(2S,3S,4S,5S)-2-((1S,2S,3R,4S,6R)-4,6-diamino-3-((2S,3R,4R,5S,6R)-3-amino-4,5-dihydroxy-6-(1-hydroxyethyl)oxan-2-yl)oxy-2-hydroxycyclohexyl)oxy-5-methyl-4-(methylamino)oxane-3,5-diol;sulfuric acid → Geneticin

The in vitro S1P1 agonist activity of the compound of the present invention was evaluated by the increase in the functional bonding activity into the G-Protein of a GTP[γ-35S] using the membrane of a human S1P1 expressing cell. A cDNA encoding a human S1P1 was cloned from a human colorectal cDNA library, and introduced to an expression vector pcDNA3.1 to construct a S1P1-pcDNA3.1. Then, by Lipofectamine 2000 (GIBCO), the S1P1-pcDNA3.1 was transfected into a CHO cell, and cultured in a Ham's F-12 culture medium containing 10% fetal bovine serum, 100 U/mL Penicillin, 100 μg/mL Streptomycin, and 1 mg/mL G418 disulfate, to obtain a stable, G418-resistant strain. The cultured human S1P1 expressing cells were isolated in a 1 mM EDTA/2Na-containing PBS, and disrupted under ice-cooling by a homogenizer made of glass in a 1 mM Tris HCl (pH 7.4) buffer solution containing 0.1 mM EDTA and a protein inhibitor. It was centrifuged at 1,400×10 min, and a supernatant was further centrifuged at 4° C. for 60 min at 100,000×g, and suspended in a 10 mM Tris HCl (pH 7.4) buffer solution containing 1 mM EDTA. The obtained membrane (0.13 mg/mL) and 50 pM GTP[γ-35S] (NEN; inactive 1250 Ci/mmol) were reacted in a 20 mM HEPES (pH 7.0) buffer solution (total amount: 150 μL) containing 100 mM NaCl, 10 mM MgCl2, 0.1% fatty acid-free BSA, and 5 μM GDP for 1 hour together with the compound of the present invention (10−12 to 10−5 M), and then a membrane was recovered on a GF-C plate with a Cell Harvester (Packard, FilterMate). The FilterMate was dried at 50° C. for 60 min, and Microscinti-o (Packard) was added thereto for measurement by a liquids scintillation counter for a microplate (Packard, TOP count). For evaluation of the human S1P1 agonist activity of the compound of the present invention and the comparative compound, the percentages with the rate of a maximum reaction to make the GTP[γ-35S] bonds saturated in the presence of the compound being set at 100%, and the rate of the reaction of the GTP[γ-35S] bonds in the absence of the compound being set at 0% was used, a non-linear regression curve was plotted, and a concentration to cause an agonist activity operating 50% of the maximum reaction was defined as an EC50 value (nM).

1. additionintroduced to an expression vector

ORD-Eintrag

ord-ecb429f915944f219d02dea9480c04ed

Quelldatensatz

uspto-grants-2011_05

DOI

10.6084/m9.figshare.5104873.v1

Patentnummer

US07951825B2