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Purity: ≥98%
Alsterpaullone (also known as 9-Nitropaullone and NSC-705701; NSC 705701) is a novel and potent ATP-competitive cyclin-dependent kinases (CDK) inhibitor with IC50s of 35 nM, 15 nM, 200 nM and 40 nM for CDK1/cyclin B, CDK2/cyclin A, CDK2/cyclin E and CDK5/p35, respectively. Moreover, Alsterpaullone (9-Nitropaullone; NSC 705701) faces competition from ATP in the 4 nM IC50 range for binding to glycogen synthase kinase-3 alpha/beta (GSK-3alpha/GSK-3beta). In addition to having antitumor activity, alsterpaullone (9-Nitropaullone; NSC 705701) may be used to treat proliferative and neurodegenerative diseases. Alsterpaullone causes a disruption in the potential of the mitochondrial membrane, which activates caspase-9 and causes apoptosis. Alsterpaullone's Apoptosis-Inducing Effect mediated toxicity in HeLa cells.
| Targets |
Cdk1/cyclin B (IC50 = 35 nM); cdk2/cyclin A (IC50 = 15 nM); CDK2/Cyc E (IC50 = 200 nM); CDK5/p35 (IC50 = 40 nM); GSK-3α (IC50 = 4 nM); GSK-3β (IC50 = 4 nM)
Alsterpaullone is identified as a cyclin-dependent kinase (CDK) inhibitor. The literature mentions it as a potent inhibitor of CDK1/cyclin B and CDK5,. |
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| ln Vitro |
Alsterpaullone (0.3, 1, 3 μM; 8 hours) causes leukemia cell lines to undergo apoptosis[2]
Alsterpaullone (5, 10, 15, 20, 25, 30 μM; 48 and 72 hours) inhibits HeLa cell growth in a time- and dose-dependent manner. Additionally, alsterpaullone treatment inhibits cell growth in a time-dependent manner[3]. Alsterpaullone (20 μM) depends on caspase activity to cause cell death[3]. Alsterpaullone reduced cell proliferation in Group 3 medulloblastoma cell lines (D425 and D458) with efficacy observed at 1 µM after 48 hours of treatment, as measured by MTS assay. It showed little effect on the proliferation of normal human fetal neural stem cells (hf5281) even at 10 µM, indicating potential selectivity for tumor cells. Western blot analysis demonstrated that Alsterpaullone induced apoptosis, evidenced by PARP cleavage in D425 and D458 cells after 48 hours of treatment. It also inhibited AKT pathway activation, shown by decreased levels of phosphorylated AKT (p-AKT) in treated cells. Genomic profiling of treated cells revealed that Alsterpaullone downregulated genes involved in the cell cycle, including the oncogene MYC. Gene Set Enrichment Analysis confirmed that the top downregulated gene sets were cell cycle-related. Querying the Connectivity Map with the gene expression profile of Alsterpaullone-treated cells yielded a high positive enrichment score (0.995), indicating its ability to reverse the Group 3 medulloblastoma gene signature. |
| ln Vivo |
Alsterpaullone (30 mg/kg, s.c., daily for 2 weeks) decreases tumor growth and improves xenograft survival for medulloblastoma[4].
In an orthotopic xenograft model using D458 cells expressing luciferase implanted in the cerebellum of nude mice, treatment with Alsterpaullone (30 mg/kg, administered subcutaneously daily for 2 weeks) significantly reduced tumor growth compared to vehicle control, as monitored by bioluminescence imaging. Treatment with Alsterpaullone also significantly increased survival in mice bearing D458 xenografts. In a separate xenograft model using D425 cells, Alsterpaullone treatment similarly increased survival and reduced tumor growth, as confirmed by histological examination (H&E staining). |
| Enzyme Assay |
GSK-3β activity was assayed using the GS-1 peptide as substrate in buffer A containing 15 µM ATP. The reaction was performed at 30°C for 30 minutes, and phosphorylation was measured by spotting aliquots onto phosphocellulose paper, followed by washing and scintillation counting.
