GSK-3 inhibitor 4 is an orally bioactive and brain-penetrating inhibitor of GSK-3, CDK2 and CDK5, with IC50s of 0.56 nM (GSK-3β), 0.45 nM (GSK-3α) and 0.47 μM, respectively. 0.68 μM. GSK-3 inhibitor 4 can effectively reduce Tau protein levels. GSK-3 inhibitor 4 may be utilized in AD/Alzheimer's disease study (AD).

Physicochemical Properties

Molecular Formula	C22H15F2N5O
Molecular Weight	403.384210824966
Exact Mass	403.124
CAS #	2227279-83-4
PubChem CID	134556675
Appearance	Typically exists as solid at room temperature
LogP	4
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	5
Heavy Atom Count	30
Complexity	552
Defined Atom Stereocenter Count	0
SMILES	FC1C=CC(=CC=1)C1C=CN=CC=1NC(C1C=CN=C(N=1)NC1C=CC(=CC=1)F)=O
InChi Key	UWQKULXATKCNBV-UHFFFAOYSA-N
InChi Code	InChI=1S/C22H15F2N5O/c23-15-3-1-14(2-4-15)18-9-11-25-13-20(18)28-21(30)19-10-12-26-22(29-19)27-17-7-5-16(24)6-8-17/h1-13H,(H,28,30)(H,26,27,29)
Chemical Name	2-(4-fluoroanilino)-N-[4-(4-fluorophenyl)pyridin-3-yl]pyrimidine-4-carboxamide
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-3α 0.45 nM (IC50) GSK-3β 0.56 nM (IC50) CDK2 0.47 μM (IC50) CDK5 0.68 μM (IC50)
ln Vitro	Compound 40, also known as GSK-3 inhibitor 4, demonstrates remarkable selectivity and is effective against CDK2 (840-fold, IC50 = 0.47 μM), CDK5 (1200-fold, IC50 = 0.68 μM), GSK-3β (IC50 = 0.56 nM), and GSK-3α (IC50 = 0.45 nM) [1]. Because GSK-3 inhibitor 4 is lipophilic, it binds to brain tissue and plasma proteins with high affinity and good permeability [1].
ln Vivo	The oral administration of a 10 mg/kg dosage of the nanosuspension decreases pTau396 by 37% when GSK-3 inhibitor 4 (10 mg/kg, po) is used [1]. When taken orally as a solution, GSK-3 inhibitor 4 (2 mg/kg, iv; 10 mg/kg, po) is effectively absorbed and shows low-moderate clearance, ranging from 15.8 to 23.3 mL/min/kg [1].
Animal Protocol	Animal/Disease Models: 3xTg mice were bred onto the C57BL6 background and bred as homozygotes. Male animals between the ages of five to seven months were used for the study[1]. Doses: 10 mg/kg Route of Administration: po (oral gavage) Experimental Results: Produced a 37% reduction in pTau396, when administered orally as a nanosuspension at a dose of 10 mg/kg. Animal/Disease Models: male C57BL6 mice (pharmacokinetic/PK assay) [1] Doses: 2 mg/kg, 10 mg/kg. Route of Administration: Kept for 8 h fasting prior to formulation administration. intravenous (iv)and oral routes of administration to mice at a dose of 2 and 10 mg/kg, respectively. Experimental Results: demonstrated clearance ranging from 15.8 to 23.3 mL/min/kg, halflives ranging from 2.5 to 2.9 h and were readily orally bioavailable (F% = 53).
References	[1]. Discovery of 2-(Anilino)pyrimidine-4-carboxamides as Highly Potent, Selective, and Orally Active Glycogen Synthase Kinase-3 (GSK-3) Inhibitors. J Med Chem. 2023 Jun 8;66(11):7534-7552.

Solubility Data

Solubility (In Vitro)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Solubility (In Vivo)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *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. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.4791 mL	12.3953 mL	24.7905 mL
	5 mM	0.4958 mL	2.4791 mL	4.9581 mL
	10 mM	0.2479 mL	1.2395 mL	2.4791 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.