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) [2][3]
- Cyclin-dependent kinase 2 (CDK2)/cyclin E (IC50 = 10 nM, human) [2][3]
- Cyclin-dependent kinase 4 (CDK4)/cyclin D1 (IC50 = 15 nM, human) [2]
- Cyclin-dependent kinase 6 (CDK6)/cyclin D3 (IC50 = 20 nM, human) [2]
- Cyclin-dependent kinase 7 (CDK7)/cyclin H (IC50 = 8 nM, human) [3]
- Cyclin-dependent kinase 9 (CDK9)/cyclin T1 (IC50 = 7 nM, human) [1][3]
- Glycogen synthase kinase-3β (GSK-3β) (activator, no direct inhibition; enhances phosphorylation by 2.3-fold at 1 μM) [1]

In MM cells, AT7519 (0–4 μM) causes dose-dependent cytotoxicity with IC50s ranging from 0.5 to 2 μM. This cytotoxicity is linked to GSK-3β activation, which is unrelated to transcriptional inhibition. AT7519 overcomes both the protective effect of BMSC and the proliferative advantage conferred by cytokines. MM cells undergo a time-dependent apoptosis in response to AT7519 (0.5 μM). Furthermore, in MM.1S cells, AT7519 (0.5 μM) partially inhibits RNA synthesis and prevents RNA polymerase II CTD from being phosphorylated[1]. Cell cycle progression in human tumor cell lines is inhibited by AT7519 (250 nM). Human tumor cell lines are also made to undergo apoptosis by AT7519[2]. In leukemia cell lines, AT7519 (100-700 nM) causes apoptosis. In human tumor cell lines, AT7519 also inhibits transcription. Additionally, AT7519 decreases the levels of antiapoptotic proteins and inhibits RNA polymerase II[3].

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AT7519 is a potent, multi-targeted cyclin-dependent kinase (CDK) inhibitor with additional GSK-3β activation activity [1][2][3]
- In human multiple myeloma cells (RPMI 8226, U266), AT7519 (0.1-5 μM) dose-dependently inhibited cell 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 [1]
- In human acute myeloid leukemia (AML) cells (HL-60, THP-1) and primary AML patient samples, AT7519 (0.05-2 μM) suppressed proliferation with IC50 values of 0.15 μM and 0.2 μM, blocked transcriptional elongation via CDK9 inhibition, and downregulated short-lived anti-apoptotic proteins (MCL-1, c-Myc) by 80% [3]
- In human solid tumor cell lines (A549, MCF-7, HCT116), AT7519 (0.2-10 μM) inhibited cell growth 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 Rb phosphorylation (Ser780) by 75% [2]
- In CDK-driven cancer cells, it exhibited synergistic antiproliferative effects with dexamethasone (1 μM) in multiple myeloma cells, increasing apoptosis by an additional 30% [1]

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

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In nude mice bearing RPMI 8226 multiple myeloma xenografts, intraperitoneal AT7519 (20 mg/kg/day for 21 days) reduced tumor volume by 60% and prolonged median survival by 35% [1]
- In HL-60 AML xenograft mice, intravenous AT7519 (15 mg/kg/day for 14 days) suppressed tumor growth by 55% and decreased intratumoral MCL-1 expression by 70% [3]
- In nude mice with HCT116 colon cancer xenografts, oral AT7519 (30 mg/kg/day for 28 days) inhibited tumor weight by 45% without significant body weight loss [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.

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Multi-CDK kinase activity assay: Recombinant human CDK1/cyclin B, CDK2/cyclin E, CDK4/cyclin D1, CDK6/cyclin D3, CDK7/cyclin H, 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 CDK7/9), and AT7519 (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 [2][3]
- GSK-3β activation assay: Human multiple myeloma cell lysates were incubated with AT7519 (0.1-5 μM) for 1 hour, then analyzed for GSK-3β Ser9 phosphorylation by Western blot to assess activation status [1]
- Transcriptional elongation assay: HeLa cell nuclear extracts were incubated with AT7519 (0.05-2 μM), ATP, and DNA template. RNA polymerase II CTD phosphorylation (Ser2/5) was detected by Western blot to evaluate CDK9 inhibitory effect [3]

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 × 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, recombinant IL-6 (10 ng/mL), or IGF-1 (50 ng/mL). 3H-TdR incorporation is then measured.

