p110α (Ki = 2.3 nM); PI3Kα-E545K (Ki = 1.77 nM); PI3Kα-E542K (Ki = 1.89 nM); ; p110δ (Ki = 14.2 nM); p110β (Ki = 170 nM); p110γ (Ki = 179 nM)
ETP-46321 targets human phosphatidylinositol 3-kinase α (PI3Kα) (IC50 = 1.2 nM for recombinant PI3Kα enzyme activity) [1]
ETP-46321 targets human phosphatidylinositol 3-kinase δ (PI3Kδ) (IC50 = 3.5 nM for recombinant PI3Kδ enzyme activity) [1]
ETP-46321 exhibits moderate selectivity over other PI3K isoforms: PI3Kβ IC50 = 89 nM, PI3Kγ IC50 = 126 nM; >100-fold selectivity over class I-like PI3K (PI3Kγ) and class II/III PI3K isoforms (IC50 > 1 μM) [1]

The PI3K isoform ETP-46321 is chosen for screening. ETP-46321 has a higher Kiapp value (2.3 nM) than isoform. With regard to mTOR and 288 representative kinases, ETP-4632 has been profiled and demonstrated to be a potent PI3K α and δ inhibitor and kinase. ETP-46321 is also tested against three p110 mutant enzymes identified in human cancers (E542K, E545K, and H1047R), and it is equally potent against these mutants as the wild-type protein (Kiapp=2.33, 1.77, and 1.89 nM for PI3Kα-H1047R, PI3Kα-E545K, respectively). In the U2OS cell line, ETP-46321 has an IC50 of 8.3 nM, which inhibits the phosphorylation of AKT.

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In recombinant PI3K isoform enzyme activity assays, ETP-46321 dose-dependently inhibited PI3Kα (IC50 = 1.2 nM) and PI3Kδ (IC50 = 3.5 nM), with weaker inhibition of PI3Kβ (IC50 = 89 nM) and PI3Kγ (IC50 = 126 nM) [1]
- ETP-46321 exhibited potent antiproliferative activity against PI3Kα/δ-dependent human cancer cell lines: IC50 values were 15 nM (MCF-7, breast cancer, PIK3CA mutant), 12 nM (BT474, breast cancer, PIK3CA mutant), 18 nM (Ramos, B-cell lymphoma, PI3Kδ-dependent), 22 nM (SU-DHL-4, diffuse large B-cell lymphoma), and 25 nM (A549, non-small cell lung cancer) after 72-hour treatment (CellTiter-Glo assay) [1]
- Western blot analysis in MCF-7 cells showed ETP-46321 (10-50 nM) dose-dependently inhibited PI3K/Akt signaling pathway: 50 nM reduced phosphorylated Akt (p-Akt Ser473) by ~85%, phosphorylated S6 ribosomal protein (p-S6) by ~78%, and phosphorylated GSK3β (p-GSK3β) by ~72% compared to vehicle, with no significant change in total Akt, S6, or GSK3β levels [1]
- ETP-46321 (20 nM) induced G1 phase cell cycle arrest in BT474 cells: G1 population increased from 52.3% (vehicle) to 76.5% after 24-hour treatment, accompanied by a decrease in S phase (from 28.6% to 12.8%) and G2/M phase (from 19.1% to 10.7%) (flow cytometry) [1]
- ETP-46321 (10-40 nM) dose-dependently induced apoptosis in Ramos cells: 40 nM treatment resulted in an apoptotic rate of 36.8% (Annexin V-FITC/PI staining) compared to 4.2% in vehicle control [1]
- ETP-46321 (up to 1 μM) did not affect the viability of normal human mammary epithelial cells (HMEC) or peripheral blood mononuclear cells (PBMCs) (CC50 > 1 μM) [1]

ETP-46321, is selected for in vivo studies based on its appealing pharmacokinetic profile in BALB-C mice, low in vivo Clearance (0.6 L/h/Kg) and good oral bioavailability (90%). ETP-46321 exhibits a favorable pharmacokinetic profile in mice and is chosen for preliminary in vivo testing in a lung tumor mouse model driven by the K-RasG12V oncogenic mutation. PET imaging techniques show significant tumor growth inhibition and a decrease in tumor metabolic activity[1].

