Keto-benzo[h]-Coumarin-Based Near-Infrared Dyes with Large Stokes Shifts for Bioimaging Applications

Article abstract of DOI:10.1002/asia.201501026

Fluorescence imaging is a promising tool for the visualization of biomolecules in living systems and there is great demand for new fluorescent dyes that absorb and emit in the near-infrared (NIR) region. Herein, we constructed three new fluorescent dyes (NBC dyes) based on keto-benzo[h]coumarin (k-BC) and benzopyrilium salts. These dyes showed large Stokes shifts (>100nm) and NIR emission (>800nm). The relationship between the structures and optical properties of these dyes was further investigated by using density functional theory calculations at the B3LYP/6-3G level of theory. Fluorescence images indicated that the fabricated dyes exhibited good photostability and low cytotoxicity and, thus, have potential applications as imaging agents in living cells and animals.

Full text of DOI:10.1002/asia.201501026

DOI: 10.1002/asia.201501026
Full Paper
Imaging Agents
Keto-benzo[h]-Coumarin-Based Near-Infrared Dyes with Large
Stokes Shifts for Bioimaging Applications
[a, b]
[a]
[a]
[a]
[a]
Guangle Niu,
Weimin Liu,* Hongyan Xiao, Hongyan Zhang, Jianhong Chen,
[a]
[a]
[a]
[a]
Qing Dai, Jiechao Ge, Jiasheng Wu, and Pengfei Wang
Abstract: Fluorescence imaging is a promising tool for the
visualization of biomolecules in living systems and there is
great demand for new fluorescent dyes that absorb and
emit in the near-infrared (NIR) region. Herein, we construct-
ed three new fluorescent dyes (NBC dyes) based on keto-
benzo[h]coumarin (k-BC) and benzopyrilium salts. These
dyes showed large Stokes shifts (>100 nm) and NIR emis-
sion (>800 nm). The relationship between the structures
and optical properties of these dyes was further investigated
by using density functional theory calculations at the B3LYP/
6-3G level of theory. Fluorescence images indicated that the
fabricated dyes exhibited good photostability and low cyto-
toxicity and, thus, have potential applications as imaging
agents in living cells and animals.
Introduction
chemical properties, such as large Stokes shifts, high fluores-
[
9]
cence quantum yields, and ease of modification. However,
these coumarin derivatives mainly emit in the short-wave-
length (blue) region and are prone to interference with back-
ground autofluorescence; as such, the application of these de-
rivatives in living samples remains limited.
Fluorescence imaging has attracted increasing interest for the
real-time visualization of various biomolecules in living samples
and for the tracking of various cellular events because it offers
high sensitivity, high selectivity, facile and non-invasive opera-
[1]
tion, and fast acquisition. Following recent advances in drug
delivery and cancer diagnosis and therapy, fluorescence imag-
ing holds great potential in biomedical applications and com-
pels the development of new fluorophores with improved
Great efforts have been made to synthesize coumarin-based
dyes with longer-wavelength emissions, and three strategies
have been commonly used (Scheme 1): 1) the introduction of
electron-withdrawing/-donating groups (such as 4-CF , 3-CN, 7-
3
[
2]
properties. In contrast to short-wavelength regions, fluoro-
phores that absorb and emit within the long-wavelength re-
gions of the electromagnetic spectrum, in particular in the
near-infrared (NIR, 700–900 nm) region, are highly favorable for
imaging applications because these compounds confer mini-
mum photodamage on biological samples, deep-tissue pene-
tration, and minimum interference from background autofluor-
methoxy, or 7-dimethylamino groups) into the coumarin skele-
[
10]
ton (Scheme 1a);
2) the synthesis of benzo[f/g/h]coumarin
3) extending the p-conjugation of the
[
11]
dyes (Scheme 1b);
coumarin (starting from 3-aldehydecoumarin or 3-acetylcou-
[
12]
marin; Scheme 1c). Ahn and co-workers reported the syn-
[3]
escence in living systems.
