Synthesis of novel taspine diphenyl derivatives as fluorescence probes and inhibitors of breast cancer cell proliferation

Article abstract of DOI:10.1002/bio.1331

Two novel taspine diphenyl derivatives (Ta-dD) were designed and synthesized by introducing different coumarin fluorescent groups into the basic structure of Ta-dD. The main advantage of these two compounds is that they can be used as fluorescence probes and inhibitors simultaneously. In the present study, the fluorescent properties of the probes were measured and their inhibition of four breast cancer cell lines was tested. Different concentrations of the fluorescence probe were added to MCF-7 breast cancer cells for fluorescence imaging analysis under normal conditions. The results suggested that both of the new compounds have not only fluorescence but also the ability to inhibit effects on different breast cancer cell lines, which indicates their possible further use as dual functional fluorescence probes in tracer analysis. Copyright

Full text of DOI:10.1002/bio.1331

Short communication
Received: 29 November 2010,
Revised: 10 May 2011,
Accepted: 12 May 2011,
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.1331
Synthesis of novel taspine diphenyl derivatives
as fluorescence probes and inhibitors of breast
cancer cell proliferation
Huaizhen He, Yingzhuan Zhan, Yanmin Zhang, Jie Zhang
and Langchong He*
ABSTRACT: Two novel taspine diphenyl derivatives (Ta‐dD) were designed and synthesized by introducing different
coumarin fluorescent groups into the basic structure of Ta‐dD. The main advantage of these two compounds is that they can
be used as fluorescence probes and inhibitors simultaneously. In the present study, the fluorescent properties of the probes
were measured and their inhibition of four breast cancer cell lines was tested. Different concentrations of the fluorescence
probe were added to MCF‐7 breast cancer cells for fluorescence imaging analysis under normal conditions. The results
suggested that both of the new compounds have not only fluorescence but also the ability to inhibit effects on different
breast cancer cell lines, which indicates their possible further use as dual functional fluorescence probes in tracer analysis.
Copyright © 2011 John Wiley & Sons, Ltd.
Keywords: taspine diphenyl derivatives; fluorescence probe; inhibitor; breast cancer cells
ligand, which targets the surface protein receptors expressed by
tumour cells. All the methods evaluate the effectiveness of an-
Introduction
Compared with normal cells, tumour cells appear to exhibit
ticancer drugs by measuring their levels of fluorescence. The
autocrine or paracrine‐stimulated growth, avoiding the normal
fluorescent probe itself does not take effect directly on the
growth‐suppressing signal (1). Breast cancer is a daunting disease
tumour cells. Therefore, it is important to develop dual‐function
and constitutes a continuing medical health problem for millionsof
women worldwide (2). Breast cancer cell lines have been the most
widely used models to investigate how proliferation, apoptosis and
fluorescence probes.
In a previous study, taspine was screened for the first time
from Radix et Rhizoma Leonticis (Hong Mao Qi in Chinese; HMQ),
migration become deregulated during the progression of breast
using cell membrane chromatography (CMC) (18,19). HMQ has
cancer (3). As treatment of breast cancer with chemotherapy is
often empirical, based on histological tumour parameters and in
many pharmacological actions, particularly anti‐cancer activity
(
20,21). Based on these data, our group designed and
the absence of specific mechanistic understanding, it has been
difficult to learn clearly from the success or failure of new drugs (4).
Therefore, it is significant to develop a target‐selective drug with
fluorescence, which can be applied in tracer analysis.
In recent years, fluorescence analysis has been applied in
medicinal preparations and research on drugs in body fluids
synthesized a series of taspine derivatives using a computer‐
aided drug design method. Through the CMC model selection,
we found that one of the taspine diphenyl derivatives Ta‐dD,
can continue to maintain taspine’s pharmacological activity,
particularly in terms of tumour cell proliferation (22,23).
