Molecular Weight (MW)

519.28

Formula

C20H20BrN3O2S.2HCl

CAS No.

130964-39-5

Storage

-20℃ for 3 years in powder form

-80℃ for 2 years in solvent

Solubility (In vitro)

DMSO: 104 mg/mL (200.3 mM)

Water: 6 mg/mL (11.6 mM)

Ethanol: <1 mg/mL

Solubility (In vivo)

1% DMSO+30% polyethylene glycol+1% Tween 80: 30mg/mL

Synonyms

Synonym: H-89; H-89 Dihydrochloride; H89; H 89 HCl.

Chemical Name: N-[2-[[3-(4-Bromophenyl)-2-propen-1-yl]amino]ethyl]-5-Isoquinolinesulfonamide dihydrochloride

InChi Key: GELOGQJVGPIKAM-WTVBWJGASA-N

InChi Code: InChI=1S/C20H20BrN3O2S.2ClH/c21-18-8-6-16(7-9-18)3-2-11-22-13-14-24-27(25,26)20-5-1-4-17-15-23-12-10-19(17)20;;/h1-10,12,15,22,24H,11,13-14H2;2*1H/b3-2+;;

SMILES Code: O=S(C1=CC=CC2=C1C=CN=C2)(NCCNC/C=C/C3=CC=C(Br)C=C3)=O.[H]Cl.[H]Cl

In vitro activity: H89 2HCl is a potent PKA (cAMP-dependent) protein kinase A inhibitor with Ki of 48 nM, exhibits 10-fold selectivity over PKG, exhibits>500-fold selectivity over PKC, MLCK, calmodulin kinase II and casein kinase I/II. Pretreatment of the cells with H-89 (30 μM) 1 h before the addition of forskolin markedly inhibits the forskolin-induced protein phosphorylation in a dose-dependent manner. H89 also inhibits several other kinases with IC50 of 80, 120, 135, 270, 2600 and 2800 nM for S6K1, MSK1, PKA, ROCKII, PKBα and MAPKAP-K1b, respectively. H89 also has activity at some cellular receptors and ion channels, including Kv1.3 K+ channels,β1AR and β2AR.

Kinase Assay: PKA enzyme activity: cAMP-dependent protein kinase activity is assayed in a reaction mixture containing, in a final volume of 0.2 mL, 50 mM Tris-HC1 (pH 7.0), 10 mM magnesium acetate, 2 mM EGTA, 1 μM cAMP or absence of cAMP, 3.3-20 μM [γ-32P]ATP (4 × 105 cpm), 0.5 μg of the enzyme, 100 μg of histone H2B, and each compound, as indicated.

Cell Assay: Levels of intracellular cAMP are determined. After 48 h in culture, PC12D cells are cultured in test medium containing 30 μM H-89 for 1 h and then exposed to fresh medium that contained both 10 μM forskolin and 30 μM H-89. Cells are scraped off with a rubber policeman and sonicated in the presence of 0.5 ml of 6% trichloroacetic acid. To extract trichloroacetic acid, 2 ml of petroleum ether is added, the preparation mixed and centrifuged at 3000 rpm for 10 min. After aspiration of the upper layer, the residue sample solution is used for determination.

H89 causes distinct modifications of protein phosphorylation, with the most robust changes in phosphorylation are fructose-1,6-biphosphatase, heterogeneous nuclear ribonucleoprotein (hnRNP), NSFL1 cofactor p47, all which have potentially regulatory connections to cAMP/PKA. H-89 (0.5, 1, 5 mg/kg) significantly attenuates prominent behavioral signs of morphine withdrawal in morphine-dependent mice.

Animal model

Mouse and rat

Formulation & Dosage

Dissolved in 100% DMSO, diluted 1:20 in 0.9% sterile saline (Rat); 1% DMSO (Mice); 20 or 200 mg/kg (Rat); 0-5 mg/kg (Mice); s.c. (rat); i.p. (mice)

References

J Biol Chem. 1990 Mar 25;265(9):5267-72; Cardiovasc Drug Rev. 2006 Fall-Winter;24(3-4):261-74; J Pharmacol Exp Ther. 2006 Aug;318(2):589-95; Sci Signal. 2008 Jun 3;1(22):re4.