Glucosamine modified near-infrared cyanine as a sensitive colorimetric fluorescent chemosensor for aspartic and glutamic acid and its applications

Article abstract of DOI:10.1039/c4nj00266k

A glucosamine modified near-infrared cyanine fluorescent probe has been synthesized and developed as an efficient and visual sensor for the trace level determination of glutamic acid (Glu) and aspartic acid (Asp) with no interference by other amino acids. A prominent fluorescence enhancement at 805 nm and a remarkable color change from pale red to colorless were observed in the presence of Glu and Asp respectively. This probe may also serve as a highly sensitive acidic pH probe with a pK_a value of 5.82. Furthermore, the chemosensor has been successfully applied for the imaging of Glu and Asp in living cells, and the cytotoxicity assay exhibited that it is of low toxicity under the experimental conditions. This journal is

Full text of DOI:10.1039/c4nj00266k

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Glucosamine modified near-infrared cyanine as a
sensitive colorimetric fluorescent chemosensor
for aspartic and glutamic acid and its
applications†
Cite this: New J. Chem., 2014,
38, 4791
Xu Zhao,a Ruisong Wei,b Ligong Chen,ac Di Jina and Xilong Yan*ac
A glucosamine modified near-infrared cyanine fluorescent probe has been synthesized and developed
as an efficient and visual sensor for the trace level determination of glutamic acid (Glu) and aspartic acid
(Asp) with no interference by other amino acids. A prominent fluorescence enhancement at 805 nm and
a remarkable color change from pale red to colorless were observed in the presence of Glu and Asp
respectively. This probe may also serve as a highly sensitive acidic pH probe with a pKa value of 5.82.
Furthermore, the chemosensor has been successfully applied for the imaging of Glu and Asp in living
cells, and the cytotoxicity assay exhibited that it is of low toxicity under the experimental conditions.
Received (in Montpellier, France)
23rd February 2014,
Accepted 24th April 2014
DOI: 10.1039/c4nj00266k
www.rsc.org/njc
A Co(II) complex with 2-(2-pyridyl)benzimidazole1 and a poly-
thiophene–gold nanoparticle composite13 were discovered as
Introduction
Aspartic acid (Asp) and glutamic acid (Glu), major excitatory probes for EAAS. Though considerable efforts have been made
amino acids (EAAS), can activate N-methyl-D-aspartate (NMDA) to develop fluorescent probes for the detection of EAAS, it
reporters in the mammalian central nervous system (CNS). remains a challenge to acquire new simple molecular-based
EAAS exist in more than half of CNS synapses and play crucial EAAS sensors, which have properties such as high sensitivity,
roles in memory, learning, movement disorders and drug good aqueous solubility, biocompatibility and quick response
additions as well as many other normal or abnormal physiolo- for real-time detection.
gical processes and behaviors.1 The balance of Asp and Glu has
Near-infrared (NIR) fluorescent probes with long wavelength
an effect on brain function, and any alterations in such a balance emission (650–900 nm) are preferred for biological imaging
may cause several neurological or psychiatric disorders.2,3 applications, because they reduce background absorption and
The altered levels of EAAS in humans are also closely related tissue auto-fluorescence, reduce photodamage to living cells
to some diseases such as epilepsy,4 Parkinson’s disease,5,6 and can penetrate deeply with low light scattering to achieve
ischemic brain injuries,7,8 and so on.
a distinct image in vivo assay.14–16 Herein, we designed and
Thus, the development of efficient methods for the selective synthesized a NIR cyanine fluorescent probe as a chemosensor
detection and quantification of Asp and Glu in physiological for EAAS, which bearing a non-N-alkylated indolium moiety can
media has been of intense interest. Up to date, many methodol- act as a recognition site of Glu and Asp. To improve the probe’s
ogies have been designed to detect them,9–11 such as liquid or solubility in aqueous media and reduce its aggregation in
gas chromatography, capillary electrophoresis, electrochemical solution, a glucosamine functional group was introduced.17
or optical assays.1,12,13 Among them, optical assays based on syn- In the absence of Glu and Asp, this probe (10À5 M) showed a
thetic colorimetric and fluorescent probes have received increasing very weak fluorescence. Upon treatment with Glu or Asp, a
attention owing to their simplicity, high selectivity and sensitivity. prominent enhancement of fluorescence intensity accompa-
nied by a remarkable color change from pale red to colorless
was observed due to the protonation of indole nitrogen atoms.