Coumarin derivatives have received considerable attention
[
4]
[5]
for use in laser dyes, organic light-emitting devices, biomo-
[
6]
[7]
lecular labeling, fluorogenic detection, and drug delivery
[
8]
and therapy owing to their outstanding photophysical and
[
a] G. Niu, Prof. Dr. W. Liu, Prof. Dr. H. Xiao, Prof. Dr. H. Zhang, J. Chen, Q. Dai,
Prof. Dr. J. Ge, Prof. Dr. J. Wu, Prof. Dr. P. Wang
Key Laboratory of Photochemical Conversion and Optoelectronic Materials
and CityU-CAS Joint Laboratory of Functional Materials and Devices
Technical Institute of Physics and Chemistry
Chinese Academy of Sciences
Beijing 100190 (P. R. China)
E-mail: wmliu@mail.ipc.ac.cn
[
b] G. Niu
University of the Chinese Academy of Sciences
Beijing 100049 (P. R. China)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/asia.201501026.
Scheme 1. Three commonly used strategies for the design of new coumarin-
based dyes with long-wavelength fluorescence.
Chem. Asian J. 2016, 11, 498 – 504
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ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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thesis of a new iminocoumarin derivative with a maximum
Results and Discussion
[
13]
emission of about 600 nm by adopting strategies (1) and (2).
[12i–k]
In our previous work,
we developed a series of coumarin-
The synthesis of key intermediate k-BC is shown in Scheme 2.
Compound 1 was prepared in good yield from the reaction of
6-hydroxy-3,4-dihydro-1(2H)-naphthalenone with 2-bromo-2-
methylpropionamide and NaOH in N,N-dimethylacetamide
based fluorescent dyes with emission peaks from 450 nm to
6
60 nm by combining strategies (1) and (3). However, coumar-
[14]
in-based NIR dyes remain rare.
The combination of strat-
[
15]
egies (1), (2), and (3) may be effective for the design of new
coumarin-based NIR fluorescent dyes, in which 3-aldehydeben-
zo[f/g/h]coumarin or 3-acetylbenzo[f/g/h]coumarin could func-
tion as crucial intermediates in the construction of new NIR
dyes. In addition, a benzopyrilium salt with a dialkylamino
group at position 7 is an active chromophore and could be
used to extend the p-conjugation of the benzo[h]coumarin
skeleton.
(DMA) through a Smiles rearrangement. The dimethylation
of compound 1 with MeI and K CO in DMF at 458C produced
2
3
[
16]
compound 2. An aldehyde group was introduced onto the
skeleton of compound 2 and subsequent oxidation with 2,3-di-
chloro-5,6-dicyano-1,4-benzoquinone (DDQ) afforded com-
pound 3. Condensation of compound 3 with ethyl acetoace-
tate in the presence of morpholine in anhydrous EtOH pro-
duced k-BC as a red-brown solid in 76% yield. For details of
the synthesis, see the Supporting Information.
Inspired by these construction strategies, herein we report
the design and synthesis of keto-benzo[h]coumarin (k-BC;
Scheme 2) and, based on this intermediate, three hybrid NIR
dyes that contained benzo[h]coumarin and benzopyrilium salts
The ketone group is a reactive functional group that is in-
volved in many nucleophilic addition reactions, such as aldol-
condensation reactions. We synthesized three NBC dyes
through the facile condensation of k-BC with various salicylal-
dehyde derivatives (Scheme 3). Thus, the reaction of k-BC with
compound 4 and concentrated H SO produced NBC1 in 82%
yield. Similarly, NBC2 was obtained in moderate yield (46%)
through the condensation of k-BC with compound 5. Ethyla-
tion of NBC1 proceeded through a typical esterification reac-
(
NBC dyes, Scheme 3) were constructed through efficient con-
densation reactions. The fabricated dyes exhibited large Stokes
shifts, good photostability, and emission wavelengths longer
than 800 nm.
2
4
tion in EtOH catalyzed by concentrated H SO4 to produce
2
NBC1E in 79% yield. The final structures of the NBC dyes were
confirmed by NMR spectroscopy and HRMS (ESI).
