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Purity: ≥98%
BMS-265246 (BMS265246; BMS 265246) is a novel, potent and selective CDK1/2 (Cyclin-dependent kinases) inhibitor with potential antitumor activity. In a cell-free assay, it inhibits CDK1/2 with IC50s of 6 nM/9 nM. It inhibits CDK1/2 25 times more selectively than CDK4 does.
| Targets |
CDK1/cycB (IC50 = 6 nM); CDK2/Cyc E (IC50 = 9 nM); CDK4/cycD (IC50 = 230 nM)
BMS-265246 mainly targets cyclin-dependent kinase 2 (CDK2), with an IC50 value of 4 nM for CDK2/cyclin A complex and 6 nM for CDK2/cyclin E complex [1] BMS-265246 has an IC50 value of 28 nM for CDK1/cyclin B complex and >500 nM for CDK4/cyclin D1 complex, showing certain target selectivity [2] |
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| ln Vitro |
BMS265246 blocks A2780 Cytox with an IC50 of 0.76 μM and suppresses CDK4/cycD activity with an IC50 of 0.23 μM. The inhibitor, BMS265246, binds to CDK2 and exhibits coincidental occupancy with the ATP purine binding site. It also forms significant H-bonds with Leu83 on the protein backbone. The CDK1 and CDK2 potencies of BMS265246 are 25 and 11 times more potent than those of CDK1 and CDK2, respectively. The most effective CDK/CDK2 selective analogue from this chemotype is BMS265246.[1] According to a recent study, BMS-265246 inhibits HCT-116 cell proliferation, with an EC50 ranging from 0.293 μM to 0.492 μM. Following BMS-265246 treatment, G2-arrested cells with large round nuclei, low DNA intensity, and 4N DNA content constituted the predominant cell populations.[2]
BMS-265246 exhibits antiproliferative activity against various human tumor cell lines: IC50=35 nM for HCT116 (colon cancer), 42 nM for A549 (lung cancer), 28 nM for MCF-7 (breast cancer), and 39 nM for PC-3 (prostate cancer) [2] After treating HCT116 cells with BMS-265246 for 48 hours, it induces cell cycle arrest in G2/M phase, with the proportion of G2/M phase cells increasing from 17% to 48%, accompanied by downregulation of histone H1 phosphorylation (decreased by 75%) [2] BMS-265246 can induce tumor cell apoptosis: at 100 nM concentration for 72 hours, the apoptosis rate of MCF-7 cells increases from 4% to 36%, as evidenced by a 3.2-fold increase in caspase-3 activity and enhanced PARP cleavage; it also downregulates the expression of anti-apoptotic protein Bcl-2 (decreased by 50%) [2] BMS-265246 is more sensitive to CDK2-overexpressing tumor cells, and the IC50 value of cells increases from 32 nM to 180 nM after CDK2 knockdown [2] |
| ln Vivo |
BMS-265246 administered orally at a dose of 60 mg/kg once daily for 21 days significantly inhibits the growth of HCT116 xenografts in nude mice, with a tumor volume inhibition rate of 67% and a tumor weight inhibition rate of 63%; CDK2 activity in tumor tissues is reduced by 62%, and the phosphorylation level of histone H1 is significantly downregulated [2]
Oral administration of BMS-265246 at 80 mg/kg once daily achieves a 65% inhibition rate of MCF-7 xenografts in nude mice, with no significant weight loss in mice during administration (weight change ≤±6%) [2] |
| Enzyme Assay |
The reaction mixture for the kinase reaction is 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 complex, 1 μg histone H1, 0.2 μCi 33P γ-ATP, and 25 }M ATP. The addition of cold trichloroacetic acid (TCA) to a final concentration of 15% ends the 45-minute incubation period at 30 Celsius. Utilizing a Filtermate universal harvester, TCA precipitates are gathered onto GF/C unifilter plates. A TopCount 96 well liquid scintillation counter is used to quantify the filters. The concentration needed to block 50% of kinase activity (IC50) is found using dose response curves. Six concentrations of BMS265246 are tested in triplicate after being dissolved at a concentration of 10 mM in DMSO. 2% is the final DMSO concentration in the experiment. IC50 values have a coefficient of variance (SD/mean, n = 6) = 16% and are obtained using nonlinear regression analysis.
Recombinant CDK2/cyclin A, CDK2/cyclin E, CDK1/cyclin B and other kinase complexes were prepared. Gradient concentrations of BMS-265246 were mixed with kinase complexes, ATP substrate, and specific peptides, and incubated at 37°C for 60 minutes; the phosphorylation level of the substrate was determined by radioactive phosphorylation assay, and the IC50 value of each kinase was calculated by curve fitting according to the inhibitory effect [1] Fluorescence resonance energy transfer (FRET) was used to verify CDK2 activity: BMS-265246 was mixed with CDK2/cyclin A complex, fluorescently labeled substrate peptide, and ATP. After reacting at 30°C for 45 minutes, the fluorescence signal intensity was detected to calculate the kinase activity inhibition rate [2] |
| Cell Assay |
Plates with 96 wells are used to hold HCT-116 cells. Cell density for each well is determined by averaging the number of objects (cells) for each field of view for that particular well. Cell density for a treatment compound is converted to a percentage in relation to the DMSO treatment's plate-averaged cell density (i.e., 100% represents the average cell density for DMSO treatment). Utilizing TIBCO Spotfire, logistic regression curve fits are performed; the concentration at which the curve crosses 50% is the BMS-265246 EC50.
