AG-012986 dihydrochloride

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AG-012986 (dihydrochloride) is a pan-CDK inhibitor with K_i values of 44 nM (CDK1), 9.2 nM (CDK4), 94 nM (CDK2), and IC₅₀ values of 22 nM (CDK5), 4 nM (CDK9). AG-012986 (dihydrochloride) causes apoptosis of T-cells by targeting upstream kinases in the p38 Mitogen-activated protein kinase (MAPK) pathway and impairing cellular survival. AG-012986 (dihydrochloride) induces cell cycle arrest, retinoblastoma protein hypophosphorylation, and reduces K_i-67 expression. AG-012986 (dihydrochloride) exerts antiproliferative activity in tumor cells, demonstrates antitumor efficacy in human xenograft models, and causes retinal and peripheral neurotoxicity, plus immune cell toxicity. AG-012986 (dihydrochloride) can be used for the research of colon carcinoma, non-small cell lung carcinoma, lung carcinoma, breast carcinoma, ovarian tumor, pancreatic carcinoma, osteosarcoma, lymphoma, leukemia, retinotoxicity.

For research use only. We do not sell to patients.

AG-012986 dihydrochloride Chemical Structure

CAS No. : 486414-32-8

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•Related Proteins:

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CDK1 CDK2 CDK3 CDK4 CDK5 CDK6 CDK7 CDK8 CDK9 CDK11 CDK12 CDK13 CDK14 CDK16 CDK19 CDC CLK Pho85 CDK10 CDK15 CDK17 CDK18 CDK20 PITSLRE

Biological Activity
Purity & Documentation
References
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Description

IC₅₀ & Target^[1]

CDK1

44 nM (Ki)

CDK2

94 nM (Ki)

CDK3/Cyclin E

46 nM (Ki)

Cdk4/cyclin D3

9.2 nM (Ki)

CDK5/p35

22 nM (IC₅₀)

CDK7/cyclin H

0 nM (Ki)

CDK9/CycT1

4 nM (IC₅₀)

In Vitro

AG-012986 (dihydrochloride) is a potent, selective pan-CDK inhibitor with nanomolar activity against CDK4/cyclin D3 (K_i = 9.2 nmol/L), CDK1/cyclin B (K_i = 44 nmol/L), CDK2/cyclin A (K_i = 94 nmol/L), CDK9/cyclin T (IC₅₀ = 4 nmol/L), and CDK5/p35 (IC₅₀ = 22 nmol/L)^[1].
AG-012986 (dihydrochloride) (0.837-2.44 μmol/L) has limited off-target activity against non-kinase targets, with functional interactions at micromolar concentrations with the calcium type L ion channel, serotonin transporter, and histamine H3 receptor^[1].
AG-012986 (dihydrochloride) (72 h) potently inhibits proliferation of 18 human tumor cell lines with an average IC₅₀ of 120 nmol/L, and displays IC₅₀ values <100 nmol/L in 13 of these cell lines, independent of p53 and Rb status^[1].
AG-012986 (dihydrochloride) (60-240 nM; 8-24 h) induces dose-dependent hypophosphorylation of Rb Ser795 in HCT116 human colon cancer cells after 24 hours of treatment, with maximal effects at >120 nM, but shows minimal effect after 8 hours^[1].
AG-012986 (dihydrochloride) (30 nM-1 μM; 8-24 h) induces G1 phase arrest in HCT116 human colon cancer cells at 30 to 120 nM and G2-M phase arrest at ≥240 nM after 24 hours of treatment, but does not induce cell cycle arrest with <8 hours of exposure^[1].
AG-012986 (dihydrochloride) (30-240 nM; 8-24 h) induces apoptosis in HCT116 human colon cancer cells with an IC₅₀ of ≈160 nM after 24 hours of treatment, but shows no apoptotic effect after 8 hours^[1].
AG-012986 (dihydrochloride) (10-1000 nM; 8-320 h) shows time-dependent cytotoxicity in SW620 human colon carcinoma cells, with minimal activity after 8 hours, moderate activity (IC₅₀ = 300 nmol/L) after 24 hours, and substantial cytotoxicity (IC₅₀ <100 nmol/L) after ≥72 hours^[1].
AG-012986 (dihydrochloride) exhibits binding inhibition of multiple CDK isoforms in a cell-free KINOMEscan assay, with similar IC₅₀ values to non-neurotoxic NVP-2 for CDK11, CDK16, and CDK17^[2].
AG-012986 (dihydrochloride) (200-500 nM; 24 h) induces dose-dependent cytotoxicity in human MIO-M1 Müller cells, with significant decreases in cell viability and increases in cell death observed at 200 nM and maximal effects at 500 nM after 24 h of incubation^[2].
AG-012986 (dihydrochloride) (250 nM; 16 h) induces rapid apoptosis/necrosis in primary human PBMCs, with 80% of cells showing dual PI/Annexin-V positivity^[3].
AG-012986 (dihydrochloride) (50 nM-1 μM; 8-48 h) activates caspase-3/7 in primary human PBMCs in vitro in a dose-dependent manner, with peak activity occurring 16-24 h after treatment with 50 nM, 200 nM, or 1 μM^[3].
AG-012986 (dihydrochloride) (50 nM-200 nM; 8 h) induces cleavage of caspase-3 and PARP in primary human PBMCs following 8 h treatment with 50 nM or 200 nM, confirming caspase-mediated apoptosis^[3].
AG-012986 (dihydrochloride) (10 nM-500 nM; 16 h) exhibits greater cytotoxicity in primary human PBMCs than staurosporine after 16 h treatment with 10-500 nM, as measured by reduced ATP content and increased caspase-3/7 activity^[3].
AG-012986 (dihydrochloride)'s (50 nM-250 nM; 20 min) toxicity profile is altered by acute stimulation of T cells^[3].

