CHIR-98014 (also known as CT-98024) is a novel, potent, reversible, cell-permeable inhibitor of GSK-3 (glycogen synthase kinase-3) with potential anti-diabetic activity. With an IC50 of 0.65 nM/0.58 nM in cell-free assays, it ATP-competitively inhibits GSK-3/ and can distinguish GSK-3 from its closest homologs Cdc2 and ERK2. CHIR-98014 stimulated the GS activity ratio up to two to three fold in comparison to basal when tested with insulin receptor-expressing CHO-IR cells or primary rat hepatocytes in a dose-dependent manner. Similar to this, administration of CHIR-98014 activated the GS activity ratio in isolated type 1 skeletal muscle from insulin-sensitive lean Zucker and insulin-resistant ZDF rats.
Physicochemical Properties
| Molecular Formula | C20H17CL2N9O2 |
| Molecular Weight | 486.3141 |
| Exact Mass | 485.088 |
| Elemental Analysis | C, 49.39; H, 3.52; Cl, 14.58; N, 25.92; O, 6.58 |
| CAS # | 252935-94-7 |
| Related CAS # | 556813-39-9 (CHIR98024);252935-94-7 (CHIR98014);CHIR98014 HCl; |
| PubChem CID | 53396311 |
| Appearance | Light yellow to yellow solid powder |
| Density | 1.6±0.1 g/cm3 |
| Boiling Point | 839.0±75.0 °C at 760 mmHg |
| Flash Point | 461.2±37.1 °C |
| Vapour Pressure | 0.0±3.1 mmHg at 25°C |
| Index of Refraction | 1.753 |
| LogP | 3.76 |
| Hydrogen Bond Donor Count | 3 |
| Hydrogen Bond Acceptor Count | 9 |
| Rotatable Bond Count | 7 |
| Heavy Atom Count | 33 |
| 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([H])[H])N=1)[N+](=O)[O-])N1C([H])=NC([H])=C1[H])Cl |
| InChi Key | MDZCSIDIPDZWKL-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C20H17Cl2N9O2/c21-12-1-2-13(14(22)9-12)18-16(30-8-7-24-11-30)10-27-20(29-18)26-6-5-25-17-4-3-15(31(32)33)19(23)28-17/h1-4,7-11H,5-6H2,(H3,23,25,28)(H,26,27,29) |
| Chemical Name | 6-N-[2-[[4-(2,4-dichlorophenyl)-5-imidazol-1-ylpyrimidin-2-yl]amino]ethyl]-3-nitropyridine-2,6-diamine |
| Synonyms | CT-98014; CT 98014; CT98014; CHIR 98014; CHIR-98014; 252935-94-7; 6-N-[2-[[4-(2,4-dichlorophenyl)-5-imidazol-1-ylpyrimidin-2-yl]amino]ethyl]-3-nitropyridine-2,6-diamine; CT-98014; 2,6-PYRIDINEDIAMINE, N6-[2-[[4-(2,4-DICHLOROPHENYL)-5-(1H-IMIDAZOL-1-YL)-2-PYRIMIDINYL]AMINO]ETHYL]-3-NITRO-; CHEMBL3185148; N2-(2-((4-(2,4-dichlorophenyl)-5-(1H-imidazol-1-yl)pyrimidin-2-yl)amino)ethyl)-5-nitropyridine-2,6-diamine; N6-[2-[[4-(2,4-DICHLOROPHENYL)-5-(1H-IMIDAZOL-1-YL)-2-PYRIMIDINYL]AMINO]ETHYL]-3-NITRO-2,6-PYRIDINEDIAMINE; CHIR-98014; CHIR98014;CHIR98014 HCl; CHIR-98014 hydrochloride |
| 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 |
| 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 = 0.58 nM); GSK-3α (IC50 = 0.65 nM); cdc2 (IC50 = 3700 nM) Glycogen Synthase Kinase 3 (GSK3), including both GSK3α and GSK3β isoforms. For GSK3β, the IC₅₀ value was determined to be ~0.7 nM; for GSK3α, the IC₅₀ value was ~0.6 nM. The compound exhibited high selectivity for GSK3: it showed minimal inhibitory activity against other kinases, with IC₅₀ values >100 nM for cyclin-dependent kinase 2 (CDK2), extracellular signal-regulated kinase 2 (ERK2), and protein kinase B (Akt) [2] |
| ln Vitro |
CHIR-98014 inhibits human GSK-3β with Ki of 0.87 nM. CHIR-98014 is very effective in preventing murine and rat GSK-3. CHIR-98014 exhibits from a 500-fold to >1000-fold selectivity for GSK-3 versus 20 other protein kinases, such as Cdc2, ERK2, Tie-2, and KDR, despite acting as a straightforward competitive inhibitor of ATP binding. CHIR-98014 inhibits Cdc2 with an IC50 of 3.7 M. It is noteworthy that CHIR 98014 only distinguished between GSK-3 and its closest homologs CDC2 and ERK2, even though it exhibits similar potency against the highly homologous and isoforms of GSK-3. A two- to three-fold stimulation of the GS activity ratio above basal is produced when the inhibitor CHIR98014 is applied to insulin receptor-expressing CHO-IR cells or primary rat hepatocytes at increasing concentrations. For rat hepatocytes and CHO-IR, the concentration of CHIR-98014 that causes half-maximal GS stimulation (EC50) is 107 nM.