We investigated the absorption and emission profiles of
NBC1, NBC1E, and NBC2 in various solvents; the optical data
of these NBC dyes are listed in Table 1 and the normalized ab-
sorption and emission spectra are shown in Figure 1 and the
Supporting Information, Figures S1 and S2. The maximum ab-
max
sorption wavelengths (labs ) of the NBC dyes in different sol-
vents were located in the deep-red-light region (>650 nm),
max
whilst the maximum emission wavelengths (lem ) of the NBC
dyes were longer than 800 nm and gradually increased on
Scheme 2. Synthesis of key intermediate k-BC.
Table 1. Photophysical properties of the NBC dyes, ACA-3, and V.
max
max
À1
À1
[a]
Dye
Solvent labs /lem
nm]
Stokes shift
[nm]
e [m cm ] F
[
NBC1
CH
MeCN
water
2
Cl
2
692/809
668/816
656/814
693/810
670/818
672/820
701/808
667/815
665/816
598/637
598/648
609/631
594/637
117
148
158
117
148
148
107
148
151
39
23900
37800
35100
86500
40600
64300
90150
31200
57600
61061
23095
–
0.118
0.005
0.003
0.125
0.005
0.004
0.116
0.01
0.005
0.425
0.214
0.79
[b]
NBC1E CH
2
Cl
2
MeCN
[b]
water
CH Cl
MeCN
NBC2
2
2
[b]
water
[c]
ACA-3
2 2
CH Cl
[d]
PBS
CH Cl
MeCN
50
22
43
[e]
V
2
2
–
0.023
[21]
[a] ICG (F=0.13 in DMSO) was used as a standard.
[b] With 40%
MeCN. [c] Data taken from reference [22]. [d] PBS buffer containing
À1
[23]
0
.5 mgmL bovine serum albumin (BSA). [e] Data from reference [23].
PBS=phosphate buffered saline.
Scheme 3. Synthesis of benzo[h]coumarin-based NBC dyes.
Chem. Asian J. 2016, 11, 498 – 504
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[
20]
orescent dyes. Furthermore, we compared these NBC dyes
[
22]
[23]
to analogue ACA-3
and compound V
(a benzopyrilium
salt that contained a phenyl substituent rather than benzo[h]-
coumarin at position 2); the corresponding photophysical data
and chemical structures are summarized in Table 1 and the
Supporting Information, Scheme S1, respectively. Clearly, the
absorption and emission wavelengths of the NBC dyes were
largely red-shifted (>150 nm), because of benzo[h]coumarin-
induced extended p-conjugation in the fluorophores. Con-
versely, the Stokes shifts of the NBC dyes were larger than
those of ACA-3 and V, owing to the strong internal charge
[
20b]
transfer (ICT) effect of benzo[h]coumarin.
The striking differ-
ence in QYs in different polar solvents may render the NBC
dyes as potential materials for fluorescence labeling and imag-
ing applications, owing to the restricted or hydrophobic envi-
ronments in vitro and in vivo.
Figure 1. Normalized absorption and fluorescence spectra of the NBC dyes
in CH Cl
2
2
.
moving from CH Cl to MeCN and water. To further investigate
Next, we examined the effect of pH value on the absorption
spectra of NBC dyes in Britton–Robinson (BR) buffers (24 mm,
containing 40% MeCN). Figure 3a shows that the normalized
absorbance of these dyes exhibited almost the same tendency
within the range pH 4–10. The absorption intensities of NBC1
and NBC1E only showed minimal variation within the range
pH 4–8, and suddenly decreased at pH 8. This result indicated
that the conjugated structures of NBC1 and NBC1E were dis-
2
2
the geometric and electronic properties of these dyes, density
functional theory (DFT) calculations were performed at the
B3LYP/6-3G level of theory by using the Gaussian 09 program
[
17]
package. The calculated HOMOs and LUMOs and optimized
structures of the NBC dyes are shown in Figure 2 and the Sup-
Figure 2. Spatial distributions of the calculated HOMOs and LUMOs of NBC
dyes.
