In a mouse model of human MM xenograft, AT7519 TFA inhibits the growth of tumors[1]. Early-stage HCT116 tumor xenografts are inhibited in their growth by AT7519 (4.6 and 9.1 mg/kg/dose). Target CDKs are also inhibited by AT7519 (10 mg/kg, i.p.) TFA in BALB/c nude mice harboring HCT116 tumors[2].

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.

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).

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.

[1]. AT7519, A novel small molecule multi-cyclin-dependent kinase inhibitor, induces apoptosis in multiple myeloma via GSK-3beta activation and RNA polymerase II inhibition. Oncogene. 2010 Apr 22;29(16):2325-36.

[2]. Biological characterization of AT7519, a small-molecule inhibitor of cyclin-dependent kinases, in human tumor cell lines. Biological characterization of AT7519, a small-molecule inhibitor of cyclin-dependent kinases, in human tumor cell

[3]. AT7519, a cyclin-dependent kinase inhibitor, exerts its effects by transcriptional inhibition in leukemia cell lines and patient samples. Mol Cancer Ther. 2010 Apr;9(4):920-8.

C18H18CL2F3N5O4

496.2678

495.07

C, 43.56; H, 3.66; Cl, 14.29; F, 11.48; N, 14.11; O, 12.90

1431697-85-6

AT7519;844442-38-2;AT7519 Hydrochloride;902135-91-5

Solid powder

C1CNCCC1NC(=O)C2=C(C=NN2)NC(=O)C3=C(C=CC=C3Cl)Cl.C(=O)(C(F)(F)F)O

XTOQTRBZWZONHQ-UHFFFAOYSA-N

InChI=1S/C16H17Cl2N5O2.C2HF3O2/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;3-2(4,5)1(6)7/h1-3,8-9,19H,4-7H2,(H,20,23)(H,21,25)(H,22,24);(H,6,7)

4-[(2,6-dichlorobenzoyl)amino]-N-piperidin-4-yl-1H-pyrazole-5-carboxamide;2,2,2-trifluoroacetic acid

AT7519 trifluoroacetate; AT-7519 trifluoroacetate; AT 7519 trifluoroacetate

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)

DMSO : ~100 mg/mL (~201.50 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.04 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.04 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.04 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)