Gemcitabine-coumarin-biotin conjugates: A target specific theranostic anticancer prodrug

Article abstract of DOI:10.1021/ja401350x

We present here, the design, synthesis, spectroscopic characterization, and in vitro biological assessment of a gemcitabine-coumarin-biotin conjugate (5). Probe 5 is a multifunctional molecule composed of a thiol-specific cleavable disulfide bond, a coumarin moiety as a fluorescent reporter, gemcitabine (GMC) as a model active drug, and biotin as a cancer-targeting unit. Upon addition of free thiols that are relatively abundant in tumor cells, disulfide bond cleavage occurs as well as active drug GMC release and concomitantly fluorescence intensity increases. Confocal microscopic experiments reveal that 5 is preferentially taken up by A549 cells rather than WI38 cells. Fluorescence-based colocalization studies using lysosome-and endoplasmic reticulum-selective dyes suggest that thiol-induced disulfide cleavage of 5 occur in the lysosome possibly via receptor-mediated endocytosis. The present drug delivery system is a new theranostic agent, wherein both a therapeutic effect and drug uptake can be readily monitored at the subcellular level by two photon fluorescence imaging.

Full text of DOI:10.1021/ja401350x

Article
pubs.acs.org/JACS
Gemcitabine−Coumarin−Biotin Conjugates: A Target Specific
Theranostic Anticancer Prodrug
Sukhendu Maiti,† Nayoung Park,† Ji Hye Han,‡ Hyun Mi Jeon,‡ Jae Hong Lee,† Sankarprasad Bhuniya,†
Chulhun Kang,*,‡ and Jong Seung Kim*,†
†Department of Chemistry, Korea University, Seoul, 136-701, Korea
‡The School of East-West Medical Science, Kyung Hee University, Yongin, 446-701, Korea
S
* Supporting Information
ABSTRACT: We present here, the design, synthesis, spectroscopic
characterization, and in vitro biological assessment of a gemcitabine−
coumarin−biotin conjugate (5). Probe 5 is a multifunctional molecule
composed of a thiol-specific cleavable disulfide bond, a coumarin moiety
as a fluorescent reporter, gemcitabine (GMC) as a model active drug,
and biotin as a cancer-targeting unit. Upon addition of free thiols that are
relatively abundant in tumor cells, disulfide bond cleavage occurs as well
as active drug GMC release and concomitantly fluorescence intensity
increases. Confocal microscopic experiments reveal that 5 is preferen-
tially taken up by A549 cells rather than WI38 cells. Fluorescence-based
colocalization studies using lysosome- and endoplasmic reticulum-
selective dyes suggest that thiol-induced disulfide cleavage of 5 occur
in the lysosome possibly via receptor-mediated endocytosis. The present
drug delivery system is a new theranostic agent, wherein both a
therapeutic effect and drug uptake can be readily monitored at the subcellular level by two photon fluorescence imaging.
Gemcitabine (GMC) is a nucleoside analogue used for an
antimetabolite of deoxycytidine8 to control nonsmall cell lung,9
pancreatic,10 metastatic breast, and recurrent ovarian cancers.11
Despite the clinical success of GMC, its short plasma half-life
(9−13 min for human plasma)12 and adverse toxicity, such as
myelosuppression, greatly limit its chemotherapeutic efficacy.
To increase its chemotherapeutic index, it would be desirable to
develop a new strategy, for example, attachment of a disulfide
linker on the GMC molecule to protect the drug from renal
clearance and prolonged circulation half-life.13 Besides, it is
hard to monitor the delivery of such a drug in vitro and in vivo,
since GMC is a nonfluorescent antitumor agent. For these
reasons, it would be a significant advance in GMC delivery to
develop a new type of GMC conjugate which is able to real-
time monitor the drug delivery and to enhance its lifetime in
circulation at the same time. Therefore, in view of demand, we
wish to design, synthesize, and spectroscopically characterize a
GMC delivery system in a specific region as well as
fluorescence-on, for in vivo and vitro use.
1. INTRODUCTION
Target-specific drug delivery, frequently called smart drug
delivery system (DDS), is an important and exciting as well as
challenging area in our science community and in modern
pharmaceutical industry.1 Its presumed advantages are the
reduction in the frequency of the dosages taken by the patient
and reduced fluctuation in circulating drug levels and drug side
effects. This method is able to provide information regarding
real-time monitoring of the release and distribution of a drug.2
Therefore, in situ monitoring of the active drug release in DDSs
could be of great benefit, particularly if the reported signal
could be detected in a noninvasive manner. On the other hand,
imaging the molecular expressions of disease may help early
detection, thus facilitating more effective early therapy or
change in lifestyle. This approach may also help in character-
izing different plaques for the purpose of treatment guidance as
well as assessing the response to therapy.
