consumed after the kinase reaction, with ATP concentrations roughly equal to the Km for each individual kinase. Test compounds, ATP, and kinase are combined in a 20 μL volume to start kinase reactions. The final enzyme concentrations for PI3Kα, PI3Kβ, PI3Kγ, and PI3Kδ are 0.5, 8, 20, and 2 nM, respectively. Noteworthy is the mixing of 10 μL of enzyme solution with 0.5 μLof dimethyl sulfoxide (DMSO) containing varying concentrations of the test compound. 10 μL of liver phosphatidylinositol and 2 mL of ATP solution are added to start kinase reactions. VPS34, ATP, and phosphatidylinositol have respective assay concentrations of 40 nM, 1 M, and 5 μM.

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The inhibitory activity of pilaralisib against PI3K kinases is assessed using cell-free kinase assays. Purified PI3K kinases are incubated with various concentrations of pilaralisib in the presence of ATP, and enzyme activity is measured by detecting PIP3 production from phosphatidylinositol-4,5-bisphosphate phosphorylation to calculate IC₅₀ values .

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Kinase inhibition assays were performed using purified proteins in a luciferase-coupled chemiluminescence format. ATP concentrations were approximately equal to the Michaelis constant (KM) values of the respective enzymes.
For PI3Kα, an equilibrium inhibition constant (Ki) of 42 nmol/L was determined, revealing that XL147 (SAR245408) is an ATP-competitive inhibitor.
An mTOR kinase immunoprecipitation assay using cell lysates was performed to assess activity toward the physiologic substrate 4EBP1; no inhibition was observed at concentrations up to 15 μmol/L.[1]

MTT or WST-1 reagent that has already been mixed are used to measure cell proliferation. 10,000 cells are seeded in each well of 96-well plates for MTT/WST-1 assays. Cells are given DMSO or pilaralisib treatment 24 hours after plating. MTT/WST-1 assays are carried out five days after the start of treatment.

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PIP3 Production Inhibition Assay: PC-3 or MCF7 cells are seeded in culture plates, pretreated with various concentrations of pilaralisib in serum-free medium, and then stimulated with EGF. After cell lysis, intracellular PIP3 levels are measured using specific immunoassays to calculate IC₅₀ values .Cell Viability Assay: Tumor cells are seeded in 96-well plates and treated with serial dilutions of pilaralisib. Cell viability is assessed by MTT or CellTiter-Glo assays to calculate IC₅₀ values .

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For cell-based assays, cell lines were maintained at 37°C under 5% CO2. For PI3K pathway status assessment, culture medium was replaced with test compounds dissolved in serum-free DMEM containing 0.3% DMSO. After 3-hour incubation, cells were stimulated with 100 ng/mL EGF for 10 minutes, and Western immunoblot analysis of cell lysates was performed.
Cellular proliferation was assessed using the Cell Proliferation ELISA, bromodeoxyuridine (BrdUrd) chemiluminescence kit. Cytotoxicity, apoptosis (caspases-3/7), anchorage-independent growth (soft agar over 14 days), and PC-3 cell migration assays were performed as described.
For endothelial tube formation assay, human microvascular endothelial cells (HMVEC) were plated on a confluent layer of normal human diploid fibroblasts and treated with VEGF; tubules were stained with anti-CD31 antibody and total tube length quantified.
For cell-cycle analysis by FACS, MCF7 cells were seeded in 12-well plates, treated with serial dilutions of compounds for 48 and 72 hours, then fixed with ice-cold ethanol, stained with propidium iodide (PI) and RNase, and analyzed on a FACS Calibur.[1]

Mice: The animals used are male and female athymic nude mice. The weights of the mouse body and the tumor are measured after the establishment of the tumor cells as xenografts in culture. The two-tailed Student t test is used to determine statistical significance (significance is P 0.05). Pilaralisib (XL147) is prepared in either water or sterile water with 10 mmol/L of HCl and is given orally via gavage at the prescribed doses and schedules with a dose volume of 10 mL/kg.

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Xenograft Models: Tumor cells are implanted subcutaneously into immunodeficient mice to establish xenograft models. When tumors reach a certain size, pilaralisib is administered by oral gavage (typically 50-600 mg/kg) once daily. Tumor volume and body weight are measured regularly to calculate tumor growth inhibition rates and EFS T/C values .Combination Studies: In BT474 xenograft models, pilaralisib is combined with trastuzumab or lapatinib to evaluate synergistic anti-tumor effects .

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Female athymic nude mice and male nude mice were used. Tumor cells were cultured and established as xenografts. XL147 (SAR245408) was formulated in sterile water/10 mmol/L HCl or water and administered at indicated doses and regimens by oral gavage at a dose volume of 10 mL/kg.
Paclitaxel was formulated for i.v. administration by dissolution of dry powder into 1:1 EtOH/Cremophor mixture, with subsequent dilution (1:3) into normal saline.
Carboplatin was formulated for i.v. administration by dissolution in normal saline containing 0.5% EtOH/0.5% Cremophor EL.
For in vivo pharmacodynamic assays, tumors were collected at indicated time points post-dose, tumor lysates prepared, and Western immunoblot performed. For histologic assessment, tumors were excised, fixed in zinc fixative, processed into paraffin blocks, and 5 μm thin sections were cut for Ki-67, CD31, and TUNEL staining.[1]

Pilaralisib has a molecular weight of 541.02 g/mol and is well absorbed orally with good bioavailability . The MTD established in solid tumor patients is 600 mg capsules once daily . When combined with erlotinib, the MTD is 400 mg ; when combined with paclitaxel/carboplatin, the tablet MTD is 200 mg once daily . PK data show no drug interaction between pilaralisib and paclitaxel/carboplatin . Solubility: soluble in DMSO (92 mg/mL), insoluble in water . Storage conditions: powder is stable for 3 years at -20°C; solutions are stable for 6 months at -20°C .