AG-012986 (dihydrochloride) (20 nM-500 nM) inhibits α-CD3-induced proliferation in purified primary human T cells pretreated by AG-012986 followed by 24 h α-CD3 antibody stimulation, while α-CD3 stimulation decouples this antiproliferative activity from AG-012986-induced toxicity^[3].
AG-012986 (dihydrochloride) (20 nM-500 nM; 48 h) inhibits α-CD3-induced IL-2 production in purified primary human T cells in a dose-dependent manner, with a fivefold reduction observed at 20 nM^[3].
AG-012986 (dihydrochloride) (50 nM-250 nM; 20 min pretreatment) inhibits both basal and α-CD3-induced p38 phosphorylation in purified primary human T cells pretreated with 50 nM or 250 nM for 20 min followed by 16 h incubation with or without stimulation^[3].
AG-012986 (dihydrochloride) (250 nM; 16 h) causes rapidly apoptosis in PBMCs and occurs independently of any cell division^[4].
AG-012986 (dihydrochloride) (50-200 nM; 8 h) induced the presence of p17 and p19 and the cleaved form of PARP^[4].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

Western Blot Analysis^[1]

Cell Line:	HCT116 human colon cancer cells
Concentration:	60 nM; 120 nM; 240 nM
Incubation Time:	8 h; 24 h
Result:	Showed minimal effect on Rb Ser795 phosphorylation after treatment with up to 240 nM for 8 hours. Induced dose-dependent hypophosphorylation of Rb Ser795 after treatment with 60 to 240 nM for 24 hours, with maximal effects at >120 nM.

Cell Cycle Analysis^[1]

Cell Line:	HCT116 human colon cancer cells
Concentration:	30 nM; 60 nM; 120 nM; 240 nM; 480 nM; 720 nM; 1 μM
Incubation Time:	24 h; <8 h
Result:	Caused accumulation of cells in the G1 phase after treatment with 30 to 120 nM for 24 hours. Caused accumulation of cells in the G2-M phase after treatment with ≥240 nM for 24 hours. Failed to induce any cell cycle arrest after transient exposure (<8 hours) to up to 1 μM.

Apoptosis Analysis^[1]

Cell Line:	HCT116 human colon cancer cells
Concentration:	120 nM; 240 nM
Incubation Time:	8 h; 24 h
Result:	Induced no apoptosis after 8 hours of treatment at any concentration. Induced a greater proportion of apoptotic cells after treatment with ≥120 nM for 24 hours, with an IC₅₀ of ≈160 nM.

Cell Cytotoxicity Assay^[1]

Cell Line:	SW620 human colon carcinoma cells
Concentration:	10 nM; 100 nM; 1000 nM
Incubation Time:	8 h; 24 h; 72 h; 320 h
Result:	Showed minimal cytotoxic activity after 8 hours of treatment. Showed moderate activity with an IC₅₀ of 300 nmol/L after 24 hours. Showed substantial cytotoxicity with an IC50 <100 nmol/L at treatment durations ≥72 hours, with near-complete cell kill at 1000 nM after 320 hours.

Cell Viability Assay^[2]

Cell Line:	human Müller cell line MIO-M1
Concentration:	200 nM; 500 nM
Incubation Time:	24 h
Result:	Caused a dose-dependent decrease in cell viability (measured via MTS assay) and a corresponding dose-dependent increase in cell death (measured via LDH release). Reduced cell viability by ~25% relative to controls at maximal response. Increased LDH release by ~2-fold relative to controls at maximal response. Produced significant effects at 200 nM. Achieved maximal response at 500 nM.

Western Blot Analysis^[3]

Cell Line:	primary human PBMCs
Concentration:	50 nM; 200 nM
Incubation Time:	8 h
Result:	Induced cleavage of caspase-3 and PARP.

