PI3Kα (IC50 = 0.5 nM); PI3Kδ (IC50 = 0.7 nM); PI3Kβ (IC50 = 3.7 nM); PI3Kγ (IC50 = 6.4 nM); mTOR (IC50 = 45 nM)
1. Phosphatidylinositol 3-Kinase (PI3K) subtypes (predominantly α and δ) - PI3Kα (p110α/p85 complex): IC50 ~0.5 nM (recombinant human PI3Kα, HTRF kinase assay)^[1]
- PI3Kδ (p110δ/p85 complex): IC50 ~0.7 nM (recombinant human PI3Kδ, same HTRF assay)^[1]
- PI3Kγ (p110γ/p101 complex): IC50 ~3.7 nM (recombinant human PI3Kγ, same assay)^[1]
- PI3Kβ (p110β/p85 complex): IC50 ~14 nM (recombinant human PI3Kβ, same assay)^[1]
2. No significant inhibition of 60+ unrelated kinases (e.g., AKT, MAPK, EGFR, JAK, mTOR) at 1 μM^[1]

BAY 80-6946 lowers pAKT levels in KPL4 cells and LPA-stimulated PC3 cells. BAY 80-6946 exhibits antiproliferative activity and induces apoptosis in a subset of human cancer cell lines with PIK3CA mutations and/or overexpression of HER2. [1] Combining HER2-targeted therapies with BAY 80-6946 inhibits growth more potently than either therapy when used independently, and it can improve cells' sensitivity to trastuzumab and lapatinib. [2]

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1. Antitumor activity in hematologic and solid tumor cells (Literature [1]): - Hematologic tumor cell lines: - Diffuse large B-cell lymphoma (DLBCL) cell lines (SU-DHL-4, OCI-Ly3): 72-hour MTT IC50 ~15 nM and ~20 nM, respectively; 50 nM Copanlisib reduced p-AKT (Ser473) by ~90%, p-S6 (Ser235/236) by ~85% (Western blot) at 24 hours; induced apoptosis in ~45% of SU-DHL-4 cells (Annexin V-FITC staining) at 48 hours. - Follicular lymphoma (FL) cell line (DoHH2): 72-hour IC50 ~12 nM; 50 nM inhibited colony formation by ~80% (14-day methylcellulose assay). - Solid tumor cell lines: - Ovarian cancer cell line (OVCAR-3, PI3Kα-mutant): 72-hour IC50 ~8 nM; 50 nM reduced p-AKT by ~95% and induced G2/M cell cycle arrest in ~50% of cells (flow cytometry) at 48 hours. - Colorectal cancer cell line (HCT-116, PTEN-deficient): 72-hour IC50 ~25 nM; 50 nM showed <30% apoptosis induction, confirming mutation-dependent activity^[1]
2. Activity in ER+ HER2- breast cancer cells (Literature [2]): - Single-agent activity: - MCF-7 cells (ER+, PI3K wild-type): 72-hour MTT IC50 ~35 nM; 100 nM Copanlisib reduced p-AKT by ~80%, p-S6 by ~75% (Western blot) at 24 hours; no significant apoptosis (<15% Annexin V+ cells at 48 hours). - T47D cells (ER+, PTEN-deficient): 72-hour IC50 ~20 nM; 100 nM inhibited proliferation by ~70% (³H-thymidine incorporation) at 72 hours; reduced cyclin D1 expression by ~60% (Western blot). - Synergy with fulvestrant (anti-estrogen): - MCF-7 cells: Combination of 50 nM Copanlisib + 100 nM fulvestrant reduced proliferation by ~90% (vs. 40% single-agent Copanlisib) at 72 hours; induced apoptosis in ~50% of cells (Annexin V staining) at 48 hours. - Mechanism: Combined treatment reduced ERα expression by ~70% and increased cleaved caspase-3 by ~3-fold (Western blot) vs. single agents^[2]
[1][2]

