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Laduviglusib (CHIR-99021; CT99021; CT 99021) is a novel, potent and orally bioavailable inhibitor of GSK-3α/β (glycogen synthase kinase 3α/β) with the potential to treat type 2 diabetes. With an IC50 of 10 nM/6.7 nM in cell-free assays, it inhibits GSK-3α/β and activates Wnt/β-catenin, which promotes autophagy. In a rodent model of type 2 diabetes, oral gavage of CHIR-99021 (30 mg/kg) improves glucose metabolism, with a maximum plasma glucose reduction of almost 150 mg/dl 3–4 hours after administration, while plasma insulin levels stay at or below control levels.
Physicochemical Properties
| Molecular Formula | C22H18CL2N8 | |
| Molecular Weight | 465.34 | |
| Exact Mass | 464.103 | |
| Elemental Analysis | C, 56.78; H, 3.90; Cl, 15.24; N, 24.08 | |
| CAS # | 252917-06-9 | |
| Related CAS # | 252917-06-9;1782235-14-6 (3HCl);2109414-84-6 (2HCl);1797989-42-4 (HCl); | |
| PubChem CID | 9956119 | |
| Appearance | Off-white to light brown solid powder | |
| Density | 1.5±0.1 g/cm3 | |
| Boiling Point | 784.1±70.0 °C at 760 mmHg | |
| Flash Point | 428.0±35.7 °C | |
| Vapour Pressure | 0.0±2.7 mmHg at 25°C | |
| Index of Refraction | 1.700 | |
| LogP | 3.98 | |
| Hydrogen Bond Donor Count | 3 | |
| Hydrogen Bond Acceptor Count | 7 | |
| Rotatable Bond Count | 7 | |
| Heavy Atom Count | 32 | |
| Complexity | 645 | |
| Defined Atom Stereocenter Count | 0 | |
| SMILES | ClC1C([H])=C(C([H])=C([H])C=1C1C(=C([H])N=C(N=1)N([H])C([H])([H])C([H])([H])N([H])C1C([H])=C([H])C(C#N)=C([H])N=1)C1=NC([H])=C(C([H])([H])[H])N1[H])Cl |
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| InChi Key | AQGNHMOJWBZFQQ-UHFFFAOYSA-N | |
| InChi Code | InChI=1S/C22H18Cl2N8/c1-13-10-29-21(31-13)17-12-30-22(32-20(17)16-4-3-15(23)8-18(16)24)27-7-6-26-19-5-2-14(9-25)11-28-19/h2-5,8,10-12H,6-7H2,1H3,(H,26,28)(H,29,31)(H,27,30,32) | |
| Chemical Name | 6-[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)pyrimidin-2-yl]amino]ethylamino]pyridine-3-carbonitrile | |
| Synonyms |
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| HS Tariff Code | 2934.99.9001 | |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
GSK-3β (IC50 = 6.7 nM); GSK-3α (IC50 = 10 nM); cdc2 (IC50 = 8800 nM) Glycogen Synthase Kinase 3 (GSK3), including both GSK3α and GSK3β isoforms. For GSK3β, the IC₅₀ value was reported to be 6.7 nM [3] |
| ln Vitro |
CHIR-99021 shows greater than 500-fold selectivity for GSK-3 versus its closest homologs CDC2 and ERK2, as well as other protein kinases. CHIR-99021 exhibits only modest inhibition of 23 nonkinase enzymes and only weak binding to a panel of 22 pharmacologically significant receptors. In CHO-IR cells that express the insulin receptor, CHIR-99021 causes the activation of glycogen synthase (GS) with an EC50 of 0.763 μM[1]. In differentiated 3T3-L1 adipocytes, treatment with Laduviglusib (CHIR99021) (at a concentration of 1 μM for 24 hours) resulted in the accumulation of β-catenin, a key downstream effector of the Wnt signaling pathway. This accumulation was accompanied by a significant inhibition of adipocyte-specific gene expression, including genes encoding peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα), which are critical regulators of adipogenesis. Additionally, the drug suppressed the expression of fatty acid synthase (FAS) and adipocyte fatty acid-binding protein (aP2), markers of mature adipocytes [4] - In mouse embryonic stem cells (mESCs) derived from refractory strains (such as CBA/Ca and 129S6/SvEvTac), culture in the presence of Laduviglusib (CHIR99021) (at a concentration of 3 μM) maintained the cells in an undifferentiated state. This was evidenced by the sustained expression of pluripotency markers, including Oct4, Nanog, and Sox2, as detected by immunofluorescence staining and quantitative real-time PCR (qPCR). Furthermore, the drug promoted the self-renewal of mESCs, as shown by increased colony formation efficiency in clonogenic assays; the number of alkaline phosphatase (AP)-positive colonies (a marker of undifferentiated ESCs) was significantly higher in