CDK1/cyclin B and CDK5/p25 activities were assayed using histone H1 as substrate in buffer C with 15 µM ATP. Reactions were run for 10 minutes at 30°C, and incorporation of radiolabeled phosphate was measured similarly using phosphocellulose paper. Kinetic studies for ATP competition were performed with varying ATP concentrations (0.5–2.5 µM) and constant GS-1 peptide, followed by linear regression analysis of double-reciprocal plots. [1] |
| Cell Assay |
Cell Line: HeLa cells
Concentration: 5, 10, 15, 20, 25, 30 μM Incubation Time: 48 and 72 hours Result: The growth of HeLa cells was inhibited in a dose-dependent manner for 48 h and 72 h ranging from 0 to 30 μM. For cell proliferation assays, D425 and D458 medulloblastoma cells were grown in suspension and seeded in 96-well plates at 10,000 cells per well. Normal human fetal neural stem cells (hf5281) were grown adherently and seeded at 4,000-5,000 cells per well. Cells were treated with various concentrations of Alsterpaullone or DMSO control for 48 hours. Cell viability was determined using the MTS assay, and absorbance was measured at 490 nm. For Western blot analysis, cells were treated with compounds for 48 hours, lysed, and proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against PARP, AKT, phospho-AKT (Ser473), and α-tubulin. For gene expression analysis, cells were treated with 5 µM Alsterpaullone for 48 hours, RNA was isolated, and profiling was performed using Human PrimeView Arrays. Gene Set Enrichment Analysis was conducted using various pathway databases. |
| Animal Protocol |
5-6 week old athymic nude mice[4]
30 mg/kg Subcutaneous injections, daily for 2 weeks Athymic nude mice (5-6 weeks old) were used. Medulloblastoma cells (250,000 D425 or D458 cells) were implanted into the right cerebellum of each mouse. Six days after inoculation, animals were randomized into treatment groups. Alsterpaullone was dissolved in vehicle (10% DMSO) and administered via subcutaneous injection at a dose of 30 mg/kg daily for 2 weeks. For D458-luciferase xenografts, tumor growth was monitored weekly using bioluminescence imaging. Animals showing progressive neurological signs or weight loss exceeding 20% were euthanized, and brains were harvested for analysis. |
| References |
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| Additional Infomation |
Asterpaullone is an organic heterotetracyclic compound, chemically named 1,3-dihydro-2H-1-benzozazepine-2-one, with its 4-5 bond linked to the 3-2 bond of 5-nitro-1H-indole. It possesses diverse activities, including as an inhibitor of EC 2.7.11.22 (cyclin-dependent kinase), EC 2.7.11.1 (non-specific serine/threonine protein kinase), EC 2.7.11.26 (tau protein kinase), an antitumor drug, an apoptosis inducer, and an anti-HIV-1 drug. It is a C-nitro compound, belonging to the organic heterotetracyclic class of compounds and caprolactams. Functionally, it is associated with a paullone. Alsterpaullone was identified as a preferred candidate drug for three groups of medulloblastomas through C-MAP analysis based on gene expression characteristics. These three groups of medulloblastomas are characterized by MYC amplification and poor prognosis. The mechanism of action of Alsterpaullone involves CDK inhibition (particularly CDK1 and CDK5), leading to cell cycle arrest, downregulation of MYC and other cell cycle genes, induction of apoptosis, and inhibition of the AKT pathway. This study provides preclinical evidence to advance Alsterpaullone as a targeted therapy for three groups of medulloblastomas.
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| Molecular Formula |
C16H11N3O3
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|---|---|
| Molecular Weight |
293.27684
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| Exact Mass |
293.08
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| Elemental Analysis |
C, 65.53; H, 3.78; N, 14.33; O, 16.37
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| CAS # |
237430-03-4
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| Related CAS # |
237430-03-4; 852529-97-0 (Alsterpaullone, 2-Cyanoethyl)
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| PubChem CID |
5005498
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.5±0.1 g/cm3
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| Boiling Point |
651.5±45.0 °C at 760 mmHg
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| Flash Point |
347.8±28.7 °C
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| Vapour Pressure |
0.0±2.0 mmHg at 25°C
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| Index of Refraction |
1.738
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| LogP |
3.55
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
0
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| Heavy Atom Count |
22
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| Complexity |
478
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=C1CC=2C3=C(NC2C4=CC=CC=C4N1)C=CC([N+]([O-])=O)=C3
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| InChi Key |
OLUKILHGKRVDCT-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C16H11N3O3/c20-15-8-12-11-7-9(19(21)22)5-6-14(11)18-16(12)10-3-1-2-4-13(10)17-15/h1-7,18H,8H2,(H,17,20)
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| Chemical Name |
9-nitro-7,12-dihydro-5H-indolo[3,2-d][1]benzazepin-6-one
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| Synonyms |
9-Nitropaullone; NSC-705701; NSC 705701; NSC705701; Alsterpaullone
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| HS Tariff Code |
2934.99.9001
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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)
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| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.4097 mL | 17.0486 mL | 34.0971 mL | |
| 5 mM | 0.6819 mL | 3.4097 mL | 6.8194 mL | |
| 10 mM | 0.3410 mL | 1.7049 mL | 3.4097 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.
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