Squaraine-based colorimetric and fluorescent sensors for Cu2+-specific detection and fluorescence imaging in living cells

Article abstract of DOI:10.1016/j.tet.2010.03.070

New squaraine-based chemosensors SQ1 and SQ2 functionalized with 2-picolyl units were first synthesized and used as highly selective and sensitive colorimetric and fluorometric dual-channel sensors for Cu²⁺-specific recognition in aqueous systems. Among a series of individual metal ions, only Cu²⁺ could result in dramatic color changes. We also evaluated their capability of biological applications and found that SQ2 could be successfully employed as a Cu²⁺-selective probe in the fluorescence imaging of living cells.

Full text of DOI:10.1016/j.tet.2010.03.070

Tetrahedron 66 (2010) 3695–3701
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Squaraine-based colorimetric and fluorescent sensors for Cu2þ-specific
detection and fluorescence imaging in living cells
,
,
b,
*
Weida Wang a, Afu Fu b, Jingsong You a b, Ge Gao a, Jingbo Lan a , Lijuan Chen
*
a Key Laboratory of Green Chemistry and Technology of Ministry of Education, College of Chemistry, Sichuan University, 29 Wangjiang Road, Chengdu 610064, PR China
b State Key laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Keyuan Road 4, Gaopeng Street, Chengdu 610041, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
New squaraine-based chemosensors SQ1 and SQ2 functionalized with 2-picolyl units were first syn-
thesized and used as highly selective and sensitive colorimetric and fluorometric dual-channel sensors
for Cu2þ-specific recognition in aqueous systems. Among a series of individual metal ions, only Cu2þ
could result in dramatic color changes. We also evaluated their capability of biological applications and
found that SQ2 could be successfully employed as a Cu2þ-selective probe in the fluorescence imaging of
living cells.
Received 4 February 2010
Received in revised form 18 March 2010
Accepted 19 March 2010
Available online 27 March 2010
Keywords:
Colorimetric chemosensor
Squaraine
Ó 2010 Elsevier Ltd. All rights reserved.
Copper ion
Fluorescent probe
Imaging agents
1. Introduction
field of colorimetric and/or fluorescent chemosensors for Cu2þ has
been obtained,5 there is still a demand to develop new indicators
The development of sensitive chemosensors capable of selective
recognition of cations, especially for metal ions with biological in-
terest, has always been of particular significance due to their po-
tential application in chemistry, biomedicine, and environmental
studies. The soft transition metal ion Cu2þ plays a pivotal part in
many fundamental physiological processes as the third most
abundant essential trace element after iron and zinc in the human
body.1 Research and development of chemosensors directed to-
ward the detection and measurement of divalent copper ion has
received much attention. Low level of Cu2þ in the cells will affect
the corresponding enzyme activity, inhibit cell normal metabolism,
and even endanger the survival of the cells, whereas Cu2þ in excess
of physiological need could also cause serious diseases, for instance
gastrointestinal disturbance, liver or kidney damage.2,3 Further-
more, Cu2þ can also act as a significant environmental pollutant
because of its widespread use in industry and agriculture. Thus, the
fast detection of Cu2þ in environmental and biological samples has
been becoming increasingly important not only due to its impor-
tant role in life but also the high toxicity to organisms under
overloading conditions.4 Even though great achievement in the
with improved properties, especially fluorescent sensors with high
efficiency in the spectral visible and near-infrared (NIR) region and
with specific selectivity toward Cu2þ over other competitive metal
ions.
Squaraines are a particularly promising class of organic NIR dyes
that exhibit unique photophysical properties, namely, a sharp and
intense absorption band in the red to NIR region. Because their
absorption and emission properties are sensitive to the surround-
ing medium, squaraine dyes have been designed and widely ap-
plied for the optical detection of metal ions such as Liþ, Naþ, Ca2þ
Mg2þ Hg2þ in recent years.6 However, there are very few
,
,
squaraine-based chemosensors designed for the Cu2þ recognition,7
and no studies pertaining to squaraine dyes as a versatile fluores-
cent scaffold for constructing bioimaging probes for Cu2þ so far.
Therefore, the design and construction of colorimetric and fluo-
rescent sensors based on squaraines for highly effective detection
of Cu2þ under either in vitro or in vivo conditions is very significant.
