GSK-3β (IC50 = 6.7 nM); GSK-3α (IC50 = 10 nM); ATM (cdc2 = 8800 nM)
Glycogen Synthase Kinase 3α (GSK3α) (Ki: 10 nM; ) [3]
- Glycogen Synthase Kinase 3β (GSK3β) (Ki: 6.7 nM;) [3]

Laduviglusib trihydrochloride inhibits human GSK-3β with Ki values of 9.8 nM[1]. Laduviglusib trihydrochloride is a tiny organic molecule that inhibits GSK3α and GSK3β by vying for their ATP-binding sites. Laduviglusib trihydrochloride specifically inhibits GSK3β (IC50=~5 nM) and GSK3α (IC50=~10 nM), with little effect on other kinases, according to in vitro kinase assays[4]. Laduviglusib trihydrochloride reduces the viability of ES-D3 cells by 24.7% at 2.5 μM, 56.3% at 5 μM, 61.9% at 7.5 μM and 69.2% at 10 μM, with an IC50 of 4.9 M[2].

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1. Enhancement of insulin-mediated glucose transport and utilization: In 3T3-L1 adipocytes and L6 myotubes, Laduviglusib (CHIR99021) trihydrochloride potentiated insulin-stimulated 2-deoxyglucose (2-DG) uptake in a concentration-dependent manner. At 1 μM, it increased insulin-induced glucose uptake by ~2-fold in 3T3-L1 adipocytes and ~1.8-fold in L6 myotubes. It also enhanced insulin activation of glycogen synthase (GS) activity, with a 2.5-fold increase in GS activity at 1 μM in 3T3-L1 adipocytes [1]
2. Activation of Wnt/β-catenin pathway in mouse embryonic stem cells (mESCs): Treatment of mESCs with Laduviglusib (CHIR99021) trihydrochloride (0.3-3 μM) increased the accumulation of cytoplasmic and nuclear β-catenin (detected by western blot) and upregulated the expression of Wnt target genes (e.g., Axin2, Lef1) measured by quantitative real-time PCR (qPCR). The pathway activation was concentration-dependent, with maximal effect at 3 μM [2, 3]
3. Promotion of mESC self-renewal: Laduviglusib (CHIR99021) trihydrochloride (3 μM) maintained the self-renewal capacity of mESCs from refractory mouse strains (e.g., CBA/Ca, 129S6/SvEv) in the absence of leukemia inhibitory factor (LIF). It increased the formation of alkaline phosphatase (AP)-positive colonies by ~3-fold compared to the control group and preserved the expression of pluripotency markers (Oct4, Nanog, Sox2) detected by immunocytochemistry and western blot [3]
4. Cytotoxicity in mESCs: Laduviglusib (CHIR99021) trihydrochloride showed low cytotoxicity to mESCs, with a 50% cytotoxic concentration (CC50) greater than 10 μM. No significant reduction in cell viability was observed at concentrations up to 3 μM (the concentration used for self-renewal and pathway activation) [2]

Laduviglusib (16 mg/kg or 48 mg/kg) trihydrochloride rapidly lowers plasma glucose in ZDF rats after a single oral dose, with a maximum reduction of almost 150 mg/dl occurring 3–4 h after administration[1]. Survival after 14.5 Gy abdominal irradiation (ABI) is significantly improved by laduviglusib (2 mg/kg) trihydrochloride given once 4 hours prior to radiation. Treatment with laduviglusib trihydrochloride significantly reduces crypt apoptosis, prevents the buildup of p-H2AX+ cells, and enhances crypt regeneration and villus height. Treatment with laduviglusib trihydrochloride boosts Lgr5+ cell survival by preventing apoptosis and successfully delays the loss of Olfm4, Lgr5, and CD44 as early as 4 h[5].

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1. Improvement of insulin sensitivity and glucose homeostasis in obese mice: In high-fat diet (HFD)-induced obese C57BL/6 mice, intraperitoneal injection of Laduviglusib (CHIR99021) trihydrochloride (10 mg/kg/day for 7 days) significantly reduced fasting blood glucose (by ~30%), fed blood glucose (by ~25%), and fasting insulin levels (by ~40%) compared to vehicle-treated mice. It also improved glucose tolerance (AUC of glucose tolerance test reduced by ~28%) and insulin tolerance (AUC of insulin tolerance test reduced by ~32%) [1]
2. Enhancement of insulin-mediated glycogen synthesis in vivo: In HFD-induced obese mice, Laduviglusib (CHIR99021) trihydrochloride (10 mg/kg/day, i.p.) increased glycogen content in skeletal muscle (by ~50%) and liver (by ~40%) under insulin-stimulated conditions, consistent with its in vitro effect on GS activation [1]

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]