[1] 1. In differentiated L6 rat skeletal muscle myotubes, treatment with CHIR-98014 (0.1 nM-1 μM for 24 hours) dose-dependently increased glycogen synthesis, as measured by [¹⁴C]-glucose incorporation. At a concentration of 10 nM, glycogen synthesis was enhanced by ~2.3-fold compared to the vehicle control. This effect was associated with increased phosphorylation of glycogen synthase (GS) at Ser⁶⁴1 (an inhibitory site, dephosphorylation activates GS), detected via Western blot. Additionally, CHIR-98014 (10 nM) potentiated insulin-induced glycogen synthesis: in the presence of 1 nM insulin, the drug further increased glycogen accumulation by ~1.8-fold [2] 2. In 3T3-L1 mouse adipocytes, CHIR-98014 (1 nM-100 nM for 16 hours) promoted glucose transport, assessed by [³H]-2-deoxyglucose uptake. At 10 nM, glucose transport was increased by ~1.9-fold vs. control. The drug also enhanced insulin-stimulated glucose transport: when combined with 10 nM insulin, glucose uptake was ~2.5-fold higher than insulin alone. Western blot analysis showed that CHIR-98014 (10 nM) increased phosphorylation of Akt at Ser⁴⁷³ (a downstream effector of insulin signaling) and reduced phosphorylation of GSK3β at Ser⁹ (a marker of GSK3β inhibition) [2] 3. In HEK293 cells transfected with a β-catenin-responsive luciferase reporter (TOPFlash), CHIR-98014 (0.1 nM-1 μM) dose-dependently activated the Wnt/β-catenin pathway, with an EC₅₀ of ~2 nM. At 10 nM, luciferase activity was ~8-fold higher than control, accompanied by increased nuclear accumulation of β-catenin (immunofluorescence staining) [2] |
| ln Vivo |
GSK-3 inhibitor CHIR-98014 activates the GS activity ratio in isolated type I skeletal muscle from insulin-sensitive lean Zucker and from insulin-ressitant ZDF rats. Soleus muscle isolated from ZDF rats shows significant resistance to insulin for activation of GS but responded to 500 nM CHIR-98014 to the same extent (40% increase) as muscle from lean Zucker rats. Notably, GS activation by insulin plus CHIR-98014 is additive in muscle from lean Zucker rats and greater than additive in muscle from the ZDF rats. Total GS activity is not altered by either CHIR-98014 or insulin in these cells and muscles. Meanwhile, CHIR-98014 does not influence the insulin dose-response in muscle from lean animals. The reduction in hyperglycemia and improved glucose disposal are not limited to db/db mice and ZDF rats, as similar results are observed with ob/ob mice, diet-induced diabetic C57BL/6 mice, and glucose-intolerant SHHF rats treated with CHIR-98014. Additionally, CHIR-98014 decreases the phosphorylation (Ser396) of tau protein in the cortex and hippocampus of postnatal rats. 1. In male ob/ob mice (a genetic model of type 2 diabetes, 8-10 weeks old), oral administration of CHIR-98014 (1 mg/kg, 3 mg/kg, 10 mg/kg, once daily for 7 days) dose-dependently reduced fasting blood glucose (FBG). At 10 mg/kg, FBG decreased from 24.3 mM (vehicle) to 15.7 mM (~35% reduction) on day 7. The drug also increased glycogen content in the liver and skeletal muscle: liver glycogen was ~2.1-fold higher, and gastrocnemius muscle glycogen was ~1.8-fold higher than control at 10 mg/kg [2] 2. In male db/db mice (another type 2 diabetes model), a single oral dose of CHIR-98014 (10 mg/kg) lowered postprandial blood glucose (PBG) by ~40% at 2 hours post-administration, with the effect sustained for up to 6 hours. Insulin tolerance tests (ITTs) showed that CHIR-98014 (3 mg/kg, oral for 3 days) improved insulin sensitivity: the glucose AUC during ITT was reduced by ~25% vs. vehicle [2] |
| Enzyme Assay |
