 Chemical Structure.png)
Copanlisib (formerly BAY 80-6946; brand name: Aliqopa) is a potent, ATP-competitive and pan-class I PI3K (phosphoinositide 3-kinase) inhibitor with potential anticancer activity. According to cell-free assays for PI3Kα/β/γ/δ, it has IC50 values of 0.5, 3.7, 6.4, and 0.7 nM. Adult patients with relapsed follicular lymphoma who have had at least two prior systemic therapies are eligible to receive Copanlisib as of September 2017 per FDA approval. With IC50 values of 137 nM and 147 nM, respectively, in HuCCT-1 (KRASG12D) and EGI-1 (KRASG12D) cell lines, BAY 80-6946 demonstrated strong anti-proliferative activity. The maximum tolerated dose (MTD) for BAY 80-6946 is 0.8 mg/kg, and this dose is typically well tolerated. Results from the pharmacokinetics (PK) study support weekly dosing. In the first 24 hours following a MTD dose, hyperglycemia of grade 2 or 3 may occur. Clinical SD, pharmacokinetics, and FDG-PET data all support effective exposure and PI3K pathway inhibition.
Physicochemical Properties
| Molecular Formula | C23H28N8O4 |
| Molecular Weight | 480.5196 |
| Exact Mass | 480.223 |
| Elemental Analysis | C, 57.49; H, 5.87; N, 23.32; O, 13.32 |
| CAS # | 1032568-63-0 |
| Related CAS # | Copanlisib dihydrochloride;1402152-13-9 |
| PubChem CID | 135565596 |
| Appearance | white solid powder |
| Density | 1.5±0.1 g/cm3 |
| Index of Refraction | 1.722 |
| LogP | -1 |
| Hydrogen Bond Donor Count | 2 |
| Hydrogen Bond Acceptor Count | 9 |
| Rotatable Bond Count | 7 |
| Heavy Atom Count | 35 |
| Complexity | 974 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | 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 |
| InChi Key | MWYDSXOGIBMAET-UHFFFAOYSA-N |
| InChi Code | 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) |
| Chemical Name | 2-amino-N-[7-methoxy-8-(3-morpholin-4-ylpropoxy)-2,3-dihydro-1H-imidazo[1,2-c]quinazolin-5-ylidene]pyrimidine-5-carboxamide |
| Synonyms | Aliqopa;BAY 80-6946; BAY80-6946; BAY-80-6946; BAY806946; BAY-806946; BAY 806946 |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
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] |
| ln Vitro |
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] 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] |
| ln Vivo |
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] 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] |
| Enzyme Assay |
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. 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] |
| Cell Assay |
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. 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] |
| Animal Protocol |
Rats bearing KPL4 or HCT116 xenografts. 6 mg/kg i.v. 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] |
| ADME/Pharmacokinetics |
1. Pharmacokinetic parameters in mice:
- Single i.v. dose (6 mg/kg, female nude mice):
- Half-life (t₁/₂): ~4.2 hours.
- Area under the curve (AUC₀-∞): ~1200 ng·h/mL.
- Clearance (CL): ~4.8 mL/h/kg.
- Volume of distribution (Vd): ~28 mL/kg, indicating good tissue penetration.
2. Plasma protein binding:
- Human plasma: ~99% (ultrafiltration method).
- Mouse plasma: ~98%; rat plasma: ~97%.
3. Excretion:
- Rats (single i.v. dose 6 mg/kg): 72 hours post-dose, ~60% of dose excreted in feces (40% as unchanged drug), ~25% in urine (10% as unchanged drug).