Squaraine dyes of the type 2,4,6-trihydroxyphenylsquaraine were
considered very important due to their potential application in se-
lective recognition of specific biomolecule.8 However, as far as we are
aware, there are very few examples9 on such a type of squaraine
investigated because it is difficult to functionalize, especially with
N-heterocycles. In continuation of our ongoing interest in the
development of N-heterocycles-based supramolecular chemistry, we
wish to design and construct novel 2,4,6-trihydroxyphenylsquaraine-
* Corresponding authors. Tel.: þ86 28 85412285; fax: þ86 28 85412203 (J.L.); tel./
fax: þ86 28 85164060 (J.C.); e-mail addresses: jingbolan@scu.edu.cn (J. Lan),
lijuan17@hotmail.com (L. Chen).
0040-4020/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.03.070

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W. Wang et al. / Tetrahedron 66 (2010) 3695–3701
attached chemosensors by incorporating N-heterocyclic groups. In-
spired by the structure of DPA (di-2-pyridylmethylamine) moiety,
which is well-known for the ability to sense zinc ion,10 we speculated
that 2-picolyl-anchored 2,4,6-trihydroxyphenylsquaraine would
provide an effective binding site for the copper ion via the synergic
coordination of N and O atoms. In this context, we present the
synthesis of 2,4,6-trihydroxyphenylsquaraine-derived compounds
SQ1 and SQ2 functionalized with 2-picolyl units (Scheme 1) and the
spectroscopic investigations upon coordination to copper(II) ion.
We also present the practical applicability of SQ2 as a fluorescent
probe for the detection of Cu2þ in living cells. Moreover, the
2,4,6-trihydroxyphenylsquaraine-derived compound SQ3 function-
alized with 4-picolyl units was synthesized for the control
experiment.
Mg2þ, Fe3þ, Co2þ, Ni2þ, Cu2þ, Zn2þ, Cr3þ, Cd2þ, Pb2þ, Mn2þ, Hg2þ
,
Agþ, La3þ, Ce3þ, Pr3þ, Nd3þ, Y3þ, Er3þ, Sm3þ, Gd3þ, and Sc3þ ions. As
depicted in Figure 1, a 10ꢀ5 M solution of SQ1 displayed a strong
pink color and Cu2þ caused a dramatic change of color from pink to
blue while other metal cations did not result in appreciable changes
under the same condition. The fact indicated the compound SQ1
exhibited a high selectivity for Cu2þ over various other metal ions.
The limit of Cu2þ by naked eye was 3
Supplementary data), which was far lower than the limit of Cu2þ
in drinking water (w20 M) set by the U.S. Environmental Pro-
mM (Fig. S1 in the
m
tection Agency (EPA). Therefore, the advantage of this assay is that
naked-eye detection of Cu2þ in water quality monitoring becomes
possible. Moreover, to explore the effects of anionic counterions on
the sensing behavior, the responses of SQ1 to different cupric salts
HO
OH
OH
HO
HO
O
2-(chloromethyl)pyridine
N
N
HO
N
N
OH
N
squaric acid
hydrochloride
2+
OH
n-butanol/toluene, 100 °C
toluene, KI, 100 °C
N
O
HO
SQ1
2
HO
HO
OH
1
HO
HO
N
N
N
N
OH
OH
O
HO
HO
N
N
4-(chloromethyl)pyridine
hydrochloride
squaric acid
2+
OH
HO
OH
n-butanol/toluene, 100 °C
toluene, KI, 100 °C
O
SQ3
3
HO
HO
O
O
2
(1) squaryl chloride, benzene
(2) CH3COOH/H2O, 2N HCl
N
OH
N
N
2+
O
N
N
n-butanol/toluene
OH
O
4
5
SQ2
Scheme 1. Synthetic routes of squaraines SQ1–SQ3.
2. Results and discussion
2.1. Synthesis
The squaraine compound SQ1 functionalized with 2-picolyl
units was successfully synthesized by a two-step reaction with
phloroglucinol and 2-(chloromethyl)pyridine hydrochloride as
starting materials. Catalyzed by iodides, phloroglucinol reacted
with 2-(chloromethyl)pyridine hydrochloride affording the yellow
compound 2,4-bis(pyridin-2-ylmethyl)benzene-1,3,5-triol (yield
19%). The condensation of 2,4-bis(pyridin-2-ylmethyl)benzene-
1,3,5-triol and squaric acid in toluene and n-butanol (v/v¼1:1) gave
rise to SQ1 (yield 78%). The unsymmetrical squaraine derivative
SQ2 was prepared by the reaction of 3-[4-(N,N-dibutylamino)-
phenyl]-4-hydroxycyclobutene-1,2-dione with 2,4-bis-(pyridin-2-
ylmethyl)benzene-1,3,5-triol (yield 60%). To understand a crucial
role which 2-picolyl unit played as a binding site for Cu2þ, we
synthesized the compound SQ3 bearing 4-picolyl units on the
phloroglucinol moiety via a similar synthetic route as SQ1.