The variable forms of drug carriers are widely used in the
targeted DDS system, such as gold nanoparticles,3 fluorescent
quantum dots,4 polymeric materials,5 and porous silica
materials.6 However, these methods suffer from extensive and
time-consuming procedures to visualize the targets concerned.
Recently, a theranostic (therapy and diagnostics) DDS
equipped with targeting and reporting abilities was introduced
by our group, where the target-specific cellular uptake of a RGD
peptide-appended therapeutic agent conjugated with campto-
thecin, and the drug release was monitored based upon
fluorescence.7
Herein, we develop a cancer-targeting theranostic DDS that
contains a GMC-loaded coumarin moiety which is coupled
with biotin as a targeting ligand. It is known that biotin
molecules or biotin conjugates can be taken up preferentially by
a cancer cell.14 Conjugate 5 is composed of the chemo-
therapeutic drug GMC, a disulfide linker cleavable by the
Received: February 5, 2013
© XXXX American Chemical Society
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intracellular thiols, a coumarin moiety, which enhances the
fluorescence intensity after disulfide bond cleavage, and biotin,
which serves as an excellent guiding molecule to tumor cells.
2. RESULTS AND DISCUSSION
Synthesis of compound 5 was performed according to the
following synthetic route shown in Scheme 1. The coumarin−
Scheme 1. Synthetic Routes of 5 and 9
Figure 1. (a) Absorption spectra of 5 (25.0 μM, 25% DMSO, and 75%
PBS buffer) recorded in the presence and absence of GSH (5.0 mM).
(b) Fluorescence spectra of 5 (5.0 μM) recorded in the presence and
absence of GSH (5.0 mM). (c) Fluorescence changes of 5 (5.0 μM)
seen upon treatment with increasing concentrations of GSH (0−60
equiv). (d) Changes in fluorescence intensity at 476 nm as a function
of GSH concentration.
stepwise addition of GSH to the solution of probe 5 (5.0 μM)
in aqueous PBS buffer, the solution’s fluorescence intensity at
λmax 476 nm gradually increased with each additional step up to
0−30 equiv of GSH (Figure 1c,d). These results indicate that
the disulfide bond of 5 can be readily cleavable by GSH, the
most abundant thiol in cells, in a dose-dependent manner,
possibly to release GMC in cells.
Whether the cleavage reaction of probe 5 is interfered by the
biological environments, such as various biochemical species or
pH, should be an important question to be answered prior to
its application to the biosystems. For the possibility of the
interference from other biologically relevant analytes, the
reactions of 5 with different thiols, nonthiol amino acids, and
metal ions were examined. Upon the addition of cysteine (Cys)
or homocysteine (Hcy) to probe 5, similar spectroscopic
changes were observed to that of GSH. In contrast, addition of
any nonthiol amino acids or biologically relevant metal ions to
the solution containing 5, no significant spectroscopic changes
were observed (Figure S2). These results indicate that the
fluorescence enhancement from the cleavage reaction of the
disulfide in 5 is induced by the thiol species. Furthermore, we
have also investigated the pH dependency studies where the
fluorescence intensity changes of 5 (5.0 μM), with the addition
of 5.0 mM GSH under different pH conditions. The large
fluorescence enhancement at λem 476 nm was observed over a
pH range of 6−9 (Figure S3). Therefore, at this stage we can
conclude that the disulfide-linked GMC−coumarin conjugates
will undergo thiol-mediated cleavage with no significant
interference from other biomolecules, such as amino acids or
metal cations, even in biological environments.
disulfide conjugates 1−3 were synthesized by previously
reported methods.15,7 The compound 3 was reacted with 4-
nitrophenyl chloroformate and diisopropylethylamine (DIPEA)
and followed by reaction with GMC to afford 4 in moderate
yield. Then, the target 5 was prepared by the Click reaction of 4
with a biotin derivative in moderate yield. The coumarin
derivative 6 as the reference compound and the biotin
derivative 9 were prepared from the previously reported
procedures.15,16 The identities of all compounds (1−6) were
1
confirmed by H NMR, 13C NMR, ESI-MS, and HRMS data
(Supporting Information, SI).
