Hypoxia-Activated Anticancer Prodrug for Bioimaging, Tracking Drug Release, and Anticancer Application

Article abstract of DOI:10.1021/acs.bioconjchem.8b00511

A novel anticancer theranostic prodrug, FDU-DB-NO₂, specifically activated by hypoxia for selective two-photon imaging hypoxia status, real-time tracking drug release, and solid tumor therapy was designed. The devised prodrug consists of an anticancer drug floxuridine (FDU), a fluorescence dye precursor 4′-(diethylamino)-1,1′-biphenyl-2-carboxylate (DB), and a hypoxic trigger 4-nitrobenzyl group. In normal cells, FDU-DB-NO₂ is "locked". Whereas in tumor cells, the prodrug is "unlocked" by hypoxia and results in fluorescent dye 7-(diethylamino)coumarin (CM) generation along with FDU release. The amounts and rates of CM formation and FDU release were controlled by hypoxic status and increased with the decreasing of the O₂ concentration. The hypoxic status, distribution of oxygen, and amount of FDU release in tumor cells, spheroids, and tumor tissue could be visualized by fluorescence. FDU-DB-NO₂ showed high cytotoxicity against hypoxic MCF-7 and MCG-803 cell lines and no cytotoxicity against normoxic BRL-3A cells and exhibited effective inhibition on tumor growth of MCF-7-cell-inoculated xenograft nude mice. This strategy may provide a promising platform for selective two-photon imaging hypoxia status, real-time tracking drug release, and personalized solid tumor treatment.

Full text of DOI:10.1021/acs.bioconjchem.8b00511

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Article
A novel hypoxia-activated anticancer prodrug for
bioimaging, tracking drug release, and anticancer application
Wei Liu, Haitong Liu, Xiaoran Peng, Guoqiang Zhou, Dandan
Liu, Shenghui Li, Jinchao Zhang, and Shuxiang Wang
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00511 • Publication Date (Web): 07 Sep 2018
Downloaded from http://pubs.acs.org on September 8, 2018
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A novel hypoxia-activated anticancer prodrug for bioimaging,
tracking drug release, and anticancer application
#a
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Wei Liu, Haitong Liu, Xiaoran Peng, Guoqiang Zhou,* Dandan Liu,
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Shenghui Li, Jinchao Zhang,* and Shuxiang Wang*
Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of
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Education, Key Laboratory of Chemical Biology of Hebei Province, College of
Chemistry and Environmental Science, Hebei University, Baoding 071002, China
KEYWORDS: hypoxiaꢀactivated theranostic prodrug, hypoxic status, oxygen
distribution, floxuridine release, twoꢀproton imaging, inhibition tumor growth
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ABSTRACT
A novel anticancer theranostic prodrug, FDUꢀDBꢀNO , specifically activated by
2
hypoxia for selective twoꢀphoton imaging hypoxia status, realꢀtime tracking drug
release, and solid tumor therapy was designed. The devised prodrug consists of an
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anticancer
drug
'ꢀ(diethylamino)ꢀ1,1'ꢀbiphenylꢀ2ꢀcarboxylate (DB), and
ꢀnitrobenzyl group. In normal cells, FDUꢀDBꢀNO is "locked". Whereas in tumor
floxuridine
(FDU),
a
fluorescence
dye
precursor
4
4
a
hypoxic trigger
2
cells, the prodrug is "unlocked" by hypoxia and result in fluorescent dye
7ꢀ(diethylamino)coumarin (CM) generation along with FDU release. The amounts
and rates of CM formation and FDU release were controlled by hypoxic status, and
increased with the decreasing of the O
of oxygen, and amount of FDU release in tumor cells, spheroids, and tumor tissue
could be visualized by fluorescence. FDUꢀDBꢀNO showed high cytotoxicity against
2
concentration. The hypoxic status, distribution
2
hypoxic MCFꢀ7 and MCGꢀ803 cell lines, no cytotoxicity against normoxic BRLꢀ3A
cells, and exhibited effective inhibition on tumor growth of MCFꢀ7ꢀcellꢀinoculated
xenograft nude mice. This strategy may provide a promising platform for selective
twoꢀphoton imaging hypoxia status, realꢀtime tracking drug release, and personalized
solid tumour treatment.
INTRODUCTION
1
ꢀ3
Chemotherapy remains the main tool for tumor treatment. However, traditional
chemotherapy always suffers from undesired purgatorial side effects due to their poor
4
,5
selectivity.
For example, 5ꢀfluorodeoxyuridine (floxuridine, FDU), an analogue of
5ꢀfluorouracil (5ꢀFUra), can be converted to 5ꢀFUra by thymidine phosphorylase and
has been widely applied in the therapy in treatment breast, colorectal, and liver
6
cancer. However, its clinical use is limited by severe side effects due to its
7
,8
nonꢀselectivity. Therefore, the design chemoꢀtherapeutic agents to selectively kill
9
,10
tumor cells represents a significant challenge for cancer therapy.
Thus, various
efforts have been made to toward the development of prodrugs for controllable active
drugs release in tumor tissues that achieve reduced side effects and enhanced
therapeutic efficacy. Hypoxia caused by tumor tissues away from the blood vessels is
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one important feature of all solid tumors, which leads to a situation that the tumors are
11ꢀ14
not sensitive to chemotherapeutic drugs, and produce drug resistance.
Hypoxia
can also cause an increase reductive stress and lead excess expression of reductases
15ꢀ18
such as nitroreductase (NTR).
As a result, hypoxia provides a new approach for
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the diagnostic or treatment of solid tumors. A lot of hypoxiaꢀresponsive probes or
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prodrugs were developed.
The strategy is an effective tool to design a theranostic
agent activated by hypoxia or NTR in hypoxic regions of the individual tumor patient,
for timely diagnosis of tumors, and decrease side effects and chemoꢀresistance.
Theranostic agents possessing both diagnostic and therapeutic capabilities in an
allꢀinꢀone platform have gained increasing attention due to their low toxicity,
decreased sideꢀeffects on patients, minimal drug resistance, and improved
23ꢀ26
biodistribution.
Therefore, theranostic agents research is an emerging and
27ꢀ29
fastꢀgrowing field and plays an important role in individual medical.
Numerous
kinds of theranostic agents response to stimuli signals were developed. The stimuli
3
0ꢀ33
34
signals may be internal stimuli (such as high acidity,
high levels of thiols, high
3
5,36
37,38
levels of reactive oxygen species,
and specific enzymes overꢀexpression
) or
39
external stimuli (such as photo irradiation ). How to improve the specificity and
sensitivity for the targeted cleavage and enhancing therapeutic effect on tumor
treatment is becoming the hotspot in cancer treatment.
On the other hand, twoꢀphoton fluorescence probes have been extensively applied
to investigate a number of biomolecular processes. Comparing to oneꢀphoton
fluorescence probes, twoꢀphoton fluorescence probes possessed some impressive
properties (such as deeper penetration depth, less photoꢀdamage to biological
40ꢀ42
specimens, and less photoꢀbleaching),
and became a very useful tool for
visualization and monitoring biological samples. Coumarin is a twoꢀphoton
fluorescent dye and has been widely applied to diagnose tumor due to its excellent
4
3ꢀ45
optical properties related to high molar absorption coefficient and quantum yield.
Herein, we designed a promising theranostic prodrug, FDUꢀDBꢀNO , specifically
2
activated by hypoxia (Scheme 1), using FDU as an active drug, 4ꢀnitrobenzyl group
as a hypoxia trigger, and 4'ꢀ(diethylamino)ꢀ1,1'ꢀbiphenylꢀ2ꢀcarboxylate (DB) as a
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fluorescence dye precursor. As supposed, FDUꢀDBꢀNO is "locked" in normal tissues.
Whereas in tumor tissues, bioreductive microenvironment of hypoxia "unlocks"
FDUꢀDBꢀNO due to the excess expression NTR. Thus, FDUꢀDBꢀNO is specifically
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2
reduced by NTR with nicotinamide adenine dinucleotide (NADH) under hypoxic
conditions and initiates fluorescent dye 7ꢀ(diethylamino)coumarin (CM, a coumarin
analogue) formation as well as FDU release. In other words, the drug released can be
tracked by the signal of fluorescence of CM, which makes it possible to detect the
distribution and accumulation of drug in situ and realꢀtime. This strategy provides a
promising way to selectively inhibit solid tumor growth, offers a noninvasively way
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to determine hypoxic status, affords a precise method to analysis of O distribution,
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and offers an approach to track drug release based on hypoxia status of individual
patient.
Scheme 1. The structure of FDUꢀDBꢀNO
RESULTS AND DISCUSSION
Synthesis and characterization. In order to prove the strategy, FDUꢀDBꢀNO
2
2
and
fluorescent dye CM were synthesized as shown in Scheme 2. First, 3ꢀmethoxyaniline
was protected by (Boc) O at r.t. to afford NꢀBocꢀ3ꢀmethoxyaniline (1). Selective
2
monoꢀbromination of compound 1 by Nꢀbromosuccinimide (NBS) in ethylene glycol
dimethyl ether at r.t. afforded NꢀBocꢀ4ꢀbromoꢀ3ꢀmethoxyaniline (2). Followed by the
deprotection of the NꢀBoc group from compound 2 using HCl (4 mol/L) in anhydrous
o
dioxane at 25 C, 4ꢀbromoꢀ3ꢀmethoxyaniline (3) was generated. Then,
4ꢀbromoꢀN,Nꢀdiethylꢀ3ꢀmethoxyaniline (4) was prepared via ethylation reaction of 3
with bromoethane. Subsequently, the demethylation and nucleophilic substitution
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reactions
of
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resulted
in
the
(5).
formation
Afterwards,
of
4ꢀbromoꢀN,Nꢀdiethylꢀ3ꢀ(4ꢀnitrobenzyloxy)aniline
methyl
1,1'ꢀbiphenylꢀ4'ꢀ(diethylamino)ꢀ2'ꢀ(4ꢀnitrobenzyloxy)ꢀ2ꢀyl acetate (6) was prepared
via Suzuki crossꢀcouping reaction between with methyl
ꢀ(dihydroxymethyl)benzoate under N atmosphere. Followed by hydrolysis of 6 in
sulfuric acid aqueous solution,
,1'ꢀbiphenylꢀ4'ꢀ(diethylamino)ꢀ2'ꢀ(4ꢀnitrobenzyloxy)ꢀ2ꢀcarboxylic acid (7) was
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afforded. Finally, in the presence of Nꢀdiisopropyl carbodiimide (DIC) and dimethyl
aminopyridine (DMAP), FDU was conjugated with 7 to generate theranostic prodrug
FDUꢀDBꢀNO . Using similar synthetic method, a control compound (FDUꢀDB)
2
containing the DB and FDU units without 4ꢀnitrobenzyl group was also synthesized,
which was constructed by conjugating of FDU and fluorescent dye precursor. Both of
the new compounds and CM were characterized by NMR, IR, and MS (Figure
S10ꢀS45, ESI†).
Scheme 2. The synthetic route for FDUꢀDBꢀNO
2
, FDUꢀDB, and CM
Mechanism of FDU-DB-NO activated by hypoxia. To demonstrate the mechanism
2
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of FDUꢀDBꢀNO
2
activated by hypoxia for generation CM and release FDU, the
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composition of a reaction mixture of FDUꢀDBꢀNO
HPLC. As shown in Figure S4a~S4c (ESI†), the retention times of FDU, CM, and
FDUꢀDBꢀNO were 3.081, 21.582, and 26.582 min, respectively. Peaks at 3.091,
1.501 and 26.408 min were observed when the mixture of FDUꢀDBꢀNO with
Na S O was stirred at 25 C for 40 mins (Figure S4d, ESI†), they were attributed to
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with Na
2
S
2
O
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was analyzed by
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anticipated products of FDU, CM, and unreacted FDUꢀDBꢀNO
that FDUꢀDBꢀNO is reduced by Na and generated CM and FDU
simultaneously. Then, the amounts of CM and FDU generated from FDUꢀDBꢀNO
2
. This result indicated
2
2 2 4
S O
2
were also analyzed by HPLC (Figure S5, ESI†). The result showed that the amount of
FDU information was nearly equal to that of CM release. Thus, the mechanism of
generation CM and release FDU is described as follows (Scheme 3): First, –NO
2
group of FDUꢀDBꢀ NO is reduced to –NH group by Na S O , and FDUꢀDBꢀNO is
2
2
2
2
4
2
converted to FDUꢀDBꢀNH
2
. Followed by 1,6ꢀrearrangementꢀelimination reaction,
, and
4ꢀmethylenecyclohexaꢀ2,5ꢀdienꢀ1ꢀimine is removed from FDUꢀDBꢀNH
2
FDUꢀDB is generated. Finally, through the rotation of the σ bond and a spontaneous
intramolecular transesterification reaction, CM was formed as well as FDU was
released.
2
Scheme 3. The mechanism of FDUꢀDBꢀNO release CM and FDU under hypoxic
conditions
Response of FDU-DB-NO
2
to hypoxia. Next, the amounts and speeds of FDU and
were investigated. Firstly, the optical properties of
CM formation from FDUꢀDBꢀNO
2
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CM and FDUꢀDBꢀNO
2
were determined in PBS buffer (0.2ꢁM, contained 0.1%
DMSO, pHꢁ=ꢁ7.4). The absorption spectra of CM and FDUꢀDBꢀNO were determined
2
(Figure 1a). As shown in Figure 1b, CM exhibited a strong green fluorescence at 530
2
nm (λex=380 nm), whereas FDUꢀDBꢀNO displayed almost no fluorescence due to
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the rotation of the σcꢀc bond and the quenching effect of 4ꢀnitrobenzyl group. As
o
expected, under N
2
atmosphere at 37 C, the mixture of FDUꢀDBꢀNO
2
incubation
with NTR (in the presence of NADH) displayed a strong green fluorescence at
ex/em=380/530 nm (Figure 1c). In addition, the fluorescence intensity increased
λ
gradually with the prolongation of reduction time, and the fluorescence intensity
reached the highest value when the mixture was incubated about 10ꢁmin (Figure 1c
and 1d), and it was about 17ꢀfold enhanced compared with that of the mixture was
incubated at 0ꢁmin. By comparison, the same mixture of NTR, NADH, and
FDUꢀDBꢀNO
2
in PBS buffer under normoxic conditions (20% O
2
) showed very weak
fluorescence. The obvious fluorescence enhancement at
λ
ex/em=380/530 nm indicated
that the 4ꢀnitrobenzyl group of FDUꢀDBꢀNO was reduced by NTR/NADH under
2
hypoxic conditions, and a fluorescence dye CM was generated. This, combined with
the experimental results of the composition of FDUꢀDBꢀNO
2
reduced by Na
Figure S4d, ESI†) and the amounts of CM and FDU generated from FDUꢀDBꢀNO
Figure S5, ESI†), we were confident that CM and FDU were generated
2 2 4
S O
(
(
2
simultaneously, the amount of FDU information was nearly equal to that of CM
release, and the fluorescence dye CM could be used as a reporter for in situ and
realꢀtime reporting the amount and the distribution of FDU in cancer cells. These
2
results collectively proved that FDUꢀDBꢀNO responded to NTR under hypoxic
conditions with high sensitivity and quickly speed (about 10 min), CM could be used
as a reporter for in situ and realꢀtime reporting the amount and the distribution of
FDU release.
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Figure 1. Absorbance (a) and fluorescence (b) of FDUꢀDBꢀNO and CM (λex=380
nm); Fluorescence intensity of FDUꢀDBꢀNO (λex/em=380/530 nm) incubated with
2
NTR (c and d) under hypoxic conditions (1% O
The selectivity and stability of FDU-DB-NO
investigated the selectivity of FDUꢀDBꢀNO
2
)
2
. According to above mechanism, we
2
to NTR by determination the changes of
fluorescence intensity. As shown in Figure S1 (ESI†), FDUꢀDBꢀNO uniquely
2
o
responded to NTR (in the presence of NADH) under N
2
atmosphere at 37 C and
produced a strong enhancement of fluorescence at 530 nm, whereas FDUꢀDBꢀNO
2
exhibited nearly no remarkable fluorescence enhancement responses to other
reductant (vitamin C), biothreitol (cysteine and glutathione), saccharides (glucose and
fructose), and amino acids (glutamic acid, aspartic acid, and histidine) under the same
condition. These results showed that FDUꢀDBꢀNO
to NTR under hypoxia conditions.
2
possessed remarkable selectivity
The stability in biological media is a key parameter of theranostic agents. To
investigate the stability of FDUꢀDBꢀNO , the changes of fluorescence intensity and
2
absorbance of a mixture solution of FDUꢀDBꢀNO (100 ꢂM) in blood serum and PBS
2
o
buffer (
V
blood serum
/
V
PBS =5/1, 37 C) was determined at different time. As shown in
Figure S2a and S2b (ESI†), negligible fluorescence intensity and absorbance change
of this mixture solution was found. These results demonstrated that theranostic
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prodrug FDUꢀDBꢀNO
2
exhibited significant stability in blood serum, and the
excellent property endowed it with a potential application in biological media.
Furthermore, the effect of pH values on the stability of FDUꢀDBꢀNO
2
was
in PBS buffer
at different pH values were tested. As shown in Figure S3a (ESI†), no obvious
fluorescence change of FDUꢀDBꢀNO in PBS buffer was found in a wide pH range 2
to 10, and no obvious fluorescence change of CM in PBS buffer in the range 4 to 10.
In addition, no obvious absorbance change of FDUꢀDBꢀNO in PBS buffer was found
evaluated. The fluorescence intensity and absorbance of FDUꢀDBꢀNO
2
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in the pH range 5ꢀ12 (Figure S3b, ESI†). These results showed that FDUꢀDBꢀNO
possessed excellent stability in PBS buffer at pH 5ꢀ10.
2
All above results suggested that FDUꢀDBꢀNO
physiological conditions, and showed remarkable selectivity to NTR.
Cellular fluorescence imaging. Subsequently, the effect of the hypoxic status on the
amount of CM released from FDUꢀDBꢀNO in tumor cells was investigated by laser
confocal microscopy. Due to the characteristics of twoꢀphoton excitation, CM could
be excited by NIR light ( ex=760 nm) with higher resolution. As shown in Figure 2,
MGCꢀ803 cells incubated with FDUꢀDBꢀNO
showed no fluorescence. However, MGCꢀ803 cells incubated with FDUꢀDBꢀNO
2
possessed excellent stability under
2
λ
2
under normoxic conditions (20% O
2
)
2
2 2 2 2
under hypoxic conditions (1% O , 3% O , 5% O , or 10% O ) showed noticeable
fluorescence, and the fluorescence intensity was increased with the increase of the
degree of hypoxia. When the content of oxygen is 1%, a strong green fluorescence
was observed, while the oxygen content is 10%, only a weak fluorescence was
observed. It indicated that the amount of CM formation was depended on the hypoxic
status. A similar result was obtained by one proton emission (
ESI†). And similar results were observed in MCFꢀ7 cells incubated with
FDUꢀDBꢀNO normoxic and hypoxic conditions (Figure S7 and S8, ESI†). These
results illustrated that FDUꢀDBꢀNO possessed reliable diagnostic capability for
detection of hypoxic status in tumor cells.
λex=380 nm, Figure S6,
2
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Figure 2. Twoꢀphoton confocal fluorescence images of MGCꢀ803 cells incubated
with FDUꢀDBꢀNO
c: 5% O , and d: 10% O
ex=577 nm, em=590 nm
Due to tumor spheres are more closely related to the heterogeneity of the tumor
microenvironment with a normoxic surface and a hypoxic core compared with
2 2 2
for 12 h under different hypoxic conditions (a: 1% O , b: 3% O ,
2
2 2
) and normoxic conditions (e: 20% O ), lysoꢀtracker red:
λ
λ
47
traditional adhere cell cultures. As shown in Figure 3a, MGCꢀ803 and MCFꢀ7
spheres cells incubated with FDUꢀDBꢀNO showed strong fluorescence in the core of
2
spheroids and weak fluorescence on the surface of spheroids. Even though the core
areas was more acidic than the exterior of spheroids, the strong fluorescence of
lysoꢀtracker red on the surface and very weak fluorescence in the core of spheroids
was found due to diffusion of dye molecules. The relative fluorescent intensity
distribution from a cross section of tumor spheres (Figure 3b) was consistant with the
content of oxygen decreased from surface to core in these tumor spheres. It illustrated
that FDUꢀDBꢀNO possessed diagnostic capability and sensitivity to monitor the
2
distribution of oxygen in the tumor microꢀenvironment.
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Figure 3. Confocal fluorescence images of MGCꢀ803 and MCFꢀ7 tumor spheres
2
incubated with FDUꢀDBꢀNO and lysoꢀtracker red for 12 h (a); the relative intensity
distribution of fluorescence in MGCꢀ803 and MCFꢀ7 tumor spheres (b)
Finally, in vivo experiments for examining the selective imaging of tumor tissue of
MCFꢀ7ꢀcellꢀinoculated xenograft nude mice injected with FDUꢀDBꢀNO
injection were performed. As shown in Figure 4, the beneficial results of the living
cell researches led us to further apply FDUꢀDBꢀNO to trace hypoxia in the living
2
via in situ
2
tumor tissue. For a control experiment (injected with saline), the tumor tissue slides
displayed almost no fluorescence (Figure S9, ESI†). In sharp contrast, tumor tissue
slides treated with FDUꢀDBꢀNO displayed green emission up to a depth of 60 ꢂm.
2
The result demonstrated that FDUꢀDBꢀNO
living tissues.
2
can detect basal levels of hypoxia in
Figure 4. Confocal fluorescence images of tumor tissue treated with FDUꢀDBꢀNO
2
(
30 ꢂM) for 1 h in different depth of 10 (a), 20 (b), 30 (c), 40 (d), 50 (e), and 60 ꢂm
f)
(
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In vitro cytotoxicity of FDU-DB-NO
2
. To investigate the inhibitory effect of
FDUꢀDBꢀNO on cancer and normal cells, the cell viability of MGCꢀ803, MCFꢀ7,
2
2
and BRLꢀ3A cells incubated with FDU, FDUꢀDBꢀNO , or FDUꢀDB alone was
evaluated via 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide (MTT)
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assay. FDU showed high cytotoxicity against MGCꢀ803 cells line under normoxic (20%
O
2
) or hypoxic (3% O
decreased along with the increasing concentration of FDU (Figure 5a and 5b). In
addition, FDUꢀDBꢀNO exhibited no cytotoxicity against MGCꢀ803 cells under
normoxic conditions (20% O ). However, FDUꢀDBꢀNO exhibited high cytotoxicity
against MGCꢀ803 cells under hypoxic conditions (3% O ), and the cell viability of
MGCꢀ803 cells decreased with the increase of FDUꢀDBꢀNO concentration. Similar
results were obtained when MCFꢀ7 cells were incubated with FDUꢀDBꢀNO under
2
) conditions, and the cell viability of MGCꢀ803 cells was
2
2
2
2
2
2
2 2
normoxic (20% O ) and hypoxic conditions (3% O ) (Figure 5c and 5d). In view of
the complexity of intracellular enzymes (such as hydrolases) in hypoxic tumors, the
cell viability of MCFꢀ7 incubated with FDUꢀDB containing the DB and FDU units
without 4ꢀnitrobenzyl group was evaluated. FDUꢀDB exhibited no cytotoxicity to
cells under normoxic (20% O
indicated that FDUꢀDB could not be significantly hydrolyzed by hydrolases, and then
nitroreductase played a major contribution to cytotoxicity of FDUꢀDBꢀNO against
hypoxic tumors. As shown in Figure 5e and 5f, incubated with FDU under normoxic
conditions (20% O ), the cell viability of BRLꢀ3A cells decreased along with the
increasing concentration of FDU. Meanwhile, the cell viability of BRLꢀ3A cells was
almost no obvious change when they were incubated with FDUꢀDBꢀNO under
normoxic conditions (20% O ). These results suggested that FDUꢀDBꢀNO possessed
2 2
) or hypoxic (3% O ) conditions (Figure 5d). The result
2
2
2
2
2
hypoxia selectivity, exhibited high cytotoxicity against hypoxic cancer cells, and
showed nonꢀcytotoxicity against normal cells. Furthermore, flow cytometry studies
were used to further verify the cell apoptosis of MGCꢀ803 cells incubated with
2 2
FDUꢀDBꢀNO under hypoxic conditions (3% O ). As shown in Figure 5, incubation
for 12 h, the late apoptotic percentage of MGCꢀ803 cells was only 10.2%, prolonging
of incubation time to 24 h, the late apoptotic percentage of MGCꢀ803 cells increases
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to 47.3% induced by FDUꢀDBꢀNO
2
under hypoxic conditions (3% O
2
). All these
results demonstrated that FDUꢀDBꢀNO
2
showed quite good therapeutic effects on
hypoxic cancer cells with nonꢀcytotoxicity to normal cells.
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Figure 5. Cell viability of MGCꢀ803 cells (a: 24 h, b: 48 h) incubated with FDU and
FDUꢀDBꢀNO under normoxic (20% O ) and hypoxic (3% O ) conditions; Cell
2
2
2
viability of MCFꢀ7 cells (c: 24 h) incubated with FDU and FDUꢀDBꢀNO
normoxic (20% O ) and hypoxic (3% O ) conditions; Cell viability of MCFꢀ7 cells (d:
8 h) incubated with FDU, FDUꢀDBꢀNO , and FDUꢀDB under normoxic (20% O )
2
under
2
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and hypoxic (3% O
2
) conditions; cell viability of BRLꢀ3A cells incubated with FDU
under normoxic (20% O ) conditions (e: 24 h, f: 48 h); apoptosis
and necrosis analysis of MGCꢀ803 cells by flow cytometry (g: control, h: incubated
with FDUꢀDBꢀNO under hypoxic conditions (3% O ) for 12 h, i: incubated with
FDUꢀDBꢀNO under hypoxic (3% O ) conditions for 24 h
In vivo tumor inhibition of FDU-DB-NO . Owing to the significant cytotoxicity of
FDUꢀDBꢀNO against hypoxic cancer cells in vitro, the in vivo anticancer potential of
and FDUꢀDBꢀNO
2
2
2
2
2
2
2
2
FDUꢀDBꢀNO
volumes of the xenograft tumours injected with FDUꢀDBꢀNO
compared with the control group (injected with saline). After the in situ injection was
2
in MCFꢀ7ꢀcellꢀinoculated xenograft mice was investigated. The
2
were recorded and
th th
th
th
th
repeated 4 times (on 0 , 4 , 8 , and 12 day), the tumor tissues were dissected on 14
day. As shown in Figure 6 and Table S1 (ESI†), the tumor growth was markedly
inhibited by intratumor injection of FDUꢀDBꢀNO compared with the control group
2
which continued to increase in size throughout the processes of the experiments. On
th
the 14 day, the mean volumes of the xenograft tumours injected with FDUꢀDBꢀNO
2
3
was only 63.810 mm with a tumor inhibiting ratio of over 86%, compared with
3
4
68.067 mm of the control group. It validated that FDUꢀDBꢀNO in hypoxic tumor
2
tissues was selectively activated and released FDU for selective inhibition tumor
growth. Meanwhile, a slight body weight increase during this period indicated that no
extreme toxicity was associated with these treatments in vivo. These results
demonstrated that FDUꢀDBꢀNO
cancer treatment with low side effects due to its hypoxia selectivity. This significant
inhibition tumor growth by FDUꢀDBꢀNO indicated that the strategy may serve a new
2
activated by hypoxia was very efficient way for
2
promising candidate for potential applications in solid tumor therapy.
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Figure 6. The relative tumor volume (a) and the weight (b) of MCFꢀ7ꢀcellꢀinoculated
xenograft mice treated with FDUꢀDBꢀNO
2
CONCLUSION
In summary, we successfully developed an anticancer theranostic prodrug
FDUꢀDBꢀNO ) for diagnosis and therapy solid tumor. FDUꢀDBꢀNO was formed by
a hypoxia response group, a fluorescent dye precursor, and an anticancer drug. It is in
lock" status in normal cells, whereas it is "unlocked" specifically by hypoxic
(
2
2
"
environment in solid tumor cells, and releases CM and FDU simultaneously. The
amounts and rates of CM formation and FDU release were controlled by the hypoxic
2 2
status, and increased with the decrease of the O concentration. FDUꢀDBꢀNO
showed high selective identification of hypoxic tumor cells and could be used to
detect the hypoxia status, distribution of oxygen, and amount of FDU release in tumor
cells, tumor spheres, and tumor tissue. It showed high cytotoxicity against hypoxia
tumor cells, and nonꢀcytotoxicity against normal cells, and significent inhibitation
tumor growth of MCFꢀ7ꢀinoculated xenograft mice. The strategy will provide a novel
and promising platform for construction theranostic prodrugs activated by hypoxia to
inhibit tumor growth, nonꢀinvasive monitor hypoxia status, distribution of oxygen,
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and active drug release, which possesses great potential to be applied in the diagnosis
and personalized solid cancer treatment.
EXPERIMENTAL SECTION
Materials. FDU, DMSO, and bovine serum albumin (BSA) were purchased from
0
1
2
3
4
5
6
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8
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Alfaꢀaesar.
mꢀAnisidine, 2ꢀmethoxycarbonyl phenylboronic acid, NBS and
tetrakis(triphenylphosphine)palladium were purchased from Energy Chemical. NTR
powder from Escherichia coli (≥100 units/mg), and NADH powder were obtained
from SigmaꢀAldrich. The lyophilized NTR was dissolved in triꢀdistilled water, which
was subsequently divided into 20 parts and stored at −20 °C. The NTR solution was
uncongealed before use, and this process did not affect the activity. MTT,
benzylpenicillin and streptomycin were obtained from Sigma. Lysoꢀtracker® red was
purchased from Life Technologies. MCFꢀ7, MGCꢀ803, and BRLꢀ3A cells were
purchased from Amoresco LLC. All cell (MCFꢀ7, MGC 803, and BRLꢀ3A) lines were
purchased from Beijing Union Cell Resource Center (Beijing, China). Fourꢀweeksꢀold
immunodeficient nude mice (BALB/c) were obtained from Beijing Vital River
Laboratory Animal Technology Co., Ltd. (Beijing, China).
Instrumentation. The IR spectra were determined with a Thermo Scientific Nicolet
iS10 spectrophotometer using KBr pellets. NMR spectra were determined with a
3 6
Bruker AVANCE III 600 NMR spectrometer (CD Cl or DMSOꢀd as the solvent).
Apoptosis was measured on Flow cytometer (BD FACS Calibur, America). MS date
were measured on 4000 Q TRAP (SCIEX, USA). Fluorescence indensity were
determined with a Hitachi Fꢀ7000 spectrophotometer (Tokyo, Japan) in 2.5×10 mm
quartz cells. Optical density (OD) was determined with
a
microplate
spectrophotometer (BD FACS Calibur, USA). All chromatography tests were carried
out with an HPLC (Agilent 1260, Agilent Techn. Germany). The HPLC system was
equipped with a binary LCꢀ20AT pump, an SPDꢀ20A UVꢀvis detector, as well as an
18
RFꢀ10AXL detector. The analytical column was a Waters Symmetry C column (4.6
mm × 250 mm, 3.5 ꢂm). Fluorescence imaging experiments were carried out with an
LSM 880 confocal microscope (Carl Zeiss AG, Germany).
Synthesis of tert-butyl (3-methoxyphenyl)carbamate (1). A mixture of 1.00 g of
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ꢀmethoxyaniline (8.26 mmol), 2.70 g of (Boc) O (12.40 mmol), and 10 mL of
dioxane was stirred at 25 °C for 10 h. After the solvent dioxane was evaporated in
vacuo to give a crude product which was purified by column chromatography
EA
(V /VPE = 1/50) to provide 1.63 g of compound 1 as a white solid in 90% yield. Mp:
1
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ꢀ1
1
5
6.6ꢀ57.6 °C. IR (KBr, cm ): 3338 (NꢀH), 3112 (ArꢀH), 2977 (CꢀH) , 1607 (C=C). H
NMR (CDCl
.8 Hz, 1 H), 6.59 (d,
NMR (CDCl , 150 MHz, ppm):
3
, 600 MHz, ppm):
δ
7.17 (t,
J
= 7.8 Hz, 1 H), 7.10 (s, 1 H), 6.84 (d,
J
=
13
7
J
= 7.8 Hz, 1 H), 6.46 (s, 1 H), 3.80 (s, 3 H), 1.53 (s, 9 H). C
3
δ
160.23, 152.62, 139.65, 129.59, 110.71, 108.81,
+
104.14, 80.53, 55.27, 28.35. MS (ESI), calcd for C12
H
17NNaO
3
,
m/z: 246.1 [M+Na] ;
+
found 246.1 [M+Na] .
Synthesis of tert-butyl (4-bromo-3-methoxyphenyl)carbamate (2). A mixture of
2.30 g of compound 1 (10.3 mmol), 1.90 g of NBS (10.8 mmol), and 30 mL of
o
ethylene glycol dimethyl ether was stirred at 25 C for 1.5 h. Then, the solvent was
evaporated in vacuo to provide a crude product which was purified by column
chromatography (VEA
/VPE=1/50) to provide 2.77 g compound 2 as a white solid in
ꢀ1
89.3% yield. Mp: 123.5ꢀ124.5 °C. IR (KBr, cm ): 3330 (NꢀH), 3110 (ArꢀH), 2979
1
(
CꢀH), 1600 (C=C). H NMR (CDCl
.31 (s, 1 H), 6.63 (d,
NMR (CDCl , 150 MHz, ppm):
02.80, 80.91, 56.23, 28.33. MS (ESI), calcd for C12
3
, 600 MHz, ppm):
δ
7.38 (d,
J
= 8.4 Hz, 1 H),
13
7
J
= 8.4 Hz, 1 H), 6.48 (s, 1 H), 3.90 (s, 3 H), 1.52 (s, 9 H). C
3
δ
156.28, 152.45, 139.00, 133.00, 111.31, 104.57,
16BrNNaO m/z: 324.0
1
H
3
,
+
+
[M+Na] ; found 324.0 [M+Na] .
Synthesis of 4-bromo-3-methoxyaniline (3). Compound 2 (3.60 g, 11.96 mmol), 15
mL of hydrochloric acid (36%), and 30 mL of 1,4ꢀdioxane were mixed and stirred at
o
25 C for 2 h. Followed by adding 100 mL of water, the mixture solution was adjusted
to pH=8 by Na CO aqueous solution. Then, the mixture was extracted thrice with 50
2
3
mL of ethyl acetate (EA). The combined organic layer was washed thrice with 50 mL
of water, dried over Na SO (anhydrous), filtered, and concentrated in vacuo to give
.94 g of compound 3 as a green solid with a yield of 81%. Mp: 94.1ꢀ95.0 °C. IR
2
4
1
ꢀ1
1
(
KBr, cm ): 3330 (NꢀH), 3005 (ArꢀH), 2972 (CꢀH), 1629 (C=C). H NMR (CDCl
3
,
600 MHz, ppm): 7.25 (d, = 3.6 Hz, 1 H), 6.26 (d, = 2.4 Hz, 1 H), 6.19 (dd, J =
δ
J
J
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Bioconjugate Chemistry
Page 18 of 41
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
13
7
1
.2, 2.4 Hz, 1 H), 3.84 (s, 3 H). C NMR (CDCl
3
, 150 MHz, ppm):
δ
156.48, 147.19,
33.44, 108.52, 99.71, 99.51, 56.01. MS (ESI), calcd for C H BrNO, m/z: 202.0
7
9
+
+
[M+H] ; found 202.0 [M+H] .
Synthesis of 4-bromo-3-methoxy-N,N-dimethylaniline (4). Compound 3 (2.4 g,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2.0 mmol), K CO (3.0 g, 22.0 mmol), and 20 mL of DMF were mixed and stirred at
2 3
1
20 °C for 9 h. After the mixture was cooled to 25 °C, 100 mL of CH
2
Cl
2
was added.
SO
anhydrous), and concentrated in vacuo to provide a crude product which was purified
The organic phase was washed thrice with 50 mL of water, dried over Na
2
4
(
by column chromatography (
V
EA
/
VPE=1/50) to afford 2.01 g compound 4 as a green
ꢀ1
1
oil in 78.2% yield. IR (KBr, cm ): 3029 (ArꢀH), 2970 (CꢀH), 1594 (C=C). H NMR
(
CDCl
dd,
3
, 600 MHz, ppm):
δ
7.27 (d,
J
= 9.0 Hz, 1 H), 6.22 (d,
J
= 9.0 Hz, 1 H), 6.17
(
J
= 9.0, 2.4 Hz, 1 H), 3.87 (s, 3 H), 3.33 (q,
J
= 7.2 Hz, 4 H), 1.16 (q, = 7.2 Hz,
J
13
6
9
H). C NMR (CDCl
3
, 150 MHz, ppm):
δ
156.48, 147.19, 133.44, 108.52, 99.71,
+
9.51, 56.01. MS (ESI), calcd for C11
H17BrNO, m/z: 257.0 [M+H] ; found 257.8
+
[M+H] .
Synthesis of 4-bromo-N,N-diethyl-3-(4-nitrobenzyloxy)aniline (5). Compound 4
2.5 g, 10 mmol), 30 mL of DCM, and 6.3 g of borontribromide (25 mmol) were
(
mixed and stirred at ꢀ20 °C for 0.5 h. Then, the temperature of the mixture was raised
to 25 °C and the reaction was continued for 1.5 h. Next, the reaction mixture was
poured into 100 mL of ice water and extracted thrice with 50 mL of DCM. The
combined organic phase was washed thrice with 50 mL of water, dried over Na
2
SO
4
(
anhydrous), and concentrated in vacuo. The obtained residue was dissolved in 15 mL
2 3
of acetonitrile. Then, 2.2 g of 4ꢀnitrobenzyl bromide (10 mmol) and 2.8 g of K CO
were added. The mixture was heated to 80 °C and stirred for 3 h. Next, the reaction
mixture was concentrated in vacuo, and the residue was purified by flash column
EA
chromatography (V /VPE=1/30) to afford 2.73 g compound 5 (yellow solid) in 64.2%
ꢀ1
yield. Mp: 88.9ꢀ90.0 °C. IR (KBr, cm ): 3096 (ArꢀH), 2975 (C=CꢀH), 1593 (C=C),
1
1
562 (ꢀNO
Hz, 2 H), 7.69 (d,
.18 (s, 1 H), 5.22 (s, 2 H), 3.29 (q,
2
), 1269 (ꢀCꢀOꢀCꢀ). H NMR (CDCl
3
, 600 MHz, ppm):
= 9.6 Hz, 1 H), 6.23 (d,
= 6.6 Hz, 4 H), 1.11 (t,
δ 8.26 (d, J = 9.0
J
= 9.6 Hz, 2 H), 7.31 (d,
J
J
= 9.6 Hz, 1 H),
13
6
J
J
= 6.6 Hz, 6 H). C
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Page 19 of 41
Bioconjugate Chemistry
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2
3
4
5
6
7
8
9
NMR (CDCl
3
, 150 MHz, ppm): δ 155.18, 148.45, 147.57, 144.51, 127.42, 123.81,
106.82, 89.77, 97.25, 69.72, 44.65, 12.42. MS (ESI), calcd for C H BrN O , m/z
:
17
20
2
3
+
+
380.6 [M+H] ; found 380.8 [M+H] .
Synthesis of methyl 1,1'-biphenyl-4'-diethylamino-2'-(4-nitrobenzyloxy)-2-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
carboxylate (6). Under N atmosphere, 200 mg of compound 5 (0.53 mmol), 8 mL of
2
DMF, 107 mg of methyl 2ꢀ(dihydroxymethyl)benzoate (0.63 mmol), 36.6 mg of
tetrakis(triphenylphosphine)palladium (0.032 mmol), 89 mg of Na
water were stirred and heated to 115 °C for 4 h. After the solution was concentrated in
vacuo, the obtained residue was purified by column chromatography ( PE=1/20)
to afford 129 mg of compound 6 (yellow solid) in 56.1% yield. Mp: 93.1ꢀ93.9 °C. IR
2 3
CO , and 2 mL of
EA
V /V
ꢀ
1
(
KBr, cm ): 3065 (ArꢀH), 2925 (C=CꢀH), 1649 (C=C), 1483 (ꢀNO
2
), 1270 (ꢀCꢀOꢀCꢀ).
= 8.4 Hz, 2 H), 7.84 (d, = 7.8 Hz, 1
= 7.2 Hz, 1 H), 7.38ꢀ7.36 (m, 4 H), 7.11 (d, = 8.4 Hz, 1 H), 6.40 (dd,
8.4, 2.4 Hz, 1 H), 6.17 (d, = 2.4 Hz, 1 H), 5.03 (s, 2 H),3.62 (s, 3 H), 3.33 (q,
6.6 Hz, 4 H), 1.13 (t, = 6.6 Hz, 6 H). C NMR (CDCl , 150 MHz, ppm):
1
H NMR (CDCl , 600 MHz, ppm):
δ
8.12 (d,
J
J
3
H), 7.50 (t,
J
J
J
=
J
J
=
13
J
δ
168.83,
55.84, 148.76, 147.24, 145.05, 139.24, 131.65, 131.38, 130.91, 129.46, 126.34,
23.57, 118.59, 105.32, 97.40, 69.58, 51.66, 44.48, 12.65. calcd for C25 m/z:
3
1
1
4
H
27
N
2
O
5
,
+
+
35.2 [M+H] ; found 434.9 [M+H] .
Synthesis of 1,1'-biphenyl-4'-diethylamino-2'-(4-nitrobenzyloxy)-2-carboxylic
acid (7). Compound 6 (100 mg, 0.23 mmol) and 15 mL of 20% sulfuric acid were
stirred and heated to 90 °C for 12 h. After being cooled to about 25 °C, the reaction
mixture was adjusted to pH=8 by Na CO aqueous solution and extracted thrice with
2
3
2
0 mL of DCM. The combined organic phase was washed thrice with 20 mL of water,
dried over Na SO (anhydrous), and concentrated in vacuo. The solid residue was
purified by column chromatography (VMethanol VDCM= 1/50) to give 57.7 mg of
2
4
/
ꢀ1
compound 7 (a green solid) in 59.6 % yield. Mp: 199.2ꢀ201.6 °C. IR (KBr, cm ):
1
3458 (OꢀH), 3072 (ArꢀH), 2965 (CꢀH), 1682 (C=O), 1522 (ꢀNO
NMR (CDCl , 600 MHz, ppm): 12.32 (s, 1 H), 8.09 (d,
7.8 Hz, 1 H) , 7.55 (t, = 7.8 Hz, 1 H) , 7.36 (t,
Hz, 1 H) , 6.98 (d, = 8.4 Hz, 1 H), 6.30 (d,
2
), 1273 (ꢀCꢀOꢀCꢀ). H
3
δ
J
= 9.0 Hz, 2 H), 7.93 (d,
J
=
J
J
= 7.8 Hz, 1 H) , 7.29 (d,
J
= 7.8
J
J
= 8.4 Hz, 1 H), 6.18 (d, J = 2.4 Hz, 1
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Page 20 of 41
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
13
H), 5.03 (s, 2 H) , 3.33 (q,
J
= 6.6 Hz, 4 H) , 1.11 (t, J = 6.6 Hz, 6 H). C NMR
173.02, 155.97, 148.99, 147.25, 144.93, 139.36, 131.85,
130.90, 130.43, 130.11, 125.45, 123.59, 105.25, 97.23, 69.54, 44.50, 12.59.calcd for
(
CDCl , 150 MHz, ppm):
δ
3
+
+
C
24
H
25
N
2
O
5
,
m/z: 421.2 [M+H] ; found 420.9 [M+H] .
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Synthesis of FDU-DB-NO . Under N atmosphere, 100 mg of compound 7 (0.24
2
2
mmol), 1 mL of DMF, 90 mg of N,Nꢀdiisopropyl carbodiimide (0.72 mmol), 2.9 mg
of 4ꢀdimethylaminopyridine (0.024 mmol), and 58.5 mg of floxuridine (0.24 mmol)
were stirred and heated to 50 °C for 18 h. After being cooled to 25 °C, the mixture
was purified by column chromatography (
FDUꢀDBꢀNO as a green solid. Mp: 90.1ꢀ91.4 °C. IR (KBr, cm ): 3411 (ꢀOH), 3068
ArꢀH), 2966 (CꢀH), 1718 (C=O), 1682 (C=C), 1556 (ꢀNO ), 1280 (ꢀCꢀOꢀCꢀ), 1074
11.78 (s,1 H), 8.12 (d, = 8.4 Hz, 2 H),
= 7.4 Hz, 1 H), 7.47 (d,
= 7.8 Hz, 1 H), 7.00 (d, = 8.4 Hz,
= 8.4 Hz, 1 H), 6.21 (s, 1 H), 6.06 (t, = 6.6 Hz, 1 H), 5.31 (s, 1 H),
.16 (s, 2 H), 4.18ꢀ4.23 (m, 2 H), 3.96 (s, 1 H), 3.79 (q, = 4.8 Hz, 1 H), 3.32 (q,
.2 Hz,4 H), 1.89ꢀ2.01 (m, 2 H), 1.05 (t, = 7.2 Hz, 6 H). C NMR (CDCl
167.74, 156.95, 156.78, 155.44, 148.86, 148.40, 146.62, 145.40,
40.58, 139.05, 138.44, 131.54, 131.40, 131.13, 130.60, 127.47, 124.30, 124.07,
23.24, 104.42, 96.56, 84.38, 83.83, 70.17, 68.32, 62.74, 43.74, 12.59. MS (ESI),
Methanol
V /VDCM=1/50) to provide 9.2 mg of
ꢀ1
2
(
(
2
1
CꢀF). H NMR (CDCl , 600 MHz, ppm):
δ
J
3
7
.73 (d,
8.4 Hz, 2 H), 7.38 (t,
1 H), 6.32 (d,
J
= 7.8 Hz, 1 H), 7.62 (d,
J
= 6.6 Hz, 1 H), 7.58 (t,
J
J
=
J
= 7.8 Hz, 1 H), 7.34 (d,
J
J
J
J
5
7
J
J =
13
J
3
, 150
MHz, ppm):
δ
1
1
+
+
4 9
calcd for C33H34FN O , m/z: 649.2 [M+H] ; found 649.4 [M+H] .
Synthesis of CM. A mixture of 100 mg of compound 6 (0.23 mmol), 200 mg of
Na (1.15 mmol), 2 mL of water, and 2 mL of methanol was stirred at 25 °C for
.5 h. Then 50 mL of water was added and extracted thrice with 50 mL of DCM. The
combined organic phase was washed thrice with 50 mL of water, dried over
anhydrous Na SO , and concentrated in vacuo. The solid residue was purified by
column chromatography ( PE=1/30) to give 58.8 mg CM (a green solid) in 92.4%
2 2 4
S O
2
2
4
EA
V /V
ꢀ1
yield. Mp: 85.1ꢀ86.6 °C. IR (KBr, cm ): 3448 (overtone of C=O), 3065 (ArꢀH), 2972
1
(CꢀH), 1708 (C=O), 1613 (C=C). H NMR (CDCl
3
, 600 MHz, ppm):
δ
J
8.30 (d,
J = 8.4
Hz, 1 H), 7.90 (d,
J
= 7.2 Hz, 1 H), 7.81 (d,
J
= 9.0 Hz, 1 H), 7.70 (t,
= 7.2 Hz, 1 H),
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Page 21 of 41
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
7
.37 (t,
H), 1.22 (t,
149.52, 136.17, 134.63, 130.44, 123.73, 120.27, 119.07, 108.81, 106.05, 98.56, 44.68,
J
= 7.2 Hz, 1 H), 6.65 (d,
J
= 9.0 Hz, 1 H), 6.56 (s, 1 H), 3.42 (q,
J = 7.2 Hz, 4
13
J
= 7.2 Hz, 6 H). C NMR (CDCl , 150 MHz, ppm): δ 162.18, 153.32,
3
+
+
1
2.52. MS (ESI), calcd for C17
Synthesis of FDU-DB.
'ꢀ(diethylamino)ꢀ2'ꢀmethoxyꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀcarboxylic acid (0.33 mmol), 1 mL of
DMF, 125 mg of N,Nꢀdiisopropyl carbodiimide (0.99 mmol), 4 mg of
ꢀdimethylaminopyridine (0.033 mmol), and 98.0 mg of floxuridine (0.40 mmol)
were stirred at 50 °C for 18 h. After being cooled to 25 °C, the mixture was purified
by column chromatography ( DCM=1/50) to provide 30 mg of FDUꢀDB as a
H18NO
2
,
m/z: 268.1 [M+H] ; found 268.2 [M+H] .
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Under N2 atmosphere, 100 mg
of
4
4
Methanol
V /V
ꢀ1
yellow solid. Mp: 189.6ꢀ190.8 °C. IR (KBr, cm ): 3083 (ArꢀH), 2969 (CꢀH), 1716
1
(C=O), 1613 (C=C), 1273 (ꢀCꢀOꢀCꢀ), 1078 (CꢀF). H NMR (DMSOꢀd6, 600 MHz,
ppm):
Hz, 1H), 7.37 (t,
(dd, J1=8.4 Hz, J2=2.4 Hz, 1H), 6.23 (s, 1H), 5.88 (t,
δ
11.86 (s, 1H), 8,16 (d,
J
=7.2 Hz, 1H), 7.70 (d,
=7.8 Hz, 1H), 6.98 (d,
=6.6 Hz, 1H), 5.30 (t,
=6.0 Hz, 1H), 3.75 (s, 1H), 3.64 (s, 3H), 3.59 (t, =4.2 Hz, 2H),
=5.4 Hz, 1H), 1.09 (t,
, 150 MHz, ppm):
J
=7.8 Hz, 1H), 7.56 (t,
=8.4 Hz, 1H), 6.31
=4.8 Hz,
J=7.2
J
=6.6 Hz, 1H), 7.28 (d,
J
J
J
J
1
3
H), 5.19 (d,
J
J
1 2
.38ꢀ3.34 (m, 4H), 2.22ꢀ2.12 (m, 1H), 2.06 (dd, J =13.8 Hz, J
13
J
1
1
=7.2 Hz, 6H), 1.00 (d,
J
=6.6 Hz, 1H). C NMR (DMSOꢀ
d
6
δ
67.56, 156.58, 148.85, 148.51, 140.73, 139.20, 138.71, 131.48, 131.31, 131.16,
30.23, 128.92, 126.03, 124.48, 124.25, 116.75, 103.87, 94.69, 84.54, 84.43, 75.07,
+
61.20, 54.72, 43.71, 36.85, 23.25, 12.40. calcd for C27
H30FN
3
O
7
,
m/z: 528.21 [M+H] ;
+
found 528.19 [M+H] .
Fluorescence intensity of FDU-DB-NO
solution of FDUꢀDBꢀNO (1 mM) was diluted into 100 ꢂM by DMSO and PBS
VDMSO/VPBS= 3/7). Then the solution was incubated with NTR (10 ꢂg/mL) and
2
incubated with NTR. DMSO stock
2
(
o
2
NADH (2 mM) at 37 C under hypoxic condition (1% O ) for different time (1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 12, 14, 16, and 18 min). Then the fluorescence intensity was detected
with a Hitachi Fꢀ7000 luminescence spectrophotometer in 2.5×10 mm quartz cells
(excited at 380 nm with 2.5/10 nm slit widths, collected from 400 to 700 nm).
2
Effects of pH value on the stability of FDU-DB-NO and CM. DMSO stock
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2
3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
solution of CM or FDUꢀDBꢀNO (1 mM) was diluted to 100 ꢂM by DMSO, and their
pH values were adjusted to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. Then the fluorescence
intensity of the solution was detected with a luminescence spectrophotometer. And the
2
absorbance of the FDUꢀDBꢀNO solution was detected with a UV spectrophotometer.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Stability of FDU-DB-NO in blood serum. PBS stock solution of FDUꢀDBꢀNO (1
2
2
mM) was diluted into 100 ꢂM by a mixture of PBS and blood serum (
VPBS/Vblood
serum=1/5). The fluorescence intensity and the absorbance of the mixture was detected
with the luminescence spectrophotometer and UV spectrophotometer.
Selectivity of FDU-DB-NO
2 2
. DMSO stock solution of FDUꢀDBꢀNO (1 mM) were
diluted into 100 ꢂM by DMSO and PBS (
V
DMSO/VPBS = 3/7). Then the solution was
incubated with NTR (10 ꢂg/mL) in presence of NADH (2 mM), or cysteine (10 mM),
glutathione (10 mM), vitamin C (10 mM), glutamic acid (10 mM), alanine (10 mM),
fructose (10 mM), glucose (10 mM), aspartic acid (10 mM), or histidine (10 mM) for
2
h, respectively. The fluorescence intensity of the mixture was detected with a
luminescence spectrophotometer.
2 2 2 4 2 2 4
The HPLC measurement of FDU-DB-NO incubated with Na S O . Na S O (10
mg) was added to a solution of FDUꢀDBꢀNO
2
(1 ꢂM) in methanol and water
o
(V
Methanol/Vwater=1/1), and the mixture was stirred at 25 C for 40 min. Followed by
filtration, the filtrate was analyzed by HPLC (Agilent 1260). The HPLC system was
performed with a binary LCꢀ20AT pump, an SPDꢀ20A UVꢀvis detector, as well as an
1
8
RFꢀ10AXL detector. The analytical column was a Waters Symmetry C
reversedꢀphase column (4.6 mm ×250 mm, 3.5 ꢂm). The solvent system was
composed of two solutions: solution A (water containing 0.05% TFA) and solution B
(acetonitrile). The 40 min gradient LC separation include 5 steps: The elution began
with a mixture of A and B (V/V=99/1), followed by a linear increase to 2.5% B over
the course of 8.0 min; then 2.5ꢀ60% solvent B for 8ꢀ15 min (linear); 60ꢀ80% solvent
B for 15ꢀ25 min; then 80% solvent B for 15 min; flow rate of 1.0 mL/min. Detection
was carried out with UV light at 254 nm.
2
The amounts of generated CM and released FDU from FDU-DB-NO . Methanol
stock solution of FDU (1 mM) and CM (1 mM) were dilute into 25, 50, 75, and 100
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ꢂM by methanol and were analyzed by HPLC (Agilent 1260). The standard curves
were linear in the range of 25~100 ꢂM. Methanol stock solution of FDUꢀDBꢀNO (1
2
mM) were diluted into 20, 40, 60, 80, and 100 ꢂM by methanol, then these solutions
2 2 2 4
of FDUꢀDBꢀNO were incubated with Na S O for 2 h. The amounts of CM generated
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1
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0
and FDU released from FDUꢀDBꢀNO were analyzed by HPLC.
2
MTT assay. MCFꢀ7 (or MGCꢀ803 and BRLꢀ3A) cells harvested at subconfluent stage
4
(
at a density of 2×10 cells/mL) were seeded in 96ꢀwell plates and incubated with
o
2
00ꢁꢂL of culture media under the cell culture conditions (5% CO
After being incubated with FDUꢀDBꢀNO
concentrations (1, 5, 10, 15, and 20 ꢂM) under normoxic (or hypoxic) conditions for
2 h, and then further incubated for 12 or 36 h. Control wells were carried out by
2
, at 37 C) for 12 h.
2
(FDUꢀDB or FDU) on different
1
adding culture medium. Banks were prepared by adding culture medium without
tumor cells. Subsequently, the cells in 96ꢀwell plates were washed twice with PBS
buffer, and 10 ꢂL of freshly prepared MTT solution (0.5 mg/mL in PBS buffer) was
added into each well and then the cells were incubated for an additional 4 h. Next, 100
ꢂL of DMSO was added into each well at 37 °C for fully dissolve the MTT formazan.
The optical density (OD) of formazan solutions was analyzed by a microplate
spectrophotometer at 570 nm. The cell viability (%) was presented as
(ODsampleꢀODblank)/(ODcontrolꢀODblank)×100.
Confocal microscopy imaging. MCFꢀ7 (or MGCꢀ803) cells were seeded in confocal
4
dish at a density of 2×10 cells/mL under the cell culture conditions (5% CO
2
, at 37
o
C) in a humidified incubator (SANYO, MCOꢀ20AIC, JPN). Then, the medium was
replaced with 10 ꢂM FDUꢀDBꢀNO
FDUꢀDBꢀNO
2
in fresh culture medium. After incubation with
o
2
under normoxic conditions (20% O
2
, 5% CO
2
, and 75% N
2
, at 37 C)
o
or different hypoxic conditions (1% O , 5% CO , and 94% N , at 37 C; 3% O , 5%
2
2
2
2
o
o
CO
2
, and 92% N
2
, at 37 C; 5% O
2 2
, 5% CO , and 90% N
2
, at 37 C; 10% O
2 2
, 5% CO ,
o
and 85% N
2
, at 37 C) in a humidified incubator (Heal Force, HF 100, CHN) for 12 h.
Then, the medium was removed and the cells were washed twice with PBS buffer.
The fluorescence imaging experiments were carried out on an LSM880 confocal laser
scanning microscope (Carl Zeiss AG, Germany) and excited with 380 nm laser light
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for oneꢀphoton imaging and 760 nm for twoꢀphoton imaging.
The cultivation of MGC 803 and MCF-7 spheres. MGC 803 (or MCFꢀ7) cells were
diluted in serum free growth medium (1000 cells/mL) and plated in 100 ꢂL in
nonꢀtissue culture coated 96ꢀwell plates. Cells were fed with 25 ꢂL of serumꢀfree
growth medium every other day, for 14 d. The culture medium consisted of
serumꢀfree DMEM/F12 supplemented with 10 U/mL heparin, 20 U/mL B27, and 20
U/mL human recombinant fibroblast growth factor. After 14 d, spheres were counted
as a tumor sphere forming unit.
0
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Animal experiment. Animal experiments were followed to the guidelines of the
Animal Welfare and Ethical Committee of Hebei University. Four weeks old Balb/c
nude mice (female, around 20 g) were fed in an animal facility (20±2 °C, 60±10%
relative humidity, 12 h light/dark cycle) with free feeding food and drinking water.
Tumorꢀbearing mice models were established by subcutaneous injection of MCFꢀ7
7
3
cells (1×10 cells each mouse). After the tumors grew to be about 50ꢀ80 mm ,
MCFꢀ7ꢀcellꢀinoculated xenograft mice were divided into two groups (n=5). The first
th
th
th
group of mice was treated with FDUꢀDBꢀNO
2
via in situ injection (on the 0 , 4 , 8 ,
th
th
1
0 , and 12 day; 10 mg/kg body weight). The second group was injected with saline
th
th
th
th
th
th
(
on the 0 , 4 , 8 , 10 , and 12 day) as the control. On the 14 day, mice were killed
by CO , the tumor tissues were dissected. The body weight and tumor volume of mice
2
were measured every two days, and the tumor volume of mice was calculated as
2
(Tumor length) × (Tumor width) /2.
MCFꢀ7ꢀcellꢀinoculated xenograft mice were killed by cervical vertebra dislocation
when the tumor grew to the right size. After washing off the blood with PBS, the
tumor tissues were cut into about 600 ꢂm thick sections. The tumor tissue slices were
incubated with FDUꢀDBꢀNO (30 ꢂM) for 1 h. After washing thrice with PBS buffer,
2
the tumor tissue slices were monitored with an LSM 880 confocal microscope excited
at 760 nm (TP mode), and the emission wavelengths were collected from 500 nm to
550 nm.
ASSOCIATED CONTENTS
Supporting Information
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The supporting Information is available free of charge on the ACS publications
websites.
AUTHOR INFORMATION
Corresponding Author
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*
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Guoqiang Zhou, Eꢀmail: zhougq1982@163.com
Jinchao Zhang, Eꢀmail: jczhang6970@163.com
Shuxiang Wang, Eꢀmail: wsx@hbu.edu.cn
Author Contributions
#
These authors contributed equally to the manuscript
Notes
The authors declare no competing financial interests.
ACKNOWLEDGEMENTS
This work was supported by the Natural Science Foundation of China (21471044,
31500812), the Key Basic Research Special Foundation of Science Technology
Ministry of Hebei Province (15962602D), and the Natural Science Foundation of
Hebei Province (B2015201213, B2016201169, B2017201226, B2017201230,
B2017201135).
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