Figure 1. (left) The color changes of SQ1 (c¼10
m
M) in (10 mM) HEPES/THF (v/v¼60/
40, pH¼7.4) upon addition of 1.0 equiv of various metal ions. From left to right and top
to bottom: Liþ, Naþ, Kþ, Ca2þ, Mg2þ, Sr2þ, Ba2þ, Fe3þ, Co2þ, Ni2þ, Cu2þ, Zn2þ, Cr3þ, Cd2þ
,
Mn2þ, Pb2þ, Hg2þ, Agþ, La3þ, Ce3þ, Pr3þ, Nd3þ, Y3þ, Er3þ, Sm3þ, Gd3þ, Sc3þ, blank.
(right) Schematic representation of the periodic table of metallic elements.
were also studied. As we expected, cupric bromide, cupric sulfate,
and cupric acetate could result in the same color change as cupric
chloride (Fig. S2). The further research revealed that the color
changes of SQ1 induced by Cu2þ were reversible as the addition of
EDTA reversed the color changes.
2.3. Spectral investigations of SQ1
The spectral responses of SQ1 (10 mM) to a range of physiological
2.2. Colorimetric assay of SQ1
and environmentally relevant metal ions were depicted in Figure 2.
SQ1 had a very strong absorption band centered at 514 nm. The UV–
vis absorption spectrum of SQ1 solution shifted largely to the red
about 161 nm when combined with Cu2þ, but no significant spec-
tral changes were observed when combined with other metals,
which probably attributed to their lower affinity with the receptor
SQ1. In particular, in the competitive experiment of SQ1, the
A small amount of chemosensor SQ1 is soluble in a mixed sol-
vent of 10 mM HEPES solution and THF (v/v¼60/40, pH¼7.4), and
further diluted for analyte detection. Figure 1 showed the color
changes of SQ1 (10
mM) upon addition of 1.0 equiv of a series of
individual metallic cations including Liþ, Naþ, Kþ, Ca2þ, Sr2þ, Ba2þ
,

W. Wang et al. / Tetrahedron 66 (2010) 3695–3701
3697
existence of alkali, alkaline earth, and other transition metal ions
did not interfere with the recognition of Cu2þ, which indicated that
SQ1 was potentially applicable to the Cu2þ sensing in complicated
environmental samples (Fig. 2).
to further evaluate the capability of SQ1 as a fluorescent probe to
image Cu2þ in living cells. Unfortunately, SQ1 could not dissolve in
cell cultured medium and even in D-Hanks containing 1% DMSO.
Figure 2. UV–vis absorption changes of SQ1 (10
m
M) in (10 mM) HEPES/THF (v/v¼60/
Figure 4. Fluorescent responses of SQ1 (C¼10 M) in (10 mM) HEPES/THF (v/v¼60/40,
m
40, pH¼7.4) in the presence of 1 equiv of different chloride salts (except for mercuric
acetate and silver nitrate) including Liþ, Naþ, Kþ, Ca2þ, Mg2þ, Fe3þ, Co2þ, Ni2þ, Cu2þ
Zn2þ, Cr3þ, Cd2þ, Pb2þ, Mn2þ, Hg2þ, Agþ, and their mixture.
,
pH¼7.4) to various metal ions (1 equiv). Excitation was at 530 nm and emission was at
626 nm.
To understand a crucial role, which 2-picolyl unit played on the
sensing behavior, we synthesized squaraine-based derivative SQ3
bearing 4-picolyl units and investigated the responses of SQ3 to
Cu2þ under the same condition as SQ1. As depicted in Figures S4
and S5, Cu2þ induced no appreciable absorption or fluorescence
spectral changes revealing that SQ3 had lower affinity for Cu2þ
compared with SQ1. The assay indicated that the nitrogen atom on
the 2-position of the pyridine group acted as an important binding
site for the Cu2þ complexation.
