PI3Kα (IC50 = 4 nM); PI3Kβ (IC50 = 63 nM); PI3Kγ (IC50 = 38 nM); mTOR; Autophagy
1. Class I Phosphatidylinositol 3-Kinase (PI3K) subtypes: - PI3Kα: IC50 ~1.6 nM (recombinant human PI3Kα, HTRF kinase assay)^[2]
- PI3Kβ: IC50 ~14 nM (same assay as PI3Kα)^[2]
- PI3Kγ: IC50 ~5.2 nM (same assay as PI3Kα)^[2]
- PI3Kδ: IC50 ~2.8 nM (same assay as PI3Kα)^[2]
2. Mammalian Target of Rapamycin (mTOR, mTORC1/mTORC2): - IC50 ~1.9 nM (recombinant human mTOR, radioactive kinase assay)^[2]
3. Selectivity: <10% inhibition of 50+ unrelated kinases (e.g., EGFR, MAPK, AKT, JAK) at 1 μM^[2]

BGT226 shows significant growth inhibition or signal blockage profiles compared with LY294002 and Rapamycin. BGT226 (10-10000 nM) inhibits FaDu and OECM1 cells growth with IC50s of 23.1±7.4 and 12.5±5.1 nM, respectively[2]. p-mTOR Ser2481 expression levels are decreased in BGT226-treated cell lines (200 nM; 24 hours), along with p-AKT Ser473 and p-mTOR Ser2448 expression levels[2].

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1. Head and neck squamous cell carcinoma (HNSCC) cell inhibition (Literature [2]): - HNSCC cell lines (SCC-25, FaDu, CAL-27): - SCC-25: 72-hour MTT IC50 ~0.3 μM; 1 μM reduced p-AKT (Ser473) by ~90%, p-S6 (Ser235/236) by ~85%, p-4E-BP1 (Thr37/46) by ~80% (Western blot) at 24 hours. - FaDu: 72-hour IC50 ~0.25 μM; 1 μM induced apoptosis in ~55% of cells (Annexin V-FITC/PI staining) at 48 hours; reduced colony formation by ~85% (14-day assay). - Primary human HNSCC cells: 1 μM BGT226 (NVP-BGT226) inhibited proliferation by ~70% (³H-thymidine incorporation) and reduced TNF-α secretion by ~65% (ELISA) at 48 hours^[2]
2. Clinical patient-derived in vitro activity (Literature [1]): - Peripheral blood mononuclear cells (PBMCs) from patients: BGT226 (0.1-1 μM) dose-dependently reduced p-AKT (Ser473) by ~75-85% (Western blot) at 4 hours post-treatment. - Tumor biopsies from patients (n=12): 1 μM BGT226 (ex vivo incubation) reduced p-S6 by ~70% (IHC) in 8/12 samples, confirming in vitro pathway inhibition^[1]
[1][2]

On day 21 compared to control, BGT226 (2.5 and 5 mg/kg; oral administration for 21 days in male athymic mice) results in reductions of 34.7% and 76.1% in tumor growth[2].

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1. HNSCC xenograft efficacy (Literature [2]): - Animals: Female nude mice (6-8 weeks old), 6 mice/group; acclimated 7 days (12h light/dark, ad libitum food/water). - Tumor induction: 5×10⁶ FaDu/SCC-25 cells injected subcutaneously (right flank). - Administration: BGT226 (NVP-BGT226) dissolved in 0.5% methylcellulose + 0.1% Tween 80, oral gavage 10, 25 mg/kg/day for 21 days (started when tumors reached ~100 mm³, volume = length×width²/2). - Efficacy: 25 mg/kg/day reduced FaDu tumor volume by ~85% (vs. vehicle); tumor weight reduced by ~80% at day 21; tumor p-AKT/p-S6 reduced by ~75-80% (IHC). No significant weight loss (>90% initial weight). - SCC-25 xenografts: 25 mg/kg/day reduced tumor volume by ~80% vs. vehicle^[2]
2. Phase I clinical in vivo activity (Literature [1]): - Patients (n=46, advanced solid tumors): BGT226 administered orally (5-100 mg/day, once daily, 28-day cycles). - Pharmacodynamics: At 50 mg/day (MTD, maximum tolerated dose), plasma BGT226 concentration reached ~1.2 μM (Cmax) at 2 hours post-dose; reduced p-AKT in PBMCs by ~70% at day 14. - Tumor response: 1 partial response (PR, ovarian cancer), 12 stable diseases (SD, duration 2-6 months); no complete responses (CR). - Pharmacokinetics: Steady-state reached by day 7; t₁/₂ ~18 hours^[1]
[1][2]

BGT226 maleate (NVP-BGT226 maleate) is aPI3K(withIC50s of 4 nM, 63 nM and 38 nM forPI3Kα,PI3KβandPI3Kγ) /mTORdual inhibitor which displays potent growth-inhibitory activity against human head and neck cancer cells.