To demonstrate whether the disulfide bond between the
coumarin and the GMC moieties of compound 5 is cleavable or
not by biologically available thiols, 5 was allowed to react with
0−60 equiv of glutathione (GSH), and the changes in its
absorption and fluorescence spectra were monitored. In the UV
spectrum, as seen in Figure 1a, 5 exhibited a strong absorption
band at λmax 400 nm, whose intensity increases by ∼3-fold upon
addition of GSH. Although the solution of 5 exhibited weak
fluorescence centered at 476 nm on excitation at 410 nm, upon
addition of 5.0 mM GSH, its intensity was 3.5-fold enhanced, as
revealed in Figure 1b. The emission spectrum of the solution
was identical with that of the reference 6 (Figure S1). On
It is quite intersting to ask whether the fluorescence
enhancement from 5 in Figure 1 means GMC release or not.
To answer the question, the temporal release of GMC from 5
and the fluorescence changes in a function of time was
measured in the presence of GSH as shown in Figures S4 and
S23. In Figure S4, it is observed that, within 10 min, the
enhancement of fluorescence intensity of 5 reached the
saturation point, implicating that it mostly dissociated. We
observed that for 5.0 mmol GSH, the reaction rate is higher
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than that of 1.0 and 3.0 mmol GSH. In contrast, in the absence
of GSH, monitoring solutions of 5 revealed neither GMC
release nor an enhancement in the fluorescence intensity at 476
nm as a function of time. Furthermore, probe 5 was treated
with 5.0 mmol GSH at 37 °C for 1 h, the aliquot was subjected
to mass analysis, and the solutions gave rise to two major peaks
associated with GMC ([M+H] = 264.2) and 6 ([M+H] =
485.3) (Figure S23). The above results lead us to confirm that
active GMC was released after the disulfide bond cleavage upon
GSH treatment.
Together, these results suggest that, by the action of GSH,
the vulnerable S−S bond cleaved and the resulting thiol
undergoes an intramolecular nucleophilic substitution at the
carabamate moiety, forming a five-membered ring thiolactone
and releasing GMC molecules in a pharmaceutically active
form, consequently inducing the fluorescence enhancement of
the product 6, which is summarized in Scheme 2. This type of
self-immolative disulfide linker can readily be applied to a
variety of tumor-targeting drug conjugates.
Figure 2. Confocal microscopy images of A549 and WI38 cells treated
with 5. The left-side panels show the confocal microscopy images of
A549 and WI38 cells treated with 10.0 μM of 5 in PBS buffer and a
total incubation period of 15 min. The right-side panels show
nonconfocal phase contrast images. Cell images were obtained using
two photon excitation at 750 nm and a long-path (420−600 nm)
emission filter.
Scheme 2. Reaction Mechanism of 5 with GSH under
Physiological Conditions
incubation of compound 5, whereas the WI38 cells show little
or no fluorescence signal under a similar experimental
condition. The result confirms that the presence of biotin
receptor only on the A549 cells causes the difference in the
fluorescence intensity for two cell lines. The difference might be
possible if the concentration of thiol species (for example,
GSH) is much lower in WI38 cells, however, it seems
unreasonable since GSH is essential to all types of eukaryotic
cells including both cell lines in this experiment. Therefore, we
conclude that the prodrug 5 can enter the cells, aided by the
interaction of its biotin moiety with a biotin receptor on the
tumor cells, possibly through receptor-mediated endosytosis.
To provide further evidence for thiol-induced disulfide bond
cleavage as well as concomitantly fluorescence-on, the probe 5
was studied in the presence of a strong thiol reacting agent, N-
ethylmalemide (NEM).18 The results obtained from these
studies are shown in Figure 3. We found that the fluorescence
Based upon the results summarized in Scheme 2, it is worth
examining whether DDS works in live cells similarly or not.
The encouraging response toward thiol-mediated active drug
recovery and concomitantly optical enhancement in vitro
solution test of theranostic prodrug 5 is committed to apply
in living cells. Apparently, the optical response of 6, using one-
photon fluorescent spectroscopy, is not worthy to use in living
systems, as the shorter wavelength light scattered and auto
absorbed by native cellular components. Therefore, we adopted
the two-photon fluorescent spectroscopy tool for cellular
imaging. It is fortuitous that the coumarin moiety in 6 showed
a two-photon absorption property.17 This tool reduced
photodamage to living biological samples and fluorophore,
minimized background absorption and scattering, and
improved the spatial resolution and sensitivity and the ability
to image thicker specimens.
For the application of probe 5 to the biological systems, we
investigated whether the biotin moiety can guide the probe
preferentially to biotin receptor-positive or biotin receptor-
negative tumor cells. Probe 5 was incubated with A549 and
WI38 cells, which are known to be biotin receptor-positive and
-negative cells, respectively. As shown in Figure 2, the A549
cells demonstrate strong fluorescence intensity within a 15 min
Figure 3. Confocal microscopy images of A549 cells treated with 5.