To understand the coordination behavior between SQ1 and
Cu2þ, the UV–vis absorption spectral titration experiment of SQ1
with Cu2þ in (10 mM) HEPES/THF (v/v¼60/40, pH¼7.4) was carried
out. As shown in Figure 3a, the original absorption band centered at
514 nm gradually decreased in intensity upon addition of Cu2þ with
the formation of a new absorption band at ca. 675 nm, generating
an isosbestic point at 565 nm, which indicated a new complex was
formed. The Job’s plot of the UV–vis titrations revealed a 1:1 stoi-
chiometric ratio between the Cu2þ ion and SQ1 (Fig. 3b). The ESI-
TOF mass spectrum of a mixture of SQ1 and CuCl2 also revealed
the formation of a 1:1 metal–ligand complex through the metal
coordination interaction where there was a major signal at m/z
2.4. The proposed binding model
The binding model of SQ1 and Cu2þ proposed was illustrated in
Figure 5. Cu2þ was coordinated by the phenolic hydroxyl oxygen
atom and two 2-picolyl nitrogen atoms in a three-dimensional way.
For squaraine SQ1, resonance-stabilized zwitterionic structure was
787.25
(calcd
787.15)
assigned
to
the
species
[SQ1(Cu)$CH3OHꢀ2H]þ (Fig. S13). The stability constant of the
newly formed complex was determined as 1.9ꢁ106 Mꢀ1 (R¼0.993)
by using nonlinear curve fitting (Fig. S3).11
Figure 3. (a) UV–vis titration curves (0–1.3 equiv) and (b) Job plot analysis of a solution of SQ1 in (10 mM) HEPES/THF (v/v¼60/40, pH¼7.4) with Cu2þ
.
The selectivity of SQ1 to Cu2þ ion was further investigated by
fluorometric detection in HEPES/THF (v/v¼60/40, pH¼7.4). Che-
mosensor SQ1 exhibited a relative strong fluorescence emission at
626 nm upon excitation at 530 nm. Upon addition of the same
amount of various metal ions, only Cu2þ quenched nearly 85 percent
fluorescent intensity of SQ1, and no new emission emerged. Com-
pound SQ1 did not respond distinctly to other metal species. The
obvious selectivity of SQ1 toward Cu2þ suggested that SQ1 could be
used as a fluorescent probe in sensing Cu2þ (Fig. 4). We had intended
preferred, while the binding of Cu2þ might localize the positive
charge to the phenolic hydroxyl group, which was opposite to the
Cu2þ-complexed binding site.12 On the basis of this hypothesis, the
symmetrical squaraine SQ1 could only bind one copper ion which
was consistent with the 1:1 stoichiometry between SQ1 and Cu2þ
from Job plot analysis. The importance of 2-picoyl groups was that
they provided additional synergic coordination sites for effective
Cu2þ recognition, which was supported by the comparative ex-
periment between SQ1 and SQ3.

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W. Wang et al. / Tetrahedron 66 (2010) 3695–3701
but no significant spectral changes were observed when other com-
OH
OH
HO
HO
O
N
HO
N
N
OH
N
peting metals were added. The UV–vis titration curves did not change
obviously any more (Fig. S9) and the absorption band peaked at
640 nm arrived at the maximum value (Fig. S10) after addition of
1 equiv of Cu2þ indicating the 1:1 complexation of SQ2 and Cu2þ. The
1:1 stoichiometry was further confirmed by the electrospray ioniza-
tion (ESI) mass spectrum analysis (Fig. S14). ESI-TOF mass spectros-
copy showed a peak at m/z¼737.33 for [SQ2(Cu)$2CH3OH$H2OþH]þ
(calcd 737.27). The related experimental results and figures were
displayed in the Supplementary data.
2+
O
1
OH
OH
HO
HO
OH
OH
HO
HO
O
O
N
HO
N
N
OH
N
N
N
OH
N
HO
N
O
2
O
3
To evaluate the possibility of SQ2 as a fluorescent probe to image
Cu2þ in living cells, we first examined the selectivity of SQ2 (10
mM)
to Cu2þ ion by fluorometric detection in DMSO/Hanks (v/v¼40/60,
pH¼7.0). As shown in Figure 7 and Figure S12, only Cu2þ quenched
nearly 90 percent fluorescent intensity of SQ2, while other
biologically relevant metal cations did not induce any discernible
changes in emission intensities. The high selectivity of SQ2 to Cu2þ
clearly established that SQ2 had the potential application of serving
as a fluorescent probe for live-cell imaging.
