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AT7519 HCl, the hydrochloride salt of AT7519 (AT-7519 ), is a potent, orally bioavailable small molecule CDK inhibitor with potential anticancer activity. It is less effective against CDK3 and CDK7 and inhibits several CDKs, including CDK1, 2, 4, 6, and 9, with an IC50 of 10-210 nM. Serine/theronine kinases called CDKs are involved in controlling the cell cycle and are overexpressed in certain cancer cell types.
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Physicochemical Properties
| Molecular Formula |
C16H18CL3N5O2
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| Molecular Weight |
418.71
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| Exact Mass |
417.052
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| Elemental Analysis |
C, 45.90; H, 4.33; Cl, 25.40; N, 16.73; O, 7.64
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| CAS # |
902135-91-5
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| Related CAS # |
AT7519;844442-38-2;AT7519 TFA;1431697-85-6
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| PubChem CID |
25033099
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| Appearance |
white solid powder
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| Density |
1.5±0.1 g/cm3
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| Boiling Point |
586.0±50.0 °C at 760 mmHg
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| Flash Point |
308.2±30.1 °C
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| Vapour Pressure |
0.0±1.6 mmHg at 25°C
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| Index of Refraction |
1.654
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| LogP |
0.95
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| Hydrogen Bond Donor Count |
5
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
26
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| Complexity |
479
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| Defined Atom Stereocenter Count |
0
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| SMILES |
0
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| InChi Key |
PAOFPNGYBWGKCO-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C16H17Cl2N5O2.ClH/c17-10-2-1-3-11(18)13(10)15(24)22-12-8-20-23-14(12)16(25)21-9-4-6-19-7-5-9;/h1-3,8-9,19H,4-7H2,(H,20,23)(H,21,25)(H,22,24);1H
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| Chemical Name |
4-[(2,6-dichlorobenzoyl)amino]-N-piperidin-4-yl-1H-pyrazole-5-carboxamide;hydrochloride
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| Synonyms |
| AT-7519 HCl; AT7519; AT 7519 |
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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 Note: Please store this product in a sealed and protected environment, 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)
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Biological Activity
| Targets |
CDK9/Cyclin T (IC50 = 10 nM); CDK5/p35 (IC50 = 13 nM); cdk2/cyclin A (IC50 = 47 nM); Cdk4/cyclin D1 (IC50 = 100 nM); cdk6/cyclin D3 (IC50 = 170 nM); Cdk1/cyclin B (IC50 = 210 nM); CDK7/Cyclin H/MAT1 (IC50 = 2400 nM); GSK3β (IC50 = 89 nM)
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| ln Vitro |
AT7519 is an ATP-competitive CDK inhibitor, and its CDK1 Ki value is 38 nM. Except for GSK3β (IC50 = 89 nM), AT7519 is inactive against all other non-CDK kinases. In several human tumor cell lines, AT7519 exhibits strong antiproliferative activity. Its IC50 values, which range from 40 nM for MCF-7 to 940 nM for SW620, are consistent with the inhibition of CDK1 and CDK2.[1] In multiple myeloma (MM) cell lines, AT7519 induces dose-dependent cytotoxicity with IC50 values ranging from 0.5 to 2 μM at 48 hours. The most resistant cell lines are MM.1R (>2 μM), while the most sensitive are MM.1S (0.5 μM) and U266 (0.5 μM). It doesn't cause peripheral blood mononuclear cells (PBMNC) to become cytotoxic. The proliferative advantage of IL-6 and IGF-1, as well as the protective effect of bone marrow stromal cells (BMSCs), are partially overcome by AT7519. A portion of the MM cell cytotoxicity induced by AT7519 is due to the fast dephosphorylation of RNA pol II CTD at serine 2 and serine 5 sites, which results in transcription inhibition. Through downregulating GSK-3β phosphorylation, AT7519 activates GSK-3β and causes apoptosis that is not dependent on transcription inhibition.[2] |
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| ln Vivo |
| In the HCT116 and HT29 colon cancer xenograft models, tumor regression is observed for both early-stage and advanced-stage s.c. tumors when AT7519 (9.1 mg/kg) is administered twice daily. (Source: ) In the human MM xenograft mouse model, treatment with AT7519 (15 mg/kg) inhibits tumor growth and increases the median overall survival of the mice in correlation with increased caspase 3 activation.[2] |
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| Enzyme Assay |