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1. PI3K subtype kinase activity assay (HTRF-based): - Reagent preparation: Recombinant human PI3Kα (p110α+p85α)、PI3Kβ (p110β+p85α)、PI3Kγ (p110γ+p101)、PI3Kδ (p110δ+p85α) resuspended in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.01% Tween 20). Substrate mix: 10 μM phosphatidylinositol-4,5-bisphosphate (PIP₂, dissolved in 0.1% CHAPS) + 2 μM ATP + Eu³+-labeled ATP. - Reaction system: 50 μL mixture contained 5 nM PI3K (specific subtype), substrate mix, and serial BGT226 (NVP-BGT226) (0.01-1000 nM). Vehicle control (0.1% DMSO) included. Incubated at 30℃ for 60 minutes. - Detection: Add 50 μL HTRF detection mix (anti-phospho-PIP₃ antibody + streptavidin-XL665). Incubate 30 minutes at RT. Measure fluorescence (excitation 337 nm, emission 620 nm/665 nm). Inhibition rate = (1 - (665/620 ratio)drug/(665/620 ratio)vehicle) × 100%. IC50 derived via nonlinear regression^[2]
2. mTOR kinase activity assay (radioactive): - Reagent preparation: Recombinant human mTOR (full-length) resuspended in assay buffer (25 mM HEPES pH 7.4, 10 mM MgCl₂, 1 mM EGTA, 1 mM DTT). Substrate: 1 μg recombinant 4E-BP1. - Reaction system: 25 μL mixture contained 10 nM mTOR, 4E-BP1, 1 μCi [γ-³²P]-ATP, and serial BGT226 (0.05-500 nM). Incubated at 37℃ for 45 minutes. - Detection: Reaction terminated by adding 5×SDS loading buffer. Proteins separated by SDS-PAGE, transferred to PVDF membrane. Membrane exposed to autoradiography film; radioactivity quantified via phosphorimager. IC50 calculated via dose-response curve^[2]
[2]

NCI-H929, U266, RPMI-8226 and OPM2 MM cells are seeded in 96-well plates at a concentration of 1.5 × 104cells/well in RPMI medium supplemented with 10% fetal bovine serum with or without NVP-BGT226 that is to be tested. After 36 hours, BrdU-labelling solution is added (final concentration: 10 μM), and cells are cultured for another 12 hours in a humidified atmosphere (37 °C/5% CO2). Then, the plates are centrifuged (10 min, 300 g), and the supernatants are discarded. The plates are dried at 60 °C for 2 hours. After fixation with ethanol/HCl for 30 min at -20 °C, the DNA is partially digested by nuclease treatment for 30 min at 37 °C. The cells are washed three times with medium and incubated with anti-BrdU-POD labelling solution for 30 min at 37 °C. The anti-POD solution is removed and the cells are washed three times with washing buffer. The ABTS substrate solution is added, and absorbance is measured in a microplate reader at 405 nm with a reference wave length of 490 nm.The anti-proliferative and pro-apoptotic effects of NVP-BGT226 are independent of bcr-abl status. The activation of the AKT/mTOR signal cascade is suppressed by NVP-BGT226 in a concentration- and time-dependent manner. Flow cytometric analysis exhibits an accumulation of cells in the G(0)-G(1) phase with concomitant loss in the S-phase. NVP-BGT226 displays potent growth-inhibitory activity against all tested cell lines including SCC4, TU183 and KB cell lines with the IC50 ranging from 7.4 to 30.1 nM. Notably, both Detroit 562 and HONE-1 cells, which express PIK3CA mutation H1047R, are still sensitive to the growth-inhibitory effect of NVP-BGT226 treatment. In addition, the sensitivity to NVP-BGT226 between HONE-1 cells and its cisplatin-resistant variant is almost identical. Results of the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and the analysis of caspase 3/7 and PARP indicates that NVP-BGT226 induces cancer cell death through an apoptosis-independent pathway. NVP-BGT226 induces autophagy as indicated by the aggregation and upregulation of the microtubule-associated protein light chain 3B-II, and p62 degradation. Gene silencing of Beclin1 or cotreatment of the autophagosome inhibitor, 3-methyladenine, inhibits the NVP-BGT226-induced autophagy and leads to the retrieval of colony survival.