Laduviglusib (CHIR99021)-treated groups compared to control groups [3] - In mESCs treated with Laduviglusib (CHIR99021) at concentrations ranging from 0.1 μM to 10 μM for 48 hours, the drug activated the Wnt/β-catenin pathway, as indicated by increased luciferase activity in a TOPFlash reporter assay (a tool for measuring Wnt pathway activation). At a concentration of 3 μM, the luciferase activity was approximately 5-fold higher than that in the vehicle control group. However, the drug also exhibited dose-dependent cytotoxicity: at concentrations ≥ 5 μM, the viability of mESCs (assessed by the MTT assay) was reduced by more than 30% compared to the control [2] |
| ln Vivo | In a rodent model of type 2 diabetes, oral administration of CHIR-99021 at 30 mg/kg improves glucose metabolism. Three to four hours after oral administration, the maximum plasma glucose reduction—roughly 150 mg/dl—occurs, and plasma insulin levels stay at or below control. In ZDF rats, oral administration of CHIR-99021 at doses of 16 or 48 mg/kg an hour prior to oral glucose challenges significantly improves glucose tolerance, with plasma glucose levels falling by 14% and 33% at the 16 mg/kg and 48 mg/kg doses, respectively. The higher dose of CHIR-99021 also lessens hyperglycemia prior to the oral glucose challenge[1]. |
| Enzyme Assay |
All kinase assays followed the same core protocol with variations in peptide substrate and activator concentrations described below. Polypropylene 96-well plates were filled with 300 μl/well buffer (50 mmol/l tris HCl, 10 mmol/l MgCl2, 1 mmol/l EGTA, 1 mmol/l dithiothreitol, 25 mmol/l β-glycerophosphate, 1 mmol/l NaF, 0.01% BSA, pH 7.5) containing kinase, peptide substrate, and any activators. Information on the kinase concentration, peptide substrate, and activator (if applicable) for these assays is as follows: GSK-3α (27 nmol/l, and 0.5 μmol/l biotin-CREB peptide); GSK-3β (29 nmol/l, and 0.5 μmol/l biotin-CREB peptide); cdc2 (0.8 nmol/l, and 0.5 μmol/l biotin histone H1 peptide); erk2 (400 units/ml, and myelin basic protein-coated Flash Plate [Perkin-Elmer]); PKC-α (1.6 nmol/l, 0.5 μmol/l biotin-histone H1 peptide, and 0.1 mg/ml phosphatidylserine + 0.01 mg/ml diglycerides); PKC-ζ (0.1 nmol/l, 0.5 μmol/l biotin-PKC-86 peptide, and 50 μg/ml phosphatidylserine + 5 μg/ml diacylglycerol); akt1 (5.55 nmol/l, and 0.5 μmol/l biotin phospho-AKT peptide); p70 S6 kinase (1.5 nmol/l, and 0.5 μmol/l biotin-GGGKRRRLASLRA); p90 RSK2 (0.049 units/ml, and 0.5 μmol/l biotin-GGGKRRRLASLRA); c-src (4.1 units/ml, and 0.5 μmol/l biotin-KVEKIGEGTYGVVYK); Tie2 (1 μg/ml, and 200 nmol/l biotin-GGGGAPEDLYKDFLT); flt1 (1.8 nmol/l, and 0.25 μmol/l KDRY1175 [B91616] biotin-GGGGQDGKDYIVLPI-NH2); KDR (0.95 nmol/l, and 0.25 μmol/l KDRY1175 [B91616] biotin-GGGGQDGKDYIVLPI-NH2); bFGF receptor tyrosine kinase (RTK; 2 nmol/l, and 0.25 μmol/l KDRY1175 [B91616] biotin-GGGGQDGKDYIVLPI-NH2); IGF1 RTK (1.91 nmol/l, and 1 μmol/l biotin-GGGGKKKSPGEYVNIEFG-amide); insulin RTK (using DG44 IR cells); AMP kinase (470 units/ml, 50 μmol/l SAMS peptide, and 300 μmol/l AMP); pdk1 (0.25 nmol/l, 2.9 nmol/l unactivated Akt, and 20 μmol/l each of DOPC and DOPS + 2 μmol/l PIP3); CHK1 (1.4 nmol/l, and 0.5 μmol/l biotin-cdc25 peptide); CK1-ε (3 nmol/l, and 0.2 μmol/l biotin-peptide); DNA PK; and phosphatidylinositol (PI) 3-kinase (5 nmol/l, and 2 μg/ml PI). Test compounds or controls were added in 3.5 μl of DMSO, followed by 50 μl of ATP stock to yield a final concentration of 1 μmol/l ATP in all cell-free assays. After incubation, triplicate 100-μl aliquots were transferred to Combiplate eight plates (LabSystems, Helsinki, Finland) containing 100 μl/well 50 μmol/l ATP and 20 mmol/l EDTA. After 1 h, the wells were rinsed five times with PBS, filled with 200 μl of scintillation fluid, sealed, left 30 min, and counted in a scintillation counter. All steps were performed at room temperature. Inhibition was calculated as 100% × (inhibited − no enzyme control)/(DMSO control − no enzyme control).[1]