OH
OH
HO
HO
O
O
N
N
H
O
N
HO
N
Figure 5. The proposed charge-localized mechanism of sensing Cu2þ
.
2.5. Selectivity of SQ2 to Cu2D
Fluorescent sensors that can selectively monitor specific metal
ions in living cells have been indispensable tools for understanding
of biological phenomena. Considering the proper amphipathicity of
a sensor was favorable for cellular uptake and intracellular fluo-
rescent imaging,13 we also prepared the 2,4,6-trihydroxy-
phenylsquaraine-based compound SQ2 with two butyl chains. We
studied the responses of SQ2 (10 mM) to various metals in (10 mM)
HEPES/THF (v/v¼60/40, pH¼7.4) and it was proved to be a colori-
metric probe for Cu2þ with high selectivity and sensitivity as SQ1
(Fig. S6). A solution of SQ2 (10
mM) displayed a bright purple color
and the color turned blue-black upon addition of 1.0 equiv of Cu2þ
,
while the same amount of other metallic ions did not interfere with
the Cu2þ detection by naked eye. The limit of Cu2þ by naked eye
was as low as SQ1 (Fig. S7) and anionic counterions had no effect on
the sensing behavior (Fig. S8).
The spectral responses of SQ2 (10 mM) to a range of physiological
and environmentally relevantmetalions were depictedinFigure 6. As
depicted in Figure 6, a strong absorption band peaked at 554 nm was
Figure 7. Fluorescent responses of SQ2 (10
to various metals (1 equiv) with excitation at 550 nm and emission at 651 nm (emis-
sion slits¼3 nm).
m
M) in DMSO/Hanks (v/v¼40/60, pH¼7.0)
observed and there was a large 86 nm red shift upon addition of Cu2þ
,
2.6. Cell imaging and cytotoxicity assay
To determine whether SQ2 could be used as a Cu2þ probe in
living cells, we carried out experiments on LL/2 and HepG2 cells
with SQ2. After LL/2 and HepG2 cells were cultured in D-Hanks
buffer containing 1% DMSO supplemented with 20 m
M SQ2 at 37 ꢂC
for 1 h and 0.5 h, respectively and washed with D-Hanks buffer to
remove the remaining dye SQ2, a clear red fluorescent image could
be observed obviously from fluorescence microscopy as shown in
Figure 8. The intracellular fluorescence became faint after addition
of Cu2þ (5 equiv) and was effectively restored by addition of EDTA.
The experimental results revealed that SQ2 permeated well
through the cell membrane and could be used as a fluorescent
probe for imaging labile Cu2þ in living biological samples. Sub-
sequently, visualization of localization within live LL/2 cells and
HepG2 cells was determined using a confocal laser scanning mi-
croscopy (CLSM). As demonstrated in Figure 9, the squaraine SQ2
preferentially accumulated in the cytoplasm, and especially around
the perinuclear region. To evaluate cytotoxicity of the probe, SQ2
was taken as an example to perform a MTT assay on HepG2 cells
Figure 6. UV–vis absorption changes of SQ2 (10
40, pH¼7.4) in the presence of 1 equiv of different chloride salts (except for mercuric
acetate and silver nitrate) including Liþ, Naþ, Kþ, Ca2þ, Mg2þ, Fe3þ, Co2þ, Ni2þ, Cu2þ
Zn2þ, Cr3þ, Cd2þ, Pb2þ, Mn2þ, Hg2þ, and Agþ
m
M) in (10 mM) HEPES/THF (v/v¼60/
with dye concentrations from 5 mM to 20 mM. The cellular viability
estimated was ca. 66% in 24 h after treatment with 20
(Fig. 10), exhibiting low toxicity to cultured cells.
mM of SQ2
,
.

W. Wang et al. / Tetrahedron 66 (2010) 3695–3701
3699
Figure 8. Brightfield and fluorescence images of LL/2 (a–d) and HepG2 (e–h) cells. (a,e) Brightfield images of LL/2 and HepG2 cells; (b,f) fluorescence images of LL/2 and HepG2 cells
stained with 20 M); (d,h) return of fluorescence after addition of EDTA
M SQ2 at 37 ꢂC for 1 h and 0.5 h, respectively; (c,g) Further supplemented with addition of CuCl2 (100
(500 M).
m
m
m
Figure 9. CLSM images of (a,b) LL/2 cells and (c,d) HepG2 cells treated with SQ2 and Hoechst 33258 (cell nuclei stained) in D-Hanks buffer containing 1% DMSO for 30 min.