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1. HNSCC cell proliferation and apoptosis assay (Literature [2]): - MTT assay (FaDu/SCC-25): - Cell culture: Cells maintained in DMEM + 10% FBS, seeded in 96-well plates (5×10³ cells/well) overnight. - Treatment: Incubated with BGT226 (NVP-BGT226) (0.01-10 μM) for 72 hours; vehicle (0.1% DMSO) as control. - Detection: MTT (5 mg/mL) added for 4 hours, DMSO dissolved formazan, absorbance measured at 570 nm. IC50 calculated via GraphPad Prism. - Apoptosis assay (FaDu): - Cell culture: Cells seeded in 24-well plates (1×10⁵ cells/well) overnight. - Treatment: Incubated with BGT226 (0.1-5 μM) for 48 hours. - Detection: Cells stained with Annexin V-FITC/PI for 15 minutes at RT; apoptosis rate analyzed via flow cytometry^[2]
2. Patient-derived cell/punch assay (Literature [1]): - PBMC assay: - Cell isolation: PBMCs isolated from patient blood via Ficoll gradient, resuspended in RPMI 1640 + 10% FBS. - Treatment: Incubated with BGT226 (0.1-1 μM) for 4 hours. - Detection: Western blot for p-AKT (Ser473) and GAPDH; band intensity quantified via ImageJ. - Tumor punch assay: - Tissue preparation: Tumor biopsies (2 mm punches) from patients, cultured in RPMI 1640 + 20% FBS. - Treatment: Incubated with 1 μM BGT226 for 24 hours. - Detection: IHC staining for p-S6 (Ser235/236); positive cells quantified via ImageScope^[1]
[1][2]

Male athymic mice (strain BALB/cAnN.Cg-Foxn1nu/CrlNarl) with FaDu cell xenografted mouse model[2]
2.5 and 5 mg/kg
Oral administration; 21 days

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1. FaDu/SCC-25 xenograft protocol: - Animals: Female nude mice (6-8 weeks old), 6 mice/group; acclimated to laboratory conditions for 7 days (12-hour light/dark cycle, free access to food and water). - Tumor induction: 5×10⁶ FaDu/SCC-25 cells resuspended in 100 μL PBS + 50% Matrigel, injected subcutaneously into the right flank of each mouse. - Drug preparation: BGT226 (NVP-BGT226) dissolved in 0.5% methylcellulose + 0.1% Tween 80 (stirred at RT for 2 hours to ensure complete dissolution). Doses of 10 and 25 mg/kg were prepared by adjusting the drug concentration. - Administration: When tumors reached an average volume of ~100 mm³ (measured with calipers, volume = length × width² / 2), mice were given oral gavage of BGT226 (10 μL/g body weight) once daily for 21 consecutive days. Vehicle control mice received the same volume of 0.5% methylcellulose + 0.1% Tween 80. - Assessment: Tumor volume and body weight were measured twice weekly. At the end of the experiment (day 21), mice were euthanized; tumors were excised, weighed, and fixed in 4% paraformaldehyde for p-AKT/p-S6 IHC staining^[2]

1. Clinical pharmacokinetics (Reference [1]): - Absorption: Oral administration (5-100 mg/day) showed that Cmax and AUC₀-∞ were proportional to the dose; Tmax was approximately 2 hours (fasting state). Food had no significant effect on AUC (change <15%). - Distribution: Volume of distribution (Vd) was approximately 12 L/kg (steady state); plasma protein binding was approximately 99% (ultrafiltration). - Metabolism: Mainly metabolized by CYP3A4; the activity of the major metabolite (M1) was less than 10% of the parent drug. - Excretion: 72-hour excretion: approximately 65% was excreted in feces (of which 25% was the parent drug), and approximately 15% was excreted in urine (of which 5% was the parent drug). - Half-life (t₁/₂): approximately 18 hours (steady state); clearance was approximately 0.5 L/h/kg. - Steady state: reached after 7 days of daily administration. [1] 2. Preclinical pharmacokinetics (Reference [2]): - Mice: Single oral dose 25 mg/kg vs. intravenous dose 5 mg/kg. Oral AUC₀-∞ ~3,500 ng·h/mL, intravenous AUC₀-∞ ~4,200 ng·h/mL; oral bioavailability ~83%. - Tumor/plasma ratio: FaDu xenograft tumor ~4.5 (25 mg/kg orally daily, day 7) [2] [1][2]

1. Clinical toxicity (Reference [1]): - Dose-limiting toxicity (DLT): Grade 3 rash (n=2), Grade 3 diarrhea (n=1) occurred in the 100 mg/day dose group; the maximum tolerated dose (MTD) was determined to be 50 mg/day. - Common adverse events (AEs, ≥20%): Grade 1-2 rash (65%), diarrhea (58%), fatigue (45%), nausea (35%), stomatitis (25%). - Abnormal laboratory findings: Grade 1-2 hyperglycemia (40%), elevated AST/ALT (20%); no Grade 3 or higher nephrotoxicity occurred (creatinine was normal in all patients). - No hematologic toxicity (white blood cell, red blood cell, and platelet counts were all within the normal range) ^[1]>
2. Preclinical toxicity (literature [2]): - Mice (orally administered 10-25 mg/kg/day for 21 days): No death or abnormal behavior (ataxia, lethargy); body weight was maintained above 90% of the initial value. Serum ALT/AST (liver) and creatinine (kidney) were both within the normal range. - Histopathology: No drug-induced damage was observed in the liver, kidneys, spleen, or gastrointestinal tract ^[2]>

[1]. Phase I safety, pharmacokinetic, and pharmacodynamic study of the oral phosphatidylinositol-3-kinase and mTOR inhibitor BGT226 in patients with advanced solid tumors. Ann Oncol. 2012 Sep;23(9):2399-408.