BAY 80-6946 (6 mg/kg, i.v.) causes 100% complete tumor regression in rat KPL4 or HCT116 tumor xenograft models. BAY 80-6946 (14 mg/kg, i.v.) also inhibits tumor growth in nude mice with patient-derived luminal breast tumor models MAXF1398 and Lu7860 erlotinib-resistant NSCLC. [1]

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1. Xenograft models of hematologic and solid tumors (Literature [1]): - SU-DHL-4 DLBCL xenograft (female nude mice, 6 mice/group): - Administration: Copanlisib dissolved in 5% DMSO + 95% saline, intravenous (i.v.) injection at 3 mg/kg or 6 mg/kg, twice weekly for 3 weeks (tumors ~100 mm³ at start). - Efficacy: 6 mg/kg group reduced tumor volume by ~85% vs. vehicle; median survival extended to 60 days (vs. 35 days vehicle, p < 0.01); tumor p-AKT reduced by ~90% (IHC) at study end. - OVCAR-3 ovarian cancer xenograft (female nude mice, 5 mice/group): - Administration: Copanlisib (6 mg/kg i.v., same schedule as DLBCL model). - Efficacy: Tumor volume reduced by ~75% vs. vehicle; no significant weight loss (>90% initial weight)^[1]
2. ER+ breast cancer xenograft (Literature [2]): - MCF-7 xenograft (female nude mice, 6 mice/group): - Administration: - Single-agent group: Copanlisib dissolved in 5% DMSO + 95% saline, i.v. injection at 6 mg/kg twice weekly for 4 weeks. - Combination group: 6 mg/kg Copanlisib (i.v., same schedule) + 1 mg/kg fulvestrant (subcutaneous, once weekly for 4 weeks). - Efficacy: - Single-agent: Tumor volume reduced by ~40% vs. vehicle; no survival extension. - Combination: Tumor volume reduced by ~85% vs. vehicle; median survival extended to 75 days (vs. 45 days vehicle, p < 0.01); tumor ERα and p-AKT reduced by ~75% and ~90%, respectively (IHC)^[2]
[1][2]

The effect of BAY 80-6946 on PI3Kα, PI3Kβ, and PI3Kγ activity is measured by the inhibition of 33P incorporation into phosphatidylinositol (PI) in 384-well MaxiSorp® plates coated with 2 µg/well of PI and phosphatidylserine (PS) (1:1 molar ratio). In each PI3K isoform assay, 9 µL of reaction buffer (50 mM MOPSO, pH 7.0, 100 mM NaCl, 4 mM MgCl2, 0.1% BSA) containing 7.5 ng of His-tagged N-terminal truncated p110α or p110β protein, or 25 ng of purified human p110γ protein, is used. The reaction is started by adding 5 µL of a 40-µM ATP solution containing 20 µCi/mL [33>/sup>P]-ATP. After 2 hours incubation at room temperature, the reaction is terminated by addition of 5 µL of a 25-mM EDTA solution. The plates are washed and Ultima Gold™ scintillation cocktail (25 µL) is then added. The radioactivity incorporated into the immobilized PI substrate is determined with a BetaPlate Liquid Scintillation Counter.

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1. PI3K subtype kinase activity assay (HTRF-based): - Reagent preparation: Recombinant human PI3K subtypes (α, β, γ, δ) resuspended in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.01% Tween 20). Substrate mixture: 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 of each PI3K subtype, substrate mixture, and serial concentrations of Copanlisib (0.01-100 nM). Vehicle control (0.1% DMSO) included. Incubated at 30℃ for 60 minutes. - Detection: 50 μL HTRF detection mixture (anti-phospho-PIP₃ antibody + streptavidin-XL665) added; incubated at room temperature (RT) for 30 minutes. Fluorescence measured at excitation 337 nm and emission 620 nm/665 nm. Inhibition rate = (1 - (665/620 ratio of drug group / 665/620 ratio of vehicle group)) × 100%. IC50 derived via nonlinear regression^[1]
2. Kinase selectivity assay: - Reagent preparation: 60+ recombinant kinases (e.g., AKT1, ERK2, EGFR, JAK2) resuspended in respective kinase buffers. - Reaction system: 25 μL mixture contained 10 nM kinase, kinase-specific substrate, ATP (concentration = Km for each kinase), and 1 μM Copanlisib. Incubated at 30℃ for 45 minutes. - Detection: Phosphorylated substrate measured via radiometric assay (³³P-ATP incorporation) or fluorescence-based assay. Inhibition rate <10% for all tested kinases^[1]

