CKIα CDK7 1.3 nM (Kd) CDK9 4 nM (Kd)
Casein Kinase inhibitor A51 selectively targets casein kinase 1 alpha (CK1α, also known as CSNK1A1), a serine/threonine protein kinase that is a component of the casein kinase 1 family. CK1α is a multifunctional kinase that regulates diverse cellular processes including Wnt/β-catenin signaling, circadian rhythm maintenance, DNA damage response, and cell cycle progression. The compound acts as a pan-specific CKI inhibitor with a Kd of 5.3 nM for CK1α. By inhibiting CK1α, Casein Kinase inhibitor A51 disrupts its downstream signaling pathways, leading to the stabilization of p53 and the induction of apoptosis in cancer cells. The selectivity profile of this inhibitor across the CKI family makes it a valuable tool for dissecting the specific roles of CK1α versus other casein kinase isoforms in cellular physiology and disease pathogenesis.

Casein Kinase inhibitor A51 (0.05-3.2 μM; 18 hours) treatment of RKO cells eliminated most of the Ser45 phosphorylation signal and the subsequent GSK3 phosphorylation cascade, which led to the stabilization of β-catenin, in a manner similar to CKIα depletion[1]. At 160 nM or less, the casein kinase inhibitor A51 is very effective at causing leukemia cells to undergo apoptosis, mostly due to its ability to stabilize p53[1]. MYC, MDM2, and the anti-apoptotic oncogene MCL1 are all eliminated by the casein kinase inhibitor A51 (0.08-2 μM; 6.5 hours). A51, a casein kinase inhibitor, significantly decreases the mRNA expression of MDM2 and MYC while increasing the expression of Wnt targets AXIN2 and CCND1 (Cyclin D1)[1].
In vitro, Casein Kinase inhibitor A51 is highly effective in inducing apoptosis in leukemia cells at concentrations of 160 nM or lower. The pro-apoptotic activity is correlated with its capacity to stabilize p53, a key tumor suppressor protein that is often dysregulated in cancer. The compound demonstrates potent inhibition of CK1α with a Kd of 5.3 nM, indicating strong binding affinity. Beyond its effects on leukemia cells, Casein Kinase inhibitor A51 exhibits broad anticancer activity, with studies showing efficacy against breast cancer, prostate cancer, and neck cancer cell lines. The compound's ability to induce apoptosis in multiple cancer types highlights its potential as a therapeutic agent for a wide range of malignancies. Detailed IC50 values and growth inhibition data across various cell lines are available from preclinical studies.

When the percentage of leukemia cells in the bone marrow is greater than 1.5% of total cells, 8 days after leukemia cell injection, oral medication (Casein Kinase inhibitor A51; 5 mg/kg/day) is started. Every mouse treated with A51 exhibits normal organ morphology, histology, and blood counts[1]. Rapid oral absorption is shown in pharmacokinetic investigations of the Casein Kinase inhibitor A51 at 20 mg/kg, with Tmax values of 0.5-2 hr, Cmax values of 1060 ng/mL, T1/2 values of 2.5 hr, and area under the curve (AUC) values of 3680 (ng*hr/mL)[1].
In vivo, Casein Kinase inhibitor A51 has demonstrated effective anti-leukemia activity and anticancer efficacy. As an orally active compound, it offers the advantage of convenient administration for in vivo studies. Preclinical studies have shown that oral administration of Casein Kinase inhibitor A51 leads to significant tumor growth inhibition in xenograft models of leukemia and other cancers. The compound's ability to stabilize p53 and induce apoptosis translates into in vivo efficacy, with treated animals showing reduced tumor burden and improved survival outcomes. Its favorable pharmacokinetic properties and oral bioavailability support its use in preclinical efficacy studies across multiple cancer models, including acute myeloid leukemia, breast cancer, and prostate cancer.

In vitro enzyme/receptor binding (non-cell) assays for Casein Kinase inhibitor A51 typically involve kinase activity assays using purified recombinant CK1α enzyme. The assays are conducted in cell-free systems where the kinase is incubated with a peptide substrate and ATP in the presence of varying concentrations of the inhibitor. Kinase activity is measured by quantifying the phosphorylation of the substrate using either radioactive (e.g., [γ-³²P]ATP) or luminescent detection methods (e.g., ADP-Glo™). IC50 values are calculated from dose-response curves. For binding affinity determination (Kd), techniques such as surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) may be employed. Selectivity profiling against a panel of kinases is performed to assess the compound's specificity for CK1α over other kinases and CKI isoforms.

Cell Viability Assay[1]
Cell Types: MV4-11 cells
Tested Concentrations: 0.08 μM, 0.6 μM, 2 μM
Incubation Duration: 6.5 hrs (hours)
Experimental Results: Abolishes the expression of MYC, MDM2, and the anti-apoptotic oncogene MCL1.
In vitro cell-based experiments for Casein Kinase inhibitor A51 are conducted using various cancer cell lines, including leukemia, breast cancer, prostate cancer, and neck cancer cell lines. Cells are seeded in multi-well plates and treated with the compound at concentrations ranging from nanomolar to micromolar for various time points (e.g., 24, 48, or 72 hours). Cell viability is assessed using standard assays such as MTT, CCK-8, or CellTiter-Glo. Apoptosis is evaluated by Annexin V/PI staining followed by flow cytometry, as well as by measuring caspase-3/7 activity and PARP cleavage. p53 stabilization is confirmed by Western blot analysis. IC50 values for growth inhibition and apoptosis induction are calculated from dose-response curves.