For the measurement of GSK3β kinase activity: Recombinant human GSK3β was incubated with a synthetic peptide substrate (sequence: YRRAAVPPSPSLSRHSSPHQpSEDEEE) in a reaction buffer containing ATP (at a final concentration of 10 μM), MgCl₂, and various concentrations of Laduviglusib (CHIR99021) (ranging from 0.1 nM to 100 nM). The reaction was carried out at 30°C for 60 minutes and was terminated by the addition of a stop buffer containing EDTA. The amount of phosphorylated substrate was detected using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay, with a donor fluorophore-labeled antibody specific for phosphorylated serine residues and an acceptor fluorophore-labeled secondary antibody. The IC₅₀ value for GSK3β inhibition by Laduviglusib (CHIR99021) was calculated based on the dose-response curve generated from the TR-FRET signal intensities [3] |
| Cell Assay |
CHO-IR cells expressing human insulin receptor are grown to 80% confluence in Hamm’s F12 medium with 10% fetal bovine serum and without hypoxanthine. In 2-ml of medium devoid of fetal bovine serum, trypsinized cells are seeded in 6-well plates at a density of 1×106 cells per well. Following 24 hours, the medium is changed to 1 ml of serum-free medium containing the GSK-3 inhibitor or a control (final DMSO concentration <0.1%) for 30 min at 37°C. Lysing and centrifuging the cells for 15 min. at 4 °C/14000g. Using the filter paper assay developed by Thomas et al., the activity ratio of GS is calculated as the difference between the activity of GS in the presence and absence of 5 mmol/l glucose-6-phosphate. Adipocyte differentiation assay in 3T3-L1 cells: 3T3-L1 preadipocytes were seeded in 6-well plates at a density of 2 × 10⁴ cells per well and cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) until confluence. Two days after confluence (day 0), differentiation was induced by adding a differentiation cocktail containing insulin, dexamethasone, and 3-isobutyl-1-methylxanthine (IBMX) to the medium. On day 2, the medium was replaced with DMEM containing 10% FBS and insulin. From day 2 onwards, cells were treated with Laduviglusib (CHIR99021) (1 μM) or vehicle control every 2 days until day 8. On day 8, cells were harvested for RNA extraction (using TRIzol reagent) to analyze gene expression by qPCR, or for protein extraction (using RIPA buffer) to detect β-catenin levels by Western blot. For qPCR, primers specific for PPARγ, C/EBPα, FAS, and aP2 were used, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the housekeeping gene. For Western blot, a primary antibody against β-catenin and a horseradish peroxidase (HRP)-conjugated secondary antibody were used, with β-actin as the loading control [4] - mESC self-renewal and pluripotency assay: mESCs from refractory strains were seeded on feeder layers of mitomycin C-inactivated mouse embryonic fibroblasts (MEFs) in DMEM supplemented with 15% FBS, leukemia inhibitory factor (LIF), penicillin-streptomycin, and β-mercaptoethanol. Cells were treated with Laduviglusib (CHIR99021) (3 μM) or vehicle control, and the medium was changed daily. After 7 days of culture, cells were fixed with 4% paraformaldehyde and stained for alkaline phosphatase (AP) using an AP staining kit to identify undifferentiated colonies. The number of AP-positive colonies was counted under a light microscope. For immunofluorescence staining, fixed cells were incubated with primary antibodies against Oct4, Nanog, and Sox2 overnight at 4°C, followed by incubation with fluorescently labeled secondary antibodies for 1 hour at room temperature. Stained cells were visualized using a fluorescence microscope. For qPCR analysis, total RNA was extracted from mESCs using an RNA extraction kit, and cDNA was synthesized using a reverse transcription kit. Primers specific for Oct4, Nanog, and Sox2 were used for qPCR, with GAPDH as the reference gene [3] - Wnt pathway activation and cytotoxicity assay in mESCs: mESCs were transfected with the TOPFlash reporter plasmid (containing TCF/LEF binding sites upstream of a luciferase gene) and a Renilla luciferase plasmid (as an internal control) using a transfection reagent. Twenty-four hours after transfection, cells