Journal of Medicinal Chemistry
BRIEF ARTICLE
OCH2), 3.85ꢀ3.89 (m, 10 H, OCH2), 3.69ꢀ3.72 (m, 10 H, OCH2),
3.56ꢀ3.66 (m, 20 H, OCH2), 3.47ꢀ3.54 (m, 10 H, OCH2), 3.35 (s, 3 H,
OCH3), 3.33 (s, 6 H, OCH3), 3.31 (s, 6 H, OCH3), 1.53 (s, 6 H, CH3).
13C{1H} NMR (100.6 MHz, CDCl3): δ 159.8, 150.4, 148.9, 145.8,
139.2, 138.8, 133.4, 130.4, 129.6, 127.4, 121.6, 117.2, 115.5, 114.3, 114.2,
83.1, 71.9, 70.9, 70.7, 70.6, 70.5, 69.8, 69.7, 69.1, 68.7, 67.6, 59.1, 59.0,
17.8 (some of the OCH2 signals are overlapped). MS (ESI): an isotopic
cluster peaking at m/z 1585 {100%, [M þ Na]þ}. HRMS (ESI): m/z
calcd for C68H95BF2I2N2NaO20 [M þ Na]þ, 1585.4521; found,
1585.4557. Anal. Calcd for C68H95BF2I2N2O20: C, 52.25; H, 6.13; N,
1.79. Found: C, 51.97; H, 6.27; N, 1.64.
’ ASSOCIATED CONTENT
S
Supporting Information. ESI mass spectra of 5 and 9;
b
absorption spectra of 6ꢀ9 in DMF; absorption spectra of 8 at
various concentrations; comparison of the rates of decay of
DPBF in DMF using 6ꢀ9 and ZnPc as the photosensitizers;
absorption and fluorescence spectra of 6ꢀ9 in DMEM; 1H NMR
spectra of 5 and 7ꢀ9; and 13C{1H} NMR spectra of 7 and 8.
This material is available free of charge via the Internet at http://
pubs.acs.org.
Distyryl BODIPY 9. According to the procedure described for 7,
BODIPY 1 (0.29 g, 0.4 mmol) was treated with benzaldehyde 5 (1.42 g,
∼1.2 mmol), glacial acetic acid (0.8 mL, 14.0 mmol), and piperidine
(1.0 mL, 10.1 mmol) in toluene (80 mL) to give 9, which was purified by
silica gel column chromatography using CHCl3/MeOH (10:1, v/v) as
the eluent, followed by size exclusion chromatography using THF as the
eluent. The product was obtained as an oily green solid (92 mg, 7%). 1H
NMR (400 MHz, CD2Cl2): δ 8.08 (d, J = 16.4 Hz, 2 H, CHdCH), 7.52
(d, J = 16.4 Hz, 2 H, CHdCH), 7.30 (d, J = 8.4 Hz, 2 H, ArH), 7.22 (d,
J = 8.4 Hz, 2 H, ArH), 7.16 (s, 2 H, ArH), 7.10 (d, J = 8.4 Hz, 2 H, ArH),
6.98 (d, J = 8.4 Hz, 2 H, ArH), 4.20ꢀ4.25 (m, 10 H, OCH2), 3.87ꢀ3.89
(m, 10 H, OCH2), 3.70ꢀ3.72 (m, 10 H, OCH2), 3.54ꢀ3.67 (m, ∼170
H, OCH2), 3.49ꢀ3.51 (m, 10 H, OCH2), 3.35 (s, 3 H, OCH3), 3.33 (s,
12 H, OCH3), 1.53 (s, 6 H, CH3). MS (ESI): an envelope of isotopic
clusters of [M þ Na]þ at m/z 2995 (37%, for n = 11), 3173 (21%, for n =
12), etc.
’ AUTHOR INFORMATION
Corresponding Author
*For P.-C.L.: phone, (852) 2696 1326; fax, (852) 2603 5057;
e-mail, pclo@cuhk.edu.hk. For D.K.P.N.: phone, (852) 2609
6375; fax, (852) 2603 5057; e-mail, dkpn@cuhk.edu.hk.
’ ACKNOWLEDGMENT
This work was supported by a Direct Grant for Research
(2010/2011) of The Chinese University of Hong Kong.
’ ABBREVIATIONS USED
BODIPY, boron dipyrromethene; DMEM, Dulbecco’s modified
Eagle’s medium; DMF, N,N-dimethylformamide; DPBF, 1,3-di-
phenylisobenzofuran; ESI, electrospray ionization; GFP, green
fluorescent protein; PBS, phosphate buffered saline; PDT,
photodynamic therapy; PI, propidium iodide; ROS, reactive oxy-
gen species; SEM, standard error of the mean; ZnPc, zinc(II)
phthalocyanine; ΦF, fluorescence quantum yield; IC50, dye con-
centration required to kill 50% of the cells
Photocytotoxicity Assay. Distyryl BODIPYs 6ꢀ9 were first
dissolved in DMF to give 1.6 mM solutions, which were diluted to 80
μM with an aqueous solution of Tween 80 (Arcos, 0.5% by volume in
these 80 μM solutions). The solutions were filtered with a 0.22 μm filter,
then diluted with the culture medium to appropriate concentrations.
The remaining steps were the same as described previously.8
Intracellular Fluorescence Studies. About 1.2 ꢁ 105 HT29
cells in the culture medium (2 mL) were seeded on a coverslip and
incubated overnight at 37 °C under 5% CO2. The medium was removed.
Then the cells were incubated with the solutions of distyryl BODIPYs
6ꢀ9 in the medium (1 μM, 2 mL) for 2 h under the same conditions.
The cells were rinsed with phosphate buffered saline (PBS) and then
viewed with a Leica SP5 confocal microscope equipped with a 633
helium neon laser. Emission signals from 650 to 720 nm (gain of 750 V)
were collected, and the images were digitized and analyzed by Leica
Application Suite Advanced Fluorescence. The intracellular fluores-
cence intensities (a total of 50 cells for each sample) were also
determined.
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Subcellular Localization Studies. About 1.2 ꢁ 105 HT29 cells
in the culture medium (2 mL) were seeded on a coverslip and incubated
overnight at 37 °C under 5% CO2. The medium was then removed. The
cells were incubated with a solution of 8 in the medium (1 μM, 2 mL) for
2 h under the same conditions. For the study using ER-Tracker, the cells
were incubated with ER-Tracker Green (Molecular Probe, 1 μM in
PBS) under the same conditions for a further 30 min. For the study using
LysoTracker and MitoTracker, the cells were incubated with LysoTrack-
er Green DND 26 (Molecular Probe, 0.2 μM in the medium) or
MitoTracker Green FM (Molecular Probe, 0.1 μM in the medium)
for a further 10 min. For all the cases, the cells were then rinsed with PBS
and viewed with a Leica SP5 confocal microscope equipped with a
488 nm argon laser and a 633 nm helium neon laser. All the Trackers
were excited at 488 nm and monitored at 510ꢀ560 nm, while 8 was
excited at 633 nm and monitored at 650ꢀ720 nm. The images were
digitized and analyzed using Leica Application Suite Advanced Fluor-
escence. The subcellular localization of 8 was revealed by comparing the
intracellular fluorescence images caused by the ER-Tracker, LysoTracker,
or MitoTracker and this dye.
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dx.doi.org/10.1021/jm101637g |J. Med. Chem. 2011, 54, 3097–3102