Colorimetric methods can be convenient in practical applications
without the aid of any advanced instruments. Especially, the che-
mosensor SQ2 can be employed as a Cu2þ-selective probe in the
fluorescence imaging of living cells. Such a sensor would become
valuable in revealing the roles of Cu2þ in biological systems under
either in vitro or in vivo conditions.
4. Experimental
4.1. General
1H NMR and 13C NMR spectra were recorded on a Bruker AV-400
(1H: 400 MHz; 13C: 100 MHz) or
a
Varian INOVA-400 (1H:
400 MHz; 13C: 100 MHz). The 1H NMR (400 MHz) chemical shifts
were measured relative to TMS (0.00 ppm) for CDCl3 or relative to
DMSO-d6 as indicated. The 13C NMR (100 MHz) chemical shifts
were given relative to DMSO-d6 and CDCl3 as indicated. High res-
olution mass spectra (HRMS) were recorded on a Waters-TOF
Premier Mass Spectrometer by positive ESI-Q-TOF. Absorption
spectra were detected on a HITACHI U-2910 spectrometer. Fluo-
rescent emission spectra were collected on a Horiba Jobin Yvon-
Edison Fluoromax-4 fluorescence spectrometer. Melting points of
compounds were determined with XRC-1 and were uncorrected.
Unless otherwise noted, all chemicals and reagents were obtained
from commercial suppliers and used without further purification.
n-Butanol was dried by heating at reflux over sodium and distilled
prior to use. Benzene or toluene was treated firstly by shaking with
concd H2SO4 until free form thiophene, then washed with water,
Figure 10. Cell viability values (%) estimated by a MTT assay on HepG2 cells.
3. Conclusion
In conclusion, we have developed highly sensitive and selective
colorimetric and fluorometric dual-channel sensors SQ1 and SQ2
for Cu2þ in aqueous solution. The two colorimetric probes can make
‘naked-eye’ detection of Cu2þ in the visible wavelength region.

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W. Wang et al. / Tetrahedron 66 (2010) 3695–3701
dilute NaOH, and water, followed by drying with sodium, and dis-
tilled prior to use. All syntheses and manipulations were carried out
under dry N2 atmosphere. The stock solutions of fluorophores and
metal chloride were freshly prepared and used for each measure-
ment. THF was either HPLC or spectroscopic grade and water was
distilled for twice. Each time a 3 mL of receptor solution was filled
in a quartz cell of 1 cm of optical path length, and the stock solution
of metal chloride was added into a quartz cell dropwise using
a micro-syringe. The volume of metal chloride stock solution added
methanol/ethyl acetate (1/7) to afford a yellow product (0.78 g,
yield 32%). Mp: 148–150 ꢂC. 1H NMR (400 MHz, DMSO-d6):
¼3.84
(s, 4H), 6.06 (s, 1H), 7.12 (d, J¼6.0 Hz, 4H), 8.34 (s, 1H), 8.36–8.38
(m, 4H), 9.16 (s, 2H) ppm. 13C NMR (100 MHz, DMSO-d6):
95.0, 104.6, 123.9, 149.2, 151.6, 154.6, 155.0 ppm. HRMS (ESI): m/z
calcd for C18H17N2O3 [MþH]þ 309.1239, found 309.1235.
d
d
¼28.2,
4.5. Synthesis of SQ3
was less than 100
changed. The excitation and emission slits of fluorescence spectra
were set at 5.0 nm if not specified.
m
L to remain the concentration of receptor un-
A flame-dried Schlenk test tube with a magnetic stirring bar was
charged with squaric acid (0.013 g, 0.11 mmol), 2,4-bis(pyridin-4-
ylmethyl)benzene-1,3,5-triol (0.070 g, 0.23 mmol), toluene (5 mL),
and n-butanol (5 mL) under nitrogen. The reaction mixture was
stirred at reflux for 57 h and then cooled to ambient temperature.
The resulting mixture was filtered and the solid was washed with
isopropanol and hexane until the filtrate was almost colorless. The
desired product was obtained as a purple solid (31 mg, yield 41%).