In this work, we used Ta‐dD as the identifying group and
(5,6). Fluorescence probes have become powerful tools for
synthesized two novel taspine diphenyl derivatives, 6‐[2‐
biochemical and cellular studies, because of their high
sensitivity, visibility and low interference in living systems (7–10).
Usually, a fluorescence probe consists of a fluorescence
chromophore and an identifying group. Currently, two methods
have been used, primarily in applying fluorescent probes to
cancer detection and visualization. The most common tech-
nique is engineering the cancer cells to express fluorescence,
such as green fluorescent protein (GFP) (11–13). Although this
method tracks the cancer cells directly, it requires gene
transfection of each type of target cancer cell line to express
the fluorescent protein (14–16). Another method is to introduce
novel telomerase‐dependent and replication‐competent ade-
novirus that can express the fluorescence to target the cancer
cells (17). However, only the presence of highly active tel-
omerase, such as that found in malignant tissues, causes bright
fluorescence. Also, researchers usually conjugate a dye to a
2
(
3‐chloro‐4‐fluorophenylamino)‐2‐oxoethoxy]‐5,5′‐dimethoxy‐N ,
2
N ′‐dimethyl‐6′‐[2‐(4‐methyl‐2‐oxo‐2H‐chromen‐7‐yloxy)ethoxy]
biphenyl‐2,2′‐dicarboxamide (F‐Ta‐dD‐dOH) and 6‐[2‐(3‐chloro‐4‐
fluorophenylamino)‐2‐oxoethoxy]‐5,5′‐dimethoxy‐N ,N ′‐dimeth-
yl‐6′‐[2‐(4‐methyl‐2‐ox‐o‐2H‐chromen‐7‐yl‐amino)‐2‐oxoethoxy]
2
2
biphenyl‐2,2′‐dicarboxamide (F‐Ta‐dD‐dNH ), which were made
2
1
by introducing different coumarin groups (R ) into the structure
*
Correspondence to: L. He, Key Laboratory of Environment and Genes
Related to Diseases, Ministry of Education, School of Medicine, Xi’an
Jiaotong University, Xi’an 710061, People’s Republic of China. E‐mail:
helc@mail.xjtu.edu.cn
Key Laboratory of Environment and Genes Related to Diseases, Ministry of
Education, School of Medicine, Xi’an Jiaotong University, People’s Republic
of China
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Copyright © 2011 John Wiley & Sons, Ltd.

H. He et al.
of Ta‐dD (Fig. 1). We studied the fluorescence properties of Ta‐dD,
the original coumarin groups, the modified coumarin groups and
the target compounds simultaneously. We also explored the
preliminary inhibitory effect of F‐Ta‐dD‐dOH and F‐Ta‐dD‐dNH2
on four breast tumour cell lines.
Synthesis of the new fluorescence probes
The new fluorescence probes were prepared as shown in
Scheme 1. On the basis of previous work, we used commercially
available isovanillin as the starting material. The key interme-
diate Ta‐dD was obtained via bromination, benzylation,
oxidation, Ulmman reaction, catalytic hydrogenation and
substitution reaction. Comprehensively utilizing the different
activated substituents in the coumarin structure, including the
hydroxyl group and amino group, we obtained the coumarin
derivatives 7‐(2‐bromoethoxy)‐4‐methyl‐2H‐chromen‐2‐one
Experimental
Instrumentation
Melting points were measured with an X‐4 digital melting
point apparatus. Infrared (IR) spectra were recorded on KBr
pellets, using a Shimadzu FT‐IR 440 spectrometer in the range
(coumarin‐OH‐Br) and 2‐chloro‐N‐(4‐methyl‐2‐oxo‐2H‐chromen‐
7
‐yl) acetamide (coumarin‐NH ‐Cl). The introduction of 1,2‐
2
dibromoethane or chloroacetyl chloride enhanced the reactivity
between coumarin derivatives and Ta‐dD.
