6492
D. K. Luci et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6489–6492
Halawa, N.; Giaid, A. Lancet 2002, 359, 1990; (c) Totsune,
Compd 12i (300 ug/kg)
UII (3 ug/kg) + iv vehicle
UII + Compd 12i
10
K.; Takahashi, K.; Arihara, Z.; Sone, M.; Satoh, F.; Ito,
S.; Kimura, Y.; Sasano, H.; Murakami, O. Lancet 2001,
358, 810.
5. (a) Jin, J.; Dhanak, D.; Knight, S. D.; Widdowson, K.;
Aiyar, N.; Naselsky, D.; Sarau, H. M.; Foley, J. J.;
Schmidt, D. B.; Bennett, C. D.; Wang, B.; Warren, G. L.;
Moore, M. L.; Keenan, R. M.; Rivero, R. A.; Douglas, S.
A. Bioor. Med. Chem. Lett. 2005, 15, 3229; (b) Douglas, S.
A.; Behm, D. J.; Aiyar, N. V.; Naselsky, D.; Disa, J.;
Brooks, D. P.; Ohlstein, E. H.; Gleason, J. G.; Sarau, H.
M.; Foley, J. J.; Buckley, P. T.; Schmidt, D. B.; Wixted,
W. E.; Widdowson, K.; Riley, G.; Jin, J.; Gallagher, T. F.;
Schmidt, S. J.; Ridgers, L.; Christmann, L. T.; Keenan, R.
M.; Knight, S. D.; Dhanak, D. Br. J. Pharmacol. 2005,
145, 620; (c) Clozel, M.; Hess, P.; Qiu, C.; Ding, S. S.; Rey,
M. J. Pharmacol. Exp. Ther. 2006, 316, 1115; (d) Caro-
tenuto, A.; Grieco, P.; Rovero, P.; Novellino, E. Curr.
Med. Chem. 2006, 13, 267.
5
0
6. Calcium mobilization. Calcium mobilization was measured
on the Molecular Devices FLIPR (Fluorescence Imaging
Plate Reader) instrument in CHOK1 cells transfected with
the rat UT receptor and loaded with Calcium Assay Kit
(Molecular Devices). The test compound was added
followed by 5 nM of U-II agonist peptide 1 and inhibition
of response seen in the absence of compound was
measured. IC50s were calculated by using a log-logit plot
of percent inhibition. See Ref. 2b for further details.
7. 4-Phenyl-piperidine-1,4-dicarboxylic acid mono-tert-butyl
ester was purchased from Arch Chemicals, Inc., and 4-(4-
chloro-phenyl)-piperidine-1,4-dicarboxylic acid mono-
tert-butyl ester was purchased from Chem-Impex
International, Inc.
8. Eastwood, P. R. Tetrahedron Lett. 2000, 41, 3705.
9. Whole cell binding. I-125 U-II binding was determined in
RMS13 cells. Compounds and I-125 U-II were co-incu-
bated for 2.5 h. Following washing, the samples were
solubilized with 1% SDS in 0.5 N NaOH and radioactivity
determined. GraphPad Prism was used to analyze the
binding data and Ki calculated via the Cheung–Prussoff
equation. See Ref. 10 for further details.
10. Qi, J.-S.; Minor, L. K.; Smith, C.; Hu, B.; Yang, J.;
Andrade-Gordon, P.; Damiano, B. Peptides 2005, 26, 683.
11. Purified by RP HPLC (gradient elution with 10–60%
acetonitrile in water, each with 0.1% TFA) and lyophilized
to yield Compound 12i as white solid (trifluroacetate salt,
0.26 g, 72%). 1H NMR (300 MHz, DMSO) d 7.58–7.53
(m, 2H), 7.37 (d, J = 8.5 Hz, 1H), 7.30 (m, 1H), 7.12–7.05
(m, 3H), 4.70 (s, 2H), 4.3–3.1 (m, 21H), 2.0–1.8 (m, 4H),
1.42 (s, 9H); MS (ES+) m/z 626.1 (M+1); Anal. Calcd for
C33H44ClN5O5–3.6CF3CO2H: C, 46.58; H, 4.63; N, 6.76.
Found: C, 46.25, H, 4.48; N, 6.73.
12. An optimized rat U-II homology model was constructed
by using bovine rhodopsin X-ray crystal structure (PDB
code 1L9H) as a template and following the same
procedure described in Ref. 2b Molecular docking into
the homology model was done using Glide (Schrodinger
LLC, Portland, OR) with OPLS_2005 force field.
Feature constraint was employed in docking, using
Asp-130 as an anchor point. A basic nitrogen feature
has to be fitted into the proximity of the carboxylic acid
oxygen atoms.
0
10
20
30
40
50
Time (min)
(N=5)
Figure 3. Effects of 12i on ear pinna temperature changes. Compound
12i was administered intravenously 15 min prior to the administration
of U-II at a dose of 300 lg/kg. Data are mean temperature changes
from baseline SEM (n = 5 per group).
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