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)
AT-7519 targets multiple cyclin-dependent kinases (CDKs), specifically CDK1, CDK2, CDK4, CDK5, CDK6, and CDK9. The IC50 values for these targets are 210 nM for CDK1/cyclin B, 47 nM for CDK2/cyclin A, 100 nM for CDK4/cyclin D1, 13 nM for CDK5/p35, 170 nM for CDK6/cyclin D3, and <10 nM for CDK9/cyclin T. It is an ATP-competitive inhibitor and has also been found to be a human RORγt agonist. AT-7519 shows minimal activity against non-CDK kinases, with the exception of GSK3β.

In MM cells, AT7519 Hydrochloride (0–4 μM) causes dose-dependent cytotoxicity with an IC50 range from 0.5–2 μM. This cytotoxicity is linked to GSK-3β activation and is not reliant on inflection point blockage. Time-induced induction of MM cells is achieved by AT7519 Hydrochloride (0.5 μM). Additionally, in MM.1S cells, AT7519 Hydrochloride (0.5 μM) partially inhibits RNA production and prevents RNA polymerase II CTD from being phosphorylated [1]. Human tumor cell lines' cell cycle progression is inhibited by AT7519 hydrochloric acid (250 nM). Human tumor cell lines likewise develop liver cancer when exposed to AT7519 hydrochloric acid [2]. Cellular effects are induced in blank cell lines by AT7519 hydrochloric acid (100-700 nM). In human tumor cell lines, AT7519 hydrochloride also suppresses transcription. Furthermore, RNA polymerase II is inhibited and antioxidant protein levels are decreased by AT7519 Hydrochronide [3].
In vitro, AT-7519 demonstrates potent antiproliferative activity across a panel of human tumor cell lines, with IC50 values ranging from 40 to 940 nM. It induces cell cycle arrest, primarily by increasing the population of cells in the G0/G1 and G2/M phases, followed by apoptosis. In MM.1S cells, this effect is time- and dose-dependent, as evidenced by an increase in the sub-G1 population, Annexin V/PI staining, and cleavage of caspases -9, -3, and -8. The mechanism of action is consistent with the inhibition of CDK1 and CDK2 in solid tumor cell lines.

In a human MM xenograft model, AT7519 hydrochloride suppresses the growth of tumors [1]. Early-stage HCT116 tumor xenografts are inhibited in their growth by AT7519 hydrochloride (4.6 and 9.1 mg/kg/dose). Target CDKs are likewise inhibited by AT7519 Hydrochloride (10 mg/kg, ip) in BALB/c nude mice with HCT116 tumors[2].
In vivo, AT-7519 inhibits tumor growth in human tumor xenograft models. Tumor regression has been observed following twice-daily dosing of AT-7519 in HCT116 and HT29 colon cancer xenograft models. The compound causes cell cycle arrest followed by apoptosis in human tumor cells, leading to inhibition of tumor growth. These in vivo findings demonstrate the compound's potential as an anticancer agent, supporting its advancement into clinical trials for the treatment of various malignancies.

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 in vitro enzyme activity assay for AT-7519 typically measures its ability to inhibit the kinase activity of recombinant CDK/cyclin complexes. These cell-free assays use purified enzymes and a peptide substrate in the presence of ATP. The compound's inhibitory potency (IC50) is determined by measuring the reduction in kinase activity, often using radioactive or fluorescence-based detection methods. These assays allow for the direct assessment of the compound's potency against specific CDK targets without the confounding effects of cellular uptake or metabolism.

The 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrasodium bromide (MTT) dye absorbance is used to measure the effects of AT7519 on the viability of MM cell lines, primary MM cells, and PBMNCs. Triiodothymidine uptake (3H-TdR) is used to quantify DNA synthesis. 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, recombinant IL-6 (10 ng/mL), or IGF-1 (50 ng/mL). 3H-TdR incorporation is then measured.
In vitro cellular assays for AT-7519 assess its antiproliferative effects and its ability to induce cell cycle arrest and apoptosis. Human tumor cell lines, such as SW620, HCT116, and HT29, are treated with various concentrations of the compound. Cell viability is typically measured using assays such as MTT or CellTiter-Glo after 72-96 hours of treatment. Cell cycle analysis is performed using flow cytometry with propidium iodide staining to determine the distribution of cells in different phases of the cell cycle. Apoptosis is assessed using Annexin V/PI staining and caspase activity assays.

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. The carrier is given to the control group separately at the same time. 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.
In vivo animal studies for AT-7519 utilize human tumor xenograft models in immunodeficient mice. Tumor cells, such as HCT116 or HT29 colon cancer cells, are implanted subcutaneously. After tumor establishment, AT-7519 is administered orally, typically twice daily. Tumor volume is measured regularly using calipers, and tumor growth inhibition is calculated by comparing tumor volumes in treated versus control groups. Endpoints include tumor regression, tumor growth delay, and assessment of apoptosis in tumor tissues by immunohistochemistry.

AT-7519 diHCl is orally bioavailable, supporting its administration as an oral therapeutic agent. As a hydrochloride salt, it exhibits enhanced aqueous solubility. However, detailed pharmacokinetic parameters such as half-life, Cmax, and AUC are not extensively detailed in the available literature. The compound's favorable oral bioavailability has enabled its evaluation in clinical trials as an orally administered anticancer agent. Its pharmacokinetic profile supports twice-daily dosing regimens in preclinical models.

Specific toxicity data for AT-7519 diHCl are not extensively detailed in the available literature. As a CDK inhibitor that affects cell cycle progression, its toxicity profile is likely related to its mechanism of action, potentially affecting rapidly dividing normal cells such as those in the bone marrow and gastrointestinal tract. Preclinical toxicology studies would typically be conducted to assess the compound's safety margin. The compound has been investigated in Phase 1/2 clinical studies, where its safety and tolerability would have been evaluated.

[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 lines.

[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.

AT-7519 is a multi-CDK inhibitor that has been investigated in Phase 1/2 clinical studies for the treatment of various cancers. Its mechanism of action involves ATP-competitive inhibition of multiple CDKs, leading to cell cycle arrest and apoptosis. The compound has demonstrated potent antiproliferative activity in a range of human tumor cell lines and efficacy in xenograft models. It is not approved for clinical use and remains an investigational agent. It is also noted to be a human RORγt agonist, suggesting potential additional pharmacological activities.

C16H18CL3N5O2

418.71

417.052608

C, 45.90; H, 4.33; Cl, 25.40; N, 16.73; O, 7.64

902135-91-5

AT7519;844442-38-2;AT7519 TFA;1431697-85-6

25033099

white solid powder

1.5±0.1 g/cm3

586.0±50.0 °C at 760 mmHg

308.2±30.1 °C

0.0±1.6 mmHg at 25°C

1.654

0.95

5

4

4

26

479

0

0

PAOFPNGYBWGKCO-UHFFFAOYSA-N

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

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

AT-7519 diHCl; AT7519; AT 7519; AT-7519

2934.99.9001

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.

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

DMSO: ~52 mg/mL (~124.2 mM)
Ethanol: ~28 mg/mL (~66.9 mM)
Water: ~43 mg/mL (~102.7 mM)

Saline: 30mg/mL (Please use freshly prepared in vivo formulations for optimal results.)