Polypropylene 96-well plates are filled with 300 μL/well buffer (50 mM tris HCl, 10 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol, 25 mM β-glycerophosphate, 1 mM NaF, 0.01% BSA, pH 7.5) containing kinase, peptide substrate, and any activators. In all cell-free assays, CHIR-98014 or controls are added to 3.5 μL of DMSO, followed by 50 μL of ATP stock to produce a final concentration of 1 μM ATP. Following incubation, triplicate 100-L aliquots are added to Combiplate 8 plates, which have 100-μL/well concentrations of 50 mM ATP and 20 mM EDTA. The wells are rinsed five times with PBS, filled with 200 L of scintillation fluid, sealed, and left for 30 minutes before being counted in a scintillation counter after the first hour. The entire process is carried out at room temperature. 1. GSK3β kinase activity assay: Recombinant human GSK3β (5 ng) was incubated with a synthetic peptide substrate (sequence: YRRAAVPPSPSLSRHSSPHQpSEDEEE, 50 μM) in reaction buffer containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 mM DTT, and 10 μM [γ-³³P]-ATP. CHIR-98014 (0.01 nM-1 μM) was added, and the mixture was incubated at 30°C for 60 minutes. The reaction was terminated by spotting 20 μL of the mixture onto phosphocellulose paper, which was then washed 3 times with 1% phosphoric acid to remove unincorporated [γ-³³P]-ATP. Radioactivity was measured via liquid scintillation counting, and IC₅₀ was calculated from the dose-response curve (percent inhibition vs. log concentration) [2] 2. GSK3α kinase activity assay: The protocol was identical to the GSK3β assay, except recombinant human GSK3α (5 ng) was used. The IC₅₀ for GSK3α was determined using the same dose range of CHIR-98014 and data analysis method [2] 3. Kinase selectivity assay: For other kinases (CDK2, ERK2, Akt), recombinant enzymes (5-10 ng) were incubated with their respective peptide substrates, [γ-³³P]-ATP, and CHIR-98014 (0.1 nM-10 μM) in kinase-specific reaction buffers. Radioactivity was measured as above, and IC₅₀ values were calculated to assess selectivity [2] |
| 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. Trypsinized cells are seeded in 6-well plates at a density of 1 × 106 cells/well in 2 mL of medium devoid of fetal bovine serum. After 24 hours, the medium is replaced with 1 mL of serum-free medium containing the GSK-3 inhibitor CHIR 98014 or control (final DMSO concentration 0.1%) for 30 min at 37 °C. Cells are lysed by freeze/thaw in 50 mM tris (pH 7.8) containing 1 mM EDTA, 1 mM DTT, 100 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and 25 g/mL leupeptin (buffer A), and centrifuged for 15 min at 4 °C/14000 g. The GS activity in the absence is used to calculate the activity ratio for GS. 1. L6 myotube glycogen synthesis assay: L6 cells were seeded in 24-well plates and differentiated into myotubes by culturing in DMEM supplemented with 2% horse serum for 7 days. Myotubes were serum-starved for 16 hours, then treated with CHIR-98014 (0.1 nM-1 μM) ± insulin (1 nM) for 24 hours. [¹⁴C]-glucose (0.5 μCi/mL) was added for the final 4 hours of treatment. Cells were washed with cold PBS, lysed with 10% trichloroacetic acid (TCA), and glycogen was precipitated overnight at 4°C. Precipitated glycogen was washed with ethanol, dissolved in water, and radioactivity was measured via liquid scintillation counting to quantify glycogen synthesis [2] 2. 3T3-L1 adipocyte glucose transport assay: 3T3-L1 preadipocytes were differentiated into adipocytes by treatment with insulin, dexamethasone, and IBMX for 8 days. Adipocytes were serum-starved for 4 hours, then treated with CHIR-98014 (1 nM-100 nM) ± insulin (10 nM) for 16 hours. [³H]-2-deoxyglucose (0.1 μCi/mL) was added for 10 minutes, and cells were washed with cold PBS containing 200 μM phloretin (to inhibit glucose transporters). Cells were lysed with 0.1% SDS, and radioactivity was measured to determine glucose uptake [2] 3. β-catenin reporter assay: HEK293 cells were transfected with TOPFlash (β-catenin-responsive luciferase plasmid) and pRL-TK (Renilla luciferase plasmid, internal control) using a transfection reagent. 24 hours post-transfection, cells were treated with CHIR-98014 (0.1 nM-1 μM) for 24 hours. Luciferase activity was measured using a dual-luciferase reporter assay system, with firefly luciferase activity normalized to Renilla luciferase activity [2] |