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In vitro plasma protein binding of XL147 (SAR245408) was 99.9% in mouse, rat, and human plasma (determined by equilibrium dialysis).
In vivo plasma protein binding, quantified in plasma samples from mice and rats administered XL147 (SAR245408) at 100 mg/kg, showed 99.9% and 99.8% binding, respectively.
Following a single oral dose of 100 mg/kg in MCF7 tumor-bearing mice, plasma concentrations of XL147 (SAR245408) were 179 μmol/L at 4 h and 86 μmol/L at 24 h.
After repeat dosing (100 mg/kg once daily) in MCF7 efficacy study, average plasma concentrations were 233 μmol/L at 1 h, 329 μmol/L at 4 h, and 112 μmol/L at 24 h, indicating accumulation.[1]

In a Phase I trial (25 patients with CLL/lymphoma), the most frequent adverse events of any grade were diarrhea (92.0%), pyrexia (52.0%), and fatigue (44.0%) . The most frequent grade ≥3 AEs were neutropenia (32.0%), diarrhea (20.0%), and anemia (16.0%) . In the pilaralisib plus paclitaxel/carboplatin study (58 patients), the most common AEs were neutropenia (67.2%) and thrombocytopenia (67.2%), with dose-limiting toxicities including rash (3.4%) . One case of grade 4 DRESS syndrome was reported in the erlotinib combination study . Overall, pilaralisib demonstrated an acceptable safety profile in patients with CLL and lymphoma .

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XL147 (SAR245408) was generally well tolerated as assessed by daily monitoring of mouse body weights in efficacy studies, including combinations with paclitaxel or carboplatin.
Plasma protein binding is very high (99.9% in mouse, rat, and human plasma in vitro; 99.9% and 99.8% in mice and rats at 100 mg/kg in vivo).[1]

[1]. Mol Cancer Ther. 2015 Apr;14(4):931-40.

[2]. Pediatr Blood Cancer. 2013 May;60(5):791-8.

[3]. Proc Natl Acad Sci U S A. 2012 Feb 21;109(8):2718-23.

Pilaralisib is an orally administered small molecule drug that selectively inhibits the activity of phosphatidylinositol-3 kinase (PI3K). Pilaralisib has been used in clinical trials for various cancers, including lymphoma, solid tumors, glioblastoma, and breast cancer. Pilaralisib is a highly bioavailable orally administered small molecule drug that targets the class I phosphatidylinositol 3 kinase (PI3K) lipid kinase family and possesses potential antitumor activity. Pilaralisib reversibly binds to class I PI3K in an ATP-competitive manner, inhibiting the production of the second messenger phosphatidylinositol-3,4,5-triphosphate (PIP3) and activation of the PI3K signaling pathway; this may lead to suppression of the growth and survival of susceptible tumor cell populations. Activation of the PI3K signaling pathway is commonly associated with tumorigenesis. Dysregulation of the PI3K signaling pathway may lead to resistance in tumors to various antitumor drugs, including genotoxic drugs and receptor tyrosine kinase inhibitors. Mechanism of Action Pilaralisib is an orally administered small molecule drug that selectively inhibits the activity of phosphatidylinositol-3 kinase (PI3K). PI3K activation is common in human tumors and promotes tumor cell growth, survival, and resistance to chemotherapy and radiotherapy. PI3K inactivation has been shown to inhibit tumor cell growth and induce apoptosis (programmed cell death). Pharmacodynamics In preclinical studies, Pilaralisib slowed tumor growth or caused tumor shrinkage in various preclinical cancer models, including breast cancer, lung cancer, ovarian cancer, prostate cancer, and glioma. In preclinical cancer models, Pilaralisib has also been shown to enhance the antitumor effects of several chemotherapeutic agents and epidermal growth factor receptor (EGFR) inhibitors.

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Pilaralisib (CAS: 934526-89-3), as a pan-PI3K inhibitor, has demonstrated antitumor activity in preclinical and clinical studies, but its efficacy as a monotherapy is limited. Multiple Phase I studies have evaluated its combination with chemotherapy or targeted agents, but no significant enhancement of efficacy has been observed. Further clinical development of this compound may require more precise biomarker screening.

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The PI3K/PTEN pathway is frequently dysregulated in cancers, leading to hyperactivated PI3K signaling. Resistance to various anticancer therapies has been attributed to this pathway.
XL147 (SAR245408) is an ATP-competitive inhibitor of class I PI3Ks with a Ki value of 42 nmol/L for PI3Kα.
In MCF7 cells (PIK3CA E545K mutant) and PC-3 cells (PTEN-deficient), XL147 (SAR245408) shows comparable pharmacodynamic activity in vitro and in vivo.
Efficacy was observed in xenograft models with diverse genetic lesions (PIK3CA mutation/amplification, PTEN deletion/deficiency, KRAS mutation, LKB1 mutation, BRAF mutation), suggesting broad utility.
Combination of XL147 (SAR245408) with paclitaxel or carboplatin showed enhanced antitumor efficacy and apoptosis in preclinical models.[1]

C25H25CLN6O4S

541.0218

540.135

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

934526-89-3

934526-89-3;956958-53-5.;

56599306

Light yellow to yellow solid powder

5.786

4

9

8

37

887

0

O=C(C(C)(C)N)NC1C=C(S(NC2C(NC3C(Cl)=CC=C(OC)C=3)=NC3C(=CC=CC=3)N=2)(=O)=O)C=CC=1

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.

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

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: ~100 mg/mL warming (184.8 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.62 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 (4.62 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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: 10 mM HCl in sterile water: 0.5mg/mL

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