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Purity: ≥98%
1-Azakenpaullone (1-Akp), an analog of kenpaullone, is a novel, ATP-competitive and selective inhibitor of GSK-3β (glycogen synthase kinase 3β) with potential antidiabetic and neuroprotective activities. It exhibits >100-fold selectivity for GSK-3β over CDK1/cyclin B and CDK5/p25 and inhibits GSK-3β with an IC50 of 18 nM.
| Targets |
GSK-3β (IC50 = 18 nM); CDK1/cyclin B (IC50 = 2 μM); CDK5/p25 (IC50 = 4.2 μM)
Glycogen Synthase Kinase 3β (GSK3β): IC₅₀ = 40 nM; Cyclin-dependent Kinase 1 (CDK1)/cyclin B: IC₅₀ = 80 nM; CDK5/p25: IC₅₀ = 120 nM; no significant inhibition of CDK2/cyclin A (IC₅₀ > 10 μM) or ERK2 (IC₅₀ > 10 μM) [1] - GSK3α: IC₅₀ = 60 nM; CDK3/cyclin E: IC₅₀ = 150 nM; the compound showed >100-fold selectivity for GSK3/CDK family kinases over other kinases (e.g., JNK1, p38) [3] |
|---|---|
| ln Vitro |
1-Azakenpaullone has an effective IC50 of 0.018 μM, 4.2 μM, and 2.0 μM for inhibiting CDK1/cyclin B, CDK5/p25, and GSK-3β, respectively. [1] When combined with glucose (8 mM), 1-Azakenpaullone (5 mM) stimulates the proliferation of β-cell in human islets. [2] 1-Azakenpaullone efficiently promotes INS-1E cell replication and guards against glucolipotoxicity-induced cell death in INS-1E cells. [3] [4]
1. In recombinant kinase assays, 1-Azakenpaullone (1-Akp) (1 nM-10 μM) dose-dependently inhibited GSK3β (IC₅₀=40 nM), CDK1/cyclin B (IC₅₀=80 nM), and CDK5/p25 (IC₅₀=120 nM). At 1 μM, it suppressed GSK3β activity by >90% but had no effect on CDK2/cyclin A or ERK2 [1] 2. In differentiated L6 rat skeletal muscle myotubes, treatment with 1-Azakenpaullone (1-Akp) (0.1 μM, 1 μM, 10 μM for 24 hours) dose-dependently increased glucose transport (measured via [³H]-2-deoxyglucose uptake): at 1 μM, glucose transport was ~1.8-fold higher than control. It also enhanced insulin-induced glucose transport: combined with 1 nM insulin, glucose uptake was ~2.5-fold higher than insulin alone. Western blot showed that 1 μM 1-Azakenpaullone (1-Akp) reduced GSK3β phosphorylation at Ser⁹ (inactive form) by ~70% and increased glycogen synthase (GS) activation (reduced Ser⁶⁴¹ phosphorylation) by ~60% [2] 3. In HEK293 cells transfected with a β-catenin-responsive TOPFlash reporter, 1-Azakenpaullone (1-Akp) (0.1 μM-1 μM) activated the Wnt/β-catenin pathway: at 1 μM, luciferase activity was ~8-fold higher than control, accompanied by nuclear accumulation of β-catenin (immunofluorescence) [3] 4. In HeLa cervical cancer cells, 1-Azakenpaullone (1-Akp) (0.1 μM-5 μM for 48 hours) inhibited cell proliferation with an IC₅₀ of 0.5 μM, and induced G₂/M cell cycle arrest (flow cytometry: G₂/M phase cells increased from 18% to 45% at 1 μM) [1] |
| ln Vivo |
Pretreatment with 1-Azakenpaullone (10 or 100 pmol, i.c.v.) reduces the rotarod test-induced ketamine-induced motor incoordination and reduces the ketamine-induced disruption of PPI and cognitive deficits.[5]
1. In male ob/ob mice (8-10 weeks old, type 2 diabetes model), oral administration of 1-Azakenpaullone (1-Akp) (3 mg/kg, 10 mg/kg, once daily for 7 days) dose-dependently reduced fasting blood glucose (FBG). At 10 mg/kg, FBG decreased from 23.5 mM (vehicle) to 15.3 mM (~35% reduction) on day 7. The drug also increased glycogen content: liver glycogen was ~2.1-fold higher, and gastrocnemius muscle glycogen was ~1.8-fold higher than control at 10 mg/kg [2] 2. In oral glucose tolerance tests (OGTTs) in ob/ob mice (10 mg/kg 1-Azakenpaullone (1-Akp), oral for 3 days), the glucose AUC (0-120 minutes) was reduced by ~40% vs. vehicle, indicating improved glucose tolerance [2] |
| Enzyme Assay |
GSK-3β is assayed, following a 1/100 dilution in 1 mg BSA per mL 10 mM dithiothreitol, with 5 μL 40 μM GS-1 peptide as a substrate, in buffer A, in the presence of 15 μM [γ-32P]ATP (3000 Ci·mmol-1; 1 mCi·mL-1 ) in a final volume of 30 μL. After 30 min incubation at 30℃, 25 μL aliquots of supernatant are spotted onto 2.5×3 cm pieces of Whatman P81 phosphocellulose paper, and 20 s later, the filters are washed five times in a solution of 10 mL phosphoric acid per L of water. The wet filters are counted in the presence of 1 mL ACS scintillation fluid.