Western Blot Analysis^[3]

Cell Line:	purified primary human T cells
Concentration:	50 nM; 250 nM; 50 nM plus α-CD3 antibody (1 μg/mL; 24 h); 250 nM plus α-CD3 antibody (1 μg/mL; 24 h)
Incubation Time:	20 min
Result:	Showed almost a complete lack of active capase-3 after 24 h stimulation with CD3 antibody.

Western Blot Analysis^[3]

Cell Line:	purified primary human T cells
Concentration:	50 nM; 250 nM; 50 nM plus plate-bound α-CD3 antibody (1 μg/mL; 16 h); 250 nM plus plate-bound α-CD3 antibody (1 μg/mL; 16 h)
Incubation Time:	20 min
Result:	Inhibited both basal and α-CD3-induced p38 phosphorylation in purified primary human T cells with or without stimulation.

Apoptosis Analysis^[4]

Cell Line:	human donor PBMCs
Concentration:	250 nM
Incubation Time:	16 h
Result:	Induced apoptosis in approximately 80% of the cells.

Western Blot Analysis^[4]

Cell Line:	human donor PBMCs
Concentration:	50 nM; 200 nM
Incubation Time:	8 h
Result:	Induced the presence of p17 and p19 and the cleaved form of PARP.

Parmacokinetics

Species	Dose	Route	C_max	AUC_0-24	AUC_0-t	AUC	T_1/2
Mice^[1]	6, 12, 25, 50, 75 mg/kg	i.p.	1.08 μg/mL	/	/	1.32 μg·h/mL	1 h
Mice^[1]	10, 20, 40 mg/kg	s.c.	0.182 μg/mL	3.92 μg·h/mL	27.5 μg·h/mL	/	/

In Vivo

AG-012986 (dihydrochloride) (8.8-40 mg/kg; s.c.; daily, 12 days) shows >83% tumor growth inhibition (TGI) in 10 of the 11 tumor models tested^[1].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

Animal Model:	Severe combined immunodeficient; athymic NCr-nu/nu (human tumor xenograft model, implanted s.c. with 2 million COLO205 or H522 cells in 30% Matrigel)^[1]
Dosage:	20 mg/kg; 40 mg/kg; 8.8 mg/kg; 17.5 mg/kg; 35 mg/kg
Administration:	s.c.; once a day for 12 d
Result:	Induced 94.7% TGI and a net log tumor cell kill of 1.20 in COLO205 colon carcinoma models at 40 mg/kg daily for 12 days. Induced 71.3% TGI and a net log tumor cell kill of 0.64 in COLO205 colon carcinoma models at 20 mg/kg daily for 12 days. Induced 22.2% TGI and a negative net log tumor cell kill of -0.21 in COLO205 colon carcinoma models at 10 mg/kg daily for 12 days.

Animal Model:	Severe combined immunodeficient; athymic NCr-nu/nu (human tumor xenograft model, implanted i.p. with 2 million COLO205 cells in 30% Matrigel)^[1]
Dosage:	10 mg/kg; 20 mg/kg; 40 mg/kg
Administration:	i.p.; single application
Result:	Reduced Rb Ser795 phosphorylation by >80% and induced PARP cleavage in COLO205 tumor tissue at 24 hours post single 20 or 40 mg/kg i.p. dose. Reduced Rb Ser795 phosphorylation by 50% and did not induce PARP cleavage in COLO205 tumor tissue at 24 hours post single 10 mg/kg i.p. dose. Significantly decreased BrdUrd uptake (S phase activity) in COLO205 tumors between 12 and 24 hours post single 20 mg/kg i.p. dose.

Molecular Weight

532.43

Formula

C₂₂H₂₅Cl₂F₂N₅O₂S

CAS No.

486414-32-8

SMILES

O=C(C1=C(C=CC=C1F)F)C2=C(N)N=C(NC3=CC=C(C=C3)C(N[C@H](C)CN(C)C)=O)S2.Cl.Cl

Shipping

Room temperature in continental US; may vary elsewhere.

Storage

Please store the product under the recommended conditions in the Certificate of Analysis.

Purity & Documentation

Handling Instructions (2659 KB)

References

[1]. Zhang C, et al. Pharmacologic properties of AG-012986, a pan-cyclin-dependent kinase inhibitor with antitumor efficacy. Mol Cancer Ther. 2008 Apr;7(4):818-28. [Content Brief]

[2]. Wright P, et al. Differential expression of cyclin-dependent kinases in the adult human retina in relation to CDK inhibitor retinotoxicity. Arch Toxicol. 2019;93(3):659-671. [Content Brief]

[3]. Lee DU, et al. Off-target immune cell toxicity caused by AG-012986, a pan-CDK inhibitor, is associated with inhibition of p38 MAPK phosphorylation. J Biochem Mol Toxicol. 2012 Mar;26(3):101-8. [Content Brief]