The cells were preincubated with media containing NEM of various
concentrations (0, 0.2, and 0.5 mM) for 30 min at 37 °C. Cell images
were obtained using two photon excitation wavelengths of 750 nm and
emission wavelengths of 525−600 nm, green signal, respectively.
intensity of 5 in A549 cells decreases as the concentration of
NEM is increased (0−0.5 mM). This observed result indicates
that fluorescence come from compound 6 generated by the S−
S bond cleavage of probe 5 by intracellular thiols.
To identify the intracellular location of GMC release from 5,
after entering the cells, colocalization experiments were
performed using fluorescent endoplasmic reticulum (ER)-
and lysosome-selective markers. As seen in the panels of
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Figure 4, the fluorescence ascribable to 5 colocalizes well with
lyso tracker (panel 1) but does not exactly match what is
most concentrations of the probes (Figure S24). From these
results, it is clearly confirmed again that the biotin moiety in 5
obviously plays as a targeting unit to tumor cells in this DDS.
3. CONCLUSIONS
In conclusion, we have reported a new theranostic agent,
GMC−coumarin−biotin conjugate (5) and its synthesis,
characterization, spectroscopic properties, and anticancer
effects as well as biological applications. Upon addition of
free thiol to compound 5, the disulfide bond cleaved, followed
by an intramolecular nucleophilic substitution at the carbamate
moiety, forming a five-membered thiolactone ring. This leads to
release of active GMC and also gives rise to fluorescence
enhancement. The colocalization experiments using commer-
cial available lysosome- as well as ER-selective dyes
demonstrated that compound 5 localized to the lysosme after
receptor-mediated endocytosis. According to confocal micro-
scopic experimental studies, our final prodrug only goes to
biotin receptor-positive A549 tumor cells, over biotin receptor-
negative WI38 cells. Therefore, our DDS could provide a
powerful new strategy for the specific tumor targeting drug
delivery and cellular imaging.
Figure 4. Confocal microscopic images of colocalized experiment in
A549 cells. (a,d) Flourescence images of A549 cells contained with 5
(10.0 μM) for 25 min. (b) Flourescence image of A549 cells incubated
with 5 (10.0 μmol) as well as lyso tracker blue DND-167 (0.05 μM)
for 25 min. (c) Overlay of the merged images of (a) and (b). (e)
Flourescence image of A549 cells incubated with 5 (10.0 μM) and ER
tracker red (0.05 μM) for 25 min. (f) Overlay of the merged images of
(d) and (e). Images of the cells were obtained using excitation
wavelengths of 750 nm, a band path (350−440 nm, blue signal),
(550−600 nm, green signal), and (650−690 nm, red signal), and
emission filters, respectively.
4. EXPERIMENTAL SECTION
Synthesis of 1. This compound was synthesized, followed by
reported procedure,15 with the yield: 50%; H NMR (DMSO-d6, 400
1
MHz): δ 6.72 (d, J = 2.2 Hz, 1H), 6.77 (d, J = 2.3, 8.5 Hz, 1H), 7.44
(d, J = 8.6 Hz, 1H), 7.55 (s, 1H), 10.53 (s, phenolic OH), ppm. 13C
NMR (DMSO-d6, 100 MHz): 102.7, 112.0, 114.5, 121.8, 128.5, 129.8,
153.4, 158.0, 161.0 ppm. HRMS: calcd for C9H6O3N3 (M+1),
204.0409; found, 204.0409.
observed in the case of ER tracker (panel 2). Therefore, it is
noteworthy that thiol-induced disulfide cleavage of 5 occurs in
the lysosome, serving to release the GMC molecule, which
presumably diffuses into the cell nucleus where it replaces one
of the building blocks of nucleic acids, cytidine, during DNA
replication. This process leads to form a faulty nucleoside,
resulting in cell apoptosis.19
Finally, we investigated the anticancer effects of probes 5
bearing biotin moiety and 4 without biotin to demonstrate the
targeting unit effect for cancer cells. Comparison of the cell
viabilities of 5 with 4 toward A549 cells as well as toward WI38
cells was conducted where they are positive and negative
regarding biotin accumulation ability, respectively.14,20 We
found that for A549 cells, compound 5 is a more potent
anticancer drug than 4, which is mostly evident in comparison
of the cell viabilities at 1.0 μM of each compound (Figure 5).