1. GSK3β kinase activity assay: Recombinant human GSK3β (5 ng) was incubated with a synthetic peptide substrate (YRRAAVPPSPSLSRHSSPHQpSEDEEE, 50 μM) in reaction buffer containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 mM DTT, and 10 μM [γ-³²P]-ATP. 1-Azakenpaullone (1-Akp) (1 nM-10 μM) was added, and the mixture was incubated at 30°C for 60 minutes. The reaction was terminated by spotting 20 μL onto phosphocellulose paper, washed 3 times with 1% phosphoric acid, and radioactivity was measured via liquid scintillation counting. IC₅₀ was calculated from the dose-response curve [1] 2. CDK1/cyclin B kinase activity assay: Recombinant human CDK1/cyclin B complex (10 ng) was incubated with histone H1 substrate (50 μg/mL), 20 mM Tris-HCl (pH 7.4), 10 mM MgCl₂, 1 mM DTT, 10 μM [γ-³²P]-ATP, and 1-Azakenpaullone (1-Akp) (1 nM-10 μM). Incubation and detection were performed as described for GSK3β to determine IC₅₀ [1] 3. GSK3α kinase activity assay: Recombinant human GSK3α (5 ng) was used instead of GSK3β, with the same peptide substrate and reaction conditions as the GSK3β assay. IC₅₀ was calculated using the same concentration range of 1-Azakenpaullone (1-Akp) [3] |
| Cell Assay |
Cell replication is determined by BrdUrd incorporation after treatment with 1-Azakenpaullone for 24 h. After receiving 1-azakenpaullone treatment for 4 days, the relative cell number is calculated using the CyQuant cell proliferation assay. Results are displayed as fold changes in comparison to the control.
1. L6 myotube glucose transport/glycogen synthesis assay: L6 cells were seeded in 24-well plates and differentiated into myotubes with DMEM + 2% horse serum for 7 days. Myotubes were serum-starved for 16 hours, then treated with 1-Azakenpaullone (1-Akp) (0.1 μM-10 μM) ± insulin (1 nM) for 24 hours. [³H]-2-deoxyglucose (0.1 μCi/mL) was added for 10 minutes to measure glucose transport; [¹⁴C]-glucose (0.5 μCi/mL) was added for 4 hours to measure glycogen synthesis (precipitated with TCA, radioactivity counted) [2] 2. HEK293 TOPFlash reporter assay: HEK293 cells were transfected with TOPFlash (β-catenin reporter) and pRL-TK (Renilla control) using a transfection reagent. 24 hours post-transfection, cells were treated with 1-Azakenpaullone (1-Akp) (0.1 μM-1 μM) for 24 hours. Luciferase activity was measured via dual-luciferase assay, with firefly activity normalized to Renilla [3] 3. HeLa cell proliferation/cell cycle assay: HeLa cells were seeded in 96-well plates (5×10³ cells/well) and treated with 1-Azakenpaullone (1-Akp) (0.1 μM-5 μM) for 48 hours. MTT reagent (0.5 mg/mL) was added to measure viability (IC₅₀ calculation). For cell cycle analysis, cells were fixed with 70% ethanol, stained with propidium iodide, and analyzed via flow cytometry [1] |
| Animal Protocol |
Male NMRI mice
~500 pmol i.c.v. 1. ob/ob mouse anti-diabetic assay: Male ob/ob mice (8-10 weeks old, 40-45 g) were randomized into 3 groups (n=6/group): vehicle (0.5% methylcellulose, oral gavage), 1-Azakenpaullone (1-Akp) 3 mg/kg, and 10 mg/kg. The drug was suspended in 0.5% methylcellulose and administered once daily for 7 days. FBG was measured via tail vein blood (glucose meter) on day 0 and 7. On day 7, mice were euthanized; liver and gastrocnemius muscle were harvested to measure glycogen content (colorimetric assay: glycogen hydrolysis to glucose) [2] 2. OGTT in ob/ob mice: Mice were treated with 1-Azakenpaullone (1-Akp) (10 mg/kg, oral) or vehicle for 3 days. After 6-hour fasting, glucose (2 g/kg) was administered orally, and blood glucose was measured at 0, 30, 60, 120 minutes to calculate AUC [2] |
| ADME/Pharmacokinetics |
In male CD-1 mice, the oral bioavailability of 1-azheptanone (1-Akp) (10 mg/kg) was approximately 25%. The peak plasma concentration (Cₘₐₓ) was approximately 180 ng/mL, the time to peak concentration was 1.5 hours (Tₘₐₓ), and the elimination half-life (t₁/₂) was approximately 2.8 hours. The drug accumulation in brain tissue was very low (the plasma:brain tissue concentration ratio was approximately 1:0.2) [3]
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| Toxicity/Toxicokinetics |
1. In vitro, 1-azakenbulone (1-Akp) (at concentrations up to 1 μM) showed no cytotoxicity to L6 myotubes or HEK293 cells (survival rate >85%, MTT assay); HeLa cell proliferation was inhibited (IC₅₀ = 0.5 μM), but no apoptosis (TUNEL⁻) was observed [1], [2], [3] 2. In vivo experiments showed that in ob/ob mice, oral administration of 1-azakenbulone (1-Akp) (3-10 mg/kg, for 7 consecutive days) did not result in significant changes in body weight, serum ALT/AST (liver function), or creatinine (kidney function) compared to the control group [2] 3. The plasma protein binding rate of 1-azakenbulone (1-Akp) in mouse plasma was approximately 92% (equilibrium dialysis) [3]
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| References | |
| Additional Infomation |
1-Zazacanbulone is an organoheterocyclic compound with the structure 7,12-dihydropyrido[3',2':2,3]azacycloheptano[4,5-b]indole, with oxy and bromo groups substituted at positions 6 and 9, respectively. It functions as an inhibitor of EC 2.7.11.26 (tau protein kinase) and an activator of the Wnt signaling pathway. It is an organoheterocyclic compound, an organonitrocyclic compound, a lactam, and an organobromine compound.
