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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.
Physicochemical Properties
| 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 |
| PubChem CID | 6538897 |
| Appearance | Light yellow to yellow solid powder |
| Density | 1.7±0.1 g/cm3 |
| Boiling Point | 648.8±50.0 °C at 760 mmHg |
| 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 |
| HS Tariff Code | 2934.99.03.00 |
| 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 |
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, oral administration of 1-Azakenpaullone (1-Akp) (10 mg/kg) showed an oral bioavailability of ~25%. Peak plasma concentration (Cₘₐₓ) was ~180 ng/mL at 1.5 hours (Tₘₐₓ), with an elimination half-life (t₁/₂) of ~2.8 hours. Minimal accumulation was observed in the brain (plasma:brain ratio ~1:0.2) [3] |
| Toxicity/Toxicokinetics |
1. In vitro, 1-Azakenpaullone (1-Akp) (up to 1 μM) showed no cytotoxicity in L6 myotubes or HEK293 cells (viability >85%, MTT assay); HeLa cells showed proliferation inhibition (IC₅₀=0.5 μM) but no apoptosis (TUNEL⁻) [1], [2], [3] 2. In vivo, 1-Azakenpaullone (1-Akp) (3-10 mg/kg, oral for 7 days) in ob/ob mice caused no significant changes in body weight, serum ALT/AST (liver function), or creatinine (kidney function) vs. vehicle [2] 3. Plasma protein binding of 1-Azakenpaullone (1-Akp) was ~92% in mouse plasma (equilibrium dialysis) [3] |
| References |
[1]. Bioorg Med Chem Lett . 2004 Jan 19;14(2):413-6. [2]. Diabetes . 2009 Mar;58(3):663-72. [3]. J Med Chem . 2008 Apr 10;51(7):2196-207. |
| Additional Infomation |
1-azakenpaullone is an organic heterotetracyclic compound that is 7,12-dihydropyrido[3',2':2,3]azepino[4,5-b]indole substituted at positions 6 and 9 by oxo and bromo groups respectively. It has a role as an EC 2.7.11.26 (tau-protein kinase) inhibitor and a Wnt signalling activator. It is an organic heterotetracyclic compound, an organonitrogen heterocyclic compound, a lactam and an organobromine compound. 1. 1-Azakenpaullone (1-Akp) is a synthetic paullone derivative that acts as an ATP-competitive inhibitor of GSK3 and CDK family kinases, with higher potency for GSK3β/α than CDKs [1], [3] 2. In metabolic tissues, 1-Azakenpaullone (1-Akp) improves glucose metabolism by inhibiting GSK3 (activating GS to promote glycogen synthesis) and enhancing insulin signaling, supporting its potential for type 2 diabetes treatment [2] 3. The activation of Wnt/β-catenin by 1-Azakenpaullone (1-Akp) (via GSK3 inhibition) suggests potential applications in stem cell pluripotency maintenance or cancer research (targeting Wnt-dependent tumors) [3] 4. Unlike non-selective paullone analogs, 1-Azakenpaullone (1-Akp) shows reduced activity against CDK2, minimizing off-target effects on normal cell cycle progression [1] |
Solubility Data
| Solubility (In Vitro) | DMSO: 66~125 mM(201.1~380.9 mM) |
| 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 |