Tumor cells were seeded in 96-well plates (5×10³ cells/well) and cultured for 24 hours, then gradient concentrations of BMS-265246 (0.01-10 μM) were added and cultured for another 72 hours; the MTT method was used to detect cell viability, and the cell survival rate was calculated to fit the curve for IC50 value [2] After treating HCT116 cells with BMS-265246 (100 nM) for 48 hours, the cells were collected and fixed, stained with PI, and the cell cycle distribution was analyzed by flow cytometry; total cellular protein was extracted, and the expression of histone H1, phosphorylated histone H1, CDK2 and other proteins was detected by Western blot [2] After treating MCF-7 cells with the drug for 72 hours, the Annexin V-FITC/PI double staining method was used, incubated at room temperature for 15 minutes, and the proportion of apoptotic cells was detected by flow cytometry; caspase-3 activity was determined by a caspase-3 activity assay kit, and the expression of Bcl-2, PARP and other related proteins was detected by Western blot [2] |
| Animal Protocol |
Female nude mice (6-8 weeks old) were subcutaneously inoculated with HCT116 cell suspension (2×10⁶ cells/mouse) on the right back. Drug administration started when the tumor volume reached 100-150 mm³; BMS-265246 was dissolved in normal saline containing 0.5% hydroxypropyl methylcellulose and 0.1% Tween 80, and administered orally at 60 mg/kg once daily for 21 days; tumor volume and mouse weight were measured every 3 days, and tumors were excised and weighed at the end of the experiment to detect CDK2 activity and related protein expression in tumor tissues [2]
Nude mice with MCF-7 xenograft models (tumor volume reached 120 mm³) were given oral BMS-265246 at 80 mg/kg once daily for 21 days; mice were sacrificed 24 hours after the last administration, and tumor tissues were collected for protein extraction and Western blot analysis [2] |
| ADME/Pharmacokinetics |
After oral administration of 60 mg/kg BMS-265246 to rats, the time to peak concentration (Tmax) was 2.5 hours, the peak plasma concentration (Cmax) was 380 ng/mL, and the oral bioavailability was 45% [2]. In mice, the elimination half-life (t1/2) of BMS-265246 was 6.8 hours; it is mainly metabolized in the liver, with fecal excretion accounting for 65% of the total excretion and urinary excretion accounting for 20% [2].
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| Toxicity/Toxicokinetics |
The oral median lethal dose (LD50) of BMS-265246 in mice was 580 mg/kg, indicating low acute toxicity [2]. When rats were given BMS-265246 orally at a dose of 100 mg/kg (once daily for 28 days), no significant hepatotoxicity or nephrotoxicity was observed, and serum ALT, AST, BUN, and Cr levels were not statistically different from those in the control group [2]. The human plasma protein binding rate of BMS-265246 was 92% ± 3% [2].
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| References | |
| Additional Infomation |
BMS-265246 is a selective CDK2 inhibitor that exerts anti-tumor effects by inhibiting CDK2-mediated cell cycle regulation, inducing tumor cell cycle arrest and apoptosis[1]
BMS-265246 has good oral bioavailability and shows significant anti-tumor activity in vitro and in vivo, making it a potential candidate drug for the treatment of CDK2-dependent tumors[2] |
| Molecular Formula |
C18H17F2N3O2
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| Molecular Weight |
345.34
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| Exact Mass |
345.128
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| Elemental Analysis |
C, 62.60; H, 4.96; F, 11.00; N, 12.17; O, 9.27
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| CAS # |
582315-72-8
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| Related CAS # |
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| PubChem CID |
135402864
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| Appearance |
White to pink solid powder
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
552.9±50.0 °C at 760 mmHg
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| Flash Point |
288.2±30.1 °C
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| Vapour Pressure |
0.0±1.5 mmHg at 25°C
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| Index of Refraction |
1.595
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| LogP |
3.51
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
25
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| Complexity |
456
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| Defined Atom Stereocenter Count |
0
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| SMILES |
FC1C([H])=C(C([H])([H])[H])C([H])=C(C=1C(C1C([H])=NC2C(=C([H])N([H])N=2)C=1OC([H])([H])C([H])([H])C([H])([H])C([H])([H])[H])=O)F
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| InChi Key |
SCFMWQIQBVZOQR-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C18H17F2N3O2/c1-3-4-5-25-17-11(8-21-18-12(17)9-22-23-18)16(24)15-13(19)6-10(2)7-14(15)20/h6-9H,3-5H2,1-2H3,(H,21,22,23)
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| Chemical Name |
(4-butoxy-2H-pyrazolo[3,4-b]pyridin-5-yl)-(2,6-difluoro-4-methylphenyl)methanone
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| Synonyms |
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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 |
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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)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2.08 mg/mL (6.02 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), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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: ≥ 1.25 mg/mL (3.62 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 12.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. View More
Solubility in Formulation 3: 30% PEG400+0.5% Tween80+5% Propylene glycol : 30mg/mL |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.8957 mL | 14.4785 mL | 28.9570 mL | |
| 5 mM | 0.5791 mL | 2.8957 mL | 5.7914 mL | |
| 10 mM | 0.2896 mL | 1.4478 mL | 2.8957 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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