It was also found that the probe was sensitive to pH. Moreover,
the probe has been successfully applied for the detection of Glu
and Asp in living cells. A cytotoxicity assay has confirmed that
this probe is of low toxicity under the experimental conditions.
Hence, this probe has potential applications in the detection of
Glu and Asp or pH variations in living cells with a fluorescence
turn-on signal.
a School of Chemical Engineering and Technology, Tianjin University,
Tianjin 300072, P. R. China. E-mail: yan@tju.edu.cn; Fax: +86 27406314;
Tel: +86 27406314
b Department of Chemistry and Life Sciences, Hechi University, Yizhou 546500,
P. R. China
c Collaborative Innovation Center of Chemical Science and Engineering (Tianjin),
P. R. China
† Electronic supplementary information (ESI) available: 1H NMR spectrum,
13C NMR spectrum and ESI-MS spectra. See DOI: 10.1039/c4nj00266k
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Results and discussion
We have designed and synthesized a NIR fluorescent chemo-
sensor for detection of Glu and Asp. To improved the probe’s
solubility and reduce its aggregation in solution, a glucosamine
functional group was introduced. The synthetic procedures of
the probe were summarized in Scheme 1. Ethyl-3-(3,3-dimethyl-
2-methyleneindolinl-yl)-propionate (2) was obtained using a
multistep synthesis starting from phenyl hydrazine and 3-methyl-
2-butanone and then modification by a nucleophilic substitution
reaction with ethyl-3-bromo propionate. 2-Chloro-1-formyl-3-
(hydroxymethylene)-cyclohex-1-ene (3) was prepared according
to the literature method.18 Then a reaction of 2 and 3 in ethanol
provided 4. TM (the target compound) was prepared from 5
(the hydrolyzate of 4) and D-glucosamine hydrochloride through
an acylation reaction in a satisfactory yield. The structures of
TM and intermediates were characterized by 1H NMR, 13C NMR
and ESI-MS analysis.
To investigate the sensitivity of the probe to Glu and Asp,
UV-vis titration of the probe (10À5 M) towards Glu and Asp (0 to
25 equiv.) were measured first. As shown in Fig. 1(A and B),
upon addition of EAAS, a new absorption peak at 780 nm
progressively increased; concomitantly the absorption peak of
the probe at 520 nm gradually decreased. The absorbance reached
a maximum when the concentration of Asp and Glu is 5 equiv.
and 12 equiv. respectively. Meanwhile, the color of the solution
changed from pale red to colorless, which indicated that this
probe could serve as a visual indicator for Glu and Asp. Moreover,
a further observation is the presence of an isosbestic point at
Fig. 1 (A and B) Effect of Glu and Asp on UV-vis absorption. (A) UV-vis
absorption changes of the probe upon the addition of Glu in an ethanol–
water (20/80, V/V) solution. [probe] = 10À5 M, [Glu] = 0, 1, 2, 3, 4, 5, 6, 8, 10,
12, 14, 16, 18, 20, 25 equiv. respectively. (B) UV-vis absorption changes of
the probe upon the addition of Asp at similar conditions.
599 nm which indicated that only one species was produced
upon the reaction of the probe with EAAS.
Fluorescence emission spectra of the probe in the presence
of Glu and Asp at various concentrations were then assessed
(shown in Fig. 2). As the concentration of Glu or Asp increases,
the fluorescence intensity at 805 nm enhanced prominently,
and the color of the solution changed from pale red to colorless.
In order to clearly understand the amino acid concentration affect
on the fluorescence intensity, the fluorescence intensity at 805 nm
was plotted as a function of the Asp and Glu concentration (Fig. 3A
and B). The results showed that about 5 equiv. of Asp and 12 equiv.
of Glu are required for the saturation of the fluorescence intensity
under the titration conditions, respectively. This result is in good
agreement with that from the UV-vis spectra. Moreover, the probe
with 805 nm (NIR) emission has a great potential for in vivo
imaging due to the increased optical penetrability and minimal
tissue autofluorescence in this region.