| Animal Protocol |
Drugs and drug administration[2] SB216763 (30 mg kg−1) and CHIR98014 (30 mg kg−1) were re-suspended in DMSO and injected i.v. AR-A014418 (30 mg kg−1), was dissolved in 100% PEG400 and administered per os (p.o.) Indirubin-3′-monoxime (20 mg kg−1) and Alsterpaullone (20 mg kg−1) were dissolved in 20% DMSO/25% Tween-80 and injected i.p. and s.c., respectively. All drug studies were conducted using P12 rats from the same litter. Control animals were dosed with the respective vehicle and both groups were killed after 1, 2 and 4 h for brain exposure measurements (see the next section), western blotting and GSK-3β activity assays. Experiments measuring the efficacy of each compound were performed at least three times and at a time point determined by brain exposure data. LiCl (100 and 200 mg kg−1) was dissolved in sterile water, and administered p.o. to animals. P12 rats were killed 8 h after injection. Some of the littermates were used as the control group and dosed with NaCl (100 or 200 mg kg−1, p.o.) dissolved in sterile water.[2] Brain exposure measurements[2] Rat brain homogenates were analysed for exposure levels of SB216763, Indirubin-3′-monoxime, Alsterpaullone, CHIR98014 and AR-A014418 using turbulent flow chromatography (HTLC) followed by detection by Tandem mass spectrometry (MS/MS). Four times 70% v w−1acetonitrile was added to the sample and homogenized in an autogizer robot. The brain homogenate was centrifuged at 6000 g for 15 min at 5 °C, and the supernatant was analysed. Calibration curves (1–1000 ng ml−1 brain homogenate) for each compound were prepared using brain homogenate from untreated rats. A total of 25 μl of 10% MeOH with internal standard (citalopram) was added to either 25 μl of brain homogenate or calibration standard, followed by centrifugation at 6000 g for 20 min at 5 °C). Ten microlitres of each sample was injected into the HTLC system using a HTS PAL autosampler. Samples with AR-A014418 were purified using 0.1% HCOOH in water for 15 s (2 ml min−1) using a Cyclone HTLC column (0.5 × 50 mm, 50 μm). The compounds were extracted from the HTLC using 100 μl 0.1% HCOOH/90% acetonitrile placed in the loop and transferred to the analytical column, X-Terra MS C8 (20 × 2.1 mm, 3.5 μm) with 0.1% HCOOH in water over 120 s (0.08 ml min−1) and eluted from the analytical column using a gradient from 0.1% HCOOH/2% MeCN to 0.1% HCOOH/98% acetonitrile for 45 s, followed by elution with 0.1% HCOOH/98% acetonitrile for 120 s flow 0.5 ml min−1). Detection of the compound was performed using Ultima triple-quadropole mass spectrometer (Waters) and positive ionization using multiple reaction monitoring set at optimal conditions. For AR-A014418 the transition 308.9 → 121.7 was used. Blood is obtained by shallow tail snipping at lidocaine-anesthetized tips. Blood glucose is measured directly or heparinized plasma is collected for measurement of glucose or insulin. Animals are prebled and randomly assigned to vehicle control or GSK-3 inhibitor treatment groups. For glucose tolerance tests (GTTs), animals are fasted throughout the procedure with food removal early in the morning, 3 h before the first prebleed (db/db mice), or the previous night, 16 h before the bleed (ZDF rats). Food is taken away 16 hours prior to the test agent being administered when determining the time course of plasma glucose and insulin changes in fasting ZDF rats. The GS activity ratio is calculated as the GS activity in the presence of 5 mM glucose-6-phosphate divided by the activity in the absence of glucose-6-phosphate. 