A R T I C L E S
Jung et al.
mL) and dried over anhydrous sodium sulfate, and the solvent was
removed in vacuo. The crude product was further purified by
column chromatography on alumina B-10 (eluant: ethyl acetate:
20 and 5 µM of 1 in H
2
O/DMSO solution (9/1, v/v) with varying
concentrations of the metal chloride salts. After the concentrations
of the free ligands and complexed forms of 1 and 2 were calculated
from the fluorescence titration experiments, the association constants
dichloromethane, 1:5 v/v) to afford a yellow solid (2.12 g, 60%):
1
16
mp 170 °C. H NMR (200 MHz, CDCl
3
(
3
): δ 1.21-1.28 (m, 6H),
were obtained using the computer program ENZFITTER.
.44-3.48 (m, 4H), 4.80 (d, 2H), 6.5 (d, 1H), 6.65 (d, 1H), 7.18
Cell Incubation. Two cell lines (LLC-MK2, and NCTC clone
1469 cells, respectively) for fluorescence imagines were used. Cell
lines were prepared from continuous culture in Dulbeccos modified
Eagles medium (GibcoBRL, USA), supplemented with 10% (v/v)
heat-inactivated fetal calf serum (HyClone), 100 µg/mL penicillin,
100 µg/mL streptomycin, and 0.25 mM L-glutamine, at 37 °C in
m, 1H), 7.30 (m, 1H), 7.43 (m, 1H), 7.65 (m, 1H), 8.60 (d, 1H),
8
2
1
1
.74 (s, 1H), 9.53 (s, 1H). 13C NMR (50 MHz, CDCl
): δ 12.42,
3
8.76, 45.07, 45.32, 96.66, 100.00, 108.41, 109.92, 110.33, 121.57,
22.09, 128.20, 131.14, 136.64, 148.24, 149.37, 152.59, 157.68,
57.74, 163.40. FAB-MS calcd for C20
found 351.00.
+
H
21
N
3
O
3
[M + H] 351.41,
2
5.0% CO humidified air. When the cells reached the logarithmic
5
Analogous procedures starting with coumarin acids with amino
derivatives gave 2 and 3.
phase, the cell density was adjusted to 1.0 × 10 per/well in culture
media. The cells were then used to inoculate in a glass bottom dish,
with 1.0 mL of cell suspension in each dish. After cell adhesion,
culture medium was removed. The cell layer was rinsed twice with
phosphate buffered saline (PBS), and then 1.0 mL of culture
medium was added in each dish.
7
-(Diethylamino)-2-oxo-N-((pyridin-3-yl)methyl)-2H-chromene-
1
3
-carboxamide (2). (2.24 g, 64%): mp 190-192 °C. H NMR (200
MHz, CDCl ): δ 1.21-1.28 (m, 6H), 3.44-3.48 (m, 4H), 4.80 (d,
H), 6.5 (d, 1H), 6.65 (d, 1H), 7.25 (m, 1H), 7.42 (d, 1H), 7.72 (d,
3
2
1
1
3
2+
H), 8.51 (d, 1H), 8.61 (s, 1H), 8.74 (s, 1H), 9.23 (s, 1H).
): δ 12.42, 34.12, 45.07, 96.48, 108.28,
09.62, 110.00, 123.46, 128.14, 131.19, 134.17, 135.38, 148.41,
48.63, 149.22, 152.65, 157.67, 162.75, 163.45, 177.86. FAB-MS
[M + H]+ 351.41, found 351.00.
-(Diethylamino)-2-oxo-N-((pyridin-4-yl)methyl)-2H-chromene-
C
Confocal Fluorescence Images with Intracellular-Cu . To
determine the cell permeability of Cu , all cell lines were incubated
2+
NMR (50 MHz, CDCl
3
1
1
with 1 (5 µM) for 1 day at room temperature. Upon addition of 5
2
+
equiv of Cu into the cell line, the fluorescence image of
2+
calcd for C20
H
21
N
3
O
3
intracellular Cu was observed under a Zeiss LSM 510 META
7
confocal microscope. Excitation wavelength of laser was 405 nm,
and emission spectra were integrated over the range 450-520 nm
1
3
-carboxamide (3). (2.00 g, 57%): mp 210-212 °C. H NMR (200
MHz, CDCl ): δ 1.21-1.28 (m, 6H), 3.44-3.48 (m, 4H), 4.68 (d,
H), 6.53 (d, 1H), 6.68 (d, 1H), 7.19 (m, 1H), 7.35 (d, 1H), 7.43
(
single channel). For all images, the confocal microscope settings,
3
such as transmission density, brightness, contrast, and scan speed,
were held constant to compare the relative intensity of intracellular
Cu fluorescence.
2
(
1
3
d, 1H), 8.56 (d, 2H), 8.74 (s, 1H), 9.30 (s, 1H). C NMR (50
MHz, CDCl ): δ 12.38, 33.93, 45.10, 96.53, 108.32, 109.52, 110.08,
22.20, 122.21, 131.27, 139.95, 147.63, 148.56, 148.56, 149.96,
49.97, 152.74, 157.74, 162.87, 163.63. FAB-MS calcd for
[M + H]+ 351.41, found 351.01.
DFT Calculations. Density functional theory (DFT) calculations
2
+
3
MTT Assay. LLC-MK2 cell lines were inoculated into a 96-
well, flat-bottomed microplate (Nunc, DNK) at a volume of 100
µL (5 × 104 cells/mL) for a stationary culture. The media were
exchanged into new fresh media. The cells were treated in the 2-fold
1
1
20 21 3 3
C H N O
2
+
down dilution series of 1 (50 µM), Cu (50 µM), and 1 (50 µM)
with Becke’s three parametrized Lee-Yang-Par (B3LYP) ex-
2
+
plus each well into Cu (50 µM) and then incubation in the 5%
CO2 at 37 °C, for 1 day. Cells were added to MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] solution (2
change functionals with 6-31G* basis sets were carried out for the
2
+
geometry optimizations of 1 and 1-Cu complex using a suite of
Gaussian 03 programs. The time-dependent DFT (TDDFT) calcula-
tions were performed to have the excitation energies and oscillator
strengths at the optimized geometries.
2
mg/mL, Sigma). After an incubation in 5% CO at 37 °C, for 4 h,
the solution was changed into 150 µL of dimethysulfoxide (DMSO:
Kanto, Japan) and shaken in a microplate mixer (Amersham, UK)
for 10 min. The optimal density (OD) value was measured by a
microplate reader (BIO-RAD 550, CA) using 540 nm wavelength.
The cell viability was calculated by the following formula: (mean
OD in treated wells ÷ mean OD in control wells) × 100.
Time-Resolved Spectroscopy. Time-resolved fluorescence was
measured by noncollinear fluorescence upconversion technique
1
5
described elsewhere. Light source was a home-built cavity
dumped Ti:sapphire oscillator pumped by a frequency doubled Nd:
4
YVO laser (Verdi, Coherent Inc.). The center wavelength of the
oscillator output was 830 nm. Pump pulses at 415 nm were
generated by the second harmonic generation in a 200 µm thick
lithium-triborate (LBO) crystal. The energy of pump pulse was
about 4 nJ at 380 kHz. The time resolution was 100 fs for the 500
µm thick ꢀ-barium borate (BBO) mixing crystal. The sample
concentration was set to 100 µM to give absorbance of 0.1 in a
Acknowledgment. This work is supported by the KOSEF Grant
funded by MOST (R11-2007-012-03002-0) (2008) and SRC (R11-
2
005-008-02001-0(2008)).
Supporting Information Available: Synthetic details, ad-
ditional NMR, UV, and emission spectral data, MTT assay, and
pH span. This material is available free of charge via the Internet
at http://pubs.acs.org.
2
00 µm cuvette.
Absorption and Fluorescence Spectra. Stock solutions (1.00
mM) of the metal chloride salts were prepared in water. Stock
solutions of 1 (0.3 mM) were prepared in H O/DMSO solution (9/
, v/v). For all measurements of fluorescence spectra, excitation
2
JA808611D
1
was at 430 nm with excitation and emission slit widths at 3.0 nm.
UV/vis and fluorescence titration experiments were performed using
(
16) (a) Association constants were obtained using the computer program
ENZFITTER, available from Elsevier-BIOSOFT, 68 Hills Road,
Cambridge CB2 1LA, UK. (b) Connors, K. A. Binding Constants;
Wiley: New York, 1987.
(
15) Rhee, H.; Joo, T. Opt. Lett. 2005, 30, 96.
2
012 J. AM. CHEM. SOC. 9 VOL. 131, NO. 5, 2009