In vivo animal experiments for Casein Kinase inhibitor A51 are performed in xenograft mouse models of cancer, including models of acute myeloid leukemia, breast cancer, and prostate cancer. Immunodeficient mice (e.g., NSG or BALB/c nude) are implanted with cancer cells subcutaneously or via tail vein injection for systemic disease models. Casein Kinase inhibitor A51 is administered orally at various doses and schedules. Tumor growth is monitored by caliper measurements or bioluminescent imaging. Body weight and clinical signs are recorded to assess tolerability. At study termination, tumors are harvested for analysis of p53 stabilization, apoptosis markers, and CK1α target engagement. Pharmacodynamic studies are performed to confirm target inhibition and pathway modulation in tumor tissue.

Casein Kinase inhibitor A51 is orally bioavailable with good systemic exposure following oral administration【0L34-L35】. The compound exhibits favorable drug metabolism and pharmacokinetic (DMPK) properties that support once-daily or twice-daily dosing in preclinical species. It has a molecular weight of approximately 400 Da and shows good solubility in DMSO (100 mg/mL). The compound's oral bioavailability and favorable half-life make it suitable for in vivo efficacy studies. Detailed pharmacokinetic parameters, including Cmax, Tmax, AUC, and half-life, are available from preclinical studies.

Preclinical toxicology studies have been conducted to evaluate the safety profile of Casein Kinase inhibitor A51. The compound is generally well-tolerated at efficacious doses in animal models, with no significant off-target toxicity reported in available literature. However, as with any kinase inhibitor, potential on-target toxicities related to CK1α inhibition (e.g., effects on Wnt signaling and circadian rhythms) should be considered. The safety profile of this compound supports its use as a research tool for studying CK1α biology and for preclinical efficacy studies in cancer models.

[1]. Small Molecules Co-targeting CKIα and the Transcriptional Kinases CDK7/9 Control AML in Preclinical Models. Cell. 2018 Sep 20;175(1):171-185.e25.

BTX-A51, a CK1α/CDK7/CDK9 inhibitor, is the dimethylbenzenesulfonate of A51, an orally bioavailable inhibitor of casein kinase 1α (CK1α) and cyclin-dependent kinases 7 and 9 (CDK7 and CDK9) with potential antitumor activity. After administration, BTX-A51 binds to and inhibits the activities of CK1α, CDK7, and CDK9. Blocking the phosphorylation and kinase activity of CK1α prevents enhanced binding of mouse dual microsome X (MDMX) to p53, the formation of the CK1α-MDM2 complex, and the resulting p53 inhibition. This induces p53-mediated cell cycle arrest, thereby slowing tumor cell proliferation. Blocking the phosphorylation and kinase activity of CDK7 and CDK9 prevents positive transcription elongation factor b (PTEFb)-mediated RNA polymerase II (RNA Pol II) activation, thereby inhibiting the transcription of genes for various anti-apoptotic proteins. This also induces cell cycle arrest and apoptosis, slowing tumor cell proliferation. CK1α, a serine/threonine kinase and a target of leukemia stem cells, exerts antitumor effects in various cancers by negatively regulating the Wnt/β-catenin signaling pathway and p53. CK1α negatively regulates p53 by phosphorylating MDMX, thereby enhancing the binding of MDMX to p53, and exerts its effect by forming a complex with MDM2. CDK7, also a serine/threonine kinase, plays a role in controlling cell cycle progression and transcriptional regulation, and promotes the expression of key oncogenes (such as c-Myc) by phosphorylating RNA polymerase II. CDK9, also a serine/threonine kinase, regulates transcriptional elongation by phosphorylating the serine 2 site (p-Ser2-RNAPII) of RNA polymerase II. CDK9 is upregulated in various tumor cell types and plays a key role in RNA polymerase II-mediated transcriptional regulation of antiapoptotic proteins. Tumor cell survival depends on antiapoptotic proteins.
Casein Kinase inhibitor A51 is also known as BTX-A51. It is a research compound primarily used for studying the role of CK1α in cancer biology and for preclinical evaluation of CK1α inhibition as a therapeutic strategy. The compound has not yet been approved for clinical use and remains an investigational agent for research purposes only. Its unique mechanism of action—inducing p53 stabilization and apoptosis through CK1α inhibition—makes it a valuable tool for exploring the therapeutic potential of targeting casein kinase 1 alpha in hematological malignancies and solid tumors.

C18H25CLN6

360.884301900864

360.182

2079068-74-7

126558497

Off-white to brown solid powder

2.9

2

5

5

25

439

0

ClC1=CN=C(N=C1C1C=NN(C)C=1CC1CC1)NC1CCC(CC1)N

RVZJFCNYSSUDCU-UHFFFAOYSA-N

InChI=1S/C18H25ClN6/c1-25-16(8-11-2-3-11)14(9-22-25)17-15(19)10-21-18(24-17)23-13-6-4-12(20)5-7-13/h9-13H,2-8,20H2,1H3,(H,21,23,24)

4-N-[5-chloro-4-[5-(cyclopropylmethyl)-1-methylpyrazol-4-yl]pyrimidin-2-yl]cyclohexane-1,4-diamine

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 (277.10 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.93 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 (6.93 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 (6.93 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.)