–
1
4
000–500 cm . Nuclear magnetic resonance (NMR) spectra
were measured using a Varian INOVA spectrometer at 400 MHz
in CDCl3 with TMS as an internal reference. Fluorescence
measurements were performed on a Hitachi F‐4500 fluorescence
spectrophotometer. The synthetic procedures were controlled by
thin‐layer chromatography (TLC) on plates precoated with silica
gel (GF‐254). The products were purified by recrystallization or
column chromatography on silica gel (53–75 µm). The fluores-
cent images were detected on an inverted phase contrast
microscope purchased from Nikon Instruments Co., Ltd. The
microplate reader was purchased from Bio‐Rad (USA). In this
study, all optical measurements were carried out at room
temperature under ambient conditions. The fluorescence inten-
sities are reported in arbitrary units; the experimental parameters
within a given experiment are the same, and the fluorescence
intensities are comparable within a given experiment.
Synthesis of F‐Ta‐dD‐dOH
Ta‐dD (5.45 g, 0.01 mol) was dissolved in anhydrous DMF
2 3
(100 mL), then anhydrous K CO (2.08 g, 0.015 mol) was added
and the mixture was stirred at 80 °C for 30 min. Then we added
coumarin‐OH‐Br (3.38 g, 0.012 mol). The reaction solution was
stirred in the same temperature for 24 h. After the reaction, the
mixture was allowed to cool and was poured into ice water. The
suspension was filtered and chromatographed on silica gel
(EtOAc:methanol, 20:1) to give F‐Ta‐dD‐dOH as a white solid; m.
−
1
p. 108–109 °C. Yield, 61%. IR (KBr) cm , 3326, 2938, 1723, 1618,
1
1
(
294, 1263, 1145, 1021. H‐NMR (400 MHz, CDCl
s, 2H), 7.477 (d, J = 8.8 Hz, 1H), 7.306 (d, J = 8.8 Hz, 1H), 7.195 (d,
J = 8.4 Hz, 1H), 7.106–7.062 (t, J = 8.8 Hz, 1H), 6.842–6.820 (m, 1H),
3
) δ(ppm): 7.683
6
6
3
.728 (d, J = 8.4 Hz, 1H), 6.640 (s, 1H), 6.425 (d, J = 4.8 Hz, 1H),
.168 (s, 1H), 4.132 (s, 2H), 4.132–4.095 (d, J = 7.6 Hz, 2H), 4.006–
.970 (d, J = 7.2 Hz, 2H), 3.816 (s, 3H), 3.759 (s, 3H), 2.803 (d,
Chemicals and materials
All chemicals used were of analytical reagent grade. Methanol,
dichloromethane, and N,N‐dimethylformamide (DMF) needed
J = 4.8 Hz, 3H), 2.629 (d, J = 4.8 Hz, 3H), 1.602 (s, 3H).
further purification. NaClO
Aldrich. Isovanillin (99%), Br
99%), chloroacetyl chloride (98.5%), H O2 (≥ 30%), sodium
2
(80%) was purchased from Sigma‐
2
(99.5%), 1‐(chloromethyl)benzene
Synthesis of F‐Ta‐dD‐dNH
2
(
2
phosphate monobasic dehydrate (≥ 99%), thionyl chloride
As mentioned earlier, Ta‐dD was treated using the same
(≥ 99%), methylamine water solution (25–30%), copper powder
(99%), Pd/C (10%), 7‐amino‐4‐methyl‐coumain (95%), resorcinol
(98%), 1,2‐dibromoethane (98%) and dimethyl sulphoxide
(98%) were purchased from Sinopharm Chemical Reagent Co.
method. We added coumarin‐NH ‐Cl (3.01 g, 0.012 mol) to the
mixture. The reaction solution was stirred at 80 °C for 24 h. After
the reaction, the suspension was filtered and chromatographed
2
on silica gel (EtOAc:methanol, 25:1) to give F‐Ta‐dD‐dNH as a
2
Ltd. Hydrogen (≥ 99.99%) and nitrogen (≥ 99.999%) were
purchased from Xi’an Weiguang gases Co. Ltd. Human breast
cancer cell lines were obtained from the Shanghai Institute of
Cell Biology in the Chinese Academy of Sciences. MTT, RPMI
grey solid under the same post‐treatment; m.p. 179–180 °C.