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1. Recombinant GSK3 protein preparation: Recombinant human GSK3α and GSK3β proteins were expressed and purified to obtain active enzymes [3]
2. Kinase activity assay setup: The assay was performed in a reaction buffer containing ATP, a specific GSK3 substrate (e.g., glycogen synthase peptide or tau protein-derived peptide), and various concentrations of Laduviglusib (CHIR99021) trihydrochloride (0.1 nM-1 μM). The reaction mixture was incubated at 30°C for 30 minutes [3]
3. Detection and analysis: The phosphorylation of the substrate was detected using a radiometric assay (incorporation of [γ-³²P]ATP) or a fluorescence-based assay. The percentage of kinase activity inhibition was calculated relative to the vehicle control, and Ki values for GSK3α and GSK3β were derived from dose-response curves [3]

The loss of cell viability is correlated with the decline in MTT activity, a reliable metabolism-based test for estimating cell viability. In LIF-containing ES cell medium, 2,000 cells are seeded overnight on 96-well plates coated in gelatin. The medium is changed to one without LIF and with reduced serum the following day. It is also supplemented with 0.1-1 μM BIO, or 1-10 μM SB-216763, CHIR-99021, or CHIR-98014. As a control, basal medium devoid of DMSO or GSK3 inhibitors is employed. Three copies of each of the tested conditions were analyzed[3].

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1. Glucose uptake assay in 3T3-L1 adipocytes and L6 myotubes :
- Cell differentiation: 3T3-L1 preadipocytes were differentiated into adipocytes over 8-10 days; L6 myoblasts were differentiated into myotubes over 5-7 days [1]
- Drug and insulin treatment: Differentiated cells were serum-starved for 4 hours, pretreated with Laduviglusib (CHIR99021) trihydrochloride (0.01-10 μM) for 1 hour, then stimulated with insulin (10 nM for adipocytes, 100 nM for myotubes) for 30 minutes [1]
- Glucose uptake measurement: [³H]-2-deoxyglucose was added to the cells and incubated for 10 minutes. Cells were washed to remove unincorporated radioactivity, lysed, and radioactivity was quantified using a scintillation counter. Glucose uptake was normalized to total cellular protein [1]
2. Glycogen synthase (GS) activity assay in 3T3-L1 adipocytes :
- Cell treatment: Differentiated 3T3-L1 adipocytes were serum-starved, pretreated with Laduviglusib (CHIR99021) trihydrochloride (0.01-10 μM) for 1 hour, and stimulated with insulin (10 nM) for 30 minutes [1]
- Enzyme extraction and assay: Cells were lysed, and GS was partially purified by centrifugation. GS activity was measured by detecting the incorporation of [¹⁴C]-UDP-glucose into glycogen. Activity was expressed as units per milligram of protein (1 unit = 1 μmol of glucose incorporated per minute) [1]
3. Wnt/β-catenin pathway activation assay in mESCs :
- Cell culture: mESCs were cultured on gelatin-coated plates in ESC medium without LIF [2]
- Drug treatment: Cells were treated with Laduviglusib (CHIR99021) trihydrochloride (0.3-3 μM) for 24 hours [2]
- β-catenin detection: Cells were lysed, and cytoplasmic/nuclear fractions were separated. β-catenin protein levels were detected by western blot using specific antibodies. Nuclear localization of β-catenin was confirmed by immunocytochemistry [2]
- Wnt target gene expression: Total RNA was extracted, reverse-transcribed into cDNA, and qPCR was performed to quantify Axin2 and Lef1 mRNA levels (normalized to GAPDH) [2]
4. mESC self-renewal and pluripotency assay :
- Cell culture: mESCs from refractory strains were cultured on gelatin-coated plates in ESC medium without LIF, supplemented with Laduviglusib (CHIR99021) trihydrochloride (3 μM) [3]
- Colony formation assay: Cells were seeded at low density (100 cells/cm²) and cultured for 7 days. Colonies were stained for alkaline phosphatase (AP), and AP-positive colonies were counted [3]
- Pluripotency marker detection: Immunocytochemistry was performed using antibodies against Oct4, Nanog, and Sox2. Protein levels were also quantified by western blot [3]
5. mESC cytotoxicity assay :
- Cell seeding: mESCs were seeded into 96-well plates at 5×10³ cells/well and cultured overnight [2]
- Drug treatment: Cells were treated with Laduviglusib (CHIR99021) trihydrochloride (0.1-100 μM) for 72 hours [2]
- Viability detection: MTT reagent was added to the cells and incubated for 4 hours. Formazan crystals were dissolved, and absorbance was measured at 570 nm. CC50 values were calculated from dose-response curves [2]

Rats[1]: Primary hepatocytes from male Sprague Dawley rats that weighed <140 g are prepared and used 1-3 h after isolation. Centrifuged and lysed by freeze/thaw in buffer A plus 0.01% NP40, aliquots of 1106 cells 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 for 30 min at 37°C in a CO2-enriched atmosphere. The GS assay is then carried out once more. Mice[4]: The mice used are 6–10 week old mice. The PUMA+/+ and PUMA-/- littermates of Lgr5-EGFP (Lgr5-EGFP-IRES-creERT2) mice are either given abdominal irradiation (ABI) or whole body irradiation (TBI) to induce radiation sickness. Two milligrams per kilogram of CHIR99021 or one milligram per kilogram of SB415286 are intraperitoneally (i.p.) injected into mice four hours prior to radiation treatment. Small intestines are removed from sacrificed mice and subjected to western blotting and histology tests. Before being sacrificed, all mice receive an intraperitoneal injection of 100 mg/kg BrdU.