However, in a parallel experiment for WI38 cell lines, two
probes fail to demonstrate such differences in cell viability at
Synthesis of 2. To a stirred solution of DMTr-protected hydroxyl
ethyl disulfide (500 mg, 1.10 mmol) and DIPEA (849 mg, 6.57 mmol)
in dry dichloromethane (DCM) (5.0 mL), a phosgene solution (429
mg, 4.38 mmol, 2.14 mL) was added. The reaction mixture was stirred
at 0 °C under argon atmosphere. After 2 h, the excess phosgene was
removed from the reaction mixture by argon purge. Then 3-azido-7-
hydroxy coumarin (111 mg, 0.55 mmol) in dry DCM was added in the
reaction mixture and was stirred overnight. After completion of the
reaction (by TLC), the reaction mixture was diluted with EtOAc,
washed twice with brine, dried over sodium sulfate, and then filtered,
and the solvent was removed under reduced pressure. The crude
product was purified by column chromatography on silica gel
(EtOAc:hexanes 1:9 to 3:7) to give 2 (460 mg, 61% yield) as a
1
yellow sticky liquid. H NMR (CDCl3, 400 MHz): δ 2.85−2.91 (m,
4H), 3.39 (t, J = 6.1 Hz, 2H), 3.77 (s, 6H), 4.46 (t, J = 6.6 Hz, 2H),
6.82 (d, J = 8.8 Hz, 4H), 7.11 (dd, J = 2.2, 8.5 Hz, 1H), 7.16−7.22 (m,
3H), 7.28 (t, J = 7.8 Hz, 2H), 7.34 (d, J = 8.8 Hz, 4H), 7.38 (d, J = 8.6
Hz, 1H), 7.45 (d, J = 7.3 Hz, 2H), ppm. 13C NMR (CDCl3, 100
MHz): 36.8, 39.9, 55.5, 62.0 67.1, 86.6, 109.8, 113.3, 113.3, 117.5,
118.6, 125.3, 126.3, 127.1, 128.1, 128.2, 128.4, 130.3, 136.2, 145.0,
151.8, 152.2, 152.9, 157.3, 158.7 ppm. ESI-MS: calcd for
C35H32N3O8S2, 685.16; found, 685.48.
Synthesis of 3. Compound 2 (450 mg, 0.65 mmol) was dissolved
in 5.0 mL DCM solvent, and 80% glacial acetic acid was slowly added.
The reaction mixture was kept for 24 h stirring to completion by TLC
(EtOAc:hexanes 1:1). Glacial acetic acid was neutralized by the
saturated aqueous sodium bicarbonate solution. EtOAc was added, the
organic phase was dried over sodium sulfate and then filtered, and the
solvent was removed under reduced pressure. The crude product was
purified by column chromatography on silica gel (EtOAc:hexanes 2:8
1
to 5:5) to give 3 (180 mg, 71% yield) as a yellow sticky liquid. H
NMR (CDCl3, 400 MHz): δ 2.87 (t, J = 5.8 Hz, 2H), 3.00 (t, J = 6.6
Hz, 2H), 3.86 (t, J = 5.8 Hz, 2H), 4.51 (t, J = 6.6 Hz, 2H), 7.11 (dd, J
= 2.2, 8.6 Hz, 1H), 7.17−7.20 (m, 2H), 7.40 (d, J = 8.6 Hz, 1H), ppm.
13C NMR (CDCl3, 100 MHz): 36.7, 41.7, 60.5, 67.0, 109.8, 117.5,
Figure 5. Cell viability of 5 and 4 on A549 cell lines. All compounds
were incubated with the cells for 72 h, and the cell viability observed
via MTT assay.
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118.6, 125.4, 126.3, 128.3, 151.7, 152.1, 152.9, 157.3 ppm. HRMS:
calcd for C14H14N3O6S2 (M+1), 384.0324; found, 384.0326.
Met, Lys, Leu, Ile, His, Gly, Gluc, Glu, Gln, Asp, Asn, Arg, Ala, Trp,
Zn(II), Na(I), Mg(II), K(I), Fe(III), Fe(II), Cu(II) and Ca(II)] were
prepared in triple-distilled water. Stock solutions of 5 were also
prepared in triple-distilled water. All spectroscopic measurements were
performed under physiological conditions (PBS buffer containing 25%
(v/v) of DMSO, pH 7.4, 37 °C). Absorption spectra were recorded on
a S-3100 (Scinco) spectrophotometer, and fluorescence spectra were
recorded using an RF-5301 PC spectrofluorometer (Shimadzu)
equipped with a xenon lamp. Samples for absorption and emission
measurements were contained in quartz cuvettes (3 mL volume).