−
1
Yield, 56%. IR (KBr) cm : 3373, 2935, 1703, 1621, 1528, 1499,
1
1407, 1146, 1020. H‐NMR (400 MHz, CDCl ) δ(ppm): 7.765–7.717
3
(m, 1H), 7.617–7.524 (m, 2H), 7.343–7.306 (m, 2H), 7.129–7.068
(m, 3H), 6.888 (d, J = 8.4 Hz, 2H), 6.211 (s, 1H), 4.457–4.405 (m,
2H), 4.204 (d, J = 14.8 Hz, 2H), 3.872 (s, 6H), 2.761 (d, J = 3.2 Hz,
6H), 2.420 (s, 3H).
1
640, DMEM and trypsin were purchased from Gibco (USA).
Gefitinib (≥ 99%) was purchased from Nanjing Ange Pharma-
ceutical Co. Ltd.
Figure 1. Structure of the compounds: (A) taspine; (B) basic taspine diphenyl derivative (Ta‐dD).
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New fluorescence probes and labelled breast cancer cells
Scheme 1. General routes for the synthesis of the fluorescence probes. The reaction conditions were: (a) Br
2
, HAc, Fe, NaAc, 23–25 °C; (b) BnCl, K
2
CO
3
, EtOH, reflux; (c)
CO
anhydrous THF, NaClO
2
, NaH
2
PO
4 2
, 30% H O
2 2
, rt; (d) CH Cl
2
, SOCl
2
; (e) CH
2
Cl
2
, CH
3
NH
2
, 0–5 °C; (f) anhydrous DMF, Cu, N
2
, reflux; (g) H , Pd/C, rt; (h) anhydrous DMF, K
2
2
3
,
8
0 °C; (i) anhydrous DMF, K
2 3
CO , 80 °C.
Figure 2. Fluorescence spectra of the analytes: (A) Ta‐dD (blue), F‐Ta‐dD‐dOH (green), coumarin‐OH‐Br (red), Coumarin‐OH (black); (B) Ta‐dD (blue), F‐Ta‐dD‐dNH
2
(green),
coumarin‐NH ‐Cl (red), coumarin‐NH (black).
2
2
chloride. However, the introduction of these flexible link
structures not only enhances the reactivity between coumarin
derivatives and Ta‐dD, but eases the influence of the original
structure of Ta‐dD on the fluorescent chromophore. Therefore,
the fluorescence properties of the target compounds can be
guaranteed.
Results and discussion
Fluorescence characterization of the new
fluorescence probes
The fluorescence spectra of the analytes were measured and the
results are shown in Fig. 2. The results indicated the feasibility of
adding fluorescence by conjugating with a fluorescence group.
We assessed the fluorescence properties from the molecular
structure. Due to the stabile conjugated π electrons and the lone
electron pairs of oxygen or nitrogen atoms, coumarin‐OH and
Biological activity analysis
The colorimetric 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetra-
zolium bromide (MTT) cell proliferation assay was used to
quantify cell viability, measuring cell survival and proliferation
spectrophotometrically. We examined the effects of the fluores-
coumarin‐NH
stability and exhibit excellent fluorescence properties. Although
their derivatives, coumarin‐OH‐Br and coumarin‐NH ‐Cl, still
2
themselves have the characteristic of strong
2
cence probes F‐Ta‐dD‐dOH and F‐Ta‐dD‐dNH
of breast cancer cell lines MCF‐7, MDA‐MB‐23, ZR‐75‐30 and SK‐
BR‐3. As shown in Fig. 3, F‐Ta‐dD‐dOH and F‐Ta‐dD‐dNH could
2
on the growth
maintain a certain fluorescence intensity, the conjugate effect
reduces via connecting with 1,2‐dibromoethane or chloroacetyl
2
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H. He et al.