4.2. Synthesis of 2,4-bis(pyridin-2-ylmethyl)benzene-
1,3,5-triol (2)
A mixture of 2-(chloromethyl)pyridine hydrochloride (4.0 g,
24.4 mmol), anhydrous phloroglucinol (1.0 g, 8.0 mmol), and
potassium iodide (0.1 g) in 8 mL of toluene was heated at 100 ꢂC
with stirring under nitrogen for 24 h and then cooled to ambient
temperature, followed by addition of methanol (16 mL), dichloro-
methane (16 mL), and sodium hydrogen carbonate (5.0 g,
59.5 mmol). The mixture was allowed to stir overnight at room
temperature. The resulting mixture was then filtered and washed
well with methanol (15 mL) and dichloromethane (15 mL). The
filtrate was concentrated under reduced pressure. The residue was
purified by column chromatography on silica gel eluting with ethyl
acetate/dichloromethane (1/6) to afford a slightly yellow product
(0.47 g, yield 19%). Mp: 193–195 ꢂC. 1H NMR (400 MHz, DMSO-d6):
Mp: >300 ꢂC. 1H NMR (400 MHz, DMSO-d6):
(s, 2H), 6.57 (br s, 4H), 7.44 (d, J¼5.2 Hz, 8H), 8.56 (d, J¼4.8 Hz,
8H) ppm. 13C NMR (100 MHz, DMSO-d6):
d
¼3.97 (s, 8H), 6.09
d
¼28.9, 95.1, 103.9, 125.2,
145.2, 154.6, 155.3, 157.6, 195.7 ppm. HRMS (ESI): m/z calcd for
C40H29N4O8 [MꢀH]þ 693.1985, found 693.1984.
4.6. Synthesis of N,N-dibutylbenzenamine (4)
A sodium hydroxide solution (20 mL, 8 N) was added dropwise
to a stirred mixture of 1-bromobutane (15 mL, 0.16 mol) and aniline
(5 mL, 53.69 mmol) at 100 ꢂC under nitrogen and the reaction
mixture was continued to react for 48 h. After being cooled to
ambient temperature, the resulting mixture was extracted with
ethyl acetate and the combined extracts were dried over anhydrous
sodium sulfate. The obtained organic layer was concentrated under
reduced pressure and the residue was purified by column
chromatography on silica gel eluting with petroleum ether/
dichloromethane (1/1) to afford a yellow liquid (8 g, yield 73%). 1H
d
¼3.96 (s, 4H), 6.00 (s, 1H), 7.16 (d, J¼7.6 Hz, 2H), 7.20–7.23 (m, 2H),
7.68–7.72 (m, 2H), 8.43–8.44 (m, 2H), 9.30 (s, 2H),10.73 (s,1H) ppm.
13C NMR (100 MHz, DMSO-d6):
d¼31.9, 95.3, 105.1, 121.6, 122.4,
137.7, 148.2, 154.8, 155.8, 161.9 ppm. HRMS (ESI): m/z calcd for
C18H17N2O3 [MþH]þ 309.1239, found 309.1231.
4.3. Synthesis of SQ1
NMR (400 MHz, CDCl3):
d
¼1.04 (t, J¼7.6 Hz, 6H), 1.41–1.50 (m, 4H),
1.63–1.71 (m, 4H), 3.34 (t, J¼7.6 Hz, 4H), 6.70–6.76 (m, 3H), 7.28–
7.32 (m, 2H) ppm. HRMS (ESI): m/z calcd for C14H24N [MþH]þ
206.1909, found 206.1906.
A flame-dried Schlenk test tube with a magnetic stirring bar was
charged with squaric acid (0.008 g, 0.074 mmol), 2,4-bis(pyridin-2-
ylmethyl)benzene-1,3,5-triol (0.045 g, 0.147 mmol), toluene
(4.5 mL), and n-butanol (4.5 mL) under nitrogen. The reaction
mixture was heated at 100 ꢂC with stirring for 20 h and then cooled
to ambient temperature. The resulting mixture was filtered and the
solid was washed with isopropanol and hexane until the filtrate
was almost colorless. The desired product was obtained as a purple
solid (40 mg, yield 78%). Mp: >300 ꢂC. 1H NMR (400 MHz, DMSO-
4.7. Synthesis of 3-[4-(N,N-dibutylamino)phenyl]-4-
hydroxycyclobutene-1,2-dione6e,14 (5)
A solution of N,N-dibutylbenzenamine (1.36 g, 6.62 mmol) in
dried benzene (30 mL) was added to a round flask charged with
squaryl chloride (1.0 g, 6.62 mmol) prepared following the reported
procedure15 and refluxed for 15 h. The resulting mixture was then
cooled to ambient temperature, dissolved in a mixture of acetic acid
(25 mL), water (25 mL), and concentrated HCl (1.3 mL) and refluxed
for 3 h. After being cooled to ambient temperature, the yellow
precipitate was obtained by filtration, washed with ether, and dried
d6):
d
¼4.06 (s, 4H), 4.14 (s, 4H), 7.42 (d, J¼8.0 Hz, 2H), 7.49–7.55
(m, 4H), 7.57–7.60 (m, 2H), 8.04 (t, J¼6.8 Hz, 2H), 8.10 (t, J¼8.0 Hz,
2H), 8.58 (d, J¼4.8 Hz, 2H), 8.61 (d, J¼5.2 Hz, 2H) 12.36 (s, 2H) ppm.