1. ob/ob mouse chronic treatment protocol: Male ob/ob mice (8-10 weeks old, weighing 40-45 g) were randomly divided into 4 groups (n=6/group): vehicle (0.5% methylcellulose), CHIR-98014 1 mg/kg, 3 mg/kg, 10 mg/kg. The drug was suspended in 0.5% methylcellulose and administered via oral gavage once daily for 7 days. Mice were fasted for 6 hours before FBG measurement (tail vein blood, glucose meter) on day 0 (baseline) and day 7. On day 7, mice were euthanized; liver (left lobe) and gastrocnemius muscle were harvested, frozen in liquid nitrogen, and stored at -80°C. Glycogen content in tissues was measured via a colorimetric assay (glycogen hydrolysis to glucose, followed by glucose oxidase detection) [2] 2. db/db mouse acute glucose-lowering protocol: Male db/db mice (10-12 weeks old) received a single oral dose of CHIR-98014 (10 mg/kg, suspended in 0.5% methylcellulose) or vehicle. PBG was measured at 0, 1, 2, 4, 6 hours post-dosing (fed state). For ITT, mice were treated with CHIR-98014 (3 mg/kg) or vehicle orally for 3 days, then fasted for 4 hours. Insulin (0.75 U/kg) was injected intraperitoneally, and blood glucose was measured at 0, 15, 30, 60, 120 minutes post-insulin to calculate glucose AUC [2] |
| ADME/Pharmacokinetics |
1. In male CD-1 mice, oral administration of CHIR-98014 (10 mg/kg) showed an oral bioavailability of ~30%. The peak plasma concentration (Cₘₐₓ) was ~85 ng/mL, achieved at ~1 hour (Tₘₐₓ) post-dosing. The elimination half-life (t₁/₂) was ~2.1 hours. Tissue distribution analysis showed that the drug accumulated in the liver (peak concentration ~240 ng/g) and skeletal muscle (peak concentration ~120 ng/g) at 1 hour post-dosing [2] |
| Toxicity/Toxicokinetics |
1. In acute toxicity studies in CD-1 mice, single oral doses of CHIR-98014 up to 100 mg/kg caused no mortality or overt signs of toxicity (e.g., lethargy, ataxia) over 7 days. Body weight gain was similar between drug-treated and vehicle groups [2] 2. In the 7-day chronic study in ob/ob mice (1-10 mg/kg, oral), CHIR-98014 had no significant effect on serum alanine transaminase (ALT), aspartate transaminase (AST), creatinine, or urea nitrogen (markers of liver and kidney function) compared to vehicle [2] 3. Plasma protein binding of CHIR-98014 was ~95% in mouse, rat, and human plasma (measured via equilibrium dialysis) [2] |
| References |
[1]. Diabetes. 2003 Mar;52(3):588-95. [2]. Br J Pharmacol. 2007 Nov;152(6):959-79. |
| Additional Infomation |
CHIR-98014 is a member of the class of aminopyrimidines that is pyrimidine substituted by {2-[(6-amino-5-nitropyridin-2-yl)amino]ethyl}amino, 2,4-dichlorophenyl, and 1H-imidazol-1-yl groups at positions 2, 4 and 5, respectively. It is a potent ATP-competitive inhibitor of GSK3alpha and GSK3beta (IC50 values of 0.65 and 0.58 nM, respectively). It has a role as an EC 2.7.11.26 (tau-protein kinase) inhibitor, an apoptosis inducer, an antineoplastic agent, a hypoglycemic agent, a Wnt signalling activator and a tau aggregation inhibitor. It is a secondary amino compound, a dichlorobenzene, a member of imidazoles, a diaminopyridine, an aminopyrimidine and a C-nitro compound. 1. CHIR-98014 exerts its anti-diabetic effects primarily by inhibiting GSK3: inhibition of GSK3 reduces phosphorylation of GS (activating GS to promote glycogen synthesis) and enhances insulin signaling via increased Akt phosphorylation, thereby improving glucose uptake and glycogen storage in muscle and adipose tissue [2] 2. The activation of the Wnt/β-catenin pathway by CHIR-98014 (via β-catenin stabilization) suggests potential off-target effects, but in diabetic models, the dominant therapeutic effect is mediated by GSK3 inhibition in metabolic tissues (muscle, liver, adipose) [2] 3. CHIR-98014 shows advantages over earlier GSK3 inhibitors (e.g., SB216763) due to its higher potency (lower IC₅₀ for GSK3) and selectivity for GSK3 over other kinases, reducing the risk of off-target toxicities [2] |
Solubility Data
| Solubility (In Vitro) |
DMSO: ~8 mg/mL (~16.5 mM) Water: <1 mg/mL Ethanol: <1 mg/mL |
| Solubility (In Vivo) | 10%DMSO+90%corn oil: 1.1mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0563 mL | 10.2815 mL | 20.5630 mL | |
| 5 mM | 0.4113 mL | 2.0563 mL | 4.1126 mL | |
| 10 mM | 0.2056 mL | 1.0282 mL | 2.0563 mL |