Kinase assays using radiometric filter binding are conducted for CDK1, CDK2, and GSK3-β. The format of the assays is ELISA for CDKs 4 and 6, and DELFIA for CDK 5. The relevant CDK and 0.12 μg/mL Histone H1 are incubated for 2 or 4 hours, respectively, in 20 mM MOPS, pH 7.2, 25 mM β-glycerophosphate, 5 mM EDTA, 15 mM MgCl2, 1 mM sodium orthovanadate, 1 mM DTT, 0.1 mg/mL BSA, 45 μM ATP (0.78 Ci/mmol), and various concentrations of AT7519. In order to test GSK3-β, the appropriate enzyme and 5 μM glycogen synthase peptide 2 are added, and the mixture is incubated for three hours at 10 mM MOPS pH 7.0, 0.1 mg/mL BSA, 0.001% Brij-35, 0.5% glycerol, 0.2 mM EDTA, 10 mM MgCl2, 0.01% β-mercaptoethanol, 15 μM ATP (2.31 Ci/mmol), all of which are tested. Millipore MAPH filter plates are used to filter the assay reactions after an excess of orthophosphoric acid is added to stop the reaction. After that, the plates are cleaned, scintillant is added, and radioactivity is determined using a Packard TopCount scintillation counting device. For a duration of 30 minutes, CDK5, CDK5/p35, 1μM of a biotinylated Histone H1 peptide (Biotin-PKTPKKAKKL), pH 7.5, 25 mM Tris-HCl, 0.025% Brij-35, 0.1 mg/mL BSA, 1 mM DTT, 15 μM ATP, and various concentrations of AT7519 are incubated. Time-resolved fluorescence at λex=335nm, λem=620nm is used to stop the assay reactions using EDTA, transfer the mixture to Neutravidin-coated plates, and quantify the phosphorylated peptide using a rabbit phospho-cdk1 substrate polyclonal antibody and DELFIA europium-labelled anti-rabbit IgG secondary antibody. Plates are coated with GST-pRb769-921 and blocked with Superblock for the CDK 4 and 6 assays. In order to initiate the reaction, ATP is added to CDK4 or 6. The incubation conditions include 15 mM MgCl2, 50 mM HEPES, pH 7.4, 1 mM DTT, 1 mM EGTA, pH 8.0, 0.02% Triton X-100, 2.5% DMSO, and various concentrations of AT7519. Reactions are halted by adding 0.5 M EDTA pH 8.0 after 30 minutes. After that, plates are cleaned and incubated for one hour with a secondary antibody (alkaline phosphatase linked anti-rabbit) and another hour with the primary antibody (anti-p-Rb Serine 780) diluted in Superblock. Fluorescence is measured on a Spectramax Gemini plate reader at excitation of 450 nm and emission of 580 nm after plates are developed using the Attophos system. Using GraphPad Prism software, IC50 values are computed from replicate curves in every scenario.
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| Cell Assay |
The assessable effects of AT7519 on the viability of primary MM cells, MM cell lines, and PBMNCs are determined by measuring the dye absorbance of 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrasodium bromide (MTT). The assay for measuring DNA synthesis uses tritiated thymidine uptake (3H-TdR). 3H-TdR incorporation is measured after MM cells (2–3 × 104 cells/well) are cultured for 24 or 48 hours at 37°C in 96-well culture plates with media and varying concentrations of AT7519 and/or recombinant IL-6 (10 ng/mL) or IGF-1 (50 ng/mL).
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| Animal Protocol |
| Dissolved in 0.9% saline; 15 mg/kg/day; i.p. injection | | In order to assess the in vivo anti-MM activity of AT7519, 5×106 MM.1S cells are subcutaneously injected into male SCID mice using 100 μL of serum-free RPMI 1640 medium. Mice are treated intraperitoneally (IP) with vehicle or AT7519 dissolved in 0.9% saline solution when tumors are detectable. Ten mice in the first group receive a daily dose of 15 mg/kg for two weeks, while the second group receives a daily dose of 15 mg/kg three times a week for four weeks in a row. At the same time, the carrier is given to the control group alone. Tumor volume is calculated using the formula V= 0.5 a × b2, where a represents the tumor's long diameter and b its short diameter. Tumor size is measured every other day in two dimensions using calipers. When a tumor is ulcerated or grows to a size of 2 cm3, the animal is killed. From the first day of treatment until death, survival and tumor growth are assessed. | |
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| References |
[1]. Mol Cancer Ther
. 2009 Feb;8(2):324-32.
[2]. Oncogene
. 2010 Apr 22;29(16):2325-36.
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Solubility Data
| Solubility (In Vitro) |
| DMSO: ~52 mg/mL (~124.2 mM) | | Water: ~43 mg/mL (~102.7 mM) | | Ethanol: ~28 mg/mL(~66.9 mM) |
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| Solubility (In Vivo) |
(Please use freshly prepared in vivo formulations for optimal results.)
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| Preparing Stock Solutions |
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1 mg |
5 mg |
10 mg |
| 1 mM |
2.3883 mL |
11.9414 mL |
23.8829 mL |
| 5 mM |
0.4777 mL |
2.3883 mL |
4.7766 mL |
| 10 mM |
0.2388 mL |
1.1941 mL |
2.3883 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. |