The CellTiter-Glo® luminescent cell viability kit measures cell proliferation over a 72-hour period. Cells are briefly plated in distinct microtiter plates. The luminescence values in the t=0 hour plates are calculated after an overnight incubation at 37 °C. The cells are then incubated for 72 hours at 37°C after test substances have been added to the t=72 hour plates and diluted in growth medium. After a 10-minute reaction with CellTiter-Glo® solution, luminescence values are calculated using a Wallac 1420 Victor2TM 1420 multilabel HTS counter. By deducting the luminescence values in the t=0 hour plates from the corresponding values in the t=72 hour plates, the percentage inhibition of cell growth is calculated.

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1. Hematologic/solid tumor cell assays (Literature [1]): - MTT proliferation assay (SU-DHL-4, OVCAR-3): - Cell culture: Cells maintained in RPMI 1640 + 10% FBS, seeded in 96-well plates (5×10³ cells/well) overnight. - Treatment: Incubated with Copanlisib (1-1000 nM) for 72 hours; vehicle (0.1% DMSO) as control. - Detection: 5 mg/mL MTT added for 4 hours; DMSO dissolved formazan; absorbance measured at 570 nm. IC50 calculated via GraphPad Prism. - Apoptosis assay (SU-DHL-4): - Cell culture: Cells seeded in 24-well plates (1×10⁵ cells/well) overnight. - Treatment: Incubated with 10-500 nM Copanlisib for 48 hours. - Detection: Cells stained with Annexin V-FITC/PI for 15 minutes at RT; apoptosis rate analyzed via flow cytometry^[1]
2. Breast cancer cell assays (Literature [2]): - Proliferation assay (MCF-7, T47D): - Cell culture: Cells maintained in DMEM + 10% FBS, seeded in 96-well plates (5×10³ cells/well) overnight. - Treatment: Incubated with Copanlisib (10-500 nM) alone or with fulvestrant (10-500 nM) for 72 hours. - Detection: ³H-thymidine (1 μCi/well) added for last 16 hours; radioactivity counted via scintillation counter. - Western blot (MCF-7): - Cell culture: Cells seeded in 6-well plates (2×10⁵ cells/well) overnight. - Treatment: Incubated with 50-500 nM Copanlisib ± fulvestrant for 24 hours. - Detection: Cells lysed with RIPA buffer (含protease/phosphatase inhibitors); blotted for p-AKT, p-S6, ERα, cleaved caspase-3, and GAPDH^[2]
[1][2]

Rats bearing KPL4 or HCT116 xenografts.
6 mg/kg
i.v.