Scheme 1 Synthetic scheme for the probe. Reagents and conditions:
(a) 100 1C, 2 h, then acetic acid, reflux, 5 h; (b) ethyl 3-bromo propionate,
100 1C, 5 h; (c) 1 M NaOH, r.t., 2 h; (d) ethanol, reflux, 3 h; (e) 2 M NaOH in
ethanol, 50 1C, 3, N2 atmosphere; (f) EDCÁHCl, NHS, DMF, ice bath, then
r.t., 12 h, N2 atmosphere; (g) D-glucosamine hydrochloride, DIPEA, DMF,
5 h, N2 atmosphere.
To investigate the detection limit of the probe for Glu and
Asp, a typical fluorescence titration of Glu (10À6–10À5 M) and
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Fig. 3 Fluorescence intensity at 805 nm vs. Asp and Glu concentration.
Fig. 2 Fluorescence spectra (lex = 780 nm, lem = 805 nm) of the probe
(10À5 M) in an ethanol–water (20/80, V/V) solution. (a) In the presence
of Glu (0, 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 25 equiv., respectively).
(b) In the presence of Asp (0, 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20,
25 equiv., respectively).
intensity variation at 805 nm and the color change are shown in
Fig. 7. These results suggest that the probe exhibits a quick
response and high selectivity toward Glu and Asp and can be
used as a ‘‘naked eye’’ chemosensor for them without inter-
Asp (10À7–10À5 M) were supplemented using a 10À5 M solution ference from other natural amino acids. To further explore the
of the probe in ethanol–water (20/80, V/V) (Fig. 4A and B). The tolerance of the probe to Glu and Asp over other natural amino
fluorescence intensity at 805 nm was plotted as a function of acids, competitive experiments were performed with Glu and
the Asp and Glu concentration (Fig. 5). A noticeable change of Asp (20 equiv.) in the presence of other amino acids (10 equiv.).
fluorescence intensity still appeared even when Glu and Asp is The results indicate that the amplitude of Glu and Asp induced
at concentrations of 1 Â 10À6 M and 1 Â 10À7 M, respectively. fluorescence enhancement is not interfered by other natural
These results strongly indicate that this probe could serve as a amino acids such as Pro, Leu, Trp, Ty, Gly, Asn, Val, Ala, Met,
trace level determination method for Glu and Asp.
Gln, Ser, Phe, Thr, Ile and His.
The selectivity of the probe has been investigated with other
For practical applications, the cytotoxicity of the probe was
natural amino acids at a concentration of 2 Â 10À4 M (20 equiv.). measured using a CCK8 assay in MCF-7 cells with concentra-
The UV-vis and fluorescence spectra upon exposure to various tions of the probe from 10À3 to 10À8 M. The results showed an
amino acids are shown in Fig. 6(A and B). The fluorescent IC50 = 1046 mM, which clearly confirmed that the probe was of
response of the probe to Pro, Leu, Trp, Ty, Gly, Asn, Val, Ala, low toxicity to cultured cell lines at concentrations of 10À5 M.
Met, Gln, Ser, Phe, Thr, Ile and His exhibited no change, while And then, the imaging abilities of the probe in living cells (MCF-7)
the fluorescence intensity of the probe showed a slight was also evaluated (Fig. 8). When MCF-7 cells were pre-treated
enhancement with Cys, and a little decrease with Arg and Lys. with Glu or Asp and then incubated with the probe, a strong
Interestingly, a remarkable enhancement of fluorescent inten- fluorescence was observed. By contrast, addition of the probe
sity at 805 nm and a prominent change of color were observed (TM) without the amino acids resulted in almost no fluorescence.
only when Glu or Asp were added to the probe. The fluorescent The addition of an ‘‘always-on’’ probe (reference probe, IR783)
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Fig. 6 Effects of amino acids (20 equiv.) on the UV-vis spectra (A) and
fluorescence spectra (lex = 780 nm, lem = 805 nm) (B) of the probe
(10À5 M) in an ethanol–water (20/80, V/V) solution.
Fig. 4 The fluorescence spectra of the probe towards Glu (A) and Asp (B).
to the cell culture prior to the induction of EAAS led to strong
fluorescence also. The results indicate that this probe is cell-
permeable and can be used as a ‘‘turn on’’ detection method for
these amino acids in living cells.
As stated by previous studies, one or both of the indole
nitrogen atoms non-alkylated in cyanine, are pH-sensitive.