Excitation was provided at 410 nm with excitation and emission slit
widths of 3 and 3 nm, respectively.
Preparation of Cell Cultures. Human lung adenocarcinoma
epithelial cells (KCLB, Seoul, Korea) were cultured in RPMI
(WelGene Inc., Seoul, Korea) supplemented with 10% FBS
(WelGene), penicillin (100 units/ml), and streptomycin (100 μg/
mL). Two days before imaging, the cells were passed and plated on
glass-bottomed dishes (MatTek). All the cells were maintained in a
humidified atmosphere of 5/95 (v/v) of CO2/air at 37 °C. For
labeling, the growth medium was removed and replaced with RPMI
without FBS. The cells were treated and incubated with ligand at 37
°C under 5% CO2 for 15 min. The cells were washed three times with
phosphate buffered saline (PBS; Gibco) and then imaged after further
incubation in colorless serum-free media for 15 min.The cells were
seeded on 24-well plates and stabilized for overnight. Compound 5
was applied to the cells to monitor their uptake and drug release, as
discussed in the main text above. In some experiments, the cells were
incubated with media containing lyso- or ERtracker prior to treatment
with 5. Then the cells were briefly washed with 1 mL of PBS and were
then treated with 5 in PBS. After incubation, residual quantities of 5
that were not taken up in the cells were removed by washing the cells
three times with PBS before the cells were placed in 1 mL of a PBS
solution. Fluorescence images were taken using a confocal laser
scanning microscope (Zeiss LSM 510, Zeiss, Oberko, Germany).
Synthesis of 4. To a DCM (5 mL) solution of 3 (100 mg, 0.26
mmol) 4-nitrophenyl chloroformate (157.7 mg, 0.78 mmol), DIPEA
(134 mg, 1.04 mmol) and a catalytic amount of pyridine were added at
0 °C and stirred for 5h at rt. Then the reaction mixture was
concentrated in vacuo. The crude residue was dissolve in 5 mL DMF.
To this solution, GMC (205 mg, 0.78 mmol) in DMF (2 mL) and
TEA (0.5 mL) were added and continued to stir for 24 h. After
completion of reaction, the reaction mixture was diluted in water. The
compound was extracted with EA. The organic layer was dried over
anhydrous sodium sulfate. The crude compound was passed through
silica column chromatography using DCM/MeOH (9:1) as eluent to
1
afford a sticky yellow liquid compound 4 (30 mg, 17% yield). H
NMR (CDCl3, 400 MHz): δ 2.95−3.06 (m, 4H), 3.78 (s, 2H), 3.99−
4.14 (m, 2H), 4.66−4.55 (m, 4H), 5.30 (s, 1H), 5.82(d, J = 6.00 Hz,
1H), 6.23(br s, 1H), 7.14 (d, J = 6.5 Hz, 1H), 7.22−7.23 (m, 1H),
7.37−7.45 (m, 2H), 7.55 (br s, 1H), 8.26 (d, J = 7.2 Hz, 1H), ppm.
13C NMR (CDCl3, 100 MHz): 36.6, 36.9, 55.1, 59.7, 65.6, 66.9, 79.0,
95.9, 109.6, 117.4, 118.5, 121.9, 125.5, 125.4, 128.2, 141.6, 151.6,
152.8, 153.8, 155.6, 156.0, 165.9, 166.1 ppm. HRMS: calcd for
C24H23F2N6O11S2 (M+1), 673.0834; found, 673.0833.
Synthesis of 5. To a MeOH (3.0 mL) solution of 4 (70 mg, 0.01
mmol), 9 (29 mg, 0.01 mmol) and sodium ascorbate (10 mol %) were
added. The reaction mixture as degassed for 15 min by purging argon
gas. Then 2 mg (0.002 mmol) of CuSO4 in 0.5 mL water was added to
the reaction mixture and stirred for an additional 3 h. The crude
reaction mixture was directly passed through silica column
chromatography using DCM/MeOH (8.5:1.5) as eluent to afford a
1
yellow sticky liquid compound 5 (25 mg, 25% yield). H NMR (300
MHz, DMSO-d6): δ 1.21−1.32 (m, 2H), 1.36−1.58 (m, 4H), 2.05 (t, J
= 7.3 Hz, 2H), 2.53−2.57 (m, 2H), 2.77−2.83 (m, 2H), 2.99−3.10 (m,
5H), 3.68 (br s, 2H), 3.79−3.82 (m, 3H), 4.08−4.12 (m, 2H), 4.26−
4.31 (m, 3H), 4.37−4.41 (m, 2H), 5.25−5.27 (m, 1H), 5.79 (d, J = 7.4
Hz, 1H), 6.18−6.25 (m, 1H), 6.36−6.42 (m, 3H), 7.43−7.45 (m, 2H),
7.64 (d, J = 7.5 Hz, 1H), 8.21−8.24 (m, 2H). 13C NMR (CDCl3, 100
MHz): 25.8, 28.3, 28.7, 28.8, 35.5, 36.9, 38.7, 55.4, 56.1, 59.8, 61.7,
65.8, 73.5, 82.0, 95.7, 101.2, 108.1, 109.0, 116.4, 116.8, 118.9, 119.5,
121.7, 124.7, 126.9, 127.0, 128.1, 130.5, 140.7, 142.1, 147.7, 153.7,
155.6, 163.3, 163.4, 166.3, 172.5 ppm. ESI-MS: calcd for
C37H42F2N9O13S3( M+1), 954.20; found, 954.21.