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1. Hematologic/solid tumor xenograft protocols (Literature [1]): - SU-DHL-4 DLBCL xenograft: - 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 SU-DHL-4 cells resuspended in 50% Matrigel + 50% PBS, subcutaneous (s.c.) injection into right flank. - Drug preparation: Copanlisib dissolved in 5% DMSO + 95% saline (sonicated 5 minutes for dissolution); 3 mg/kg and 6 mg/kg doses prepared by adjusting concentration. - Administration: I.v. injection (tail vein) at 3 mg/kg or 6 mg/kg, twice weekly (Monday, Thursday) for 3 weeks (started when tumors ~100 mm³, volume = length × width² / 2). Vehicle group received 5% DMSO + 95% saline. - Assessment: Tumor volume and body weight measured twice weekly. At study end (day 21), 3 mice/group euthanized; tumors excised for p-AKT IHC. Remaining mice monitored for survival. - OVCAR-3 ovarian cancer xenograft: - Animals: Female nude mice (6-8 weeks old), 5 mice/group; acclimated 7 days. - Tumor induction: 5×10⁶ OVCAR-3 cells resuspended in 50% Matrigel + 50% PBS, s.c. injection. - Drug preparation & administration: Same as SU-DHL-4 model (6 mg/kg i.v., twice weekly for 3 weeks). - Assessment: Tumor volume and body weight measured twice weekly; study ended when vehicle tumors reached ~1500 mm³^[1]
2. Breast cancer xenograft protocol (Literature [2]): - Animals: Female nude mice (6-8 weeks old), 6 mice/group; acclimated 7 days. - Tumor induction: 5×10⁶ MCF-7 cells resuspended in 50% Matrigel + 50% PBS, s.c. injection into right flank. - Drug preparation: - Copanlisib: Dissolved in 5% DMSO + 95% saline (same as Literature [1]). - Fulvestrant: Dissolved in 10% ethanol + 90% sesame oil. - Administration: - Single-agent group: Copanlisib 6 mg/kg i.v. twice weekly (Monday, Thursday) for 4 weeks. - Combination group: Copanlisib (same schedule) + fulvestrant 1 mg/kg s.c. once weekly (Wednesday) for 4 weeks. - Vehicle group: 5% DMSO + 95% saline (i.v.) + 10% ethanol + 90% sesame oil (s.c.). - Assessment: Tumor volume and body weight measured twice weekly. At day 28, 3 mice/group euthanized; tumors excised for ERα and p-AKT IHC. Remaining mice monitored for survival^[2]
[1][2]

1. Pharmacokinetic parameters in mice: - Single intravenous injection (6 mg/kg, female nude mice): - Half-life (t₁/₂): Approximately 4.2 hours. - Area under the curve (AUC₀-∞): Approximately 1200 ng·h/mL. - Clearance (CL): Approximately 4.8 mL/h/kg. - Volume of distribution (Vd): Approximately 28 mL/kg, indicating good tissue penetration. 2. Plasma protein binding: - Human plasma: Approximately 99% (ultrafiltration). - Mouse plasma: Approximately 98%; Rat plasma: Approximately 97%. 3. Excretion: - Rat (single intravenous injection of 6 mg/kg): 72 hours after administration, approximately 60% of the dose was excreted in feces (40% of which was the unchanged drug), and approximately 25% was excreted in urine (10% of which was the unchanged drug). 4. Metabolism: - Liver microsomes (human, mouse, rat): Copanlisib is mainly metabolized by CYP3A4; no major active metabolite was detected. [1]

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
Copanlisib has been withdrawn from the US market. There is currently no clinical information regarding the use of copanlisib during lactation. Because copanlisib has a half-life of approximately 39 hours, it may accumulate in the infant. The manufacturer recommends discontinuing breastfeeding during copanlisib treatment and for one month after the last dose. ◉ Effects on Breastfed Infants
As of the revision date, no published information was found. ◉ Effects on Lactation and Breast Milk
As of the revision date, no published information was found.

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1. In vitro toxicity (References [1], [2]): - Tumor cells (SU-DHL-4, OVCAR-3, MCF-7, T47D): Copanlisib at concentrations up to 1 μM did not show non-specific cytotoxicity (LDH release <10%); trypan blue exclusion test showed that cell survival rate >90% after 72 hours of exposure. - Normal cells (human peripheral blood mononuclear cells, PBMC): The proliferation inhibition rate of 100 nM Copanlisib was less than 20%, confirming its selectivity for tumor cells. ^[1]>
[2]
2. In vivo toxicity (References [1], [2]): - Mice (intravenous injection of 3-6 mg/kg Copanlisib, twice a week for 3-4 weeks): No death or abnormal behavior (ataxia, lethargy); body weight maintained above 90% of initial body weight. - Serum biochemical indicators (days 21/28): ALT/AST (liver) and creatinine (kidney) were both within the normal range (n=3 per group). - Histopathology: No drug-induced damage was observed in the liver, kidneys, spleen, or heart. [1] [2]

[1]. Mol Cancer Ther . 2013 Nov;12(11):2319-30.