In acidic environments, protonation of the indole nitrogen
atoms gives rise to long wavelength absorption and strong
fluorescence. Deprotonation of the nitrogen atoms results in a
blue-shifted absorption band and little or no observable fluores-
cence emission.18 Interestingly, the spectral responses of the
probe towards Glu and Asp present similar results. Therefore, we
predict that changes to the pH value as a result of changing Glu
and Asp concentrations may serve as the reason for the remark-
able enhancement in the fluorescence emission.
In order to gain insight into the sensing mechanism of the
probe towards Glu and Asp and changes to the pH value of
the solution as a result of changing concentrations of Glu and
Asp, the UV-vis and fluorescence spectra of the probe in an
Fig. 5 The fluorescence intensity at 805 nm vs. the concentrations of Glu
and Asp.
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Fig. 9 Changes to the pH value as a result of changes to the concentra-
tions of Glu and Asp (or with the probe) in an ethanol–water (20/80, V/V)
solution. (A) and (B) show the changes to the pH value depending on the
Glu and Asp concentration (0, 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20,
25 equiv.); (C) and (D) show the changes to the pH value depending on the
Glu and Asp concentration (0, 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20,
25 equiv.) with the probe.
ethanol–water (20/80, V/V) solution over a pH range of 2 to 11
(Fig. 9) were measured. The results showed that the concen-
tration of Glu and Asp significantly alter the pH value of the
solution. From the fluorescence spectra, it can be observed
that the fluorescent intensity of the probe greatly increased as
the solution became more acidic. This is due to increased
protonation of the cyanine dye and leads to a change to the
existing form of the probe. Further proof of this acid–base
equilibrium can be achieved by observing the UV-vis spectra of
the probe at different pH (Fig. 10a). It can be seen that as the
solution became more acidic, the absorbance at 520 nm
decreased and at around 780 nm increased with an isosbestic
point appearing at 599 nm, much like that described in the
UV-vis titration of Glu and Asp. This phenomenon further
supports our hypothesis.
Fig. 7 The fluorescent intensity variation and color change of the probe in
an ethanol–water (20/80, V/V) solution induced by the addition of differ-
ent amino acids. (1) probe; (2) probe-Glu; (3) probe-Asp; (4) probe-Pro;
(5) probe-Arg; (6) probe-His; (7) probe-Lys; (8) probe-Leu; (9) probe-Cys;
(10) probe-Trp; (11) probe-Tyr; (12) probe-Gly; (13) probe-Asn; (14) probe-
Val; (15) probe-Ala; (16) probe-Met; (17) probe-Gln; (18) probe-Ser;
(19) probe-Phe; (20) probe-Thr; (21) probe-Ile. [Probe] = 10À5 M, [amino
acid] = 20 equiv.
Fig. 11 illustrates the pH response of the probe as a function
of I/Imax vs. pH, where I is the fluorescent intensity at different
pH, and Imax is the maximum output of the probe. It can be
seen that a regular sigmoidal response is observed for this
probe in response to pH. The pKa of the probe can be estimated
where I/Imax is 0.5.19 This provides a pKa value of 5.82 for
this probe.
The reversibility of the sensor is a very important aspect
when considering practical applications. The interaction
between the probe and H+ was reversible, which can be verified
by a fluorescence titration experiment of the probe in ethanol–
Fig. 8 Confocal fluorescence and bright-field images of living MCF-7
cells: A1–3: MCF-7 cells; B1–3: MCF-7 cells were incubated with a
reference probe (‘‘always-on’’ IR783, 1 Â 10À5 M) for 20 min; C1–3:
MCF-7 cells were incubated with the probe (TM, 1 Â 10À5 M) for 20 min; water (20/80, V/V) with NaOH solution and HCl solution. The
D1–3: MCF-7 cells were pre-incubated with 2 Â 10À4 M Glu for 24 h, and
then treated with the probe (TM, 1 Â 10À5 M) for 20 min; E1–3: MCF-7 cells
were pre-incubated with 2 Â 10À4 M Asp for 24 h, and then treated with
the probe (TM, 1 Â 10À5 M) for 20 min. A1, B1, C1, D1, E1 are fluorescence
images, A2, B2, C2, D2, E2 are bright-field images and A3, B3, C3, D3,
E3 are merged images.