Figure 3. Inhibition of F‐Ta‐dD‐dNH
2
and F‐Ta‐dD‐dOH on breast tumour cell proliferation in different cell lines was evaluated using the MTT assay. Gefitinib was used as
a positive control drug. (A) Breast tumour cell lines MCF‐7 and MDA‐MB‐2; (B) breast tumour cell lines ZR‐75‐30 and SK‐BR‐3.
take effect with the target directly and inhibit cell growth to
different degrees. The activity of F‐Ta‐dD‐dOH against ZR‐75‐30
recognized. Labelled MCF‐7 cells were incubated in vitro. When
–5
the concentration of the probe was < 5 × 10 mol/L, a small
number of tumour cells were labelled and had no obvious
changes in shape (Fig. 4B, C). As the probe concentration
increased, the visual field was brighter and the shapes of the
MCF‐7 cells appeared to have irregular defects (Fig. 4A). In
addition, we simultaneously examined the staining capability of
and the activity of F‐Ta‐dD‐dNH against SK‐BR‐3 were greatest.
2
According to the above results, we can see that the introduction
of the fluorescence chromophore not only retained the
backbone structure of taspine biphenyl derivatives and phar-
macological activity, but also produced target compounds with
fluorescence properties.
F‐Ta‐dD‐dNH for the normal cell line HEK 293. Compared with
2
the control group (normal cells without probes), there was no
obvious staining behaviour (Fig. 4D–F). The data demonstrated
that the dual‐function molecules had selectivity for cancer cells
over normal ones. In short, combined with the MTT assay, it can
Fluorescence micrographs of the breast cancer cells labelled
by F‐Ta‐dD‐dNH2
We investigated the staining behaviour of the fluorescence
2
be seen that F‐Ta‐dD‐dNH may label MCF‐7 cells directly and
probe F‐Ta‐dD‐dNH
2
on the living breast cancer cell line MCF‐7
has some inhibitory effect on this cell line.
and the normal cell line HEK 293. The measurements of
fluorescence scanning and image collection were carried out
after washing with PBS three times to get rid of excess
compound. We did not find an obvious phenomenon of
fluorescence quenching prior to clearing the excess probe from
the cell. Fluorescence signal was detected using an inverted
phase‐contrast microscope. The images were taken using a
fluorescence imaging system and analysed by NIS BR image
processing software. All operations were carried out at room
temperature. Figure 4 shows the staining capability of F‐Ta‐dD‐
Conclusions
In this study, two novel compounds, F‐Ta‐dD‐dOH and F‐Ta‐dD‐
2
dNH , both able to fluoresce and inhibit the growth of breast
tumour cells, have been synthesized. The fluorescence spectra of
the target compounds and the change of relative fluorescence
intensity were studied. The effects of F‐Ta‐dD‐dOH and F‐Ta‐dD‐
dNH
examined by an MTT assay. The staining capability of F‐Ta‐dD‐
dNH for MCF‐7 cells was also analysed, which would offer a new
2
on the growth of different cell lines of breast cancer were
dNH
2
for MCF‐7 cells, where selective stainability was not
2
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New fluorescence probes and labelled breast cancer cells
2
Figure 4. Fluorescence microscopy images of the breast cancer cell line MCF‐7 and normal cell line HEK 293, each labelled with F‐Ta‐dD‐dNH in different concentrations:
–4
–5
–5
–4
–5
(
A) MCF‐7, 1 × 10 mol/L; (B) MCF‐7, 5 × 10 mol/L; (C) MCF‐7, 1 × 10 mol/L; (D) HEK 293, 1 × 10 mol/L; (E) HEK 293, 5 × 10 mol/L; (F) control (normal cells without
probes). Magnification, ×200.
method in the study of mechanism of drug action. Although the
new probes are not exceptionally bright, the introduction of these
fluorescence chromophores does not affect the skeleton structure
or the recognition of tumour cells.
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This work was financially supported by the National Natural
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