ASSOCIATED CONTENT
* Supporting Information
■
S
Additional spectra (UV−vis absorption, fluorescence, NMR,
ESI-MS, HRMS) and cell viability data. This material is
available free of charge via the Internet at http://pubs.acs.org.
Synthesis of 6. To a MeOH (3.0 mL) solution of 1 (200 mg, 0.98
mmol), 9 (277 mg, 0.98 mmol) and sodium ascorbate (10 mol %)
were added. The reaction mixture as degassed for 15 min by purging
argon gas. Then 5 mol % of CuSO4 in 0.5 mL water was added to the
reaction mixture and stirred for 4 h. Then the crude reaction mixture
was directly passed through silica column chromatography using
DCM/MeOH (8.5:1.5) as eluent to afford a yellow solid 9 (270 mg,
AUTHOR INFORMATION
Corresponding Author
kangch@khu.ac.kr; jongskim@korea.ac.kr
Notes
■
The authors declare no competing financial interest.
1
57% yield). H NMR (400 MHz, DMSO-d6): δ 1.21−1.30 (m, 2H),
ACKNOWLEDGMENTS
1.39−1.56 (m, 4H), 2.08 (t, J = 6.7 Hz, 2H), 2.51 (br s, 1H), 2.74−
2.78 (m, 1H), 3.03−3.07 (m, 2H), 4.07−4.10 (m, 1H), 4.25−4.27 (m,
1H), 6.32 (s, 1H), 6.39 (s, 1H), 6.81 (br s, 1H), 6.87 (d, J = 6.3 Hz,
1H), 7.71 (d, J = 7.8 Hz, 1H), 8.32 (br s, 1H), 8.37−8.39 (m, 1H),
8.54 (br s, 1H). 13C NMR (CDCl3, 100 MHz): 25.9, 28.7, 28.9, 34.6,
35.7, 39.7, 56.1, 59.9, 61.7, 102.8, 111.0, 115.0, 120.0, 124.3, 131.6,
136.7, 146.1, 155.3, 157.0, 163.2, 163.4, 172.8 ppm. HRMS: calcd for
C22H25N6O5S (M+1), 485.1607; found, 485.1606.
■
This work was supported by the CRI project (20120000243)
(J.S.K.) and by Basic Science Research Program
(2012R1A1A2006259) (C.K.) through the National Research
Foundation of Korea funded by Ministry of Education, Science
and Technology
REFERENCES
Synthetic Materials and Methods. Chemicals used in this
project were purchased from Aldrich, Alfa-Aesar, Carbosynth, TCI,
and Ducsan without further purification. Silica gel 60 (Merck, 0.063−
0.2 mm) was used for column chromatography. Analytical TLC was
performed using Merck 60 F254 silica gel (precoated sheets, 0.25 mm
■
(1) (a) Garnett, M. C. Adv. Drug Delivery Rev. 2001, 53, 171−216.
(b) Jaracz, S.; Chen, J.; Kuznetsova, L. V.; Ojima, I. Bioorg. Med. Chem.
2005, 13, 5043−5054. (c) Yokoyama, M. J. Artif. Organs 2005, 8, 77−
84. (d) Torchilin, V. P.; Schafer-Korting, M. Handbook of Experimental
Pharmacology; Springer: Berlin, 2010; p 3.
1
thick). H and 13C NMR spectra were collected in CDCl3, DMSO
(2) Andersen, E. S.; Dong, M.; Nielsen, M. M.; Jahn, K.; Subramani,
R.; Mamdouh, W.; Golas, M. M.; Sander, B.; Stark, H.; Oliveira, C. L.