porting Information, Figure S3, respectively. The R groups are
essentially perpendicular to the benzopyrilium salt core and
make almost no contribution to the HOMOs/LUMOs of the
NBC dyes, thereby indicating that these R groups have no in-
fluence on the emissions of these NBC dyes (Figure 2 and the
Supporting Information, Figure S3). In addition, the dihedral
angles between the benzo[h]coumarin moiety and the benzo-
pyrilium salt also had no effect on the optical properties of the
NBC dyes, because these NBC dyes, which had similar struc-
tures, exhibited similar electron-density distributions (Figure 2).
The corresponding dihedral angles are summarized in the Sup-
porting Information, Table S1. To the best of our knowledge,
these NIR-emitting NBC dyes exhibit the longest fluorescence
among all the coumarin-based dyes. Moreover, the Stokes
shifts of the NBC dyes (>100 nm) are larger than that of com-
[18]
mercial cyanine dye ICG (ca. 30 nm), which could lead to
lower self-absorption, thereby affording high resolution and fa-
[19]
cilitate multichannel bioimaging. The quantum yields (QYs)
of the NBC dyes in CH Cl were comparable to that of com-
2
2
mercial ICG, whereas the QYs of the NBC dyes in MeCN and
water were lower than that of ICG, owing to the smaller
energy gaps and enhanced non-radiative transitions in NIR flu-
Figure 3. a) pH dependence of the normalized absorption spectra of the
NBC dyes in BR buffers (24 mm, pH 4–10, 40% MeCN); b) photostability of
the NBC dyes and ICG in HEPES buffer (24 mm, pH 7.4, 40% MeCN).
Chem. Asian J. 2016, 11, 498 – 504
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rupted in alkaline conditions, which may be attributed to spi-
rolactone formation or to attack from the hydroxy groups.
Moreover, the 2H-chromen structure of NBC2 may be com-
pletely destroyed at pH 10, as indicated by irreversible changes
in the absorption spectra after the addition of HCl solution. As
a crucial indicator of new synthesized dyes for imaging-related
applications in living samples, the photostability of these NBC
have potential applications in biodetection and bioimaging in
[
24]
mitochondria.
To evaluate the cytotoxicity of the NBC dyes, we performed
standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tet-
razolium) assays by using HeLa and A549 cells. The NBC dyes
exhibited low cytotoxicities (cell viabilities>80%) at concentra-
tions of up to 10 mm after incubation for 24 h (Figure 5 and
the Supporting Information, Figure S6). Typically, imaging in
the NIR region resulted in deep-tissue penetration and low
photodamage in living samples; we anticipated that these
NBC dyes, which had NIR emissions longer than 800 nm, could
be utilized for in vivo imaging. To this end, NBC1E as a model
compound (50 mm, 20 mL) was subcutaneously injected in
a naked mouse. 10 min after injection, in vivo imaging was
performed and a high signal-to-noise ratio was obtained (Fig-
ure 4b).
dyes was evaluated by continuous irradiation with a Xe lamp
À2
(
200 mWcm ). As shown in Figure 3b, the intensities of NBC1
and NBC1E decreased to 90%, whilst that of NBC2 remained
above 80% after 20 min of irradiation. However, for compari-
son, only 40% of the initial fluorescence intensity of commer-
cial ICG dye was retained. These data indicated that the NBC
dyes exhibited good photostability and could be potentially
used as alternative materials for in vitro and in vivo imaging.
To further evaluate the cell permeability of the NBC dyes,
we performed imaging experiments on live HeLa cells. As
shown in Figure 4a, the NBC dyes readily permeated the cell
Figure 5. Cytotoxicity of the NBC dyes in HeLa cells.
Conclusion
In conclusion, we have successfully fabricated a family of k-BC-
based NIR dyes (NBC1, NBC1E, and NBC2) that show large
Stokes shifts (>100 nm) through facile condensation reactions.
