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Flavopiridol (also known as Alvocidib, NSC 649890 HCl; HMR1275; L868275; HMR 1274; NSC649890), a broad spectrum and ATP-competitive inhibitor of CDKs with potential antineoplastic activity. It inhibits CDKs, such as CDK1, CDK2, CDK4, and CDK6, with an IC50 of less than 40 nM, by competing with ATP. The selectivity of flavopiridol for CDK1, 2, 4, and 6 is 7.5 times higher than that of CDK7. Synthetic N-methylpiperidinyl chlorophenyl flavone compound: alvocidib. By inhibiting cyclin-dependent kinase and downregulating the expression of cyclin D1 and D3, alvocidib, an inhibitor of cyclin-dependent kinase, causes cell cycle arrest in the G1 stage and apoptosis.
Physicochemical Properties
| Molecular Formula | C21H20CLNO5 | |
| Molecular Weight | 401.84 | |
| Exact Mass | 401.102 | |
| Elemental Analysis | C, 62.77; H, 5.02; Cl, 8.82; N, 3.49; O, 19.91 | |
| CAS # | 146426-40-6 | |
| Related CAS # | 131740-09-5 (HCl);146426-40-6; | |
| PubChem CID | 5287969 | |
| Appearance | Light yellow to yellow solid powder | |
| Density | 1.4±0.1 g/cm3 | |
| Boiling Point | 603.6±55.0 °C at 760 mmHg | |
| Flash Point | 318.8±31.5 °C | |
| Vapour Pressure | 0.0±1.8 mmHg at 25°C | |
| Index of Refraction | 1.673 | |
| LogP | 1.92 | |
| Hydrogen Bond Donor Count | 3 | |
| Hydrogen Bond Acceptor Count | 6 | |
| Rotatable Bond Count | 2 | |
| Heavy Atom Count | 28 | |
| Complexity | 628 | |
| Defined Atom Stereocenter Count | 2 | |
| SMILES | O=C1C2=C(C=C(C([C@]3([H])[C@H](O)CN(C)CC3)=C2OC(C4=CC=CC=C4Cl)=C1)O)O |
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| InChi Key | BIIVYFLTOXDAOV-YVEFUNNKSA-N | |
| InChi Code | InChI=1S/C21H20ClNO5/c1-23-7-6-12(17(27)10-23)19-14(24)8-15(25)20-16(26)9-18(28-21(19)20)11-4-2-3-5-13(11)22/h2-5,8-9,12,17,24-25,27H,6-7,10H2,1H3/t12-,17+/m0/s1 | |
| Chemical Name | 2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methylpiperidin-4-yl]chromen-4-one | |
| Synonyms |
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| 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 Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. |
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| 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 | CDK1/Cyc B1 (IC50 = 0.31 nM); CDK4/Cyc D1 (IC50 = 0.83 nM); HDAC6 (IC50 = 71.8 nM); HDAC8 (IC50 = 877 nM) | |
| ln Vitro |
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| ln Vivo |
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| Enzyme Assay | In the CDK1/cyclin B1 kinase assay, kinase reactions comprise 50 μL of kinase buffer (50 mM Tris, pH 8.0, 10 mM MgCl2, 1 mM EGTA, 0.5 mM DTT), 100 ng of baculovirus-expressed GST-CDK1/cyclin B1 (human) complex, 1 μg histone HI, 0.2 μCi [γ-33P]ATP, and 25 μM ATP. The CDK2/cyclin E kinase assay uses the following components: 50 μL of kinase buffer (50 mM Hepes, pH 8.0, 10 mM MgCl2, 1 mM EGTA, and 2 mM DTT), 5 ng of baculovirus-expressed GST-CDK2/cyclin E (human) complex, 0.5 μg GST-RB fusion protein (amino acids 776-928 of retinoblastoma protein), 0.2 μCi [γ-33P]ATP, and 25 μM ATP. The steps involved in the CDK4/cyclin D1 kinase assay are as follows: 50 μL of kinase buffer (50 mM Hepes, pH 8.0, 10 mM MgCl2, 1 mM EGTA, 2 mM DTT), 150 ng of baculovirus-expressed GST-CDK4/cyclin D1 (human), 280 ng of Stag-cyclin D1, 0.5 μg GST-RB fusion protein (amino acids 776-928 of retinoblastoma protein), 0.2 μCi [γ-33P]ATP, 25 μM ATP. The reactions are stopped by adding cold trichloroacetic acid (TCA) to a final concentration of 15% after being incubated for 45 minutes for CDK1 and CDK2 and 1 hour for CDK4 at 30 °C. A Filtermate universal harvester is used to gather TCA precipitates onto GF/C unifilter plates, and a TopCount 96-well liquid scintillation counter is used to quantify the filters. Dimethylformamide (DMF) is used to dissolve flavonidol at a concentration of 10 mM. Six concentrations of flavonidol are tested in triplicate. In the assay, the final DMF concentration was 2%. IC50 values have a coefficient of variance of 16% and are obtained through nonlinear regression analysis. A filter-binding assay is developed to measure flavopiridol activity on CDK6. The reaction mixture combines the following ingredients: 2 microliters (0.7 mg/μL) of CDK6, 5 microliters (six milligrams/mL) of histone H1, 14 microliters of kinase buffer (60 mM β-glycerophosphate, 30 mM p-nitrophenyl phosphate, 25 mM MOPS (pH 7.0), 5 microliters of EGTA, 15 milliliters of MgCl2, 1 millisecond of DTT, 0.1 milliliters of Na-vanadate), 3 microliters of escalating concentrations of Flavopiridol diluted in 50% DMSO, and 6 microliters of 33P-ATP (1 mCi/mL) in nonradioactive ATP at 90 μM concentration (final concentration: 15 μM). 