were treated with various concentrations of Laduviglusib (CHIR99021) (0.1 μM to 10 μM) or vehicle control for 48 hours. Luciferase activity was measured using a dual-luciferase reporter assay system, with the firefly luciferase activity normalized to Renilla luciferase activity. For cytotoxicity assessment, mESCs were seeded in 96-well plates at a density of 5 × 10³ cells per well and treated with the same concentrations of Laduviglusib (CHIR99021) for 48 hours. MTT reagent was added to each well, and the plates were incubated at 37°C for 4 hours. The formazan crystals formed were dissolved in dimethyl sulfoxide (DMSO), and the absorbance was measured at 570 nm using a microplate reader. Cell viability was calculated as the percentage of absorbance in drug-treated wells relative to control wells [2] |
| Animal Protocol |
Female db/db mice; Male ZDF rats 8-48 mg/kg Oral administration [1] Primary hepatocytes from male Sprague Dawley rats that weighed <140 g are prepared and used 1-3 h after isolation. Aliquots of 1×106cells in 1 mL of DMEM/F12 medium plus 0.2% BSA and CHIR 99021(orally at 16 or 48 mg/kg) or controls are incubated in 12-well plates on a low-speed shaker for 30 min at 37°C in a CO2-enriched atmosphere, collected by centrifugation and lysed by freeze/thaw in buffer A plus 0.01% NP40; the GS assay is again performed. Mice[4] Mice 6-10 weeks old are used. The PUMA+/+ and PUMA-/- littermates on C57BL/6 background (F10) and Lgr5-EGFP (Lgr5-EGFP-IRES-creERT2) mice are subjected to whole body irradiation (TBI), or abdominal irradiation (ABI). Mice are injected intraperitoneally (i.p.) with 2 mg/kg of CHIR99021 4 h before radiation or 1 mg/kg of SB415286 28 h and 4 h before radiation. Mice are sacrificed to collect small intestines for histology analysis and western blotting. All mice are injected i.p. with 100 mg/kg of BrdU before sacrifice[2]. |
| Toxicity/Toxicokinetics |
In mESCs, Laduviglusib (CHIR99021) showed dose-dependent cytotoxicity. After 48 hours of treatment, concentrations of 5 μM and 10 μM resulted in a reduction in cell viability (assessed by MTT assay) of 32% and 58%, respectively, compared to the vehicle control group. No significant cytotoxicity was observed at concentrations ≤ 3 μM [2] |
| References |
[1]. Selective glycogen synthase kinase 3 inhibitors potentiate activation of glucose transport and utilization in vitro and in vivo. Diabetes. 2003 Mar;52(3):588-95. [2]. Cytotoxicity and activation of the Wnt/beta-catenin pathway in mouse embryonic stem cells treated with four GSK3 inhibitors.BMC Res Notes. 2014 Apr 29;7:273. [3]. Pleiotropy of glycogen synthase kinase-3 inhibition by CHIR99021 promotes self-renewal of embryonic stem cells from refractory mouse strains. PLoS One. 2012;7(4):e35892. [4]. Regulation of Wnt signaling during adipogenesis. J Biol Chem. 2002 Aug 23;277(34):30998-1004. [5]. Pharmacologically blocking p53-dependent apoptosis protects intestinal stem cells and mice from radiation. Sci Rep. 2015 Apr 10;5:8566. |
| Additional Infomation |
CHIR 99021 is a member of the class of aminopyrimidines that is 2-aminopyrimidine substituted at positions N2, 5 and 6 by (5-cyanopyridin-2-yl)ethyl, 4-methylimidazol-2-yl and 2,4-dichlorophenyl groups respectively. It has a role as an EC 2.7.11.26 (tau-protein kinase) inhibitor. It is a member of imidazoles, a dichlorobenzene, an aminopyrimidine, an aminopyridine, a cyanopyridine, a secondary amino compound and a diamine. Background: Inhibition of glycogen synthase kinase-3 (GSK-3) improves the efficiency of embryonic stem (ES) cell derivation from various strains of mice and rats, as well as dramatically promotes ES cell self-renewal potential. β-catenin has been reported to be involved in the maintenance of self-renewal of ES cells through TCF dependent and independent pathway. But the intrinsic difference between ES cell lines from different species and strains has not been characterized. Here, we dissect the mechanism of GSK-3 inhibition by CHIR99021 in mouse ES cells from refractory mouse strains. Methodology/principal findings: We