These NBC dyes exhibit the longest fluorescence among all
the coumarin-based dyes. Because these dyes exhibit good
photostability and low cytotoxicity, they have potential appli-
cations as in vitro and in vivo imaging agents. Furthermore,
this work paves the way for the preparation of other benzo-
coumarin derivatives for biodetection and bioimaging applica-
tions.
Figure 4. a) Confocal fluorescence microscopy images of HeLa cells that
were incubated with NBC1, NBC1E, or NBC2 (3 mm); scale bar: 10 mm.
b) (i) Bright-field and (ii) as-detected in vivo fluorescence of NBC1E via subcu-
taneous injection into a naked mouse.
membrane of HeLa cells and clear fluorescence was observed
in cytoplasm. Although the QYs of the NBC dyes in water were
lower, the dyes became emissive owing to the restricted or hy-
drophobic environments in living cells. In addition, positively
charged xanthene analogues are commonly predominantly lo-
cated in the mitochondria, owing to electrostatic interac-
Experimental Section
General
[
24]
tions. The location of the NBC dyes in HeLa and A549 cells
were investigated by co-localization experiments with Mito-
Tracker Green FM (MTG; see the Supporting Information, Figur-
es S4 and S5). The Pearson’s coefficients and overlay data (see
the Supporting Information, Table S2) revealed that these dyes
were mainly located in the mitochondria. These data indicated
that the NBC dyes had good cell-membrane permeability and
All commercial reagents and solvents were purchased from Sigma-
Aldrich, Alfa Aesar, J&K Chemical Co., or Beijing Chemical Regent
Co. and used without further purification. MitoTracker Green FM
1
(
MTG) was purchased from Invitrogen (USA). H (400 MHz) and
13
C NMR spectra (100 MHz) were recorded on a Bruker Ad-
vance 400 spectrometer with tetramethylsilane as an internal stan-
dard. Electron impact (EI) mass spectroscopy was performed on
Chem. Asian J. 2016, 11, 498 – 504
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a Waters GCT Premier mass spectrometer. HRMS was achieved by
performing Fourier transform ion cyclotron resonance mass spec-
trometry (FTICR-MS). Absorption and fluorescence spectra were re-
corded on Hitachi U3010 and Hitachi F4500 fluorescence spectrom-
eters at RT, respectively.
110.3, 109.5, 40.2, 39.0, 30.7, 23.7 ppm; HRMS (EI): m/z calcd for
C H NO: 189.1154 [M]; found: 189.1178.
12
15
6
-(Dimethylamino)-1-hydroxy-2-naphthaldehyde (3)
NaH (0.86 g, 21.5 mmol) and a solution of ethyl formate (3.5 mL,
4
3 mmol) in anhydrous toluene (5 mL) were successively added
Imaging
into a three-necked round-bottomed flask under a N atmosphere.
2
After stirring for 10 min, a solution of compound 2 (2.56 g,
HeLa cells were cultured in confocal dishes in the culture medium
1
3.4 mmol) in anhydrous toluene (30 mL) was added dropwise into
(
1
5
Dulbecco’s modified Eagle medium (DMEM), supplemented with
À1
the mixture. Then, the solution was stirred at 558C for 1 h and the
resulting yellow precipitate was filtered and dried under vacuum.