33P-ATP is added to start the experiment. At 30°C, the reaction is incubated for twenty minutes. The supernatant is then spotted onto Whatman P81 phosphocellulose paper using a 25 μL aliquot. Five times, filters are cleaned in a 1% phosphoric acid solution. In the presence of one milliliter of scintillation fluid, wet filters are tallied. A mixture of 50 nM recombinant Cdk9/cyclin T in 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT, 3 μM Na3VO4, 150 μM RNA polymerase CDT peptide, and 80 μM ATP is used to measure Cdk9 activity. The Cdk7 assay is conducted in the same buffer with 10 μM myelin binding protein as a substrate and 37 nM purified kinase in the presence of 200 μM ATP. Either a scintillation proximity assay or a strong anion exchanger (Dowex 1-X8 resin, formate form)-based assay is used to assess the potency of flavopiridol toward CDK9 and CDK7. The dose-response curves are used to calculate the IC50 values. | |
| Cell Assay | Following a 72-hour exposure to different flavoperidol concentrations, cells are treated with a combination of phenazine methosulfate and MTS, a tetrazolium dye. The number of viable cells is directly proportional to the absorbency, which is measured at 492 nm after three hours. IC50 values are used to express the results. Cells are fixed in paraformaldehyde and ethanol for cell cycle analysis. They are then rinsed, resuspended in a staining solution containing TdT enzyme and FITC-dUTP, and stained with PI after being treated with RNase. Flow cytometry is used to analyze the cells. | |
| Animal Protocol |
Female Balb/c×DBA/2J F1 mice inoculated ip with P388 ascites leukemic cells, and Balb/c nu/nu nude mice subcutaneous implanted with A2780, Br-cycE, or A431 cells ~7.5 mg/kg/day Injection i.p. |
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| ADME/Pharmacokinetics |
Metabolism / Metabolites Flavopiridol has known human metabolites that include (2S,3S,4S,5R)-6-[2-(2-chlorophenyl)-5-hydroxy-8-[(3S,4R)-3-hydroxy-1-methylpiperidin-4-yl]-4-oxochromen-7-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid. |
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| References |
[1]. J Med Chem . 2000 Nov 2;43(22):4126-34. [2]. J Med Chem . 2005 Feb 10;48(3):737-43. [3].Nat Chem Biol . 2008 Jun;4(6):357-65. [4]. Cancer Res . 1996 Jul 1;56(13):2973-8. [5]. J Med Chem . 2002 Aug 29;45(18):3905-27. [6]. J Immunol . 2008 Feb 1;180(3):1954-61. [7]. TClin Cancer Res . 2001 Dec;7(12):4209-19. [8]. Nat Chem Biol . 2008 Jun;4(6):357-65. [9]. Crit Rev Oncol Hematol . 2001 May;38(2):139-70. |
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| Additional Infomation |
Alvocidib is a synthetic dihydroxyflavone that is 5,7-dihydroxyflavone which is substituted by a 3-hydroxy-1-methylpiperidin-4-yl group at position 8 and by a chlorine at the 2' position (the (-)-3S,4R stereoisomer). A cyclin-dependent kinase 9 (CDK9) inhibitor, it has been studied for the treatment of acute myeloid leukaemia, arthritis and atherosclerotic plaque formation. It has a role as an antineoplastic agent, an EC 2.7.11.22 (cyclin-dependent kinase) inhibitor, an antirheumatic drug and an apoptosis inducer. It is a dihydroxyflavone, a hydroxypiperidine, a member of monochlorobenzenes and a tertiary amino compound. It is a conjugate base of an alvocidib(1+). Alvocidib is a synthetic flavonoid based on an extract from an Indian plant for the potential treatment of cancer. It works by inhibiting cyclin-dependent kinases, arresting cell division and causing apoptosis in non-small lung cancer cells. Alvocidib is the free base form of a synthetic N-methylpiperidinyl chlorophenyl flavone compound. As an inhibitor of cyclin-dependent kinase, alvocidib induces cell cycle arrest by preventing phosphorylation of cyclin-dependent kinases (CDKs) and by down-regulating cyclin D1 and D3 expression, resulting in G1 cell cycle arrest and apoptosis. This agent is also a competitive inhibitor of adenosine triphosphate activity. Drug Indication Investigated for use/treatment in esophageal cancer, leukemia (lymphoid), lung cancer, liver cancer, and lymphoma (unspecified). Mechanism of Action Inhibits cyclin-dependent kinases, arresting cell division and causing apoptosis in non-small lung cancer cells. |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.22 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (6.22 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Solubility in Formulation 3: 5% DMSO+30% PEG 300+ddH2O: 2.5mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.4886 mL | 12.4428 mL | 24.8855 mL | |
| 5 mM | 0.4977 mL | 2.4886 mL | 4.9771 mL | |
| 10 mM | 0.2489 mL | 1.2443 mL | 2.4886 mL |