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1. Insulin sensitivity and glucose homeostasis study in HFD-induced obese mice :
- Mouse model preparation: C57BL/6 mice were fed a high-fat diet (60% kcal from fat) for 12 weeks to induce obesity and insulin resistance [1]
- Grouping and dosing: Mice were randomly divided into vehicle control and treatment groups (n=8 per group). Laduviglusib (CHIR99021) trihydrochloride was dissolved in 0.9% saline containing 0.1% DMSO, and administered via intraperitoneal injection at 10 mg/kg/day for 7 days. The vehicle group received the same volume of 0.9% saline with 0.1% DMSO [1]
- Sample collection and detection: Fasting blood glucose (after 16-hour fast) and fed blood glucose were measured daily using a glucometer. Fasting insulin levels were detected by ELISA. Glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed on day 6 and 7, respectively [1]
2. Glycogen content measurement in mice :
- Tissue collection: After the 7-day treatment, mice were euthanized, and skeletal muscle (gastrocnemius) and liver tissues were harvested [1]
- Glycogen extraction and quantification: Tissues were homogenized in perchloric acid, and glycogen was precipitated with ethanol. Glycogen content was measured using a colorimetric assay based on the reaction with anthrone reagent, and normalized to tissue weight [1]

1. In vitro cytotoxicity: Laduvigrusib (CHIR99021) trihydrochloride showed low cytotoxicity to mouse embryonic stem cells (mESCs) (CC50 > 10 μM), and no significant cytotoxicity to 3T3-L1 adipocytes or L6 myotube cells at concentrations up to 10 μM [1, 2] 2. In vivo toxicity: In high-fat diet (HFD) induced obese mice, intraperitoneal injection of 10 mg/kg/day of Laduvigrusib (CHIR99021) trihydrochloride for 7 consecutive days did not cause significant changes in body weight, organ weight (liver, kidney, heart), or serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), or creatinine levels, indicating no significant acute toxicity [1]

[1]. Selective glycogen synthase kinase 3 inhibitors potentiate insulin 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.

CHIR 99021 belongs to the aminopyrimidine class of compounds. Its structure is 2-aminopyrimidine, with N2, 5, and 6 positions substituted by (5-cyanopyridin-2-yl)ethyl, 4-methylimidazol-2-yl, and 2,4-dichlorophenyl, respectively. It is an EC 2.7.11.26 (tau protein kinase) inhibitor. It belongs to the imidazole, dichlorobenzene, aminopyrimidine, aminopyrimidine, cyanopyrimidine, secondary amine, and diamine classes of compounds.

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1. Laduvigluxibuline hydrochloride (CHIR99021) is a highly selective ATP-competitive GSK3α and GSK3β inhibitor[3]
2. GSK3 is a serine/threonine kinase that negatively regulates the insulin signaling pathway by phosphorylation and inhibition of glycogen synthase, and negatively regulates the Wnt/β-catenin pathway by phosphorylation of β-catenin to facilitate its degradation by the proteasome[1, 3]
3. The dual mechanism of action of laduvigaluxibuline hydrochloride (CHIR99021) (enhancing the insulin signaling pathway and activating the Wnt/β-catenin pathway) makes it a potential drug for the treatment of type 2 diabetes and a valuable tool. Stem cell research (promoting ESC self-renewal) [1, 3]
4. Laduviglusib hydrochloride (CHIR99021) showed no significant cross-reactivity with other kinases (e.g., CDK2, ERK1, JNK1) at concentrations up to 10 μM, confirming its selectivity for GSK3 [3]
5. In mouse embryonic stem cells (mESCs), the self-renewal promoting effect of latoveglusib hydrochloride (CHIR99021) was independent of LIF, therefore it can be used to maintain ESC strains insensitive to conventional LIF culture systems [3]

C22H21CL5N8

574.72

572.033

C, 45.98; H, 3.68; Cl, 30.84; N, 19.50

1782235-14-6

Laduviglusib;252917-06-9;Laduviglusib monohydrochloride;1797989-42-4

78243722

White to yellow solid powder

6

7

7

35

645

0

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.Cl[H].Cl[H].Cl[H]

DSFVSCNMMZRCIA-UHFFFAOYSA-N

InChI=1S/C22H18Cl2N8.3ClH/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);3*1H

6-[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)pyrimidin-2-yl]amino]ethylamino]pyridine-3-carbonitrile;trihydrochloride

CHIR-73911 3HCl; CHIR911;CHIR 911; CT- 99021; CT-99021; CHIR73911; CHIR 73911 trihydrochloride; CHIR-911; CT- 99021; GSK 3 inhibitor XVI; GSK 3IXV; CHIR99021; CHIR 99021

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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.62 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (3.62 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 20.8 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: ≥ 2.08 mg/mL (3.62 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.

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