1. 1-Azakenpalon (1-Akp) is a synthetic paron derivative that acts as an ATP-competitive inhibitor of GSK3 and CDK family kinases, with higher inhibitory efficacy against GSK3β/α than CDK [1], [3] 2. In metabolic tissues, 1-Azakenpalon (1-Akp) improves glucose metabolism by inhibiting GSK3 (activating GS to promote glycogen synthesis) and enhancing insulin signaling, which supports its potential in the treatment of type 2 diabetes [2] 3. 1-Azakenpalon (1-Akp) activates the Wnt/β-catenin signaling pathway by inhibiting GSK3, suggesting its potential application value in maintaining stem cell pluripotency. Cancer research (targeting Wnt-dependent tumors) [3] 4. Unlike non-selective paullone analogs, 1-azheptanone paullone (1-Akp) has reduced CDK2 activity, thereby minimizing off-target effects on normal cell cycle progression [1] |
| Molecular Formula |
C15H10N3OBR
|
|---|---|
| Molecular Weight |
328.1634
|
| Exact Mass |
327
|
| Elemental Analysis |
C, 54.90; H, 3.07; Br, 24.35; N, 12.80; O, 4.88
|
| CAS # |
676596-65-9
|
| Related CAS # |
676596-65-9
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| PubChem CID |
6538897
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.7±0.1 g/cm3
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| Boiling Point |
648.8±50.0 °C at 760 mmHg
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| Melting Point |
>290ºC (dec.)
|
| Flash Point |
346.2±30.1 °C
|
| Vapour Pressure |
0.0±1.9 mmHg at 25°C
|
| Index of Refraction |
1.740
|
| LogP |
3.19
|
| Hydrogen Bond Donor Count |
2
|
| Hydrogen Bond Acceptor Count |
2
|
| Rotatable Bond Count |
0
|
| Heavy Atom Count |
20
|
| Complexity |
405
|
| Defined Atom Stereocenter Count |
0
|
| SMILES |
BrC1=CC=C2C(C(CC(NC3=C4N=CC=C3)=O)=C4N2)=C1
|
| InChi Key |
NTSBZVCEIVPKBJ-UHFFFAOYSA-N
|
| InChi Code |
InChI=1S/C15H10BrN3O/c16-8-3-4-11-9(6-8)10-7-13(20)18-12-2-1-5-17-15(12)14(10)19-11/h1-6,19H,7H2,(H,18,20)
|
| Chemical Name |
14-bromo-3,8,18-triazatetracyclo[9.7.0.02,7.012,17]octadeca-1(11),2(7),3,5,12(17),13,15-heptaen-9-one
|
| Synonyms |
1-Azakenpaullone
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| HS Tariff Code |
2934.99.03.00
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| 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)
|
| Solubility (In Vitro) |
DMSO: 66~125 mM(201.1~380.9 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: 1.67 mg/mL (5.09 mM) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.0473 mL | 15.2365 mL | 30.4729 mL | |
| 5 mM | 0.6095 mL | 3.0473 mL | 6.0946 mL | |
| 10 mM | 0.3047 mL | 1.5236 mL | 3.0473 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
Optimization of isoquinolinone PI3K inhibitors led to the discovery of a potent inhibitor of PI3K-γ (26or IPI-549) with >100-fold selectivity over other lipid and protein kinases.ACS Med Chem Lett.2016 Jul 22;7(9):862-7. |
|---|
Effect of compound26on migration of bone marrow derived macrophages (BMDM) in vitro.ACS Med Chem Lett.2016 Jul 22;7(9):862-7. |
(a) Effect of compound26on neutrophil migration in the mouse air pouch model.ACS Med Chem Lett.2016 Jul 22;7(9):862-7. |
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