P.; Pedersen, J. S.; Birkedal, V.; Besenbacher, F.; Gothelf, K. V.; Kjems,
J. Nature 2009, 459, 73−76.
(3) (a) Brown, S. D.; Nativo, P.; Smith, J.-A.; Stirling, D.; Edwards, P.
R.; Venugopal, B.; Flint, D. J.; Plumb, J. A.; Graham, D.; Wheate, N. J.
J. Am. Chem. Soc. 2010, 132, 4678−4684. (b) Voliani, V.; Signore, G.;
(Cambridge Isotope Laboratories, Cambridge, MA) on Varian 300
and 400 MHz spectrometers. All chemical shifts are reported in ppm
value using the peak of residual proton signals of TMS as an internal
reference. The mass spectra were obtained on an Ion Spec HiRes mass
spectrometer.
Spectroscopic Materials and Methods. Stock solutions of
biologically relevant analytes [thiols, Val, Tyr, Thr, Tau, Ser, Pro, Phe,
E
dx.doi.org/10.1021/ja401350x | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
Nifosí, R.; Ricci, F.; Luin, S.; Beltram, F. Recent Pat. Nanomed. 2012, 2,
1−11.
(4) Jung, J.; Solanki, A.; Memoli, K. A.; Kamei, K.-I.; Kim, H.; Drahl,
M. A.; Williams, L. J.; Tseng, H.-R.; Lee, K. Angew. Chem., Int. Ed.
2010, 122, 107−111.
(5) Francis, M. F.; Cristea, M.; Winnik, F. M. Pure Appl. Chem. 2004,
76, 1321−1335.
(6) (a) Yang, P.; Gai, S.; Lin, J. Chem. Soc. Rev. 2012, 41, 3679−3698.
(b) Singh, N.; Karambelkar, A.; Gu, L.; Lin, K.; Miller, J. S.; Chen, C.
S.; Sailor, M. J.; Bhatia, S. N. J. Am. Chem. Soc. 2011, 133, 19582−
19585.
(7) Lee, M. H.; Kim, J. Y.; Han, J. H.; Bhuniya, S.; Sessler, J. L.; Kang,
C.; Kim, J. S. J. Am. Chem. Soc. 2012, 134, 12668−12674.
(8) Plunkett, W.; Huang, P.; Gandhi, V. Anti-Cancer Drugs 1995, 6,
7−13.
(9) Noble, S.; Goa, K. L. Drugs 1997, 54, 447−472.
(10) Friberg, G.; Kindler, H. Curr. Oncol. Rep. 2005, 7, 186−195.
(11) Zhang, J.; Visser, F.; King, K.; Baldwin, S.; Young, J.; Cass, C.
Cancer Metastasis Rev. 2007, 26, 85−110.
(12) Reid, J. M.; Qu, W.; Safgren, S. L.; Ames, M. M.; Krailo, M. D.;
Seibel, N. L.; Kuttesch, J.; Holcenberg, J. J. Clin. Oncol. 2004, 22,
2445−2451.
(13) Moog, R.; Burger, A.; Brandl, M.; Schuler, J.; Schubert, R.;
Unger, C.; Fiebig, H.; Massing, U. Cancer Chemother. Pharmacol. 2002,
49, 356−366.
(14) Chen, S.; Zhao, X.; Chen, J.; Chen, J.; Kuznetsova, L.; Wong, S.
S.; Ojima, I. Bioconjug. Chem. 2010, 21, 979−987.
(15) Sivakumar, K.; Xie, F.; Cash, B. M.; Long, S.; Barnhill, H. N.;
Wang, Q. Org. Lett. 2004, 6, 4603−4606.
(16) Cordero, F. M.; Bonanno, P.; Chioccioli, M.; Gratteri, P.;
Robina, I.; Vargas, A. J. M.; Brandi, A. Tetrahedron 2011, 67, 9555−
9564.
(17) Kim, H. -C.; Kreiling, S.; Greiner, A.; Hampp, N. Chem. Phys.
Lett. 2003, 372, 899−903.
(18) Gregory, J. D. J. Am. Chem. Soc. 1955, 77, 3922−3923.
(19) Cerqueira, N. M. F. S. A.; Fernandes, P. A.; Ramos, M. J.
Chem.Eur. J. 2007, 13, 8507−8515.
(20) Heo, D. N.; Yang, D. H.; Moon, H. -J.; Lee, J. B.; Bae, M. S.;
Lee, S. C.; Lee, W. J.; Sun, I. -C.; Kwon, I. K. Biomaterials 2012, 33,
856−866.
F
dx.doi.org/10.1021/ja401350x | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX