Conjugated 5-fluorouracil with mitochondria-targeting lipophilic cation: Design, synthesis and biological evaluation

Article abstract of DOI:10.1039/c6md00268d

5-Fluorouracil (5-FU) was linked with F16 by vulnerable bonds to selectively target cancer mitochondria which resulted in conjugated compounds, including F16-5-FU, F16-OOC-FU, F16-NHOC-FU and F16-SS-FU. F16-OOC-FU decreased the antiproliferative activity of 5-FU on the nontumor cell line, and the cytotoxicity of F16-SS-FU significantly increased when administered with dithiothreitol (DTT).

Full text of DOI:10.1039/c6md00268d

MedChemComm
View Article Online
View Journal
RESEARCH ARTICLE
Conjugated 5-fluorouracil with mitochondria-
targeting lipophilic cation: design, synthesis and
biological evaluation†‡
Cite this: DOI: 10.1039/
c6md00268d
Jia Wang, Xiao-Yang Fan, Li-Yun Yang, Huan He, Rong Huang,
Feng-Lei Jiang and Yi Liu*
Received 17th May 2016,
Accepted 21st July 2016
5-Fluorouracil (5-FU) was linked with F16 by vulnerable bonds to selectively target cancer mitochondria
which resulted in conjugated compounds, including F16–5-FU, F16–OOC-FU, F16–NHOC-FU and F16–SS-
FU. F16–OOC-FU decreased the antiproliferative activity of 5-FU on the nontumor cell line, and the cyto-
toxicity of F16–SS-FU significantly increased when administered with dithiothreitol (DTT).
DOI: 10.1039/c6md00268d
www.rsc.org/medchemcomm
7
As pluripotent organelles, mitochondria constitute the most
prominent source of ATP and are implicated in multiple
anabolic and catabolic circuitries, controlling cell death as
necrosis. Moreover, F16 shows excellent optical properties
7
with fluorescence emission in the visible region. Consider-
ing the specific mitochondria-targeting ability of F16, cou-
pling 5-FU with F16 may improve the selectivity of 5-FU be-
tween tumor and nontumor cells and provide the novel
compounds with optical imaging ability.
The first conjugated compound designed and synthesized
by our team was F16–5-FU, and the structure is shown in
1
well as several aspects of cell survival. The pioneering work,
which started in the early 1990s, demonstrated that
mitochondria-localized antiapoptotic proteins could be suc-
2
cessful targets for cancer therapy. Since cancer cell mito-
chondria are structurally and functionally different from their
3
8
normal counterparts, mitochondria-targeting agents emerge
Scheme 1. However, the toxicity of F16–5-FU on the human
as a means to selectively target cancer for therapeutic
purpose.
gastric carcinoma SGC-7901 cell line was reduced and did
not show selectivity between tumor and nontumor cell lines
(Table 1), which was beyond our initial expectation. The
mechanism of cytotoxicity of 5-FU has been ascribed to the
misincorporation of fluoronucleotides into RNA and DNA
and to the inhibition of the nucleotide synthetic enzyme
4
5
-Fluorouracil (5-FU) was introduced in 1957 as a ratio-
5
nally designed anti-cancer agent. It has been widely used in
the treatment of a range of cancers, including colorectal,
breast and head and neck cancers, and cancers of the
aerodigestive tract. However, 5-FU displays nonspecific cyto-
toxicity for tumor cells and poor distribution to tumor sites.
The clinical use of 5-FU would result in inhibition of rapidly
dividing tissues such as the bone marrow hematopoietic cells
and the gastrointestinal mucosal cells. Well described side ef-
fects of 5-FU include myelosuppression, stomatitis, nausea,
6
emesis, and diarrhea.
In order to reduce the side effects of 5-FU and enhance its
selectivity towards cancer cells, we conjugated 5-FU with F16
((E)-4-(1H-indol-3-ylvinyl)-N-methylpyridinium
iodide),
a
delocalized lipophilic cationic (DLC) compound exhibiting
mitochondria-specific accumulation in a variety of cancer
cells and resulting in cytotoxicity by triggering apoptosis or
State Key Laboratory of Virology & Key Laboratory of Analytical Chemistry for
Biology and Medicine (MOE), College of Chemistry and Molecular Sciences,
Wuhan University, Wuhan 430072, PR China. E-mail: yiliuchem@whu.edu.cn;
Fax: +86 27 6854067; Tel: +86 27 68756667
†
‡
The authors declare no competing interests.
Scheme 1 Conjugating 5-fluorouracil (5-FU) with F16 by three kinds
of vulnerable bonds to facilitate the release of 5-FU after using F16 as
a carrier for selectively targeting mitochondria in cancer cells.
Electronic supplementary information (ESI) available. See DOI: 10.1039/
c6md00268d
This journal is © The Royal Society of Chemistry 2016
Med. Chem. Commun.

View Article Online
Research Article
MedChemComm
Table 1 Cytotoxicity of F16–OOC-FU, F16–NHOC-FU, F16–SS-FU, F16–5-
FU and 5-FU on the tumor cell line SGC-7901 and the nontumor cell line
GES-1. The ratio represents the cell viability of GES-1 compared to that of
SGC-7901
SGC-7901
GES-1
Drug treatment
48 h)
(
Cell viability
Cell viability
Ratio
DMSO
1 ± 0.035
0.707 ± 0.029
0.597 ± 0.042
0.977 ± 0.042
0.888 ± 0.041
1.108 ± 0.043
1.021 ± 0.043
1.019 ± 0.043
0.986 ± 0.055
0.600 ± 0.022
1 ± 0.044
0.822 ± 0.030
0.561 ± 0.051
1.055 ± 0.052
1.069 ± 0.034
0.978 ± 0.049
0.895 ± 0.036
1.072 ± 0.053
0.733 ± 0.031
0.530 ± 0.028
1
1
5
1
5
1
5
5
1
5
0 μM F16–OOC-FU
0 μM F16–OOC-FU
0 μM F16–NHOC-FU
0 μM F16–NHOC-FU
0 μM F16–SS-FU
0 μM F16–SS-FU
0 μM F16–5-FU
0 μM 5-FU
1.16
0.94
1.08
1.20
0.88
0.88
1.05
0.74
0.88
0 μM 5-FU
Fig. 1 The cell viability of SGC-7901 cells treated with various con-
centrations of F16–OOC-FU, F16–NHOC-FU, F16–SS-FU, F16–5-FU or
5-FU for 48 h or 72 h. An asterisk indicates P < 0.05 compared to the
control group (ANOVA). *, P < 0.05; ***, P < 0.001.
9
thymidylate synthase (TS), while F16 concentrates in the mi-
tochondrial matrix and is capable of inhibiting oxidative
phosphorylation, inducing mitochondrial transmembrane de-
7
polarization and permeability transition. Since the action
mechanisms of 5-FU and F16 are different, it may be reason-
able to uncouple 5-FU from F16 after using F16 as a carrier
for the selective delivery of 5-FU to cancer cells. Therefore,
based on optimizing the structure of F16–5-FU, we designed
and successfully synthesized three other conjugated com-
pounds linked by vulnerable bonds, including F16–OOC-FU
linked by an ester bond, F16–NHOC-FU linked by an amido
bond and F16–SS-FU linked by a disulfide bond.
inherited F16's fluorescent characteristic and exhibited close
absorption and emission wavelengths, while they displayed
red shifts of the maximum absorption wavelength and de-
creased absorption compared to F16. We took advantage of
the optical properties to investigate the cellular localization
of these compounds. The results are shown in Fig. 2B and
S2.‡ The green fluorescence signal representing F16–OOC-FU
overlapped well with the red fluorescence signal produced by
The antiproliferative activities of the four conjugated com-
pounds were preliminarily investigated by testing their anti-
proliferative effects on human gastric carcinoma (SGC-7901)
and human fetal gastric epithelial (GES-1) cell lines. The fig-
ures in Table 1 show the viability of SGC-7901 and GES-1 cells
with the treatment of these compounds. It was indicated that
F16–OOC-FU and F16–NHOC-FU exhibited stronger anti-
proliferation ratios in SGC-7901 than in GES-1 cells, com-
pared to 5-FU. Moreover, F16–OOC-FU showed the strongest
cytotoxicity among these compounds (Fig. 1).
2
the mitochondrion-selective dye MitoTracker® Red CM-H -
XRos, indicating the mitochondrion-targeting property. F16–
SS-FU also exhibited selective accumulation in the mitochon-
dria. On the other hand, the green fluorescence signals
representing F16–5-FU and F16–NHOC-FU could hardly be
To investigate the stability of the four conjugated com-
pounds, F16–5-FU, F16–OOC-FU, F16–NHOC-FU and F16–SS-
FU were incubated in PBS or DMEM (supplemented with
FBS) for various hours, and then the fluorescence intensity
was tested and compared to that for 0 h (Fig. S1‡). It was ob-
served that the four compounds were stable in PBS for as
long as 72 h. Moreover, F16–5-FU, F16–OOC-FU and F16–
NHOC-FU were relatively stable for 48 h in DMEM, as their
fluorescence intensities didn't change much. Meanwhile, the
stability of F16–SS-FU in DMEM might be influenced by pro-
teins and the fluorescence intensity obviously increased as
the incubation time extended. This indicated that prolonged
treatment time might not be beneficial for conjugates
displaying activities.
Fig. 2 A: UV-vis absorption and fluorescence spectra of F16, F16–5-
FU, F16–OOC-FU, F16–NHOC-FU, and F16–SS-FU (in water). B:
Colocalization assay. MCF-7 cells were pre-incubated with 2 μM F16
The optical properties of these synthetic compounds were
studied at a concentration of 10 μM in water. Based on the
spectra shown in Fig. 2A, the four conjugated compounds all
(green) or 5 μM F16–OOC-FU (green), and then were stained with
MitoTracker® Red CM-H XRos (red) and Hoechst 33342 (blue).
2
Med. Chem. Commun.
This journal is © The Royal Society of Chemistry 2016

View Article Online
MedChemComm
Research Article
observed in the SGC-7901 cells, indicating that the two com-
pounds might not enter into cells.
The tumor cell uptake of these conjugated compounds
was studied by incubating SGC-7901 cells with the com-
pounds for various hours and then analyzing the cellular
fluorescence signal intensity by using a flow cytometer
the fact that F16–5-FU and F16–NHOC-FU were predicted to
have weaker lipophilicities (clog P −1.25 and −1.65, respec-
tively) compared to F16–OOC-FU and F16–SS-FU (clog P −1.04
and 0.01, respectively).
The above experiments revealed that F16–OOC-FU was the
most promising compound among the four conjugates, thus
we focused on F16–OOC-FU and further investigated its cyto-
logical effect. The IC50 values of F16–OOC-FU on two tumor
and two nontumor cell lines are listed in Table S1.‡ The cyto-
toxicity of F16–OOC-FU was not very strong, while it still
showed selectivity for the tumor cell lines. Fig. 4A shows the
cytotoxicity of F16–OOC-FU on GES-1 cells with 5-FU serving
as the control. F16–OOC-FU displayed less antiproliferative
effect on GES-1 cells compared to 5-FU. The distinction was
especially obvious at low concentrations. Annexin V-PE in
conjunction with the viability dye 7-AAD was used to stain
the cells, monitored using a flow cytometer to indicate cell
death under treatment with F16–OOC-FU. Increasing the con-
centration of F16–OOC-FU resulted in an increase in cell
death, indicated by a decrease in viable, nonapoptotic cells
(Fig. 3). Like F16, F16–OOC-FU displayed good uptake after
incubating with SGC-7901 cells for 4 h, and F16–SS-FU
showed a similar performance. F16–5-FU displayed poor up-
take, and F16–NHOC-FU almost could not be found in the
SGC-7901 cells. The result is identical with that of cellular lo-
calization. The difference in uptake might be attributed to
(
PE-/7-AAD-) and an increase in the Annexin V-PE positive cell
population (Fig. 4B).
Our previous study has shown that F16 would induce an
increase in the number of cells in the S phase in SGC-7901
cells, while 5-FU resulted in G1 arrest. The effect of F16–
OOC-FU on the cell cycle in SGC-7901 cells seemed to be a
mixture of those of F16 and 5-FU. When the treatment con-
centration of F16–OOC-FU was lower than 20 μM, the per-
centage of cells in the G1 phase increased. However, as the
treatment concentration of F16–OOC-FU further increased,
the number of cells in the G1 phase decreased. The number
of cells in the S phase was consistently increasing compared
Fig. 4 A: Cytotoxicity of F16–OOC-FU on GES-1 cells in comparison
with that of 5-FU. B: Annexin V-PE and 7-AAD double staining on
SGC-7901 cells untreated or treated with various concentrations of
F16-OOC-FU for 48 h. C: Cell cycle analysis of SGC-7901 cells after
treating with increasing concentrations of F16–OOC-FU for 24 h. D:
The addition of dithiothreitol (DTT) increased the cytotoxicity of F16–
SS-FU on SGC-7901 cells. An asterisk indicates P < 0.05 compared to
the control group (ANOVA). *, P < 0.05; **, P < 0.01; and ***, P <
0.001.
Fig. 3 The uptake of F16 (B), F16–OOC-FU (C), F16–5-FU (D), F16–
NHOC-FU (E) and F16–SS-FU (F) in SGC-7901 cells at various time
points analyzed by using a flow cytometer.
This journal is © The Royal Society of Chemistry 2016
Med. Chem. Commun.

