1
Merclineritant data: H NMR (500 MHz, d6-DMSO): 12.39 (1H,
quadruplicate. Drug concentrations were made from serial
dilutions in media, from a stock solution of the drug dissolved in
an organic solvent or water vehicle. Controls were set as the
highest vehicle concentration used, typically 0.1% solvent. At the
relevant time points, CellTiter 96® Aqueous One Solution
reagent (Promega) (20 μL) was added directly to cultured wells
with minimal exposure to light. Plates were incubated for 1-2 h
(37 °C, 5% CO2) and the formazan absorption was measured at
492 nm using an anthoslucy2 spectrometer. The mean
absorbance of drug-treated wells was displayed as optical density
or expressed as a percentage of control to estimate the
proliferation status:
br. s, C-1’’ COOH), 8.91 (1H, d, J = 4.3 Hz, H-9), 8.15 (1H, d, J
= 1.1 Hz, H-16), 7.78 (1H, d, J = 8.9 Hz, H-13), 7.68 (1H, dd, J =
1.1, 8.9 Hz, H-14), 7.54 (1H, br. s, C-2’’NH), 7.28 (1H, d, J = 4.3
Hz, H-10), 7.24 (1H, t, J = 8.4 Hz, H-1), 6.92 (1H, s, H-6), 6.53
(2H, d, J = 8.4 Hz, H-2), 3.42 (6H, s, H-3’), 2.59 (2H, s, H-3’’),
2.10 (2H, m, H-4’’/6’’), 1.99 (2H, m, H-4’’/6’’), 1.77 (2H, m, H-
5/8), 1.60-1.70 (4H, m, H-7’’/4’’/6’’), 1.58 (2H, m, H-4’’/6’’);
LCMS: Rt = 5.09, [MH]+ = 587.38; IR (neat) cm-1: 3405.7 (w),
2910.8 (m), 2860.2 (w), 1728.5 (m), 1666.9 (s), 1592.5 (m);
HRMS: Calcd. for [C32H32N4O5Cl]+ = 587.2061, found 587.2065,
= 0.7 ppm
1
Data as the iso-propyl solvate: H NMR (500 MHz, d6-DMSO):
Percentageofcontrol(%)=(Absorbancetreatmentgroup-
12.37 (1H, br. s, C-1’’ COOH), 8.92 (1H, d, J = 4.6 Hz, H-9),
8.15 (1H, d, J = 2.1 Hz, H-16), 7.78 (1H, d, J = 9.1 Hz, H-13),
7.68 (1H, dd, J = 2.1, 9.1 Hz, H-14), 7.59 (1h, br. s, C-2’’NH),
7.29 (1H, d, J = 4.6 Hz, H-10), 7.24 (1H, t, J = 8.5 Hz, H-1), 6.93
(1H, s, H-6), 6.53 (2H, d, J = 8.5 Hz, H-2), 4.34 (1H, d, J = 4.1
Hz, i-PrOH), 3.77 (1H, m, i-PrOH CH), 3.42 (6H, s, H-3’), 2.59
(2H, s, H-3’’), 2.10 (2H, d, J = 12.0 Hz, H-4’’/6’’), 1.99 (2H, d, J
= 12.5 Hz, H-4’’/6’’), 1.77 (2H, m, H5’’/8’’), 1.60-1.70 (4H, m,
H-7’’/4’’/6’’), 1.59 (2H, d, J = 12.5 Hz, H-4’’/6’’), 1.04 (6H, d, J
= 6.1 Hz, i-PrOH CH3). 13C NMR (125 MHz, d6-DMSO): 173.3
(C-1’’), 160.1 (C-8), 157.5 (C-3), 151.8 (C-9), 149.1 (C-17),
148.0 (C-7), 143.4 (C-11), 139.2 (C-5), 134.7 (C-15), 131.9 (C-
1), 128.0 (C-14), 127.7 (C-16), 125.9 (C-13), 122.2 (C-12), 118.5
(C-10), 109.7 (C-6), 105.7 (C-4), 104.0 (C-2), 62.7 (C-2’’), 62.0
(HOCH(CH3)2), 55.4 (C-3’), 37.3 (C-7’’), 33.4 (C-4’’/6’’), 32.6
(C-4’’/6’’), 31.7 (C-3’’), 26.4 (C-5’’/8’’), 26.1 (C-5’’/8’’), 25.5
(HOCH(CH3)2); IR (neat) cm-1: 3403.3 (w), 2924.7 (m), 2860.6
(w), 1724.2 (m), 1673.9 (s), 1591.1 (m); HRMS: Calcd. for
[C32H32N4O5Cl]+ = 587.2061, found 587.2084, = 3.9 ppm. The
Absorbanceblank)(Absorbancecontrol-Absorbanceblank)x100%
Absorbance treatment group: HUVECs + medium + Drug +
MTS reagent
Absorbance blank: medium + MTS reagent
Absorbance control: HUVECs + medium + MTS reagent
Values were expressed as the mean ± the standard deviation
(SD). Where necessary, one obvious outlying result was
removed.
4. Acknowledgments
We gratefully acknowledge funding from the EPSRC (MOK),
the BP 1702 endowment (SVL), and the Royal Society (IRB). X-
ray crystal structures were determined by Dr John Davies at the
Department of Chemistry, University of Cambridge.
References and notes
structure
was
unambiguously
confirmed
by
X-ray
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crystallography and the structure deposited at the CCDC with the
unique reference 913975.
3.2. Biology
3.2.1. Materials and Methods
Unless otherwise stated, all chemicals were purchased from
Sigma-Aldrich. Casodex® (Bicalutamide) was purchased from
Toronto Research Chemicals Inc., SR142948 was purchased
from Tocris biosciences, LNCaP cells were available from
CRUK cell line bank and the American Type Culture Collection
(ATTC)®. LNCaP C4-2 and C4-2b were a kind gift from Dr D.
Prowse, Centre for Cutaneous Research, Barts and The London,
Queen Mary's School of Medicine and Dentistry. LNCaP-
bicalutamide resistant (LNCaP-Bic) cells were a kind donation
from Prof. Z. Culig, Department of Urology, University of
Innsbruck.
5. Dobner, P. R. Peptides 2006, 27, 2405–2414.
6. a) McCann, S. M., Vijayan, E., Koenig, J., Krulich, L. Ann. N. Y.
Acad. Sci. 2006, 400, 160–171; b) Aronin, N., Coslovsky, R.,
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Cancer 2001, 33, 1–9.
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Attoub, S., Gespach, C., Gompel, A., Forgez, P. Cancer Res.2006,
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1998, 42, 546–550.
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3.2.2. Cell culture
LNCaP cells were maintained in RPMI-1640 with L-
Glutamine media (Invitrogen™) supplemented with 10% FBS
(Hyclone). Frozen cells were thawed in a 37°C water bath,
resuspended in pre-warmed (37°C) media and centrifuged (1,300
g, 3 min, 21°C). The supernatant was discarded and the pellet
was resuspended thoroughly before seeding into culture flasks.
Cells were routinely subcultured 1:2 or 1:3 2-3 times a week
using .05% Trypsin-EDTA (Invitrogen™). Cell counts were
conducted using a haemocytometer.
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16. Agreement for a standard contraction to represent the neurotensin
receptors has yet to be reached. Numerous studies contract
Neurotensin to NTS and Neurotensin receptor 1 to NTSR1.
3.2.3. CellTiter 96® AQueous One Solution Cell
Proliferation assay
Cells were seeded at a density of 3,000 cells / 100μL / well. 24 h
after seeding, the media was aspirated and replaced with the
relevant treatments. All experiments were produced in