13C NMR data were not recorded due to poor solubility. HRMS (ESI):
m/z calcd for C40H31N4O8 [MþH]þ 695.2142, found 695.2136.
(yield 71%). 1H NMR (400 MHz, DMSO-d6):
d¼0.88 (t, J¼7.2 Hz, 6H),
4.4. Synthesis of 2,4-bis(pyridin-4-ylmethyl)benzene-1,3,5-
triol (3)
1.27–1.36 (m, 4H), 1.49 (s, 4H), 3.39 (s, 4H), 4.94 (br s, 1H), 6.87
(s, 2H), 7.86 (s, 2H) ppm. HRMS (ESI): m/z calcd for C18H23NO3Na
[MþNa]þ 324.1576, found 324.1573.
A mixture of 4-(chloromethyl)pyridine hydrochloride (4.0 g,
24.4 mmol), anhydrous phloroglucinol (1.0 g, 8.0 mmol), and po-
tassium iodide (0.1 g) in 8 mL of toluene was heated at 100 ꢂC with
stirring under nitrogen for 12 h and then cooled to ambient tem-
perature, followed by addition of methanol (16 mL), dichloro-
methane (16 mL), and sodium hydrogen carbonate (5.0 g,
59.5 mmol). The mixture was allowed to stir overnight at room
temperature. The resulting mixture was then filtered and washed
well with methanol (15 mL) and dichloromethane (15 mL). The
filtrate was concentrated under reduced pressure. The residue was
purified by column chromatography on silica gel eluting with
4.8. Synthesis of SQ2
A flame-dried Schlenk test tube with a magnetic stirring bar
was charged with compound 2 (0.102 g, 0.33 mmol), compound 5
(0.100 g, 0.33 mmol), toluene (8 mL), and n-butanol (8 mL) under
nitrogen. The reaction mixture was stirred at reflux for 34 h and
then cooled to ambient temperature. The solvent was removed
under reduced pressure and the residue was purified by column
chromatography on silica gel eluting with methanol/dichloro-
methane (1/20) to afford a blue solid (0.12 g, yield 60%). Mp: 134–

W. Wang et al. / Tetrahedron 66 (2010) 3695–3701
3701
136 ꢂC. 1H NMR (400 MHz, CDCl3):
d
¼0.92–0.99 (m, 6H), 1.33–1.42
Acknowledgements
(m, 4H), 1.58–1.65 (m, 4H), 3.36 (t, J¼7.6 Hz, 4H), 4.07 (s, 4H), 6.67
(d, J¼8.8 Hz, 2H), 7.07 (s, 2H), 7.41 (s, 2H), 7.62 (s, 2H), 8.05
(d, J¼8.0 Hz, 2H), 8.34 (s, 2H), 12.93 (s, 3H) ppm. 13C NMR
This work was supported by grants from the National Natural
Science Foundation of China (No. 20772086). We thank the Centre
of Testing and Analysis, Sichuan University for NMR measurements.
(100 MHz, CDCl3):
d
¼13.8, 20.2, 29.5, 31.4, 51.1, 108.1, 108.7, 112.3,
117.6, 121.4, 123.5, 132.3, 137.8, 147.4, 160.9, 161.4, 182.9 ppm.
HRMS (ESI): m/z calcd for C36H38N3O5 [MþH]þ 592.2811, found
592.2803.
Supplementary data
Supplementary data associated with this article can be found in
online version at doi:10.1016/j.tet.2010.03.070. These data include
MOL files and InChiKeys of the most important compounds
described in this article.
4.9. Cell culture
Human hepatocellular carcinoma cell line HepG2 and Lewis
lung carcinoma cell line LL/2 were obtained from the American
Type Culture Collection (ATCC) and cultured in DMEM containing
References and notes
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