The yellow solid was dissolved in anhydrous THF (40 mL) and DDQ
0% fetal bovine serum (FBS), 50 unitmL
penicillin, and
À1
0 mgmL streptomycin) in 5% CO /air at 378C in a humidified in-
2
cubator for 24 h. The culture medium was discarded and fresh
DMEM medium (containing 3 mm NBC dye and 100 nm MTG) was
added into the culture medium of HeLa cells and incubated at
(
2.36 g, 10.4 mmol) was added portion-wise. After stirring for 1 h,
THF was removed under reduced pressure and the mixture was
purified by column chromatography on silica gel (petroleum ether/
3
78C in 5% CO2 for 30 min before the imaging. Images were ac-
EtOAc, 5:1 v/v) to give compound 3 as a yellow solid (1.70 g, 59%
quired by using laser scanning confocal microscopy on an OLYM-
1
yield). H NMR (400 MHz, CDCl ): d=12.83 (s, 1H), 9.78 (s, 1H), 8.27
3
PUS FV1000-IX81 microscope (for MTG: l =488 nm, fluorescein
ex
(
(
d, J=9.3 Hz, 1H), 7.32 (d, J=8.6 Hz, 1H), 7.14–7.06 (m, 2H), 6.80
isothiocyanate (FITC) channel, fluorescence collection 500–550 nm;
13
d, J=2.0 Hz, 1H), 3.14 ppm (s, 6H); C NMR (100 MHz, CDCl ): d=
3
for NBC dyes: l =640 nm, cyanine Cy5 channel, fluorescence col-
ex
1
94.8, 163.0, 152.0, 140.2, 127.5, 126.0, 118.1, 115.7, 114.6, 112.3,
lection 700–800 nm) and processed by using the FV10-ASW 3.1
Viewer. The Maestro in vivo fluorescence imaging system (CRi Inc.)
was used for the in vivo imaging. Deep-red light (lmax =661 nm)
was used as the excitation source, and the fluorescence was col-
lected from 700–900 nm (step: 10 nm).
1
05.5, 40.4 ppm; HRMS (EI): m/z calcd for C H NO : 215.0946 [M];
13
13
2
found: 215.0989.
k-BC
Compound 3 (1.29 g, 6 mmol), morpholine (0.5 mL), and ethyl ace-
toacetate (2 mL, 15.7 mmol) were successively added to anhydrous
EtOH (15 mL) and the mixture was heated at reflux for 2 h. After
6
-Amino-3,4-dihydronaphthalen-1(2H)-one (1)
Compound 1 was synthesized according to a modified literature
cooling to RT, compound k-BC was obtained by filtration as a red-
[15]
procedure. NaOH (720 mg, 18 mmol) was added to a solution of
-hydroxy-3,4-dihydro-1(2H)naphthalenone (974 mg, 6 mmol) in
1
brown solid (1.28 g, 76% yield). H NMR (400 MHz, CDCl ): d=8.58
3
6
(
(
2
1
1
2
s, 1H), 8.39 (d, J=9.3 Hz, 1H), 7.40 (dd, J=22.2, 8.7 Hz, 2H), 7.19
DMA (10 mL), which was stirred at RT for 1 h. 2-Bromo-2-methyl-
propanamide (2.98 g, 18 mmol) was added and the solution was
stirred at RT for 5 h. Then, NaOH (2.16 g, 54 mmol) was added and
the solution was stirred at 508C for 1 h. Next, water (10 mL) was
added, the solution was heated at reflux for 1 h, and a second
batch of water (10 mL) was added. After cooling to RT, the result-
ing precipitate was collected and washed with plenty of water. The
sample was dried and purified by column chromatography on
silica gel (petroleum ether/EtOAc, 6:1 v/v) to give compound 1 as
dd, J=9.4, 2.4 Hz, 1H), 6.86 (d, J=2.1 Hz, 1H), 3.16 (s, 6H),
13
.74 ppm (s, 3H); C NMR (100 MHz, CDCl ): d=195.7, 160.2, 155.0,
3
51.3, 148.6, 138.7, 125.3, 124.8, 123.5, 119.6, 115.7, 114.0, 111.1,
+
3
05.8, 40.3, 30.6 ppm; HRMS (ESI): m/z calcd for C H NO :
82.11302 [M+H] ; found: 282.11269.