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Multiple myeloma cell apoptosis assay: RPMI 8226 cells were seeded in 24-well plates, treated with AT7519 (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). Caspase-3/7 activity was measured by luminescent assay [1]
- AML cell proliferation assay: HL-60 cells and primary AML patient cells were seeded in 96-well plates, treated with AT7519 (0.05-2 μM) for 72 hours. Cell viability was measured by MTT assay, and IC50 values were calculated [3]
- Solid tumor cell cycle assay: HCT116 cells were treated with AT7519 (0.5 μM) for 24 hours, stained with propidium iodide, and cell cycle distribution was analyzed by flow cytometry. Rb phosphorylation was detected by Western blot [2]
- Anti-apoptotic protein expression assay: THP-1 cells were treated with AT7519 (0.1-2 μM) for 12 hours. MCL-1 and c-Myc protein levels were detected by Western blot and quantified [3]

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. The carrier is given to the control group separately at the same time. 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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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 dissolved in 10% DMSO + saline was administered intraperitoneally at 20 mg/kg/day for 21 days. Tumor volume and survival time were monitored [1]
- HL-60 AML xenograft model: Male nude mice (20-25 g) were subcutaneously inoculated with HL-60 cells (3×10⁶ cells/mouse). Tumor-bearing mice received intravenous AT7519 (15 mg/kg/day) for 14 days. Tumor growth and MCL-1 expression were evaluated [3]
- HCT116 colon cancer xenograft model: Female nude mice (18-22 g) were subcutaneously inoculated with HCT116 cells (4×10⁶ cells/mouse). AT7519 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 [2]

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

Acute toxicity: Oral LD50 in rats > 400 mg/kg; in mice > 350 mg/kg [2] - Intraperitoneal LD50 in rats = 120 mg/kg; in mice 100 mg/kg [2] - 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 (reduction in white blood cell count ≤10%) occurred at 60 mg/kg/day [2] - In xenograft mice, therapeutic doses (15-30 mg/kg/day) did not cause significant behavioral abnormalities or organ damage [1][3] - Drug interactions: Can be inhibited by potent CYP3A4 inhibitors (e.g., ketoconazole), increasing AUC by 1.9-fold; no interaction with dexamethasone [1][2]

[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.

4-(2,6-Dichlorobenzoamide)-N-(piperidin-4-yl)-pyrazole-3-carboxamide belongs to the pyrazole class of compounds. Its structure is 4-amino-1H-pyrazole-3-carboxylic acid, in which the primary amino group is acylated by a 2,6-dichlorobenzoyl group, and the carboxylic acid group undergoes a condensation reaction with the primary amino group of 4-aminopiperidine to form amide. It is an EC 2.7.11.22 (cyclin-dependent kinase) inhibitor and an antitumor drug. It is a secondary formamide, belonging to the pyrazole, dichlorobenzene, and piperidine classes of compounds. AT7519 is a compound that selectively inhibits certain cyclin-dependent kinases (CDKs), leading to tumor regression. This drug was developed by Astex for the treatment of solid tumors and hematologic malignancies. The CDK inhibitor AT7519 is a small molecule drug with high oral bioavailability and potential antitumor activity. AT7519M selectively binds to and inhibits cyclin-dependent kinases (CDKs), thereby leading to cell cycle arrest, inducing apoptosis, and inhibiting tumor cell proliferation. CDKs are serine/threonine kinases involved in cell cycle regulation and may be overexpressed in certain types of cancer cells.

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Drug Indications
It has been investigated for the treatment of leukemia (unspecified), lymphoma (unspecified), myelodysplastic syndromes, and solid tumors.

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Mechanism of Action
AT7519 is a selective inhibitor of certain cyclin-dependent kinases (CDKs) that can lead to tumor regression.

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AT7519 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][3].
- Its core mechanisms include inhibiting CDK1/2/4/6/7/9-mediated cell cycle progression and transcriptional elongation, activating GSK-3β to enhance apoptosis, and downregulating short-lived γ-lactamases. Anti-apoptotic proteins (MCL-1, c-Myc) [1][3]
- Therapeutic applications include multiple myeloma, acute myeloid leukemia, and solid tumors (colon cancer, lung cancer, breast cancer) [1][2][3]
- It has shown synergistic effects with glucocorticoids (dexamethasone) in multiple myeloma, supporting combination therapy strategies [1]
- Good tumor and hematopoietic tissue distribution and manageable toxicity support its potential for clinical development [2][3]

C16H17CL2N5O2

382.24

381.075

C, 50.28; H, 4.48; Cl, 18.55; N, 18.32; O, 8.37

844442-38-2

AT7519 TFA;1431697-85-6;AT7519 Hydrochloride;902135-91-5

11338033

White to off-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

4

4

4

25

479

0

O=C(C1C(Cl)=CC=CC=1Cl)NC1C(C(NC2CCNCC2)=O)=NNC=1

OVPNQJVDAFNBDN-UHFFFAOYSA-N

InChI=1S/C16H17Cl2N5O2/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)

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

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.54 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 (6.54 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 (6.54 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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Solubility in Formulation 4: 30% Propylene glycol , 5% Tween 80 , 65% D5W: 30 mg/mL

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