J. Xiang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2865–2869
2869
carbons (5) and carbon types were confirmed through
direct carbon observation and DEPT analysis. The chem-
References and notes
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1
17. All final compounds were characterized by H NMR and
either HRMS, LC/MS, or CHN.
18. CHO cells were stably transformed with plasmid contain-
ing the full-length sequence of human 11b-HSD1 (Locu-
slink i.d. 3290) to generate the CHO-HSD1 cell line.
CHO cells were stably transformed with plasmid con-
taining the full-length sequence of human 11b-HSD2
(Locuslink i.d. 3291) to generate the CHO-HSD2 cell
line. Non-transformed CHO cells do not have detectable
11b-HSD1 or 11b-HSD2 activity. Cell-based assays for
11b-HSD1 were performed using the CHO-HSD1 cell
line. The effect of test article on 11b-HSD1 activity was
assayed by determination of the conversion of radioactive
cortisone to cortisol. In this assay, 50,000 CHO-HSD1
cells were plated in 24 well-plates and incubated over-
night. The cells were washed once with ethylene glycol
dimethyl ether and 197.5 lL ethylene glycol dimethyl
ether was added to each well. Appropriate drug concen-
trations (2.5 lL) were added to each well and the cells
were incubated for 30 min at 37 °C/5% CO2. [1,2-3H]-
cortisone was diluted to a concentration of 200 nM in
ethylene glycol dimethyl ether and 50 lL of the 200 nM
solution was added to each well. Final cortisone concen-
tration in the assay was 40 nM. Cells were incubated for
2 h at 37 °C/5% CO2. At the end of the incubation,
steroids in media were extracted with 3 vol of ethyl
acetate. The organic phase was transferred to a fresh
tube, dried, resuspended in 5 lL methanol, and spotted
˚
on a 60 A silica TLC plate. The plate was run in 92%
chloroform/8% ethanol. The plate was dried and scanned
using an AR-2000 TLC Imaging Scanner (BioScan Inc.,
Washington DC). IC50 values were determined by plot-
ting cortisol formed against inhibitor concentration and
fitting the data to using Origin 7.0 software (Origin Lab
Corporation, Northampton, MA).
Inhibition of 11b-HSD2 was determined using the same
assay, except that the CHO-HSD2 line was used and
[1,2-3H]-hydrocortisone was used as the substrate.
19. Male C57 mice were orally dosed with the testing
compound at a single dose of 50 mg/kg in 2% Tween 80
and 0.5% methyl cellulose. Plasma concentrations of the
testing compound were measured by LC–MS/MS. The
area under curve (AUC) was calculated by a non-
compartmental method with WinNonlin Software (ver-
sion 4.1).
16. Structure determination of (1-(5-oxo-2,5-dihydrofuran-3-
yl)urea) 4: H NMR (400 MHz, DMSO-d6): 5.10 (s, 2H),
1
5.33 (s, 1H), 6.51 (br s, 2H), 9.60 (s, 1H). 13C NMR
(DMSO-d6): 69.4, 91.3, 154.8, 163.6, 174.8. The number of