found that CHIR99021, a GSK-3 specific inhibitor, promotes self-renewal of ES cells from recalcitrant C57BL/6 (B6) and BALB/c mouse strains through stabilization of β-catenin and c-Myc protein levels. Stabilized β-catenin promoted ES self-renewal through two mechanisms. First, β-catenin translocated into the nucleus to maintain stem cell pluripotency in a lymphoid-enhancing factor/T-cell factor-independent manner. Second, β-catenin binds plasma membrane-localized E-cadherin, which ensures a compact, spherical morphology, a hallmark of ES cells. Further, elevated c-Myc protein levels did not contribute significantly to CH-mediated ES cell self-renewal. Instead, the role of c-Myc is dependent on its transformation activity and can be replaced by N-Myc but not L-Myc. β-catenin and c-Myc have similar effects on ES cells derived from both B6 and BALB/c mice. Conclusions/significance: Our data demonstrated that GSK-3 inhibition by CH promotes self-renewal of mouse ES cells with non-permissive genetic backgrounds by regulation of multiple signaling pathways. These findings would be useful to improve the availability of normally non-permissive mouse strains as research tools.[3] Laduviglusib (CHIR99021) exerts its effects primarily through the inhibition of GSK3, which leads to the stabilization and accumulation of β-catenin. The accumulated β-catenin translocates to the nucleus, where it binds to T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors, thereby activating the expression of Wnt target genes. This mechanism is critical for its role in maintaining the self-renewal of mESCs and inhibiting adipogenesis [2], [3], [4] - In the context of adipogenesis, the inhibition of GSK3 by Laduviglusib (CHIR99021) disrupts the normal differentiation process of preadipocytes into mature adipocytes by suppressing the expression of key adipogenic transcription factors (PPARγ and C/EBPα) and downstream adipocyte markers. This suggests that GSK3 inhibitors like Laduviglusib (CHIR99021) may have potential applications in the regulation of adipose tissue development and related metabolic disorders [4] - For mESCs from refractory strains, which typically have low self-renewal capacity and are prone to differentiation in standard culture conditions, Laduviglusib (CHIR99021) (at 3 μM) effectively maintains their pluripotency and promotes self-renewal. This property makes the drug a valuable tool for the in vitro culture and manipulation of mESCs from genetically diverse mouse strains, which is important for studies involving genetic models of disease [3] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.47 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 20.8 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (4.47 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 3: ~3.5 mg/mL (7 mM) in 4%DMSO+30%PEG 300+ddH2O, clear solution Solubility in Formulation 4: ~5 mg/mL (10.7 mM) in 0.5% CMC-Na/saline water, suspension solution Solubility in Formulation 5: ≥ 2.1 mg/mL (4.5 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + + 45% Saline, clear solution For example, if 1 mL of working solution is to be prepared, you can take 100 μL of 21 mg/mL of DMSO stock solution and add tO + 400 μL of PEG300, mix well (clear solution); Then add 50 μL of Tween 80 to the above solution, mix well (clear solution); Finally, add 450 μL of saline to the above solution, mix well (clear solution). Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Solubility in Formulation 6: ≥ 2.1 mg/mL (4.5 mM) in 10% DMSO + 90% Corn oil, clear solution For example, if 1 mL of working solution is to be prepared, you can take 100 μL of 21 mg/mL of DMSO stock solution and add to 900 μL of corn oil, mix well (clear solution). Solubility in Formulation 7: 5 mg/mL (10.74 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 8: 5 mg/mL (10.74 mM) in 20% SBE-β-CD adjusted to pH 4-4.5 with 1 N acetic (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1490 mL | 10.7448 mL | 21.4897 mL | |
| 5 mM | 0.4298 mL | 2.1490 mL | 4.2979 mL | |
| 10 mM | 0.2149 mL | 1.0745 mL | 2.1490 mL |