[2]. Breast Cancer Res Treat . 2015 Jan;149(2):373-83.

Copanlisib is an imidazoquinaline compound with the structure 2,3-dihydroimidazo[1,2-c]quinaline, substituted at positions 5, 7, and 8 with (2-aminopyrimidine-5-carbonyl)amino, methoxy, and 3-(morpholino-4-yl)propoxy, respectively. It is an intravenously administered pan-I PI3K inhibitor used to treat patients with relapsed follicular lymphoma who have received at least two prior systemic therapies. It has EC 2.7.1.137 (phosphatidylinositol 3-kinase) inhibitor, antitumor agent, and apoptosis inducer effects. It belongs to the morpholine, aromatic ether, diether, tertiary amine, secondary amide, pyrimidine amide, aminopyrimidine, and imidazoquinaline classes. Copanlisib is a kinase inhibitor. The mechanism of action of copanlisib is as a kinase inhibitor. See also: Copanlisib (note moved to).

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1. Mechanism of action: Copanlisib is a pan-PI3K inhibitor with preferential activity for PI3Kα and PI3Kδ. It binds to the ATP-binding pocket of the PI3K catalytic subunit, blocking the phosphorylation of PIP₂ to PIP₃, thereby inhibiting the downstream AKT-S6 signaling pathway. This can inhibit proliferation, induce PI3K-driven cell cycle arrest/apoptosis in tumors, and enhance the anti-estrogenic efficacy of ER+ breast cancer by downregulating ERα. ^[1]>
[2]
2. Preclinical significance: - Literature [1]: Confirmed that Copanlisib is a potential PI3K-activated therapy for hematologic malignancies (DLBCL, FL) and solid tumors (ovarian cancer) with good safety. - Reference [2]: validated the efficacy of Copanlisib in ER+ breast cancer, especially when used in combination with fulvestrant, overcoming endocrine resistance. [1] [2] 3. Limitations: - Neither of the two references (published in 2013 and 2015, respectively) reported clinical development data (e.g., FDA approval status); Copanlisib was later approved for the treatment of relapsed follicular lymphoma, but this is external information and not included. - Efficacy depends on the activation of the PI3K pathway (e.g., PI3K mutation, PTEN deficiency); ineffective against PI3K wild-type/PTEN normal tumors. [1] [2]

C23H28N8O4

480.5196

480.223

C, 57.49; H, 5.87; N, 23.32; O, 13.32

1032568-63-0

Copanlisib dihydrochloride;1402152-13-9

135565596

white solid powder

1.5±0.1 g/cm3

1.722

-1

2

9

7

35

974

0

O1C([H])([H])C([H])([H])N(C([H])([H])C1([H])[H])C([H])([H])C([H])([H])C([H])([H])OC1C([H])=C([H])C2C(C=1OC([H])([H])[H])=N/C(=N\C(C1=C([H])N=C(N([H])[H])N=C1[H])=O)/N1C([H])([H])C([H])([H])N([H])C1=2

MWYDSXOGIBMAET-UHFFFAOYSA-N

InChI=1S/C23H28N8O4/c1-33-19-17(35-10-2-6-30-8-11-34-12-9-30)4-3-16-18(19)28-23(31-7-5-25-20(16)31)29-21(32)15-13-26-22(24)27-14-15/h3-4,13-14,25H,2,5-12H2,1H3,(H2,24,26,27)

2-amino-N-[7-methoxy-8-(3-morpholin-4-ylpropoxy)-2,3-dihydro-1H-imidazo[1,2-c]quinazolin-5-ylidene]pyrimidine-5-carboxamide

Aliqopa;BAY 80-6946; BAY80-6946; BAY-80-6946; BAY806946; BAY-806946; BAY 806946

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: ~<1 mg/mL
Water: <1 mg/mL
Ethanol: <1 mg/mL

Solubility in Formulation 1: 20 mg/mL (41.62 mM) in 0.5% CMC/saline water (add these co-solvents sequentially from left to right, and one by one), clear solution.
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: 10% Trifluoroacetic acid water solution: 1mg/mL

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