Molecular Weight (MW)

453.52

Formula

C21H23N7O3S

CAS No.

486424-20-8

Storage

-20℃ for 3 years in powder form

-80℃ for 2 years in solvent

Solubility (In vitro)

DMSO: 7 mg/mL (15.43 mM)

Water: <1 mg/mL

Ethanol: <1 mg/mL

Solubility (In vivo)

30% PEG400+0.5% Tween80+5% propylene glycol: 30 mg/mL

Synonyms

AZD-2858; AZD2858; AZD 2858

Chemical Name: 3-amino-6-[4-[(4-methyl-1-piperazinyl)sulfonyl]phenyl]-N-3-pyridinyl-2-pyrazinecarboxamid

InChi Key: FHCSBLWRGCOVPT-UHFFFAOYSA-N

InChi Code: InChI=1S/C21H23N7O3S/c1-27-9-11-28(12-10-27)32(30,31)17-6-4-15(5-7-17)18-14-24-20(22)19(26-18)21(29)25-16-3-2-8-23-13-16/h2-8,13-14H,9-12H2,1H3,(H2,22,24)(H,25,29)

SMILES Code: NC1=NC=C(C2=CC=C(S(N3CCN(C)CC3)(=O)=O)C=C2)N=C1C(NC4=CC=CN=C4)=O

In Vitro

In vitro activity: AZD2858 is a selective GSK-3 inhibitor with an IC50 of 68 nM, inhibits tau phosphorylation at the S396 site, activates Wnt signaling pathway. AZD2858 treatment (1 μM, 12 h) on primary isolated human osteoblast-like cells results in a 3-fold increase of β-catenin levels. AZD2858 causes β-catenin stabilisation in human and rat mesenchymal stem cells, stimulates hADSC commitment towards osteoblasts and osteogenic mineralisation in vitro.

Kinase Assay: Tau phosphorylation assay: NIH-3T3 cells expressing 4-repeat Tau are used to assess functional activity of AZD2858 in vitro. The cells are grown in DMEM media and 2 mM L-glut, and 10% HiFCS, and plated at a concentration of 6×105 cells/well in 6-well plates. In each experiment, AZD2858 is dosed in triplicates at a concentration of 1, 10, 100, 500, 1000, 2000 and 10,000 nM. Cells are treated for 4 h prior to cell lysis using 100 μL ice cold lysis buffer (0.5% NP-40, 10 mM Tris, pH 7.2, 150 mM NaCl, 2 mM EDTA). A suspension is made with addition of protease and phosphatase inhibitors: 50 mM NaF, 0.2 mM NaVO4 and Cocktail Protease inhibitors. The solution is then snap frozen at – 80 °C for at least 1 h, before thawing on ice and lysate clarification by centrifugation, followed by Western blot according to standard protocols. After blocking, the blots are exposed to the primary antibody, Phospho-Ser396-tau (1:1000) over night, washed and incubated with the secondary antibody (donkey anti-rabbit, 1:5000), followed by a final wash. For re-probing, the primary antibody Tau5 (1:200) and the secondary horseradish peroxidase linked antibody (sheep anti-mouse, 1:10000) are used. All blots are developed using ECL Western blot detection reagents, Kodak X-ray films, quantified using densitometric analysis, and the ratio of S396 tau to total tau (tau5) is calculated.

Cell Assay: Human adipose derived stem cells and rat MSCs (isolated from bone marrow of Sprague Dawley rats at less than 8 weeks after gestation) are cultured in a basal media of DMEM containing 5% FBS and 2 mM GlutaMax. Cells are seeded in basal media into 96-well plates (3–5000 cells/well) for 18 h before treatment with AZD2858 (0.3 nM to 20 mM). After 24 h, β-catenin stabilisation is measured.

In Vivo

In rats, oral AZD2858 treatment causes a dose-dependent increase in trabecular bone mass compared to control after a two-week treatment with a maximum effect at a dose of 20 mg/kg once daily (total BMC: 172% of control). A small but significant effect is also seen at cortical sites (total BMC: 111% of control). AZD285 treatment (30 μmol/kg) on rats daily for up to 3 weeks shows an increase in both mineral density (of 28% at 2 weeks and 38% at 3 weeks) and mineral content (of 81% at 2 weeks and 93% at 3 weeks) in the calluses. AZD285 treatment makes the fractures heals more rapidly, with a bony callus without an obvious endochondral component. AZD2858 produces time-dependent changes in serum bone turnover biomarkers and increases bone mass over 28 days exposure in rats. After 7 days, AZD2858 increases the bone formation biomarker P1NP, and reduces the resorption biomarker TRAcP-5b, indicating increased bone anabolism and reduced resorption in rats.

Animal model

Rat

Formulation & Dosage

Dissolved in water adjusted to pH 3.5 ; 30 mg/kg; Oral gavage

References

Bone. 2012 Mar;50(3):619-27;Toxicol Appl Pharmacol. 2013 Oct 15;272(2):399-407; Bone. 2013 May;54(1):126-32.