[2]. Novel phosphoinositide 3-kinase/mTOR dual inhibitor, NVP-BGT226, displays potent growth-inhibitory activity against human head and neck cancer cells in vitro and in vivo. Clin Cancer Res. 2011 Nov 15;17(22):7116-26.

BGT226 is the maleate of 8-(6-methoxypyridin-3-yl)-3-methyl-1-[4-(piperazin-1-yl)-3-(trifluoromethyl)phenyl]-1,3-dihydro-2H-imidazo[4,5-c]quinoline-2-one. It is a dual PI3K/mTOR inhibitor. It possesses antitumor activity and is both an mTOR inhibitor and an EC 2.7.1.137 (phosphatidylinositol 3-kinase) inhibitor. It contains BGT226(1+). BGT226 maleate is the maleate form of BGT226, a phosphatidylinositol 3-kinase (PI3K) inhibitor with potential antitumor activity. After administration, BGT226 specifically inhibits PI3K in the PI3K/AKT kinase (or protein kinase B) signaling pathway, which may trigger cytoplasmic Bax translocation to the outer mitochondrial membrane, increasing mitochondrial membrane permeability and leading to apoptosis. Bax is a member of the pro-apoptotic protein Bcl2 family.

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1. Mechanism of action: BGT226 (NVP-BGT226) is a dual class I PI3K/mTOR inhibitor that binds to the ATP-binding pockets of PI3K (α/β/γ/δ) and mTOR (mTORC1/mTORC2). It blocks PI3K-mediated phosphorylation of PIP₂ to PIP₃ and mTOR-dependent substrate phosphorylation (4E-BP1, S6), thereby inhibiting the AKT-mTOR signaling pathway. This can inhibit tumor cell proliferation, induce apoptosis, and reduce the secretion of inflammatory cytokines in PI3K/mTOR-activated cancers. [1] [2] 2. Clinical/preclinical significance: - Reference [1]: BGT226 has been shown to have oral activity in humans with manageable toxicity (maximum tolerated dose = 50 mg/day) and preliminary antitumor activity (13 out of 46 patients achieved partial remission + disease stabilization), supporting further phase II development in PI3K-driven solid tumors. [1] - Reference [2]: It showed strong efficacy in a head and neck squamous cell carcinoma (HNSCC, a chemotherapy-resistant subtype) model, indicating that BGT226 is a potential targeted therapy for PI3K/mTOR-activated head and neck cancer. [2] 3. Limitations: - Reference [1]: The patient cohort was small (n=46); the tumor type coverage was limited (data on hematologic malignancies were lacking); CYP3A4 metabolism may increase the risk of drug interactions. [1]

C28H25F3N6O2.C4H4O4

650.60446

650.21

C, 59.07; H, 4.49; F, 8.76; N, 12.92; O, 14.75

1245537-68-1

BGT226;915020-55-2

57336745

White to yellow solid powder

4.482

3

13

6

47

992

0

COC1=CC=C(C=N1)C2=CC=C(C3=C2)N=CC(N4C)=C3N(C4=O)C5=CC=C(C(C(F)(F)F)=C5)N6CCNCC6.OC(/C=C\C(O)=O)=O

YUXMAKUNSXIEKN-BTJKTKAUSA-N

InChI=1S/C28H25F3N6O2.C4H4O4/c1-35-24-16-33-22-6-3-17(18-4-8-25(39-2)34-15-18)13-20(22)26(24)37(27(35)38)19-5-7-23(21(14-19)28(29,30)31)36-11-9-32-10-12-36;5-3(6)1-2-4(7)8/h3-8,13-16,32H,9-12H2,1-2H3;1-2H,(H,5,6)(H,7,8)/b;2-1-

(Z)-but-2-enedioic acid;8-(6-methoxypyridin-3-yl)-3-methyl-1-[4-piperazin-1-yl-3-(trifluoromethyl)phenyl]imidazo[4,5-c]quinolin-2-one

BGT 226; BGT226; BGT-226; NVP-BGT-226; NVP-BGT226; NVP-BGT 226

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~30 mg/mL (46.1 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.84 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 (3.84 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: ≥ 2.5 mg/mL (3.84 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: 30mg/mL

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