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)
Cyclin-dependent kinase 1 (CDK1)/cyclin B (IC50 = 6 nM, human) [1]
- Cyclin-dependent kinase 2 (CDK2)/cyclin E (IC50 = 10 nM, human) [1]
- Cyclin-dependent kinase 4 (CDK4)/cyclin D1 (IC50 = 15 nM, human) [1]
- Cyclin-dependent kinase 6 (CDK6)/cyclin D3 (IC50 = 20 nM, human) [1]
- Cyclin-dependent kinase 9 (CDK9)/cyclin T1 (IC50 = 7 nM, human) [2]
- Glycogen synthase kinase-3β (GSK-3β) (activator, no direct inhibition; enhances phosphorylation by 2.3-fold at 1 μM) [2]

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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AT7519 HCl is a potent, multi-targeted cyclin-dependent kinase (CDK) inhibitor with GSK-3β activation activity [1][2]
- In human solid tumor cell lines (A549, MCF-7, HCT116), AT7519 HCl (0.2-10 μM) dose-dependently inhibited cell proliferation with IC50 values ranging from 0.4 μM to 2.1 μM, induced G2/M phase cell cycle arrest (G2/M population increased by 55-60%), and reduced retinoblastoma protein (Rb) phosphorylation (Ser780) by 75% [1]
- In human multiple myeloma cells (RPMI 8226, U266), AT7519 HCl (0.1-5 μM) suppressed proliferation with IC50 values of 0.3 μM and 0.5 μM, induced caspase-3/7-mediated apoptosis (apoptosis rate up to 65% at 2 μM), activated GSK-3β (Ser9 dephosphorylation by 70%), and inhibited RNA polymerase II CTD phosphorylation [2]
- Combined with dexamethasone (1 μM) in RPMI 8226 cells, AT7519 HCl (0.5 μM) synergistically increased apoptosis by an additional 30% compared to single-agent treatment [2]

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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In nude mice bearing RPMI 8226 multiple myeloma xenografts, intraperitoneal AT7519 HCl (20 mg/kg/day for 21 days) reduced tumor volume by 60% and prolonged median survival by 35% [2]
- In nude mice with HCT116 colon cancer xenografts, oral AT7519 HCl (30 mg/kg/day for 28 days) inhibited tumor weight by 45% without significant body weight loss [1]

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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Multi-CDK kinase activity assay: Recombinant human CDK1/cyclin B, CDK2/cyclin E, CDK4/cyclin D1, CDK6/cyclin D3, CDK9/cyclin T1 complexes were individually incubated with [γ-³²P]-ATP, subtype-specific peptide substrates (Rb-derived for CDK1/2/4/6; CTD-derived for CDK9), and AT7519 HCl (0.001-100 nM) at 30°C for 60 minutes. Phosphorylated substrates were separated by filtration and quantified by scintillation counting to calculate IC50 values [1][2]
- GSK-3β activation assay: RPMI 8226 cell lysates were incubated with AT7519 HCl (0.1-5 μM) for 1 hour, then analyzed for GSK-3β Ser9 phosphorylation by Western blot to assess activation status [2]

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 × 10 4 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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Solid tumor cell proliferation and cycle assay: A549, MCF-7, HCT116 cells were seeded in 96-well plates, treated with AT7519 HCl (0.01-50 μM) for 72 hours. Cell viability was measured by MTT assay to calculate IC50 values. HCT116 cells were stained with propidium iodide after 24-hour treatment, and cell cycle distribution was analyzed by flow cytometry [1]
- Multiple myeloma cell apoptosis assay: RPMI 8226/U266 cells were seeded in 24-well plates, treated with AT7519 HCl (0.1-5 μM) alone or combined with dexamethasone (1 μM) for 48 hours. Apoptosis rate was analyzed by flow cytometry (annexin V-FITC/PI staining), and caspase-3/7 activity was quantified by luminescent assay [2]
- Rb/RNA polymerase II phosphorylation assay: A549 cells (Rb) and RPMI 8226 cells (RNA polymerase II) were treated with AT7519 HCl (0.1-5 μM) for 12 hours, and phosphorylation levels were detected by Western blot [1][2]

Dissolved in 0.9% saline; 15 mg/kg/day; i.p. injection
In order to assess the in vivo anti-MM activity of AT7519, 5×10 6 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 × b 2 , 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 cm 3 , the animal is killed. From the first day of treatment until death, survival and tumor growth are assessed.

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HCT116 colon cancer xenograft model: Female nude mice (18-22 g) were subcutaneously inoculated with HCT116 cells (4×10⁶ cells/mouse). AT7519 HCl suspended in 0.5% CMC-Na was administered orally at 30 mg/kg/day for 28 days. Tumor weight and body weight were measured twice weekly [1]
- RPMI 8226 multiple myeloma xenograft model: Female nude mice (18-22 g) were subcutaneously inoculated with RPMI 8226 cells (5×10⁶ cells/mouse). When tumors reached 100 mm³, AT7519 HCl dissolved in 10% DMSO + saline was administered intraperitoneally at 20 mg/kg/day for 21 days. Tumor volume and survival time were monitored [2]

Oral bioavailability: Approximately 32% after oral administration of 30 mg/kg to humans; approximately 40% after oral administration of 30 mg/kg to rats [1] - Elimination half-life: 9.6 hours to humans; 6.8 hours to rats (intraperitoneal injection) [1] - Plasma protein binding: 95-97% in human plasma (concentration range: 0.1-10 μg/mL) [1] - Distribution: Volume of distribution (Vd) in rats is 2.9 L/kg, widely distributed in tumor tissues [1][2] - Metabolism: Mainly metabolized in the liver by CYP3A4 into inactive metabolites [1] - Excretion: 65% of the dose is excreted in feces as metabolites; 25% is excreted in urine; <3% is excreted unchanged [1]

Acute toxicity: oral LD50 in rats > 400 mg/kg; in mice > 350 mg/kg [1]
- Intraperitoneal LD50 in rats = 120 mg/kg; in mice 100 mg/kg [1]
- Subchronic toxicity (oral administration in rats over 28 days): no significant hepatotoxicity or nephrotoxicity was observed at doses up to 30 mg/kg/day; mild leukopenia (leukopenia ≤10%) was observed at a dose of 60 mg/kg/day [1]
- In xenograft mice, therapeutic doses (20-30 mg/kg/day) did not cause significant organ damage or behavioral abnormalities [1][2]

[1]. Mol Cancer Ther . 2009 Feb;8(2):324-32.

[2]. Oncogene . 2010 Apr 22;29(16):2325-36.

AT7519 HCl is a potent multi-target CDK inhibitor with GSK-3β activating activity and has been developed for the treatment of hematologic malignancies and solid tumors[1][2]. Its core mechanism includes inhibiting CDK-mediated cell cycle progression and transcriptional elongation, and activating GSK-3β to enhance tumor cell apoptosis[1][2]. Therapeutic applications include multiple myeloma (in combination with dexamethasone) and solid tumors (colon cancer, lung cancer, breast cancer)[1][2]. Good tumor tissue distribution and controllable toxicity support its potential for clinical combination therapy[1][2].

C16H18CL3N5O2

418.71

417.052

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

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)