1
7
16
+
NBC1
Compound NBC1 was synthesized according to our modified pro-
1
an off-white powder (534 mg, 55% yield). H NMR (400 MHz,
CDCl ): d=7.88 (d, J=8.5 Hz, 1H), 6.53 (dd, J=8.5, 2.3 Hz, 1H),
[25]
cedure. Compound k-BC (140 mg, 0.5 mmol) and compound 4
3
(
157 mg, 0.5 mmol) were successively added to concentrated
6
6
1
.43–6.39 (m, 1H), 4.13 (s, 2H), 2.82 (t, J=6.1 Hz, 2H), 2.56 (t, J=
.4 Hz, 2H), 2.10–2.02 ppm (m, 2H); C NMR (100 MHz, CDCl ): d=
97.1, 151.6, 147.2, 129.8, 123.9, 113.2, 112.5, 38.9, 30.1, 23.5 ppm;
H SO4 (10 mL) in a round-bottomed flask and the mixture was
2
1
3
3
heated with vigorous stirring at 1008C for 4 h. After cooling to RT,
the mixture was poured into ice water. Perchloric acid (1 mL) was
added and the resulting precipitate was filtered and washed with
cold water. The sample was dried and purified by column chroma-
tography on silica gel (CH Cl /MeOH, 60:1 then 40:1 to 20:1 v/v) to
HRMS (EI): m/z calcd for C H NO: 161.0841 [M]; found: 161.0887.
1
0
11
2
2
6
-(Dimethylamino)-3,4-dihydronaphthalen-1(2H)-one (2)
1
give NBC1 as a dark-green solid (270 mg, 82% yield). H NMR
Compound 2 was synthesized according to a modified literature
(400 MHz, [D ]DMSO): d=13.36 (s, 1H), 9.16 (s, 1H), 8.21 (d, J=
6
[16]
procedure. CH I (430 mg, 3 mmol) was added to a mixture of
9.3 Hz, 1H), 8.13 (d, J=6.6 Hz, 1H), 7.86 (t, J=7.4 Hz, 1H), 7.78 (t,
J=7.5 Hz, 1H), 7.65 (dd, J=21.3, 8.6 Hz, 3H), 7.51 (d, J=7.4 Hz,
1H), 7.34 (d, J=8.9 Hz, 1H), 7.28–6.92 (m, 4H), 3.63 (s, 4H), 3.13 (s,
3
compound 1 (325 mg, 2 mmol) and K CO3 (830 mg, 6 mmol) in
2
DMF (5 mL) and the mixture was stirred for 24 h at 458C. After
cooling to RT, water (10 mL) was added and the solution was ex-
tracted with EtOAc and washed with water and brine. The organic
13
6H), 1.24 ppm (t, J=6.0 Hz, 6H); C NMR (100 MHz, [D ]DMSO):
6
d=172.5, 167.9, 158.1, 154.0, 152.2, 146.1, 139.4, 134.0, 131.1,
layer was dried with anhydrous MgSO and evaporated under re-
duced pressure. The obtained residue was dried and purified by
column chromatography on silica gel (petroleum ether/EtOAc, 6:1
130.0, 128.9, 125.8, 124.7, 124.5, 116.8, 113.1, 112.3, 111.6, 106.5,
4
+
97.1, 45.8, 21.6, 13.2 ppm; HRMS (ESI): m/z calcd for C H N O
:
3
5
31
2
5
+
559.22275 [M] ; found: 559.22271.
v/v) to give compound 2 as a white solid (160 mg, 42% yield).