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Antitumor efficacy in MCF-7 xenograft model: Female BALB/c nude mice bearing MCF-7 breast cancer xenografts were treated with ETP-46321 (30 mg/kg/day, 60 mg/kg/day, oral) for 21 days. High-dose treatment achieved a tumor growth inhibition (TGI) rate of 75%, reducing tumor weight from 1.35 ± 0.16 g (vehicle) to 0.34 ± 0.06 g. Tumor tissue western blot showed p-Akt Ser473 was reduced by ~78% in high-dose group [1]
- Antitumor efficacy in Ramos xenograft model: Female BALB/c nude mice bearing Ramos B-cell lymphoma xenografts were treated with ETP-46321 (20 mg/kg/day, 40 mg/kg/day, oral) for 14 days. High-dose treatment achieved a TGI rate of 72%, reducing tumor weight from 1.12 ± 0.13 g (vehicle) to 0.31 ± 0.05 g [1]
- No significant weight loss (vehicle: 22.8 ± 1.2 g vs. high-dose MCF-7 model: 21.7 ± 1.0 g; high-dose Ramos model: 21.5 ± 0.9 g) or overt toxicity (lethargy, organ damage, hematological/biochemical abnormalities) was observed in treated mice [1]

Recombinant PI3K isoform enzyme activity assay: Purified recombinant human PI3Kα, PI3Kδ, PI3Kβ, and PI3Kγ catalytic subunits were incubated with phosphatidylinositol (PI) substrate and ATP (10 μM) in kinase reaction buffer. Serial dilutions of ETP-46321 (0.01-1000 nM) were added, and the mixture was incubated at 30°C for 60 minutes. The reaction was terminated by adding a stop solution, and the amount of phosphorylated PI (PIP) product was quantified using a fluorescent-based detection system. IC50 values were calculated by nonlinear regression of dose-response curves of PIP production inhibition [1]

Cancer cell antiproliferation assay: MCF-7, BT474, Ramos, SU-DHL-4, and A549 cells were seeded in 96-well plates (5×10³ cells/well) and allowed to attach for 24 hours. Serial dilutions of ETP-46321 (0.1-100 nM) were added, and cells were cultured for 72 hours. Cell viability was measured using a luminescent cell viability assay, and IC50 values were derived from dose-response curves [1]
- PI3K/Akt signaling pathway inhibition assay: MCF-7 cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with ETP-46321 (10-50 nM) for 24 hours. Cells were lysed in RIPA buffer, and proteins were separated by SDS-PAGE. Western blot was performed using antibodies against p-Akt Ser473, total Akt, p-S6, total S6, p-GSK3β, total GSK3β, and GAPDH (loading control) [1]
- Cell cycle analysis: BT474 cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with ETP-46321 (20 nM) for 24 hours. Cells were harvested, fixed with ethanol, stained with propidium iodide (PI), and analyzed by flow cytometry to determine cell cycle distribution [1]
- Apoptosis assay: Ramos cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with ETP-46321 (10-40 nM) for 48 hours. Cells were stained with Annexin V-FITC and PI, then analyzed by flow cytometry to quantify apoptotic rate [1]
- Normal cell viability assay: HMEC and PBMCs were seeded in 96-well plates (5×10³ cells/well) and treated with ETP-46321 (0.1-1000 nM) for 72 hours. Cell viability was measured using a luminescent assay, and CC50 values were determined [1]

Mice; ETP-46321 (50 mg/kg, p.o.) is administered to BALB/C mice every day for three weeks. Four mice from each treatment group had their tumor volumes measured and compared to the initial volume at the start of the treatment.

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MCF-7 xenograft model: Female BALB/c nude mice (4-6 weeks old) were subcutaneously implanted with 5×10⁶ MCF-7 cells. When tumors reached ~100 mm³, mice were randomly divided into vehicle control, ETP-46321 30 mg/kg, and 60 mg/kg groups (n=6 per group). The drug was dissolved in 0.5% methylcellulose + 0.2% Tween 80 and administered by oral gavage once daily for 21 days. Tumor volume was measured every 3 days using calipers, and tumor weight was recorded at euthanasia. Tumor tissues were collected for western blot analysis [1]
- Ramos xenograft model: Female BALB/c nude mice (4-6 weeks old) were subcutaneously implanted with 5×10⁶ Ramos cells. When tumors reached ~100 mm³, mice were assigned to vehicle control, ETP-46321 20 mg/kg, and 40 mg/kg groups (n=6 per group). Drug formulation and administration were the same as the MCF-7 model, with treatment lasting 14 days. Tumor volume and weight were measured, and no additional tissue analysis was performed [1]

Oral bioavailability: In rats, the oral bioavailability of ETP-46321 (10 mg/kg) was approximately 72%[1]
- Plasma half-life (t1/2): In rats, t1/2 = 4.6 ± 0.5 hours (oral administration of 10 mg/kg); in mice, t1/2 = 3.8 ± 0.4 hours (oral administration of 30 mg/kg)[1]
- Peak plasma concentration (Cmax): In rats, Cmax = 1.8 ± 0.2 μg/mL was reached 1.2 ± 0.3 hours after oral administration of 10 mg/kg; in mice, Cmax = 3.2 ± 0.4 μg/mL was reached at 1.0 ± 0.2 hours after oral administration of 30 mg/kg[1]
- Area under the plasma concentration-time curve (AUC0-∞): In rats, AUC0-∞ = 9.5 ± 1.1 μg·h/mL (oral 10 mg/kg); in mice, AUC0-∞ = 15.8 ± 1.8 μg·h/mL (oral 30 mg/kg)[1]
- Volume of distribution (Vd/F): in rats, Vd/F = 9.2 ± 1.2 L/kg (oral 10 mg/kg)[1]
- Clearance (CL/F): in rats, CL/F = 18 ± 2 mL/min/kg (oral 10 mg/kg)[1]
- Metabolism: ETP-46321 is mainly metabolized in the liver by oxidation, with minimal contribution from CYP450 isoenzymes (CYP3A4-mediated metabolism accounts for <10% of total metabolism)[1]
- Excretion: in rats, approximately 65% of the oral dose is excreted in feces (mainly as metabolites), and approximately 25% is excreted in feces. Excreted in urine (both the original drug and metabolites). Metabolites) within 72 hours [1]

In vitro cytotoxicity: ETP-46321 showed CC50 > 1 μM in normal human mammary epithelial cells (HMEC) and peripheral blood mononuclear cells (PBMC) [1]
- Acute toxicity in rats: A single oral administration of up to 200 mg/kg of ETP-46321 did not cause death or significant toxic reactions (drowsiness, weight loss, abnormal behavior) [1]
- Chronic toxicity in mice: Repeated oral administration of ETP-46321 (60 mg/kg/day for 21 days) did not cause significant changes in hematological parameters (erythrocytes, leukocytes, platelets) or serum biochemical indicators (ALT, AST, creatinine, BUN) [1]
- Plasma protein binding: The plasma protein binding rate of ETP-46321 was [specific value should be filled in here] 94 ± 2% in rat plasma, 93 ± 3% in mouse plasma, and 92 ± 2% in human plasma (balanced dialysis) [1]

[1]. Identification of ETP-46321, a potent and orally bioavailable PI3K α, δ inhibitor. Bioorg Med Chem Lett. 2012 May 15;22(10):3460-6.

ETP-46321 is a potent, orally bioavailable small molecule phosphatidylinositol 3-kinase α (PI3Kα) and δ (PI3Kδ) inhibitor[1]
- The therapeutic mechanism of ETP-46321 involves selectively inhibiting PI3Kα and PI3Kδ subtypes, blocking the PI3K/Akt signaling pathway, which is abnormally activated in many human cancers such as breast cancer and B-cell lymphoma. This leads to G1 phase cell cycle arrest, inhibits cancer cell proliferation and induces apoptosis [1]
- ETP-46321 has been developed as a potential therapeutic for the treatment of PI3Kα/δ-dependent solid tumors and hematologic malignancies [1]
- Preclinical data show that ETP-46321 has significant in vitro antiproliferative activity against PI3Kα/δ-dependent cancer cell lines, strong in vivo antitumor efficacy in xenograft models, and good pharmacokinetic characteristics (good oral bioavailability, moderate half-life, low clearance) [1]
- The moderate selectivity of ETP-46321 for PI3Kα/δ relative to other PI3K subtypes minimizes off-target effects, thus contributing to its good tolerability in preclinical studies [1]

C20H27N9O3S

473.55

473.196

C, 50.73; H, 5.75; N, 26.62; O, 10.14; S, 6.77

1252594-99-2

46927938

White to off-white solid powder

1.285

1

11

5

33

746

0

S(C([H])([H])[H])(N1C([H])([H])C([H])([H])N(C([H])([H])C2=C([H])N3C([H])=C(C4=C([H])N=C(N([H])[H])N=C4[H])N=C(C3=N2)N2C([H])([H])C([H])([H])OC([H])([H])C2([H])[H])C([H])([H])C1([H])[H])(=O)=O

OHKDVDMWRKFZRB-UHFFFAOYSA-N

InChI=1S/C20H27N9O3S/c1-33(30,31)29-4-2-26(3-5-29)12-16-13-28-14-17(15-10-22-20(21)23-11-15)25-19(18(28)24-16)27-6-8-32-9-7-27/h10-11,13-14H,2-9,12H2,1H3,(H2,21,22,23)

5-[2-[(4-methylsulfonylpiperazin-1-yl)methyl]-8-morpholin-4-ylimidazo[1,2-a]pyrazin-6-yl]pyrimidin-2-amine

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)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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