View Article Online
Research Article
MedChemComm
to the control without drug treatment. Despite inducing apo-
ptosis and cell cycle arrest, F16–OOC-FU could also increase
the intracellular reactive oxygen species (ROS) level, as indi-
cated by right shifts of the fluorescent signal peaks of di-
not show obvious synergistic effects, which may be ascribed
to the fact that conjugation causes an effect on the structure
of F16 as well as 5-FU, suggesting the importance of design-
ing the structure of the conjugate so as to achieve high cyto-
toxicity on cancer cells. This study will not only develop a
novel way of enhancing the selectivity of 5-FU towards cancer
cells, but also expand the usage of F16 as a probe for mito-
chondrion-targeting.
We gratefully acknowledge financial support from the Na-
tional Natural Science Foundation of China (21225313 and
21473125), Hubei Natural Science Foundation of China
(2014CFA003), and Fundamental Research Funds for the Cen-
tral Universities (2042014kf0287).
hydroethidium (DHE) and MitoTracker® Red CM-H XRos
2
(Fig. S3‡).
In the cellular uptake and localization experiments, the
performance of F16–SS-FU was very similar to that of F16–
OOC-FU, as they both showed good uptake in SGC-7901 cells
and selectively accumulated in the mitochondria, while the
antiproliferative effect of F16–SS-FU on the SGC-7901 cell line
was not as obvious as that of F16–OOC-FU. Since the cleavage
of a disulfide bond needs reduced sulfhydryl, we suspected
that enhancing the level of reduced sulfhydryl in the mito-
chondria might stimulate the breakage of the disulfide bond
in F16–SS-FU and thus increase its toxicity. Therefore, we
chose to administer dithiothreitol (DTT) with F16–SS-FU
Notes and references
1
L. Galluzzi, O. Kepp, C. Trojel-Hansen and G. Kroemer, Circ.
Res., 2012, 111, 1198–1207.
(Fig. 4D). The SGC-7901 cells were incubated with F16–SS-FU
first, and then DTT was added 4 h later to avoid reacting with
F16–SS-FU in the medium. The results showed that DTT
could strengthen the toxicity of F16–SS-FU and led to a strik-
ing decrease in cell viability. Compared to adding F16–SS-FU
and DTT separately, it was found that the increased cytotoxic-
ity was not the sum of the effects of the two compounds, but
might result from DTT cleaving the S–S bond in compound
F16–SS-FU.
2
3
S. W. Fesik, Nat. Rev. Cancer, 2005, 5, 876–885.
V. Gogvadze, S. Orrenius and B. Zhivotovsky, Trends Cell
Biol., 2008, 18, 165–173; J. S. Modica-Napolitano and K. K.
Singh, Mitochondrion, 2004, 4, 755–762.
S. Fulda, L. Galluzzi and G. Kroemer, Nat. Rev. Drug
Discovery, 2010, 9, 447–464; S. E. Weinberg and N. S.
Chandel, Nat. Chem. Biol., 2015, 11, 9–15.
C. Heidelberger, N. K. Chaudhuri, P. Danneberg, D. Mooren,
L. Griesbach, R. Duschinsky, R. J. Schnitzer, E. Pleven and J.
Scheiner, Nature, 1957, 179, 663–666.
F. Fata, I. G. Ron, N. Kemeny, E. O'Reilly, D. Klimstra and
D. P. Kelsen, Cancer, 1999, 86, 1129–1134; F. J. Ansfield,
J. M. Shroeder and A. R. Cucreri, JAMA, 1962, 181, 295–299.
V. R. Fantin, M. J. Berardi, L. Scorrano, S. J. Korsmeyer and
P. Leder, Cancer Cell, 2002, 2, 29–42; V. R. Fantin and P.
Leder, Cancer Res., 2004, 6, 329–336.
4
5
6
7
We used F16 as a mitochondria-targeting group and
linked it with 5-FU to enhance the cancer cell specificity of
5
-FU, and thus successfully synthesized four conjugated com-
pounds. These conjugates all inherited the fluorescent prop-
erties of F16, which were used to study the cellular uptake
and localization status. In particular, F16–OOC-FU acquired
the mitochondria-targeting ability of F16, which resulted in
cell death, cell cycle arrest and an increased cellular ROS
level in tumor cells and decreased the antiproliferative activ-
ity of 5-FU on the nontumor cell line GES-1. Also, the cytotox-
icity of F16–SS-FU would significantly increase when adminis-
tered with DTT. One thing to note is that these conjugates do
8
9
C. Xiang, D. W. Li, Z. D. Qi, F. L. Jiang, Y. S. Ge and Y. Liu,
Luminescence, 2013, 28, 865–872.
D. B. Longley, D. P. Harkin and P. G. Johnston, Nat. Rev.
Cancer, 2003, 3, 330–338.
Med. Chem. Commun.
This journal is © The Royal Society of Chemistry 2016