CDK1/Cyc B1 (IC50 = 0.31 nM); CDK4/Cyc D1 (IC50 = 0.83 nM); HDAC6 (IC50 = 71.8 nM); HDAC8 (IC50 = 877 nM)

For seven days, 7.5 mg/kg of flavopiridol was administered to mice with P388 murine leukemia, which showed a slight antitumor activity with a %T/C value of 110. It also produced 1.5 log cell kill (LCK) when human A2780 ovarian carcinoma was implanted sc in nude mice.[5] Treatment with flavonopiridol at 1-2.5 mg/kg for 10 days drastically reduces mice's collagen-induced arthritis in a dose-dependent manner by preventing joint degradation and synovial hyperplasia, while serum levels of anti-collagen type II (CII) Abs and the proliferative reactions to CII are left unchanged.[6] Administration of CPT-11 (100 mg/kg) and Flavopiridol (3 mg/kg) 7 and 16 hours later significantly inhibits tumor regression by 86% and 82%, respectively, in the p21-intact Hct116 xenografts in nude mice. This shows a >2 fold inhibition compared with CPT-11 alone by 40%. Compared to CPT-11 alone, which yields no complete response rate (CR), the combination results in ~30% CR.[7]

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.

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.

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.

[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.

C21H20CLNO5

401.84

401.103

C, 62.77; H, 5.02; Cl, 8.82; N, 3.49; O, 19.91

146426-40-6

131740-09-5 (HCl);146426-40-6;

Solid powder

CN1CC[C@@H]([C@@H](C1)O)C2=C(C=C(C3=C2OC(=CC3=O)C4=CC=CC=C4Cl)O)O

BIIVYFLTOXDAOV-YVEFUNNKSA-N

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

2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methylpiperidin-4-yl]chromen-4-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

5% DMSO+30% PEG 300+ddH2O: 2.5mg/mL (Please use freshly prepared in vivo formulations for optimal results.)