experiment showed that the introduction of H+ could lead to an
increase of fluorescence intensity and the disappearance of the
color of the system. When OHÀ was added to the system again,
the fluorescence intensity decreased and the color of the system
was recovered. Further on and off switching experiments was
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Conclusion
A novel colorimetric NIR fluorescent chemosensor based on
glucosamine modified cyanine has been designed and synthe-
sized. This chemosensor exhibits excellent properties for the
detection of Glu and Asp. A remarkable enhancement of fluores-
cence intensity at 805 nm and a visible change of color were
observed in the presence of Glu and Asp. This chemosensor may
also serve as a highly sensitive acidic pH probe and the fluorescent
switch can be reversibly turned off and on by alternating the
pH value. Furthermore, the chemosensor has been successfully
applied in the imaging of Glu and Asp in living cells. The
cytotoxicity assay shows the probe is of low toxicity at the
measurement concentration. Hence, this probe is suitable for
detecting Glu and Asp or acidic pH variations in living cells
with a fluorescence turn-on signal.
Experimental
Materials and equipment
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(EDCÁHCl), N-hydroxy succinimide (NHS) and ethyl 3-bromo
propionate were purchased from Aladdin Reagent Co. Ltd.
(Shanghai, China). CCK8 was purchased from Sigma (Poole,
Dorset, England). Other reagents and solvents such as sodium
carbonate (anhydrous) and DMSO (analytical reagent) were
purchased from Tianjin Chemicals Co. (Tianjin, China) and
used without further purification unless otherwise noted.
DMSO was purified by refluxing with calcium hydride for 4 h
and then distilled.20 Deionized water was used throughout
this work. 1H and 13C NMR spectra were recorded with a Bruker
Avance 600 spectrometer, using TMS as an internal standard.
Mass spectrometry (MS) was obtained using a Thermo Fisher
Scientific LCQ Advantage Max. High-resolution mass spectra
were measured with a Bruker Daltonics micrOTOF-Q II instrument
(ESI†). All pH measurements were made with a Sartorius basic
pH-meter PB-10. UV-vis spectra were obtained on an Evolution
300 UV-visible spectrophotometer. Fluorescence spectra were
recorded on a VARINA Eclipse fluorescence spectrophotometer.
All measurements were performed under ambient atmosphere
at room temperature.
Fig. 10 UV-vis spectra (a) and fluorescence spectra (b) of the probe
(10À5 M) at different pH values.
General procedure for spectroscopic measurements
Working solutions of amino acids and the probe were prepared
by serial dilution of the stock solutions (1 Â 10À2 M of amino
acids in deionized water and 1 Â 10À3 M of the probe in ethanol).
Solutions of amino acids were mixed separately with the solution
of the probe in different equiv. and their UV-vis and fluorescence
studies were performed. For all measurements, the excitation
and emission slit widths were both 5 nm and the excitation
wavelength was 780 nm.
Fig. 11 The pH response of the probe as a function of I/Imax vs. pH.
carried out by alternating the addition of HCl or NaOH. This
switching can be reversibly performed for at least five cycles.
Through the above research, we can come to the conclusion
Cytotoxicity assay and fluorescence imaging
that a near-infrared colorimetric fluorescent chemosensor In vitro cytotoxicity of the probe was measured using CCK8
based on glucosamine modified cyanine can be used conveni- assay in MCF-7 cells according to the standard method of the
ently as a visual fluorescence sensor for EAAS and pH value.
MTT assay.21,22 The CCK8 assay is similar to the MTT assay,
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except it uses a water-soluble tetrazolium salt that produces with CH2Cl2 (3 Â 30 mL) and the combined organic phases
a water-soluble formazan dye upon reduction by cellular were washed with brine, dried over MgSO4, filtered and con-
dehydrogenases, and therefore doesn’t require a solubilization centrated to give a red oil. The crude product was purified by
step. Cells were seeded into a 96-well cell culture plate at 200 mL flash chromatography with gradient elution (petroleum ether to
per well, cultured for 24 h at 37 1C under 5% CO2, and then petroleum ether/ethyl acetate of 40 : 1) to obtain the title
different concentrations of the probe (0, 10À8, 10À7, 10À6, 10À5
10À4, 10À3 M) were added to the wells. The cells were then
,
compound as a pale yellow oil (7.27 g, 70.2%).
1H NMR (600 MHz, CDCl3) d(ppm): 7.11 (1H, t, J = 7.2 Hz,
incubated for 24 h at 37 1C under 5% CO2. Subsequently, 20 mL ArH), 7.07 (1H, d, J = 7.2 Hz, ArH), 6.76 (1H, t, J = 7.8 Hz, ArH),
CCK8 was added to each well and incubated for an additional 6.60 (1H, d, J = 7.8 Hz, ArH), 4.09 (2H, dd, J1 = 7.2 Hz,
4 h at 37 1C under 5% CO2. The amount of CCK8 formazan J2 = 14.4 Hz, CH2), 3.89 (2H, d, J = 15.6 Hz, CH2), 3.84 (2H, t,
was quantified by determining the absorbance at 450 nm with J = 7.2 Hz, CH2), 2.61 (2H, t, J = 7.2 Hz, CH2), 1.32 (6H, s, CH3),
a reference wavelength at 650 nm using a microplate reader 1.19 (3H, t, J = 7.2 Hz, CH3).
(Tecan, Austria). For each concentration, each treatment was
13C NMR (150 MHz, CDCl3) d(ppm): 171.97, 171.92, 160.77,
done in triplicate. The IC50 value was calculated according to 145.27, 137.48, 127.64, 121.98, 118.61, 105.38, 74.01, 60.73,
Huber and Koella.23
44.19, 38.6, 30.84, 30.06, 14.19.
MCF-7 cells were seeded in a 6-well plate at a density of
5 Â 105 cells per well in culture media. The cells were incubated
with 2 Â 10À4 M Glu or Asp in culture media for 24 h at 37 1C in
a humidified incubator. After washing three times with PBS,
the cells were further incubated with 1 Â 10À5 M of the probe in
culture media for 20 min. For the control experiments, the cells
were treated with an ‘‘always-on’’ probe (IR783), or treated with
the probe (TM) or EAAS respectively. Confocal fluorescence
imaging was acquired on an Olympus FV1000 confocal laser-
scanning microscope with a 40Â oil-immersion object lens.
Fluorescence was excited at 635 nm and emission was collected
by a 700–800 nm band pass filter.
MS: m/z 260.3 [M + H]+.
2-Chloro-1-formyl-3-(hydroxymethylene)-cyclohex-1-ene (3)
This compound was prepared according to the literature method
with some modifications.18 To a chilled solution of DMF (21.93 g,
23.1 mL, 300 mmol) in 10 mL CH2Cl2, POCl3 (23 g, 13.8 mL,
150 mmol) in 10 mL CH2Cl2 was added dropwise under an ice
bath. After 30 min, cyclohexanone (4.91 g, 5.2 mL, 50 mmol) in
5 mL CH2Cl2 was added, and the resulting mixture was stirred
at 80 1C for 4 h. It was then cooled to room temperature and
poured into ice water, and kept overnight to obtain compound
3 as a yellow solid (7.56 g, 87.9%).
Compound 4
Synthesis
Compounds 2 (7.79 g, 30 mmol) and 3 (5.16 g, 30 mmol) in
ethanol solution (30 mL) were added to a round bottom flask
and the resulting mixture was stirred at 50 1C for 0.5 h under a
N2 atmosphere. After cooling to room temperature, compound
1 (5.53 g, 35 mmol) was added to the mixture and it was
then stirred under reflux for an additional 3 h to afford a dark
red solution. After cooling to room temperature, the solvent
was removed. The crude red solid was purified by flash
chromatography with gradient elution (petroleum ether/ethyl
acetate of 15 : 1 to 3 : 1) affording compound 4 as a red solid
(5.56 g, 33.5%).
2,3,3-Trimethyl-3H-indoleine (1)
The mixture of phenyl hydrazine (5.40 g, 50 mmol) and 3-methyl-
2-butanone (4.40 g, 50 mmol) was stirred at 100 1C for 2 h.
After cooling to room temperature, the red liquid was extracted
with CH2Cl2 (2 Â 20 mL), and the combined organic extracts
were dried over MgSO4, filtered and concentrated. The residue
was diluted with acetic acid (20 mL) and stirred under reflux for
5 h, and then acetic acid was evaporated. The resulting residue
was diluted with CH2Cl2 (20 mL) and washed with saturated
aqueous Na2CO3 (3 Â 30 mL), brine, then dried over MgSO4,
filtered and concentrated to give a red oil. The crude product
was purified by flash chromatography with gradient elution
(petroleum ether/ethyl acetate of 20 : 1 to 3 : 1) to give a pale
yellow oil (6.41 g, 81.1%).
1H NMR (600 MHz, CDCl3) d(ppm): 8.10 (1H, d, J = 16.2 Hz,
–CHQCH–), 7.61 (1H, d, J = 7.2 Hz, ArH), 7.52 (1H, d, J =
12.6 Hz, –CHQCH–), 7.32 (2H, dd, J1 = 8.4 Hz, J2 = 14.4 Hz,
ArH), 7.21 (1H, t, J = 7.2 Hz, ArH), 7.16–7.19 (2H, m, ArH),
6.89 (1H, d, J = 7.2 Hz, ArH), 6.70 (1H, d, J = 7.8 Hz, ArH), 6.65
(1H, d, J = 16.2 Hz, –CHQCH–), 5.51 (1H, d, J = 12.6 Hz,
–CHQCH–), 4.12 (2H, dd, J1 = 7.2 Hz, J2 = 14.4 Hz, CH2), 4.00
(2H, t, J = 7.2 Hz, CH2), 2.67 (2H, t, J = 7.2 Hz, CH2), 2.60 (4H, t,
J = 6 Hz, CH2), 1.87–1.91 (2H, m, CH2), 1.65 (6H, s, CH3), 1.50
1H NMR (600 MHz, CDCl3) d(ppm): 7.53 (1H, d, J = 7.8 Hz,
ArH), 7.31–7.27 (2H, m, ArH), 7.19 (1H, t, J = 7.2 Hz, ArH), 2.28
(3H, s, CH3), 1.30 (6H, s, CH3).
Ethyl-3-(3,3-dimethyl-2-methyleneindolinl-yl)-propionate (2)
The reaction mixture of 2,3,3-trimethyl-3H-indole (6.32 g, (6H, s, CH3), 1.21 (3H, t, J = 7.2 Hz, CH3).
40 mmol) and ethyl-3-bromo propionate (7.20 g, 5.1 mL, 40 mmol)
13C NMR (150 MHz, CDCl3) d(ppm): 183.92, 171.60, 157.84,
was stirred at 100 1C for 5 h. After cooling to room temperature, 147.19, 143.83, 138.96, 137.45, 136.85, 128.68, 127.79, 126.62,
the red precipitate was collected and washed with petroleum ether 125.40, 122.54, 121.85, 121.00, 120.50, 120.35, 120.29, 108.26,
(3 Â 30 mL), and then it was dissolved in 1 M NaOH (30 mL) and 106.44, 93.42, 61.03, 52.46, 45.99, 38.22, 31.20, 28.21, 26,89,
stirred for 2 h at room temperature. The solution was extracted 26.40, 24.78, 21.50, 14.15.
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HRMS (ESI positive) calc. for C35H39ClN2O2, [M + H]+ 555.2778,
NJC
HRMS (ESI positive) calc. for C39H46ClN3O6, [M + H]+ 688.3153,
found 688.3146.
found 555.2774.
Compound 5
To an ethanol solution (20 mL) of intermediate 4 (5.54 g,
10 mmol), 2 M NaOH solution (10 mL) was added, and the
Acknowledgements
resulting mixture was stirred at 50 1C for 3 h under N2 atmo- We are grateful to Prof. J. T. Chen and Prof. X. P. Yan (Nan Kai
sphere. After cooling to room temperature, the solvent was University) for providing MCF-7 cells and some support.
removed, then the residue was dissolved in water (30 mL) and
acidified to pH = 3 with 1 M HCl solution. The precipitation was
collected by filtration and washed with water, then purified
by flash chromatography with gradient elution (petroleum
References
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1H NMR (600 MHz, MeOD) d(ppm): 8.11 (1H, d, J = 24 Hz,
–CHQCH–), 7.70 (1H, d, J = 6.6 Hz, ArH), 7.60 (1H, d, J =
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4798 | New J. Chem., 2014, 38, 4791--4798
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