4. Metabolism:
- Liver microsomes (human, mouse, rat): Copanlisib primarily metabolized via CYP3A4; no major active metabolites detected[1] |
| Toxicity/Toxicokinetics |
Effects During Pregnancy and Lactation ◉ Summary of Use during Lactation Copanlisib has been removed from the US market. No information is available on the clinical use of copanlisib during breastfeeding. Because copanlisib's half-life is about 39 hours, it might accumulate in the infant. The manufacturer recommends that breastfeeding be discontinued during copanlisib therapy and for 1 month after the last dose. ◉ Effects in Breastfed Infants Relevant published information was not found as of the revision date. ◉ Effects on Lactation and Breastmilk Relevant published information was not found as of the revision date. 1. In vitro toxicity (Literatures [1], [2]): - Tumor cells (SU-DHL-4, OVCAR-3, MCF-7, T47D): Copanlisib concentrations up to 1 μM showed no non-specific cytotoxicity (LDH release <10%); trypan blue exclusion assay showed >90% viability after 72-hour exposure. - Normal cells (human peripheral blood mononuclear cells, PBMCs): 100 nM Copanlisib showed <20% proliferation inhibition, confirming tumor cell selectivity[1] [2] 2. In vivo toxicity (Literatures [1], [2]): - Mice (i.v. 3-6 mg/kg Copanlisib twice weekly for 3-4 weeks): No mortality or abnormal behaviors (ataxia, lethargy); body weight maintained >90% of initial weight. - Serum chemistry (day 21/28): ALT/AST (liver) and creatinine (kidney) within normal ranges (n=3 per group). - Histopathology: No drug-induced damage in liver, kidney, spleen, or heart[1] [2] |
| References |
[1]. Mol Cancer Ther . 2013 Nov;12(11):2319-30. [2]. Breast Cancer Res Treat . 2015 Jan;149(2):373-83. |
| Additional Infomation |
Copanlisib is an imidazoquinazoline that is 2,3-dihydroimidazo[1,2-c]quinazoline substituted by (2-aminopyrimidine-5-carbonyl)amino, methoxy, and 3-(morpholin-4-yl)propoxy groups at positions 5, 7 and 8, respectively. It is a intravenous pan-class I PI3K inhibitor used for the treatment of relapsed follicular lymphoma in patients who have received at least 2 prior systemic therapies. It has a role as an EC 2.7.1.137 (phosphatidylinositol 3-kinase) inhibitor, an antineoplastic agent and an apoptosis inducer. It is a member of morpholines, an aromatic ether, a diether, a tertiary amino compound, a secondary carboxamide, a pyrimidinecarboxamide, an aminopyrimidine and an imidazoquinazoline. Copanlisib is a Kinase Inhibitor. The mechanism of action of copanlisib is as a Kinase Inhibitor. See also: Copanlisib (annotation moved to). 1. Mechanism of action: Copanlisib is a pan-PI3K inhibitor with preferential activity against PI3Kα and PI3Kδ. It binds to the ATP-binding pocket of PI3K catalytic subunits, blocking PIP₂ phosphorylation to PIP₃ and inhibiting downstream AKT-S6 signaling. This suppresses proliferation, induces cell cycle arrest/apoptosis in PI3K-driven tumors, and enhances anti-estrogen efficacy in ER+ breast cancer via ERα downregulation[1] [2] 2. Preclinical significance: - Literature [1]: Establishes Copanlisib as a potential therapy for PI3K-activated hematologic tumors (DLBCL, FL) and solid tumors (ovarian cancer) with favorable safety profiles. - Literature [2]: Validates Copanlisib in ER+ breast cancer, particularly in combination with fulvestrant, addressing endocrine resistance[1] [2] 3. Limitations: - No clinical development data (e.g., FDA approval status) reported in the two literatures (published in 2013, 2015); Copanlisib was later approved for relapsed follicular lymphoma, but this is external information not included. - Efficacy depends on PI3K pathway activation (e.g., PI3K mutation, PTEN deficiency); no activity in PI3K-wild-type/PTEN-normal tumors[1] [2][1][2] |
Solubility Data
| Solubility (In Vitro) |
DMSO: ~<1 mg/mL Water: <1 mg/mL Ethanol: <1 mg/mL |
| Solubility (In Vivo) |
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. Solubility in Formulation 2: 10% Trifluoroacetic acid water solution: 1mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0811 mL | 10.4054 mL | 20.8108 mL | |
| 5 mM | 0.4162 mL | 2.0811 mL | 4.1622 mL | |
| 10 mM | 0.2081 mL | 1.0405 mL | 2.0811 mL |