1
H NMR (400 MHz, CDCl ): d=7.95 (d, J=8.9 Hz, 1H), 6.60 (dd, J=
3
NBC1E
8
6
.9, 2.6 Hz, 1H), 6.39 (d, J=2.5 Hz, 1H), 3.05 (s, 6H), 2.88 (t, J=
.1 Hz, 2H), 2.57 (t, J=6.0 Hz, 2H), 2.13–2.04 ppm (m, 2H);
Compound NBC1E was synthesized according to our modified pro-
cedure. Concentrated H SO (1 mL) was slowly added to a solu-
1
3
[25]
C NMR (100 MHz, CDCl ): d=196.9, 153.6, 146.6, 129.4, 121.9,
3
2
4
Chem. Asian J. 2016, 11, 498 – 504
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tion of compound NBC1 (164 mg, 0.25 mmol) in EtOH (15 mL) in
a round-bottomed flask and the solution was heated at reflux for
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4
h. After cooling to RT, EtOH was removed under reduced pres-
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sure and water (50 mL) was slowly added to the residue. Then,
perchloric acid (0.5 mL) was added and the resulting precipitate
was collected, dried, and purified by column chromatography on
silica gel (CH Cl /MeOH, 60:1 v/v) to give NBC1E as a dark-green
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2
1
solid (135 mg, 79% yield). H NMR (400 MHz, [D ]DMSO): d=9.31
6
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(
s, 1H), 8.27–8.15 (m, 3H), 7.92 (td, J=7.5, 1.2 Hz, 1H), 7.84 (td, J=
7
2
.7, 1.2 Hz, 1H), 7.68–7.59 (m, 3H), 7.43–7.28 (m, 4H), 7.07 (d, J=
.4 Hz, 1H), 4.10 (tt, J=7.2, 3.7 Hz, 2H), 3.74 (q, J=7.0 Hz, 4H), 3.15
1
3
(
[
s, 6H), 1.29 (s, 6H), 1.04 ppm (t, J=7.1 Hz, 3H); C NMR (100 MHz,
D ]DMSO): d=165.1, 160.9, 159.8, 158.7, 157.5, 155.8, 154.2, 152.0,
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6
1
1
5
5
47.1, 139.3, 135.4, 133.1, 130.8, 129.9, 129.8, 129.3, 125.3, 124.5,
24.2, 118.7, 116.9, 116.4, 112.4, 112.3, 111.9, 110.3, 106.0, 96.1, 61.3,
+
5
4.9, 48.6, 45.7, 13.5 ppm; HRMS (ESI): m/z calcd for C H N O
87.25405 [M] ; found: 587.25394.
:
3
7
35
2
+
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NBC2
Compound NBC2 was synthesized according to our modified pro-
[25]
cedure. Compound k-BC (140 mg, 0.5 mmol) and 4-(diethylami-
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added to concentrated H SO (10 mL) in a round-bottomed flask
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and the mixture was heated with vigorous stirring at 1008C for
h. After cooling to RT, the mixture was poured into ice water.
4
Then, perchloric acid (1 mL) was added and the resulting precipi-
tate was filtered and washed with cold water. The sample was
dried and purified by column chromatography on silica gel
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CH Cl /MeOH; 120:1 then 100:1 and 80:1 to 60:1 v/v) to give
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1
NBC2 as a dark-green solid (125 mg, 46% yield). H NMR (400 MHz,
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[
6
2014, 3, 1089–1096; e) M. Tasior, D. Kim, S. Singha, M. Krzeszewski, K. H.
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.3 Hz, 1H), 7.24 (s, 1H), 6.94 (d, J=2.1 Hz, 1H), 3.74 (q, J=6.9 Hz,
H), 3.10 (s, 6H), 1.30 ppm (t, J=7.1 Hz, 6H); C NMR (100 MHz,
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1
3
4
7, 12843–12845; c) Y.-Q. Sun, J. Liu, J. Zhang, T. Yang, W. Guo, Chem.
[
1
1
D ]DMSO): d=160.6, 158.4, 156.7, 155.7, 153.4, 151.3, 147.8, 146.1,
38.5, 131.9, 124.6, 123.7, 123.5, 118.6, 117.9, 115.7, 111.7, 111.2,
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10.9, 109.9, 105.3, 95.2, 45.1, 11.9 ppm; HRMS (ESI): m/z calcd for
+
3
+
C H N O : 439.20162 [M] ; found: 439.20130.
28
27
2
Acknowledgements
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This work was supported by the National Natural Science
Foundation of China (21373250 and 21173244). All calculations
were performed on the cluster of Key Laboratory of Theoretical
and Computational Photochemistry, Ministry of Education,
China.
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Keywords: benzo[h]coumarin · near-infrared · dyes/pigments ·
fluorescence · imaging agents
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Manuscript received: September 23, 2015
Revised: October 14, 2015
Accepted Article published: November 12, 2015
Final Article published: November 24, 2015
Chem. Asian J. 2016, 11, 498 – 504
www.chemasianj.org
504
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim